Journals on Biomedical Science |BJSTR

Alzheimer’s Disease: Nanotherapeutics

Editorial

Nanotherapeutics is forefront of new strategies that is nanotechnology [1] due neurons size of nanotools and devices which fall in the range of a billionth of a meter, are being considered to treat and diagnose neurodegenerative diseases [2,3]. Irregularities in the natural functioning of neurons, initiate brain health disorders. Therefore, to treat these neurons is a very complicated task and even their locations are beyond the common approach. Nanotherapeutics [4] is quite capable to do the aforesaid chore and can be a good choice to be a therapy for neurodegenerative diseases, such as Parkinson’s disease (PD) and Alzheimer’s disease (AD) [5]. Various nanoparticles have been reported that can target brain in-vitro and in-vivo. Recently, the association of microglia, the brain-immune cells and uncontrolled inflammation has been discovered by the researchers of Rutgers University. Microglia tackled pathological changes within the brain to identify the undesired and foreign particles, so that it be cleared in a fast mode. The neurodegenerative disorders are very complex physiological, to resolve this issue, it is not a good approach to solely have a concern with searching therapy for neurons, but there are needs to search the mediators and those should be treated first, like in this case microglia, the mediator of neuron physiology and it be targeted first as this new strategy. This finding is considered as a breakthrough and could be a potential therapeutic target [6]. Such an approach may further reduce brain inflammation.

According to described literature, nanoparticles as a therapy and source of diagnosis of Alzheimer disease can cross the BBB and eliminate the Aß burden [7]. Finally, this therapy can slow the long-term progression of the disorder. The abnormal structure present in the brain of AD patients are senile plaques (deposits of the protein) and neurofibrillary tangles (neuron exposure) and both of them tied with Aß. By removing Aß, these disorders will be healed. It is very unfortunate that no researcher can get success in it. By applying nanotechnology, a few nanoparticles and nanomaterials have been designed that can simultaneously cross over BBB and hit the desired target [8]. Moreover, nanotechnologically developed tools and devices can perform diagnosis and imaging of brain diseases, including brain ischemia, stroke, and inflammation of the nervous system [9]. More interestingly, nano-biosensors have been developed that have a high grade of sensitivity for earlier detection of AD. The synthesis of biologically active HDL-based nanomaterials was reported through a microfluidic technology [10]. Lipid, protein, gold, quantum dot, and iron oxide, are the key components of aforesaid nanoparticles [11] These specifically designed nanoparticles can interact with microglia and this is what required. These interactions will explore new avenue for designing those nanomedicine platforms that can enable targeted delivery with zero toxicity. [12] Nanotherapeutics prescribed for curing Alzheimer’s disease that explores various the applications of nanotechnology in clinical neuroscience for developing innovative therapeutic for treating AD [1].

Nanomedicine-based immunotherapy summarize to be considered for developing nanomedicine-based therapy to overcome the pathways of BBB and that can mimic the innate immune actions [13]. Nanorobotics are automatic molecular machines that can travel and monitor different compounds, and can store that information [14]. Nanotherapeutic that can combat excitotoxicity during stroke, TBI, and hypoxia–ischemia and deal with mitigating oxidative stress, was described [15]. Nanomedicines were reported that can replace or enrich diseased or injured cells and tissue and replenish the local environment or can halt further degeneration and hence capable in dissolve translational challenges for treating neurological disorders [16]. Generally, microglia protect neurons by removing pathological agents, but if a long-term exposure of microglia stimulants initiate neuronal damage because of secretion of pro-inflammatory agents, resulting neuroinflammation and neurodegeneration [17] Designing microglia-targeting nanotherapeutics for the treatment of neurodegenerative disorders was presented [18]. Intranasal delivery of nanotherapeutics and nano biotherapeutics were designed as an engineered nanocarriers as a smart delivery system for treating AD and highlighted as a proficient approach [19]. In order to assess the potential applications of the medicinal plants, their derived components, and Phyto-Therapeutic, nano medicinal strategies have been developed that can transport aforesaid components for treating AD [20].

Flavonoid-based nanomedicines inhibit the accumulation of large aggregates state of amyloid-beta 42 (Aβ42), and hindered the formation of neurofibrillary tangles of hyper-phosphorylated forms of microtubule-associated tau protein, during AD [21]. The stem cell and nano-based therapies were applied as novel therapeutic strategies for treating AD, associated pathogenesis and pathogenic trajectories [22]. Nanomedicine [23] can offer impressive resolutions as a tiny machine for the healthcare that will cure a lot of life threatening diseases [24] Nanocarriers having polysaccharides as a component are remarkable as nanotherapeutics for curing Alzheimer disease and increase the transportation of the desired molecules [25]. Exploring recent advances in nanotherapeutics being tiny molecules of immunotherapeutic have many advantages over biological drugs regarding complexity, tissue penetration, manufacturing cost [26] Extracellular vesicles were also applied to treat neuroinflammation and are very helpful for pathogenesis, diagnosis, and therapy [27] Nanotherapeutics medicine of extracellular vesicles performed as theragnostic platform for biogenesis, membrane trafficking, and oxidative stress [28]. Highly bioactive zeolite imidazole framework-8–capped were applied as nanotherapeutics to suppresses inflammation- and the activation of astrocytes and secretion of proinflammatory cytokines, and notified as neuroprotective therapy. [29] Nanotools can detect the degeneration of amyloid-β peptide (Aβ) oligomers, and act as suppressor of oxidative stress of free radicals. [30].

Advances in personalized nanotherapeutics is a as state-of-theart, that can be applied in various strategies and developed a novel nano-enabling therapeutic schemes to cure and monitor diseases. [31] The emergence of nanotherapeutics from laboratory to clinic comprehensively discussed and the persisted shortcomings for delivery of classical (small) drugs, macromolecular therapeutics, and recombinant vaccine were illustrated. [32,33] Nanomedicine overcome the obstacles and improve diagnostic and therapeutic outcomes in the healing neurodegenerative disorders. [34] Nano neuro medicines can be applied for neuroimaging as well as for therapeutic applications for degenerative, inflammatory and infectious disorders of the nervous system. [35] Small interfering RNAs, displayed remarkable promises and underlined as potential therapeutics option for curing AD. [36] Precision nanomedicine was reported as target-specific agents and are able to bypass the blood-brain barrier rapidly, and ultimately become a novel therapeutic modality to treat neurodegenerative diseases. [37] Potential use of nanomedicine for treating anti-inflammatory neurodegenerative diseases, (AD and PD) was underlined and that have ability of penetration of the BBB to achieve a therapeutic potential in the field of therapy that can be used for healing of neurodegenerative diseases. [38] Macrophage targeted theragnostic as personalized nanomedicine were discovered that can be successful as a theragnostic in macrophage ablation, phenotype modulation and inhibition of their inflammatory activity [39].

Selective targeting capabilities of nanomedicine have been reported that can deal with the pathological permeability of the blood– brain barrier during neurovascular inflammation [40]. Comparatively to conventional formulations, a number of nano formulations were recommended for improving the pharmacodynamics of the encapsulated drug for healing inflammatory diseases that can improve the quality of therapeutic treatment for curing diseases like AD and PDs. [41] Published article covered the promising aspects of ongoing developments in the diagnosis and management of Alzheimer’s disease by applying nanotechnology. [42,43] Emerging role of nanotechnology and different nanoparticle (Gold, quantum dots, polymeric, liposomes, carbon nanotubes, and fullerenes) were highlighted for treating neurological disorders, including AD, PD and Huntington’s disease and has shown great potential to overcome the problems. Phosphorus dendrimers that have potential to fight against cancers, neurodegenerative diseases, and inflammation, both in vitro and in vivo, were described. [44] A book, entitled “Nanomedicine for Inflammatory Diseases” covered different aspects of translational medicine for clinicians and disclosed various aspects for treating inflammatory disease and bridging the gap between the laboratory bench top and the clinical bedside [45].

Acknowledgements

One of the authors, Rajiv Kumar, gratefully acknowledges his younger brother, Bitto.

Consent for Publication

Not Applicable.

Conflict of Interest

The authors declare no conflict of interest, financial or otherwise.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data Availability Statement

Due to the nature of the research, [ethical, legal/commercial] supporting data is not applicable and thus not available.

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Open access clinical and medical journal |BJSTR

Educating to Generate Self-Learning

Introduction

Universities continue to reject a large number of students who, despite having previously completed the official and necessary school cycles (preschool, primary, secondary and baccalaureate), did not manage to acquire solid and sufficient knowledge in a period of at least sixteen years or more of schooling to pass the evaluations that are applied to them as a requirement to be accepted as new students to higher education. Entering a school becomes, and is seen by the student and society, a mere formality, a matter full of bureaucratic-administrative processes and tasks that take up a lot of time, which at the time is carried out with a superficial character, out of obligation and without conviction or depth, when it should be a procedure for the school authorities, Teachers and parents and the applicants themselves would know their strengths and weaknesses in terms of their knowledge and knowledge and therefore be able to apply themselves to continue strengthening or correct their deficiencies. In addition, learning and consequently the knowledge and knowledge of students is a variable that is almost always taken into account by employers who demand certain competencies from their future employees. This situation is notorious due to the gap between the competencies certified by the professional degree and the real skills of the university graduate, this difference is a serious problem since it is thought that the graduate is less and less prepared to face the new professional and labor standards that are required. On the other hand, the concerns and complaints of students in this regard are also abundant, for them the educational system does not teach them correctly to solve problems, to make decisions, to think and to learn how to self-learn.

Most teachers are expert information processors, however they are primarily concerned with the results they must achieve, as well as the number of topics they have to cover in tight periods of time and more concerned with teaching than with student learning; To be able to decipher why the teacher, even in these times, continues to think that teaching is more of a priority than learning is very difficult because his thinking and that of the student is a black box, an enigma that is difficult to unravel. The presentation of the contents of a topic in higher education should not depend mainly on the erudition capacity of the teacher, since this situation does not ensure that the group follows it and understands the explanation, these classes can provide in general terms to the group of students congruent, structured and valuable information in general and particular, But there will always be students who are not able to understand and assimilate the information because they do not have the knowledge, nor the level of understanding that the teacher has reached around them, all of which determines that the information presented is not internalized and amalgamated with the information they already have, the information received is dispersed in a series of unconnected data, isolated, fragments of information, unrelated to previous knowledge or situations of daily or professional life. In terms of learning, it is still tied to intuitive practices of common sense, to the old popular affirmations and beliefs about the process of teaching and learning, that it is the obligation of the teacher to teach and the student to learn from him, rigid strategies and tactics are used and formats as old as the rote and arbitrary reception of reading by the student and relying on memory for an evaluation or a In addition, the student trusts in his ability and ability to copy and the teacher in silencing his conscience by making him see nothing, all this and more is in part what irremediably condemns what he has learned to oblivion, although in these cases that is not the important thing, what matters is to obtain a passing grade (Granados López H, et al. [1]).

Self-learning, which, unlike teacher-conditioned learning, can lead the student to move towards academic independence and thus be in the future a better professional who really contributes to the solution not only of the problems of society in general, but also of his, for this the teaching of study strategies in students is a primary factor to achieve better learning based on self-learning and thus facilitate their school transition and increase the chances of academic success.

Educating to Stop Self-Learning

Experimental studies and achievements in the fields of artificial intelligence and cognitive psychology of learning have made it possible to formulate new perspectives and redefine the laws that regulate human learning, which change the logic of education. When we talk about difficulties at school, we immediately think of bad grades, long and tedious class sessions, failed exams, threats of expulsion, failure, stress, depression, etc., school demands are a fact that we can only accept exist; If the student does not comply in the way that the teacher expects or the institution demands, then other means are resorted to make him work harder, even if the parents, teachers or coordinators and/or those responsible for his education do not seek the remedy directly on what produces the problem. Rather, they act on the causes that originate the problem, with this the students remain subject to the set of obligations established by the educational system, within which studying to learn and know in a more satisfactory and broader way, represents for the student one of the most punitive and most volitional obligations. It would be interesting to ask ourselves about what educational demands cost the student and how much guilt and responsibility teachers have in the fatigue, instability, anxiety, blockage, deception and repudiation of study originated in the student; This consideration is not only about asking simple and plain questions, without foundation, that provoke reactions of indifference, it is about creating a current situation that gives rise to situations and the establishment of a link between education or teaching and educational research, where the action of students, teachers and parents improve the conceptions that are held in this regard, intending in the end to qualitatively sensitize the student about the importance of learning and knowledge for the achievement of each student’s particular life project.

When talking about strategies, techniques and study habits and their effect on learning, it is common to start by rescuing that motivation is an essential factor to achieve academic success as a result of the effort of study strategies and learning as an inherent instrument to achieve the proposed goals, the strengthening of self-esteem and confidence in one’s own competence as essential traits to obtain a good school performance and to be able to face the social commitment that the student contracts when being educated, giving society as a professional all the accumulation of knowledge and skills as homogeneous as heterogeneous that were achieved through their course through school institutions. Nowadays it is not enough to learn to read, write and solve simple calculations, it is also important to analyze and address in a timely manner those aspects that determine many of the problems related to high failure rates and low terminal efficiency. It is known that motivation is an essential and necessary factor for satisfactory academic achievement, for the orientation towards success and in the valuation of effort and skills as an inherent instrument to achieve the proposed goals, as well as the strengthening of self-esteem and confidence in one’s own competence as essential traits. The quality of education depends, to some extent, on the knowledge and skills it fosters corresponding to what the student requires and what their environment demands, for this it is necessary to strengthen self-learning thanks to study or learning strategies. Study strategies are currently considered as any behavior or thought that facilitates coding in such a way that it improves the integration and retrieval of knowledge; More specifically, these thoughts and behaviors constitute organized plans of action designed to achieve a goal.

From the new cognitive conceptualization of study strategies, new research began to be carried out with a different approach, since previously studies on human learning were dominated by the behaviorist school of thought, this resulted in a total dependence on the effects of external events as determinants of what was learned. With the increase in interest in organizational mental processes and in the transformation of information that takes place within the student, interest was refocused on what was known as the black box of the human mind (Barca Lozano A, et al. [2]). At the end of the 20th century, there was a reassessment of the role of educational systems, investment in human resources, in more and better training of professionals and citizens has once again been considered a primary and priority objective to promote development and social and individual progress.

These new approaches are due to the fact that in the last thirty years the changes, trends and professional, labor and social demands advance at a dizzying pace, being the most important trends that have had the greatest impact on the educational, professional and labor world the following: The internationalization of the economy that is transforming social relations, the dominant culture and values according to the rules of the market economy; the globalization of communication and information, with increasingly agile and powerful systems displacing traditional media; The information society is already a fact in which progress is made day by day with innumerable consequences in social life, in the advancement of knowledge and in the organization of work; scientific and technological development, which extends to all areas of knowledge and has a decisive influence on the social field, which poses significant challenges to the structure of curricular content and its development; the disappearance of barriers between nations and the attractiveness of developing countries are producing migratory movements with enormous impacts; cultural, religious and ethnic integration, avoiding cultural assimilation, is one of the most important challenges for national education systems; demographic and family changes, among which four main trends can be highlighted: the ageing of the population, the relative ageing of employed workers as the recruitment of young people is reduced, the increase in single-parent families posing new responsibilities for educational establishments that care for children at an early age, and the increasing incorporation of women into the world of work, However, there is still significant gender inequality in the labour market. The diversification of employment where the time of stable and long-term work at the end of studies has ended, the present situation is characterized by the difficulty of finding employment, by part-time work, by the increase in self-employment and by the need for constant changes in work according to new demands. the continuation of unemployment or underemployment, where there is no reasonable prospect that it will fall in the immediate future and where the most affected groups are those who cannot obtain a job due to their limited qualifications or because of the difficulties in accessing the specialization required by the new jobs; the ideological and moral pluralism of society, where the changes that are taking place in society as a whole are separating the consensus around the values to be transmitted; There is a risk that the fragmentation of values will lead only to pragmatic criteria, which will serve as a reference point for human behaviour.

It is possible that a new society has been taking shape for some time, a society saturated with information and knowledge, impregnated with science and technology, open to the world, a society characterized by the situation of the various individual situations, marked by the variety of their rhythms, a society eager for capacities in perpetual renewal, a society that could also be called a society of education or training, and for this new society it is necessary to The emergence of a new educational system based on self-learning and meta-learning, where students are taught the importance and value of knowledge and the impact that educational convictions and study strategies can have on success or failure, not only at school, but also professionally. If the purpose is for the student to learn how to learn, to be able to generate his own style of self-learning, that is, to be the manager of his own learning, to be active, participatory, dynamic expository, creative, innovative and transformative of his own reality, it is necessary that he be taught, guided and educated how to get there, and for that obviously a competitive teaching staff is required. active, participatory, dynamic, expository, creative, innovative and transformative of their own reality. Undoubtedly, if it is necessary to really have a student who becomes a student, it is necessary to teach him how to be one and for this the strategies are vast and diverse, both to be used by the teacher and by the student.

The use of strategies in students is handled as a constructivist conception of learning, where the arguments that support the way of teaching are illustrated, in this regard it can be said that it is the idea that the individual maintains in cognitive and social aspects of behavior, as well as in affective aspects where it is not the product of the environment or the results of his internal dispositions. rather, it refers to the reconstruction that takes place day by day (Castillo Claure Víctor, et al. [3]). The human being builds his own knowledge where he uses the schemes he already has, to then relate it to the new knowledge that at a certain moment provokes a conflict to then establish a balance; In order for the construction process to be developed, it is necessary to point out two fundamental aspects: What previous knowledge the student has regarding the new information or the activity or task to be solved and what external activities or activities are carried out. must be carried out by the student. Constructivism in school learning seeks the idea of fulfilling the purpose of the education provided by the school, that of promoting the processes of personal growth of the learner. The characteristics of students’ prior knowledge differ from one subject to another, some knowledge is more conceptual and others more procedural; some are more descriptive, others more explanatory; some more general, others more specific, etc., the student must be provided with general knowledge to help him modify his previous ideas, it is necessary that the presentation of school knowledge is based on situations of daily life, this so that the student takes more interest in the effort made in the school (Diaz Barriga Arceo F, et al.[4]).

Influence of the Cognitive Approach

The main interest of the cognitive approach has been focused on describing and analyzing various processes, such as perception, attention, comprehension, thinking, knowledge representation, memory, problem solving, among others, based on the human information processing approach, which currently constitutes the central current of thought in both psychology and education. The emphasis is located on the study of mental processes and on the examination of the structures of knowledge that can be deduced from the different and varied forms of human behavior, this current of thought has tried to explain some aspects that were not studied by associationist approaches, such as the effects of the characteristics of the learner on learning, its role as a processing organism, the ways of thinking at a high level, what it processes during a learning situation, the consequences of certain forms of processing, and the way learning outcomes are measured. Consequently, it can be said that the cognitive approach has influenced the theory and research on human learning in a significant way, regarding this approach the following can be said: It conceives learning as an active and constructive process, emphasizes the presence of high-level processes in learning, points out that learning is a cumulative process in which prior knowledge plays a fundamental role. In other words, learning is a process that consists of the accumulation of information which is organized in our cognitive structures or schemas in such a way that they are enriched and structured until they reach levels of refinement that are characteristic of expert subjects, this approach tries to determine the way or ways in which knowledge is represented and organized in memory. Some say that representation is in the form of images, but others say that it is in the form of verbal propositions or statements and point out that the organization is hierarchical and others that it is in the form of networks (Serrano González Tejero J M, et al. [5]).

Learn to Build

Learning contributes to development to the extent that learning is not copying or reproducing reality; For the constructivist conception, learning occurs when one is able to elaborate a personal representation about an object of reality or content that one intends to learn. This elaboration implies approaching the object or content in order to apprehend it; It is not an empty approach, from nothing, but of the previous experiences, interests and knowledge that one has, we could say that with the meanings that have been constructed one comes into contact with a new aspect that only sometimes seems new, but that in reality can be interpreted perfectly with the meanings that one already possesses. while at other times it will be a challenge to which an attempt will be made to respond by modifying the meanings already provided in such a way as to account for the new content, phenomenon or situation. In this process, not only is the known modified, but the new is also interpreted in a peculiar way, so that it is registered and made one’s own; when this process occurs, it is said that one learns meaningfully, constructing one’s own personal meaning for an object of knowledge that objectively exists (Erausquin C, et al. [6]).

Effective Study and Learning Strategies

Studying is a process that consists mainly of activities carried out by students in order to prepare themselves to take tests, exams or other types of tasks, with the purpose of passing academic courses in educational institutions. Many students finish their high school studies with no or very few study strategies, so universities and other higher education institutions have found it necessary to offer training programs in this type of strategy, with a remedial or compensatory perspective. The area of study strategies was once known as study habits. This name was mainly due to the preponderance of associationist and behaviorist models of learning, which pointed out that in order to learn something it was necessary to internalize it, through repetition and practice creating habits. It could be pointed out, then, that such positions promoted study habits related mainly to external aspects of the student such as: organizing and establishing a study schedule, choosing a place to study, among others; With the change of orientation in the approach to learning processes from an associationist and/ or behaviorist perspective to a cognitive perspective, study began to be conceived as a set of internal and external processes carried out by students to acquire knowledge and develop skills and abilities that allow them to pass academic courses in the educational institutions of the educational system. It is important, in order to understand the difference between habit and strategy, to distinguish between studying and other forms of learning, depending on the purposes and context, learning is not the same as studying; Learning can be the result of a set of processes that can occur anywhere, we can learn in the street, watching television, reading a book, visiting a museum or exercising in a gym, but we also learn in educational institutions called schools, being that the difference between one type of learning and another is that in schools it is academic learning and in other places it is everyday learning.

Carrying out one or more study strategies implies the performance of a set of explicit or implicit operations that students perform during the study process, it basically refers to all those activities that allow them to learn the information contained in materials or texts in the form of prose; To do this, the strategies can be diverse, such as copying the material, taking notes, underlining or highlighting the important parts of the material and summarizing the information, among others. The purpose of study strategies is to help the student pay attention to the important aspects of the study material, to make sure to transfer the material to the working memory because as we know if this information is elaborated, that is, worked, we can store it in our permanent memory system or else it will be lost. The influence that these strategies can have on the study can be as follows:

They direct the student’s attention to certain relevant parts of the information contained in the text, limit the amount of attention the student pays to the information in the text, stimulate the condition by allowing the reader to add information to the information already stored in his or her memory system, allow the establishment of internal relationships, forcing the student to construct an outline or give a coherent organization to the material, and allow the construction of information in the text. external relations, encouraging the student to add his/her comments or reactions to the information contained in the text (Mauri T, et al [7]).There are general strategies, such as broad activities related tocific skills or resources we use when performing a task. On the other hand, there are strategies of repetition, elaboration, organization, regulation and affective, considering each of them in basic learning tasks and in complex learning tasks. Most of them can be considered as micro or macro learning strategies; Micro strategies are more specific to each task, are more related to specific knowledge and skills and can be taught, macro learning strategies aim at the knowledge and understanding of the subject’s own learning mechanisms, have a high degree of transfer and are difficult to teach.

reasoning and thinking, and mediation strategies, which are the specific skills or resources we use when performing a task. On the other hand, there are strategies of repetition, elaboration, organization, regulation and affective, considering each of them in basic learning tasks and in complex learning tasks. Most of them can be considered as micro or macro learning strategies; Micro strategies are more specific to each task, are more related to specific knowledge and skills and can be taught, macro learning strategies aim at the knowledge and understanding of the subject’s own learning mechanisms, have a high degree of transfer and are difficult to teach.

Micro Strategies

A) Repetition strategies (require a minimum degree of cognitive control), operate on the memorization of data and aim to select and acquire units of information for transfer to working memory. Repetition strategies in complex learning tasks help students to pay attention to the most valuable aspects of a lesson and transfer that material to working memory for later study: we could cite recording practices, copying, repetition, etc.
B) Elaboration strategies (they favor the connections between the knowledge previously learned by the student and the new contents, the level of cognitive control would still be low).
• Elaboration strategies in basic learning tasks which intervene in the coding processes for the construction of associations in the materials to be learned.
• Elaboration strategies in complex learning tasks serve to complete previous knowledge with new information. This group would include note-taking and notes, diagrams and summaries, concept maps, etc.

Macro Strategies

Go to

A) Organizational strategies (they look for an internal structure or organization in the learning material that gives it a meaning of its own). Hierarchy and classification would be clear examples of organizational strategies, but also thinking and problem-solving skills could be included in this category. Cognitive control is superior.
• Organizational strategies for basic learning tasks, which help the student to organize the materials in such a way as to facilitate further learning, require the student to be able to group or categorize the contents.
• Organizational strategies for complex learning tasks, these strategies would be responsible for the selection of information to be transferred to working memory and the construction of relationships between ideas in that memory.

Regulatory Strategies

They are applied by the student when he sets goals in his learning and assesses the degree to which he has achieved those goals. It would encompass the use of metacognitive skills: meta attention, meta comprehension and meta memory (Meza A, et al. [8]).

Meta-Cognition

Regarding meta-cognition, since 1976 it has been defined and described as the knowledge of oneself and concerning one’s own cognitive processes and products to everything related to it. Metacognition indicates, among other things, the active examination and consequent regulation and organization of these processes in relation to the cognitive objects they deal with, usually in the service of some specific end or object. Within meta-cognition there are affect-motivational strategies which are related to the control that the student exercises over his or her learning environment, they are strategic used consciously it is a personal style of learning, these strategies demand a high level of control. Macro strategies based on meta-knowledge are responsible for re-establishing the parameters of a task, locating errors, determining the most appropriate intervention techniques and methods, controlling their application and making decisions based on the results obtained, although the direct resolution of the task corresponds to micro strategies through specific operations or procedures. They are classified into four broad types of basic or primary strategies for learning: acquisition, retention, recovery, and support. There are so-called support strategies that favor study and are precisely those that should be taught to the student; Among them is the planning of study time, attitude in class, how to develop a topic, study conditions, differentiated study by subjects, methods, etc. All strategies involve a lot of information at different levels, so there are also different types of classifications, for example: Linguistic strategies that are those that involve superficial structures such as texts or sentences with their underlying semantic representations; cognitive strategies that involve the use of knowledge about the world and other types of episodic and cognitive information such as attitudes, beliefs, aspirations, etc.

All strategies are flexible, operate at different levels of analysis at the same time, operate with different types of input data, can be executed even when the information processed is incomplete and can combine data-driven processes, are context-sensitive depending on the student’s interest, attention, goals, beliefs, and attitudes, are non-deterministic, and tend to produce a large number of options that vary in plausibility. There are hierarchical relationships between strategies, where some dominate the others, those that work with global information act as top-down processing strategies in the understanding of local information; there may be local-level strategies, which place constraints on subsequent meanings, as well as retroactive strategies, which explain the meaning of expressions to which only a partial interpretation has been assigned; There are those to use knowledge, such as retrieval and interpretation, which allow us to obtain the relevant information in each state or point, leaving the more general information at the level of a general control scheme. There are preoperative strategies, even before having read or heard the learning material, they are communicative and contextualizing; they specify purposes, tasks, difficulties in materials, etc., determining the subsequent choice of more local or global strategies, they are anticipatory and have a determining role in learning (Bonilla Traña M, et al. [9]). A cognitive strategy is also the set of procedures that a learner can use to acquire, retain, and retrieve different kinds of performance and knowledge, these cognitive strategies include the learner’s ability to represent (drawing, writing, imagining), selection ability (attention and intention), and self-direction ability (self-programming and self-monitoring).

With this conception, it is assumed that the composition of a cognitive strategy is a guiding cognitive task (a method for inducing the student to perform certain kinds of operations) and a capacity for selection, representation and self-direction. On other occasions, it is necessary to provide a specific guiding task to induce the use of the relevant learning strategies, as would be the case of elaboration strategies that add color to a given representation, which improves its recall, or organizational strategies that undress the representation of any external referent but that structure the information to be learned. in such a way that they integrate its various components, improving their comprehension and memorability. According to this point of view, an instructional sequence is designed to help students develop and organize internal mediation processes, it is about teaching them, in addition to the assistance of cognitive strategies, an appropriate use of them, so it would facilitate the acquisition, retention, integration and recovery of what has been learned (Córdoba Urbano D L, et al. [10]).

Conclusion

The simple transit through higher education schools in an inertial, passive way, with disinterest and without intrinsic and extrinsic motivations does not guarantee professional, economic and social success, on the contrary it practically condemns the student during his stay in these institutions to an endless number of problematic situations, stress, sanctions and even delay in graduation or desertion and for those who manage to graduate practically condemn themselves to underemployment or unemployment. accompanied by frustrations and waste of time. That is why it is important that from the first educational levels the student begins to be taught the importance of studying, what study strategies are and what they are for, so that they have the basic foundations in this regard and that they are gradually strengthened and enriched as they advance in the educational levels, until they achieve self-learning and metacognition.

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Generalization of the Law of Wave Refraction at the Interface of Mobile Environments

Introduction

Waves are the most common physical process. Almost all natural processes take place with the participation of waves of various natures, the basic properties of which are largely similar [1]. During the interaction of waves propagating in media at the interfaces, reflection and refraction of waves arise – a fundamental phenomenon of physics, which has found great application in various fields of technology and in natural processes. Wave reflection occurs when waves are reflected from the interface between media, propagate in the same medium and change their direction without changing the parameters of the medium [2]. In this case, the angle of reflection is equal to the angle of incidence, the incident and reflected rays lie in the same plane with the normal to the surface. Wave refraction occurs when waves move from one medium to another with different parameters and at the same time change their speed and direction. According to the law of refraction, the wave vector of the refracted wave lies in the same plane with the wave vector of the incident wave and the normal to the interface drawn at the point of incidence. The ratio of the sine of the angle of incidence to the sine of the angle of refraction is equal to the ratio of the velocities of sound waves in the first and second media c1 and c2 (Snell’s law). When a wave passes from a medium with a higher density (lower speed of wave propagation) to a medium with a lower density (accordingly, with a higher speed of wave propagation at a certain angle of incidence, it may not pass into the second medium and be completely reflected from the interface between the media. This effect is called total internal reflection and is widely used in optical technology for channeling optical radiation. The laws of reflection and refraction are well studied for stationary parameters of contacting media [3,4]. In the nonstationary state of the parameters of the adjacent media, there are features of the laws of reflection and refraction that must be taken into account in measuring technology [5-7]. This paper examines the influence of the motion of adjacent media parallel to the interface between media on the laws of reflection and refraction.

Simulation

The passage of waves through the interface of two media moving along the interface 0z (for simplicity, we assume that 0 ) (Figure 1) in the general case with different velocities u1 and u2 is considered. The speed of propagation of waves in the first stationary

The solution for the incident, reflected and transmitted waves in each of the regions is sought in the form of functions:

where in the general case, the wave numbers of the forward and backward waves in the first medium and the forward wave in the second semi-infinite medium differ due to the difference in the velocities of these waves, which is created by the movement of the media: where in the general case, the wave numbers of the forward and backward waves in the first medium and the forward wave in the second semi-infinite medium differ due to the difference in the velocities of these waves, which is created by the movement of the media:

Main Results and Discussion

Substitution of the field components into the boundary conditions for Ey leads to the relation:

where z – components of wave numbers are determined by the relations:

since the boundary conditions must be satisfied at any point z, then.

The angle of refraction is determined through the angle of incidence by the relation.

which in the absence of mutual displacement of the adjacent media ( u1 = u2 ) transforms into the known relation [1-3]:

Thus, the law of reflection does not change, but the law of refraction is transformed, which is associated with a change in the ratio of wave velocities in media during their relative motion. Figure 2 shows the dependence of the angle of refraction on the angle of incidence for various ratios of media parameters.

Figure 2

biomedres-openaccess-journal-bjstr

When a wave passes an interface from a denser medium to a less dense one (for example, from moving water into air), the critical angle of total internal reflection at is determined by the relation.

and depends on the relative difference in the velocities of the media to the wave speed c1.2 in a denser layer (in Figure 3). An increase in the angles of total internal reflection is observed when the direction of motion of the medium changes. The movement of the adjacent media also affects the polarization effect upon reflection. The angle of total polarization (Brewster angle from the condition T 0 2 π ) is determined from the equation.

A change in the Brewster angle due to the movement of media is observed when the difference in media velocities changes. For example, changing from -0.2 to 0.2 changes the Brewster angle from 1.05 to 1.15 rad.

Figure 3

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Conclusion

The movement of media adjacent to the interface does not lead to a change in the law of wave reflection, but significantly changes the law of refraction, the angle of total internal reflection and the Brewster angle, which can be used to control these parameters or to measure the velocities of media.

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Acidic and Basic Proteins from Spider Latrodectus Pallidus Venom that Induce Formation of Non-Bilayer Phase and Increase Proton Capacity in Model Myelin Membranes Feature High Sequence Homology to Isoforms of Myelin Basic Protein: Pharmacological Relevance

Introduction

Spiders evolved over 300 million years ago, and through their long evolutionary history, spiders developed into animals with wellequipped venoms comprising a great variety of biochemical compounds designed to paralyze and/or kill their prey, rendering spiders the most successful venomous creatures in evolution [1]. Proteins constitute a major portion of spider venoms that display diverse pharmacological activities, representing vast evolutionary-edited natural pharmacopoeias that attract research efforts leading to the potential development of novel pharmaceuticals [2,3]. A few spider venom protein toxins have been used as invaluable ligands targeting various pharmacological targets. Some of these venom protein toxins are now undergoing preclinical and clinical studies for the treatment of multiple sclerosis, diabetes, and cardiovascular pathologies [4]. Spider venom proteins are also used in fundamental studies as crucial tools for probing electrophysiological processes in biological membranes [2]. For example, hanatoxins from Grammostola rosea and agatoxins from Agelenopsis aperta have been used as specific ligands of voltage-gated potassium and calcium channels [5,6]. In addition, spider venoms comprise a variety of stable small proteins with strong insecticidal activity that cause paralysis and/or lethality of insects by affecting ion channels, receptors, and enzymes [7]. This kind of research on the insecticidal activity of spider venom proteins holds high potential for the development of novel bioinsecticides, which are finding applications in green agricultural biotechnology [8,9].

The venoms of Latrodectus widow spiders, a genus of spiders in the family Theridiidae erected by Charles Athanase Walckenaer in 1805 [10], are probably the most studied spider venoms due to their extraordinary potent neurotoxins [11,12], which draw not only medical interest but also the general interest of scholars who use Latrodectus neurotoxins in studies of molecular mechanisms in neurobiology and pharmaceutical research [13]. The spiders of the Latrodectus genus comprise 32 species spread around the globe [14-16], with most of the species found in South and North Americas [10,17]. The most potent Latrodectus neurotoxin is α-latrotoxin, which targets the vertebrate central nervous system by attacking the presynaptic membrane of neurons, leading to increased intracellular Ca2+ concentration that triggers elevated exocytosis of neurotransmitters in the intermembrane synaptic cleft [13,18]. The less potent Latrodectus neurotoxins are α, β, γ, δ, ε-latroinsectotoxins, which bind to specific receptors on the presynaptic membrane of neuronal cells of insects, and α-latrocrustatoxin, which attacks the presynaptic membrane of crustaceans [18].

Overall, the neurotoxic activity of ‘latro-insecto-crusta-toxins’ is based on the binding of toxins to neuronal cells, which promotes the release of neurotransmitters either via stimulating exocytosis or through the formation of transmembrane tetrameric pores promoting specific Ca2+ permeability [18]. Noteworthy, it has been reported earlier that α-latroinsectotoxin from Latrodectus mactans venom promotes the conductivity of bilayer lipid membranes in the ion channel manner for divalent cations in the following order of decreasing permeability Ba2+ > Ca2+ > Mg2+ > Cd2+ > Zn2+ [19]. Besides the neurotoxins, Latrodectus venoms have been reported to contain metalloproteases, serine proteases, chitinases, hyaluronidases, venom allergen antigen 5-like proteins, etc. [13]. In addition, cationic proteins with antibacterial activity have been detected in Latrodectus geometricus venom [20]. In a recent study, the chromosome-level genome assembly of Latrodectus elegans spider venom with 55 identified toxin genes has been reported [21], which is more than the number of different types of venom proteins identified in all studied species of the Latrodectus genus. This finding suggests that there are still more unknown proteins to be identified in the venom of spiders of the Latrodectus genus.

The venom of Latrodectus pallidus, the white widow spider, which inhabits the deserts of Central Asia, is the least studied venom among species of the Latrodectus genus [22]. In 2014, the venom from Latrodectus pallidus was fractionated to reveal that, in addition to the types of proteins commonly found in venoms of the Latrodectus genus, such as α-latrotoxin, latrodectin, latroinsectotoxin, phospholipase D, hyaluronidase, and serine protease, there are two proteins, one acidic and another basic, both of 18.5 kDa, in the Latrodectus pallidus venom. These proteins have amino acid sequences which are not homologous to the amino acid sequences of proteins previously identified in venoms of the Latrodectus genus [22]. Interestingly, the amino acid sequences of the two proteins of 18.5 kDa from the Latrodectus pallidus venom are highly homologous to the sequences of myelin basic protein isoforms. In the present study, we used the Latrodectus pallidus venom acidic and basic proteins of 18.5 kDa as the protein component of the model myelin membrane, which included, as the lipid component, the major phospholipids of the myelin membrane, such as sphingomyelin, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and phosphatidylcholine, to examine the proton absorbing capacity and the polymorphic phospholipid transitions in the model myelin membrane. The results obtained in the present study support the innovative proposal that outlines the novel biological function of the myelin membrane related to the accumulation of protons on the myelin membrane surface to serve a function of proton capacitor, a novel concept suggested by Professor Alessandro M. Morelli [23]. The results of the present study also propose new tentative physiological roles for phospholipids and acidic and basic proteins of the myelin membrane.

Materials and Methods

Chromatography

The lyophilized venom from the Central Asian white widow spider Latrodectus pallidus was a gift from Prof. L. Ya. Yukelson (Institute of Biochemistry, Uzbekistan Academy of Sciences). The venom (200 mg) was dissolved in 1.5 ml of 5.0 mM Tris-HCl, pH 8.0 buffer and applied onto the cation-exchange CM Sephadex C-50 (Nanjing Duly Biotech Co. Ltd, Nanjing, China) borosilicate column (1.5 × 35 cm) equilibrated overnight in 5.0 mM Tris-HCl, pH 8.0 buffer at 25°C. The KCl gradient in the same buffer was administered as shown in Figure 1. Acidic protein was collected from the shaded area of F1 peak (Figure 1), then dialyzed against water in 1.0 kDa cutoff dialysis tube (Sigma Aldrich, Saint Louis, USA) and lyophilized on the vacuum freeze-drying lyophilizing machine YTLG-10 (Shanghai Yetuo Co., Ltd., China) at 2 Pa and –60°C for 12 hours. A sample from shaded area in F7 peak (Figure 1) was dialyzed and lyophilized as described above and then sub-fractionated by cation-exchange HPLC in a Syn- Chropak S300 column. A 20 mg/ml sample was dissolved in 0.02 M Bis-Tris, pH 7.3 and injected into a SynChropak S300 column and a linear gradient was established with 0.5 M HCl buffer 5 min after the sample was injected. Basic protein was collected after 17 min elution time as shown by shaded area in Figure 2. The basic protein was dialyzed and lyophilized as described above.

Electrophoresis

SDS-polyacrylamide gel electrophoresis was carried out on the Mini-PROTEAN Tetra Vertical Electrophoresis Cell using Mini-PROTEAN TGX precast 12% density gels (Bio-Rad Laboratories Co., Ltd., Shanghai, China). Protein samples were dissolved in 20mM Tris-HCl pH 7.0 buffer with 2 mM EDTA, 5% SDS, 10% DDT, 0.01% Bromophenol Blue, 2% glycerol at a final protein concentration 2 mg/ml and boiled for 10 min. A 15 μl portion of each protein sample and low molecular weight markers for SDS-PAGE (Thermo Fisher Scientific Inc., Shanghai, China) were applied into wells of the precast 12% density gel. The running buffer was 500 ml 1×TAE (Tris-acetate-EDTA) pH 7.0 including 7.2 g glycine and 0.2 g SDS. Electrophoresis proceeded for 50 minutes at 100 V. Isoelectric focusing (IEF) was carried out on the horizontal plate Isoelectric Focusing apparatus DYCP-37B (Beijing Liuyi Biotechnology Co., Ltd., China) using the Ready Gel Precast Gel with ampholyte of pH range 3–10.5 and the protein markers for IEF with pI range of 4.65–10.6 (Shanghai Yeyuan Biotechnology Co., Ltd., China). The pH gradient in the precast IEF gel was adjected by running electric power at 75 V for 30 minutes after which 5 μl of each protein sample (1mg/1ml H2O) and IEF protein markers were applied onto the precast IEF gel and the power of 90 V was run for 120 minutes. Amino acid residues sequencing: The detailed protocol for resolving the sequences of amino acid residues of acidic and basic proteins purified from the Central Asian white widow spider Latrodectus pallidus was described previously [22]. Briefly, the amino acid composition of proteins was determined with the High-Speed Amino Acid Analyzer LA8080 AminoSAAYA (Hitachi High-Tech, Shanghai Co., Ltd., China). Reduction and carboxymethylation of proteins were conducted as previously described [24]. Proteins were hydrolyzed for 5 hours at 37°C with trypsin at 1:50 w/w protein to trypsin ratio. The carboxymethylated protein hydrolysate was chromatographed on Chromo- Beads resin (Koram Biotech Corp., Seul, Korea) and then further purified by paper electrophoresis and chromatography. The amino acid sequence of the protein fragments was determined as described previously [25]. The carboxymethylated proteins were incubated for 20 hours at 25°C in 70% formic acid. The cyanogen bromide cleavage was done using a 100-fold excess of reagent. The products of the carboxymethylation and cyanogen bromide cleavage were purified on Sephadex G-25 in 0.1 M NH4–HCO3 buffer. Paper chromatography was used to isolate the chymotryptic peptides of the cyanogen bromide fragment of proteins.

The N-terminal sequences of carboxymethylated proteins and the cyanogen bromide fragment forming the C-terminus of the original proteins were resolved on a Beckman model 890C sequencer (Beckman Instruments Inc., Fullerton, CA, USA) and the C-terminus sequence was resolved by means of carboxypeptidase A. Total lipid extract isolation and purification: Wistar rat liver was homogenized in a Warning Blender 700G for 3 minutes in 300 ml chloroform and 200 ml methanol, then100 ml of methanol and 200 ml dd-H2O was added and the mixture was blended for another 5 minutes. Homogenate was centrifuged at 200 g for 20 minutes and aqueous layer of methanol and dd-H2O was discarded. The chloroform phase with lipids was washed with 300 ml saline and centrifuged at 200 g for 15 minutes after which saline layer was discarded, and the chloroform phase was washed two more times. The lipids in chloroform were filtrated through the Whatman No.1 filter paper and the lipids were washed from the filter paper with 30 ml methanol.

Preparation of phospholipid specific polyvalent antibodies: Polyvalent antibodies specific for the selected phospholipids were developed using adult male Wistar rats. The adjuvant-primed animals received injections of the synthetic polar heads of phospholipids: one animal was injected with one type of phospholipid polar head. The synthetic polar heads of phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, phosphatidylserine and sphingomyelin were from NOF America Co., White Plains, USA. Ascites fluids were collected from the peritoneal cavity of animals two weeks after injection. The polyvalent antibodies from ascites fluids were purified on a Protein-A-Sepharose column (Pharmacia, Uppsala, Sweden). Purification of individual phospholipids by immunoaffinity chromatography: The borosilicate columns for immunoaffinity chromatography were loaded with the Sephadex G-25 resin containing covalently linked antibodies specific to the polar heads of either phosphatidylserine, sphingomyelin, phosphatidylinositol, phosphatidylcholine or phosphatidylethanolamine with each column containing only one type of antibodies, so overall five different immunoaffinity columns were prepared.

The antibodies were covalently linked to the Sephadex G-25 resin using Pierce™ Traut’s Reagent 2-iminothiolane Kit and N-succinimidyl 3-(2-pyridyldithio)propionate Kit (Thermo Fisher Scientific Inc., Shanghai, China) according to the manufacture’s protocol. The Sephadex G-25 resin with linked antibodies was equilibrated in 1.0 mM Tris-HCl, pH 7.0 buffer at 25°C for three hours. A 5 ml portion of the concentrated total lipid extract in methanol was applied into each of five immunoaffinity columns and the individual phospholipid fractions eluted from the columns were pulled into tubes with 15 ml chloroform and centrifuged at 200 g for 15 minutes to discard aqueous layer. Chloroform from the tubes was removed by drying with vacuum pump for one hour. The mass of phospholipid obtained was determined as the different in mass between the tube with dry phospholipid and the same empty tube. The concentration of stock solutions of phospholipids in chloroform of 0.1 M was prepared assuming molecular masses of phospholipids: 716 g/mol for phosphatidylethanolamine, 887 g/mol for phosphatidylinositol, 768 g/mol for phosphatidylcholine, 792 g/mol for phosphatidylserine and 734 g/ mol for sphingomyelin.

Preparation of unilamellar liposomes: The unilamellar phospholipid liposomes for the studies of ability of liposomal membrane to absorb protons, that is to serve as protons capacitor, were prepared by the treatment of aqueous lipid dispersions with ultrasonic wavelength frequency. Aliquots of phospholipids in chloroform containing molar ratio 4 to1 of phosphatidylcholine and either phosphatidylethanolamine, phosphatidylserine, sphingomyelin or phosphatidylinositol were dried with vacuum pump for 5 hours at 25°C for complete removal of chloroform, that is until the chloroform free phospholipid films were formed. Phospholipid films were then hydrated with dd- H2O pH 7.0 at phospholipid concentration 10–5 M and the resulted phospholipid dispersion was sonicated with the Ultrasonic Dispenser Yt-JY96-IIN (Shanghai Yetuo Technology Co., Ltd., China) at 22 kHz in helium atmosphere for 15 minutes at 4°C. Liposomes were then incubated for 15 hours at 15°C. The unilamellar phospholipid liposomes modified by acidic and basic proteins were prepared by adding equal molar amounts of acidic and basic proteins into the liposome sample at a total protein concentration 2×10–7 M.

To prevent electrostatic interaction of acidic and basic proteins in solution, the acidic protein was added first to liposome samples and incubated for 15 minutes with the continuous stirring of liposome solution with the magnetic stirrer JB-3 (Shanghai INESA Scientific Instruments Co. Ltd., China). Then the basic protein was added to the liposomes which were already modified by acidic protein and liposomes were incubated for another 15 minutes. The pH readings were taken in pure dd-H2O, in liposome samples and in liposome samples modified by the acidic and basic proteins. For the sake of uniform pH readings, all liposome samples treated or untreated with the proteins were continuously stirred with the magnetic stirrer. The proton concentration in pure dd-H2O and liposome samples treated or untreated with the proteins were determined from the measured pH values by using the following conversion: [H+] = 10–pH. 1H-NMR studies of unilamellar liposomes: For the 1H-NMR studies 2H2O was used instead of dd-H2O to prepare sonicated unilamellar liposomes according to protocol described above. Phospholipid concentration in liposomes for the 1H-NMR studies was 1.4 × 10–2 M and protein concentration was 1.75 × 10–4 M. Liposomes were treated with 10 μl of saturated K3Fe(CN)6 solution per 1 ml of liposomes. 1H-NMR spectra from N+(CH3)3 groups of unilamellar liposomes were recorded using a Varian XL-200 spectrometer (USA) at an operating frequency of 200 MHz at 25°C. The width of the 90° pulse was 8.7 μs, the relaxation delay was 50 μs and the acquisition time for free induction signal was 1 s. The integral intensity of 1H-NMR signals from choline groups was measured in triplicate and variation among triplicate readings was never higher than 6%.

Statistics: For each data point coming from the pH readings of dd-H2O and the liposome samples, they were prepared in triplicate, and the standard deviation between the readings was within 0.09% of the means. The pH readings coming from the four types of liposome samples and from the dd-H2O sample were statistically compared by using an ANOVA test. For statistical comparison of pH readings between liposome samples and liposome samples treated with proteins, the T-test was used. The p-values less than 0.05 were considered as statistically significant differences.

Results

We fractionated the Central Asian spider Latrodectus pallidus venom by the cation-exchange column chromatography to purify acidic and basic proteins. We used one column to purify acidic protein and two columns to purify basic protein. The chromatograms derived from the two column fractionations are shown in Figure 1. Acidic protein was collected from the shaded area of the F1 peak in the chromatogram obtained by fractionation on the CM Sephadex C-50 column (Figure 1A). To isolate the basic protein, a fraction of proteins was collected from the shaded area of the F7 peak (Figure 1A), and this fraction of proteins was then sub-fractionated by cation- exchange HPLC in a SynChropak S300 column. The basic protein was isolated from the shaded area of the fraction shown in Figure 1B. Both the acidic and basic proteins were homogeneous by SDS-PAGE and isoelectric focusing electrophoresis (Figure 2). The molecular mass of both proteins was somewhat below 21.1 kDa, and the pI values were around 4.65 and slightly above 10.6 for the acidic and basic proteins, respectively. The amino acid sequences of the acidic and basic proteins, which were resolved according to the protocol described in the Materials and Methods, are aligned in Figure 3.

Figure 1

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Figure 2

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Figure 3

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Amino acid sequences of both proteins are highly homologous to the human myelin basic protein isoforms [26]. In addition, both acidic and basic proteins show 100% sequence homology to the myelin basic protein isoforms from various species in the highly conserved amino acid sequence regions [27] (Figure 3). The calculated molecular mass and the isoelectric points of the Latrodectus pallidus venom acidic and basic proteins match exactly the most abundant isoforms in the adult human myelin with molecular mass 18.5 kDa [26]. These findings prompted us to use acidic and basic proteins in the studies of electrophysiological and structural properties of model myelin membrane. We have incorporated acidic and basic proteins from the Latrodectus pallidus venom into the model myelin membrane to investigate whether the model myelin membrane can absorb protons on its surface from the bulk water and undergo polymorphic phospholipid transitions.

The myelin membrane in the vertebrate central nervous system is mostly made of five phospholipids: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), and sphingomyelin (SM) [28-31]. In terms of the protein component of the membrane, the isoforms of myelin basic protein are one of the most abundant components of the myelin membrane, the primary role of which is to maintain a compact myelin sheath [32]. PC is the dominant phospholipid that stabilizes the bilayer structure in most, if not all, biological membranes, including the myelin membrane [32-34]. The role of the other four phospholipids is not clear, but it has been suggested that each of those phospholipids plays a particular purpose in the functional activities of the myelin membrane [32]. In the present work, for the studies of proton absorption by the membrane surface, we have prepared four types of model myelin membranes, each of which includes PC and one of the four phospholipids of a particular function with the molar percentage ratio of 80% for PC and 20% for either PE, PS, PI, or SM, with a total phospholipid concentration of 10–5 M.

For the protein component of the four types of model myelin membrane, the total protein concentration of acidic and basic proteins of an equal molar ratio was 2×10–7 M. For the studies of polymorphic transitions of phospholipids in the model myelin membranes, we prepared the same four types of myelin membranes with the molar percentage ratio of 80% for PC and 20% for one of the phospholipids of a functional group, but we used different total phospholipid and protein concentrations, which were 1.4 × 10–2 M for phospholipids and 1.75 × 10–4 M for proteins. The model membrane system for myelin membranes was unilamellar liposomes, both for the studies of proton absorption by the membrane surface and for the studies of polymorphic phospholipid transitions. To assess the absorbance of protons by the myelin membrane liposomes, we measured the difference between pH values in pure dd-H2O and in dd-H2O solutions of liposomes from four models of myelin membranes, which did not include acidic and basic proteins (Table 1).

Table 1: The pH readings taken in triplicate at 25°C in dd-H2O and in dd-H2O solutions of liposomes made of either phosphatidylcholine (PC) + phosphatidylserine (PS), phosphatidylcholine + phosphatidylinositol (PI), phosphatidylcholine + phosphatidylethanolamine (PE) or phosphatidylcholine + sphingomyelin (SM). The mean values of pH readings, standard deviations (SD) and the ANOVA p-value are also given in the Table 1. The concentration of phospholipids in all samples is 10–5 M.biomedres-openaccess-journal-bjstr

Preparations of liposomes in dd-H2O produced basic pH values (pH above 7.0), which is a result of protein absorption by the surface of the liposomal membrane. As shown in Table 1, the PS-containing membrane (PC+PS) demonstrates the highest change in pH towards basic values (7.18), indicating that the PC+PS membrane is the strongest proton capacitor among the four models of myelin membranes. This is due to the two acidic moieties, carboxyl and phosphate groups, in the PS polar head (Figure 4). Although the other three model myelin membranes each contain one of the ‘functionally-driven’ phospholipids (PI, PE, or SM), all of which have only one acidic moiety (phosphate group) in their polar heads, their proton absorption abilities differ and decrease in the following order: PI – pH 7.15, PE – pH 7.12, and SM – pH 7.09. The highest ability of PI to absorb protons among the three ‘functional’ phospholipids can be explained by the polar hydroxyl groups of the inositol moiety in the polar head of PI (Figure 4), which have the potential to absorb protons via the formation of coordinate bonds with the lone pair of electrons on the oxygen atoms of the hydroxyl groups.

Figure 4

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The higher ability of PE to absorb protons compared to SM can be attributed to the larger size of the choline group (–N+(CH3)3) in SM versus the amino group (–N+H3) in PE (Figure 4). The smaller size of the amino group allows better access for protons to the phosphate group of PE than the choline group of SM. It should be noted that the ANOVA p-value for the statistical differences in pH values is very small within the group including pure dd-H2O and dd-H2O solutions of the four models of myelin liposomal membranes, indicating that the differences are statistically significant. The effect of the equimolar mixture of acidic and basic proteins (Pr) incorporated into the four models of myelin liposomal membranes is shown in Table 2. In all four membrane models, the incorporation of Pr resulted in a further increase in pH values, demonstrating that Pr contributes to the proton absorption of the myelin membrane. Thus, Pr enhances the myelin membrane’s ability to act as a proton capacitor. The largest increase in pH value (1.05) triggered by Pr was observed in the PS-containing membrane. The second largest increase in pH value (1.03) was observed in PE-containing membranes.

Table 2: Comparison of pH values in samples of liposomes made of either phosphatidylcholine (PC) + phosphatidylserine (PS), phosphatidylcholine + phosphatidylinositol (PI), phosphatidylcholine + phosphatidylethanolamine (PE) or phosphatidylcholine + sphingomyelin (SM) and the same liposomes treated with a mixture of acidic and basic proteins (Pr). The pH readings mean values, standard deviations (SD) and the T test p-values are also given in the Table 2. The concentrations of phospholipids and Pr are 10–5 M and 2×10–7 M respectively.biomedres-openaccess-journal-bjstr

The increase in pH value triggered by Pr in PC+PI and PC+SM was significantly lower, at 0.74 and 0.67, respectively. The very low T-test p-values comparing the statistical differences between pH values in model myelin membranes not containing Pr and those containing Pr confirm that the increases in pH values triggered by Pr are statistically significant. To better present the differences in proton capacitor power between the four models of myelin membranes in the absence and presence of Pr, the concentrations of absorbed protons are shown in Figure 4 as the negative logarithm of [H+] absorbed by the model myelin membranes. It should be noted that the lower the negative logarithm of [H+] value, the higher the concentration of H+ ions absorbed. Thus, by comparing the heights of the bars in Figure 4, one can conclude that the myelin membranes containing Pr exhibit higher proton capacitor power than myelin membranes without Pr. Additionally, one can conclude that among the myelin membranes containing Pr, the PC+PS and PC+PE membranes have a higher proton absorbing capacity than the PC+PI and PC+SM membranes (Table 3).

Table 3: The concentration of H+ ions in dd-H2O (initial H+ concentration), concentration of H+ ions after adition of proteins, liposomes or liposomes modified with proteins to dd-H2O (final H+ concetration) and concentration of H+ ions absorbed by proteins, liposomes or liposomes modified with proteins (absorbed H+ concentration). Concentrations of phospholipids and proteins in liposome samples are 10–5 M and 2 × 10-7 M respectively. Pr – mixture of acidic and basic proteins, PC – phosphatidylcholine, PS – phosphatidylserine, PE – phosphatidylethanolamine, PI – phosphatidylinositol, SM – sphingomyelin. Column –log [H+] shows negative logarithm values of absorbed [H+].biomedres-openaccess-journal-bjstr

To study the permeability and polymorphic transitions of phospholipids in the four models of myelin membranes in the absence and presence of Pr, we used 1H-NMR spectroscopy of unilamellar liposomes in the presence of K₃[Fe(CN)₆]. The methodology and advantages of unilamellar liposome 1H-NMR spectroscopy with paramagnetic Fe(CN)₆³⁻ ions are described in detail in our previous publications [35-38]. Briefly, Fe(CN)₆³⁻ ions interact with the choline (N+(CH₃)₃) groups of the outer monolayer of the liposomal membrane, shifting the choline signal of the outer monolayer to higher values of the applied magnetic field, while the signal of the choline groups from the inner monolayer remains unchanged. This results in the splitting of 1H-NMR signals from the outer and inner monolayers of the liposomal membrane. Should the liposomal membrane become permeable to Fe(CN)₆³⁻ ions, which would allow interaction with the choline groups of the inner membrane, the signal of the choline groups from the inner monolayer would shift to the field of the outer monolayers, causing the inner monolayer signal to visually disappear.

As shown in Figure 5, all four model myelin liposomal membranes in the absence of Pr were not permeable to Fe(CN)₆³⁻ ions. The higher intensity of the 1H-NMR signals in the PC+SM liposomes compared to the other three types of liposomes is due to both PC and SM having a choline group in their polar heads, while in the other three types of liposomes, only PC has a choline group. The addition of Pr to the liposome samples resulted in the broadening of 1H-NMR signals due to the restriction of molecular mobility of lipids by Pr. However, the addition of Pr did not make the membrane in any of the four liposome samples permeable to Fe(CN)₆³⁻ ions, as the signals from the inner monolayers remained intact. Notably, in the PC+PS+Pr and PC+PE+Pr liposomes, a small signal appeared in the higher field from the signal of the outer monolayer (Figure 5). This signal originated from the formation of a non-bilayer lipid phase [35-38] and was more pronounced in the PC+PE+Pr membrane. The formation of the non-bilayer lipid phase in model membranes containing PS and PE, triggered by interaction with basic proteins, was observed previously [35,38-41]. It is evident that the non-bilayer lipid phase in model myelin membranes containing PS or PE was induced by the basic protein from the white widow spider venom (Figures 5 & 6).

Figure 5

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Figure 6

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Discussion

It has been suggested that physiologically active proteins in animal venom have evolved by mimicking the structure and functions of body proteins with important functional activity [42]. For example, many phospholipases isolated from snake and insect venoms resemble structurally and functionally the phospholipases that regulate inflammatory processes and lipid metabolism in biological membranes [42,43]. Rattlesnake venom metalloproteinases have been reported to act similarly to body metalloproteinases involved in controlling the homeostasis of blood and intercellular fluids [42,44]. Cationic proteins isolated from cobra venom have been shown to phenocopy the membranotropic activities of the C-8 protein from the Fo sector of ATP synthase [45-47]. In this research study, we present acidic and basic proteins isolated from the venom of the white widow spider Latrodectus pallidus, with amino acid sequences highly homologous to the isoforms of myelin basic proteins of the vertebrate central nervous system [25,26]. It is therefore reasonable to assume that the physiological activities of these acidic and basic proteins from Latrodectus pallidus venom resemble those of the myelin basic proteins of the vertebrate central nervous system.

Myelin basic proteins exist as isoforms that differ in size and charge [48]. The size isoforms are produced by alternative splicing of an mRNA transcript [49], while the charge isoforms result from post-translational modifications that decrease the basic charge, affecting the functional activities of the myelin basic proteins [26]. The size isoforms of human myelin basic proteins include proteins of 17.2, 18.5, 20.2, and 21.5 kDa, with the 18.5 kDa isoform being the most abundant and the most studied [26,49]. There are eight charge isoforms of the 18.5 kDa myelin basic protein, termed C1–C8. The C1 isoform has the highest positive charge and is the least modified after post-translational modification [26]. The charge isoforms C2–C6 are modified by phosphorylation, deamidation, and deamination, while C8 is predominantly modified by peptidyl arginine deiminase, which converts positively charged arginine to neutral citrulline. This conversion can involve up to 11 arginine residues, leading to an overall loss of positive charge of +11 [50]. The isoelectric points of the 18.5 kDa isoforms range from 4.5 for C8 to 11 for C1 [26], which is very similar to the isoelectric points of acidic and basic proteins of 18.5 kDa from the venom of the Latrodectus pallidus spider. This suggests that acidic and basic proteins from spider venom may phenocopy the functions of the C1 and C8 isoforms of the 18.5 kDa myelin basic protein. The myelin sheath is a repetitive multilayer of tightly packed myelin membrane bilayers held together by proteins in myelin membranes [26]. The most long-lived proteins in the body are myelin basic proteins [51,52]. This suggests the importance of myelin basic proteins in the stability of the supramolecular structure of the myelin sheath [26]. It is believed that the C1 isoform functions by tightly attaching to acidic phospholipids of myelin bilayer membranes, stabilizing the tight packing of the myelin sheath [26]. Overall, C1, C2, and C3 are most likely responsible for myelin stability [26], while C8, which is abundant in childhood and scarce in adults, seems to play a role in development and is important in the formation of myelin rather than its stability [50]. However, the exact molecular mechanism of the functional role of C8 is not understood. Notably, the pathogenesis of multiple sclerosis (MS) is linked to abnormal changes in the 18.5 kDa isoform composition caused by an abnormal increase in the activity of peptidyl arginine deiminases, leading to the loosening of the tight packing of the myelin sheath [26,53-56].

The common explanation of the role of the myelin sheath is that it serves as an insulator of neuronal axons to promote rapid and saltatory conduction of nerve impulses [26,57]. However, nerve impulses are not transmitted through nerve fibers in the same way electrons move through a metal wire [57]. It is not clear how myelin insulates neurotransmission or what the insulation of nerve impulses means. It is obvious that even if myelin serves as an insulator in some way, it is far more than just an insulator [57]. A breakthrough discovery revealed aerobic ATP synthesis in the myelin sheath, which was unexpected as myelin is devoid of mitochondria [58-61]. However, oligomycin titration experiments have determined the presence of F1 subunits of ATP synthase in myelin [59], and the presence of respiratory complexes in lipid rafts of myelin was also established [62,63]. These discoveries suggest a way for delivering mitochondrial respiratory components to the lipid rafts in myelin.

Professor Alessandro Morelli of Genoa University proposed that mitochondria, which have their own DNA for respiratory complexes and ATP synthase to drive oxidative phosphorylation, deliver all the protein complexes necessary for ATP synthesis to the myelin sheath with the help of the endoplasmic reticulum (ER) [64], with which mitochondria are closely associated in cytoplasmic space and function [65,66]. It has been determined that mitochondria produce necessary vesicles providing the ER with the necessary oxidative phosphorylation complexes [67,68]. Aerobic ATP synthesis has also been observed in structures devoid of mitochondria such as rod outer segment discs [69-72], platelets [73], cell plasma membranes [74-78], exosomes, and microvesicles [79], strongly implying that the oxidative phosphorylation machinery is transferred to these extra-mitochondrial sites via the ER [80].

It has been shown by freeze-fracture X-ray crystallography that particles of 8.6 nm, the dehydrated F1 subunits of ATP synthases, are exposed on both sides of the myelin sheath [81,82]. This bi-faced orientation of F1 subunits in the myelin sheath does not agree with Mitchell’s concept of delocalized proton coupling, where the gradient of proton concentrations in bulk water across the inner mitochondrial membrane drives the proton movement through ATP synthases with the F1 subunits exposed only on the matrix side. However, the orientation of F1 subunits on both sides of the myelin sheath agrees with the concept of localized coupling, where protons are absorbed and move along the surface on both sides of the myelin membrane, establishing a proton circuit built entirely inside a major dense line of myelin [80]. Professor Morelli suggests that extra-mitochondrial synthesis of ATP can take place on any single membrane as long as the membrane surface can localize (absorb) protons to act as a ‘proton capacitor’, so the accumulated positive charge can support a proton circuit in which protons move along the membrane surface by the Grotthuss mechanism [83].

From the respiratory complexes to the F1 subunit of ATP synthase, and in the middle of the F1 subunit, protons turn to the hydrophobic center of the membrane through which protons move back to the respiratory complexes [23]. Thus, the proton circuit, taking place entirely inside a single membrane, couples respiration with ATP synthesis [23,64,80]. In our view, the experimental evidence described above supports localized proton coupling, which is based on the membrane’s ability to absorb protons on its surface. However, we note that protons cannot move as charge through the low dielectric environment in the center of the membrane, and there must be a vehicle to transfer protons through the hydrophobic environment to the respiratory complexes.

The experimental results of our study on model myelin membranes with acidic and basic spider proteins, acting as isoforms of myelin basic proteins, showed that these membranes absorb protons on the membrane surface.

The protons are absorbed by both the phospholipid polar heads and the spider proteins, primarily by acidic proteins rather than basic ones. The ability of phospholipids to absorb protons differs depending on the physical-chemical properties of the phospholipid polar head. Additionally, we observed the formation of a non-bilayer phase in model myelin membranes made of PC+PS+Pr and PC+PE+Pr. We have previously observed the formation of a non-bilayer phase through the interaction of PS with basic proteins. The non-bilayer propensity of PE was also described previously, and it is strongly enhanced by interaction with basic proteins. Therefore, we can conclude that the non-bilayer phase in model myelin membranes was triggered by the interaction of spider basic protein, but not by spider acidic protein, with PS and PE. It should be noted that the highest ability to absorb protons was observed in PC+PS+Pr and PC+PE+Pr membranes, which contain the non-bilayer phase. In our previous studies, we have demonstrated that the non-bilayer phase in the inner mitochondrial membrane, triggered by basic proteins, stabilizes the intermembrane association between adjacent crista membranes, facilitating higher activity of ATP synthase [35,46,84,85].

One of the mechanisms for enhancing ATP synthase activity facilitated by the non-bilayer phase was recently proposed by us, in which inverted micelles transfer protons across the hydrophobic environment of the crista membrane in the inner aqueous volume of inverted micelles [35,86]. The non-bilayer phase observed in our study in model myelin membranes likely exists in the form of inverted micelles, which transport protons in the myelin membrane’s hydrophobic environment, not across the membrane but through the membrane center from the F1 subunit to the respiratory complexes to complete the proton circuit inside a single membrane, as proposed by Professor Morelli [64,80].

Conclusion

The experimental results of our study support the new concept of the physiological role of the myelin sheath, related to the accumulation of energy through membrane action as a proton capacitor, as proposed by Professor A.M. Morelli. The stored energy is utilized for the production of ATP synthase through localized proton circuits that couple respiratory complexes with ATP synthases. According to Professor A.M. Morelli’s proposal, the generated ATP molecules in the myelin sheath could be used by ionic pumps in neuronal axons for the rapid transmission of nerve impulses. Our results suggest that the acidic isoforms, but not the basic isoforms, of myelin basic proteins, along with phospholipid polar heads, are responsible for the absorption of protons on the membrane surface. Additionally, our results indicate that basic isoforms of myelin proteins interact with acidic PS and neutral PE to promote the formation of a non-bilayer phase. The non-bilayer phase may promote the tight association of adjacent myelin membranes and facilitate the transport of protons through the hydrophobic environment in the center of the myelin membrane to couple respiratory complexes with ATP synthase.

The results obtained in this study warrant further investigation of acidic and basic proteins from the Latrodectus pallidus spider venom in model myelin membrane systems. This research could lead to a better understanding of the molecular mechanisms of neurotransmission and neurodegeneration, as well as the potential development of novel pharmaceutical products that may alleviate or halt the process of myelin sheath degradation in aging and diseases.

Acknowledgement

Dr. Anwaar S Chaudary is grateful to Prof. L. Ya. Yukelson of the Institute of Biochemistry, Uzbekistan Academy of Sciences for Latrodectus pallidus spider venom given as a gift. The research project was supported by the start-up grant from the Beijing Chaoyang Kaiwen Academy to the STEM Research Center.

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Systematization of the Types of Adaptations Requested in Pedestrian Routes: Human Strategies in Sport, Tourism and other Critical Situations

Introduction

Man moves, using the same instruments and strategies and even the same models of action, adapting to the different problems faced in different journeys. It is always the same man and, of course, the same individuality.

It is the ‘self’, its identity, that is called into question, a ‘self’ that has adapted itself in its evolutionary process, where it has been transformed and built (in its morphology, in its physiology, in its psyche, etc.) gaining functionality in the actions it undertakes, see Gordon Childe (1947) in his work, already a classic, of “Man makes himself”. They may seem to be mere one-off arrangements (we only see what we are capable of perceiving) but it is, permanently, the expression of a whole that we must try to understand and not lose the details (important, no doubt, but which it is essential to situate) that hide the overall vision and the ability to relate, namely by the “tunnel effect”, and the ability to relate (let’s not forget what culture it is, today, the ability to find bridges and relationships in a world of dialectics). Thus, for example, a pedestrian route can be considered a mere muscular exercise, a simple activation of cardiopulmonary function, going in search of new smells or (more or less) spectacular views, a form of socialization with a group, a subjection to local or more universal fashions and customs, an occasion to take photographs, to review stories (or even history), …, and so many other things and pretexts that, being there, we can (consciously or unconsciously) have the opportunity to live or that may not even serve for more than ‘telling friends’ on nights of pleasant talks or bragging about ‘adventures’.

However, to enjoy circumstances is always to build culture (the possibility of understanding and exercising relationships) which, paradoxically, always passes through the capacity of the ‘culture’ that we already possess (a virtuous circle) that enriches (more or less, according to the cultural tool we have) the experiences and amplifies (more or less) the value obtained from what is experienced. In this way, we can situate a pedestrian route as a privileged situation where we leave the habits and vices of everyday life, which, due to the change of context, the rhythm and hurry of ‘normal’ life and the vulgar conditioning where we are integrated. A situation, therefore, that disposes (it may even predispose, if we are capable) to reflection and that sharpens sensitivity (it is known that a repeated stimulus loses the ability to generate sensations), creating development potentials for those who experience it.

To understand the Pedestrian Route

The transition from an understanding/explanation mode, which we have tried to frame above in an accessible way , to an operational mode, implies making choices and options in order to select the variables and parameters that can be more efficient in a cost/benefit relation according to the resources and intentions in question. We must also not forget that when we move from the orientation of an activity, whatever it may be, sport, tourist, etc., to a structure in which the simple transmission of techniques considered useful and based on experience, fundamentally by imitation, is made to the management of existing functionalities, as we implicitly defend here, it is a profound structural rupture. This rupture that we propose consists in following, predominantly, a strategy in which we confront contradictory evolutionary tendencies in the search for the most favourable balances that we can, in an expeditious way, categorize as a passage from a casuistic empiricism to science, which it is not up to us to explain here, but has marked implications that we should not ignore. In this way, we will frame and explain the operational strategy, which we will later show how to operationalize through decision trees. The most common classifications of the difficulty of pedestrian routes, given the frame of reference on which they are based, are fundamentally on variables inherent to the context, such as distance, unevenness, altitude, duration, among other factors essentially focused on morphological, geographical and physical characteristics of the terrain itself [1-3].

This type of characterization is important, but it is not enough for it to be possible to integrate these variables into models that consider the characteristics of the individual who performs the activity and define the difficulty that the chosen route will have for the individual or choose the training/experience that best fits with his objectives. In a previous work, we proposed as a way to overcome this type of difficulties the characterization of pedestrian routes based on the adaptations they require from the individual who performs them [4]. The advantages of this type of characterization is that it makes it possible to broaden the understanding and explanation of the phenomenon, restructuring the frame of reference used in such a way that, in addition to the attributes of the context, it allows us to face the whole process that is triggered in the individual to achieve his or her path, that is, the cycle “aggression » reaction » adaptation » transformation (ARAT)” [5]. In this way, a change is made in the ‘knowledge’ tool used, giving new dimensions and increasing the possibilities of efficiency, and even the dimension of what we can achieve with the use of a more accurate and precise reference framework. In this line we will delve into some examples of adaptations, such as the uneven terrain and the sloping terrain (downhill), identifying in the context, in the situation, and in the individual, the main variables to be taken into account and, subsequently, how to frame them in a Sports or Tourism perspective.

Uneven Terrain

The terrain with irregularities, whether stones, vegetation, sinuosity’s of the terrain or other types of obstacles, can prevent a regular displacement (which may have its advantages when we focus attention on the modification of the individual, on his training, but which will certainly have more costs in the displacement itself – a proof of the new capabilities of the new reference framework of the knowledge tool used) (Figure 1), modifying, in particular, the rhythm, the distance between steps, the cadence of the pace, the times of the pace, the direction of the route, among other variations that can be caused by these characteristics. There is a consensus in the literature that the variation in the gait pattern leads to an increase in the energy cost necessary to carry out a given route [6-9]. According to O’Connor, Xu, & Kuo [9] the energy cost increase is mainly due to two reasons, for one hand, active movement corrections require a muscular effect, on the other hand steps variability (width) is less energy-efficient. All these constraints will imply that the individual is permanently reading the terrain, making decisions about where to place the steps and the best strategy to carry it out, as well as the necessary motor adjustments to carry out the displacement.

Figure 1

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This constant adaptation has energy costs at the motor level, but also at the nervous level [7]. In these circumstances, it is important to identify the possibilities that arise to overcome the irregularities of the terrain in order to systematize not only the decisions that the individual can make, but also to understand what type of adjustments are possible to provoke (Figure 2), that is, the inherent demands. Concretizing the choices that are made at the time we move, if an irregularity is identified in the terrain in the place where we would naturally place the next step, it can be solved by: changing the direction of the step, deviating and/or bypassing it; change the pace length to overlap it (when the height of this irregularity does not disturb the normal pace height); change the height of the pace in order to vertically contour the irregularity; perform the step on the irregularity by adjusting the way the support is placed to the characteristics of the irregularity and the effects we expect it to have on it (we may have to increase the stability of the joints if the deformation of the tread is expected, we may have to adjust the direction of the force in order to maximize the compression force and minimize the slip force, If a decrease in friction is expected, we may have to change the characteristics such as direction, length, height, among other possibilities). The decisions to be made must be considered according to the conditions of the context, but also to the characteristics of each individual, in order to opt for that situation in which he is more proficient and consequently will have less change in his performance regarding the circumstances.

We highlight that in some situations we can have options that happen simultaneously, for example, the change in the direction of the step can happen together with the change in the length of the pace, thus “adding” the effects that each of these changes will have and, of course, understanding the interactions that can be established between the variables in question. As mentioned by Wong, Selinger & Donelan [6] several parameters can influence the gait “… it may not only have a different step frequency and speed but also a different step width, toe clearance, and maximum knee angle. In addition, at the muscle level, the new step may be accomplished with muscle and motor unit activity patterns that differ in many ways from the average step. Some of these changes to coordination, if performed in isolation, may increase energetic cost, while others may decrease it.”. To define which are the most profitable options, it is necessary to consider how each one of them influences the cycle and rhythm of the pace, the path of the Center of Mass (CM), knowing that there may be options that have higher energy costs than others and that there are differentiated energy costs (with incidence at the nervous or motor level, but not only). In addition to the effects at the motor level, it is also important to understand that the energy sources used in movement (anaerobic alactic, lactic and aerobic system) have different regeneration timings and the decisions made can influence their alternations as well as the need to use each of them. It is also essential to understand the framework of this set of variables in the whole of the journey to be taken, since, depending on its total duration, the point where we are and the demands that are still expected, there are options that may be more appropriate than others taking into account the circumstances of the moment we are facing.

Figure 2

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Sloped Terrain (Downhill)

Routes on sloping terrain, in the case of descents, allow a part of the force of gravity to be used to generate the movement of displacement itself. Knowing how to use this force in an efficient, systematic and less flawed way (as opposed to a more empirical process made up of mere experiential situations) implies understanding the variables inherent to movement, but also being able to operationalize the appropriate techniques so that the benefits are greater than the costs of their use in displacement. Several difficulties in moving downhill are mentioned, which often overlap with the search for energy efficiency that the descent of a slope can provide, namely, the constant braking of movement and impacts on joints [10-12] and the search for stability to the detriment of gravity [13]. The need to adapt to the context and situation is fundamental to achieve the desired objectives in a cost-effective way [11]. Thus, knowing how to consider the possible strategies for displacement and what are the expected implications in the decisions we make is essential to manage the activity. AS mentioned by Wong, Selinger, & Donelan [14] it is necessary to have a global notion of the problem and of the interactions that different variables establish between themselves “Furthermore, whether a particular change in coordination will gain an energetic benefit depends on the walking task: decreasing step frequency, for example, may be beneficial when walking downhill but not when walking on the level.

The task to be solved by the nervous system when initiating optimization is to determine which of these many candidate changes to coordination are responsible for sensed reductions in energetic cost and in which contexts.”. Controlling the changes that a slope implies in the individual’s displacement involves first of all, within the constraints of the context itself, making decisions by choosing the slope that we intend to face through the selected path (Figure 3). The path can be in the direction of the slope, thus subjecting us to the existing slope, or we can opt for an oblique path in relation to the slope, which the farther it is from the direction of the slope, the lower the slope will have (a relationship involving the effort – slope/space covered – similar to what happens in a lever in the relationship between the dimensions of the force arms/force intensities). This choice of trajectory in relation to the slope has, of course, implications for the space that will need to be covered to get from one point to the other, and these two variables have contradictory evolutionary trends, and as such, the greater the slope, the smaller the space to be covered. The costs of each option depend not only on the strategy chosen (leading to the choice of the route), but also on the individual’s ability and mastery of the technique used (the individual’s potential and the way to use them).

Figure 3

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In the case of lateral sliding, usually used in terrain with little friction and deformable, such as snow, small stone or sand floors, the individual, in order to control the pressure exerted on the ground (ratio between the force exerted / area) regulates the position of the supports on the slope, by adjusting the dimension (in some cases using equipment that facilitates this type of strategy such as skis) in order to slide down the slope while maintaining the projection vertical of its Center of Mass within the boundaries of the Support Base. A sudden braking of the supports by not keeping them in the position that allows them to slide, will lead to the individual being projected (by the action of the existing inertia) towards the slope downwards. On the other hand, reactions such as fear, which are associated with a defensive strategy such as the retreat of the pelvis and/or the search for protective positions such as the approach of the body to the slope, lead to an increase in the weight component that acts on “the slip” and, consequently, to a reduction in the compressive force on the slope. This can result in an uncontrolled slippage where the very position of contact with the ground makes the situation difficult. The frontal progression (in addition to the slope of the terrain) also implies a control of the position of the Center of Mass, which must necessarily be balanced with the positioning of the steps in the pace (through the frequency of the steps and amplitude). When the individual cannot keep up with the speed “that the CM gains” with his stride, which can result in losing balance, due to not being able to adjust the position of the CM in relation to the support base. The difficulties in regulating this set of variables can have repercussions on the joints or, conversely, if there is not a sufficient advance of the Center of Mass to enhance the use of the Force of gravity in the displacement, the energy expenditure of the descent can be equal to or greater than that of the ascent.

From Sport to Tourism

If in Sport the immediate objective is to maximize the performance of the athlete, in Tourism the challenge is to provide the best possible experience to the tourist, through the activity that is offered [12,13], being (and should be), however, the mediate objective, in both situations, the transformation of Man (that is, the same general objective through different means). To train and evolve, the individual has to approach his performance to his limits, without, however, exceeding them (which would cause injuries) because only in this way is it possible to transform himself (see ARAT), the management of contradictory evolutionary tendencies mentioned above. The same happens (should happen) in tourism, if only so that we don’t have boring situations that don’t motivate (whatever the purpose, immediate objective – take a picture, integrate into the group … as we indicated before) and where there are still challenges to face (even if it’s just to tell in friends talk nights…).

However, in the orientation of activities of this nature, where we focused on the displacement of man, there are specific differences between these two aspects. In sport, it becomes possible to plan continuously to provide a “training effect”. In a “touristic activity” we must create motivations and ‘ways of selling’, also according to the intended objectives and in a personalized way, but where the imaginaries pursued by the customer follow, namely, other “models to imitate”, in a different framework and with different time constraints. A reflection to be carried out, a debate to be held and the search for new answers, which, in order to be efficient, must integrate the new possibilities and resources that are available to us today. As we can see, as we stated at the beginning of this work, in order for this intervention to happen successfully, it is necessary not only to understand the stimuli we intend to provide, but also the processes of adaptation to these stimuli and the transformations that can result from them. We would also like to make a few notes in this regard. We must also consider that in the “sports training” facet, it usually happens that there is a continuity of contact between the coach and the athlete. This generates the possibility of an in-depth knowledge of the athlete, his personal characteristics, limitations and potentialities. A continuous work that is being permanently adjusted to the evolution of the athlete and to the stipulated goals.

On the other hand, in sports activities framed in Tourism, the contact between the advisor and the tourist is usually more fortuitous, and it is frequent that they only establish a first contact already in the activity. These circumstances mean that the diagnosis has to be much faster than in a “normal” training situation. Being able to interpret some indicators, even before the activity, is fundamental and essential to be able to have a quality intervention. Sometimes, looking at the equipment they use is enough to get a first impression of their mastery of the context, it is not uncommon to see people who present themselves with shoes that are totally inappropriate for the walk that awaits them, but we also have the opposite, such as people with an elite equipment, but too new not to denounce that it has never been used before. It is in this balance of indicators that the manager must seek to situate himself in order to prepare for orientation. Also, in terms of intervention, it is necessary to understand that in a tourism situation,

Where the individual is only expected to perform one activity, the prescription will have to be based on variables that we can act on and have almost immediate effects, that is, in a period compatible with that foreseen for our intervention with the individual. In a training context, the mentality has to be different, we can sometimes opt for prescription solutions that will only have effects in the medium/long term, but that may be more advantageous in the evolution of the individual. In short, in the same frames of reference, but in different contexts, it is necessary to break with the traditional ways of looking at these activities, so that the resources we have today (knowledge, equipment, communication facilities, conceptual, etc.) can be optimized. This implies structural, normative, methodological, and organizational changes, among others. It is not enough to make mere oneoff arrangements, it is essential to find the new coherences, which are already possible today. Evolution, let us even say the enrichment of man, requires more, much more than simple changes in appearances and speeches.

Final Reflection

Understanding the movement of Man on a pedestrian route or in any other context is a complex process. It is necessary to have a comprehensive view not only of the motivations inherent to this activity, but also of the various conditionings that can influence this process. The strategies of action and the instruments used for displacement imply a global vision of the individual, conditioned by the evolutionary process of the human species itself and which involves the profitability of a set of structures (nervous, muscular, energetic, technical, psychological, cultural, etc.) and that it is necessary to understand this amplitude in order to be able to act. In this work, we sought to define a comprehensive and integrative frame of reference for the various aspects of human displacement, but at the same time, to exemplify through the decision trees presented, how it is possible to create models to understand the adaptations that are placed on the individual during a journey. We exemplified through two types of adaptations, Uneven Terrain and Sloped terrain (downhill) how it is possible to unfold some of the variables that we consider fundamental to understand this phenomenon, to structure future research, where several options are quantified that allow us to understand the evolutionary trends of the variables and their interactions, but also to be able to act coherently with the intended objectives both in the sport and tourism.

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Veterinary Vaccines- Harmonization of Veterinary Vaccines Monographs of Indian Pharmacopoeia (IP)

Introduction

Vaccines are a diverse group of pharmaceuticals that contain immunogenic ingredients that can trigger a specific, protective immune response to infectious diseases. These could be created using toxins from bacteria, viruses, parasites, or other appropriate species. As antigens, vaccines may include live, attenuated, avirulent, inactivated, or dead microorganisms. Some vaccinations are made up of antigenic fractions or chemicals that are made by the same pathogenic bacteria, but are rendered harmless while still being immunogenic. The microorganisms used to make vaccines can come from a single species, two species, or more. Recombinant DNA technology could be used to produce the antigen. Vaccines may be produced following the procedure detailed in the designated monograph or by using an alternative approach, assuring that the integrity of the antigen is maintained and that the products are devoid of any microbial impurities or extraneous materials. When vaccinations are made, appropriate adjuvants can be added. Antibiotics are often only added to materials used in cell culture media, material taken from skin or other tissues, and egg innocula during the production process. Vaccines may, if necessary, be preserved or treated with an appropriate bactericide. The finished goods are distributed under aseptic conditions and then sealed to prevent the entry of any foreign bacteria. However, as mentioned in the monograph, the final vaccine can be dispensed in single-dose or multiple-dose vials [1].

Veterinary biological products include vaccines, serums, immunosera, and other similar products that are formulated to induce active or passive immunity, assess the level of immunity, or detect diseases or health issues in animals. The procedure of immunizing a household, livestock, or wild animal is known as animal vaccination. The practice is related to veterinary medicine [2]. Louis Pasteur developed the initial vaccine for chicken cholera in 1879, marking a significant milestone in the history of animal vaccines [3]. Vaccines are classified into traditional and advanced generation vaccines [4]. Implementing animal vaccination has produced remarkable results, establishing it as the primary and lasting strategy for combating infectious veterinary diseases [5]. India has a significant resource of livestock and poultry, which plays a crucial role in improving the socioeconomic status of rural farmers. Monitoring health, diagnosing diseases and controlling them through effective vaccines are the key to increasing livestock productivity and reducing losses from disease morbidity and mortality. Ongoing research and development in veterinary biologicals have led to the successful eradication of major diseases such as rinderpest, African horse sickness, and contagious bovine pleuropneumonia in the country [6]. Various bacterial and viral diseases that affect cattle, buffaloes, sheep, goats, pigs, and poultry are being controlled through prophylactic vaccines created by the Animal Science Institutes of ICAR. These biologicals have been distributed to commercial manufacturers and state biological production units to ensure sufficient availability nationwide [7].

Indian Pharmacopoeia

Pharmacopoeia, a term derived from the Greek words “Pharmakon” (meaning drug) and “Poiea” (meaning to make), refers to an authorized and official publication issued by the government-appointed authorities in each country. As stated by the World Health Organization, there are currently 68 different Pharmacopoeias being effectively used in 66 countries worldwide [8]. It is an official book documenting medications or other pharmacological substances, including their usage, production, and control. It is legally binding scientific reference work that details the criteria and requirements for medications used in a particular nation or region and were created by a regional or national authority [9]. They are official publications that include international and national regulations and procedures that must be adhered to scientific and legal techniques, including the qualitative and quantitative measurement of active ingredients and excipients used in the manufacture of pharmaceuticals. The Pharmacopoeia is instrumental in safeguarding public health and ensuring the quality of drugs. It achieves this by combining recommended analytic procedures and specifications for excipients, pharmaceuticals, and dosage forms in its general sections and special monographs.

By translating scientific findings into common practice through the use of pharmacopoeial regulations, pharmaceutical analysis offers cutting-edge research which can directly impact the efficacy, quality, and safety of medicines [10]. Monographs in the pharmacopoeia detail the definition, description, appearance, production, identification, recognition-diagnostic analysis, physicochemical properties (such as solubility, boiling point, and melting point), quantification, biological properties (including biological activity and definition), packaging, and storage conditions of chemical, biological and biotechnological active and auxiliary substances, herbal/animal drugs and preparations, finished and medicinal products.

The Drugs and Cosmetics Act

The Indian parliament laid down Drugs and Cosmetics Act, 1940 which regulates the import, manufacture, and distribution of drugs in India [11]. The main goal of the act is to ensure the safety, efficacy, and compliance with the state quality standards of the drugs and cosmetics sold in India. In 1940, the legislation was approved and initially referred to as the Drug Act. The original act was prepared in accordance with the suggestions made by the 1930-formed Chopra Committee. The related Drugs Rules were passed in 1945. Since 1940, the act has undergone several amendments and is now known as the 1940 Drugs and Cosmetics Act [12].

The act is now referred to as the Drugs and Cosmetics some other goals:

  • Licensing-based regulation of import, sale and distribution of drugs and cosmetics.
  • Incorporation of exclusively qualified individuals in the distribution, import, and sale of medications and skincare items.
  • Maintain high standards for medical care by preventing the appearance of substandard drug quality.
  • Manufacturing and distribution of Unani, Siddha and Ayurvedic remedies.
  • Establishment of the Drug Technical Advisory Board (DTAB) and Drug Consultative Committees (DCC) for allopathic and allied drugs, along with cosmetics [13].

The Drugs and Cosmetics Rules 1945

The Drug and Cosmetics Act of 1940 gave rise to the Drugs and Cosmetics Rules of 1945, which were subsequently adopted by the Indian government. These regulations provide instructions for the sale, exhibition, storage, and prescription of each schedule of pharmaceuticals, which are categorised according to specific schedules [12,14]. License requirements are described in detail in Rule 67. The labelling requirements can be found in Rule 97 [15]. The rules have also undergone periodic revisions in order to address changing needs and address any flaws that were found when they were being put into practise.

History of Indian Pharmacopoeia

The Indian Pharmacopoeia Commission (IPC) is entrusted with the task of releasing the Indian Pharmacopoeia (IP) in adherence to the provisions outlined in the Drugs and Cosmetics Act, 1940 and Rules 1945. This responsibility is carried out on behalf of the Ministry of Health & Family Welfare, Government of India. IP is acknowledged as the official standard-setting document for drugs produced and/or commercialized in India. IP includes several authoritative methods for analyzing pharmaceuticals and defining their identity, purity, and potency. The regulatory agencies implement the authorized IP standards to guarantee the high quality of drugs in India. The IP requirements are deemed legally acceptable during the process of quality assurance and in the event of a legal dispute. The history of the Indian Pharmacopoeia (IP) begins in 1833, when a commission suggested that a pharmacopoeia be published. The majority of the remedies included in this pharmacopoeia, which was first published in 1844, are commonly used indigenous remedies. The pharmaceuticals from the British Pharmacopoeia and the indigenous remedies used in India were both covered in a later version published in 1868. In 1885, India officially ratified the BP. The publication of a National Pharmacopoeia was recommended in 1927 by a drug inquiry committee appointed by the government. The Indian Pharmacopoeia Committee was formed in 1948, after independence, with the publication of IP serving as its primary duty [16]. https://ipc.gov.in).

The Government of India established the IP Committee on November 23, 1948. The Central Indian Pharmacopoeia Laboratory (CIPL) was founded in 1965 as a subordinate office/laboratory of CDSCO under the Directorate General of Health Services (DGHS), Ministry of Health & Welfare. Drugs Controller was appointed as Member Secretary and Director CIPL was member of IP Committee. To help the IP committee, several other subcommittees were created. Along with other regulatory initiatives including Zonal testing of drug samples from India’s northern zone, CIPL actively participated in the publication of IP-1985, IP-1996, and its Addendum in 2000 and 2002. The Ministry of Health and Family Welfare, Govt. of India, submerged the existing Central Indian Pharmacopoeia Laboratory along with the Indian Pharmacopoeia Commission. IPC is an autonomous fully funded agency located in the Ghaziabad NCR region as of January 1, 2009. The publication of the Indian Pharmacopoeia serves the purpose of advancing the goals set by the Indian Pharmacopoeia Commission. The objectives revolve around the formulation of all-encompassing drug monographs that will be highlighted in the Indian Pharmacopoeia. This encompasses dosage forms, active pharmaceutical ingredients, medical devices, pharmaceutical aids, and the regular revision of these monographs. The goal of the IPC is to advance public health in India by developing authoritative and formally recognized standards for the quality of drugs used by patients, consumers, and healthcare professionals, including dosage forms, active pharmaceutical ingredients and excipients.

The Vision of the Commission is

Guaranteeing the highest standard of pharmaceuticals for both humans and animals, while considering the practical constraints of current manufacturing and analytical technologies [17].

Mission

To improve public health and animal health in India through the establishment of recognized and officially approved guidelines for the quality of drugs, such as active pharmaceutical ingredients, excipients, and dosage forms. These standards are intended for use by healthcare professionals, patients, and consumers [18].

Mandate

  • Publication of new editions and addendums of the Indian Pharmacopoeia
  • Publication of the National Formulary of India (NFI).
  • Certification and distribution of IP Reference Substances. • Serve as a National Coordination Centre (NCC) for running Pharmacovigilance Programme of India (PvPI).
  • Foster collaborative partnerships with similar institutions both nationally and internationally.
  • To coordinate educational programs, skills development initiatives, and research activities, fostering a collaborative environment for growth and innovation [19].

Status of Veterinary Products Standards

A pharmacopoeial monograph provides a reliable framework for conducting an impartial assessment of the quality of a pharmaceutical substance. IP 1996, its Addendum 2000, Supplement 2000 for Veterinary Products and Addendum 2002 were developed because of the ongoing and fast expansion of the variety of pharmaceuticals produced in India. With the veterinary supplement 2000 to IP 1996, veterinary items were highlighted for the first time. For ease of access, a distinct volume of veterinary items was introduced in IP 2014.

Global Market Status of Veterinary Vaccines

Veterinary immunizations improve overall productivity and guarantee animal health in livestock farming. To meet the needs of an anticipated global population of 9.1 billion people, it is predicted that total food production will have to expand by 70% from 2005 to 2050. Vaccinations that protect animal health and increase supply are essential elements in achieving this goal [20]. The increased demand for efficient immunization solutions and the increasing frequency of various infectious epizootic diseases are the two main factors driving the global market for veterinary vaccines. Along these lines, substantial government investments in the pharmaceutical industry to improve cutting-edge production techniques for veterinary

vaccines are acting as another growth-inducing factor. The growing demand for veterinary vaccinations to stop the spread of disease-carrying bacteria from livestock-based goods, such as milk, meat, leather, eggs, and wool, is also propelling the market’s expansion. Other elements, such as major advancements in healthcare infrastructure, ongoing R&D initiatives and strategic alliances amongst leading players to improve the effectiveness of veterinary vaccinations, are fostering an optimistic outlook for the market [21].

The market for animal and veterinary vaccines, which was valued at $ 10.69 billion in 2022, is projected to grow to $ 18.23 billion by 2030, with a CAGR of 6.9% from 2023 to 2030.

Some of the most common veterinary vaccinations are those against rabies, foot and mouth disease, and the equine influenza virus, which function by reproducing naturally acquired immunity to stop the spread [22]. In 2022, North America dominated the global market for veterinary vaccinations. The market for veterinary vaccinations was second largest in the Asia-Pacific region. Western Europe, Asia-Pacific, North America, Eastern Europe, South America, the Middle East, and Africa are the regions included in the veterinary vaccinations study. The 12 nations covered by the veterinary vaccines market include nations such as France, Germany, India, Indonesia, Japan, Australia, Brazil, China, Russia, South Korea, the United Kingdom, and the United States [23]. According to market research data, the Indian animal vaccination market is expected to reach $232 million by 2026. Given the increase in the prevalence of various animal diseases caused by the significant expansion of the poultry and animal industries, the rise of pet ownership, the adoption of scientific husbandry procedures in the large animal industry, and numerous government-led initiatives to improve animal husbandry and health, it is imperative that vaccine development continue. There is already and will continue to be a significant demand for animal vaccines as a result of these changes. More crucially the government of India’s new strategy for animal husbandry and animal health requires a vigorous and broad vaccination program for livestock animals to immunize against diseases like Brucellosis and Foot & Mouth Disease (FMD). Government-run programs have also given animal vaccine producers a boost. Government funding and subsidies for the development of veterinary vaccines will be an additional benefit of this massive One Health initiative. In conclusion, it will be important to keep an eye on the market for animal vaccines. Animal immunization will be required everywhere in the world as new animal diseases endanger the future of our planet. For the successful implementation of One Health and the sustainability of our planet, urgent issues must be addressed quickly and sustainably [24].

Status of Veterinary Products Monographs

An individual volume specifically for veterinary monographs has been created as Volume IV of the 9th edition of the Indian Pharmacopoeia in order to give individuals responsible with the quality control of veterinary medicines extensive information. This volume includes a variety of chemical monographs as well as monographs on surgical supplies, diagnostic tools, and veterinary vaccines. IP 2022 volume IV comprises standards for following- General notices, Veterinary general monographs (Intramammary infusions, intrauterine preparations, Veterinary diagnostics, Veterinary immunosera, Veterinary liquid preparation for cutaneous applications, Veterinary oral liquid, oral pastes, oral powders, parental preparations, tablets and boluses, Veterinary vaccines: General Requirements), Veterinary dosage forms, drug substances, and pharmaceutical aid monographs, Veterinary biological monographs, Veterinary diagnostic monograph, Veterinary Immunosera monographs, and Veterinary surgical immunographs. The commission has also asked stakeholders to provide feedback at regular time intervals on these veterinary monographs.

A few monographs and common chapters have moreover been changed, in addition, to overhaul them as per current worldwide prerequisites and to harmonize with other pharmacopoeias like USP, BP, EP, etc. [25]. Whereas the immunizations for veterinary utilize is experiencing harmonization and are beneath talk with specialists and stakeholders. The harmonization of benchmarks with worldwide measures is anticipated to offer assistance IP getting recognized and acknowledged in outside nations. At the show, IP is acknowledged and recognized in Afghanistan, Ghana, Mauritius, Nepal, and presently Suriname. Afghanistan was the first nation to recognize IP [26]. The IP guidelines are lawfully enforceable and definitive. It is a command for drugs manufactured and showcased in India to follow to the measures endorsed in the IP, disappointment that may render the sedate not of standard quality and may result in punishments under the Drugs and Makeup Act.

Status of Veterinary Vaccines Standards in IP 2022

The Veterinary Biological Monograph in IP 2022, Vol. 4, consists of 50 veterinary vaccine monographs (Figure 1).

Figure 1

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Need for Harmonization

The responsibility of the Pharmacopoeia is to establish or guarantee the quality of pharmaceutical standards. Quality standards (drug quality, safety, and efficacy) are crucial in the context of marketing authorization and market surveillance to ensure quality & safe standards to protect public health and facilitate the free movement and commerce of medicines between nations and regions. Therefore, the necessity for developing global quality standards for drugs, which ultimately aim at harmonizing the pharmacopoeia, is expanding as a result of globalization and the expansion of international trade [27]. The need to create global quality standards for pharmaceuticals is increasing as a result of globalization and the increase in international trade. Synchronization of standards, requirements, and regulations is crucial for international trade. Harmonization enables companies to take advantage of the essential needs of global markets, facilitating the production of market-specific products. Numerous public, private, and government entities are actively involved in harmonizing standards. Companies expanding into new markets and countries witnessing economic growth stand to benefit financially from these collaborative efforts [28,29].

Harmonization is needed since pharmacopoeial standards are an essential tool for marketing authorization, market surveillance, and the free flow of medicines between regions and nations. The advantages of establishing international pharmacopoeia standards to ensure consistent pharmaceutical quality appear evident. Currently, the bio-pharmaceutical business has a globalised manufacturing and supply chain. Having uniform standards to adhere has advantages for the industry. It is advantageous for regulators to be able to examine drug applications and pharmaceutical facilities anywhere without being hindered by inconsistent pharmacopoeia standards. Most importantly, there is a worldwide patient population today. Patients all over the world will ultimately benefit from being able to access medicines that are of the same quality, assessed against uniform standards found in all pharmacopoeias, regardless of where the product or its components were made [30]. Need for Pharmacopoeial Harmonization-

  • The purpose of creating a drug product that can satisfy the various market requirements is to develop
  • By eliminating duplication of effort, we reduce the overall cost of pharmaceutical research globally.
  • Update of the monographs with new Science and Technology inputs
  • As a result, cutting down on the time required for a) the availability of new medicines, b) the maintenance of international quality standards for marketed drugs (life cycle maintenance of marketed medicinal products), and c) the broadening of established medications to various regions (as part of the geographical expansion of medicinal products)

The Indian Pharmacopoeia Commission understands the value of collaborating with other pharmacopoeial bodies to provide uniform general chapters and monographs. The streamlining and rationalization of quality control systems and licensing procedures are just two advantages of this harmonization, which is completely aligned with the Commission’s stated goals. Since some of the developed guidelines rely on pharmacopoeial general chapters for their application, such harmonization also increases the advantages of the work of ICH and VICH. The restructuring of the monographs has been undertaken for the following reasons.

  • The up gradation of these monographs is required to meet global requirements.
  • There is a lack of uniformity within the IP monographs.
  • Restructuring of monographs along with harmonization with international standards helps in increased globalisation of domestic products.

Development of Restructured Veterinary Vaccine Monograph

(Figure 2).
Figure 2

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Background of the Structure for Veterinary Vaccines in IP

Purpose of the Structure of the Veterinary Vacuum

This structure guide provides guidance to contributors of Indian Pharmacopoeia monographs on veterinary vaccines. The aforementioned applies specifically to the

  1. Group of experts and higher approval authorities for vaccines for veterinary use.
  2. The National Control Laboratory (NCL) is also subject to these provisions.
  3. Manufacturers of vaccines and immunosera for veterinary use are also included in this category.
  4. Additionally, analytical laboratories (both public and private) that work for any of the entities mentioned above are also covered by these regulations [29].

The official standards for medicinal products are established by the monographs and general chapters of the Indian Pharmacopoeia. Should there be any uncertainty or disagreement, the Indian Pharmacopoeia text holds the ultimate authority (Figures 3-6).

Figure 3

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Figure 4

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Figure 5

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Figure 6

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Content of the Monographs on Vaccines for Veterinary Use

The General Notices state: “All statements contained in the monograph, except where a specific general notice indicates otherwise and with the exceptions given here after, constitute standards for the official articles. Exceptions to the General Notices do exist, and where they do, the wording in the individual monograph or an appendix takes precedence and specifically indicates directions or the intent. Thus, the specific wording of standards, tests, assays, and other specifications is binding wherever deviations from the General Notices exist. Likewise, where there is no specific mention to the contrary, the General Notices apply” [16]. The requirements for a particular preparation may not be fully comprehensive; additional requirements could be specified in the individual monograph. Drug products covered in an individual monograph must also meet the tests described in general monographs. The drug products specified in an individual monograph must also meet the requirements set forth in the general monographs.

Sections of the Monographs

The restructured IP monographs are designed under the following sections.

Title of the Monograph: The title of the monograph consists of

  1. Vaccine and the type i.e. live or inactivated
  2. Name of the disease for which it will be used
  3. ‘for veterinary use’ (where the vaccine for human use also exists) should also be mentioned, and
  4. the target species where necessary, for example. Brucella Abortus (S19 Vaccine) Vaccine, Live, Canine Adenovirus Vaccine, Inactivated
  5. Synonym of the vaccine.

Definition: The monograph outlines the extent of its coverage and its significance for the products accessible on the market. It establishes the authoritative reference point for all products falling under this description. Furthermore, the product composition is succinctly detailed in each individual monograph. For example, a vaccine (live / inactivated) is a formulation of one or more appropriate strains [bacteria / virus]. This monograph is applicable to vaccines designed for active immunization (Live Vaccines) or those that are inactivated but retain sufficient immunogenic properties (inactivated vaccines).

Production: The content of this section is primarily directed towards manufacturers. The document covers the principles and details related to vaccine production, the anticipated tests throughout the product’s creation, the regular and ongoing tests that producers may perform, and the batch tests executed to guarantee the pharmaceutical quality of the product. Manufacturers are provided with direction on how to demonstrate the clinical value and efficacy of their products through developmental testing. The production section of the general and individual monographs contains a blend of requirements and information related to specific aspects of the manufacturing process, such as source materials, process validation and control, and in-process testing. These elements are crucial in showcasing the consistency of the manufacturing process.

This subsection in the individual monograph includes specific requirements for:

  • Preparation of the Vaccine: This subsection discusses the various techniques used in the vaccine preparation, including the propagation and harvest of bacterial and viral antigens, the inactivation process, and the preparation of the final bulk and batch. The production of the vaccine is based on a seedlot system, which ensures consistent and reliable manufacturing. For inactivated bacterial vaccines, the seed lot is cultured in a suitable medium to promote optimal growth under specific incubation conditions. After culture, the bacterial suspensions are collected and subsequently rendered inactive using a suitable technique. In the case of live vaccines, they can be lyophilized, liquid, or frozen. The formulation may also include stabilizers, buffers, excipients, and an adjuvant to enhance its effectiveness. The vaccine virus is typically grown in embryonated hens’ eggs or in cell culture.
  • Substrate for Virus Propogation: Embryonic eggs. The eggs of the embryonated hens are sourced from SPF flocks when the vaccine virus is cultivated in them. Cell cultures. Alternatively, cell cultures must meet the standards for the production of veterinary vaccines if used for virus growth. In the case of continuous cell lines, they must originate from a seed-lot system. When primary chicken cells are used, they should come from SPF flocks.
  • Seed Lots

a. Extraneous Agents

The master seed lot satisfies the criteria for the presence of foreign agents in the seed lots. It passes the test if it does not induce the production of antibodies against the agents tested. During the examination of the master seed lot, the organisms utilized are within 5 passages from the original seed lot at the start of the tests.

b. Choice of Vaccine Composition and Choice of Vaccine Strain: This section pertains to the safety and effectiveness assessments that must be performed while developing a vaccine, as outlined in Sections (2.7.17) and (2.7.12). Normally, these assessments are performed just once throughout the vaccine development process. Unless specified otherwise, the testing procedures provided for confirming these attributes and the acceptable limits, when applicable, are included as illustrative examples of suitable methods and the corresponding limits. However, developmental evaluations must be performed in a manner that ensures that the product meets pharmacopoeial standards [29].

c. Safety: Compliance with the detailed requirements specified in the chapter (Evaluation of safety in veterinary vaccines and immunosera) is essential. The individual monograph may provide technical insights into various tests to assist in establishing appropriate protocols. To assess the effectiveness of animal vaccination methods and routes of administration in all relevant categories, it is essential to carry out tests using animals that are below the minimum vaccination age.

d. Test for Reversion to Virulence/Increase in Virulence: For live vaccines, for example, details are usually provided for the conduct of the test for increase in virulence. Carry out the test using an animal / SPF flock if it is chicken free from antibodies against [virus]. The vaccine virus is in accordance with the test if there are no indications of increased virulence in the organism obtained from the final passage when compared to the material used in the first passage.

e. Immunogenicity or Potency: The administration of vaccination tests should be performed for each recommended route and method unless otherwise specified in the monograph. The age of the subjects used in the tests should not exceed or fall below the minimum or suitable age recommended for vaccination. The quantity of the vaccine strain administered to each animal should not exceed the minimum number of live bacteria stated on the label, and the strain present in a batch of vaccine should be at its most attenuated passage level. The specific procedure for testing will be defined for each individual vaccine.

Manufacturer’s Tests: This segment focuses on the examinations that the manufacturer may carry out as part of the testing process to demonstrate that each batch meets the required quality standards. These tests are specifically designed to ensure that the batch complies with the pharmacopoeial requirements outlined in this section. This section covers a wide range of test variations which are tailored to the specific characteristics of the product. The tests carried out by the manufacturer, which are specific to each product, are compiled in the dedicated section of the individual monographs. There are no specific numerical limits provided, as the manufacturer needs to establish these limits based on the observed values from batches of vaccines that have been proven to be safe and effective. The most frequently listed tests in individual monographs are as follows: • Identification: Identification of the vaccine virus is accomplished by using appropriate molecular biology, biochemistry, cell culture, and immunochemical techniques.

  • Batch Potency Test: Conducting the relevant potency test or tests for every batch of the vaccine is not required if a batch with a minimum potency has already undergone the test. In the absence of these tests, an alternative validated method is utilized, with acceptance criteria determined based on a batch of vaccines that has shown satisfactory results in the potency test.
  • Residual Live Virus: A test is performed to determine the presence of any remaining live viruses. The amount of inactivated virus used must be equal to or greater than x doses of vaccine. If a live virus is not detected, the inactivated virus harvest is considered to meet the test requirements. In the case of live vaccines, analysis must be performed to determine the concentration of virus or the number of bacteria as specified in the relevant individual monographs and general monographs. An appropriate acceptance criterion for this test is expected, which takes into account the following points: – The minimum acceptable virus concentration or bacterial count should be determined during development studies, based on the vaccine sets used in the efficacy test or other efficacy studies. – The loss observed in stability studies should be added to this value to ensure that the content remains above the minimum acceptable concentration or amount at the end of the shelf life. The concentration or amount equal to or greater than the calculated value must be checked after the release of each batch. For inactivated vaccines, it is assumed that instead of a potency test, an appropriate group efficacy test will be developed for routine use. Acceptance criteria must be determined on the basis of the correlation of the results of the set that passed the performance test. This information is generally presented in a comprehensive manner and may suggest different methods. For inactivated vaccines, it is recommended to consider the use of in vitro methods during development, provided that the most important in-process parameters are defined and monitored and in-process control tests and the target composition of the final product are considered.

Batch Tests: This section focuses on tests that a manufacturer can perform as part of the testing process to demonstrate that each lot meets the required quality standards. The purpose of these tests is to verify that the batch complies with the criteria outlined in the pharmacopoeia. The manufacturer’s test section in individual monographs contains specific tests based on the nature of the product. It provides information on tests that must be performed regularly and can be applied to many vaccines. The general monograph section contains guidelines and qualification scores for tests such as free for maldehyde, phenol, identification, sterility, foreign body mycoplasma, safety and efficacy. The individual monographs in this section describe the tests and requirements that all product lots must meet during their shelf life. This means that all lots on the market must meet these requirements when tested by an independent analyst. In order to exempt the batch manufacturer, these tests do not need to be carried out for each batch, if the tests carried out during production or other tests on the final product provide an equal or better guarantee of conformity or if alternative tests have been validated according to the pharmacopoeial method. Except for a few cases, individual monographs consistently have a section called Power. This usually involves performing the test described in the Immunogenicity section. The monograph incorporates an efficiency test that can be conducted on any batch, thereby utilizing a single recommended method of administration.

  • Identification: The vaccine strain is determined through the use of suitable methods. Eachindividual strain within the vaccine is identified through various methods such as morphological, serological, molecular, immunochemical, or biochemical techniques, along with culture on selective medium.
  • Bacterial and Fungal Contamination / Sterilization (2.2.11): To evaluate the efficacy of live vaccines, it is essential to quantify the population of live bacteria in a suitable solid medium for the cultivation of bacterial strains. The vaccine is deemed compliant if each dose contains no less than the minimum number of live bacteria specified on the label. Similarly, inactivated vaccines, together with the applicable reconstitution diluent, must meet the sterility test outlined in the general requirement.
  • Mycoplasmas (2.7.8 or 2.7.9): The vaccine meets the requirements for mycoplasma testing.
  • Water (2.3.43) Percent Moisture Content if it is a Lyophilized Formulation.
  • Extraneous Agents: For live viral vaccines, it is essential to use a monospecific antiserum to neutralize the vaccine virus before inoculating it into cell cultures that are susceptible to animal pathogens. The vaccine is deemed acceptable if there are no cytopathic effects observed and there are no haemagglutinating or haemadsorbing agents present. Furthermore, the batches of finished products undergo tests to ensure that the vaccine meets the required criteria for extraneous agents.
  • Safety: Each recommended route and method of administering the vaccine to animals in relevant categories or laboratory animals should undergo testing. Animals selected for the test must be within the recommended minimum age for vaccination, as outlined in the individual monograph. The test should be conducted using an attenuation level equal to or less than the commercial batch. Furthermore, the test should be performed with the highest expected dose level or a microbial count that is [number] times higher than a single dose. When in vivo batch tests are performed on target animals for purposes other than the target animal safety test, such as potency tests, and these tests involve the collection of safety information, such as mortality data, it is recommended that manufacturers make use of these tests to gather additional safety data for the vaccine in the target species. 2. Note: General requirements shall be referred to regarding omission of the batch safety test.
  • Potency: The vaccine meets the requirements of the immunogenicity test when administered by the recommended route and method. It is not necessary to conduct the potency test for every batch of vaccine if it has already been performed on a representative batch using a vaccine dose that contains no more than the minimum bacterial count indicated on the label. An alternative in vitro method can be utilized as a potency test for batch release if a correlation between the potency test and the alternative test has been established. In the batch test, the virus titer can be used as a substitute for in vivo potency testing if a correlation between the virus titer and potency has been established [31].

Labelling: The labeling requirements outlined in the general monograph are applicable to all veterinary vaccines. Additional information may be required for specific vaccines, which will be incorporated in the individual monograph in the Labeling section. This supplemental information complements the general requirements of the monograph. The label must clearly indicate that the vaccine is 1. for veterinary use only, 2. the recommended routes of administration, 3. instructions for use, 4. the targeted animal species, 5. storage temperatures, 6. Batch Number, Manufacturing date, date of expiry, 7. precautions for pregnant animals, 8. total volume and number of doses, and 9. a statement in compliance with the approval document [29].

Major Highlights of Restructuring

Indian Pharmacopoeia Commission (IPC) has taken initiatives to implement 3R through the Indian Pharmacopoeia (IP). Through deletion of animal tests at final lot for biologicals and scope of reduction in number of animals used where deletion of the animal test is not possible and refining the tests causing the minimum suffering to the animals are explored.

Target Animal Batch Safety Test

Blanket waive off for all vaccines may have a risk involved considering the fact of potential safety risk involved. Therefore, the provision for conditional waiver for the target animal batch safety test is included in all veterinary vaccine monographs and general requirements for veterinary vaccines along with a note are included in the revised ‘General requirement-Veterinary vaccines’ for the waiver of TABST. Note: The batch safety test using target animal can be omitted if 1] safety test has been performed with satisfactory results in the master seed lot and 2] the consistency of the manufacturing process has been well established to the satisfaction of the National Regulatory Authority and 3] at least 10 consecutive production batches have been produced and comply with the safety test. Significant changes to the manufacturing process may require resumption of routine safety testing to reestablish consistency.

Batch Potency

Hemorrhagic Septicemia Vaccine, Inactivated: For batch test, mice potency test will be included in addition to cattle as was mentioned earlier in IP 2010 & IP 2014.

Immunogenicity

Along with sheep, a laboratory model (guinea pig or rabbit) is also included as a model for the immunogenicity testing under production.

Safety

Canine parvovirus, activated – reduction in the number of target animals, ie, dogs, from six to two in the safety test under production.

Challenges While Harmonizing the IP Veterinary Vaccine Monographs

Pharmacopoeial harmonization assists global patients who depend on these medications to prolong and improve their lives by improving support for international regulatory organizations and addressing the global nature of bio-/pharmaceutical manufacturing and delivery. In addition, there are still several obstacles in the way of the total harmonization of these IP monographs. First, the safety profiles and therapeutic efficacy of finished pharmaceutical products (FPPs) are greatly influenced by their quality. Production is becoming more multi-national because of global trade and pharmaceutical firm mergers, which upset national restrictions. However, the failure to follow Good Manufacturing Practices by manufacturers and ineffective quality control measures may result in quality faults. Although various manufacturers may use various control mechanisms, experience has shown that some quality control and manufacturing procedures are insufficient to guarantee the creation of high quality medications. Despite the various measures implemented to address these deficiencies, additional rigorous protections must be established. However, it should be noted that more stringent regulations could lead to higher expenses and additional challenges for both producers and regulators, who are already facing resource constraints. Second, there is diversity among the antigen strains found locally. Since antigenically variable pathogens (AVPs) are the primary cause of infectious diseases today, they bear a heavy cost. The biggest challenge in creating new or better vaccines is high genetic and antigenic heterogeneity. In addition, there is a growing public awareness of the positive impacts of veterinary vaccines on animal health. Ultimately, if vaccines are to be used, they must be produced in such a way as to ensure consistent and high-quality performance. Since vaccines are those biological products that exhibit intrinsic variability and therefore, again making this harmonization challenging at the grass root level [30].

Conclusion

The Indian Pharmacopoeia (IP) is the official document defining the standards for drugs in India and contains detailed information on the quality, purity, and potency of medicines available in the country. The role it plays is essential to guarantee the quality, safety, and effectiveness of medications in India and is an important tool for the pharmaceutical industry, regulatory authorities, and healthcare professionals. Pharmacopoeial standards are an essential cog in the proper operation of marketing authorization and market surveillance processes, as well as the free movement and commerce of drugs worldwide. Harmonization of global pharmacopoeia would improve access to high-quality pharmaceuticals for people all over the world. These continuous harmonization efforts are supported by the industry’s position on the ideal pharmacopoeia and methods for achieving compendial globalisation. The industry benefits from having consistent standards with which to comply. Regulators gain from the ability to assess drug applications and visit pharmaceutical facilities anywhere, without the complications of various pharmacopoeia standards. Most importantly, the patient population today is global. Finally, patients around the world will benefit from receiving medications of the same quality, measured against standard requirements included in all pharmacopoeias, regardless of where the product or its ingredients were created.

Data Availability

The research data is not presented in the article.

Declaration of Conflicting Interests

The author declared that she has no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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Borderline Aspects of Biomedicine

Mini Review

I, as a professional analytical physicist who began to study Science, however, from the optional school of the mathematician Pontryagin (60 years ago), am interested, first of all, in the borderline aspects of Biomedicine with the Fundamental Physical Laws, which I have been actively combing in recent years. But these latest works of mine did not begin out of nowhere, but as the result of a piecewise continuous research method, which I have been using for half a century at the Academy of Sciences. The essence of this method is simple – in the search for technical solutions to current Problems, experiments are carried out on the basis of known Models that can provide answers that existing Theories cannot provide answers to. And as a result of PRECISION experiments, you come to the need for clarification of standard Theories, and even for redefining the Fundamental Concepts of Physics. This is how I have previously been able to create devices and technologies that can radically improve the performance characteristics of devices and materials. I created (with different people-organizations) ultra-low-noise Electronics elements, ultra-sensitive Radiation Detectors, ultra-selective Radiation Sources, precision broadband polarizers and a number of unique materials, for which I received the title “Inventor of the USSR”. In recent years, I have concentrated on Corrections in the FOUNDATIONS of Physics. These latest works of mine have already received about one hundred thousand views in scientific journals, and on their basis I prepared the book “FOUNDATIONS OF QUANTIZATION” for publication. Next up, after completing the Redefinition of the Magnetic Field, is the book “FOUNDATIONS OF RELATIVITY”.

The key word for the experiments I conduct is PRECISION. And this level of my experiments far exceeds what I saw in such advanced medical centers as the Bekhterev Brain Institute, where I was invited for a consultation, and at the Almazov Cardiology Center, where I ended up as a patient. But the laxity of experimental medical measurements may not be the main drawback of Medical Research. The no longer young and elderly man himself, having fallen into the clutches of doctors after an injury, saw not only a lot of technical errors (which even my worst graduate students would not have made), but also a METHODOLOGICAL Error in the artificial blocking of the information channel of communication between the doctor and the INDIVIDUAL, who is being turned into faceless, average patient [1].

Thank God at the Almazov Center there were adequate young doctors and showing my heart on the screen, they consulted with me about inserting a manipulator into it and helped me to rehabilitate, and did not harm me like the previous doctor who asked me: “How do I feel?” and when I answered, then shouted: “Who is treating whom— me you or you me?!” To which I replied: “I am used to making precision measurements and I simply described to you my precise sensations and their UNDERSTANDING!”But the point, of course, is not in these demos. In addition to the above-mentioned General Medical Issue, the pandemic has raised a number of Current Medical Issues [2], the solution of which is only possible with the involvement of Sciences related to Medicine [3-7]. Specifically, Biomedicine, combining two independent disciplines, is borderline not only phenologically, but also in terms of considering LIFE as a borderline state [8]. Indeed, the processes she studies relate either to the emergence (birth) of life, or to its stagnation (dying). These processes and their speed of development were predetermined by Nature itself. And a person, with the help of Medicine, either corrects failures at these stages, or tries to slightly modify them. At the inception stage, there are known cases of acceleration of processes due to external conditions.

Thus, on Sakhalin at the initial stage, the processes of the emergence of life proceed faster than on the mainland, both in plants and in animals and people. There, it is not uncommon, but the norm, not only the rapid growth of plants, but also the birth of seven-month-old children. Apparently the “clean” atmosphere of the ocean also affects the acceleration of development. It also influences the slowing down of stagnation processes in the body in old age, which is clearly manifested, other things being equal, in higher life expectancy in Japan. A “clean” atmosphere apparently varies; in the mountains it promotes life expectancy, but there are no confirmed statistics on accelerating fetal development. Thus, we can identify a common parameter, the change of which divides the area of origin and stagnation of the organism – this is the rate of cell division. If we move from the integral characteristics of the stages of life to consideration of the speed of chemical processes, then, I think, we will not see such a critical change in it even in the young Schwarzenegger, nor his old one.

But in his youth, under loads, his muscle mass, after micro-tears, only increased, and in old age, not only does it not increase, but shrinks and even tears, not having time to heal between workouts. Not everyone is given the opportunity to become champions, but chemistry, the speed of chemical reactions, from the comparison of the processes of emergence-stagnation and restoration of muscle mass, rather influences the extreme achievements of the human body as a background. Like a certain limiting “speed of sound”, while its achievement can be achieved by “effort of will”, as, for example, in sports, and in science, and in art. So, achieving this “speed of sound” determines the speed of information transmission, which is determined not only and not so much by the speed of transmission of nerve impulses (which also acts as a kind of background parameter), but by the HARMONY of the Dynamic Element of LIFE [9]. Even before the Nobel laureates in physics showed us a quiet adagio “performed” by an individual living cell – Harmonic Sounds – NOTES transforming into other NOTES without violating Harmony, Ali Engalychev and I used vibration during a solid-state phase transition – growing disproportionate crystals from a highly symmetrical solid crystal as it passes through a temperature gradient. Thus, it was shown that the rate of phase transformation increased by orders of magnitude even in the solid state.

And since living tissue is in no way solid, phase transformations in it are guaranteed to accelerate even from simple vibration. Ali and I also did a playful experiment with crystals, playing symphonic music in them, but we didn’t have enough time to obtain reliable results at temperatures above a thousand degrees. Whereas at the temperature of a living cell, its reaction to Harmonic Sounds is not particularly difficult to study. Many, as I have already described, magnificent Ideas are buried under the “cultural layer” [10]. But the pandemic hit us over the head and some of them surfaced. So Lev Zilbert, back in the middle of the last century, developed the concept of the viral nature of cancer. So, almost 100 years have passed and only now they remembered Belozersky’s Idea of the emergence of LIFE – “It seems that RNA, associated with the most general manifestations of life, was formed at an earlier stage in the development of life, while the emergence of DNA is associated with the formation of narrower and phylogenetically later properties of organisms.” (A. N. Belozersky, Moscow International Symposium “The Origin of Life on Earth”, 1957). These luminaries in their reasoning did not proceed from consequences, as is customary (say, the proliferation of cancer cells), but from causes. The fact is that the growth of a LIVING Cell is not just a chemical reaction or a chemical process. Purely chemical growth would last indefinitely. The growth of a LIVING cell comes from already prepared Fragments, from non-living fragments of LIFE, but not living ones.

Those. in fact, from the same level of fragments as viruses. And pathogenic viruses replace the fragments necessary for a LIVING Cell to reproduce. And certain viruses are embedded in such a way that the defective Cell retains the ability to reproduce. And these considerations can be supplemented, at the modern level, by the fact that the conclusions about studies of canonized DNA and RNA are in many ways a decoding of “texts” on burnt paper. Whereas it is more informative and more gentle to use IR spectroscopy, which directly provides information not only about the structure, but also about the DYNAMICS, about the Music of LIFE of a LIVING CELL, the NOTES of which do not include the pathogenic virus fragment. Moreover, even at the dawn of the last century, for ELEMENTARY reasons, in principle, an experiment on the Characteristic Frequencies of Viruses was correctly carried out (Table 1). If we discard the semi-mystical theory- interpretation of the Characteristic Frequencies of R. Rife (these experiments can be quite correctly described by concentration models), and take into account that the frequencies he used are modulation frequencies of the carrier frequency of 330 MHz, then we can assume that he “felt” low-level Harmony viruses (not yet the Music of LIFE of a Living Cell, but Harmonic vibrations that do not coincide in frequency with the NOTES OF LIFE). The fact that official Medical Science tried to discredit Ralph’s experiments reflects only its then tendentious attitude towards technical innovations. As I already mentioned, I, as a high-level technician, found a lot of errors in the most advanced modern medical experiments. And Ralph’s measuring technique needs to improve. But the IDEA itself of using simple Harmonic Oscillations to influence fragments of LIFE – viruses, deserves close consideration.

Table 1: CT Exam Protocol.

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Love Addiction: “I Can’t be with You, I Can’t be without You.” Psychological Aspects and Clinicals Intervention

Introduction

Love as an Addiction

Romantic love is a universal phenomenon that every individual seeks during his life, for the purposes of reproduction and survival. Love inevitably influences the quality of life of each subject, as it can bring joy and happiness, but also pain and suffering. It has been the object of interest of artists, singers, poets and philosophers, who described it as a deep and intense feeling, capable of causing both euphoria and despair. In this regard, Ovid wrote: “I cannot live without you or with you”; in Plato’s Symposium, Aristophanes argued that, in the beginning, man was a perfect and complete being, a creature with four arms, four legs and one head with two faces; there was, therefore, no distinction between man and woman. To reduce the superiority of men, Zeus, envious of their perfection, decided to divide them in two, condemning them to the continuous search for their half, to recover the lost perfection and feel complete again. From this, it emerges the common conviction of having to find one’s own soul mate, “the other half of the apple” that can fill its own void, a conviction that influences people’s behavior and how they conceive love. The English term “addiction” comes from the Latin “addictus” which means “ enslave “. The expression is also used for the use and abuse of chemicals. In recent years, the scientific community has also treated behavioral addictions that are obsessively repeated, these addictions however, are socially accepted and refer to “new addiction” and dependent love, can take on the connotation of slavery.

The boundary between love and emotional dependence is thin, in the early stages of a relationship is physiological the need to want to spend more time with your partner, the addiction to the loved one and his idealization, are part of the physiological process, in a healthy relationship, these aspects gradually fade with time, leading to seeing and accepting the partner in its uniqueness, despite differences; while, in emotional dependence, idealization persists excessively and one-way, The desire of the other becomes a compulsive and necessary need to function, and you continue to remain in a relationship despite the negative consequences. In the literature, there is no unanimous agreement on the relationship between love and addiction; in this regard, Earp and colleagues [1] have proposed two perspectives: the narrow view and the broad view. The narrow view considers love as an affective dependence, only in some conditions, in reference to the phases of falling in love and the most extreme and pathological behaviors. Advocates of this view consider aspects such as: sexual compulsions, pedophilia, toxic or abusive relationships, abnormal attachment and lack of tolerance of the negative outcomes of life and relationships [2,3]. According to neuroscientists and psychiatrists, the prevailing model of substance dependence assumes that drugs generate addiction, as they gradually trigger abnormal and non-natural functioning patterns [4], therefore, addictive behaviors are the result of brain processes not present in the brain of unrelated subjects.

Volkow and colleagues [5] have shown how the intake of substances activates neurotransmitters in the brain to create reward signals, learning patterns occur in the brain cellular adaptation that would not occur if there were no intake of substances. The addict who continuously searches for drugs acts a typical drug addict behavior. Other researchers have, instead, found behavioral and neurological similarities between, for example, those who take food uncontrollably and those who use substances. The narrow view of affective dependence, therefore, argues that one can be dependent on love, but only in the presence of abnormal brain processes, analogous to those that form when taking illicit drugs. The lover suffers from addiction if he engages in sexual behavior or abnormal attachment, probably related to abnormal brain processes, therefore his search for love:

1) Interferes with his ability to carry out daily life activities.
2) Prevents healthy relationships.
3) It has negative consequences for themselves or for others. The broad view considers love an addiction, i.e. being in love is equivalent to being addicted, with similar chemical and behavioral processes [6,7]. Proponents of this view place emphasis on the similarities of experience between addicts and those involved in a phase of falling in love. Such analogies are found as stated by Fisher and colleagues [6] in “mood swings, desire, obsession, compulsion, distortion of reality, emotional dependence, personality changes, risk taking and loss of self-control”.

Burkett and Young [7] hypothesize that there is “a deep and systemic concordance between the brain regions and the neurochemicals involved in both addiction and social attachment”. Love produces a gratification, similar to that obtained from drugs, food, gambling, or sex [4,8,9]. Addictions are appetites that each individual possesses and such appetites if they are not satisfied for a long time, manifest themselves in a more intense way. Therefore, each individual is dependent on something, food, sex or love; what differs is the intensity with which it manifests itself [1].

Affective Dependence

Affective addiction [love addiction], a term introduced for the first time by Peele and Brodsky [10] in Love and addiction, is a widespread problem, especially in women. Affective dependence is a new addiction that refers to a series of dysfunctional behaviors, in which the object of dependence is not a substance, but a socially accepted behavior or activity; however, it has not yet been included in any nosographic system, therefore, there are no recognised diagnostic criteria for diagnosis, nor a precise definition. Despite this, several scholars, including Reynaud and collaborators [3] have proposed a number of criteria similar to those for substance dependence. They define emotional dependence as “A maladaptive or problematic love relationship pattern leading to clinically significant impairment or distress, as manifested by three [or more] of the following [the first five of which occur at any time in the same 12-month period]” [3]:

1. Presence of withdrawal syndrome in the absence of a loved one, characterized by significant suffering and compulsive need of the other.
2. A considerable amount of time invested in the relationship [or with thought].
3. Reduction of important social, professional, or recreational activities.
4. Persistent desire or unsuccessful efforts to reduce or control the relationship.
5. Continuation of the report despite the existence of problems created by the report.
6. Presence of attachment difficulties, as demonstrated by at least one of the following:
a) Repeated love relationships exalted without any lasting period of attachment.
b) Repeated painful love relationships characterized by an insecure attachment.

The “women who love too much” [Robin Norwood] tend to enter relationships with distant people, cold, unable to enter into intimacy, with the belief that with their love they can change them; a vicious circle is thus established that confirms the belief that we are not worthy of being loved, increasing their suffering. In addition, the attitude of the other is justified, attributing it to traumatic or painful situations experienced during childhood. The relationship, despite causing pain, is not interrupted, as it has become the center of their life: the desire to spend as much time as possible with the loved one increases, when this does not happen you think obsessively about the relationship; his absence is experienced as intolerable; you lose interest in everything that was important before; friends, family, work/ study take second place. The partner is idealized, and it is thought that without him his existence has no meaning. Their goal is to take care of the partner, putting them completely aside, until they cancel themselves. One’s well-being depends on one’s partner and they tend to entrust responsibility for one’s own happiness to the other.

Psychobiology of Affective Dependence

Studies have shown that all forms of dependence are based on a common substrate, the mesolimbic reward system. It consists of a network of brain structures which are activated in response to pleasant stimuli, rewarding and that interact with each other for the regulation of motivated behavior, for associative learning and for the attribution of salience. This system consists of some main areas:

1. The ventral tegmental area [VTA] is a group of neurons located at the base of the midbrain. Dopaminergic cells of the Mess corticolimbic system originate in VTA, which play a crucial role in the reward system.
2. The nucleus accumbent is a brain region located in the basal forebrain, and also plays a fundamental role in the reward system. It is involved in motivated behavior, the regulation of pleasure and gratification associated with specific stimuli and, therefore, in learning by reinforcement.
3. The prefrontal cortex, is localized in the anterior portion of the frontal lobe of the brain, precedes the primary motor cortex and the premotor cortex. It is a cortex involved in several higher cognitive functions, such as planning, motivation, cognitive control, regulation of social and emotional behavior; specifically, the frontal orbiting cortex is involved in decision-making processes. In relation to addictions, it has been observed that the activity of the prefrontal cortex increases during abstinence, thus demonstrating that it has a role in the reiteration of addictive behaviors [11]. Furthermore, by being implicated in executive control over decision-making processes, Goldstein and Volkow [12] suggest that an alteration in the way it works may result in the loss of impulse behavior control, Decisions are therefore taken based on an immediate reward; at the expense of the negative consequences of one’s conduct.

4. The limbic system, responsible for motivation and reward, regulates emotions and stores emotionally significant events. It is formed by two structures, amygdala, and field, important for emotions and memory. In particular, the amygdala is in the anterior portion of the medial temporal lobe of the brain and plays a crucial role in the processing and regulation of emotions especially negative ones [such as fear, sadness, anxiety] it is also involved in the formation and storage of memories related to emotional experiences, associating emotions with events. The hippocampus is localized in the medial portion of the temporal lobe and is implicated in learning and memory processes, especially in the encoding of memories and emotional valences related to such memories [13].

At the neurobiological level, studies of biochemistry, functional neuroimaging and genetics highlight a close relationship between behavioral addictions and substance dependence [14], as, for example, food, gambling, Affective dependence are activities that activate the circuits responsible for gratification, like those caused by the use of drugs [15]. Goodman [16] hypothesized that behavioral addictions are the basis of a common “additive process” resulting from the alteration of three functional systems: “motivation-gratification”, “regulation of affections” and “behavioral inhibition”. the negative consequences of their conduct.The alteration of the motivation-gratification system causes unpleasant sensations in the subject, this causes the conducts capable of activating the gratification system to acquire greater reinforcement [16]. The alteration of the regulation of affections leads the individual to avoid painful emotions, experienced as intolerable and which he is unable to control [16]. The alteration of the mechanisms of behavioral inhibition, finally, determines in the individual the need to perform a behavior able to activate the gratification system, without thinking about the negative consequences that it determines in the long term [16].

Pathological Addictions: Maccanisms

In recent years, the idea is spreading that the term addiction can also refer to the recurrence of behaviors, which we feel the need to implement compulsively. The characteristic of these behaviors is that they are socially accepted, which are part of their daily life; in this case, we speak, therefore, of behavioral dependence or new addiction defined as “a group of heterogeneous disorders […] that imply an involvement in a repetitive and persistent habit, aimed at changing the state of consciousness of the individual, and that in the long term involve a significant impairment of the sphere of work, affective-relational and social of the subject” [15]. These include pathological gambling, compulsive shopping, addictions to work, study, technology, sex, exercise, food, and relationships. However, the scientific community recognizes only pathological gambling, including it in the DSM-V among the “disorders related to substances and addiction disorders” [APA, 2013]. The English terms “dependence” and “addiction” are both translated, in Italian, with a single term “dependence”; however, these English terms refer to two different phenomena: the first indicates the physical dependence on the substance that the body needs to function; the second indicates, instead, psychological dependence on a pleasant stimulus. A subdivision of addictions that is based on social regulators is due to Francisco Alonso-Fernández [17], who distinguishes “social or legal addictions” that include legal drugs [such as alcohol, drugs, tobacco, etc.] and socially accepted activities [such as eating, working, shopping, playing], “antisocial or illegal addictions” that include drug addiction and illegal activities [such as cocaine, heroin, cannabis, non-consensual sex] [17].

From a symptomatologic point of view, there are many similarities between behavioral and substance use addictions:

• The individual is unable to control his or her behavior by acting compulsively, which gives him or her pleasure.
• Shows craving, defined as “an attraction, of varying intensity, towards certain psychotropic substances of abuse[…] at high levels of intensity, intense and serious psychophysical alterations appear that lead the subject to think only about the substances from which he is attracted and the means by which to obtain them” [18];
• Can develop abstinence in case of absence of the object of dependence and tolerance that is, the rewarding effects are no longer due to the repeated use of the same dose of the substance or the compulsive implementation of an activity and consequently, the individual is forced to increase the dose or activity to achieve the initial effect.
• The individual persists in behavior despite the negative consequences it entails.

In addition to symptomatology, drug and behavioral addictions have similar neurobiological mechanisms. Several studies have shown that when the subject acts a behavior that causes pleasure and gratification, this system is activated, resulting in a strengthening of the behavior itself, as it is encoded in memory as positive. It is a physiological system that is usually activated by adaptive behaviors, necessary for survival, such as eating, drinking etc.; however, the alteration of this circuit is also at the base even addictive behavior [18]. A malfunction of the brain mechanisms involved in gratification and motivation involving the meso-cortico-limbic circuits, seems to be the basis of both substance and behavioral addictions. The regulation of these mechanisms is made possible by the interaction of different neurotransmitter systems; in particular, in addiction a crucial role is played by the dopaminergic system, which regulates the motivation that guides the search for rewarding stimulus [20]. Dopamine produces different effects on motivated behaviors depending on how it reacts to two different motivational stimuli: appetitive stimuli, which push the individual to get a reward, and consumer stimuli, that keep the subject’s proximity to the reward. Both stimuli activate the dopaminergic neurons of the neocortical pathway; whereas, the dopaminergic neurons of the mesolimbic pathway, considered the pleasure pathway, are activated only by consumer stimuli [19].

In addition to the dopaminergic system, other systems involved in addiction mechanisms are:

• The serotonergic system, the alteration of which appears to be related to impulsive behavior, typical of addiction [15]. The authors of this theory believe that the basis of the phenomenon of craving and relapses is the process of incentive sensitization [15,20,21]. In addition, such process simple modifications of the synaptic plasticity in the nucleus accumbent and in the cortex of neurotransmitter systems such as serotonin, glutamate, noradrenaline, and GABA [15]. Some authors [15,20,21] believe that similar alterations of the circuits involved in the incentive salience are also the basis of dysfunctional behaviors typical of new addiction; Therefore, stimuli and contexts related to compulsive behavior could assume abnormal incentive-motivational properties. However, to date, there are insufficient experimental tests to support this hypothesis.

Etiopathogenesis of Affective Dependence

It has been seen how neurobiological aspects favor the onset and maintenance of affective dependence. However, it cannot be explained solely in biological terms since affective dependence is a complex and multifactorial phenomenon. For an exhaustive understanding of it, therefore, one must also consider psychological factors [such as attachment, emotional dysregulation] and sociocultural factors [such as media influence and cultural expectations], all factors that interact with each other in the determination of this type of dependence.

Psychological Factors: Attachment

John Bowlby developed the theory of attachment [22] which emphasizes the importance of the mother-child relationship and how these early relationships can influence the child’s social and emotional development. Bowlby criticizes the prevailing theories of the time, according to which the child would seek the proximity of the reference figure only to satisfy the need for nourishment. He believes that the child has an innate tendency to establish strong emotional ties with the attachment figure and seeks its proximity to gain protection and security. He calls attachment behavior “any form of behavior that leads a person to achieve or maintain closeness with another individual”, usually the mother. It is activated by distance from the reference figure and is inhibited by its proximity. Bowlby’s theory has found important confirmation in Mary Ainsworth [23], who invented the strange Situation, a standardized observational procedure that allows to evaluate the attachment of the child from twelve months of age. It is an experimental situation consisting of 8 micro episodes, lasting about three minutes each, in which the behavior of the child is observed in the presence of the mother, when it is separated from her and at the time of reunification (Figure 1). Other scholars have added other styles of attachment, notably:

• At Main and Solomon [24,25] we owe the introduction of the disorganized/disoriented attachment observed in children with mothers who did not process a trauma or mourning during their childhood [26];
• Crittenden has introduced avoidant/ambivalent attachment that seems to correspond to a defensive strategy against abusive mothers [26].

Figure 1

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Several studies have examined the relationship between attachment styles and obsessive love, Hazan and Shaver [27] were the first to broaden the theory of attachment to adult romantic relationships, suggesting that the anxious-attachment style could be a risk factor for the development of obsessive love. Feeney and Noller [28] conducted a study involving a sample of 374 university students [162 males and 212 females] aged 17-58 and almost all single. A series of questionnaires were administered that evaluated the attachment style and the love style and the results showed that the anxious-ambivalent attachment group reported high levels of dependence, the desire to be involved in romantic relationships, although these were characterized by less stability. They also highlighted a greater propensity for obsessive concern, idealization of the partner to rely on, and emotional dependence. Otherwise, subjects with safe and avoidant attachment were characterized by greater self-confidence and others, stable and positive relationships, the latter by the tendency to avoid intimacy, little or no intense relationships. This study seems to confirm that the style of attachment affects adult romantic relationships and that the anxious-ambivalent attachment style may be the basis for the development of love addiction. The study of Ahmadi, et. al. [29] aimed to investigate the relationship between obsessive love and attachment styles. It was conducted on a sample of 290 students [117 females and 173 males]. They were given the Passionate Love Scale [PLS] and the Adult Attachment Inventory. The results showed a significant relationship between anxious-ambivalent attachment style and obsessive love. The child, with such a style of attachment, manifests separation anxiety because of a reference figure who tends to have an inconsistent attitude. As a result, as adults these individuals show a constant concern for their relationships and fear separation, rejection and betrayal by the partner, becoming dependent on them.

This study also confirms that the anxious-ambivalent attachment style can determine the development of obsessive love. A more recent study aimed at investigating the relationship between attachment styles and obsessive love style is that of Honari and Saremi [30]. The sample consisted of 306 students, who were given the following questionnaires: Adult Attachment Style [AAQ] and Love Attitude Style [LAS]. From such a study emerged a significant association between the obsessive love style and the anxious-ambivalent attachment style; while the same could not be said about the relationship between safe and insecure-avoiding attachment styles and obsessive love. This study also concludes that the anxious-ambivalent attachment style can predict obsessive love style. In this regard, the authors stress that evaluating attachment styles is important as it allows prediction of people’s love styles. Borgioni [31] also associated affective dependence with anxious-ambivalent attachment. Subjects with this style of attachment had a caregiver present intermittently and often assumed the role of “adult child”, as they were the ones to take care of the parents. In adult romantic relationships these subjects tend to unconsciously recreate the same dynamics that were created with the parent. Research in the literature seems to confirm that anxious-ambivalent attachment is at the origin of affective dependence, also given the overlap of their typical characteristics. For example, concern, vigilance, rumination are factors that are observed in romantic love [32,33] and obsession [American Psychiatric Association, 2000], but are also linked to anxious attachment. It should be noted, however, that some research indicates that attachment is not stable over time and may be influenced by further relationships. For example, Belsky, et al. [34] indicate that, after three months from the initial administration of the attachment assessment questionnaires, about fifty percent of people have a different attachment style. However, the research has shown that the first experiences with the caregiver have quite important effects on the development of affective dependence.

Adult Attachment and Self-Esteem

According to Bowlby’s theory, the first relationships with the reference figures are internalized by the child and constitute the internal operating models [MOI]. The latter are mental representations of self and attachment figure that are formed during childhood, but remain over time, as they influence future interpersonal relationships, orient the subject’s worldview, behavior, and his beliefs and expectations [27]. In accordance with this concept of Bowlby, Bartholomew, and Horowitz [35] developed an “adult attachment model”. From the combination of two dimensions, the “model of self” and the “model of the other”, or the positive or negative perception that each has of oneself and the other, they have identified four different styles of adult attachment: The secure attachment style manifests itself in subjects who have a positive perception of both themselves, as they feel lovable, and others, as they expect people to be available. In addition, they are comfortable with intimacy and autonomy, and possess adequate emotional regulation strategies; The insecure, concerned attachment style is observed in subjects who, having a negative view of themselves, feel inadequate, while others are assessed positively. Such individuals need support, love, and seek the approval of others; The insecure attachment style of the buffer manifests itself in subjects who have a positive image of themselves and a negative view of others, who are considered unreliable. In addition, these subjects tend to avoid intimate relationships; Fearful insecure attachment style is typical of subjects who have a negative view of both them and others. Such individuals desire contacts and intimacy, however, given the lack of trust in others and the fear of being rejected, they avoid intimate relationships. Individuals with concerned attachment and those with fearful attachment, therefore, tend to manifest greater dependence on others.

In this regard, Gori, et. al [36] conducted research with the aim of analyzing variables that may be associated with affective dependence, with particular attention to adult attachment patterns and self-esteem. The research involved a sample of 300 individuals engaged in a love relationship. Subjects were given the following questionnaires: Love Addiction Inventory – Short form [LAI – SF], Relationship Questionnaire [RQ] and Rosenberg self-esteem Scale [RSES]. The results of such research showed, first, a significant relationship only between affective dependence and concerned and fearful adult attachment; Secondly, it emerged that self-esteem plays a mediating role in the relationship between adult attachment patterns and emotional dependence. Individuals who present these attachment styles have a negative view of themselves [37], as a result they show low levels of self-esteem, factors that can lead to dysfunctional relationships characterized by excessive dependence on the partner [38]. In the literature there are studies that examined the relationship between self-esteem and attachment, the results, showed that there is an association between insecure attachment and low self-esteem levels [39,40]; it is also shown that low levels of self-esteem can lead to the onset of interpersonal dependence [38]. However, the authors of this research believe it is necessary to emphasize that it may be the affective dependence itself that influences the self-esteem of the subject, thus determining a vicious circle.

Trauma, Dissociation and Emotional Dysregulation

Several studies have shown that pathological addictions are closely related to both childhood trauma and to emotional dissociation and dysregulation [41,42]. Exposure to childhood relational trauma, such as experiences of emotional deprivation, physical, sexual and/ or psychological abuse, can lead to conditions such as dissociation and emotional dysregulation. Dissociation is defined as a “disintegration of normally integrated functions of consciousness, memory, identity, or perception of the environment” [43]. It is a defense mechanism that isolates from his consciousness a painful experience and the emotions connected to it; It has, therefore, the aim of protecting the ego from pain through the alteration of the state of consciousness, inhibiting information experienced as intolerable and creating a more positive reality in which to take refuge [44]. Dissociation is not a dysfunctional aspect; however, a pathological recourse to this type of mechanism can lead to an impoverishment of the personality of the subject, a compromise of the sense of continuity of one’s identity, detachment from reality, causing a deficit in mentalization and self-regulation of emotions [42]. The child, in his first years of life, does not have the ability to independently regulate their emotional states; Therefore, it is necessary the intervention of the caregiver to help him manage his emotions and at the same time allow him to learn the ability to manage them independently [45]. The “good enough” caregiver Winnicott [46] will be able to provide adequate responses to the emotional states of the child and this will allow him to develop a “theory of mind” [47] becoming aware of his own emotions and those of others; This happens, therefore, in the case of a secure attachment with the parent. The child thus gradually learns and develops the ability to regulate his emotions independently [45].

Otherwise, if the child has experienced an emotionally neglectful caregiver, who is not able to regulate his emotions, he will not develop the ability to mentalize and regulate his emotions [45]. Rather, he will find himself in a state of emotional dysregulation, that is, he will not be able to regulate his emotions on his own and will have difficulty tolerating negative emotional states. As a result, the subject will most likely have to resort to an external regulator that allows him to return to a state of rest. Such a regulator can be identified in a substance or dependent behavior, the use of which represents a dysfunctional strategy of emotional self-regulation; this can lead to the onset of a pathological dependence, where the object of dependence would represent an external regulator of one’s emotional states [44]. In the literature there are several studies by Caretti and collaborators that highlight the significant correlation between dissociation and emotional dysregulation, resulting from relational trauma, and the different forms of pathological dependence. About love addiction, Stavola, et al. [46] conducted research to assess the predisposing factors of this type of addiction. They hypothesized that the dissociation and emotional dysregulation that occur as a result of a childhood relational trauma and insecure attachment style are the basis of emotional dependence. The results confirmed what the authors had assumed, as a significant association emerged between trauma, attachment, dissociation, and emotional dysregulation, confirming them as predisposing factors to love addiction. child has experienced an emotionally neglectful caregiver, who is not able to regulate his emotions, he will not develop the ability to mentalize and regulate his emotions [45]. Sociocultural factors in addition to neurobiological aspects and psychological factors, there are also sociocultural factors underlying affective dependence. The mass media seem to have a significant influence on the development of affective addiction [47]: movies, TV series, songs, novels often tend to represent love in a pathological way [48,49].

Vannini and Myers [50] conducted a study to analyze the lyrics of albums belonging to the musical genre “teen pop”, a genre of music that is aimed primarily at a teenage audience. The main theme of these songs is romantic love, which is described as an intense desire, “an overwhelming and painful experience”. Described lovers tend to have obsessive thoughts and feelings and idealize their partner. Often the lyrics of these songs convey the idea that you cannot live without the other person, now indispensable. These kinds of songs can have a significant impact on young teens, who use the meaning of lyrics to build their identity, to understand how relationships work. These texts convey a kind of pathological love, which is represented as ideal love, can affect the way young adolescents conceive love and what they expect from relationships. Even movies often transmit dysfunctional relational dynamics, and this can lead teenagers to search for those same dynamics, considered right. In addition, affective dependence seems to prevail in women, for example, Miller [50] claims that 99% of affective dependent subjects are female. According to Sussman [47] gender would affect the ways in which emotional dependence manifests itself. In this a key role is played by culture, which seems to strengthen affective dependence: the gender stereotype sees women as too sensitive, naive, weak, passive, not very rational, dependent, dedicated to the care of others, putting aside herself, submissive to the will of others; while, man tends to be seen as more rational, stronger and independent. Such stereotypes can influence the behaviour of women, who, to conform to cultural expectations, risk entering into dysfunctional, potentially dangerous relationships.

Anxious and Depressive Symptoms

Affective dependence can significantly affect the psychological and physical well-being of the person. For the affective dependent subjects, the relationship with the partner becomes of fundamental importance; consequently, everything that does not concern the relationship passes into the background: they lose interest in activities that they loved to practice previously, are unable to concentrate on work and study, friendships and family relationships are neglected. All this can lead to social isolation. Moreover, the fear of losing a partner, of being abandoned or rejected, can make the affective employee experience strong anxious and depressive states. Excessive worry and anxiety can lead to the development of a real generalized anxiety disorder that is characterized by the presence of “physical symptoms [muscle tension, restlessness, easy fatigue], neurovegetative [tachycardia, dyspnea, tremors, dizziness] and psychological [difficulty concentrating, irritability]” [19]. The affective employee may also develop a “major depressive disorder” [16] whose main symptoms are: depressed mood, loss of interest or pleasure in previous activities, impaired weight and appetite, loss of energy and fatigue, insomnia or hypersomnia, feelings of guilt and self-assessment, difficulty in contracting and suicidal thoughts [16]. Fisher and colleagues [51] point out that romantic rejection can compromise the subject’s psychological health, inducing clinical depression and in the most extreme cases can lead to suicide. It can also impair physical health, as an increase in blood pressure and a lowering of the immune system may occur, due to severe stress [52]. Even more serious consequences occur when the partner, to whom you are emotionally dependent, decides to end the relationship or when you remain in a dysfunctional relationship, despite being dangerous to your life.

Intimate Partner Violence

Affective addicts tend to enter relationships with abusive, abusive, violent, manipulative and detached persons [3]. A peculiar feature of affective dependence is that, despite suffering, the dependent subject cannot end the relationship and causes the behavior of the partner to be attributed to the traumas of his childhood [53]. The “women who love too much” have the dysfunctional belief that it is only by sacrificing and dedicating themselves totally to the other, cancelling sexless, that they can help the partner. This belief only feeds the permanence in the relationship; permanence that can prove lethal for one’s life. They do not manage to separate from their partner, as it would be too painful for them and prefer, rather, to cling to the illusion and hope that the partner changes, a wait that turns out to be useless as this will never happen. The risk that the affective addicted may face is that of entering abusive relationships, becoming a victim of domestic violence, which in English is called intimate partner violence. The UN defines IPV as “the behavior of an intimate partner or ex-partner that causes physical, sexual or psychological harm, including physical aggression, sexual coercion, psychological abuse or controlling behavior” [UN, 1993]. In addition, it is very important to understand the dynamic between the affective addicted and the abusive and violent partner, because it becomes even more difficult for her to end the relationship. In this regard, Lenore Walker [54] has developed a model of “cycle of violence” that defines “the progressive and ruinous vortex in which the woman is swallowed by the continuous, systematic, and therefore cyclical violence, by the particular party, the author believes that there are three phases of domestic violence: “phase of construction of tension”, “explosion or violence phase” and “reconciliation/ honeymoon phase” [55].

The first phase of construction of tension [“tension building phase”] is characterized by the progressive increase of tension in the relationship, due to frequent conflicts and verbal violence against the victim. The latter perceives that the partner is always nervous and irritated; therefore, to avoid further conflicts, the victim will tend to take a pandering attitude, trying to appease the partner. In the second phase of explosion or violence [“explosion phase”] there occurs the loss of control by the aggressor that results in real violence. This may include physical, psychological and/or sexual abuse of the victim. In the third phase of reconciliation/honeymoon [“honeymoon phase”] the aggressor, can show repentance, seeming, kind, makes romantic gestures, apologizes and promises that it will not happen anymore, that will change, manipulating thus the victim, who will sincerely believe his aggressor, and will continue to hope that the relationship will improve and that he can really change [56]. However, unfortunately, this will never materialize in fact, following the “honeymoon” phase, the cycle starts again from the first phase. This pattern of behavior can help to strengthen the dependency of the victim, who will feel responsible and take the blame for the situation, believing that she deserves it, further undermining the already low self-esteem. Moreover, as the dysfunctional relationship continues, these phases will occur more frequently, more intensely and more rapidly, to the point that psychological, physical, economic, and sexual violence leads to feminicide. November 25 is the international day against violence against women, “wounded, massacred, humiliated women who carry in their body and soul the bruises of what was thought to be love” “When being in love means suffering, we are loving too much. [..] When we justify her moods, her bad temper, her indifference, or consider them the consequences of an unhappy childhood and try to become her therapist, we love her too much. […] When our relationship with him jeopardizes our emotional well-being, and perhaps even our health and safety, we are loving too much” [54]. The criminal analysis service reports that from 1 January to 3 September 2023 there were 225 murders, of which 77 are female victims, 61 killed in the family/ affective; 38 of these died because of the partner or former partner (Ministry of the Interior, 2023).

Individual Therapies

According to some authors, among individual therapies, cognitive behavioral therapy and psychodynamic treatment could be beneficial in the treatment of affective dependence [55]. Some authors have suggested that cognitive behavioral therapy (CBT) may have a potential benefit in treating affective dependence. Automatic thoughts and cognitive distortions (such as generalization, catastrophization) could play a decisive role in maintaining affective dependence [55]. Cognitive behavioral therapy could be effective in addressing these distortions, through cognitive restructuring: fundamental is, therefore, to make the patient aware of his patterns of negative thoughts and adopt appropriate strategies to modify them. However, again, there are no studies on the use of CBT in the treatment of affective dependence [55].

Drug Treatment

Although there is no scientific evidence in the literature to prove the efficacy of a drug treatment of affective dependence, Sanches, and John [55] suggest that the phenomenological characteristics and neurobiological mechanisms involved in this condition, suggest that the use of drugs may be beneficial for the treatment of affective dependence. However, the bioethical implications must be considered; in particular, Earp and colleagues [57] have highlighted some situations in which it would be justified to use drug treatment for affective dependence. According to these authors, it can be considered to treat love using drugs only when this causes pain and suffering to the patient, as in the case of domestic violence, or when psychological treatments are not sufficient [56]. Sanches and John [55] hypothesize that affective dependence can be treated using drugs such as antidepressants, mood stabilizers, antipsychotics, and exogenous neuropeptides. Since obsessive-compulsive disorder (OCD) and some cases of affective dependence have similar phenomenological characteristics, the authors hypothesize that antidepressants, specifically selective serotonin reuptake inhibitors [SSRIs] may also play a role in the management of affective dependence, given the effectiveness of SSRIs in the treatment of DOC [55]. SSRIs may be useful in reducing obsessive thoughts, typical of both phenomena [56] In addition, affective dependence and impulsiveness are strongly correlated, which suggests that mood stabilizers may play a role in managing this condition [55]. Further drugs that could be useful in the treatment of affective dependence are antipsychotics, due to their function as dopamine antagonists. Antipsychotics could modulate the dopaminergic activity that is activated during falling in love [55]. As for exogenous neuropeptides, studies have shown that vasopressin and oxytocin are involved in the attachment system. Finally, it could be hypothesized that drugs used to treat addictions may be beneficial for the treatment of love addiction, given the similarity between the two conditions: for example, naltrexonee buprenorphine [55]. The authors stress the importance of further studies to investigate whether these drugs can benefit the treatment of affective dependence [55].

Group Therapy

Group therapy is considered by many authors as the treatment of choice for affective dependence [55]. Yalom [56] identified several factors that appear to be therapeutic within a group, such as: “the infusion of hope, universality, information, altruism, corrective recapitulation of the family primary group, the development of socialization techniques, imitative behavior, interpersonal learning, group cohesion, catharsis and existential factors” [56]. Within a group, therefore, a climate of trust will be created that will allow patients to tell each other without shame. This will allow members of the group to find emotional support, to feel understood and less alone, as they all share the same issue. In addition, listening to the story of others can stimulate important reflections in the patient who faces that same pain. However, there are no studies showing the effectiveness of these selfhelp groups [55].

Conclusion

During this review, we have deepened the affective dependence and we have seen that it is a complex phenomenon that involves several factors and significantly influences the psychological well-being of the people involved. The findings from this work emphasize the importance of the quality of primary relationships, as it was seen as an anxious attachment-ambivalent, having experienced relational traumas in childhood are actors predisposed to affective dependence. Studies have shown that at the base of love addiction there are neurobiological mechanisms analogous to those that occur in substance dependence. It also emerged that society and culture play a crucial role in maintaining this condition, suggesting the importance of primarily cultural change. Subjects with affective dependence tend to enter into abusive relationships, with the risk of becoming victims of domestic violence, and this makes us reflect on the importance of paying more attention and gaining greater awareness of this issue. “The general objective that every woman must achieve is the conquest of independence and emotional autonomy, without suffering the bullying of those who want to prevail by inducing feelings of guilt. And do not call it love, love is another thing, because to love is to give oneself in a perspective of reciprocity and sharing”[57].

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Lactic Acid, Magnesium, Metformin and Lactic Acidosis

Introduction

About Lactic Acid Production and Metabolism

Lactic acidosis is a medical condition characterized by a buildup of lactate [La] (especially l-lactate) in the body, with formation of an excessively low pH in the bloodstream. It is a form of metabolic acidosis, in which excessive acid accumulates due to a problem with the body’s oxidative metabolism [1]. Wikipedia gave 36 causal agents for lactic acidosis, including metformin, but without magnesium deficiency [1]. PubMed Search [2] [“lactic acid” metformin magnesium] gave only one result, which was not associated with body magnesium status.

It is generally known that the production of lactic acid (often named by its anion “lactate” [3]) increases when the demand for ATP and oxygen exceeds supply, as occurs during intense exercise and ischaemia [4]. Nowadays is known that lactate also serves as a major circulating carbohydrate fuel and enables the uncoupling of carbohydrate- driven mitochondrial energy generation from glycolysis [4]. Lactate and pyruvate together serve as a circulating redox buffer that equilibrates the NADH/NAD ratio across cells and tissues [4]. During fermentation, the NADH is used to reduce pyruvate to lactate, which is then excreted [4]. This process results in a net yield of two ATP and two lactate molecules per glucose, without consuming any oxygen [4]. In respiration, the NADH electrons and pyruvate generated by glycolysis are shuttled into the mitochondria, where they are consumed and subsequently produce copious usable energy (approximately 25 ATP molecules per glucose) [4].

Lactate turnover reflects lactate release primarily from muscle, gut, adipose, and erythrocytes and uptake by the liver and kidney, primarily for the purpose of energy production (Krebs (TCA) cycle) while the remainder is used for gluconeogenesis (Cori cycle) [5]. Healthy mitochondria function well, even at low O2 levels such that dysoxia is rare and low O2 is likely to be a minor factor in the increasing concentrations of lactate [6]. Catecholamines promote glycogen breakdown to lactate [6]. In the (old) crossroad from pyruvate to a) lactate or b) ATP via TCA cycle the b) route can be hindered e.g. by mitochondrial diseases [6], low mitochondrial density [6] or thiamine deficiency [7], so promoting lactate production.

Observations on magnesium

Magnesium has an important role in ATP synthesis [8]. (ATP: Adenosine triphosphate, a nucleotide that provides energy to drive and support many processes in living cells, such as muscle contraction [9]). Magnesium deficiency (defined by S-Mg & lt; 0.8 mmol/) was detected by 23 % of critically ill patients, this proportion was (1.6 – 2.0- fold) higher by patients with increased S-lactate content, diabetics and alcoholics [10].

Study on Serum Metformin, Lactate, Blood pH and Mortality In a study on databases of cases by metformin overdose with documented mortality data and values of serum lowest (“nadir”) pH, highest (“peak”) lactate level, and metformin concentrations, with outlines of their treatments [11]. The databases searched included MEDLINE from 1950 to June 2007, EMBASE from 1974 to June 2007, CINAHL from 1982 to June 2007, and TOXNET from 1965 to June 2007; these ranges encompassed the maximal time range limits of each database.

Number of cases fulfilling inclusion criteria was 22, five of them died. The represented data are from Table 1 in [11], partially analyzed by the author with IBM SPSS Statistics, version 29 and MS Excel. Ranges, (means & SD) of the parameters: metformin (Met): 0.3 – 188 (67.2 & 58.1) μg/ml; peak lactate: 1.4 – 47 (17.0 & 14.1) mmol/L; (nadir) pH: 6.59 – 7.49 (7.1 &0.3). Mortality got values: 1 for death, 0 for survivors. Four of non-survivors had got renal transplantation therapy. None of the intentional overdose patients whose serum nadir pH was over 6.9, maximum lactate concentration less than 25 mmol/L, or maximum metformin concentration less than 50 μg/mL died. Regression of death by [Met, lactate and pH] represents 61 % of the total variation of death (p < 0.001). Beta coefficients of this regression equation were: [-0.0035;+0.162;-0.626 – respectively]. Beta coefficient of Met was weak, but negative. According to one pilot study the average plasma Met (prescribed 500 – 3 000 mg Met/d) concentrations was 1846 ng/mL (≦LoQ-5560 ng/mL) and independent of the prescribed daily dose [12]. By three cases in [11] plasma Met was below (5.6 μg/ml) 5600 ng/ml.

The same data [11] were selected by the highest values of Met including all (5) non-survivors. The number of cases was 12. The ranges by parameters were: Met 50.9 – 188 μg/ml, lactate 4.2-47, nadir pH 6.59 – 7.49. Regression of death by different factors: Met predicted death by 1 % (p 0.762, i.e, non-significantly), (nadir) pH by 56.4 % (p = 0.005) and lactate by 53.6 % (p = 0.007). Regression of Death by [Met, lactate and pH] explained deaths by 59.1% (p = 0.056, trend like association). Beta coefficients: Met -0.162; Lactate +0.244; Nadir pH -0.571, i.e. association of death was, as expected, negative with pH and positive with lactate and (N.B.) negative with Met. Strength of associations evaluated by single absolute Beta coefficient values divided by their sum was: Met 16.6, Lactate 25.0 and Nadir pH 58.5 %. Strongest detrimental association was with pH and protective association with Met. These results are accordant with that Met therapy improves the altered levels of magnesium and GSH in diabetic rats [13].

Experiments by Magnesium and Metformin on lactate and pH

Lactate increase of sportsmen can be prophylactically inhibited by Mg [14]. Dietary experiment has shown that serum lactic acid content can be increased by low-magnesium diet, without muscle exhaustion, increasingly, after 40-60 days ad 6.3 (vs. control 4.6) mmol/L (and base excess reduced below -4 mmol/l [15]). Even by Escherichia coli Mg-deficiency has increased production of lactate and decreased respiration [16]. In the magnesium experiment of Nuoranne et al. [15] Serum Mg: Ca ratio and duration of low-Mg diet [time (T, d)] explained blood pH by equation, pH: 6.98 – 0.0017*T+ 1.25*(Mg:Ca) (P.time ≦ 0.001, P.(Mg:Ca) ≦ 0.01). “The equation represents 62.0% of the total variation of blood pH.” A thirteen weeks experiment by rats showed that the no observable adverse effect level (NOAEL) of metformin was 200 mg/kg/day [17], i.e. 14 000 mg for a human weighting 70 kg, which responds 5 x the human maximal daily dose, 3g [18]. Minimal metabolic acidosis and lactate increase was observed with metformin level 600 mg/kg/day.

Temperature

When “energy aimed for ATP formation is disturbed, the excessive energy is changed to warmth ” [19]. In the feeding experiment of Nuoranne, et al. [15] regression of body temperature (BT, C⁰) by time (T, d, days) and serum lactate (mmol/L) gave equation: BT = 38.49 – 0.00064*T + 0.017 *lactate content (P.time non-sig, *P.lact ≦ 0.05) [15]. Nuoranne wrote that he had become accustomed to regard elevated skin temperature and sweating as signs of Mg deficiency [19]. The temperature of a sow can be increased ad 41-42 centigrade. The only acute treatment by 100 ml 20 % MgSO4 subcutaneously has been sufficient (veterinary surgeons are accustomed with high doses of magnesium). The result was to be seen even within one hour, after that treatment continued by usual oral Mg supplementation (Nuoranne 1977) [19]. Even by grass tetany temperature can increase ad 41-42 centigrade [19]. Symptoms like Delirium tremens (alcohol withdrawal syndrome) can be achieved if S-Mg declines to about 0.5 mmol/L [20]. Delirium tremens [21], (associated with Mg deficiency, e.g. [10]) “include shaking, shivering, irregular heart rate, and sweating. People may also hallucinate. Occasionally, a very high body temperature or seizures may result in death [21].” Metformin, which accumulates and increase lactate production in intestine, anyhow reduces blood lipopolysaccharides and its initiated low-grade inflammation [22]. Mg works like an on-demand antioxidant, but this ability is decreased in Mg-deficiency [23], which can increase ROS (Reactive Oxygen Species), IL-6 and TNF-α production [24] and temperature (fever).

Discussion

The understanding of the role of lactate has been increased remarkably during the last 50 years [4,5,6]. The role of magnesium in lactate formation is known, but because Mg is mainly intracellular cation and its extracellular measurement data (in serum less than 1 %) are unsatisfactory. We have need of easily available method for determination of body or muscle Mg status, [25-27]. Heart muscle damages were detected by all pigs with the lowest dietary Mg level [19], {why Nuoranne considered that Mg can have a role in the etiology of human heart diseases, too [19] as Professor Pentti Halonen taught in the 1970’s: “heart infarct is myocardial necrosis, which has two etiological factors: one is the deficiency of oxygen, the other someone (unknown) metabolic factor” [28]. Possibly the metabolic factor is lactic acid as a consequence of the weakness in ATP production [23]. In situations, where lactate concentration is 6 mmol/L at rest, as in [15], the benefits of lactate [29] have been lost. The dietary Mg experiment produced severe lactic acidosis (lactate content was 6 mmol/L) [15], c.f. a given reference for severe lactic acidosis is serum lactate concentrations of > 4 mmol/L [10]. Significance of association between body temperature (BT, C⁰) and serum lactate (mmol/L) could have been higher, if time had been removed from the regression [15]. Met can increase intestinal lactate production in epithelial cells and microbiome [22], which is possibly beneficial [30].

Conclusion

Managed lactate seems to be a beneficial power and health product, but unmanaged increased lactate predicts mortality risk. Deficiency of magnesium, associated with reduced ATP production and unmanaging of inflammation, is enriched with the diseases associated with lactic acidosis, as diabetes, which is associated with metformin medication. The role of metformin seems to be a bystander, in situations, where its help is not sufficient. Even in such situations relative Met content can associated with decreased mortality risk. Solution to Mg deficiency needs methodological (and statistical) measures.

Acknowledgement

I am grateful to Professor Osmo Hänninen and veterinary surgeon Seppo Haaranen for several discussions.

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The Urban Environment and Non-Communicable Diseases (NCDs)

Introduction

Non-communicable diseases are the leading cause of death and disability around the world. They are a category of illnesses primarily not caused by an acute infection but have long-term health implications and frequently necessitate long-term therapy and care (WHO [1], CDC [2]). Cancer, cardiovascular disease, diabetes, and chronic lung ailments are part of NCDs. NCDs originate from a mix of genetic, physiological, environmental, and behavioural variables (WHO [1], CDC [2]). According to the CDC and WHO, NCDs kill 41 million people a year, accounting for more than seven out of every ten deaths worldwide. Every year, 17 million individuals die from a non-communicable disease before the age of 70. In total, 86% of these premature fatalities occur in low- and middle-income nations, with 77% occurring exclusively in these countries (WHO [1], CDC [2]).

The NCD epidemic has been exacerbated by changing social, economic, and structural factors such as increased urbanization and the spread of unhealthy lifestyles CDC [2]. Urbanization, which is the increase in the proportion of people living in towns and cities, is a worldwide trend. Urban communities across the globe including Africa are growing rapidly. Urbanization may be seen as a double‐edged sword, that is, while its beneficial economic effects are widely acknowledged, it is commonly alleged to produce adverse side effects for NCD‐related health outcomes (Zhang [3]). The urban environment has been found to significantly impact non-communicable diseases.

This is because, the way cities are built, their density, how easy it is to get around, their building design, and overall layout have been found to have a significant impact on how much people exercise, what they eat, air quality, blood pressure, and even obesity rates. In other words, the design of cities has made it harder for people to stay healthy and avoid NCDs (Fazeli Dehkordi [4]). Research has indicated that the prevalence of non-communicable diseases (NCDs) is likely to rise in suburban and urban areas, concomitant with an increased intake of prepackaged foods and cooking ingredients (Mahawan [5]). In a study, it was reported that an increase in the death rate from coronary heart disease has been connected to the cultural shift brought about by urbanization (Tyroler & Cassel [6]). Additionally, it has been discovered that urbanization raises average body mass index and cholesterol levels especially in low- and middle-income nations and this has been linked to certain urban behavioural patterns, including altered eating habits and inactivity (Gunapala, et al. [7,8]). By 2050, two-thirds of the world’s population is predicted to reside in urban regions, up from the current 50% of people who live in cities (National Geographic Society [9]). The provision of services for people living in metropolitan areas is already a challenge for local governments. Every industry is under pressure, including the health system. The people’s health demands cannot be met by the lack of adequate medical personnel and modern facilities in the cities.

Inadequate waste management and high pollution rates has resulted in poor air and water quality, which negatively impact public health by causing respiratory disorders, cardiovascular problems, and other health complications. Countries with lower living standards are seen to be more affected by urbanization’s effects on public health than those with higher living standards. The primary determinants of disease are mainly economic and social; therefore its remedies must also be economic and social. To address NCD prevention and control, effective evidence-based multi-sectoral policies must be implemented, with an emphasis on the primary risk factors (Juma, et al. [10]). NCD policies must be integrated into urban planning. This includes designing and developing parks and recreational facilities such as pedestrian and cycling tracks. Zoning walkways where motorized vehicles are not permitted should also be established, and incentives for non-motorized vehicle use should be provided. Fiscal strategies and regulatory measures to prevent unhealthy eating environments in African metropolitan centers are required to limit the ever-increasing food marketing by food businesses (Juma, et al. [10]). It is also important to create awareness and sensitize people on the effects urbanization has on health and how it could predispose them to NCDs (Olowoyo, et al. [11]). This will foster a culture of responsibility towards personal and community health and empower individuals to make informed decisions about their well-being, enabling them to adopt healthier lifestyles and preventive measures [12].

Conclusion

The rise of non-communicable diseases in urban areas presents a pressing public health challenge that demands urgent action. As urbanization continues unabated, cities must prioritize interventions aimed at preventing and controlling NCDs. This necessitates a holistic approach that integrates health considerations into urban planning processes, promoting environments that support healthy lifestyles. From designing walkable neighbourhoods to regulating food marketing, policymakers have a crucial role to play in shaping urban environments conducive to health. Moreover, efforts to raise awareness and educate the public about the impact of urbanization on health are essential for fostering community engagement and promoting behaviour change. By addressing the root causes of NCDs through economic and social interventions, societies can work towards creating healthier and more equitable urban environments for all.

Conflict of Interest

There is no form of conflict of interest to declare.

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