Participation of Various Nuclei of the Amygdala in the Implementation of the Drinking Conditioned Reflex

Introduction

The amygdaloid region (almond-shaped complex, amygdala) is a complex formation of the brain that is part of the limbic system. Being its integral part, it has a modulating and corrective effect on the activity of the main brain stem formations, through which the formation of a motivational-emotional state is carried out. The amygdala receives information from all the senses [1]. This is possible due to its communication with a structure called the entry gate in the brain – the thalamus. Direct communication with the amygdala is necessary for a quick response to external stimuli [2]. In addition, emotional responses include the occurrence of precipitating factors simply by thinking or imagining [3]. To do this, the amygdala has a connection with associative zones – areas of the cortex, covering the assembly of incoming information into whole images, as well as with the hippocampus. In addition, the tonsils are found with subcortical structures such as the basal ganglia and the accessory nuclei. With most structures, the amygdala is connected by bilateral connections, and above all, with the prefrontal cortex. For example, the amygdala influences the prefrontal cortex (PFC) in risk/reward risk assessment in animals [4], and stimulation of the projection pathways of certain regions of the PFC to the amygdala can increase or decrease prosocial behavior [5].

Violation of the connections between the amygdala and PFC can be detected in a number of pathological conditions-post-traumatic stress disorder (PTSD), depression, etc. [6]. The amygdala is also associated with the hypothalamus, which plays an important role in the regulation of stress reactions, sexual behavior, and aggression [7]. Different nuclei of the amygdala are involved in different behaviors. So, the lateral nucleus plays a role in reactions that are associated with fear [8]; the basolateral nucleus contains groups of neurons that respond to both negative stimuli (fear, anxiety) and positive ones (reward) [9]. CEA is the main exit gate of the amygdala. This part is responsible for reactions to emotional stimuli. The medial nucleus of the amygdala in animals is especially important in sexual behavior. In humans, MeA mainly reacts to pungent odors [10] and takes part in their memorization [11]. Considering all of the above, the main goal of this study was to study the effect of electrical stimulation and temporary switching off of the basolateral and central nuclei of the amygdala on the performance of the developed drinking conditioned reflex skill.

Methods

Experiments were performed using 20 rabbits breeds of “Chinchilla” in weight 2-3kg trained to perform a conditioned operant drinking behavior reflex. The animals were subjected to water deprivation for 24 and 48 hours, after which they were trained in the instrumental drinking skill. In response to the sound signal, the rabbit pressed the pedal, as a result of which the door opened, jumped over the barrier from the starting compartment of the chamber to its target section to receive water in a strictly defined dose (5-10 ml) and then returned back to the starting section cameras. The conditioned stimulus was applied at regular intervals (every 45 s) from 10 to 15 times during the experiment. During the experimental day, the animals received an average of 100-120 ml of water. The experiments were carried out under conditions of 100% reinforcement. In the study of behavioral reactions, the time from the moment the signal was given to the start of the jump (latency period), the time of running, lapping, and also the time spent on returning to the starting box of the camera were recorded. Electrical stimulation of the basolateral nucleus of the amygdala – AB (А-1; L5; Н18) and central nucleus of the amygdala – AC (А- 1; L5; Н16) of the brain was carried out using a universal electro stimulator ESU-1. Temporary shutdown of the studied areas of the CNS was carried out with a 10% novocaine solution. To control the rabbits in the study area was injected with saline in a volume equal to the injected solutions.

Results and Discussion

Our research revealed the functional differentiation of the amygdala complex in relation to the execution of the developed conditioned reflex skill. So, electrical stimulation AB nucleus of the amygdala at low stimulation parameters of 10-50 μA; 5 Hz, 0.5 ms) did not lead to a violation of the drinking habit. The rabbits responded in a timely manner to the sound signal, making a running jump from the starting section of the chamber to the target section to receive reinforcements (water 5–10 ml) and then returning back to the starting section of the experimental chamber. The time parameters of the conditioned reflex skill remained at the level of background indicators: latent period – 1.23±0.07 sec, jogging – 3.27±0.07 sec, lapping – 20.23±0.12 sec, reverse return 4.23±0.07 sec. An increase in stimulation parameters (50–100 μA; 5–10 Hz msec) also did not lead to a violation of the drinking habit. In comparison with intact rabbits, from the first minutes after stimulation, there was an increase in behavior of an emotionally positive type – the rabbit is animated, sniffing the starting compartment of the experimental chamber, licking, washing, sometimes chewing movements appear. At the same time, despite this increase motor activity, when a sound signal was given, the animal promptly responded to the given sound signal by jumping and running from the starting compartment of the chamber to its target box, followed by lapping.

The latent period of the reaction remained at the level of background indicators (1.2±0.07 sec), while the time spent on jumping and running due to the occurrence of a search reaction increased from 3.27±0.07 sec to 7, 5±0.1 sec. The time spent on the consumption of 5 ml of water did not change and amounted to 20.17±0.11 sec. In connection with the occurrence of the reaction of licking, washing, sniffing the experimental chamber, the time for the animal to return to the starting box of the chamber slightly increased (the animal was easily distracted, looked around, sniffed the barrier of the chamber) – from 4.23 ± 0.07 seconds to 8, 57±0.08 sec. An increase in stimulation parameters led to the occurrence of the reactions described above directly during stimulation and continued after the cessation of the stimulus. In connection with prolonged washing, the time of the latent period of the reaction to the conditioned stimulus increased from 1.2±0.07 to 3.1±0.07 sec. An increase in stimulation parameters did not affect the amount of water drunk. A subsequent increase in current strength to 300 μA led to rage.

Recording the amount of water consumed showed that it remained at the level of background indicators. Electrical stimulation of the AC nucleus of the amygdala at low stimulation parameters (10-50 μA; 5 Hz; 0.5 ms) did not affect execution drinking developed skill. The rabbits were completely calm, all parameters of the conditioned reflex skill remained at the level of background indicators. An increase in stimulation parameters (50-100 μA; 5-10 Hz; 0.5 ms) did not lead to drastic changes in the behavior of the animals. The rabbits were alert, an increase in general motor activity was noted, which was accompanied by an increase in the time of the latent period of the reaction to the conditioned stimulus (from 1.23±0.07 to 3.4±0.09 sec). The remaining parameters of the execution of the conditioned reflex skill remained at the level of background indicators: jump-run – 3.27±0.07 sec, lapping – 20.23±0.15 sec, reverse return – 4.25±0.29 sec.

A further increase in the parameters of the stimulating current – current strength up to 100-200 μA, frequency – 10-50 Hz, stimulus duration 0.5 ms led to the emergence of a state of an aggressivedefensive nature: the animal became shy, it was difficult to pick it up, shuddered at the applied conditioned stimulus, was very mobile. This state could be attributed to an emotionally excited state. The execution of skills was not disturbed: the animal reacted to an undelivered signal, performed all components of the conditioned drinking reflex. It should be noted a slight increase in the time of the latent period of the reaction to the conditioned stimulus from 1.23 ± 0.07 to 3.53 ± 0.09 sec and the return return – from 4.2 ± 0.07 to 8.73 ± 0.07 sec, and the time spent on jumping and lapping remained at the level of background indicators and, respectively, was equal to: 3.23 ± 0.07 and 20.2 ± 0.15 seconds.

Recording the amount of water consumed showed that it remained unchanged. Observations of animal behavior have shown that under conditions of electrical stimulation of the amygdala nucleus AB, an orienting-exploratory reaction of an emotionally positive type is noted, accompanied by licking, washing, salivation, while exposure to similar parameters of the irritating current on the AC nucleus of the amygdala provokes the appearance of aggressivedefensive behavior. Our data are consistent with the studies of some authors, who also observed the appearance of orientingexploratory behavior upon stimulation of the nucleus AB and an aggressive defensive reaction upon stimulation of the AC nucleus of the amygdala complex [12].

At the same time, some scientists noted the onset of an alert reaction, turning into an orienting reaction, with threshold stimulation of the amygdala. With an increase in the parameters of stimulation of AL and AC of the amygdala nuclei, the alertness reaction could be replaced by a fear reaction, and when stimulated by AB, by a rage reaction [13]. In addition, in experiments on rabbits, stimulation of the amygdala caused changes in the emotional state of various nature. Motor acts of sniffing, licking, chewing and salivation were noted by scientists during stimulation of the anteromedial part of the amygdala and refer these symptoms to sexual and parental activity [14].The effects of electrical stimulation of AB and AC nuclei of the amygdala showed that its different nuclei are related to opposite manifestations of emotional states, which can be divided into three groups: search behavior of an emotionally positive type (licking, sniffing, salivation), reaction of alertness and aggression.

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Acute Appendicitis with Pyogenic Liver Abscesses

A 51-year-old male presented in our emergency department with a 4-day history of fever up to 39.0°C. He did not complain of pain, denied any nausea or diarrhea, and had no urinary symptoms. He had no trouble breathing, no coughing and no throat pain. A Covid-19- PCR-Test was negative. He presented with normal blood pressure, tachycardia with a pulse rate of 126/min, a body temperature of 37.7°C, an oxygen saturation of 97% and a breathing rate of 20 bpm. The patient had no personal history of illnesses, no previous operations and did not take regular medication. A blood sample revealed a leucocyte count of 21.48 G/l with predominantly neutrophil granulocytes (18.85 G/l) and a thrombocytosis of 731 G/l. The Acute-phase proteins were significantly high with a C-reactive protein of 237 mg/l, Procalcitonin of 10.48 ng/ml and Ferritin of 4593 μg/l. He also showed elevated liver enzymes (GOT 97 U/l, GPT 87 U/l) and elevated cholestasis parameters (y-GT 472 U/l, ALP 242 U/l and Bilirubin 47.7 μmol/l) upon which we performed a sonography of the abdomen.

The ultrasound scan showed no evidence of cholecystitis or cholezystolithiasis, but multiple liver lesions, which warranted further investigation by computer tomography. The findings of the CT scan corresponded with the ultrasound images and showed multiple liver lesions and a possible appendicitis. The lesions were located in segment VI (6.4 x 7.2 x 6.1 cm) and subcapsular in the segments VII/VIII (11.0 x 12,1 x 7.3 cm) and were suggestive of liver abscesses. The working diagnosis of acute appendicitis with pyogenic liver abscesses was established. The patient was hospitalized. Blood samples with bacterial cultures, serology for parasites (amoeba, echinococcosis) and viruses (HIV, HAV, HBV, HCV) were obtained and the patient underwent surgery. The diagnostic laparoscopy showed an acute appendicitis without perifocal abscess formation. The appendix was removed at the cecal pole using a linear stapling device (Endo-GIA, 45 mm).

Upon inspection and palpation of the liver perforation of the caudal abscess with purulent discharge occured. A microbiotic sample was obtained and a drainage was placed in the abscess cavity, which quickly collected 300ml of pus. The cranial (subdiaphragmal) liver abscess was left untouched for later interventional drainageplacement. The blood culture showed growth of Streptococcus milleri, which coincided with the findings of the intraoperative sample from the abscess. An intravenous antibiotic regiment with Metronidazol and Ceftriaxon was initiated. 7 days after drain removal the antibiotic treatment with Ceftriaxon was changed to Amoxicillin and Probenecid according to the antibiogramm results.

The histology of the appendix showed pyogenic inflammation with no signs for malignancy, further supporting the working diagnosis of acute appendicitis with hematogenous liver abscesses. The serology (viruses and parasites) results all turned out to be negative. Upon interdisciplinary discussion and according to well established treatment practice [16], the cranial abscess was drained percutaneously (25cm, 10 French), which immediately released 135ml of pus. Further bacteriological samples were obtained, which again confirmed Streptococcus milleri. After a few days the drain was removed after complete drainage.During the hospitalization period, the patient remained afebrile and without any symptoms apart from general fatigue. The patient was discharged from hospital after 15 days with an oral antibiotic regiment containing Metronidazol, Amoxicillin and Probenecid.

Investigation Images (Figures 1 & 2)

Figure 1: 6 weeks course with evident size reduction of the liver abscesses.

Figure 2: Intraoperative situs: Top left and right: Spontaneus perforation of the caudal liver abscess with consecutive drainage insertion. Bottom left: Appendix (stapled and resected, apex on the right, basis on the left). Bottom right: View of the subdiaphragmal abscess with adhesions

Treatment

Antibiotic Treatment

Ceftriaxon i.v. 2g, 17.05-01.06.2020
(total duration 14d).
Metronidazol i.v. 1500mg, 17.05-20.05.2020
(total duration 3d).
Metronidazol p.o. 500mg, 20.05-08.07.2020
(total duration 49d).
Amoxicillin p.o. 3000mg, 20.05-08.07.2020
(total duration 49d).
Probenecid p.o. 1000mg, 20.05-08.07.2020
(total duration 49d).

Surgical Treatment

1. 17.05.2020: Laparoscopic appendectomy and drainage of liver abscess (total duration of drainage: 8 days).
2. 20.05.2020: Ultrasound-guided drainage of liver abscess (total duration of drainage: 6 days).

Supportive Treatment

1. Respiratory physiotherapy

Outcome and Follow-Up

The patient was seen weekly for follow-up examinations over the course of 6 weeks after discharge. During this period, the patient presented himself once with tachycardia and episodes of sweating in the emergency department. Computer tomography ruled out a pulmonary embolism but showed a thrombus of the medial hepatic vein. Oral anticoagulation with Rivaroxaban 20mg daily was established for a planned total duration of 3 months. The antibiotic treatment was continued until the abscesses could no longer be identified in the CAT scan. The patient remained afebrile and did not show any symptoms, apart from fatigue, during followup. The antibiotic treatment could be discontinued as planned, 7 weeks after initiation.

Discussion

The treatment of pyogenic liver abscesses consists of abscess drainage and antibiotic therapy [17-20]. The therapeutic approach depends on the number and size of liver abscesses. For single, unilocular abscesses smaller than 5cm in diameter, a percutaneous drainage is indicated. This can either be performed through needle aspiration or catheter placement, both bearing similar success rates [17,21-23]. However, needle aspiration has to be repeated in approximately half of the cases [22-24]. In the case of a single, unilocular abscess bigger than 5cm in diameter, a percutaneous drainage with a catheter placement is recommended. With needle aspiration of these larger abscesses the results show slower abscess drainage, longer time to clinical improvement and more often the need for surgical intervention compared to patients with catheter placement [17]. This management should also be applied to very large abscesses (larger than 10cm in diameter), so called “giant abscesses”. With a size of over 10cm in diameter, the risk of complications, including drainage failure and even sepsis leading to death, become significantly higher [8,24,25]. There have been attempts to manage these abscesses surgically, which have shown a lower rate of treatment failure.

However, no change in mortality, duration of clinical manifestation or rate of complications could be identified in the comparison between percutaneous and surgical drainage [26,27]. The therapeutic approach to multiple abscesses should be made by a multidisciplinary team according to the various capabilities and experiences. The successful percutaneous drainage of multiloculated and multiple abscesses has been described [28], which led to a shift in treatment strategy from surgical to an interventional approach. The empiric antibiotic regiment with ceftriaxone and metronidazole is recommended, such that Streptococci, enteric gram-negative bacilli and anaerobes are covered. Alternative regiments should be applied according to local resistances or probable infection pattern. Antibiotic regiments should generally be continued for a total duration of 4-6 weeks, in patients with incomplete drainage the antibiotic application should be intravenously for the whole duration, whereas patients that showed a positive response to drainage can continue the antibiotic therapy orally after 2-4 weeks of parenteral application [21,29,30].

In the case of our patient, the more caudal abscess perforated spontaneously during a laparoscopy. A drainage was inserted, and microbiological samples were taken, which helped identify the responsible pathogen. According to the stated guidelines, we initiated the antibiotic treatment and inserted a percutaneous drainage of the cranial abscess, which was left untouched during surgery. Our case shows the importance of a multidisciplinary approach and shows the efficacy of surgical as well as interventional percutaneous drainage of liver abscesses.

Take Home Messages

1. Acute appendicitis is a medical chameleon: although one of the more common pathologies, it can have very diverse clinical manifestations.
2. The clinical findings and symptoms do not always correlate with the gravity of an illness.
3. Acute appendicitis, even if abdominally asymptomatic, can lead to extensive pyemia and abscess formation
4. The minimally invasive management of liver abscesses with antibiotics and drainage has shown to be effective with minimal risk to the patient.

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Novel Anesthesia Technique for Clavicle Fracture with Autologous Bone Graft: A Case Report

Introduction

Surgical treatment of clavicle fractures is becoming more frequent as it reduces the nonunion rate, shortens the time to union and also provides better esthetic outcomes [1]. Acute postoperative pain is moderate to severe requiring opioids for its´ management, so planning for adequate perioperative pain management is essential. Clavicle surgery is usually performed under general anesthesia combined with interscalene plexus block, where phrenic nerve paralysis can reach up to 70% [2] depending on the technique which can be a relevant unwanted effect in patients with pulmonary disease, obesity or with concomitant rib fractures as in polytrauma [3]. We describe a novel anesthesia management of this fracture which has been made possible by advances in the use of ultrasound for guidance of locoregional anesthesia.

Case Description

A 32-year-old man suffered an isolated right middle third clavicle fracture after a motorcycle accident. He was proposed for osteosynthesis with autologous bone graft harvested from the iliac crest (Figure 1). The patient had no relevant past personal medical history, ASA physical status I, weight 75 kg. As part of the anesthetic plan, the patient underwent general anesthesia (intravenous induction: Fentanyl 200 mcg + Propofol 200mg + Rocuronium 50 mg; maintainance: Sevoflurane in O2/air) combined with ultrasound-guided locoregional anesthesia performed after general anesthesia was established: clavipectoral fascia block (Ropivacaine 0.5% 10ml 50mg), superficial cervical plexus block (Ropivacaine 0.5% 10ml 50mg) and transverse plane abdominal (TAP) block (Ropivacaine 0.5% 20ml 100mg). No intraoperative complications occurred and the patient was extubated immediately at the end of the procedure. He did not require any intraoperative opioids after general anesthesia induction nor during the postoperative period. The postoperative analgesic regimen was mantained with intravenous Paracetamol 50 mg q 8 hours alternating with Dexketoprofen 50mg q 8 hours, achieving pain VAS (visual analogue score) <2. The patient was discharged home uneventfully after 48 hours (Figure 2).

Figure 1: Preoperative x-ray where the fracture is seen.

Figure 2: Postoperative radiographic control illustrating satisfactory osteosynthesis.

Discussion

While the cutaneous innervation of the skin above the clavicle is supplied by the supraclavicular nerve of the superficial cervical plexus, the sensory innervation of the clavicle is complex and unclear [4], it seems to be provided by terminal branches of the braquial plexus such as subclavian, long thoracic and suprascapular nerves, passing through the plane between fascia and clavicle [5]. The combination of interscalene plexus block and superficial cervical plexus block reach a good level of postoperative analgesia for clavicle fractures. However, supplementation with intraoperative opioids is necessary because the skin and medial clavicle sensory innervation is not blocked. Also, various side effects have been described, the most relevant being phrenic nerve paralysis. In this context, the clavipectoral block is a good alternative for pain management being an easy and safe technique when performed with ultrasound guidance.

Clavipectoral block was described by Valdes- Vilches in 2017. The clavipectoral fascia covers the clavicular site of the pectoralis major muscle. Compared with interscalene plexus block, clavipectoral fascia block offers a more lateral and superficial plane, far from neurovascular structures, the clavicle being a natural backstop during injection. When properly done, no specific adverse effects of this technique have been described [3]. The literature on the clavipectoral plexus block combined with deep cervical plexus block being used as a single anesthetic modality for surgery of the clavicle is scarce [3]. Combining it with superficial cervical plexus block is beneficial for providing analgesia of the skin incision. In this case, it was necessary to harvest bone graft from the iliac crest. There is high incidence of severe postoperative pain (39%) [6] with this procedure. The TAP block is a good solution for pain control. Regional anesthesia results in less overall opiod consumption decreasing nausea, vomiting and possible hyperalgesia and immunodepressive effects [7]. It enables early deambulation and patient discharge.

Technique

Clavipectoral fascia block: in supine position, the superficial cervical plexus was blocked. Then the linear transducer probe (11 MHz) was placed on the anterior surface of the medial third of the clavicle. A 20-gauge block needle (35 mm) was inserted in a caudal to cephalic direction. An injection of local anesthetic under ultrasound guidance was made between the clavipectoral fascia and the periosteum on the medial and lateral area of clavicle injury [5] (Figure 3). The TAP block was performed in supine position, with a 22-gauge block needle (80 mm), the injection of local anesthetic was made between the internal oblique muscle and transversus abdominis muscle.

Figure 3: Ultrasound landmarks to identify clavipectoral fascia.

Conclusion

Combined general anesthesia with clavipectoral fascia block and deep cervical block is an excellent alternative for patients undergoing clavicular fracture surgery, being a successful and effective regional anesthesia method. We present it as an alternative to interscalene brachial plexus block, eliminating intra and postoperative opioid consumption and possible phrenic nerve paralysis. This case illustrates the efficacy of clavipectoral fascial block as a peripheral nerve block in clavicle surgery. Additional studies are required in order to clarify the anatomy, innervation and distribution of the sensory blockade and the efficacy and safety of the clavipectoral block in this context.

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Insight into Secondary Metabolites of Circinella, Mucor and Rhizopus the Three Musketeers of Order Mucorales

Introduction

Fungi are rich sources of biologically active natural compounds, which are used in the manufacturing of wide range of clinically important drugs. Fungi produce important antibiotics such as the beta-lactam antibiotics members, penicillin and cephalosporin, which and their derivatives are dominating the most important antibiotic market until now [1-5]. Fungi generally and endophytic ones specifically represent future factories and potent biotechnological tools for production of bioactive natural substances which could extend healthy life span of humanity, as done by penicillin from centuries, and considered promising alternatives for some high costly produced chemicals and drugs [6- 12]. The present review highlights some bioactivity of secondary metabolites, produced by Circinella, Mucor, and Rhizopus, involved in medical, pharmaceutical, agricultural, and industrial applications. Zygomycetes constitute a remarkable group of microscopic fungi. These fungi are mainly soil inhabitants living as saprobes and decomposers of organic matter and herbivorous feces. Zygomycota, represent the most basal terrestrial phylum of the kingdom of Fungi [13].

Zygomycetes have an integral role in the development of microbial ecosystems, a property which has the potential to be converted for biotechnological and industrial applications ranging from food technology to drug development [14]. The Mucorales, which is classified into the subphylum Mucormycotina [15], is the largest order of fungi. Members of this group (Circinella, Mucor and Rhizopus) are abundant saprophytes in nature. They are commonly found in soil and decaying vegetation, and can also be found in grains [16,17]. Mucorales members (Example; Circinella, Mucor and Rhizopus) grow and invade quickly on easily digestible substrates, such as those containing starches, sugars, and hemicelluloses. Future studies should investigate Circinella, Mucor and Rhizopus their ability to produce extracellular enzymes and potential applications in biotechnology.

Circinella, Mucor and Rhizopus Description and Ecology

The Genus Circinella belonging to Phylum: Mucoromycota; Class: Zygomycetes; Order: Mucorales; Family: Syncephalastraceae. Colonies mucraceous, usually up to few mm high; sporangiophores erect, branching sympodially and each branch recurved (Circinate) terminated with a sporangium; sporangia globose, columellate, many-spored, covered with non-diffluent wall, incrusted with calcium oxalate, breaking into pieces; columellae globose, subglobose or cylindro-conic; sporangiospores globose or oval, smooth; heterothallic; zygospores when produced, born on erect hyphae, with more or less similar gametangia and suspensors. Colonies fast growing, attaining 7.5 cm after 7 days on malt extract agar at 25C. The most common Circinella species is Circinella muscae. Circinella colonies color is brownish and common present in soil (Figure 1) [11,13]. The Genus Mucor belonging to Phylum: Mucoromycota; Class: Zygomycetes; Order: Mucorales; Family: Mucoraceae. Colonies fast growing, often up to several cm in height, white to yellow becoming dark grey with age; mycelium non-septate, occasionally septa produced in old cultures; sporangiophores erect arising singly from the mycelium without rhizoids, forming a dense mat, unbranched or branched monopodially or sympodially, sometimes the branches recurved, bearing terminal sporangia; sporangia globose, many-spored, columellate, without apophysis; sporangial wall deliquescent, incrusted with needles of calcium oxalate; columellae of various shapes, colourless or coloured; sporangiophores colourless or colored, greyish or brownish, globose or elliptical, wall thin smooth; chlamydospores produced in some species , terminal, hyaline, smooth walled; zygospores produced in the aerial mycelium and arising from copulation of two straight gametangia.

Figure 1: Circinella spp., different species.

Figure 2: Mucor spp., different species.

Mucor is one of the largest genera of Order: Mucorales and a number of its species are worldwide. Mucor species are common in soil and also occur organic matter. Colonies fast growing, filling the plate of malt extract agar after 3 days at 25C (Figure 2). The most common Mucor species is Mucor circinelloides, Mucor hiemalis and Mucor racemosus [11,13]. The Genus Rhizopus belonging to Phylum: Mucoromycota; Class: Zygomycetes; Order: Mucorales; Family: Mucoraceae. Colonies fast growing, filling the whole plate after 2 days on malt extract agar at 25C, at first white, later becoming pale or dark grey brown. Mycelium non-septate, occasionally septa produced in old cultures, differentiated into stolons, rhizoids arising from stolon ends and erect unbranched sporangiophores sporouting opposite the rhizoids; sporangia generally globose, apophysate, columellate, many-spored; sporangiospores short ellipsoidal, brownish, striate in many species; most species heterothallic; zygospores naked, dark-coloured; wall strongly roughened, suspensors straight, large and swollen. Rhizopus species are common in soil and soil containing various organic matter. Rhizopus species are impotent spoilage organisms. The most common Rhizopus stolonifer, Rhizopus arrhizus and Rhizopus oryzae (Figure 3) [11,13].

Figure 3: Rhizopus spp., different species.

The Genus Circinella as a Biotransformation Agent

Enzymatic reactions and biotransformations catalyzed by fungal enzymes and used in industry, agriculture, food technology, and medicine have increased in importance tremendously in recent years. It was later demonstrated that filamentous fungi from several genera, including Circinella, Mucor and others, are able to hydroxylate dibenzofuran, forming 1-, 2-, 3-, or 4-hydroxybenzofuran and some other more hydrophilic products [18]. Yan et al., [19], mentioned that, nine hydroxylated and glycosylated metabolites, of which two have not been reported previously, were obtained and identified after incubation by Circinella muscae. The specific 7β, 15α, and 21β hydroxylated products yielded from this bio-process by Circinella muscae. In addition, the selectively glycosylation at C-28 was another main reaction type. It was also observed that the 3β-OH group was selectively dehydrogenated into carbonyl group. These reactions may be difficult to achieve by chemical synthetic means. In vitro biological tests indicated that compounds 3β, 7β dihydroxyolean-12-en-28-oic acid; 3β, 7β, 15α-trihydroxyolean- 12-en-28-oic acid, and 3β, 7β, 15α-trihydroxyolean-12-en- 28- oic acid-28-Oβ-D-glucopyranosyl ester showed significant antiinflammatory activities, which suggested that hydroxylation at C-7 and glycosylation at C-28 by Circinella muscae had benefit effects [19]. Macromycete Circinella muscae AS 3.2695, catalyzed the oxidation of Oleanolic Acid [20]. Biotransformation of ursolic acid by Circinella muscae CGMCC 3.2695 was investigated by Chu et al., [21].

Scaled-up biotransformation reactions yielded ten metabolites. Their structures were established based on extensive NMR and HR-ESI-MS data analyses, and four of them are new compounds. Circinella muscae could selectively catalyze hydroxylation, lactonisation, carbonylation and carboxyl reduction reactions. Also, all the identified metabolites were evaluated for their anti-neuroinflammatory activities in LPS-induced BV-2 cells. Most metabolites displayed significant inhibitory effect on nitric oxide (NO) production and the results suggested that biotransformed derivatives of ursolic acid might be served as potential neuroinflammatory inhibitors [21]. Biotransformation of betulinic acid was carried out with Circinella muscae CGMCC 3.2695 and Cunninghamella echinulata CGMCC 3.970 by Chen et al., [22], yielded six previously undescribed hydroxylated metabolites and four known compounds. Circinella muscae could catalyze the regioselecitve hydroxylation and carbonylation at C-3, C-7, C-15 and C-21 to yield seven products. Cunninghamella echinulata could catalyze the C-1, C-7 and C-26 regioselecitve hydroxylation and acetylation to yield five metabolites. The structures of the metabolites were established based on extensive NMR and HR-ESIMS data analyses and most of the metabolites exhibited significant inhibitory activities on lipopolysaccharides-induced NO production in RAW264.7 cells [22].

The Genus Mucor Source of Biologically Active Compounds

Species of Mucor ramosissimus produce extracellular enzymes, such as endopolygalacturonase and lipase, and secondary metabolites, such as phytoalexin elicitor [23,24]. Lately, several studies have focused on applying Mucorales members to produce ethanol and biomass by-product. Particularly, Mucor ramosissimus has been reported as a potential ethanol-producing mold [25]. Mucor circinelloides has a great industrial potential since it started producing bioactive compounds of interest, such as microbial lipids, carotenoids or sterols [26,27]. Mucor circinelloides is able to produce microbial lipids which could be useful for the production of biodiesel using the common way to produce fatty acid methyl esters, due to the beneficial properties of these compounds, new sustainable and more environmentally friendly methods have emerged in order to increase their production [28]. Three important strains of Mucor circinelloides grown in specific media for specified period (72 h, 120 h and 168 h) under submerged fermentation conditions were investigated by Hameed et al., [29], for their potential antioxidants/secondary metabolite production and found that all mycelial extracts demonstrated effective antioxidant activities in terms of β-carotene/linoleic acid bleaching, radical scavenging, reduction of metal ions and chelating abilities against ferrous ions [29].

Antioxidant property of all three important strains of Mucor circinelloides extracts were duo to their phenolic and condensed tannin contents. Strains Mucor circinelloides MC277.49 was found to be the biggest producer of secondary metabolites under nutritional stress condition in late exponential phase. These Mucor strains prove to be rich sources of antioxidants and secondary metabolites, which could be used in the development of nutraceuticals and natural antioxidants [29]. The new cyclic heptapeptide unguisin F and the known congener unguisin E, were obtained from the endophytic fungus Mucor irregularis, isolated from the medicinal plant Moringa stenopetala, collected in Cameroon studied by [30]. The structure of the new compound was determined on the basis of one- and two-dimensional NMR spectroscopy as well as by high-resolution mass spectrometry. Heptapeptide unguisin F and congener unguisin E were evaluated for their antibacterial and antifungal potential, but failed to display significant activities [30]. Biotransformation is an important tool for the structural modification of organic compounds, especially natural products with complex structures, which are difficult to achieve using ordinary methods [31].

Mucor genus is widespread in nature and some species are used extensively in biotechnology for enzyme and useful compound production. Biotransformation can be used as a very convenient way of producing compounds, particularly when the structure is complex and they can neither be isolated as metabolites nor chemically synthesized [31]. It is safe to conclude that biotransformation by Mucor species is of great importance due to their wide-ranging use in the stereospecific production of compounds of commercial interest, and because they simplify the study of the metabolism of such compounds in order to obtain novel agents with many interesting biological activities [31]. The biotransformation of 1R-(−)-camphorquinone, achieved by growing cells of Mucor plumbeus isolated from soil. Results were found for M. plumbeus, which was only able to perform monoreduction of camphorquinone when cultivated on a glucose– peptone–yeast extract medium. Large-scale experiments were set up and the camphorquinone biotransformation products formed by Mucor plumbeus were purified by column chromatography and identified by 1H and 13C nuclear magnetic resonance (NMR). Mucor plumbeus could be of great use for the selective reduction of camphorquinone and related compounds [32].

The Genus Rhizopus Source of Biologically Active Compounds

Secondary metabolites is one of the characteristic features of microorganisms. More than 50,000 bioactive compounds have been isolated from the extracts of microorganisms with a diversified arrangement of chemical structures, which showed antimicrobial, antitumor and agrochemical activity and others [33]. The ethyl acetate extract of Rhizopus stolonifer has potent cytotoxic activity against brine shrimps, suggesting that it could serve as a lead compound for anticancer compounds. Its antifungal activity also indicates its potential for development into an antifungal product. Furthermore, its phytotoxicity against Lemna minor weed demonstrates that it contains compounds with herbicidal activity. More studies on Rhizopus stolonifer is needed to isolate and structural characterization of its constituents which could lead to the development of pharmaceutical and agricultural products [34].

From previous studies, it was concluded that the extracts of Rhizopus species have compounds which have ability to inhibit the growth of fungal and bacterial strains [35]. Chitosan was isolated from the fungus Rhizopus oryzae by using yeast peptone glucose broth medium, showed antimicrobial activity against Escherichia coli, Staphylococcus aureus and Candida albicans and reach to 20, 15 and n 15 mm as inhibition zone respectively [36]. Nanotechnology is a field that is increasing day by day, making an impact in all spheres of human life. Biological methods of synthesis nanoparticles called “greener synthesis” of nanoparticles and these have proven to be better methods due to slower kinetics, they offer better manipulation and control over crystal growth and their stabilization [37]. The extracellular synthesis of silver nanoparticles by Rhizopus stolonifer and its efficacy against multidrug resistant (MDR) strains isolated from burnt cases from hospitals at Gulbarga region, Karnataka, India is reported by Rathod and Ranganath, [38], the biosynthesized nanosilver showed excellent antibacterial activity against multidrug resistant Pseudomonas aeruginosa isolated from burnt infections. Biologically synthesized nanosilver showed zone of inhibition (mm) of two isolates about 33mm and 30.5mm in diameter strains of Pseudomonas aeruginosa respectively [38].

This study reports the extracellular synthesis of silver nanoparticles by Rhizopus stolonifer and its efficacy against multidrug resistant (MDR) Escherichia coli and Staphylococcus aureus isolated from Khwaja Bande Nawas Hospital, Gulbarga, Karnataka was carried by Banu and Rathod, [39] and synthesis of silver nanoparticles (AgNPs) was carried out by using fungal filtrate of Rhizopus stolonifer and an aqueous solution of AgNO3. Biotransformations of sesquiterpenoids by Rhizopus species have been used to provide new derivatives with potential biological activities. Fungal transformation processes by Rhizopus species, has been the introduction of hydroxyl groups into remote positions of the molecules, which is difficult to achieve by chemical means. Other reactions carried out with stereo-selectivity by these microorganisms have been: epoxidations of double bonds, hydrogenations of the exocyclic C-C double bond of the lactone rings, and reductions of carbonyl groups giving Salcohols. Several reactions have also been performed by these Rhizopus species with regio-selectivity, such as oxidations of hydroxyl groups and deacetylations [40]. The enttrachyloban diterpene ent-18, 19-dihydroxytrachylobane was biotransformed by Rhizopus stolonifer, and produced the new ent- 11β, 18, 19-trihydroxytrachylobane, and the new ent-kaurene diterpenes ent-16α, 18, 19-trihydroxykaur-11-ene and ent- 18, 19-dihydroxy-16α-methoxykaur-11-ene. The formation of derivative ent-11β,18,19-trihydroxytrachylobane, involved the first hydroxylation of C-11 of ent-trachyloban diterpene skeleton by fungus, and compounds ent-kaurene diterpenes ent-16α, 18, 19-trihydroxykaur-11-ene and ent-18,19-dihydroxy-16α- methoxykaur-11-ene were probably produced by backbone rearrangement of ent-11β,18,19-trihydroxytrachylobane, these results, verified the potential application of microbial transformation (Especially By Mucorales Rhizopus species) of natural products for the formation of new compounds [41].

Conclusion

Fungi in general are important source of unique natural products with a high level of biodiversity and also yield several compounds having different industrial applications and pharmaceutical activities, which is currently attracting scientific researches. Every study conducted on Circinella, Mucor and Rhizopus resulted in discovery of new metabolites that may have an important applications, which made these genera potential source of pharmaceuticals and attracted attention for further investigations of their important bioactivities properties. Circinella, Mucor and Rhizopus are known for their capability of producing various bioactive compounds with medical applications as antineuroinflammatory, antiinflammatory, and enzymes producers and as biotransformation agents.

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A Novel 3 Dose Rabies Vaccine

Introduction

Rabies is a zoonotic viral disease that is caused by rabies virus (RABV) and claims the highest fatality among all infectious diseases. Rabies is found in more than 100 countries and territories [1]. To prevent rabies, timely and complete postexposure prophylaxis (PEP) is necessary. Currently five doses of rabies vaccine are administered on days 0, 3, 7, 14, and 28 but the studies have shown that the compliance to complete course was only 40% [2]. Therefore, an effective way to address these limitations is the reduction of PEP doses in humans and a novel vaccine with improved immunological outcomes through an accelerated PEP schedule is desirable. In this regard, Cadila Pharmaceuticals Ltd., Ahmedabad, India has developed a novel recombinant nanoparticle-based rabies G protein vaccine (Thrabis®) prepared by using Virus Like Particle technology (VLP).

Recombinant Nanoparticle-Based Rabies G Protein Vaccine

For generation of recombinant rabies G protein vaccine using VLP platform; genetic sequences encoding the rabies G protein sequence are selected. The genes are then cloned into baculovirus and the baculovirus was made to infect insect cells (sf9). The target antigens were expressed in the sf9 cells which were purified using various chromatographic techniques. The purified target antigen exists as an assembly of polypeptides that is present in multiple copies in subunit antigens in well-ordered arrays with defined orientations [3]. This can potentially mimic the repetitiveness, geometry, size, and shape of the natural host-pathogen surface interactions. Such nanoparticles offer a collective strength of multiple binding sites (avidity) and can provide improved antigen stability and immunogenicity [4,5]. Safety is a major advantage of VLPs. Like traditional vaccines, VLPs are immunogenic and excellent to control infectious diseases. VLPs cannot replicate, recombine or undergo reassortment because they do not contain infectious DNA or RNA material; therefore, VLPs are safer than traditional vaccines. VLPs possess several advantages over the products that are produced by chemical syntheses such as smaller size, which ranges from 10 to 2000 nm, availability of high-resolution threedimensional (3D) models of their structure, construction flexibility, high-production yields, and structural uniformity of each type of virus or VLP [6].

Clinical Study

A multi-centric, open label, assessor blind, centre-specific block randomized, parallel design, phase III clinical study was conducted among 800 subjects. The eligible subjects were randomized in 2:1 ratio for recombinant rabies G protein vaccine and the reference vaccine. Subjects in recombinant rabies G protein vaccine arm received 3 doses of vaccine on days 0, 3 and 7; while subjects in reference vaccine arm received 5 doses of WHO pre-qualified vaccine on days 0, 3, 7, 14 and 28. The primary objective was to demonstrate the non-inferiority of the test vaccine on day 14 after first dose relative to the reference vaccine in terms of seroprotection rate (RVNA titer of ≥0.5IU/mL). The secondary endpoints were the seroprotection rate on day 42 post first dose of the study vaccine and the frequency of solicited and unsolicited adverse events (AEs) were reported between day 0 & 180. On day 14, 99.24% in the test vaccine arm and 97.72% in the reference vaccine arm were seropositive; the difference was statistically non-significant. Likewise, on day 42, 98.69% of the subjects in the test vaccine arm and 100.00% in the reference vaccine were seropositive, the difference was statistically non-significant. A statically significant higher number of participants in the reference arm had adverse events (AEs) compared to test arm (17.2% vs 9.9%, P=0.0032). All the AEs were mild to moderate in nature, which resolved without any complications. The most frequently observed local AEs were pain, redness and swelling at the injection site. The systemic AEs were fever, headache, ear pain, urticaria, joint pain and nausea [3].

Conclusion

The novel 3 dose recombinant rabies G protein vaccine (Thrabis®) was found to be safe & immunogenic and was comparable to 5 doses of WHO pre-qualified vaccine in simulated post exposure prophylaxis. The reduced number of vaccine doses leads to a reduction in number of visits and travel cost; as well as increases the compliance, which is important to prevent rabies and ultimately help in eliminating dog mediated human rabies by 2030 [7].

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Application of High-Flow Oxygen Therapy in Acute Pancreatitis Complicated with Acute Respiratory Dysfunction

Introduction

Pulmonary complications of acute pancreatitis (AP) characterized by increased permeability of the pulmonary microvasculature and alveolar spaces filled with leaked proteinrich exudate were frequent with morbidity about 75%, [1,2] including acute lung injury (ALI) or ARDS, atelectasis, pleural effusion, alteration in diaphragmatic function, i.e. [3-5]. It usually resulted in a need of endotracheal intubation and mechanical ventilation, in addition, the mechanisms were complex and still to be discovered [6]. It was reported that 30%-60% of death was related to pancreatitis- associated ALI and ARDS [7-9]. Furthermore, nearly one-third of death of acute pancreatitis prior to admission to hospital were associated with ALI [10]. Studies have revealed that acute respiratory dysfunction (ARD) was an independent prognostic factors for hospital mortality in severe acute pancreatitis (SAP) along with the age, chronic health situation, and organ failures, which was associated with 60% of death within first week [11,12]. Therefore, more attention have been paid on treatment of pancreatitis-associated respiratory complications for reducing early death.

HFNC, which is increasingly used in intensive care unit (ICU) and non-ICU wards was considered as an alternative to Noninvasive Positive Pressure Ventilation (NPPV) and conventional oxygen therapy (COT) with better tolerated and decreasing work of breathing [13,14]. It could provide heated and humidified air and reduce airway secretion and atelectasis with high-flow rates of up to 60 L/min as well as high FiO2 from 0.21 to 1.0 [15,16]. Previous studies have revealed that HFNC has the capability of generating low levels of positive end-expiratory pressure and decreasing physiological dead space through flushing expired carbon dioxide into the upper airway [17]. It has been applied to various diseases, including various respiratory failure, cardiogenic pulmonary edema, postextubation, postoperative patients, and infants [15,16,18-20]. However, the efficiency of HFNC in acute pancreatitis is unclear. Our research is intended to illustrate the value of HFNC in acute pancreatitis complicated with acute respiratory failure.

Material and Methods

Trial Design

All acute pancreatitis complicated with ARD admitted to ICU were included in this retrospective study from January 2014 to June 2019. This trial was approved by the ethical committee of our hospital (approval number:KY030-01) and was registered on the Chinese Clinical Trial Registry (www.chictr.org.cn/, registration number: ChiCTR2000029202) and written informed consent was obtained from patients or their families before HFNC treatment.

Patients

Computed tomography (CT) combined with supportive specific laboratory data were used in the diagnosis of AP. All AP were enrolled if they followed one of the following criteria: PaO2/FiO2 < 300 mmHg; respiratory rate > 25/min; dyspnea or accessory muscle use; asynchronous or paradoxical breathing; required >5 L/min O2 to maintain SpO2 >92%.Exclusion criteria: need for immediate intubation, central nervous system disorder, cannot answer questions, contraindications for the use of the mask (noncooperative patient).Patients were asked to grade their dyspnea score [21] (improvement; no change; deterioration) and comfort score [22] (poor; acceptable; good) 1 hour after intervention.

Study Intervention

All patients were treated with COT though face mask or nasal cannula before enrolled. In the COT group, oxygen therapy was applied continuously through face mask or nasal cannula at a flow rate up to 10 liters per minute. The rate was adjusted to obtain SpO2>92% until patients recovered or were intubated. High flow humidified oxygen (37°C and 44mgH2O/L) was delivered continuously through a nasal cannula with Optiflow (Fisher and Paykel Healthcare) with a primal flow rate of 50L/min, a primal FIO2 of 50%, and dynamic adjustments to obtain SpO2>92%. HFNC was switched to COT if SpO2 was ≥95% at ≤5 L/min O2 or the PaO2:FIO2 was at least 300. Intubation and mechanical ventilation decisions were made by the physicians with the criteria: respiratory arrest; respiratory pauses with loss of consciousness or gasping respiration; encephalopathy; cardiovascular instability; unmanageable secretions; respiratory fatigue; refractory hypoxemia (HFNC:SpO2≤ 88% with FIO2 = 100%; COT: SpO2≤88% with at least 10L/min), or respiratory acidosis (pH<7.30 and PaCO2 ≥50mmHg).

Primary and Second Outcomes

The primary outcomes are the early intubation rate, defined as percentage of intubation and mechanical ventilation in 10 days and the median time to intubation from patients enrolled. The second outcome includes changes in physiological parameters as well as arterial blood gases and grade of dyspnea, comfort score, regression of respiratory failure after 1 hour intervention, adverse events (atelectasis, pleural effusion and abdominal distension), early mortality defined as mortality in 10 days and ICU stay length.

Statistical Analysis

The Chi-square test or Fisher test was used in the comparisons for categorical variables while the unpaired Student’s t test or Mann-Whitney U test were performed for continuous variables. The analysis of the data obtained before and after intervention from each patient was made with the paired Wilcoxon test. Variables associated with respiratory failure regression were assessed by univariate and multivariate logistic- regression analyses. Variables suspected to be associated with regression of respiratory failure with a P<0.10 after univariate analysis were accounted into the multivariate analysis. All statistical analyses were performed using IBM SPSS, Version 19.0 (IBM Corp., Armonk, NY, USA) or GraphPad Prism version 6.0 (GraphPad Software Inc., San Diego, CA, USA). A p value (two-tailed) <0.05 was considered statistically significant.

Results

Patients

A total of eighty-one patients with AP were admitted to ICU. Seven were intubated immediately at admission and five did not meet the classification criteria at last. Ultimately, sixty-nine patients were included in this retrospective study. 30 out of 69 were treated with conventional oxygen through nasal cannula or face mask, while 39 were treated with HFNC. Baseline characteristics of patients, including demographic data, severity of illness (APACHE II), etiology, severity of AP (Ranson score and Balthazar score) and associated comorbidities were recorded in Table 1. Vital signs and arterial blood gases before and after enrollment (1h) were collected in Table 2. We also evaluated the incidence of atelectasis and pleural effusion in the two groups by CT.

Table 1: Patients Characteristics.

Note: HFNC: High-Flow Oxygen though Nasal Cannula; COT: Conventional oxygen therapy

Table 2: Primary outcome.

Note: Median ± SD, h

HFNC: High-Flow Oxygen though Nasal Cannula; COT: Conventional oxygen therapy

Primary Outcome

The early intubation rate was 25.6% (10 of 39 patients) in the HFNC group, 56.7% (17 of 30) in the COT group (P=0.013) Table 3, Figure 1. The median time to intubation was 64.25h in the HFNC group, 7.75h in the COT group (P<0.001) Table 3. Meanwhile, we compared the intraabdominal pressure (cm H2O) before intubation of intubated patients in the two groups (HFNC 21.7 vs COT 19.1; P=0.103; not shown).

Table 3: Physiological parameters and arterial blood gases at baseline and 1hour after intervention.

Note: HFNC: High-Flow Oxygen though Nasal Cannula; COT: Conventional oxygen therapy; a: Baseline VS 1 Hour in HFNC; b: Baseline VS 1 Hour in COT; c: Changes in the HFNC group VS that in the COT group 1hour after intervention.

Figure 1: Comparison of intubation rate between the two groups.

Note: HFNC: High-Flow Oxygen though Nasal Cannula; COT: Conventional oxygen therapy.

Changes in Physiological Parameters and Arterial Blood Gases

After 1 hour of intervention, patients treated with HFNC had decreased respiratory rate (breaths/min), heart rate (beats/min) (27 vs 21, P<0.001; 121 vs 104, P=0.008) and increased PaO2 (mmHg) (64 vs 110; P< 0.001), while PaCO2 (mmHg) and PaO2/ FiO2 (mmHg) had no changes (37 vs 42, P=0.087; 189 vs 210, P=0.539) compared with baseline. However, in the COT group, after 1 hour of intervention, respiratory rate, heart rate, and PaO2/FiO2 were similar (30 vs 26, P=0.166; 114 vs 109, P=0.486; 192 vs 201, P=0.142) with baseline, whereas PaCO2 and PaO2 were higher (38 vs 50; P< 0.001; 64 vs 83; P<0.001) Table 2. There was no difference in physiological parameters and arterial blood gases at baseline between the HFNC group and the COT group. Compared with the COT group, patients treated with HFNC had decreased respiratory rates, PaCO2 (21 vs 26, P=0.020: 42 vs 50, P<0.001) and increased PaO2 (110 vs 83, P=0.003) 1 hour after intervention. Patients showed greater improvement in PaO2 in the HFNC group than that in the COT group. (P<0.001) Table 2.

Dyspnea Grade, Comfort Score, and Respiratory Dysfunction Regression

The comfort score was similar between the two groups (P=0.596, Table 2). There was a higher proportion of patients felling improvement in dyspnea in the HFNC group (87.2% vs 56.7%, P=0.006) Figure 2. Improved in respiratory dysfunction was defined as respiratory rate <25/min and improvement in breathing effort, including accessory breathing muscle activity and/or paradoxical breathing. Finally, 34 out of 69 patients (26 in the HFNC group vs 8 in the COT group; P=0.001; Figure 3) got respiratory failure regression 1 h after intervention. In the univariate analysis the following parameters were associated with respiratory failure improvement: APACHE II score, PaO2 at baseline and Oxygen strategies. Higher APACHE II score, lower PaO2 at baseline and conventional oxygen therapy exhibited an increased risk for poorer regression of respiratory failure (OR =12.250, 95% CI 1.268-118.361, P=0.030; OR=8.000, 95% CI 1.252-51.137, P= 0.028; OR=6.429, 95% CI 1.026-40.261, P=0.047). In multivariate logistic-regression analyses, HFNC and APACHE II score were independent predict factors to regression of respiratory failure (OR =20.381, 95% CI 1.177-351.911, P=0.038; OR=36.827, 95% CI 1.529-887.083, P=0.026) Table 4.

Figure 2: Comparison of dyspnea score improvement between the two groups 1 hour after intervention.

Note: HFNC: High-Flow Oxygen though Nasal Cannula; COT: Conventional oxygen therapy

Figure 3: Comparison of respiratory failure regression between the two groups 1 h after intervention.

Note: HFNC: High-Flow Oxygen though Nasal Cannula; COT: Conventional oxygen therapy

Table 4: Univariate and Multivariate analysis of risk factors to regression of respiratory failure.

Adverse Events and Clinical Outcomes

No significant differences were found for adverse events between the two groups. Early mortality in the HFNC group had trend to be significantly lower than that in the COT group (17.9% vs 40.0%, P= 0.058). Patients treated with COT had much longer ICU stay length (19.5±13.4 vs 7.8±4.7, P=0.009) Table 5.

Table 5: Adverse Events and Clinical Outcomes.

Note: HFNC: High-Flow Oxygen though Nasal Cannula; COT: Conventional oxygen therapy

Discussion

Accumulating research have revealed some advantages of HFNC application in pneumonia-associated respiratory dysfunction, including decreased intubation rate, lower 90-day mortality, and increased ventilator-free days compared with NPPV and/or COT [23-26]. Despite this, COT with a face mask or nasal cannula is still the first-line treatment for those lacking studies about HFNC application in AP associated respiratory dysfunction. However, COT is not able to satisfy some patients with severe hypoxemia. Therefore, to our knowledge, this study illustrated the efficiency of HFNC in acute pancreatitis complicated with acute respiratory dysfunction for the first time. In this study, eliminating the effect of secondary infection on intubation, we compared the early intubation rate and the median time to intubation of the two groups in combination with the pathophysiological characteristics of pancreatitis. Severe acute pancreatitis has two representative phases. The first stage is in the first ten days, with characteristics of the systemic inflammatory response syndrome (SIRS). The second usually comes up during the second week and is marked by infectious manifestations [27]. In the end, we found patients in the HFNC group had a lower early intubation rate and a longer median time to intubation.

We also compared other outcomes, including changes in physiological parameters and arterial blood gases, comfort score, regression of dyspnea and respiratory dysfunction, adverse events, early mortality, and ICU stay length between the two groups and found that first, HFNC was superior to COT in improving dyspnea, decreasing respiratory rate and heart rate, preventing carbon dioxide retention, and had a stronger effect on improving PaO2. However, we did not find a difference in PO2/FiO2 between the two groups, indicating that HFNC could improve PO2 with higher FiO2, but it could not improve physiopathologic changes. Second, HFNC was independently associated with the regression of respiratory dysfunction. Third, the comfort score of HFNC was similar to that of COT. At last, patients treated with HFNC had shorter ICU stay length and lower early mortality. We hypothesize that the reason why the early mortality between the two groups did not reach statistical significance would be due to the limited number of patients in our study. Above all, we would come to the conclusion that HFNC is prior to COT in acute pancreatitis complicated with acute respiratory dysfunction. There are some limitations to our reach. First, this study is a single center with a small number of patients. Second, we did not compare HFNC and NPPV. Hence, in the future, we should compare HFNC, NPPV and COT in multicenter studies with large samples.

Conclusion

HFNC is a more efficient option in acute pancreatitis complicated with acute respiratory failure and may be an alternative treatment to COT.

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How Can We Track COVID-19 Hotspots and Prevent its Spread?

The outbreak of novel coronavirus disease 2019 (COVID-19) was declared a public health emergency by the World Health Organization (WHO) on 30th January 2020, due to its spread across the globe [1,2]. Because of continued waves of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it is essential to introduce efficient monitoring and screening techniques Virus containment remained challenging in spite of the various advancements in the research field. The most common modes of virus transmission include droplet, contact or fomite, and fecal transmission [3,4]. Fecal transmission can be a serious risk for both humans and animals in case of aerosolization of fecal waste contaminated with the virus [5]. In diarrhea patients, severe acute respiratory syndrome coronavirus (SARS-CoV) was found stable in feces at room temperature for a minimum of 1-2 days and could survive for up to 4 days in the stool [6]. This detection increases the chance of fecal-oral transmission [7] because flushing may aerosolize fecal matter and cause airborne transmission [8]. This type of transmission could be high where toilets are shared for example in quarantine centers and hospitals. From these toilets, flushed water enters into sewerage systems. Consequently, the sewerage system becomes a carrier of this virus. Countries are trying hard to maximize the testing of the virus to avoid community spread of the COVID-19 and after around two years it looks still difficult. Therefore, it is crucial to locate the COVID-19 hotspots to plan mitigation strategies And sewerage system monitoring could be an efficient strategy for finding the virus hotspots. This probable route of virus transmission may worsen the problem of community transmission [9]. Though, this environmental surveillance, the magnitude, and duration of the virus spread may be measured in specific populations. Further, bacterial and viral community interactions can be easily studied in wastewater systems. And this model has already been successfully working for monitoring of poliovirus and Aichi virus towards elimination [10,11].

Coronaviruses can survive up to 2–3 days in sewage water and up to10 days in tap water at 23 °C [10]. Further, it was also observed that temperature, organic matter levels, and the presence of antagonistic bacteria and oxidants such as chlorine may affect the virus survival [10]. Some studies reported the presence of ribonucleic acid (RNA) of SARS-CoV-2 in sewage water [12], however, the persistence of the virus in the sewage system is not yet exactly determined [13]. Fecal matter’s chemical components are mostly organic in nature and may stimulate the extended survival of the virus in the system [14]. Previous studies on SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), determined that coronaviruses (single-stranded RNAs) are less resistant and more fragile to water treatment procedures. It was found that the virus can be grown with the help of bacteriophage in cell culture media for propagation. After the RNA isolation, it was tested for SARS-CoV-2 activity by using a real-time reverse transcriptionpolymerase chain reaction (RT-PCR) assay and untreated samples tested positive. However, the treated samples showed the presence of viral RNA, it was unclear whether the virus retained infectious properties after the routine treatment [11].

Standard methods are not so far established for COVID-19 detection in wastewater, however, environmental surveillance of sewersheds helps to track COVID-19 hotspots in different areas [15]. Factors that affect the efficiency of monitoring tools include geographical location, general sanitary, climatic conditions, sampling methods like trap sampling, precipitation methods, charge-based filters, and detection methods [11]. The main challenge is that the virus’ genetic markers may easily get lost during the flow of sewage [15]. Water treatment plants perhaps impact the virus signals. Researchers are investigating the procedures to understand the data collected from sewage samples. These results help to create an accurate map of how the virus is spreading and show the emergence of the next wave of the pandemic. Sampling and sample storage is a very important part; it can be dangerous for the wastewater workers due to exposure to the virus. This would help in to make policy decisions [15] find out the virus hotspots, and identify the communities with virus carriers to prevent further spread of COVID-19, and also for surveillance purposes.

Arthropods such as cockroaches and houseflies which are major vectors of some pathogens may play a role in transmitting coronaviruses mechanically by contact with contaminated surfaces and/or with the feces of infected individuals [4]. Though, SARSCoV- 2 spread to healthy individuals through inhalation of droplets of infected individuals’ coughs and sneezes. To investigate and measure the potential role of houseflies and cockroaches in the transmission of COVID-19 is crucial in the countries with open sewerage systems because these arthropods feed on human feces, wastes, and carcasses [16,17] and can mechanically carry microbes including viruses in their moth parts and with their legs to transmit to a healthy individual. Previously, through nested RT-PCR, coronavirus was detected in surface wipe to study the spread of coronaviruses by cockroaches [18]. Hence, in an open wastewater or sewerage system, these arthropods could carry and transmit SARS-CoV-2.

Therefore, it is crucial to study the survival rate of SARSCoV- 2 in both surface wastewater and sewage water because of asymptomatic infections. For virus surveillance, existing wastewater treatment systems and evaluation methods should be improved. Consequently, the sensitivity of tools is very important, so that these can capture even the smallest amount of viral infection during the initial stages to prevent the spread of viral particles. In wastewater pre-treatment, the use of nanofiber filters especially electrospun nanofiber membranes can screen diseasecausing pathogens. Virus detection methods such as enzyme-linked immunoassay (ELISA), RT–PCR, multiplex PCR, complementary deoxyribonucleic acid (cDNA) microarrays, isothermal nucleic acid amplification-based methods, or the newly discovered paperbased device for coronaviruses could be used in these wastewater treatment facilities. Metagenomic approaches could also be helpful to study the entire microbiota of wastewater towards management strategies. Owing to the physical stability of coronaviruses in the environment, the absence of protective immunity in humans, infection control against SARS-CoV-2 remains the primary means to prevent person-to-person transmission. Further, there is a possibility of the re-emergence of SARS-CoV-2 and other novel viruses; therefore, there is a need for preparedness for the next pandemic.

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Cardiac Arrest with Multiple Coronary Occlusions….!!!

We report the case of 59 years-old man with history of arterial hypertension, heavy smoking and obesity. The patient has presented effort angina two weeks before he had a cardiac arrest while effort (preceded by severe chest pain). The patient immediately received two defibrillations for ventricular tachycardia and cardiopulmonary resuscitation. Then, the prehospital thrombolysis was administrated. The low flow time was 30 min. The ECG post resuscitation showed a right bundle branch block with premature ventricular complexes. In coronary angiography, double simultaneous culprit occlusion of the proximal left anterior descending (LAD1) coronary artery and the distal left circumflex (LCX 3) associated to chronic total occlusion of the proximal right coronary artery (RCA) were found (Figures 1 & 2). A double angioplasty with stenting of LAD 1 and LCX 3 was performed. Transthoracic echocardiography showed left ventricule ejection fraction of 30% with extensive akinesia of anterior wall. The post resuscitation care was continued including a therapeutic hypothermia (36°) to optimize the neurological prognosis and hemodynamic optimization with inotropic and vaso-pressive agents. The evolution was unfavorable with anoxic encephalopathy and death at 9 days after admission. The simultaneous coronary occlusions resulting in acute myocardial infarction remain rare (1.3-2.5% in PCI vs 25-50% in autopsy). Many factors are thought to be behind the multiple coronary occlusion such inflammatory response, hyper-catecholaminergic secretion, arterial hypotension, hyper-coagulopathy, coronary embolism and coronary artery spasm. An urgent angiography within 2 hours with revascularization of all culprit coronary lesions should be considered in survivors of cardiac arrest, including unresponsive survivors, when there is a high index of suspicion of ongoing infarction such as the presence of chest pain before arrest, a history of established coronary artery disease, and abnormal or uncertain ECG results.

Figure 1: The coronary angiogram in left anterior oblique projection 30°(A) showing chronic total occlusion of the proximal right coronary artery (RCA) and right anterior oblique projection with caudal angulation (B) showing occlusion of the proximal left anterior descending (LAD1) coronary artery and the distal left circumflex (LCX 3).

Figure 2: The coronary angiogram in anteroposterior cranial projection 30° showing successful angioplasty of the double occlusion of LAD 1 (white arrows) and LCX3 (red arrows) with coronary reflow TIMI 3.

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Responses of Chronic Obstructive Pulmonary Disease (COPD) Patients to Healthy Individuals Using Cardio-Pulmonary Exercise Testing (CPET) – A Case-Control Study

Introduction

Chronic obstructive pulmonary disease (COPD) is a progressive inflammatory lung disease, which according to the World Health Organization is the third leading cause of death worldwide [1]. Common symptoms of COPD include dyspnea, cough and sputum production [2]. Multiple physiological mechanisms in COPD patients lead to them often having a reduced exercise tolerance. We aim to compare peak exercise tolerance of patients with moderate COPD against healthy individuals using Cardio-Pulmonary Exercise Testing (CPET). The purpose of our recommendations is to try and improve cardiopulmonary fitness of COPD patients with lifestyle and exercise interventions.

Methods

Study Population

The study consisted of 8 COPD patients (8 male) and 8 healthy individuals (6 male, 2 female) as a control. All COPD patients had to have a Global Initiative for Chronic Obstructive Lung disease (GOLD) grade of 2B or worse. Exclusion criteria included <93% SpO2, other medical conditions or an exacerbation in the past 6 months. The control group had no health conditions.

CPET

Both groups completed the following exercise protocol on a cycle ergometer to exhaustion.

i. 3 minutes resting sat on the cycle.

ii. 3 minutes unloaded warmup at 60 rotations per minute.

iii. Individualised ramp protocol. Ramp protocol to be terminated at exhaustion.

iv. 5-minute rest.

The variables in Table 1 (excluding age, BMI, SpO2), VO2, end tidal CO2 (PETCO2) and VE/VCO2 were measured during exercise with recordings at Anaerobic Threshold (AT) and Peak exercise. AT was measured at the point VCO2 starts increasing at a greater rate than VO2. Peak exercise was determined when the work-rate curve was at its highest. Maximal effort was regarded as reached when either Respiratory exchange ratio (RER) > 1.15 or maximal HR > 85% predicted (220 – age).

Statistical Analysis

Variable’s means and standard deviations (SD) were calculated. Normality of data distribution was assessed using the Shapiro- Wilk test. The Independent T-test or Kruskal-Wallis test was used to compare the two groups. SPSS software was used with P < 0.05 considered significant (Figures 1 & 2).

Figure 1: Comparing mean VO2/kg between COPD (blue) and Healthy (green) group at different stages.

Figure 2: Comparing PETCO2 between COPD and Healthy group at different stages.

Results

Patient Characteristics

No statistical difference in BMI between groups but a large difference in age between the populations.

Maximal Effort

Using the study’s criteria none of the COPD group achieved maximal effort compared to five of the healthy group. Peak RER values for the COPD group were significantly lower than the Healthy group. No COPD participants had an RER over 1.15 compared to five in the healthy group. Neither group reached a HR value equal to 220-age. Peak WR was significantly lower in COPD compared to the Healthy group as seen in Table 1.

Ventilatory: Significant difference with lower peak minute ventilation (VE), peak tidal volume (VT) and peak respiratory rate (RR) values in the COPD group compared to the Healthy group as seen in Table 1.

Table 1: CPET Results Comparing COPD to Healthy Groups.

Cardiovascular

Table 1 shows peak HR was significantly lower in the COPD group compared to the Healthy group. Mean SpO2 at rest (95.16 ± 1.92 vs97.9 ± 0.64 P = 0.015) was significantly lower in COPD groups compared to Healthy population. Rest VO2/kg values between groups were not significantly different (3.74 ± 0.53 vs 5.36 ± 2.11 P = 0.053). However, AT (10.31 ± 1.78 vs 19.75 ± 7.67 P = 0.004) and peak (12.27 ± 3.05 vs 42.38 ± 15.25 P = <0.001) values are significantly different.

Gas Exchange

Peak VE/VCO2 is significantly higher in the COPD population compared to the Healthy group as seen in Table 1. PETCO2 mean values for COPD vs Healthy at rest (30.68 ± 5.64 vs 35.32 ± 1.93 P = 0.045) and peak (112.03 ± 4.96 vs 110.95 ± 6.46 P = 0.714) were not statistically different. Whereas AT values (108.030 ± 6.59 vs 96.76 ± 4.95 P = 0.001) were statistically different.

Discussion

The inability of the COPD group to reach maximal effort is likely due to ventilatory limitations making it difficult to facilitate high gas exchange. This is also one of the mechanisms causing exertional dyspnea [3]. The lower RER and HR values in COPD patients suggest a high level of exercise intensity was not achieved [3]. Another reason it can be difficult for COPD patients to reach higher exercise intensities is that the disease causes hypoxic conditions in cells inducing reactive oxygen species production. This results in upregulation of inflammatory mediators responsible for muscle degeneration. The reduced skeletal muscle mass makes intensiveexercise difficult to maintain [4].COPD patients have an increased risk of coronary artery disease; a reduced left ventricular end-diastolic volume, stroke volume and cardiac output. Additionally, inflammation and damage to the pulmonary vasculature as well as reduced muscle mass all contribute to a low VO2 [4]. Increased amounts of CO2 are produced at higher exercise intensities due to increased anaerobic metabolism and buffering.

Ventilatory limitations seen with reduced Peak VT, VE and RR suggest reduced respiratory flow in COPD. This along with increased dead space and small airway inflammation make it harder to expel CO2 from the system [4]. The limit in respiratory mechanics results in difficulty performing exercise at higher intensities For longer. VE/ VCO2 is a measure of the ventilatory need to expel a certain amount of CO2 which has been produced due to activity. Higher peak VE/ VCO2 suggests ventilatory inefficiency, caused by lung damage in the form of bronchitis and emphysema likely to be present in more severe COPD [4]. Exercise induced secondary hypoventilation which occurs in COPD patients often results in a raised VE/VCO2. This reflects the presence of an elevated dead space volume/tidal volume ratio [4]. COPD patients need a higher minute ventilation to expel the same amount of CO2 as the healthy population. The VE/ VCO2 slope from a patient’s CPET is an indication of the severity of their COPD [3].

Interestingly the PETCO2 for the Healthy group increases after AT, following the COPD trend rather than falling which we would expect. Possibly due to random error or lower exercise intensity. The increase in PETCO2 after AT highlights ventilatory-perfusion (V/Q) limitations as you would expect PETCO2 to drop after the respiratory compensation point and VE increases in a healthy population [3]. The V/Q mismatch can also lead to hypoxia which can be partially seen with the lower resting SpO2 [5]. A limitation of this study is the differing ages and sexes of the two groups.

Conclusion

Several physiological mechanisms prevent COPD patients from exerting themselves during exercise as can be seen from CPET results. Recommendations to improve quality of life for COPD sufferers include smoking cessation, pharmacological therapy, pulmonary rehabilitation, education and nutritional support.

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Methods for Determining the Energy Function of Mitochondria

Introduction

Energy exchange in the cell is associated with mitochondria, which play an important role in vital processes, participating not only in the formation of ATP but also in the storage and transmission of hereditary information, apoptosis and plastic processes [1,2]. Mitochondria are very mobile and plastic organelles that constantly change their shape, merge, and then separate again. The movement of mitochondria in the cytoplasm is associated with microtubules, which determines their orientation and distribution in the cell. In some cells, mitochondria form long mobile filaments or chains, while in others, they are fixed near the places of consumption of ATP [3,4]. Each mitochondrion contains highly specialized membranes that play a key rolein its activity. The membranes form two isolated mitochondrial compartments: the inner matrix and the narrow intermembrane space. Each section contains a unique set of proteins [5,2]. The outer membrane contains the protein porin, which forms wide hydrophilic channels in the lipid bilayer, resulting in a membrane-like sieve, permeable to all molecules weighing less than 10,000 daltons.

These molecules can penetrate the intermembrane space, but most of them are unable to pass through the impermeable inner membrane [1,6]. The main functional part of mitochondria is the matrix and the surrounding inner membrane. The matrix of mitochondria has a more viscous consistency than the cytoplasm of the cell. It contains enzymes, mitochondrial DNA, ribosomes, organic compounds, ions, including calcium and magnesium. Matrix enzymes are involved in the Krebs cycle, oxidative phosphorylation, pyruvate oxidation, and beta-oxidation of fatty acids [2]. The inner membrane forms a complex system of folds in the mitochondrial matrix – cristae, which significantly increase its area. For mitochondrial cristae in cells of various organs, morphological features and different enzymatic compositions are characteristic [7]. The most characteristic feature of organelles that the presence of enzyme complexes involved in oxidative phosphorylation and energy supply to the cell.

Most of the enzyme proteins are components of the electron transport chain that maintains a proton gradient across the membrane. Another large protein complex is the enzyme ATP synthase, which catalyzes the synthesis of ATP [8]. In mitochondria, oxidative metabolism takes place, the substrate for which is mainly fatty acids and pyruvate, formed as a result of glycolysis in the cytosol. These substances are transported from the cytosol to the mitochondrial matrix, where they break down into twocarbon groups, combined with acetyl coenzyme A (acetyl-CoA). In the composition of the acetyl-CoA molecule, each acetyl group is included in the Krebs cycle as a source of high-energy electrons. Electrons are transferred to the respiratory chain of the inner mitochondrial membrane, where energy is generated as a result of their transfer (Figure 1).

Figure 1: Complexes of the mitochondrial respiratory chain.
Note: complex I (NADH-ubiquinone oxidoreductase; NADH-dehydrogenase), complex II (succinate dehydrogenase; succinate-ubiquinone reductase), complex III (cytochrome bc1 complex; ubiquinone-cytochrome c oxidoreductase, complex IV (cytochrome c oxidase, complex V (mitochondrial ATP synthase) [9]

Disorders of energy metabolism in the cell are one of the key links in many diseases. This makes it necessary to study the work of the electron transport chain of mitochondria, both of its complexes and the entire chain as a whole [9]. The purpose of this review is to analyze and systematize literature data on methodological approaches to the study of the energy function of mitochondria.The Oxidative Phosphorylation System (OxРhoS), localized in the inner mitochondrial membrane, consists of five membrane enzymes. Four of the five protein complexes make up the “respiratory chain” and are involved in the transfer of electrons, which at three points is coupled with the translocation of protons across the inner mitochondrial membrane. The resulting proton gradient is used by the ATP synthase complex (the fifth enzyme complex) to phosphorylate ADP [10,11,1]. For a long time, a fluid-state model has been used to describe the organization of the OxPhoS system. According to this model, the complexes of the respiratory chain freely diffuse in the membrane, and the transfer of electrons occurs as a result of random chaotic collisions.

This model is based on the fact that all protein complexes of the OxPhoS system can be isolated while maintaining enzymatic activity [8,9]. In the last decade, there is more and more evidence indicating stable interactions of OxPhoS complexes in the form of supercomplexes. It is assumed that the OxPhoS supercomplexes and their single complexes coexist in the inner mitochondrial membrane. The association of complexes into super-complexes and the dissociation of super-complexes into OxPhoS complexes is a dynamic process that dependson the physiological state of the cell. Recent studies of mitochondria show that ATP synthase in mitochondrial membranes is organized into long strips of dimers and mitochondrial cristae act as proton traps, and ATP synthase can optimize its activity when there is a lack of protons [12,13]. Complex I, or NADH dehydrogenase, is the main entry point for electrons into the respiratory chain. Complex I oxidizes NAD-H, taking two electrons and reducing one ubiquinone Q molecule, which is released in the membrane. Ubiquinone Q is lipid-soluble; inside the membrane, it diffuses to complex III.

Complex I plays a central role in cellular respiration and oxidative phosphorylation, providing up to 40% of the proton gradient for ATP synthesis. During the oxidation of one NADH molecule, the NADH-dehydrogenase complex transfers four H+ protons from the matrix to the intermembrane space of the mitochondria through the membrane. The formation of reduced NADH is associated with the conversion of malate to oxaloacetic acid and glutamate to α-ketoglutarate. The transfer of electrons through the I complex is associated with the release of 3 ATP molecules [14,1]. Complex II, or succinate dehydrogenase, is another entry point for electrons into the respiratory chain, it is not associated with the translocation of protons across the membrane, transfers electrons from succinate to ubiquinone, and directly binds the Krebs cycle with the respiratory chain. In this case, succinate is oxidized to fumarate with subsequent reduction of ubiquinone Q.

Electrons from succinate are first transferred to flavinadenine dinucleotide, and then through Fe-S clusters to ubiquinone Q. Electronic transport of the II complex is not accompanied by the creation of a proton gradient. The H + protons formed during the oxidation of succinate remain in the matrix and then are reabsorbed during the reduction of the quinone. Complex II works as a carrier of electrons with the formation of 2 ATP molecules [15,1].Respiration of mitochondria and the work of complexes I and II are assessed by registering the rate of oxygen consumption in a polarographic cell using a built-in Clarke electrode (volume 3 ml, at a temperature of 25 ° C (Figure 2).To study the operation of complexes I and II, the rat brain is removed in the cold (0-4 ° C), dried with filter paper, and homogenized in an isolation medium (0.32 M sucrose, 10 mM Tris-HCl, 1 mM ethylenediaminetetraacetic acid, pH 7.4 in the ratio 1:10) using a Potter-Evelheim homogenizer with a Teflon pestle according to a modified method [16,17].

Figure 2: Polarographic cell for studying the respiratory activity of mitochondria
1. Cell
2. Thermostatically controlled chamber
3. Clarke electrode
4. Magnetic stirrer
5. Sealing plug
6. Channels for dosed anaerobic administration of substrates and ADP
7. A sealing ring
8. Channel for removing air and excess liquid
9. Fitting for connection to an ultrathermostat.

Mitochondria are isolated by differential centrifugation. The nuclear fraction is separated by centrifugation of the brain homogenate at 600 g for 10 min (4 ° C). The resulting supernatant is centrifuged at 8500 g for 10 min (4 ° C), the mitochondrial pellet is washed twice in the isolation medium and resuspended to a protein concentration of 35-40 mg/ml in the isolation medium and stored in a short tube on ice. Protein concentration is determined by the Lowry method [16]. A concentrated suspension of mitochondria is introduced into a thermostatted sealed polarographic cell with an incubation medium (0.17 M sucrose, 40 mM KCl, 10 mM Tris-HCl, 5 mM KH2PO4, 8 mM KHCO3, 0.1 mM ethylenediaminetetraacetic acid, pH 7.4) in an amount providing a final protein concentration in the cell of 1 mg/ml. Registration of changes in oxygen tension (pO2) in the mitochondrial suspension is carried out using an electronic recorder. The Clarke electrode is calibrated by sequentially blowing ai) and gaseous nitrogen through the cell.
Basal respiration is assessed, as well as respiration stimulated by the introduction of substrates: malate/glutamate to assess the work of complex I and succinate to assess the work of complex II. The following indicators of mitochondrial respiration are recorded: V1 – basal respiration rate, V2 – substrate-dependent respiration rate, V3 – respiration rate associated with phosphorylation (after ADP introduction), V4 – respiration rate after completion of added ADP phosphorylation. Indicators characterizing the conjugation of oxidation and phosphorylation processes in mitochondria are determined: the acceptor control coefficient (V3 / V2), the respiratory control coefficient (V3 / V4), and the phosphorylation coefficient – ADP / O. After recording the rate of basal (endogenous) respiration in the absence of a substrate (V1), respiration substrates (malate – 2 mM/glutamate – 5 mM or succinate – 5 mM) are alternately introduced into the mitochondrial suspension, and then ADP in an amount of 200 nmol/ml.
The obtained polarograms are used to calculate the respiration rate of mitochondria in different metabolic states and the coefficients characterizing the conjugation of oxidation and phosphorylation processes. The use of solutions of substrates of succinate and malate/glutamate complex makes it possible to assess the degree of functional activity of complexes I and II of the electron transport chain [16]. The central component of the OxPhoS system is cytochrome c-reductase, or complex III, which functions as a dimer. It transports electrons from reduced ubiquinone (ubiquinol) to cytochrome c, a small mobile electron carrier bound to the outer surface of the inner membrane. This multiprotein transmembrane complex is encoded by the mitochondrial (cytochrome b) and nuclear genomes [18]. Electronic transport in complex III is associated with the transfer of protons from the matrix to the intermembrane space and the generation of a proton gradient on the mitochondrial membrane. Cytochrome c is a component of the electron transport chain, the function of which is to transfer electrons between complex III (ubiquinonecytochrome c-reductase or cytochrome bc complex) and complex IV (cytochrome c-oxidase).
For every two electrons passing along the chain of transfer from ubiquinone to cytochrome c, two photons are absorbed from the matrix, and four more protons are released into the intermembrane space. The reduced cytochrome c moves along the membrane in an aqueous medium and transfers one electron to the next respiratory complex, cytochrome oxidase [19,9]. It is the only peripheral protein that interacts with the outside of the inner mitochondrial membrane.Cytochrome c is a water-soluble protein of low molecular weight (about 12,000 Da), the primary structure of which contains about 100 amino acids. Cytochrome c can catalyze hydroxylation and aromatic oxidation reactions and has peroxidase activity by oxidizing various electron donors [20]. Cytochrome c is a metalloprotein that functions in electron transfer reactions and contains heme c (or several hemes) as a prosthetic group, covalently bound to a protein molecule through one or two thioether bonds between the cofactor and the sulfhydryl group of cysteine squirrel. The ligand in the 5th coordination position of the iron ion is always histidine.
Cytochrome c is localized in the intermembrane space. All cytochromes c can be divided into four classes [10].The first class is the low-spin form of soluble cytochrome c of mitochondria and bacteria, in which histidine bound to the heme is located in the N-terminal part of the protein molecule, and the ligand in the 6th coordination position – methionine is located 40 residues towards The C-terminus of the molecule [18].The second class is the high-spin form of cytochrome c and several low-spin forms (for example, cytochrome c 556), which have heme-binding sites in the C-terminal region of the protein molecule. The protein contains four helical regions of the polypeptide chain [19]. The third class includes cytochromes containing several hemes and having a low redox potential (cytochrome c7 (three hemes), c3 (four hemes), and high molecular weight cytochrome c (hexadecahaem)), in which there are 30-40 residues per heme molecule. Hemes, coordinated by two histidines, are structurally and functionally nonequivalent and are characterized by different redox potentials from 0 to 400 mV [19,18].
The fourth class is necessary to maintain complex proteins with heme and other prosthetic groups, for example, flavocytochrome c, cytochromes cd. Cytochromes of this class are proteins containing four hemes, which in the 5th and 6th coordination positions have either two histidines or histidine and methionine. Mitochondrial cytochrome c is one of the three redox subunits of the third complex of the respiratory chain (cytochrome bc complex) [11,19]. Mitochondrial cytochrome c is anchored in the membrane by one membrane segment near the C-terminus. A water-soluble cytochrome C1 preparation can be obtained by removing the hydrophobic C-terminal region. Water-soluble cytochrome c1 cannot participate in the assembly of the bc complex [11,18]. Cytochrome c, in addition to the function of a carrier of electrons in the respiratory chain, can separate from the inner mitochondrial membrane, be transported into the cytoplasm of the cell, and trigger a chain of events in the cytosol, which accelerates apoptosis [11].
The ability of cytochrome c to exhibit various functions inside mitochondria and in the cytosol is associated with the cellular localization of the hemoprotein. Cytochromes are an important class of metal proteins involved in electron transfer and redox catalysis. Redox enzymes and metal proteins play an important role in signaling processes, are responsible for the regulation of genes and their expression, provide the conversion of energy in the processes of respiration and photosynthesis. Thus, cytochrome c in the electron transport chain of mitochondria acts as an electron acceptor for complex III (cytochrome c reductase) and an electron donor for complex IV (cytochrome oxidase) [20,21]. Determination of the activity of complex III The activity of complex III is determined spectrophotometrically at a wavelength of 550 nm by antimycin A-sensitive reduction of ferri- and ferrocytochrome with decylubiquinone in the presence of Tween 20, albumin, and sodium azide.To measure the activity of complex III, the mitochondrial suspension is diluted to a concentration of 0.05 mg/ml of protein in a medium containing 35 mM KH2PO4, 2 mM NaCN, 0.5 mM ethylenediaminetetraacetic acid, pH 7.25. The samples are then sonicated in a water bath for 30 s.
The activity of the complex is determined by the rate of antimycin-sensitive reduction of cytochrome c (550 nm, molar extinction coefficient 18,500 M4 cm4) with the addition of 60 μM decilubiquinone, 50 μM cytochrome c, and 5 mM MgCl2 to a suspension of mitochondria exposed to ultrasound (the content of mitochondrial protein in the sample is 0.01 mg/ ml). Spectrophotometric determination of the concentration of cytochromes c and b in aqueous solutions. The molar extinction coefficients for cytochrome c A vost-oxide = 18,500 M4 cm-1 at 550 nm. Recovery is carried out with ascorbate or dithionite. In the presence of cytochrome b, the content of cytochrome c was determined from the differential spectra of the forms reduced by ascorbate, minus the forms oxidized by ferricyanide. Cytochrome b is determined from the differential spectra of the forms reduced by dithionite minus the spectra of the forms reduced by ascorbate. The activity of enzyme systems seems to be the most important parameter characterizing the work of the electron transport chain of mitochondria and the bioenergetic status of the cell.
Extraction of cytochrome c from the mitochondrial membrane and the reconstruction of the respiratory chain makes it possible to assess the transfer of electrons from complex III to IV. The principle of the method for extracting cytochrome c from the mitochondrial membrane consists of the destruction of the outer membrane using detergents or hypotonic processing and extraction of proteins with saline solutions. Reagents for isolation of mitochondria include: 0.25 M sucrose solution, 0.15 and 0.015 M KS solutions, 5 mM potassium succinate solution, pH 7.4, 5 mM potassium glutamate solution, pH 7.4, 0.005 M solution 2.4 dinitrophenol, pH 7.4, incubation medium (0.15 M sucrose, 0.075 M KC1, 0.01 M potassium phosphate, pH 7.4), cytochrome c solution – 1 mg / ml and reagents for protein determination. All solutions are prepared with bidistilled water. Isolated mitochondria are suspended in 3 ml of 0.25 M sucrose solution. Three conical flasks are filled with 40 ml of solutions:
1) 0.25 M sucrose;
2) 0.15 M KC1
3) 0.015 M KC1. 1 ml of a thick suspension of mitochondria is added to each flask. The contents of all flasks are gently mixed for 10 minutes at 0 ° C and then transferred to three 50 ml centrifuge beakers.
The mitochondria are separated by centrifugation at 10,000 g, suspended in 0.5 ml of 0.15 M KC1, and transferred into three flasks containing 40 ml of 0.15 M KC1. The contents of the flasks are again stirred in the cold for 10 minutes and centrifuged again under the same conditions to separate the mitochondria. The resulting sediments of mitochondria are resuspended in 0.3-0.5 ml of 0.3 M sucrose solution. The rates of oxygen consumption are determined polarographically, using succinate and glutamate (5 mM) as substrates, and after 1-2 minutes dinitrophenol (50-100 μM) is added. For a preparation washed with a hypotonic solution of KCl, the dependence of the rate of succinate oxidation on the amount of cytochrome c added to the incubation medium (concentration from 0 to 100 μg / 2 ml, 5-6 points) is determined [15,4 ,11]. Complex IV, or cytochrome c oxidase, is the final complex of the respiratory chain. Cytochrome c oxidase catalyzes the transfer of electrons from cytochrome c to molecular oxygen, reducing the latter to water.
Complex IV is the terminal oxidase of the aerobic respiratory electron transport chain, which catalyzes the transfer of electrons from cytochrome c to oxygen to form water. Complex IV sequentially oxidizes four cytochrome c molecules and, accepting four electrons, reduces O2 to H2O. During O2 reduction, four H+ are captured from the mitochondrial matrix to form two H2O molecules, and four more H+ are actively pumped across the membrane. Thus, cytochrome oxidase contributes to the creation of a proton gradient for ATP synthesis and is part of the oxidative phosphorylation pathway [11,4]. Complex IV activity Cytochrome oxidase activity is assessed by the polarographic method according to the rate of oxygen consumption by mitochondria. It should be noted that all procedures for the isolation, operation, and storage of mitochondria must be carried out observing the temperature regime: the samples must be stored in ice, the media and the instrument must be pre-cooled to a temperature of 0-4 ° C. A suspension of intact mitochondria or mitochondria destroyed by detergent is introduced into a polarograph cell containing a medium of the following composition: 0.125 M sucrose, 60 mM KCl, 10 mM Tris-HCl, 0.1 mM 2,4-dinitrophenol, 40 μM cytochrome c.
The content of mitochondrial protein in a polarographic cell is 0.5-1.0 mg/ml. The cytochrome oxidase reaction is started by introducing a solution of ascorbic acid neutralized to pH 7.4, creating a concentration of 10 mM in the sample. Measure the rate of oxygen uptake and calculate the activity of cytochrome oxidase in nanomoles of absorbed 02 for 1 min per 1 mg of protein [17]. ATP synthase, complex V, uses the resulting proton gradient to synthesize ATP. ATP synthase is an integral protein of the inner mitochondrial membrane that carries out the reaction of ATP formation from ADP [20]. Mitochondrial ATP synthase plays an important role in stem cell differentiation, promotes the maturation of mitochondrial cristae by dimerization and specific regulation. The enzyme belongs to the alpha/beta ATP synthase family. It consists of two structural domains (F1 – extramembrane catalyst and F0 – membrane proton channel), connected by a central rod, consisting of γ, δ, and ε subunits, and together with the membrane subunit oligomer representing the rotary domain of the enzyme [15].
Determination of the content of ATP synthase (V complex) is carried out by the immunohistochemical method using monoclonal antibodies. For this purpose, after decapitation and extraction of the brain, the material is fixed in zinc-ethanol-formaldehyde at + 4 ° C (overnight), then embedded in paraffin [22]. Paraffin sections with a thickness of 5 μm are prepared using a microtome, mounted on glass slides. The preparations are processed according to the protocol of immunocytochemical reaction for light microscopy, excluding the procedure of thermal unmasking of antigens. To determine the immunoreactivity of the molecular marker of mitochondria ATP synthase (complex V, which forms ATP from ADP), primary monoclonal antibodies (Anti-ATP5A antibody) are used at a dilution of 1: 2400 at + 4 ° C, with an exposure of 20 h in a humid chamber [22]. Thus, the above methods for studying the activity of the electron transport chain of mitochondria, especially their use in combination, can significantly detail the understanding of the pathogenesis of disorders of cell energy exchange that occurs in various diseases, which will improve the prevention and correction of mitochondrial dysfunction.

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