Why Cancer Occurs

Introduction

Written by Theresia Indah Budhy, cancer is a term for malignant tumors, until now the incidence of cancer is still very high and the problem has not been solved properly. This is because the occurrence of a cancer is very complex and the factors involved in it are also heterogeneous so this case is still quite high. Some factors related to cancer can originate from humans itself (host) endogenous or factors from outside/exogenous. Endogenous factors can be in the form of regulatory genes/cell regulators, hormones, immune and psychic systems, also hereditary. While exogenous factors are more complex because there are so many, among others biological factors can be bacteria, viruses, fungi; chemical factors can come from food, drinks, cosmetics, cleaning materials, air, environmental sanitation; whereas physical factors such as electronics, radiation; although radiation is also divided into several types, for example solar radiation and X-rays. After reading this book hopefully adds more insight about cancer. Remembering the incident of a cancer is very complex, the discussion of the cancer problem still needs to be added.

In this book only briefly discuss things related to cancer, starting from the definition of cancer, the classification of the names of each cancer type, the different clinical manifestations of each type of cancer and some of the factors that cause its occurrence as well as the pathobiology of cancer. In giving a name to cancer based on the cell of origin or cancer parenchyma for example, in cancer that occurs in epithelial cells, the word Carcinoma will be added to its name, if it originates from mesenchymal cells, it will be added Sarcoma. The description of the clinical manifestations of a cancer varies from both types of carcinoma and sarcoma. In the type of cancer, carcinoma usually presents as an ulcer or a nodule exophytic resembles a cauliflower, while the sarcoma type has a shape like a lump or liquid when it occurs in the blood. The occurrence of a cancer when reviewed from the causative factor sometimes also gives a different form and location, as an example of a factor the cause of the sun’s rays are many attacks in the epidermal or skin area, the type that occurs because the most exposure to sunlight is Basal cell carcinoma.

While other cases are caused by a virus usually gives a picture like the condition of a network infected by a virus, in the form of a nodule and ulcer and its various types can be a carcinoma or sarcoma. Examples caused by viral infections are nasopharyngeal carcinoma, lymphoma maligna where the main causative factor is Epstein Barr virus. In other types caused by viruses such as Kaposi’s sarcoma, where many are associated with HIV virus infection in AIDS patients. When the occurrence of a cancer is reviewed from its pathobiology then many are involved in it, especially regulatory genes on cell growth. There is a concept that the pathobiology of cancer is influenced by four main things, namely the proliferation regulator gene (oncogene) as an example that has been mentioned in the previous chapter is race; c-myc; apoptosis regulator genes (gene suppressors) such as the p53 gene; genes regulating the repair of damaged cells and the immune system. Currently other related concepts are also developing with cancer progression, namely angiogenesis, because factors in the formation of new blood vessels have a very important role in the ability to survive and the development and spread of a cancer.

As it is known that blood vessels can provide nutrition and oxygenation in the cells, so that the cancer cells become stronger and live in addition to the blood flow throughout the body allowing cancer cells to move to a place further away from the primary cancer. Considering that a lot of research has been done related to angiogenesis in cancer. To determine the stage of cancer can also be performed through the clinical stage with the concept of T (tumor cross-section), N (nodule, lymphnode involved) and M (metastasis). Besides based on that, the most important thing in determining the diagnosis is histopathological examination or examination with microscope. Through histopathological examination is the Gold Standard for the diagnosis of a cancer, it can be known the type of cancer and the stage of cancer cells differentiation, so that it can correctly determine the therapy plan and know the prognosis (estimated cure) of a cancer. Histopathology inspection becomes very important for the final diagnosis of a cancer because from the results of the examination many things will be obtained such as the type of cancer cells, the shape of cancer cells, even the genetic markers of a cancer.

In conclusion after reading this book hopefully adds more insight about cancer. Remembering the incident of a cancer is very complex, the discussion of the cancer problem still needs to be added. In this book only briefly discuss things related to cancer, starting from the definition of cancer, the classification of the names of each cancer type, the different clinical manifestations of each type of cancer and some of the factors that cause its occurrence as well as the pathobiology of cancer [1].

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Long-Term Cryopreservation of an Autologous Immunocomplex of Dendritic Cells and Cancer Antigen (HiDCV-OS1) that Stimulates Antitumor Immunity Against Chemotherapy-Resistant Ovarian Cancer

Introduction

In recent years, therapies that use cellular products made from autologous tissues have been developed [1]. In Japan, regenerative medicine is practiced in accordance with two regulations [2]. Ovarian cancer (OC) has a longer time to recurrence than other cancers, which has been extended in recent years with the arrival of novel drugs such as PARP inhibitors [3]. However, when OC relapses, it undergoes repeated remissions and progressions even after treatment is restarted, and eventually resistance develops; therefore, new and improved treatments are needed. Therefore, we established a novel therapeutic strategy using an autologous immunocomplex of dendritic cells (DCs) and cancer antigen (HiDCV-OS1). At the time of the initial surgery, the patient’s tumor cells and DCs were fused with HVJ-E for high efficiency and without antigen destruction [4,5], and the immunocomplex was cryopreserved. HiDCV-OS1 was thawed and used when OC relapsed. HiDCV-OS1 was used in patients suffering from chemotherapy- resistant OC (jRCTc051190054) in Japan. This is the first report on the long-term cryopreservation of vaccine dendritic cells for cancer treatment.

Materials and Methods

Production of HiDCV-OS1 using Patient-Derived OC and Mononuclear Cells

Pre-treatment of Tumor Tissue: The OC was cut into small pieces (14.6 g ± 2.5 g) and dissociated completely using a gentleMACS octo Dissociator with Heaters (Miltenyi Biotec, K.K., Tokyo, Japan). A cloudy cell layer was obtained using the Ficoll–Paque centrifugation method. OC was isolated using Dynabeads CD45 (Thermo Fischer Scientific K. K., Tokyo, Japan). 1.2 x 10⁶ cells of OC with STEM-CELLBANKER GMP grade (Nippon Zenyaku Kogyo Co.,Ltd. Fukushima, Japan) was added to 1.8 mL cell cryopreservation tubes and frozen at 80°C. The cells were transferred to -150.0°C ± 15.0°C for storage within 3 days. The cells were thawed and irradiated with 50-Gy radiation (TX-2500, Nanogray, inc. Osaka, Japan).

Mononuclear Cell Isolation and DC Maturation: Apheresis (COBE-Spectra, Terumo BCT, Tokyo, Japan) was performed to obtain mononuclear cell components 1.3 ± 0.14 x 10⁹ cells) and incubated for approximately 7 days. Immature DCs were then collected.

Promotion of DC Maturation: AIM-V medium (Thermo Fischer Scientific K. K., Tokyo, Japan) and 25 mNAU/mL of HVJ-E were added to DC and incubated for 24 h for DC maturation.

Cryopreservation of DC: DC was suspended in STEM-CELLBANKER GMP grade at a rate of 2 million cells per 1.2 mL, placed in 1.8 mL cell cryopreservation tubes, and frozen at 80 °C. The cells were transferred to -150.0°C ± 15.0°C for storage within 3 days.

Preparation of HiDCV-OS1: Mixed at a ratio of 1 x 106 mature dendritic cells to 5 x 105 OC cells with 250 mNAU of HVJ-E and then shaken with a shaker (75 rpm) at 4°C for 10 min. The cells were then shaken with a shaker (75 rpm) for 20 min at 37°C.

Cryopreservation of HiDCV-OS1: One million cells/mL of HiDCV- OS1 STEM-CELLBANKER GMP grade was added to 1.8 mL cell cryopreservation tubes and frozen at 80°C. The cells were transferred to -150.0°C ± 15.0°C for storage.

Assessment of the HiDCV-OS1 Quality

Viability: Cells stored for variable periods (3, 12, 24, 96 months) were tested for viability using the trypan blue exclusion test.

Fusion rate of HiDCV-OS1: The percentage of HiDCV-OS1 cells was calculated by detecting CD11c- and CD326- or CD90-positive cells using flow cytometric analysis (Table 1).

Table 1: List and addition volume of the antibodies.

Note: APC, allophycocyanin; CD, cluster of differentiation; Cy, carboxylic acid; EpCAM, epithelial-specific cell adhesion molecule; IgG, immunoglobulin G; PE, phycoerythrin; Thy1, thymus cell antigen 1.

Results

The viability and fusion rate of cryopreservation start date and results up to 4 years after storage are presented in Table 2, and the HiDCV-OS1 subpopulation is shown in Table 3.

Table 2: Viability and fusion rate of HiDCV-OS1 after long-term storage.

Table 3: Subpopulation (%) of HiDCV-OS1.

Note: EpCAM, epithelial-specific cell adhesion molecule; Thy1, thym.

Discussion

We showed the viability and cell fusion rates of HiDCV-OS1 after four years of cryopreservation. The specification criteria were the quality of HiDCV-OS1 after cryopreservation based on previous reports on antitumor efficacy [6]. In fact, all HiDCV-OS1 stocks met these criteria. To produce HiDCV-OS1, we used HVJ-E, which has high fusion efficiency between DCs and cancer cells, high antigen-presenting ability, and high antitumor effect [6]. While OC has a prolonged time between initial treatment remission and relapse [3], there is a lack of treatment options when the disease recurrence. Therefore, the use of HiDCV-OS1 after recurrence is expected to have an antitumor effect as a novel therapeutic tool. It is thought to be difficult to obtain sufficient tumor volume to produce the necessary amount of HiDCV- OS1 at that time, and the general condition of relapsed patients is often poor, and sufficient monocytes cannot be collected to differentiate into dendritic cells. There is a concern that the properties of tumor cells may change during recurrence and HiDCV-OS1 production. However, tumor cells that have acquired resistance to chemotherapy probably derived from cancer stem cells and express Thy-1. Because HiDCV-OS1 contains cancer antigens derived from Thy-1-positive cells, it can be expected to generate antitumor immunity even after recurrence [7,8]. Therefore, it is reasonable to prepare and store cell preparations for future administration in case of recurrence when the patient is well. HiDCV-OS1, which was stored for approximately 4 years, was used in patients with recurrent OC. There were no serious adverse events, and some efficacy was observed [9,10]. The relapsed patient was alive 387 days after the administration of HiDCV-OS1.

Conclusion

HiDCV-OS1, consisting of autologous tumor tissue and dendritic cells with HVJ-E, was cryopreserved for four years, after which the thawed cells maintained their fusion rate and showed a certain anti- tumor effect, suggesting that they may have antigen-presenting potential.

Acknowledgement

We are grateful to the Centre for Translational Research, Osaka University Hospital, for their kind support in the production of HiDCV- OS1 and the conduct of the clinical trial.

Authorship Contribution

Conceptualization, Y. K. and K. S.; data curation, M. S., T. N. and K. S.; investigation, T. N., CY. C., J. F., H. H., K. O., K. S. and T. K.; writing-original draft preparation, M. S.; writing-review and editing, K. S.; project administration, T. N., K. S. and T. K.; supervision, Y. K.

Conflict of Interests

Conflicts of interest related to this study have been reviewed and appropriately managed by the Institutional Conflicts of Interest Committee.

Funding

This research was supported by AMED under Grant Number JP21bk0104104, a joint research grant from Ishihara Sangyo Kaisha, LTD., and a Grant-in-Aid for Exploratory Research (23659671).

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Selective Peroxisome Proliferator-Activated Receptor-α Modulator K-877 Stimulated the Expression of ABCA1 in Pancreatic Beta Cells in a Glucotoxic State

Introduction

Peroxisome proliferator activated receptors (PPARs) are proteins of the so-called nuclear receptor superfamily that regulate gene transcription in response to ligands. Three subtypes of PPARs have been identified: PPARα, PPARγ, and PPARβ/δ. Among these, PPARα is activated by fibrates, and its expression is found mostly in the liver, pancreas [1], brown adipose tissue, heart, and kidney [2]. Free fatty acids and other physiological ligands activate the PPARα pathway. This activation of PPARα decreases blood triglyceride levels and increases High-Density Lipoprotein (HDL) levels. Exogenous ligands include fibrate drugs, such as bezafibrate and clofibrate, and most target genes are related to lipid metabolism and are primary targets of hypertriglyceridemia-modifying drugs. Selective PPAR modulator alpha (SPPARM alpha) has been newly developed and is a next-generation lipid-lowering agent that selectively regulates PPARα-mediated gene transcription. K-877, which is SPPARM alpha, increases the benefits and reduces the risks of conventional PPARα agonists [3]. Moreover, K-877 has a superior PPARα activation activity, higher degree of subtype selectivity (by > 1000-fold), and lower EC50 value compared with other fibrates [4]. ATP-binding cassette protein A1 (ABCA1) is an important membrane protein that is very necessary for reverse cholesterol transport [5]. ABCA1 is expressed on the plasma membrane and regulates the steps in the efflux of intracellular cholesterol and phospholipids to HDL.

Recently, Brunham, et al. [6] reported on insulin secretion and sensitivity in mice lacking ABCA1 in pancreatic beta cells. They reported that the pancreatic β cell specific ABCA1 knockout mice had normal insulin sensitivity but caused reduced insulin secretion and impaired glucose tolerance. These results suggest that cholesterol accumulation in pancreatic β cells may cause β cell dysfunction. This is known as “pancreatic lipotoxicity”. We have previously reported the effects of K-877 on the expression of pancreatic β-cell ABCA1 [7]. Our results suggest that K-877 may regulate ABCA1 expression and affect insulin secretion in β cells. Another study demonstrated that treatment of rat islets with Free Fatty Acid (FFA) decreases PPARα expression and inhibits insulin secretion in response to glucose stimulation [8]. Such secretion of insulin in response to glucose stimulation is called GSIS (Glucose Stimulated Insulin Secretion). The fact that insulin secretion is altered by the expression level of PPRAα in the pancreas suggests that PPARα may play an important role with regard to the mechanism of insulin secretion in the pancreas. However, the detailed mechanisms, such as how PPARα agonists affect insulin secretion, are not fully elucidated. On the other hand, “glycotoxicity” is a very important clinical issue in diabetes. Persistent hyperglycemia causes dysfunction of pancreatic beta cells. In addition, insulin action is reduced in insulin target organs such as skeletal muscle, adipose tissue, and liver, resulting in insulin resistance.

This phenomenon is called “glucotoxicity,” and is widely known clinically as a factor that aggravates diabetes. Expression of the insulin gene in pancreatic β-cells is suppressed, insulin secretion is decreased, and increased apoptosis is observed in the state of pancreatic β-cell glucotoxicity. Various mechanisms related to glucotoxicity have been reported, one of which potentially involves calmodulin kinase (CaMK). Calmodulin (CaM) is a calcium-binding protein that senses calcium levels and functions as an intermediary molecule to transmit information to various proteins. The CaMK family is one of the most well-defined families of enzymes. Among them, binding to the Ca2+/ CaM complex activates calcium/calmodulin-dependent protein kinase IV (CaMKIV). In addition, it is activated by being phosphorylated by CaM kinase kinase (CaMKK) [9] and plays various roles in intracellular signalling. Previously, we investigated the relationship between glucotoxicity and CaMKIV in pancreatic β cells [10]. The expression of CaMKIV was decreased when β cells were cultured under conditions of high concentrations of glucose. This suggested that CaMKIV may be involved in glucotoxicity. In this study, we analyzed GSIS in β cells in a glucotoxic state. In addition, we examined the effect of K877 on insulin secretion and ABCA1 expression in pancreatic β cells under chronic hyperglycemic states (diabetes / glucotoxicity).

Materials and Methods

Cell Culture

INS-1 cells, which developed by the Division of Biochimie Cliniqe (courtesy of C. B. Wollheim, Geneva, Switzerland), were used in this study. The cells are derived from the rat insulinoma cell line. RPMI- 1640 medium (SIGAMA, Tokyo, Japan) was used to culture these cells. This medium contained 11.2 mmol/l and 22.4 mmol/l glucose, respectively, supplemented with 10% heat-inactivated fetal bovine serum (Dainippon Pharmaceutical Co., Tokyo, Japan). In addition, 0.1 mg/ml streptomycin,100 U/ml penicillin, and 50 μmol/l 2-mercaptoethanol are added. The cells were exposed to chronic hyperglycemic conditions for 7 days at 37°C in humidified atmosphere containing 5% CO2. We changed the medium daily to keep the glucose concentration in the medium constant. For the K-877 group (K-877, provided by Kowa Company, Ltd. (Nagoya, Japan)), the cells were treated with 1 μM K-877 for 7 days and then collected for cholesterol measurement, mRNA extraction, and protein extraction.

Glucose Stimulate Insulin Secretion

Each experiment was performed using cells cultured under various conditions described below. GSIS was performed after culturing INS-1 cells at 11.2 mM, 22.4 mM and 22.4 mM+K-877 for 7 days. INS-1 cells were then withdrawn into Krebs-Ringer bicarbonate (KRB) buffer and incubated at 37°C while starved for 1 hour. The KRB buffer was supplemented with 0.1% bovine serum albumin (pH 7.4), 25 mM NaHCO3, 2.5 mM CaCl2, 1.1 mM MgCl2, 5 mM KCl and 120 mM NaCl. Cells were then incubated with a new KRB buffer supplement. This KRB buffer varied the glucose concentration as follows: stimulatory: 16.7 mM, basal: 3.3 mM. After 1 h of incubation at a controlled temperature of 37 °C, we collected the supernatant of these cells. The collected supernatant was assayed for insulin levels using an ELISA kit (Shibayagi, Japan). All incubations were performed in a 5% CO2 incubator at a controlled temperature of 37°C.

Real-Time Polymerase Chain Reaction

RNA-Bee-RNA isolation reagent was used to isolate total cellular RNA. Absorbance at 260 nm was used to quantify this total cellular RNA. For reverse transcription, 6 μg of total RNA was used. With regard to sequence, the forward and reverse primer sequences for rat ABCA1 were 5′-CCCGGCGGAGTAGAAAGG-3′ and 5′-AGGGCGATGCAAACAAAGAC- 3′. GAPDH was used as the housekeeping gene.

Western Blot Analysis

Proteins were separated by using 7.5% sodium dodecyl sulfate polyacrylamide gels. Then, the proteins were transferred to a polyvinylidene difluoride membrane for immunoblotting. 7.5% skim milk was prepared from a solution of 0.1% Tween 20 in PBS. Using this solution, the membranes were blocked overnight at a controlled temperature of 4 degrees for 24 hours. The membrane was incubated with either anti-ABCA1 antibody (1:1000; Santa Cruz), anti-GAPDH antibody (1:5,000; Biomol Research), anti-CaMKIV (1:2000; Abcam), and anti-phospho-CaMKIV Thr196 antibody (1:1000; Santa Cruz). After overnight incubation with the primary antibody, incubation was continued with the secondary antibody at 4 degrees for 1 hour (HRP-labeled anti-rabbit IgG; 1:2000). Membranes were then washed again. This was done three times for 10 minutes each. Then, ECL (GE Healthcare) was used to visualize antigen-antibody complexes. Under Luminescent image analyzer LAS-1000 Plus (Fuji Film, Japan), protein bands in western blot analysis were obtained.

Cholesterol Efflux Assay

The method reported by Shahnaz, et al. [11] was adopted to measure intracellular cholesterol ester concentrations. This method performs various reactions in the presence of an indicator substrate. This reaction enzymatically hydrolyzes cholesterol esters to free cholesterol and enzymatically oxidizes the free cholesterol. This cholesterol assay is a fully automated method that produces a fluorescent product.

Oil Red O Stain

First, INS-1 cells were seeded on coverslips and subjected to various treatments. 4% paraformaldehyde (PFA) was then used to fix the cells. This was done at room temperature and for 30 minutes. The cells were washed three times with phosphate buffered saline (PBS; pH 7.2). The fixed cells were then incubated with Oil Red O solution for 15 min. After washing three times with PBS, cells were incubated with hematoxylin solution for 30 seconds to stain nuclei. After washing three times with PBS, cells were mounted. Pictures were taken using an upright microscope (Olympus BX-51/DP-72).

Statistical Analysis

In multi-treatment experiments, group mean differences were tested by ANOVA for comparisons. An unpaired t-test was performed to compare data between the two groups. Data was expressed as mean ± SE. *P<0.05 indicates statistical significance in all figures.

Results

K-877 Enhanced the Expression of ABCA1 in INS-1 Cells We analyzed the effect of K-877 on the expression of ABCA1. Western blotting was performed to measure the protein expression of ABCA1 in INS-1 cells. The results show that ABCA1 protein expression is reduced when INS-1 cells are cultured under high glucose conditions (Figure 1). We further examined changes in these cells when exposed to K-877 for 7 days. We have exposed cells to various concentrations of K-877 (0, 10 nM, 100 nM and 1 μM) to investigate the optimal K-877 concentration for ABCA1 expression in INS-1 cells. As exposure to 1 μM K-877 significantly increased ABCA1 expression, we chose 1 μM as the concentration of K-877 to proceed with our experiments. Then, compared to control cells, treatment with K-877 significantly increased endogenous ABCA1 protein (Figure 1). The expression of ABCA1 mRNA was also assessed using real-time PCR. The expression of ABCA1 mRNA was elevated by K-877 compared to cells cultured under glucotoxic conditions (Figure 2). Surprisingly, K-877 also stimulated the expression of ABCA1 even when cultured under non-glucotoxic condition. These results clearly show that K-877 increases ABCA1 expression not only under non-hyperglycemic conditions but also under hyperglycemic conditions.

Figure 1

Figure 2

Cholesterol Accumulation in INS-1 Cells was Improved by K-877

ABCA1 is an important factor for intracellular cholesterol efflux. Based on this, the accumulation of cholesterol was assessed by measuring the intracellular cholesterol content. To stain intracellular lipids such as triglycerides and cholesteryl esters in cells, we performed Oil Red O staining. Figure 3 shows that intracellular lipid droplets were increased and enlarged in INS-1 cells cultured in glucotoxic state, whereas K-877 treatment reversed this effect. It also shows that treatment of cells with K-877 results in fewer intracellular lipid droplets. In addition, the size of the lipid droplets was also reduced. Intracellular cholesterol accumulated in INS-1 cells cultured under high glucose conditions; however, addition of K-877 under these high glucose conditions reduced the intracellular cholesterol content.

Figure 3

K-877 Improved Glucose Stimulated Insulin Secretion

We next investigated the effect of K-877 on GSIS under chronic high glucose conditions. INS-1 cells were first stimulated with a low glucose concentration (3.3 mM) and then with a high concentration of glucose. ELISA was used to measure insulin levels secreted from β cells into the medium. A comparison of insulin secretion from INS- 1 cells in the groups incubated under normal and high glucose conditions is shown in Figure 4. Figure 4 shows that the differences in insulin levels secreted under low- and high- glucose stimulation were significantly reduced in the glucotoxic group. In particular, insulin levels did not increase in response to increasing glucose concentrations in a group of glucotoxic cells cultured at 22.4 mM. On the other hand, treatment of this glucotoxic group with K-877 clearly improved the amount of insulin secreted in response to increased glucose concentration. These K-877-induced changes were particularly pronounced at high glucose concentrations of stimulation. K-877 treatment also showed a non-significant improvement in insulin secretory capacity at low glucose concentrations (3.3 mM). These results indicate that the addition of K-877 to INS-1 cells may have a positive effect on GSIS.

Figure 4

Effect of K-877 on the Phosphorylation of CaMKIV Under Glucotoxic Conditions

As previous reports indicated that CaMKIV is involved in the pathogenesis of glucotoxicity in pancreatic β cells [10,12], we focused on culture conditions, such as glucotoxicity, to determine whether phosphorylation of CaMKIV is altered. As represented in Figure 5, CaMKIV phosphorylation was lower in cells cultured with 22.4 mmol/L glucose for seven days than in cells cultured with 11.2 mmol/L glucose. The results so far indicate that high glucose concentration may decrease the expression of CaMKIV in pancreatic β cells. Furthermore, the expression of CaMKIV was higher in K-877-treated cells cultured under high glucose conditions for seven days than in control cells (Figures 5a & 5b). K-877 enhanced CaMKIV / phospho- CaMKIV expression regardless of glucose concentration (Figures 5a & 5b). Collectively, these results indicate that K-877 enhances the CaMKIV expression and induces the phosphorylation of CaMKIV in INS-1 cells despite high glucose conditions (Figure 6).

Figure 5

Figure 6

Discussion

In this study, we found that exposure of INS-1, a pancreatic beta cell line, to high glucose conditions for seven days suppressed ABCA1 expression, while K-877 reversed this effect. Furthermore, chronic hyperglycemia caused lipid accumulation in INS-1 cells, as shown by cholesterol content analysis and Oil Red O staining but was ameliorated by K-877 treatment. Additionally, GSIS, which was suppressed under high glucose conditions, was ameliorated by K-877 treatment. As for the intracellular signaling pathway, the expression of CaMKIV was reduced under chronic high glucose conditions but improved by K-877 treatment. Various effects of intracellular lipid accumulation associated with ABCA1 deficiency on GSIS have been reported. Interestingly, in pancreatic beta cell selective ABCA1 knockout mice, the lipid content in pancreatic beta cells was increased and GSIS was impaired (6). Previously, we reported that TNF-α, angiotensin II, and oxidized low-density lipoprotein increase intracellular cholesterol content and reduce the expression of ABCA1 in pancreatic β cells [13- 15]. We also reported that GSIS is decreased in INS-1 cells with accumulated cholesterol. In other words, lipid accumulation in pancreatic β-cells owing to decreased ABCA1 expression reduces GSIS. In a previous study, we have reported that K-877 is closely involved in the expression of ABCA1 in pancreatic beta cells. K-877 enhanced ABCA1 mRNA and protein expression by enhancing the promoter activity of the ABCA1 gene.

In addition, K-877 suppressed cholesterol content in pancreatic beta cells as a result of ABCA1 induction [7], which may contribute to decreased pancreatic beta cell lipotoxicity. In addition, the relationship between PPARα and ABCA1 was investigated. When mice were fed a high-fat diet, the expression of PPARα in the pancreas was reduced. On the other hand, administration of K-877 to mice fed an HFD restored the reduced PPARα expression. These findings suggest that PPARα may be closely related to the regulation of ABCA1 expression by the action of K-877.We have also previously reported on the relationship between Vascular Smooth Muscle Cells (VSMCs) and glucotoxicity: in VSMCs, ABCA1 expression is down-regulated by exposure to high glucose conditions [16]. In that experiment, intracellular signaling pathways were also examined. Under high glucose conditions, ABCA1 expression is suppressed. In relation to this, it was suggested that activation of the p38 mitogen-activated protein kinase (p38 MAPK) pathway may be partly responsible for this suppression of ABCA1. In our study, we found that ABCA1 expression is down-regulated in pancreatic beta cells under conditions of glucotoxicity, while K-877 induced ABCA1 expression. Previous reports showed that activation of p38 MAPK induces PPARα phosphorylation. In addition, it has been mentioned that the transcriptional activity of PPARα is inhibited by a tripartite interaction between PPARα, p38 MAPK, and ZIP/p62 [17]. Although the mechanism by which K-877 affects the suppression mechanism of PPARα by p38 MAPK activation during hyperglycemia is unknown, K-877 might affect the crosstalk of the intracellular signaling system p38 MAPK-PPARα.

Additionally, although K-877 is a selective regulator of PPARα-mediated gene transcription, this pathway functioned even under glucotoxic conditions. In this study, K-877 promoted GSIS and improved CaMKIV expression, even under glucotoxic conditions. Interestingly, K-877 significantly induced CaMKIV expression at both 22.4 mM and 11.2 mM glucose concentrations in the cell culture. In addition to the improvement in CaMKIV expression, which was decreased by glucotoxicity, the possibility of K-877 having a direct effect on CaMKIV should be considered. There are various potential mechanisms responsible for the relationship between K-877 and CaMKIV. First, K-877 has been reported to inhibit cell apoptosis. Wei, et al. [18] reported that K-877 inhibits the expression of caspase and may be involved in the regulation of apoptosis in mitochondria. Sugiyama, et al. [10] reported that, under high glucose conditions, calpain, an important protease involved in apoptosis, is activated and CaMKIV expression is decreased, and this change was completely blocked by calpain inhibitors. Compared to calpain activity in INS-1 cells under normal conditions, calpain activity in INS-1 cells under glucotoxic conditions was shown to be higher. In contrast, CaMKIV was downregulated. These findings suggest that calpain is involved in the reduction of CaMKIV protein levels in glucotoxic conditions. These also emphasize the importance of calpain in the regulation of basic signaling pathways in diabetes. Based on these reports, K-877 may improve CaMKIV expression by inhibiting calpain activation.

Second, PGC-1α (PPAR-gamma coactivator-1 alpha) is a factor involved in the regulation of CaMKIV and PPARα. PGC-1α is a transcriptional coactivator that binds to various transcription factors, mainly nuclear receptors, and regulates the expression of target genes. For example, CaMKIV is activated by exercise stimulation, which in turn activates PGC-1α [19]. Furthermore, PGC-1α binds to the PPARα and RXR (retinoid X receptor) complex to affect its function [20] and is also involved in apoptosis [21]. These findings suggest that PGC-1α may play an important role in the action mechanism of K-877, which binds to PPARα and stimulates CaMKIV expression. Third, K-877 improved insulin secretion by increasing ABCA1 expression and might reduce lipotoxicity in the pancreas; however, it is also possible that the autocrine effects associated with the action of insulin itself improved insulin secretion. Leibiger, et al. [22] reported an important mechanism for insulin biosynthesis. They elucidated that in pancreatic beta cells, insulin secreted under physiological stimuli promotes transcription of the insulin gene in an autocrine fashion. They showed that secreted insulin acts on the cells themselves by mediating insulin receptors and upregulating insulin gene transcription via the CaMK pathway. This indicates that insulin acts on pancreatic beta cells to activate CaMK. Furthermore, we previously reported that activation of the CaMK pathway activates CaMKIV and promotes insulin synthesis in pancreatic β cells [23].

We investigated the mechanism that stimulates insulin gene transcription by focusing on the effects of changes in glucose concentration. Exposure of INS-1 cells to 11.2 mmol/l glucose increased insulin promoter activity, which in turn increased CaMKIV activity. The constitutively active form of CaMKIV was then transfected into pancreatic β cells. We checked the changes under these conditions and found that insulin promoter activity was upregulated. In addition, transfection of CaMKIV dominant-negative mutants into INS-1 cells markedly suppressed glucose-induced insulin promoter activity. These results indicate that the CaMK pathway may be essential in the transcriptional activation of insulin genes in response to changes in glucose concentration. In view of the above, it is necessary to examine markers associated with glucotoxicity in order to further develop the present study. In addition to apoptosis markers such as caspase and calpain, AGEs, reactive oxygen species, inflammatory markers and Endoplasmic Reticulum (ER) stress markers, which may serve as markers for the development of glucotoxicity, could be examined in more detail in relation to glucotoxicity and ABCA1. In addition, the relationship between ABCA1 and CaMKIV requires further investigation. We have previously reported on a direct relationship between ABCA1 and CaMKIV in hepatocytes [24]: the CaMKK / CaMKIV / PREB signalling pathway regulates ABCA1 expression in the liver and the GLP-1, Excendin-4 treatment enhanced ABCA1 expression via the CaMKIV pathway and suppressed cholesterol accumulation in hepatocytes.

In the present study, we showed that K-877 is involved in the activation of both CaMKIV and ABCA1. Based on these findings, it is likely that K-877 directly activates CaMKIV. On the other hand, as a further issue, it is very important to check how the loss of CaMKIV function affects the expression of ABCA1 and how it results in changes in GSIS. This study has several important clinical implications. Previous clinical trials have shown that K-877 has little effect on glucose intolerance. Araki, et al. [25] examined the efficacy of pemafibrate for 52 weeks in hypertriglyceridemic patients with concomitant type 2 diabetes. As a result of this report, pemafibrate improved the lipid profile associated with patients with type 2 diabetes, including hypertriglyceridaemia and hypo-HDL cholesterolaemia, while it did not significantly alter glucose metabolism. Based on these findings, the efficacy of pemafibrate should be carefully evaluated when used in clinical practice. On the other hand, K-877 has recently been reported to be effective in reducing postprandial hyperglycemia in mice [26]. The improvement in GSIS by K-877, which we clarified in this study, may be involved in the improvement of postprandial hyperglycemia. In conclusion, our results suggested that K-877 may affect insulin secretion by regulating ABCA1 expression in pancreatic β-cells in a glucotoxic state. This finding provides a new therapeutic target for glucotoxicity in diabetes mellitus.

Acknowledgments

We thank Miss A. Sugimoto for her excellent technical assistance.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author, K.F., upon reasonable request.

Conflict of Interest

The authors declare that they have no conflict of interest.

Funding

This work was supported by JSPS KAKENHI Grant Numbers 21K08531 to K.M. (Grant-in-Aid for Scientific Research(C)), 21K16342 to K.F.( Grant-in-Aid for Early-Career Scientists).

Author Contributions

Conceptualization, K.F.., J.L., H.I. and K.M.; methodology, K.F., J.L., H.I., J.W., T.K., T.S., T.Y., S.S., and N.S.; validation, J.L., H.I., J.W., T.K., T.S., T.Y., and S.S.; investigation, K.F. and J.L.; resources, K.F., J.L., H.I., J.W., T.K., T.S., T.Y., and S.S.; data curation, K.F.; writing-original draft preparation, K.F. and K.M.; writing-review and editing, K.F. and K.M.; supervision, H.I. and K.M.; project administration, K.M.; funding acquisition, K.M., and K.F.. All authors have read and agreed to the published version of the manuscript.

Compliance with Ethical Standards

This article does not contain any studies with human or animal subjects performed by any of the authors.

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C-Reactive Protein in Solid Tumors: Clinically Meaningful Change

Introduction

C-Reactive Protein (CRP), part of the innate immune response, is produced mainly by the liver [1]. In healthy individuals, median CRP concentration is 0.8 mg/L (range 0-10 mg/L) [2]. The wide range is explained by genetic factors (50%), [3] age, body mass index, physical inactivity, race, and tobacco smoking [4,5]. CRP levels ≥10 mg/L are associated with acute infection, autoimmune diseases, inflammation, trauma, and tumors [6]. Although Elevated CRP may persist in chronic conditions, it remains stable over time in healthy individuals making it a candidate for tumor screening [7,8] Indeed, elevated CRP in healthy subjects was associated with later cancer development [9-11]. This association was strongest in Asians (breast cancer), and in men (colorectal cancer) [12,13]. We previously examined the relationship between a single CRP assessment and survival (N=4971); higher CRP values were associated with earlier death, even among those with higher normal levels [14]. Also, tumor expressed CRP when present, was independently associated with survival [15]. In cancer, high CRP was prognostic in 90% of 271 studies and associated with recurrence [16]. Hybrid scores with albumin were created: CRP/Albumin Ratio, Glasgow Prognostic Score (GPS), and modified GPS [17]. Despite the association between CRP and later cancer development, shorter survival, and cancer recurrence, it is used inconsistently in routine practice. A major challenge is the inability to interpret changes in CRP levels. The objective of this study was to determine when a change in CRP is clinically meaningful.

Material and Methods

Study Design/Population/Measures

This is a retrospective cohort study of Electronic Medical Records (EMR; My Practice/EPIC, Epic Systems Corporation, WI, USA). The Cleveland Clinic IRB approved the protocol and waived informed consent. Consecutive subjects presented, to the Taussig Cancer Institute, between 2006-2012 with a solid tumor, and at least two CRP measurements post-diagnosis were included. We excluded age <18, CRP assessments <7 days apart, hematologic malignancy, or those with missing data. We used the first CRP value present after diagnosis (baseline) and the second value reported thereafter. In 2020, we retrieved death date from the EMR or Social Security Death Index. The endpoint was Overall Survival (OS), defined as months from tumor diagnosis to death. Detailed description of data elements was reported elsewhere [16]. Subjects were divided into baseline: high CRP (bHCRP; CRP≥10 mg/L) and normal CRP groups (bNCRP; CRP<10 mg/L) because these groups were biologically different (Table 1).

Table 1: Patients’ Demographic and Baseline Characteristics.

Note: a. Other than Aspirin
Numbers rounded to the nearest whole number and p-values to one significant figure.
p-value <0.05 is considered statistically significant.

Statistical Analysis

We report mean and Standard Deviation/Error (SD/SE) or Median and Range (R) for continuous variables; and counts and percentages (%) for categorical variables. Percentages were rounded to the nearest whole number and numbers to one significant figure, unless otherwise specified. Categorical variables were compared by the Chi-square test or Fisher Exact test, and continuous variables by appropriate parametric and nonparametric tests. Percentage change in CRP (%ΔCRP) was defined as ((second CRP assessment– baseline CRP assessment) /baseline CRP assessment)) *100. Cut-off points for %ΔCRP was determined using literature reports, median and quartile range, and/or Receiver Operator Curve analysis, when an appropriate sample size was available [18]. We determined the Cut-off points to be 50% decrease or a 2-fold increase in CRP. To confirm cut off points we used Kaplan-Meier survival plots, log-rank test, and constructed Cox Proportional Hazard Model (CPHM) for bHCRP and bNCRP groups separately. Models were adjusted to account for potential confounders (Age; Body Mass Index; Cancer Site and Stage; Cancer Treatment; Comorbidities: arthritis, gastro-intestinal, heart, inflammatory, liver, and thromboembolic diseases; Gender; Metastatic Disease; Race; White Blood Cell Count (proxy for inflammation and infection)). Results are shown as Hazard Ratios (HR) with 95% Confidence Intervals (CI). We used Goodness-of-Fit to assess CPHM. Variables significant on univariate analysis or of known clinical significance were included in the models. A clinically meaningful survival benefit was reported to be two months or more [19]. Sample size calculation was not done due to the exploratory nature of this study. Statistical tests were two-sided and a p-value<0.05 indicated statistical significance. Analyses were performed with SAS software (SAS® On Demand for Academics. Cary, NC: SAS Institute Inc.).

Results

Demographic

7716 presented with a solid tumor (2006–2012).1473 had at least two CRP assessments ≥7 days apart. Those in the bNCRP group(n=530) were more likely to be female, breast or skin cancer, lower BMI, and longer OS. The bHCRP group(n=943) was more likely gastrointestinal cancers, higher total white blood cell count, liver disease, metastatic disease, and prior surgery (Table 1).

Kaplan Meier Survival Estimation

OS in bHCRP was, mean (SE), 87(2) and 81(4) months for subsequent ≥50% and <50% CRP decrease; and in bNCRP, 90(3) and 105(3) months for ≥2-fold and < 2-fold increase (Figures 1A & 1B).

Cox Proportional Model Analysis

In bHCRP, CRP increase did not predict OS, but a ≥50% decrease had a 40% lower mortality risk compared to <50%. In bNCRP, a CRP decrease did not predict OS, but a ≥2-fold increase doubled the mortality risk compared to a lower increase (Table 2).

Figure 1

Table 2: Cox Proportional Hazard Models for Overall Survival in baseline high C-Reactive Protein Group and Baseline Normal CRP Group.

Note: Model adjusted for Age; Body Mass Index; Cancer Site and Stage; Cancer Treatment: Chemotherapy, Surgery; Comorbidities: arthritis, gastro-intestinal, heart, inflammatory, liver, and thromboembolic diseases; Gender; Metastatic Disease; Race; White Blood Cell Count (proxy for infection)
DF: Degrees of Freedom; HR: Hazard Ratio
p-value <0.05 is considered statistically significant.

Discussion

We were able to quantify “how much change in CRP is significant” after cancer diagnosis. At least a 2-fold increase after a bNCRP and a 50% decrease in bHCRP was associated with OS. That remained statistically and clinically meaningful after adjustment for confounders. No prior studies, to our knowledge, examined longitudinal CRP changes post cancer diagnosis. Two studies evaluated the risk of de novo cancer development. In a Danish general population (N=10,408; follow up for16 years) the risk of new cancer development was 2-fold for lung cancer in the highest versus lowest CRP quintiles [9]. Similarly in another study (N=592), there was a 2-fold greater risk of de novo cancer development.10 Although these studies lacked post diagnosis longitudinal CRP assessment, they lend support to use of a 2-fold CRP increase as clinically important.

Limitations

Unknown indication for CRP assessment; although we accounted for multiple conditions an unknown confounder may still bias the results, dividing subjects reduced final subgroups’ sample sizes. Future studies should conduct a more comprehensive evaluation in a larger prospective design to confirm our findings and confirm their generalizability. CRP is a cheap, readily available, non-invasive biomarker. It could be used in multiple solid tumors using our approach to screen for disease progression or regression. We present a novel approach to interpret CRP changes, in a large sample, of mixed solid tumors, representative of those typically presenting to a cancer center. We did not incorporate complex CRP and albumin algorithms in favor of a simple method easily incorporated into practice. We defined parameters for clinically meaningful change in CRP in cancer patients. This will reduce healthcare disparities in cash-strapped systems.

Conclusion

In solid tumors, after a baseline normal CRP an increase of at least 2-fold reflects shorter OS, while a decrease of at least 50% after a baseline high CRP, was associated with longer OS. Serial CRP measurement after diagnosis may accurately reflect disease progression or regression. Quantification of clinically meaningful CRP change could eliminate a barrier to more effective CRP use as a biomarker and prognostic indicator and aid therapeutic decision making. Use of cheap biomarkers like CRP will reduce health disparities especially in developing countries. A large prospective study is needed to confirm our findings.

Acknowledgment

We acknowledge Aynur Aktas, MD for her involvement in data acquisition.

Conflict of Interest

• Wael Lasheen: No conflict of interest to disclose.
• Declan Walsh: No conflict of interest to disclose.

Financial Disclosure

• Wael Lasheen: No financial disclosures to report.
• Declan Walsh: No financial disclosures to report.

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Optimizing Injera’s Nutritional Value: A Strategy for Starch Reduction

Introduction

Ethiopians have a profound fondness for injera, a dietary staple deeply ingrained in their culinary culture. With Ethiopia’s population estimated at approximately 120 million, alongside an additional 10 million in the diaspora, it is common for a significant portion of the populace to incorporate injera into their daily meals, often regarding it as indispensable. However, amidst its widespread popularity, it’s imperative to recognize the prevalence of diabetes, which impacts over 5% of Ethiopia’s adult population aged between 20 and 79 years (Berhe, [1]). Injera is a significant source of carbohydrates, fiber, and various nutrients. Nevertheless, responsible consumption is paramount. According to the International Diabetes Federation (IDF), as of 2019, only about half of individuals with diabetes adequately manage their condition (IDF, [2]). Approximately 50% of people with diabetes are not successful in controlling their blood sugar levels through medication, lifestyle modifications, and other interventions. Studies also show that diabetes was responsible for an estimated 4.2 million deaths globally in 2019 (IDF, [2]). This figure includes deaths directly attributed to diabetes as well as deaths from complications related to the disease.

Disparities in healthcare access and quality, as well as challenges in diabetes management, can contribute to preventable deaths from diabetes-related complications. In managing type II diabetes globally, meticulous evaluation of starch levels in staple foods is crucial, particularly concerning ingredients like teff, the fundamental element of injera. UV-visible spectroscopy has become an essential analytical technique in this field. Here, we use absorption spectra analysis to explore the complex dynamics of starch content in teff, concentrating on the 520, 590, and 700 nm wavelengths. Additionally, our investigation establishes the starch concentration at 5%, complemented by a distinctive iodine-to-potassium iodide ratio of 2:1. Amylase is a critical facilitator in the efficient breakdown of starch molecules in the human digestive system (Brown, [3]). Amylase is produced in the salivary glands and various microorganisms and acts as a starch-reducing agent, catalyzing the hydrolysis of starch molecules into simpler sugars. There are two primary types of amylases: alpha-amylase and beta-amylase. Alpha-amylase is chiefly responsible for breaking down starch into shorter carbohydrate chains such as maltose and glucose by cleaving the alpha-1,4 glycosidic bonds in starch molecules. Beyond its biological role, amylase is extensively utilized in industrial processes, particularly within the food industry (Brown, [3]). Amylase plays a critical role in converting starch into sugars while producing various food products, and it is widely accepted as safe. For instance, in baking, the incorporation of amylase enzymes into dough facilitates the breakdown of starch into simple sugars. Yeast ferments these sugars, leading to the production of carbon dioxide gas, which effectively leavens the injera batter. In this paper, amylase is a crucial enzyme both in biological contexts and the food industry, facilitating the breakdown of starch molecules into smaller, more readily eliminated components. Once amylase breaks down starch, the resulting fragments, comprised of monosaccharides and disaccharides, dissolve in water and can be siphoned out, leaving behind a gluten-free paste that contains little to no carbohydrates. Adding amylase to teff offers a straightforward and practical method of preparing injera with a lower glycemic index suitable for consumption by type II diabetic patients and individuals mindful of their dietary intake and producing low carb teff dietary options for individuals managing diabetes [4].

Methodology

To prepare the teff solution, fine flour powder was weighed according to the experiment’s requirements. The teff was then dissolved in water in a test tube, ensuring thorough mixing to achieve a homogeneous solution. Specific amylase enzyme concentrations were measured and added to the teff solution, followed by stirring to ensure even enzyme distribution. The solution was left to react for approximately 30 minutes, allowing the enzyme to catalyze the hydrolysis of starch molecules into simple sugars. Control samples of pure starch with known concentrations served as a reference for comparison. Iodine solution, made of I2/KI in a 1:2 ratio, was added to the control samples and the teff solution [5]. Starch molecules react with iodine to form a blue-black complex, indicating the presence of starch. The intensity of the color change correlates with the concentration of starch in the sample. A spectrophotometer compared the color intensity between the experimental and control samples to assess amylase’s effectiveness in hydrolyzing teff starch. Solution absorbance of the solution was measured across a range of wavelengths (520, 590, and 800 nanometers).

We suggest the following steps for employing the α-amylase enzyme for injera starter preparation and starch reduction. The α-amylase enzyme, traditionally used in brewing and baking, finds additional application in injera preparation. To prepare the ersho or injera starter, thoroughly combine a small amount of teff flour with water in a large mixing bowl. A small amount of amylase enzyme was incorporated into the mixture to facilitate fermentation [6]. The prepared mixture can then be transferred into a closed container for anaerobic fermentation. The mixture was allowed to ferment undisturbed for three days, enabling the natural fermentation process to develop the ersho or injera starter. After fermentation, the liquid was drained from the fermented batter to remove excess sugars, ensuring the desired consistency and flavor for the injera. Teff flour may be blended with water to prepare the teff batter in a separate mixing dish. The ersho is mixed with batter; water is added to obtain the desired consistency.

Results

UV-visible spectroscopy was employed as the primary analytical method to assess the starch levels present in teff, the critical ingredient of injera. Analysis of absorption spectra, particularly at wavelengths of 520 nm, 590 nm, and 700 nm, yielded significant insights. The solubility of starch in water varies based on factors like temperature, pH, and starch type, typically ranging from 1 to 10 mg per liter at room temperature. Starch concentration in the presence of amylase and iodine was evaluated through the spectrophotometer. Figure 1 and Table 1 summarize the absorbance readings obtained at different starch concentrations. Figure 2 and Table 2 show absorbance readings of starch with amylase in an iodine solution. (Figure 3) The data demonstrate a consistent increase in absorbance readings across all three wavelengths as the starch concentration escalates from 0.03125 mg/L to 1 mg/L (Table 3). This indicates a proportional surge in the interaction between starch, amylase, and iodine with heightened starch concentrations. Furthermore, a general decrease in absorbance readings is observed as the wavelength increases, with the peak absorption occurring at 520 nm and the lowest at 700 nm. This behavior aligns with the expected characteristics of iodine-starch complexes, where maximal absorption is observed at shorter wavelengths.

Table 1: Mass of teff before and after solubility assessment.

Table 2: Absorbance readings of pure starch with amylase solutions.

Figure 1

Figure 2

Figure 3

Table 3: Absorbance readings of teff with amylase solutions.

Conclusion

Based on the comprehensive analysis conducted in this study, it is evident that using amylase to reduce starch levels in teff holds significant promise. UV-visible spectroscopy has provided invaluable insights into the starch content of teff. Particularly at the 520, 590, and 700 nm wavelengths, a noticeable pattern of starch reduction with the introduction of amylase is observed. These findings firmly establish the feasibility of using amylase to effectively lower starch concentrations in teff, which could have profound implications for various applications in food processing and dietary management. Moreover, exploring starch-amylase interactions further clarifies the potential benefits of employing amylase to modulate starch levels. The significant trends in absorbance readings across different wavelengths underscore the intricate interplay between starch concentration, enzymatic function, and iodine complex formation.

The data indicates a proportional surge in interaction between starch, amylase, and iodine, reinforcing the notion that heightened amylase concentrations correspond to amplified starch breakdown. With transmission levels stabilizing after reaching a certain threshold of amylase concentration, there appears to be a saturation point in the effect of amylase on starch reduction. These findings emphasize the importance of carefully optimizing amylase concentration to achieve desired outcomes, paving the way for further research and development in utilizing amylase for starch reduction in teff and beyond. Through continued exploration and innovation, the incorporation of amylase in starch reduction processes holds immense potential for addressing dietary concerns and enhancing food quality and nutritional value.

Acknowledgment

We thank Lydia Moges for her participation.

Below adding link

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The Surgical Anatomy of the Pyramidal Lobe and Its Significance in Thyroid Surgery

Background

Currently, the preferred surgical techniques for thyroid gland problems are hemithyroidectomies or complete thyroidectomies. When it comes to autoimmune and malignant conditions in particular, the extent of resection is crucial. Following surgical resection, leftover tissue may make it more difficult to treat specific ailments and to follow up following surgery. Incomplete thyroid tissue removal due to differences in the gland’s anatomy might occasionally lead to a recurrence of the condition [1,2]. One classic example of anatomical difference impacting the completion of thyroidectomy is the existence of the Pyramidal Lobe(PL). A thyroid surgeon has to be completely knowledgeable about the architecture of the thyroid gland, including any changes that may be acquired, congenital, or embryological. The PL is characterized as an embryologically derived thyroid tissue remnant situated in the pretracheal area, between the hyoid bone and the isthmus, during the intrauterine descent of the fetal lingual thyroid to its anatomically proper position. According to published reports, its prevalence might range from 12% to 80% [3,4]. Failure to remove the PL completely, if it is present, usually precludes a thyroidectomy from being successful and increases the risk of disease recurrence.

Finding out the frequency and anatomical characteristics of the PL in our patients who had thyroid surgery was the goal of this prospective investigation.

An endocrine organ located in the anterior neck, within the pretracheal fascia, is the thyroid gland [5]. It consists of two lobes (left and right), an isthmus that connects them, and a pyramidal lobe (PL), which is present in up to 65% of instances [6]. During fetal development, it marks the distal end of the thyroglossal duct, which forms along the thyroid gland’s migration path [7]. In the third week of pregnancy, the thyroid gland near the base of the tongue starts to grow. The thyroid diverticulum migrates in the midline downward ahead of the laryngeal cartilage and hyoid bone, and settles at the level of the C5–T1 vertebrae in the anterior neck. The thyroglossal duct joins the gland to the base of the tongue during its descend. Only the foramen cecum remains as a reminder of the thyroglossal duct’s presence at the base of the tongue when it degenerates through apoptosis by the tenth week of intrauterine life. There are situations where the duct is not completely obliterated, leaving behind vestiges along its course, the most frequent of which is the pyramidal lobe [8]. Understanding this structure’s anatomy, morphology, variations, and frequency is essential for thyroid surgery, as it may serve as the primary site for a single or multiple malignant diseases, or it may be the site of recurrent thyroid disease following hemi- or total thyroidectomy.

Materials and Methods

This prospective analysis included 16 patients (mean age 34.5; range 11–63) who had thyroid gland problems surgically treated between January 2022 and December 2023 with hemi and complete thyroidectomy. Surgical Technique: To accomplish complete excision of the gland, a typical procedure for dissecting the lateral lobes was followed. After total surgical dissection, the lateral lobe was medially mobilized. Prior to the gland’s removal, the anterior cervical (pretracheal) region between the gland’s isthmus and the hyoid bone was thoroughly examined, seen, and searched for thyroidal tissue. If thyroidal tissue was found, it was entirely dissected from the isthmus up to the hyoid bone. To guarantee that the thyroidectomy was performed completely, the lateral lobe, the isthmus, and the PL (if any) were all completely removed and sent for histopathological examination. The frequency of the PL was found in individuals who had either a partial or full thyroidectomy. It was determined which sexes made up the thyroidectomy patients with or without PL. It’s worth mentioning that the PL base originates from the primary thyroid gland. We identified the origin of the PL on thyroidectomy tissues. Following the gland’s removal, the size and length of the PL were measured on fresh tissue, and it was determined to be short (≤5 mm), medium (6–31 mm), or long (≥43 mm).

Results

Out of sixteen patients who underwent hemi or total thyroidectomy, fourteen (87.5%) showed pyramidal lobes. The prevalence of PLs was 66% in men and 92.3% in women (Table 1). Twelve of the sixteen (75%) individuals with PLs were female. In 78.5% of patients, the PL began in the isthmus, followed by the left side (Tables 1-3). The PL originated from the isthmus in 81.8% of female patients and 18.2% of male patients (Table 2). The mean length of the PL was 24.1mm (range 5–49) mm in our present series, and 50% of the PLs were longer than 30 mm. We found PLs of various lengths with origin at various sites on the thyroid gland (Figures 1- 4). The location of the pyramidal lobe’s origin on the top border of the isthmus was carefully considered when analysing the anatomical structure of the structure during the procedure. Pyramidal lobes, if they existed, were eliminated in every instance. The lobes were dissected cranially until all discernible thyroid tissue was gone, starting at the branching level from the isthmus. The pyramidal specimen underwent further pathological analysis, which involved measuring using a standard ruler and doing both macro- and microscopic analyses (Figures 5 & 6). The incidence, position, size, and pathological involvement of the pyramidal lobe was compared with the sex, age, and type of thyroid disease of the patient. Three men (age 27 – 63, median 47.3) and thirteen women (age 11 – 54, median 50) were operated on and examined. Table 3 displays the frequencies of all thyroid diseases linked to the pyramidal lobe in this dataset. Numerous statistical techniques were applied, including the ANOVA test, t-test, chi-square test, and Spearman’s test.

Table 1:

Table 2:

Table 3:

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Discussion

The pyramidal lobe exhibits variations in size, shape, and location. Many shapes have been described, including string, flat, triangle, and pyramidal shapes. Depending on where it originated on the upper edge of the isthmus, it points upward in the midline or slightly to the left or right. It might have fibrous tissue securing it to the thyroid cartilage. The origin may be found on the upper poles, the medial border of the lateral lobes, or the upper border of the isthmus [9]. According to the majority of writers, the left thyroid lobe or the left side of the isthmus represent the most common location (40–60%) of the pyramidal lobe origin [1,10-12]. A pyramidal lobe’s morphologic appearance can be used to categorize it as either bulge (with a rounded bulge at the end of the conical elongation), ectopic (when the lobe is not a continuation of the thyroid gland proper), broad (involving both lobes and having a broad originating base), or parallel (conical elongation arising from either or both lobes) [13] Its shape might be a nodule, a thread, or an inverted Y [14]. There is debate over whether gender matters when it comes to the pyramidal lobe’s prevalence, however it has been demonstrated that women are more likely to have them since they have a higher frequency of thyroid diseases [15,16].There are wide differences in the information on the pyramidal lobe’s length.

While Braun et al [1] reported a median length of 24.1 mm with a range of 3 – 63 mm and larger lobes in females, Filho et al [17]. found that lobe length varied from 10 to 50 mm. There were no pyramidal lobes larger than 20 mm, according to Geraci et al [3]. In addition to having longer lobes in females, we also identified a length range of 8 to 40 mm with a median length of 20.13 mm, which matches the range reported in the current literature. The majority of sources characterize the pyramidal lobe’s extent in terms of surrounding structures: Marshall [18] asserts that the structure extends over 50% of the time, Braun [1] notes that it does so in 25% of cases, and Harjeet et al [19]. say that it reaches the hyoid bone in 5% of cases. Recent literature continues to refer to the pyramidal lobe as a common site for the recurrence of benign diseases following total and hemi-thyroidectomy, despite the fact that it is a normal component of the thyroid gland with varying positions and sizes and possible pathological modifications in all thyroid diseases [20-24] Additionally, following surgical excision of differentiated thyroid carcinoma, it is the most often detected location for radio-iodine uptake on postoperative scinti scans [25] (Figures 7-9). When performing thyroid surgery, the pyramidal tract of the thyroid gland should always be inspected; in the case of a complete thyroidectomy, its removal is required for a number of reasons:

• To lessen the chance that benign illnesses may reoccur locally
• Considering the high rate of multifocality of these tumors, it is likely to permanently cure 90% of patients with differentiated thyroid carcinoma [26].
• To enhance the use of adjuvant radioiodine therapy for differentiated thyroid carcinoma [27].
• In individuals with differentiated thyroid carcinoma, to raise the sensitivity of serum thyroglobulin [26].

Figure 7

Figure 8

Figure 9

Conclusion

The pyramidal lobe should always be checked during thyroid surgery and should always be removed in total and hemi thyroidectomies since it is an integral part of the thyroid gland that can occur in a variety of locations and sizes as well as exhibit pathological alterations in benign and malignant illnesses. The preferred surgical techniques for thyroid gland problems are hemi or total thyroidectomies. When it comes to autoimmune and malignant conditions in particular, the extent of resection is crucial. Following surgical resection, leftover tissue may make it more difficult to treat specific ailments and to follow up following surgery. Incomplete thyroid tissue removal due to differences in the gland’s anatomy might occasionally lead to a recurrence of the condition.

Compliance with Ethical Standards

The procedure performed in this case report was in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Funding

This study is not funded by any resources.

Conflict of Interest

The author (s) declares no potential conflicts of interest with respect to the research, authorship, and/or publication of this paper.

Ethical Approval

For the purpose of publishing this case report, the patient’s written informed consent was obtained.

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To study the Effect of Practical Teaching with Demonstration on Urine Analysis in Second Year Medical Students

Introduction

• According to the latest guidelines laid by National Medical Council, a new pattern of assessment has been made which gives priority to psychomotor, communication and affective domains which were not included in the traditional method of assessment.
• These domains are given weightage according to Miller’s pyramid [1].

Miller’s Pyramid

Basic Model of Miller’s Pyramid:

• Millers pyramid is way of ranking clinical competence both in educational settings and in the workplace.
• As a framework it distinguishes between knowledge at the lower levels and action in the higher levels.
• Miller’s ideas to define education by its outputs and not by its inputs and so at the end of any teaching intervention we are interested in what learners can do, which is not the same as what we have taught them. The higher levels of learning have greater professional authenticity.
• Miller’s pyramid is usually described as having 4 levels; knows, knows how, shows how and does. Additional levels before these have been added to suggest that learners need to have heard about and have awareness of before knowing (Figure 1).

Figure 1

Millar’s Prism (Modified Millar’s Pyramid):

• Millar’s prism further integrates knowledge, skills and attitude which is used in clinical settings.
• Every aspect of the prism has its own importance and any individual aspect alone is not sufficient while assessing a student (Figure 2).

Figure 2

Methodology

• A group of 110 students were taught theoretically about urine analysis.
• Pre questionnaire was given to analyze their understanding based on the knowledge they have gained.
• A practical demonstration was given to them.
• Thereafter they themselves performed that practical.
• A post questionnaire was given to analyze their knowledge gained through practical work and theoretical class.

About the Topic Urine Analysis:

• It is one of the basic tests with great clinical and pathological importance.
• Further urinalysis consist of physical, questionnaire and microscopic examination which helps us to assess an individual on different grounds of understanding.

About the Questionnaire:

• Topic: URINE ANALYSIS
• The questions were based on:
• What student learnt (Know).
• How much he understood the topic (Know how).
• What interpretation is he able to make when demonstration was shown (Show and Show how).
• Is he able to demonstrate the same tests himself (Perform).
• These questions were also graded as (Table 1).

Table 1: CT Exam Protocol.

Result

Performance in following type of questions (Tables 2 & 3).

Table 2: CT Exam Protocol.

Table 3: CT Exam Protocol.

Conclusion

• The pre questionnaire and post questionnaire used as tool of understanding showed that post questionnaire done after practical demonstration had better results than pre questionnaire done after theoretical teaching.
• Only theoretical teaching can be a boredom and very monotonous on a long run. Practical understanding adds a uniqueness to the learning process.
• Henceforth, it concludes that “hands on experience” has a significant difference in understanding of a topic as well as retention of knowledge [2].

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Mode-of-Action of Antimicrobial Peptides

Background

The mechanism of action of the preparation consists of: stimulation of recognition of viral antigens and affected cells by natural killers (NK), neutrophils and other effector systems of natural (innate) immunity; and stimulation of interferon synthesis. Insects show a specific resistance to infectious agents either through its natural structures (the cuticle made of rigid chitin) or through a mechanism still incompletely determined, but which seems to be mediated by receptors that recognize structural elements: proteins, glycoproteins and polysaccharides. One such receptor, called GNBP (Gram negative binding protein) has been identified in the silkworm. It turned out to be part of the family of scavenger receptors, which in humans achieves the clearance of oxidized lipoproteins in the blood. Nonshelf recognition stimulates the phagocytic activity of hemocytes and triggers two proteolytic cascades: one responsible for hemolymph coagulation, and the second leading to prophenoloxidase activation [1]. This enzyme produces melanin deposits on the surface of the microbe, being also responsible for the formation of free radicals and other cytotoxic compounds, such as: quinones, semiquinones, reactive oxygen species, etc. At the same time, the synthesis of Pps with antimicrobial activity is induced in the body of the insect, which is the functional counterpart of the cat, and in certain hemocytes. The kinetics of this process is similar to the acute phase reaction observed in humans in case of inflammation.

The AMPs exhibit antibacterial, antifungal, antiviral and antiprotozoal action, including against strains resistant to antibiotics and chemotherapy. Some Pps are characterized by selective action on gram-positive or gram-negative flora. Others fight bacteria, fungi, viruses or protozoa [2]. In the process of evolution, organisms acquired the property of producing substances that change the permeability of the membrane of other organisms, which were called poroformers– proteins capable of incorporating into the foreign membrane with the formation of pores. A common primary effect of these proteins, different in chemical structure, is reduced to the coupling with the cell membrane and the formation in it of a hydrophilic channel that facilitates, depending on the size of the pores, the transport through the hydrophobic part of the membrane of various substances (ions, carbohydrates and even proteins). The formation of additional pores initiates various mechanisms of cell death [3]. The cytoplasmic membrane is not directly accessible to PAMs. Thus, in gram-positive bacteria it is protected by the peptidoglycan layer, and in gram-negative ones by the outer membrane and peptidoglycan. In this context, some PAMs can be effective only against gram-positive flora, others against gram-negative flora, a fact that is explained by their specific recognition. Thus, gram-negative bacteria are identified by their lipopolysaccharide content. The AMPs pass through external structures substituting calcium and magnesium ions due to cationic properties with increased permeability that facilitate the penetration of other molecules [1]. After penetrating the outer membrane, PAMs get the opportunity to be absorbed on the surface of the cytoplasmic membrane, and subsequently their behavior can be determined by several mechanisms:

• The formation of permanent barrel-type channels (barrel- staves), the walls of which are formed by the aggregation of several Pps. This type is characteristic of the few Pps (melitins, pardoxins, alamethicins) that are not strong cations and have the role of forming hydrophobic bonds that reduce the selectivity towards bacteria acting on macroorganism cells (erythrocytes);
• The formation of toroid-type pores (toroidal pore), the walls of which are made up of the polar parts of PAMs and hydrophilic lipids. This type of pore is characteristic for some PAMs with an alpha-helix structure (mageinins).
• The formation of carpet-type pores (carpet model), when the interaction between PAMs and the membrane does not lead to the formation of permanent or temporary channels, but to the substitution of lipids with the amphipathic molecules of PAMs on a limited sector with the destruction of membranes, similar to the action of detergents. The respective mechanisms are not self-exclusive, but can complement–1 and 2 with 3 [4,5].

Target Cell Death Can Occur According to the Following Hypotheses:

• Deregulation of the membrane state by: changing the fluidity of the lipid layer; deregulation of the stability of intercalated protein complexes; stop breathing; disruption of membrane potential and energy balance; affecting membrane barrier functions; uncontrolled influx of water into the cell; loss of essential metabolites;
• Penetration of PAMs into the cytoplasm and coupling with intracellular structures (less elucidated mechanism), for example, with cellular polyanions (such as DNA and RNA) resulting in the arrest of protein biosynthesis and cell death [6]. It is also believed that PAMs, due to their positive charge, would form a film covering the membrane with negative charges, which subsequently leads to the destruction of the bacteria. In experimental studies, although a clear correlation between the structure and the mechanism of action of PAMs was not determined, some trends can still be stipulated: the positive correlation between the ability to act aggressively on the membrane (structure and functional parameters) and low selectivity (action on bacteria and eukaryotic cells) [7];
• The repeat of the segment of 3–6 AAs for PAMs with alpha- helix is important for the interaction with the membrane, especially if the PAMs are amphipathic and the hydrophobic domain is larger. The smaller the polar angle in PAMs, the more actively they form pores and the more stable they are [8];
• Presence or substitution with proline contributes to change the mechanism–from pore formation–to intracellular targets. Linear Pps with alpha helix (spirals) also bear the generic name of cecropins, since the first compounds of this type were identified through experimental infections of the pupae of the butterfly Hyalophora cecropia. Currently, 21 such Pps are known both from diptera: Drosophilla melanogaster (contains 3 isoforms), Sarcophaga peregrina (flesh weevil), Aedes albopictus (a species of American mosquito), and from lepidoptera: Hyalophora cecropia, Manduca sexta, Bombyx mori. Cecropins have been determined in the hemolymph of various insects (sarcotoxin A in flies, cecropin in butterflies, spinigerin in termites) and are eliminated as toxins with the function of cytolysins. It has been found that they exhibit antimicrobial action, weak hemolytic and are a component of the immune system of insects that ensures their protection from microorganisms [9]. Cecropin Pps are secreted in an inactive form, having a signal Pps and a sequence at the N-terminal end which are then removed by proteases, and at the C-terminal end they always present a glycine residue which under the action of a specific monooxidase (starch enzyme) is removed, the second amino acid remaining with an amido-terminal group [10]. The biologically active part comprises 36–40 AAs that adopt a secondary structure consisting of an amphipathic alpha helix, followed by a linker region and a shorter hydrophobic alpha helix towards the C-terminus.

Initially, both helices are oriented parallel to the cell membrane, so that the N-terminal helix, positively charged, interacts electrostatically with the negative groups of the membrane lipids, and the C-terminal helix infiltrates the membrane with the formation of pores. It was found that binding to lipid membranes seems to be favored by anionic phospholipids (phosphatidylserine) and hindered by cholesterol, an effect that is not seen in the case of other types of sterols. This would justify the selectivity with which cecropins lyse prokaryote membranes, their fluid composition permissive for their mechanism of action, unlike eukaryotic membranes. Tests on liposomes loaded with fluorescent dyes and in which a difference in transmembrane potential was achieved, showed that cecropins cause membrane potential deregulation in just a few minutes, and only after about an hour the lysis of the membrane and the release of the dye are observed. In vitro cecropins have been shown to be active especially on gram-negative germs, less on gram-positive and almost non-toxic on mammalian cells [11]. The PAMs represent a class of antimicrobial preparations, the further development of which is justified by the multiple advantages: they act on numerous germs, including polyresistant ones; the mechanism of action makes resistance unlikely; show minimal toxicity to mammals [12]. The AMPs have a role in the immune defense against infections, being present both in plants and insects as well as in higher vertebrates. Each species synthesizes, following antigenic stimulation, specific AMPs, different from each other.

In humans and mammals, there is a large number of AMPs, which intervene in non-specific defense, both through the antibacterial, antifungal, antiviral microbicidal effect, as well as through the modulation of immunity. Natural AMPs generally have a small molecular mass, that is, a relatively small number of AAs, being the simplest weapons of cellular defense, belonging to the non-specific immune system. Their structure is amphipathic, presenting two regions, one hydrophilic and one hydrophobic on one side and the other of the Pps structure, and the net electric charge is intensely positive [13]. The hydrophobic region can interact with lipids in the microbial membrane, and the hydrophilic region interacts with water or negatively charged structures in the target cell membrane, but does not typically affect normal cells that have a membrane rich in cholesterol and neutral lipids. The cationic and amphipathic structure allows the creation of strong electrostatic bonds with the cell membranes of bacteria, fungi or with the envelope of enveloped viruses [14]. In addition to the antibacterial activity and modulation of the immune response that these Pps have, recent studies have shown that some of these cationic Pps have an important cytotoxic activity on cancer cells and do not act on normal mammalian cells. Most of the available anticancer drugs allow tumor growth to be controlled only at concentrations that also affect healthy cells, resulting in undesirable side effects. Thus, it is imperative to find new products with innovative mechanisms of action, and one of the current research directions is represented by the use of cytotoxic AMPs [15]. Several hypotheses could be made regarding the biological effect of the Pps that did not influence the viability and proliferation of the studied tumor cell lines:

• Membrane pore formation: probably the membrane pores formed are small in size or the density of membrane pores is low, so that cell death by necrosis or apoptosis cannot be induced as a result of caspase activation. We can assume that cells sensed the presence of Pps on the cell membrane, which led to the stimulation of some cellular activation pathways with the appearance of secondary messengers with the final effect of stimulating cell proliferation. Therefore, the activation of a certain pathway will lead to the stimulation of proliferation with the probable tendency to strengthen the ability of the cells to expel the Pps from the cell membrane [16,17].
• The transformation of an antagonist into an agonist: it was found that 24 hours after incubating the tumor cells with the Pps, there was a transient inhibition that was removed after 48 and 72 hours, respectively, and cell proliferation was stimulated. It is likely that the Pps could be processed (coupled) by the tumor cell so that its insertion into the cell membrane does not lead to cell death [18,19]. Since the molecular mechanisms of interaction between proteins and membranes are at an important frontier of cell biology, investigations on these Pps could bring useful information on the mechanism of interaction between small Pps and cell membranes as well as on the possibilities in which this process can be modulated [20]. Although the exact mechanism of action of AMPs remains a matter of controversy, there is a consensus that these Pps selectively disrupt cell membranes, and the amphiphatic structural arrangement of the Pps is believed to play an important role in this mechanism.

Concluding Remarks and Future Perspectives

Considering all these aspects from the specialized literature, in these experimental studies the hypothesis was verified according to which cytotoxic Pps known as antimicrobials have tumoricidal potential whose intensity depends both on the nature of the Pps used and on its concentration in the living environment of the cells, but also the type of cell line used experimentally, in vitro. It can be stated that the information is relevant for medical practice because, in addition to cytostatics commonly used in the therapy of various types of cancer, other compounds could be used, as is the case with these cytotoxic Pps that have been shown to have tumoricidal potential. The cytotoxic effect on tumor cells of these Pps must also be evaluated in vivo on experimental animal models because it is important for oncological medical practice, to test and permanently improve therapeutic pathways, by finding new compounds with tumoricidal potential such as those from the category of AMPs. These compounds could lead to new therapeutic possibilities, that is, to provide significant tumoricidal effects with minimal side effects, in the sense of minimal cytotoxicity for normal cells.

Funding

Not applicable.

Conflicts of Interest/Competing Interests

The authors declare no conflict of interest.

Availability of Data and Material

Not applicable.

Code Availability
Not applicable.

Authors’ Contribution
Conceptualization, M.D.C.; I.G. and M.B.; data curation, M.D.C. and M.B.; writing—original draft preparation, M.B.; writing—review and editing, M.D.C. I.G. and M.B.; visualization, M.B.; supervision, I.G and M.B. All authors have read and agreed to the published version of the manuscript.

Ethics Approval

Not applicable.

Consent to Participate

Not applicable.

Consent for Publication
Not applicable.

Data Availability Statement
The data presented in this study are available on request from the corresponding author.

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Effect of Egg Weight on Water Loss and Chicks Yield of Broiler Breeders

Introduction

Poultry products are a great source of both revenue and protein (Hussain, et al. 2015). The poultry business employs Thousands of veterinarians to treat diseases and ensure the quality of chicken products (Anonyms, 2011). Broiler hatchability is influenced by the caliber of the incubation and hatching circumstances for eggs (Jabbar, et al. [1]). During incubation, temperature and humidity are crucial environmental factors that must be considered (Lourens, et al. [2]). Water loss and chick production of chicken eggs are influenced by several variables (Alsobayel [3]). Numerous studies have demonstrated that the viability of the embryo and post-hatch performance are influenced by the quality of the breeder’s egg (Yoho, et al. 2008). According to Alsobayel [3], the age of the hen may have an impact on hatchability, embryonic mortality, and fertility. As chickens get older, their hatchability and fertility decline (Insko, et al. 1947). One of the most crucial environmental variables during incubation is temperature and humidity (Lourens, et al. [2]). Due to the embryo’s increased heat generation near the conclusion of the incubation phase, the eggshell temperature and water loss rise (Hussain, et al. [2,4]). Various stages and ages of the embryo require varying temperatures and humidity levels (Lourens, et al. [2]). Because the embryo is producing more heat during the third week of incubation, the eggshell temperature rises (Yousaf, Lourens, et al. [2,5]).

Because of their genetic selection for rapid growth and high meat yield (Balog, 2003; Arce-Menocal et al. 2009), modern broiler chickens are more susceptible to metabolic disorders like ascites, which can lead to decreased visceral organ development, ideal chick yield, and water loss (Havenstein, et al. 2003). As chickens get older, their hatchability and fertility decline (Yousaf, et al. [6,7]). Depending on the stage and age of the embryo, different temperatures and humidity levels are needed (Yousaf, et al. [2,8]). Water loss, chick weight, chick yield, hatchability, and temperature have all been shown to be tightly correlated (Jabbar, et al. [1]). Since temperature influences embryo development, temperature, humidity, and water loss are the three most important factors to consider while managing egg hatching (Yousaf, et al. [9]). Because hatchability and eggshell quality are related, maintaining hatching eggshell quality is crucial. The main determinants of eggshell quality are chickens’ age, housing, nutrition, environment, and genetics. The quality of eggshells may also be related to eggshell color. This study aims to examine hatchery performance by taking into account key variables including water loss, chick weight, and chick yield of broiler breeders.

Materials and Methods

Sit Selection

The study was carried out at Salman Poultry (Pvt) Limited, Chakri Hatchery, Rawalpindi. The hatchery is situated 05km from the Chakri Interchange motorway (M2) to the west. It contains the latest HVAC automation, having ISO 1900-2000 certified. This is South Asia’s largest egg capacity hatchery, which produces the best quality chicks through a single-stage incubation system (Avida G4 chick master USA).

Group Classification

Eggs from broiler breeders (Ross 308) were classified into four groups age-wise of breeder hens, such as A (Young age 24-31 weeks, 51.27±1.64g), B (Prime age 32-50 weeks, 57.81±1.48g), C (50 + weeks, 61.23±1.67g) and D (combine, 57.32±1.49g) (Table 1). Group D was served as control, while rests of three groups were treated with different age wise incubation program in commercial single stage setters (Avida G4, Chick Master USA).

Table 1: Weight Of Ross-308 Breeders Eggs.

Eggs Selection

High-quality hatching eggshells were smooth, without ridges or small lumps of calcified material (pimples). The color of eggs within a flock was uniform. Young flocks produce eggs with thicker shells and when the flock is older the shell becomes thinner, and the incidence of abnormal shells increases. Hatchable eggs were selected based on shell quality, weight, and color. All the hatchable eggs were graded through the egg grading machine MOBA 9A. Only oval-shaped good-quality intact eggs were selected for hatching. Substandard eggs such as cracked, misshapen, blood-stained, dirty, and elongated were rejected (Khan, et al. [10]).

Fumigation Process

These eggs were collected at the farm and stored at 20°C and 75% relative humidity until used in the hatching trail. Before, trail eggs were fumigated with 20 g KMnO4, 40 ml formalin (40%), and 40 ml of water for 100ft3 areas for 15 minutes.

Incubation Programme

Each experimental group consisted of (n=4,03,920) eggs. All four groups were pre-heated at 82oF for 05 inside individual incubators. After completion of the pre-heating stage, the incubator started automatically age-wise stage profile (Recommended by Chicks Master USA). After completing 456 hours (19 days) in an incubator the weight of eggs was performed through digital balance to check the water loss during incubation. All the infertile eggs were removed from the tray and just fertile eggs were shifted to hatchers for the next 50 hours (2 days and 8 hours). Hatch pulling was performed after 506 hours. Upon hatching chicks’ body weights, hatchability percentage, and hatching yield were determined immediately to calculate as described by (Sahin, et al. 2009).

Water Loss & Yield Measure

Water loss was measured through the formula (full tray weight at setting-full tray weight at transfer ÷ full tray weight at setting-empty tray weight x 100), while chick yield was measured through formula weight of chicks at hatch ÷ weight of eggs at time of setting x 100.

Results

After ten successful hatches out for individual groups, hatchability, Yield, (dead in shell) DIS, and water losses were recorded. Differences in terms of hatchability, yield, DIS, Water loss, and Chick weight were found for groups A, B, and C which were incubated agewise program and D combined incubation program (control) respectively. Group B showed better hatchability percentage was recorded (89.02±0.41) as compared to A and C which are significantly P<0.005 same (88.66±0.33), (88.80±0.65) and D (control) (86.25±1.22) (Table 1). In term of chicks yield C significantly remain same (69.28±0.54) as compare to A (69.17±0.54) and B (69.99±0.54), While highly yield was recorded for D (71.45±0.54) (Table 2). Dead in the shell (DIS) was significantly the same for A (6.18±0.29), B (6.20±0.37), and C (6.13±0.60) than D (7.70±0.67) which contain high DIS percentage (Table 3). In terms of eggs, water loss was significantly the same for A (11.93±0.26), B (12.24±0.26), and C (12.31±0.26) which was recorded as ideal than D (10.16±0.26) which contained low water loss percentage (Table 2). Similarly, as the age of breeder increase size of egg also increase and chick weigh will also increase. The result in terms of the chick’s weight significant no difference recorded as B (prime) (40.71±0.96) and D (combine/control) (40.02±0.78), while the chick’s weight was less for A (35.46±0.89) than C (42.24±0.87) was recorded (Table 1).

Table 2: Effect of Agewise Incubation Programme on yield & water loss.

Discussion

Age-wise wise incubation stage program has three different incubation temperatures and humidity set points for the eggs given by different ages of breeders i.e. prime, young, and old which provides uniform temperature and humidity for growing embryos, helps to achieve good quality chicks, and ideal water loss. Uniform temperature and humidity of every stage for developing embryos enhances the water loss (Jabbar, et al. [11,12]). Similarly, the age-wise breeder eggs incubation regimen had a high-water loss and better chick quality as compared to the combined incubation program. To achieve better hatchability ideal water loss of 10-12% is necessary (Jabbar, et al. [1,13]). It was documented that the best egg fertility and hatchability in hens aged between 31 and 50 weeks (Jabbar, et al. [14,15]) as shown in result that group B is better. Hatchability and good weight of chicks are critical factors to assess the performance of hatchery (Marandure, et al. [16]). Ideal water loss provides ideal chick yield and helps to achieve good quality chicks (Tona, et al. [17]). Hatchability, water loss, chick yield, and DIS were found better for groups A, B, and C than D but overall group B was found better due to prime-age production (Tables 1-4). So, incubation of eggs with their age-wise temperature and humidity set points provides better results as compared to incubation of different ages eggs at the same temperature and humidity set points. The finding of the current study tended to show that age-wise incubation stage program to broiler breeder eggs enhances the hatchability. The high level of DIS and maximum water loss was recorded in age age-wise incubation system (Nowaczewski, et al. [18-20]) as shown in (Tables 2 & 3).

Table 3: Dead In Shell Analysis Report.

Note: a-b denotes difference in columns (P<0.05).

Table 4: Effect of Agewise Incubation Programme on Hatchability & Chicks Weight.

Note: a, b, c denotes difference in columns (P<0.05).

Conclusion

Age-wise incubation stage program is a good factor, which may be used to enhance the Chicks quality, hatchability, water loss, and chick yield.

Conflict of Interests

The authors declare that they have no conflict of interest concerning the research, authorship, and/or publications of this article.

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Approximation of the Impact of the Implementation of Work at Home in the Reduction of GHG Greenhouse Gases in the City of Bogotá – Colombia

Introduction

Through this study, it is hoped to examine the effects that the adoption of the “Work at Home” modality, a remote work modality, has had on employees of Colombian organizations, on air quality, in particular on GHG greenhouse gases, from the declaration of mandatory and preventive quarantine or social isolation brought on by the COVID pandemic 19. That is how telework, remote work, or work at home is done [1,2]. Its implementation in Colombia resulted from the declaration of a Health Emergency in the country. It has allowed several companies to continue developing their activities, taking into account the mandatory preventive asylum that the National Government applied for in several cities of the Colombian territory. In this sense, non-attendance at the offices managed to prevent the spread of the COVID-19 virus by maintaining social isolation and simultaneously generated an indirect effect on the reduction of environmental impacts, such as the mitigation of Greenhouse Gases worldwide and, of course, in Colombia.

Literature Review

In the first instance, the definition of GHG Greenhouse Gases should be brought up, which are the gaseous components of the atmosphere that are characteristic of absorbing and emitting radiation at specific wavelengths. These are those mentioned below: H₂O, CO₂, N₂O, CH₄, and O₃ [3]. It is essential to bring up some definitions stipulated in Colombia to implement the different methodologies related to remote work. Thus, according to Law 2121 of 2021 of the Congress of the Republic of Colombia, remote work was defined like this:

“The employment relationship must be carried out remotely through information and telecommunications technologies or another means or mechanism from its inception to its termination. During the contractual relationship, the employer and employee do not physically interact. In any case, this form of execution does not share the constituent and regulated elements for teleworking and work at home and the rules that modify it” [4,5]. In the case of work at home, the Colombian Government defined it under Law 2088 of 2021 as follows:

“It is understood as work at home the room to the public servant or private sector worker to temporarily perform their functions or activities outside the place where they usually perform them, without modifying the nature of the contract or employment relationship, legal and regulatory respective” [6]. Some authors state that the COVID-19 virus indirectly affected social life since there may be some negative impacts on people’s physical activity, physiological state, and ability to master the effects that confinement to homes can cause in the short and long term [7,8].

GHG Greenhouse Gases cause a more significant proportion of global warming, which is generated by agriculture and the consumption of fossil fuels by industry and automobiles [9]. According to the GHG Emissions Inventory report in Bogotá, a total of emissions for the year 2017 of 11,421,724.32 tons of CO₂ was registered, which generated 48% by the transport sector [10]. That is to say, of the total value, the transport sector generated 5,419,433 tons of carbon dioxide for that year [11]. However, it is essential to mention that although CO₂ is the gas with the highest atmospheric concentration, it is not the one with the most significant warming potential, which corresponds to sulfur hexafluoride [12]. Likewise, the average generation of Carbon Dioxide and the total GHG Greenhouse Gases are highlighted, which was 0.994 for 2019, according to the United States Environmental Protection Agency (EPA) [13]. According to that entity, this is how the amount of CO₂ emitted per gallon of burned engine gasoline is 8.89*10-3 metric tons for the United States of America.

Design and Methods

The present study focused on analyzing the impact of” Work at Home” implementation due to the COVID-19 virus pandemic under an approach to reducing GHG Greenhouse Gases. It is important to emphasize that the application of the “Work at Home” modality during the contingency originates from the issuance of Circular No. 0021 of March 17, 2020, where work at home is mentioned as a mechanism to be adopted in companies to avoid contagion [14]. The research approach is of a mixed type [15,16]. This type of research is based on three elements: conceptualization and operationalization of the variables, the degree of intervention or application by the researcher, and a solution proposed to a practical problem. In this case, is the approach to the qualitative determination of the reduction of Greenhouse Gases GHG in the workers of several companies when performing their duties from home.

To determine an approximation to the reduction of negative environmental impacts, specifically the decrease in Greenhouse Gases GHG, by the implementation of work at home that over time generated the possibility of alternation, which implied working some days at home and others in the offices, a survey was applied through Google Forms, eleven questions that allowed to investigate aspects such as means of transport used and the type of fuel used, travel time, use of main roads, travel mileage, among others. The sample corresponds to 402 people between employees and independent workers. This survey was applied virtually for two months (October and November) in 2021, considering Colombia was under a health emergency due to the COVID-19 pandemic following Decree 1026 of 2022 [17]. Subsequently, the annual GHG Greenhouse Gas emissions per passenger vehicle were determined, highlighting that these vehicles are those with two axles and four tires, such as cars, vans, and some buses of the SITP Integrated Public Transport System [18]. For this, equation (1) proposed by the Federal Highway Administration of the United States FHWA [C] was taken to determine the gallons of gasoline consumed per vehicle per year:

Where,
VMT = average vehicle miles traveled in units of miles/year
CO2 = Carbon Dioxide
CH4 = Methane
N2O = Nitrous Oxide [18]
It is important to note that the applicable values for Colombia were used according to Colombia’s First Useful Energy balance and the Quantification of related energy Losses and the energy efficiency gap of 2019 [19]. Likewise, the air quality map of the city of Bogotá was consulted, according to the data provided by the Environmental Observatory of Bogotá OAB and the District Secretariat of Environment [20].

Results

According to the questionnaire, the following information was obtained: 20% of the respondents correspond to independent workers (80 people), and the remaining 80% are employees or workers. It is worth highlighting that 205 people stated that they own a vehicle with a gasoline-based operation, while ten people own hybrid cars and 02 people use an electric vehicle. Likewise, according to the adoption of alternation by several companies to reactivate their mission processes, this modality was implemented according to the results of the respondents; 62% had to attend the office at least two times a week (Figures 1-4). In comparison, 30% never heard, and only 7.7% attended thrice weekly. Now, concerning the determination of Greenhouse Gases GHG for the research project in Bogotá, we proceeded to calculate the average value of miles traveled per vehicle according to the answers obtained from the surveys. In this context, taking into account that the result of the question “Number of kilometers traveled or traveled by 2-axle 4-wheeled motor vehicle” was contemplated considering the following options:

Between 0 -10 Km
Between 10 Km – 20 Km
Between 20 Km – 30 Km
Between 30 Km – 40 Km
More than 40 Km

Figure 1

Figure 2

Figure 3

Figure 4

A calculation was made for the average value of each of the selected distance options. Subsequently, the average gasoline performance established by the UPME was calculated [18], taking the values of cars, vans, taxis, buses, Busta, and Microbus. To determine the amount of Carbon dioxide produced by the combustion of gasoline, it was carried out as stipulated by the EPA [13], taking a standard conversion factor of 8,887 grams of CO₂ emissions per gallon of gasoline consumed, if the car models range between the years 2012 and 2016. By having this value in grams, the conversion to metric tons is carried out, so a value of 8.87 * 10-3 metric tons of CO₂ / gallon of gasoline is obtained (Table 1). Now, concerning the use of main roads that you should use to move from your place of residence to work, it is essential to highlight that 98 people say they use Avenida Boyacá, a high vehicular traffic road [21]. Of course, the use of main roads is mentioned considering that these corridors have high concentrations of atmospheric pollutants, as is the case of Avenida 68, the South Highway

Where:
DPUT: Average distance (km) in one way
DPIR: Average distance (km) in round trip
VMT: Average miles traveled or traveled in units of miles/year.
RPG: Average gas mileage (miles/gallon)
CCO₂G: CO₂ equivalent emission (metric tons CO₂/vehicle/year)

Table 1: Calculation of Greenhouse Gas emission GEI.

ECO₂ NO₂ and CH₄ = Emission of CO₂ Equivalents (Metric Tons CO₂ E /vehicle/ year) of Nitrous Oxide and Methane. It is essential to highlight that, to calculate the emission of Carbon Dioxide Equivalents of Nitrous Oxide and Methane, Carbon Dioxide emissions were divided by the proportion of CO₂ emissions and total GHG emissions. It is also highlighted that the average value of gasoline corresponds to the information reported by the Mining Energy Planning Unit for Colombia [18]. Likewise, it is highlighted that of the total respondents, 23.6% work in companies dedicated to technological services, while 18.2% are linked to advisory and consulting entities. In this sense, according to the object or economic activity of the organization, it is feasible to increase or maintain remote work, thus continuing with a low contribution from the said guild in the generation of Greenhouse Gases GHG. In the same way, when mentioning the type of company, it is possible to show that 32.1% belong to Simplified Joint Stock Companies, while 18.4% are from the state sector. That is to say, there is a greater probability of continuing with remote work, whether teleworking, working at home, or remote work in the private sector since in the public sector, fundamental activities such as customer service and other series of actions that have currently led to the majority of the state sector have returned face-to-face to the facilities of the public sector must be guaranteed.

Discussion

The adoption of Work at Home worldwide has presented a variable in implementing alternation by organizations, as highlighted in a study conducted by WeWork in 2020 [2]. In this sense, an appreciable percentage of workers want to return to company facilities at least three days a week since, according to Oracle and Workplace [17], 78% of 12,000 employees indicated that quarantine negatively affected their mental health. In several countries of the world, the measurement of CO2 is carried out through the Carbon Monitor [11]; for example, in the year 2020, when the first wave of the COVID-19 pandemic occurred, a decrease in Carbon dioxide was identified in the countries of the United States, France, and India [21]. According to the results obtained, it is essential to highlight that Colombia issued Law 2169 on December 22, 2021[22]. “Using which the Low-Carbon Development of the Country is promoted through the establishment of minimum goals and measures in the field of Carbon Neutrality and Climate Resilience and other provisions are dictated,” where article 12 makes references to the measures of the Transport Sector, among which the following can be mentioned:

. “Actions to accelerate the transition to electric mobility, designing and implementing policies to establish regulatory and technical standards for the commercialization and operation of electric vehicles with 2, 3, and 4 wheels or more, as well as the promotion of financial instruments that encourage the entry of electric cars.
2. Development of financial instruments that generate enabling conditions for the circulation of at least 600,000 electric vehicles in the country by 2030” [21].

Now, when comparing the percentage of vehicles by category that transit in Bogotá, it is essential to analyze the statistics presented by the District Secretariat of Environment in the annual Air Quality report for the year 2019 for the city of Bogotá [20], approximately 90% of the vehicles that transit in Bogotá is cars (cars, campers, and vans), which represents almost 1,601,744 of the total number of cars that move around the Colombian capital.

A lower generation of CO2 Equivalent Emissions is highlighted at a shorter travel distance of employees from their homes or places of residence to companies, as exposed by the concentration emitted up to 10 km away (Figure 5). It is also important to note that at a longer distance, there is a higher concentration of metric tons of Carbon Dioxide, so it can be deduced that a smaller number of vehicles can emit a lower amount of Greenhouse Gases GHG.

Conclusion

The adoption of remote working modalities, such as working at home, teleworking, and remote work at the time of starting the mandatory preventive isolation in several countries to prevent the spread of COVID-19 has allowed for a reduction in the generation of Greenhouse Gases by avoiding the displacement of officials or employees from the place of residence to offices or workplaces. In this sense, it is essential to highlight that according to the Bogotá Cómo Vamos Foundation, approximately 2,400,000 vehicles are circulating in the Colombian capital, of which 50% correspond to cars, 20% to motorcycles and 14% to vans, while only 5% is related to public transport [23]. Likewise, according to the Climate Action Plan for Bogotá, it is contemplated ithin the zero (0%) reduction goals for reducing vehicle kilometers traveled. In other words, in the face of the Paris agreement. Today, in Bogotá, there are about 2’400.000 vehicles circulating. Of these, 50% are cars, 20% are motorcycles, and 14% are pickup trucks. Only 5% corresponds to public service transport and 2% to yellow taxis. This means that there is a motor vehicle for every three inhabitants, and for every motorcycle, four cars. In other words, given that 125 of the respondents own the vehicle above, motorbikes are the group where the output of greenhouse gases has decreased most significantly, according to the report. In the last five years, the car fleet has grown by 24%, pickup trucks by 62%, and motorcycles by 23%. In contrast, public service vehicles have barely grown by 2 (Bogotá cómo vamos, 2022) [23].

Although it is true that in Colombia and especially in the city of Bogotá, the use of electric skateboards as a means of transport has increased, it is essential to highlight that their use is still reduced considering the safety factor and transport distances. Since the generation of guidelines by the National Government of Colombia, the use of electric vehicles has been encouraged to reduce the generation of Greenhouse Gases GHG. However, it must establish a policy that allows tax relief and access to facilities for people with limited economic resources.

Acknowledgement

We want to thank the Minuto de Dios University Corporation for the support provided during the research.

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