Uncovering the impact of SARS-CoV2 spike protein variants on human receptors: A molecular dynamics docking and simulation approach.

BACKGROUND: The SARS-CoV-2 pandemic, caused by the novel coronavirus, has posed a significant global health threat since its emergence in late 2019. The World Health Organization declared the outbreak a pandemic on March 11, 2020, due to its rapid global spread and impact on public health. New variants have raised concerns about their potential impact on the transmission of the virus and the effectiveness of current diagnostic tools, treatments, and vaccines. This study aims to investigate the effect of new variants in Pakistani virus strains on human receptors, specifically ACE2 and NRP1. In-silico analysis provides a powerful tool to analyze the potential impact of new variants on protein structure, function, and interactions. OBJECTIVES: The SARS-CoV-2 virus is evolving quickly. After being exposed in Wuhan, SARS-CoV-2 underwent numerous mutations, leading to several variants' emergence. These variants stabilize the interaction of spike protein with human receptors ACE2 and NRP1. The study aims to check the molecular effect of these variants on human receptors using the in-silico approach. MATERIAL AND METHODS: We use in-silico mutational tools to analyze new variants in SARS-CoV-2 and to check the molecular interaction of spike protein with human receptors (ACE2 and NRP1). Genomic sequences of 41 SARS-CoV-2 strains were sequenced using Ion Torrent (NGS) and submitted to the GISAID database. Spike protein of SARS-CoV-2 sequence trimmed and translated into a protein sequence using ExPasy. We used multiple sequence alignments to check for variants in the spike protein of strains. We utilized mutation tools such as Mupro, SIFT, SNAP2, and Mutpred2.3D structures of SARS-CoV-2 spike proteins (wild and mutated) to analyze further the mutations, ACE2 and NRP1 modelled by the ITASSER protein modelling server. Interactions of spike proteins (wild and mutant) analyzed by MD Docking, Simulation, and MMGBSA RESULTS: Variants I210T, V213G, S371F, S373P, T478K, F486V, Y505H, and D796Y were identified in SARS-CoV-2 Pakistani strains' spike protein. Variant Y505H were found to affect protein function. MD Docking, MMGBSA and MD simulation revealed that these variants increased spike protein's binding affinity with human receptors (ACE2 and NRP1). MD simulation revealed that mutated spike protein stabilized earlier than wild when interacting with ACE2 after 40 ns and interaction with NRP1 stabilized after 30 ns for mutated spike protein compared to wild. CONCLUSION: These variants in Pakistani strains of SARS-CoV-2 are increasing the stability of spike protein with human receptors. These findings provide insight into how the SARS-CoV-2 virus evolves and adapts to human hosts. This information may help develop strategies to control the virus's spread and develop effective treatments and vaccines in the future.

Population-level median cycle threshold (Ct) values for asymptomatic COVID-19 cases can predict the trajectory of future cases.

BACKGROUND: Recent studies indicate that the population-level SARS-CoV-2 cycle threshold (Ct) values can inform the trajectory of the pandemic. The presented study investigates the potential of Ct values in predicting the future of COVID-19 cases. We also determined whether the presence of symptoms could change the correlation between Ct values and future cases. METHODS: We examined the individuals (n = 8660) that consulted different sample collection points of a private diagnostic center in Pakistan for COVID-19 testing between June 2020 and December 2021. The medical assistant collected clinical and demographic information. The nasopharyngeal swab specimens were taken from the study participants and real-time reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect SARS-CoV-2 in these samples. RESULTS: We observed that median Ct values display significant temporal variations, which show an inverse relationship with future cases. The monthly overall median Ct values negatively correlated with the number of cases occurring one month after specimen collection (r = -0.588, p <0.05). When separately analyzed, Ct values for symptomatic cases displayed a weak negative correlation (r = -0.167, p<0.05), while Ct values from asymptomatic cases displayed a stronger negative correlation (r = -0.598, p<0.05) with the number of cases in the subsequent months. Predictive modeling using these Ct values closely forecasted the increase or decrease in the number of cases of the subsequent month. CONCLUSIONS: Decreasing population-level median Ct values for asymptomatic COVID-19 cases appear to be a leading indicator for predicting future COVID-19 cases.

Regulation of Lipid Metabolism Under Stress and Its Role in Cancer.

Within the tumor microenvironment, cancer cells are often exposed to oxygen and nutrient deficiency, leading to various changes in their lipid composition and metabolism. These alterations have important therapeutic implications as they affect the cancer cells' survival, membrane dynamics, and therapy response. This chapter provides an overview of recent insights into the regulation of lipid metabolism in cancer cells under metabolic stress. We discuss how this metabolic adaptation helps cancer cells thrive in a harsh tumor microenvironment.

Cancer Awareness Measure (CAM) and Cancer Awareness Measure MYthical Causes Scale (CAM-MY) scores in Pakistani population.

Lifestyle modifications could prevent almost one-third to one-half of all cancer cases. The awareness of cancer risk factors could motivate people to make such changes in their behaviors and lifestyles. This work aims to investigate the cancer awareness level in the Pakistani population. Telephone interviews of 657 individuals in Pakistan were carried out using the Cancer Awareness Measure (CAM) and Cancer Awareness Measure-MYthical Causes Scale (CAM-MY). We observed that participants scored significantly better on the CAM scale than the CAM-MY scale, and CAM scores were negatively associated with CAM-MY scores. Years of formal education or a biology major at undergraduate or graduate level did not affect our population's cancer awareness levels. Age displayed a weak but statistically significant negative association with CAM scores. Most participants failed to identify modifiable cancer risk factors, e.g., low physical activity. Efforts should be made to improve awareness of modifiable risk factors. We observed that brief training sessions could markedly improve people's understanding of cancer risk factors and myths.

Factors associated with cycle threshold values (Ct-values) of SARS-CoV2-rRT-PCR.

BACKGROUND: Presented work studies the association of COVID-19 severity, patient demographics, and clinical history with cycle threshold (Ct) values of SARS CoV2-rRT-PCR. We studied the Ct values for Orf1ab, N, and RdRp genes in association with all the factors mentioned above. METHODS AND RESULTS: We examined the individuals (n = 6331) that consulted two private diagnostic centers for COVID-19 testing. SARS-CoV-2 was detected by RT-PCR assays using different commercial kits. Clinical and demographic information was collected by the attending health care professional. Ct values were not associated with the age, sex, or clinical history of the patient. Orf1ab and N genes Ct values were only weakly associated with symptoms at the time of the SARS-CoV-2 RT-PCR test. Also, the distributions of Ct values in SARS-CoV-2 positive patients are very similar irrespective of symptomatology. CONCLUSION: We conclude that the Ct values may have limitations in reliably predicting COVID-19 severity and should be used or reported with caution.

Assessment of biosafety implementation in clinical diagnostic laboratories in Pakistan during the COVID-19 pandemic.

Laboratory diagnostic capacity is crucial for an optimal national response to a public health emergency such as the COVID-19 pandemic. Preventing laboratory-acquired infections and the loss of critical human resources, especially during a public health emergency, requires laboratories to have a good biorisk management system in place. In this study, we aimed to evaluate laboratory biosafety and biosecurity in Pakistan during the COVID-19 pandemic. In this cross-sectional study, a self-rated anonymous questionnaire was distributed to laboratory professionals (LPs) working in clinical diagnostic laboratories, including laboratories performing polymerase chain reaction (PCR)-based COVID-19 diagnostic testing in Punjab, Sindh, Khyber Pakhtunkhwa, and Gilgit-Baltistan provinces as well as Islamabad during March 2020 to April 2020. The questionnaire assessed knowledge and perceptions of LPs, resource availability, and commitment by top management in these laboratories. In total, 58.6% of LPs performing COVID-19 testing reported that their laboratory did not conduct a biorisk assessment before starting COVID-19 testing in their facility. Only 31% of LPs were aware that COVID-19 testing could be performed at a biosafety level 2 laboratory, as per the World Health Organization interim biosafety guidelines. A sufficiently high percentage of LPs did not feel confident in their ability to handle COVID-19 samples (32.8%), spills (43.1%), or other accidents (32.8%). These findings demonstrate the need for effective biosafety program implementation, proper training, and establishing competency assessment methods. These findings also suggested that identifying and addressing gaps in existing biorisk management systems through sustainable interventions and preparing LPs for surge capacity is crucial to better address public health emergencies.

Too complex to fail? Targeting fatty acid metabolism for cancer therapy.

Given the central role of fatty acids in cancer pathophysiology, the exploitation of fatty acid metabolism as a potential antineoplastic therapy has gained much attention. Several natural and synthetic compounds targeting fatty acid metabolism were hitherto identified, and their effectiveness against cancer cell proliferation and survival was determined. This review will discuss the most clinically viable inhibitors or drugs targeting various proteins or enzymes mapped on nine interconnected fatty acid metabolism-related processes. We will discuss the general significance of each of these processes and the effects of their inhibition on cancer cell progression. Moreover, their mechanisms of action, limitations, and future perspectives will be assessed.

COVID-19 severity: Studying the clinical and demographic risk factors for adverse outcomes.

BACKGROUND: The primary goal of the presented cross-sectional observational study was to determine the clinical and demographic risk factors for adverse coronavirus disease 2019 (COVID-19) outcomes in the Pakistani population. METHODS: We examined the individuals (n = 6331) that consulted two private diagnostic centers in Lahore, Pakistan, for COVID-19 testing between May 1, 2020, and November 30, 2020. The attending nurse collected clinical and demographic information. A confirmed case of COVID-19 was defined as having a positive result through real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay of nasopharyngeal swab specimens. RESULTS: RT-PCR testing was positive in 1094 cases. Out of which, 5.2% had severe, and 20.8% had mild symptoms. We observed a strong association of COVID-19 severity with the number and type of comorbidities. The severity of the disease intensified as the number of comorbidities increased. The most vulnerable groups for the poor outcome are patients with diabetes and hypertension. Increasing age was also associated with PCR positivity and the severity of the disease. CONCLUSIONS: Most cases of COVID-19 included in this study developed mild symptoms or were asymptomatic. Risk factors for adverse outcomes included older age and the simultaneous presence of comorbidities.

Antitumor effects of sodium selenite on acute lymphocytic leukemia.

Selenium is obligatory for proper functioning of body as it is the part of enzyme protection system. Its both organic and inorganic forms are thought to be active as an antitumor agent. We trialed the different dosages (0 × 106 M, 2.7 × 106 M, 5.4 × 106 M, and 8.1 × 106 M) of sodium selenite given to the acute lymphocytic leukemia cell lines incubated for 24, 48, and 72 h. The ratios of dead cells to live cells when treated with sodium selenite were very high as compared to the control with no treatment. This dosage-dependent apoptosis increased with the incubation time.

Abundance, fatty acid composition and saturation index of neutral lipids in colorectal cancer cell lines.

Lipid droplets, the dynamic organelles that store triglycerides (TG) and cholesterol esters (CE), are highly accumulated in colon cancer cells. This work studies the TG and CE subspecies profile in colon carcinoma cell lines, SW480 derived from primary tumor, and SW620 derived from a metastasis of the same tumor. It was previously reported that the total TG and CE content is dramatically higher in SW620 cells; however, TG and CE subspecies profile has not been investigated in detail. The work presented here confirms that the total TG and CE content is significantly higher in the SW620 cells. Moreover, the fatty acid (FA) composition of TG is significantly altered in the SW620 cells, with significant decrease in the abundance of saturated triglycerides. This resulted in a significantly decreased TG saturation index in the SW620 cells. The saturation index of CE was also significantly decreased in the SW620 cells.

Expression of lipid transport-associated genes in lipid-deprived cancer cells.

Cancer cells are known to significantly alter their lipid profiles in response to changes in extracellular lipid availability. Recent studies have shown that in response to lipid deprivation, cancer cells display significant changes in their cellular lipid homeostasis. These changes have been linked to the modulation of de novo lipid synthesis pathways that are markedly altered under lipid-deprived growth conditions. However, the effects of such environment on intracellular lipid trafficking-that could also affect cellular lipid homeostasis-have not been widely investigated. The presented work studies the effect of lipid deprivation on expression of genes for lipid transport proteins (LTPs) in cancer cell lines.

Revisiting the association between human leukocyte antigen and end-stage renal disease.

Multiple works have studied possible associations between human leukocyte antigen (HLA) alleles and end stage renal disease (ESRD) showing, however, contradictory and inconsistent results. Here, we revisit the association between ESRD and HLA antigens, comparing HLA polymorphism (at HLA-A, -B, -C, -DRB1, -DQB1 and DQA1 loci) in ESRD patients (n = 497) and controls (n = 672). Our data identified several HLA alleles that displayed a significant positive or negative association with ESRD. We also determined whether heterozygosity or homozygosity of the ESRD-associated HLA alleles at different loci could modify the prevalence of the disease. Few HLA allele combinations displayed significant associations with ESRD, among which A*3_26 combination showed the highest strength of association (OR = 4.488, P≤ 0.05) with ESRD. Interestingly, the age of ESRD onset was not affected by HLA allele combinations at different loci. We also performed an extensive literature analysis to determine whether the association of HLA to ESRD can be similar across different ethnic groups. Our analysis showed that at least certain HLA alleles, HLA-A*11, HLA-DRB1*11, and HLA-DRB1*4, display a significant association with ESRD in different ethnic groups. The findings of our study will help in determining possible protective or susceptible roles of various HLA alleles in ESRD.

Prevalence of Hepatitis C Virus Genotypes in Pakistan: Current Scenario and Review of Literature.

Hepatitis C virus (HCV) is a major public health concern globally, resulting in liver-related complications. Approximately 6% population of Pakistan is infected with HCV. HCV is error prone, due to which it is classified into 7 genotypes and 67 subtypes. HCV genotype determination is critical for treatment and therapy response. In this study, 3,539 samples were collected from 2015 to 2019 from all over Punjab. RNA was extracted from samples using QIA Amp Viral RNA MINI kit (Qiagen, Germany) and viral genotyping was performed. Furthermore, a systemized literature search (2009-2018) was done to analyze the HCV genotype distribution pattern in Pakistan. In Punjab, genotype 3a (86.46%) is most prevalent, followed by untypable (7.17%) and genotype 1a (3.84%) and 3b (1.04%). Mixed genotype constitutes only 0.67% of total infections. Genotype 2a, 2b, 3c, and 4 were found to be rare. Data available from literature review when compiled showed that HCV genotype 3a (58.16%) was predominant in Pakistan, followed by genotypes 3b (9.05%), 2a (6.70%), 1a (6.22%), and 1b (2.39%). The frequency of mixed genotypes was found to be 4% and 12% of untypable HCV variants. This study highlights the HCV genotype distribution pattern in different regions of Pakistan. Therapy response and disease management depend on genotype, so HCV genotype determination is crucial. In Pakistan, the most prevalent genotype is 3a, followed by untypable genotype. Both interferon and sofosbuvir are effective against genotype 3a, but treatment with sofosbuvir has comparatively high sustained virological response, less adverse effects, and more tolerability.

Cancer cell lipid class homeostasis is altered under nutrient-deprivation but stable under hypoxia.

BACKGROUND: Cancer cells modify the balance between fatty acid (FA) synthesis and uptake under metabolic stress, induced by oxygen/nutrient deprivation. These modifications were shown to alter the levels of individual triglyceride (TG) or phospholipid sub-species. To attain a holistic overview of the lipidomic profiles of cancer cells under stress we performed a broad lipidomic assay, comprising 244 lipids from six major classes. This assay allowed us to perform robust analyses and assess the changes in averages of broader lipid-classes, stratified on the basis of saturation index of their fatty-acyl side chains. METHODS: Global lipidomic profiling using Liquid Chromatography-Mass Spectrometry was performed to assess lipidomic profiles of biologically diverse cancer cell lines cultivated under metabolically stressed conditions. RESULTS: Neutral lipid compositions were markedly modified under serum-deprived conditions and, strikingly, the cellular level of triglyceride subspecies decreased with increasing number of double bonds in their fatty acyl chains. In contrast and unexpectedly, no robust changes were observed in lipidomic profiles of hypoxic (2% O2) cancer cells despite concurrent changes in proliferation rates and metabolic gene expression. CONCLUSIONS: Serum-deprivation significantly affects lipidomic profiles of cancer cells. Although, the levels of individual lipid moieties alter under hypoxia (2% O2), the robust averages of broader lipid classes remain unchanged.

Lipid metabolism in cancer cells under metabolic stress.

Cancer cells are often exposed to a metabolically challenging environment with scarce availability of oxygen and nutrients. This metabolic stress leads to changes in the balance between the endogenous synthesis and exogenous uptake of fatty acids, which are needed by cells for membrane biogenesis, energy production and protein modification. Alterations in lipid metabolism and, consequently, lipid composition have important therapeutic implications, as they affect the survival, membrane dynamics and therapy response of cancer cells. In this article, we provide an overview of recent insights into the regulation of lipid metabolism in cancer cells under metabolic stress and discuss how this metabolic adaptation helps cancer cells thrive in a harsh tumour microenvironment.

Correction to: Anthropometric and metabolic indices in assessment of type and severity of dyslipidemia.

After the publication of this work [1] an error was noticed in one of the formulas.

Anthropometric and metabolic indices in assessment of type and severity of dyslipidemia.

BACKGROUND: It has been shown that obesity is associated with increased rates of dyslipidemia. The present work revisits the association between plasma lipid levels and classical indicators of obesity including body mass index (BMI). The significance of various anthropometric/metabolic variables in clinical assessment of type and severity of dyslipidemia was also determined. Recently described body indices, a body shape index (ABSI) and body roundness index (BRI), were also assessed in this context. METHODS: For the present cross-sectional analytical study, the participants (n = 275) were recruited from the patients visiting different health camps. Participants were anthropometrically measured and interviewed, and their fasting intravenous blood was collected. Plasma lipid levels were accordingly determined. RESULTS: The values for different anthropometric parameters are significantly different between dyslipidemic and non-dyslipidemic participants. Receiver operating characteristics curve analyses revealed that all the tested variables gave the highest area under the curve (AUC) values for predicting hypertriglyceridemia in comparison to other plasma lipid abnormalities. BRI gave slightly higher AUC values in predicting different forms of dyslipidemia in comparison to BMI, whereas ABSI gave very low values. CONCLUSIONS: Several anthropometric/metabolic indices display increased predictive capabilities for detecting hypertriglyceridemia in comparison to any other form of plasma lipid disorders. The capacity of BRI to predict dyslipidemia was comparable but not superior to the classical indicators of obesity, whereas ABSI could not detect dyslipidemia.

Lipid-load in peripheral blood mononuclear cells: Impact of food-consumption, dietary-macronutrients, extracellular lipid availability and demographic factors.

Lipid-load in peripheral blood mononuclear cells (PBMCs) has recently gained attention of the researchers working on nutritional regulation of metabolic health. Previous works have indicated that the metabolic circuitries in the circulating PBMCs are influenced by dietary-intake and macronutrient composition of diet. In the present work, we analyzed the impact of diet and dietary macronutrients on PBMCs' lipid-load. The overall analyses revealed that dietary carbohydrates and fats combinatorially induce triglyceride accumulation in PBMCs. On the other hand, dietary fats were shown to induce significant decrease in PBMCs' cholesterol-load. The effects of various demographic factors -including age, gender and body-weight- on PBMCs' lipid-load were also examined. Body-weight and age were both shown to affect PBMC's lipid-load. Our study fails to provide any direct association between extracellular lipid availability and cholesterol-load in both, freshly isolated and cultured PBMCs. The presented work significantly contributes to the current understanding of the impact of food-consumption, dietary macronutrients, extracellular lipid availability and demographic factors on lipid-load in PBMCs.

Leukemia cells display lower levels of intracellular cholesterol irrespective of the exogenous cholesterol availability.

BACKGROUND: Different types of cancer cells are previously shown to accumulate intracellular cholesterol. However, the data on intracellular cholesterol levels in leukemia cells provide contradictory evidence. Various previous works indicate either increase, decrease or no difference in total cholesterol levels between leukemia cells and healthy peripheral blood mononuclear cells (PBMCs). METHODS: We studied the intracellular cholesterol levels in acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL) cells and compared with that in PBMCs from the healthy subjects. RESULTS: We observed that the PBMCs from AML (n=7) and ALL (n=7) patients displayed significantly lower intracellular levels of total cholesterol in comparison to PBMCs from the healthy subjects (n=26). Consistent with the patient data the ALL (CCRF-CEM and MOLT-3) and AML (KG-1 and THP-1) cell lines also displayed significantly lower intracellular levels of total cholesterol. We confirmed this observation using multiple methodological approaches. Both ALL and AML cell lines also displayed significantly lower levels of free cholesterol and cholesteryl ester contents in comparison to normal hematopoietic cells. We observed that >90% of the total cholesterol in leukemia cells as well as in normal PBMCs was present in the form of cholesteryl esters. It was also observed that the lower levels of cholesterol in leukemia cells are not affected by exogenous cholesterol availability. CONCLUSIONS: Present study provides convincing evidence to prove that the cellular free cholesterol and cholesteryl ester content is significantly reduced in leukemia cells in comparison to normal hematopoietic cells in circulation. Moreover, it was shown that the lower levels of cholesterol in leukemia cells are not affected by exogenous cholesterol availability.

Atypical plasma lipid profiles in leukemia.

Numerous studies have reported alterations in the plasma lipid profiles of leukemia patients. However, there are several inconsistencies in these reports. The present review highlights and compiles findings from different research groups regarding association of plasma lipoprotein levels with the risk of developing leukemia. We have also discussed the clinical significance of plasma lipid profiles in management of leukemia. Furthermore, the potential role of plasma lipids in promoting leukemogenesis is also highlighted.