Research Articles (Chemical Pathology)

Permanent URI for this collectionhttp://hdl.handle.net/2263/2658

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Maternally inherited diabetes and deafness with a variable presentation across three generations within a pedigree, South Africa
(AOSIS, 2024-05) Makgopa, Herbert; Kemp, Tanja; Meldau, Surita; Honey, Engela M.; Chale-Matsau, Bettina
INTRODUCTION: Maternally inherited diabetes and deafness (MIDD) is caused by the m.3243A>G pathogenic variant in maternally inherited mitochondrial DNA. Diabetes is prevalent in our setting; however, MIDD is rarely diagnosed. This study, undertaken in Pretoria, South Africa, highlights the variable presentation of MIDD in different patients within the same family. CASE PRESENTATION: A 45-year-old man (proband) with hearing impairment was referred to the endocrine unit in July 2015 due to poor glycaemic control (HbA1c = 13%). His clinical and biochemical features were in keeping with MIDD. A genetic study of accessible maternal relatives was pursued. His mother had difficulty hearing and reportedly died from an unspecified cardiovascular cause. Two sisters with diabetes and deafness died of cardiac-related conditions. One nephew had diabetes (HbA1c = 7.7%), hearing loss and tested positive for m.3243A>G. A third sister tested positive for m3243A>G, but aside from bilateral mild hearing loss in higher frequencies, showed no other signs of target organ damage. Her daughter developed end-stage kidney failure necessitating a transplant, while her son had no biochemical abnormalities and was negative for m.3243A>G. MANAGEMENT AND OUTCOME: A multidisciplinary team managed and screened for complications of the patient and his maternal relatives. Proband died prior to genetic testing. CONCLUSION: Most MIDD patients initially present with symptoms of diabetes only, and it is probable that many cases remain undiagnosed. A high index of suspicion is necessary when encountering a family history of both diabetes and impaired hearing, and screening should be offered to the patient’s maternal relatives. WHAT THE STUDY ADDS: This study demonstrates the importance of proper assessment when evaluating a patient with diabetes and a family history of hearing loss.
A scoping review evaluating the current state of gut microbiota research in Africa
(MDPI, 2023-08) Pheeha, Sara M.; Tamuzi, Jacques L.; Chale-Matsau, Bettina; Manda, S.O.M. (Samuel); Nyasulu, Peter S.; bettina.chale-matsau@up.ac.za
The gut microbiota has emerged as a key human health and disease determinant. However, there is a significant knowledge gap regarding the composition, diversity, and function of the gut microbiota, specifically in the African population. This scoping review aims to examine the existing literature on gut microbiota research conducted in Africa, providing an overview of the current knowledge and identifying research gaps. A comprehensive search strategy was employed to identify relevant studies. Databases including MEDLINE (PubMed), African Index Medicus (AIM), CINAHL (EBSCOhost), Science Citation index (Web of Science), Embase (Ovid), Scopus (Elsevier), WHO International Clinical Trials Registry Platform (ICTRP), and Google Scholar were searched for relevant articles. Studies investigating the gut microbiota in African populations of all age groups were included. The initial screening included a total of 2136 articles, of which 154 were included in this scoping review. The current scoping review revealed a limited number of studies investigating diseases of public health significance in relation to the gut microbiota. Among these studies, HIV (14.3%), colorectal cancer (5.2%), and diabetes mellitus (3.9%) received the most attention. The top five countries that contributed to gut microbiota research were South Africa (16.2%), Malawi (10.4%), Egypt (9.7%), Kenya (7.1%), and Nigeria (6.5%). The high number (n = 66) of studies that did not study any specific disease in relation to the gut microbiota remains a gap that needs to be filled. This scoping review brings attention to the prevalent utilization of observational study types (38.3%) in the studies analysed and emphasizes the importance of conducting more experimental studies. Furthermore, the findings reflect the need for more disease-focused, comprehensive, and population-specific gut microbiota studies across diverse African regions and ethnic groups to better understand the factors shaping gut microbiota composition and its implications for health and disease. Such knowledge has the potential to inform targeted interventions and personalized approaches for improving health outcomes in African populations.
A framework for implementing best laboratory practices for non-integrated point of care tests in low resource settings
(International Federation of Clinical Chemistry and Laboratory Medicine, 2023) Jafri, Lena; Ahmed, Sibtain; Majid, Hafsa; Ghani, Farooq; Pillay, Tahir S.; Khan, Aysha Habib; Siddiqui, Imran; Shakeel, Shahid; Ahmed, Shuja; Azeem, Saba; Khan, Adil
The method we respond to pandemics is still inadequate for dealing with the point of care testing (POCT) requirements of the next large epidemic. The proposed framework highlights the importance of having defined policies and procedures in place for non-integrated POCT to protect patient safety. In the absence of a pathology laboratory, this paradigm may help in the supply of diagnostic services to low-resource centers. A review of the literature was used to construct this POCT framework for non-integrated and/or unconnected devices. It also sought professional advice from the Chemical Pathology faculty, quality assurance laboratory experts and international POCT experts from the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC). Our concept presents a comprehensive integrated and networked approach to POCT with direct and indirect clinical laboratory supervision, particularly for outpatient and inpatient care in low-resource health care settings.
Impact of potassium test sample rejections on routine laboratory service, South Africa
(AOSIS, 2023-12-12) McAlpine, Sarah; Chale-Matsau, Bettina
BACKGROUND : Accurate potassium measurements are necessary for effective clinical management of hyperkalaemia. Pre-analytical factors may affect laboratory measurements, leading to erroneous results and inappropriate patient management and negatively impact the efficiency and finances of laboratories and hospitals. OBJECTIVE : This study evaluated the impact of rejected potassium test requests on laboratory service. METHODS : We conducted a retrospective descriptive study to assess potassium test data at a public laboratory in Pretoria, Gauteng, South Africa, using samples collected from an academic hospital, peripheral hospitals, and outpatient clinics between January 2018 to December 2018. We assessed the relationship between reasons for rejection and health facility type, as well as financial implications for the laboratory. RESULTS : The potassium result rejection rate was 15.1% (29 806 samples), out of the 197 405 requests received. The most common reasons for rejection were old sample (> 1 day old) (41.4%; 12 348 rejections) and haemolysis (38.2%; 11 398 rejections). The most frequent reason for rejections at the central, academic hospital was haemolysis (42.0%), while old sample was the most common reason for rejection at peripheral hospitals (43.4%; 4119/9493 requests) and outpatient health facilities (57.2%; 7208/12 605 requests) (p = 0.022). The total cost of potassium sample rejection over the study period was substantial, given the resource constraints in this setting. CONCLUSION : Peripheral hospitals and outpatient departments accounted for the majority of rejected potassium testing results, possibly resulting from delays in transportation; causing substantial financial impact on the laboratory. Improved sample collection, handling, and expedited transportation are recommended. WHAT THIS STUDY ADDS : This study highlights the importance of appropriate sample collection and handling and the undesirable consequences of non-adherence to these pre-analytical considerations.
25-Hydroxycholecalciferol inhibits cell growth and induces apoptosis in SiHa cervical cells via autocrine vitamin D metabolism
(MDPI, 2023-03) Punchoo, Rivak; Dreyer, Greta; Pillay, Tahir S.; rivak.punchoo@up.ac.za
Preclinical studies show that the anticancer actions of vitamin D metabolites are mediated by apoptosis, inhibition of cell proliferation and induction of cell cycle arrest. Cervical cancer cells express an autocrine vitamin D metabolising system (VDMS) comprised of a vitamin D receptor, vitamin D catabolic enzyme (CYP24A1), and the activating enzyme of 25-hydroxycholecalciferol (25(OH)D3), CYP27B1. We assessed the anticancer effects of 25(OH)D3 at clinically relevant concentrations on a cervical squamous cell cancer cell line, SiHa. We evaluated cell health parameters (cell count, viability, and cell cycle), cell death modes (apoptosis, autophagic-dependent death, and necrosis by flow cytometry and transmission electron microscopy), and autocrine VDMS gene and protein expression by qPCR and Western blot, respectively. Our study demonstrates that physiological and supraphysiological doses of 25(OH)D3 inhibit cell growth and viability and induce biochemical and morphological apoptosis in SiHa cells. These growth effects are mediated by alteration in the VDMS gene and protein expression, with prominent negative feedback at supraphysiological treatment dose. These data identify promising therapeutic potential of 25(OH)D3 in cervical cancer, which warrants further clinical translational investigations.
Laboratory considerations for reporting cycle threshold value in COVID-19
(International Federation of Clinical Chemistry and Laboratory Medicine, 2022-08) Punchoo, Rivak; Bhoora, Sachin; Bangalee, Avania; rivak.punchoo@up.ac.za
The Coronavirus Disease 2019 (COVID-19) pandemic is caused by the SARS-CoV-2 RNA virus. Nucleic acid amplification testing (NAAT) is the mainstay to confirm infection. A large number of reverse transcriptase polymerase chain reaction (RT-PCR) assays are currently available for qualitatively assessing SARS-CoV-2 infection. Although these assays show variation in cycle threshold values (Ct), advocacy for reporting Ct values (in addition to the qualitative result) is tabled to guide patient clinical management decisions. This article provides critical commentary on qualitative RT-PCR laboratory and clinical considerations for Ct value reporting. Factors contributing to Ct variation are discussed by considering relevant viral life-cycle factors, patient factors and the laboratory total testing processes that contribute to the Ct variation and mitigate against the reporting of Ct values by qualitative NAAT.
The added value of molecular-based diagnostics in the African forensic medical setting
(Clinics Cardive Publishing, 2022-09) Van Deventer, B.S.; Makhoba, Aubrey Musa; Du Toit-Prinsloo, L.; Van Niekerk, Chantal
Sudden unexpected infant death (SUDI) is reported to be an extraordinarily high burden in sub-Saharan Africa, with the incidence rate in South Africa among the highest in the world. It is common for the cause of many such infant deaths to remain unexplained even after a full medico-legal death investigation, and then to be categorised as a sudden unexplained infant death (SUID). Fortunately, advances in molecular- based diagnostics allow researchers to identify numerous underlying inherited cardiac arrhythmogenic disorders in many SUDI cases, with a predominance of variants identified in the SCN5A gene. Such cardiac arrhythmogenic-related sudden deaths generally present with no structural alterations of the heart that are macroscopically identifiable at autopsy, therefore highlighting the importance of post mortem genetic testing. We report on a significant genetic finding that was made on a SUDI case in which the cause was ascribed to an acute bacterial pneumonia but it was still subjected to post mortem genetic testing of the SCN5A gene. The literature shows that many SUDI cases diagnosed with inherited cardiac arrhythmogenic disorders have demonstrated a viral prodrome within days of their death. It is therefore not uncommon for these cardiac disorders in infants to be mistaken for flu, viral upper respiratory tract infection or pneumonia, and without the incorporation of post mortem genetic testing, any other contributory causes of these deaths are often disregarded. This study highlights the need for research reporting on the genetics of inherited cardiac disorders in Africa.
Neonatal presentation of a child with Liddle syndrome, South Africa
(AOSIS, 2023-04-14) Steyn, Nicolene; Chale-Matsau, Bettina; Abera, Aron B.; Van Biljon, Gertruida; Pillay, Tahir S.
INTRODUCTION : Liddle syndrome is an autosomal dominantly inherited disorder usually arising from single mutations of the genes that encode for the alpha, beta and gamma epithelial sodium channel (ENaC) subunits. This leads to refractory hypertension, hypokalaemia, metabolic alkalosis, hyporeninaemia and hypoaldosteronism, through over-activation of the ENaC. CASE PRESENTATION : We describe a 5-day old neonate who presented with severe hypernatraemic dehydration requiring admission to Steve Biko Academic Hospital in South Africa in 2012. Further evaluation revealed features in keeping with Liddle syndrome. Two compound heterozygous mutations located at different subunits encoding the ENaC were detected following genetic sequencing done in 2020. The severe clinical phenotype observed here could be attributed to the synergistic effect of these known pathological mutations, but may also indicate that one of the other variants detected has hitherto undocumented pathological effects. MANAGEMENT AND OUTCOME : This child’s treatment course was complicated by poor adherence to therapy, requiring numerous admissions over the years. Adequate blood pressure control was achieved only after the addition of amiloride at the end of 2018, which raised the suspicion of an ENaC abnormality. CONCLUSION : To our knowledge, this is the first Liddle syndrome case where a combined effect from mutations resulted in severe disease. This highlights the importance of early recognition and management of this highly treatable genetic disease to prevent the grave sequelae associated with long-standing hypertension. Whole exome sequencing may assist in the detection of known mutations, but may also unveil new potentially pathological variants. WHAT THIS STUDY ADDS : This study highlights the importance of developing a high index of suspicion of tubulopathy such as Liddle syndrome for any child presenting with persistent hypertension associated with hypokalaemic metabolic alkalosis.
Structure-based identification of galectin-1 selective modulators in dietary food polyphenols : a pharmacoinformatics approach
(Springer, 2022-06) Bhowmick, Shovonlal; Saha, Achintya; AlFaris, Nora Abdullah; ALTamimi, Jozaa Zaidan; ALOthman, Zeid A.; Aldayel, Tahany Saleh; Wabaidur, Saikh Mohammad; Islam, Md Ataul
In this study, a set of dietary polyphenols was comprehensively studied for the selective identification of the potential inhibitors/modulators for galectin-1. Galectin-1 is a potent prognostic indicator of tumor progression and a highly regarded therapeutic target for various pathological conditions. This indicator is composed of a highly conserved carbohydrate recognition domain (CRD) that accounts for the binding affinity of β-galactosides. Although some small molecules have been identified as galectin-1 inhibitors/modulators, there are limited studies on the identification of novel compounds against this attractive therapeutic target. The extensive computational techniques include potential drug binding site recognition on galectin-1, binding affinity predictions of ~ 500 polyphenols, molecular docking, and dynamic simulations of galectin-1 with selective dietary polyphenol modulators, followed by the estimation of binding free energy for the identification of dietary polyphenol-based galectin-1 modulators. Initially, a deep neural network-based algorithm was utilized for the prediction of the druggable binding site and binding affinity. Thereafter, the intermolecular interactions of the polyphenol compounds with galectin-1 were critically explored through the extra-precision docking technique. Further, the stability of the interaction was evaluated through the conventional atomistic 100 ns dynamic simulation study. The docking analyses indicated the high interaction affinity of different amino acids at the CRD region of galectin-1 with the proposed five polyphenols. Strong and consistent interaction stability was suggested from the simulation trajectories of the selected dietary polyphenol under the dynamic conditions. Also, the conserved residue (His44, Asn46, Arg48, Val59, Asn61, Trp68, Glu71, and Arg73) associations suggest high affinity and selectivity of polyphenols toward galectin-1 protein.
Ketamine : friend or foe?
(BMJ Publishing Group, 2023-09) Steyn, Nicolene
Ketamine, a dissociative anaesthetic drug, acts on the central nervous system (CNS) primarily through antagonism of the n-methyl-d-aspartate (NMDA) receptor. Methods of administration include intravenous, intramuscular, snorting and smoking. Intranasal use, which has a very rapid onset of action, is common among recreational users.
Practical tips to using formalin-fixed paraffin-embedded tissue archives for molecular diagnostics in a South African setting
(AOSIS, 2022-06-23) Van Deventer, Barbara Stroh; Du Toit-Prinsloo, L.; Van Niekerk, Chantal
BACKGROUND : Formalin-fixed paraffin-embedded (FFPE) tissue archives in hospitals, biobanks, and others offer a vast collection of extensive, readily available specimens for molecular testing. Unfortunately, the use of tissue samples for molecular diagnostic applications is challenging; thus, the forensic pathology FFPE tissue archives in Africa have been a largely unexploited genetic resource, with the usability of DNA obtainable from these samples being unknown. INTERVENTION : The study, conducted from January 2015 to August 2016, determined the usefulness of FFPE tissue as a reliable source of genetic material for successful post-mortem molecular applications and diagnostics. Formalin-fixed paraffin-embedded tissue samples were collected and archived from autopsies conducted over 13 years in the forensic medicine department of the University of Pretoria (Pretoria, South Africa). Deoxyribonucleic acid from FFPE tissue samples and control blood samples was amplified by high-resolution melt realtime polymerase chain reaction before sequencing. The procurement parameters and fixation times were compared with the quantity and quality of the extracted DNA and the efficiency of its subsequent molecular applications. LESSONS LEARNT : This study has shown that FFPE samples are still usable in molecular forensics, despite inadequate sample preparation, and offer immense value to forensic molecular diagnostics. RECOMMENDATIONS : FFPE samples fixed in formalin for more than 24 h should still be used in molecular diagnostics or research, as long as the primer design targets amplicons not exceeding 300 base pairs.
Postmortem genetic testing in young individuals : what clinical medical practitioners need to know
(Health and Medical Publishing Group, 2022-12) Van Deventer, Barbara Stroh; Du Toit-Prinsloo, Lorraine; Van Niekerk, Chantal; u26376645@tuks.co.za
The death of a young person is most often a tragic occurrence, more so when this death was unexpected. Forensic pathologists are mandated to investigate such deaths, and there has been a strong move internationally towards genetic testing as an additional investigative tool. The aim of our article is to bring the advantage of implementing the so-called molecular autopsy in a local setting to the attention of medical practitioners. When a multidisciplinary approach is taken in cases of sudden unexpected death, the benefits to family members, and society as a whole, are irrefutable.
Cardiovascular deaths : what do the genes say?
(Health and Medical Publishing Group, 2022-12) Van Deventer, Barbara Stroh; Du Toit-Prinsloo, Lorraine; Van Niekerk, Chantal; u26376645@tuks.co.za
Cardiovascular diseases (CVDs) are ever-increasing, and as such are considered to be one of the most concerning public health burdens worldwide. They remain the leading cause of death across the world (~17.7 million deaths were reported in 2015), accounting for 31% of all global deaths.
Decline in antibody responses to SARS-CoV-2 post-vaccination poses a risk to health care workers
(Elsevier, 2022-09) Worsley, Catherine M.; Van der Mescht, Mieke Adri; Hoffmann, Daniel; Meyer, Pieter Willem Adriaan; Ueckermann, Veronica; Rossouw, Theresa M.; u05116602@up.ac.za
No abstract available.
Identification of selective Lyn inhibitors from the chemical databases through integrated molecular modelling approaches
(Taylor and Francis, 2021) Shetve, V.V.; Bhowmick, Shovonlal; Alissa, Siham A.; Alothman, Z.A.; Wabaidu, S.M.; Asmary, F.A.; Alhajri, Hassna Mohammed; Islam, Md Ataul
In the current study, the Asinex and ChEBI databases were virtually screened for the identification of potential Lyn protein inhibitors. Therefore, a multi-steps molecular docking study was carried out using the VSW utility tool embedded in Maestro user interface of the Schrödinger suite. On initial screening, molecules having a higher XP-docking score and binding free energy compared to Staurosporin were considered for further assessment. Based on in silico pharmacokinetic analysis and a common-feature pharmacophore mapping model developed from the Staurosporin, four molecules were proposed as promising Lyn inhibitors. The binding interactions of all proposed Lyn inhibitors revealed strong ligand efficiency in terms of energy score obtained in molecular modelling analyses. Furthermore, the dynamic behaviour of each molecule in association with the Lyn protein-bound state was assessed through an all-atoms molecular dynamics (MD) simulation study. MD simulation analyses were confirmed with notable intermolecular interactions and consistent stability for the Lyn protein-ligand complexes throughout the simulation. High negative binding free energy of identified four compounds calculated through MM-PBSA approach demonstrated a strong binding affinity towards the Lyn protein. Hence, the proposed compounds might be taken forward as potential next-generation Lyn kinase inhibitors for managing numerous Lyn associated diseases or health complications after experimental validation.
Spinal epidural abscess caused by Aspergillus spp masquerading as spinal tuberculosis in a person with HIV
(Elsevier, 2021-11) Rule, Roxanne; Mitton, Barend C.; Govender, Nelesh P.; Hoffmann, Daniel; Said, Mohamed
Spinal epidural abscess caused by Aspergillus spp is a debilitating form of invasive aspergillosis that can easily be misdiagnosed as spinal tuberculosis due to shared risk factors and clinical features. In this Grand Round, we describe a case of thoracic aspergillus spinal epidural abscess in a patient with underlying HIV infection. The initial diagnostic consideration was that of spinal tuberculosis. Consequently, despite positive microbiological cultures of Aspergillus fumigatus, antifungal therapy was delayed until histopathological evaluation of the affected tissue confirmed the presence of fungal hyphae. The patient showed an initial favourable response after surgical removal of the infected focus, but unfortunately never returned to premorbid functioning. This case highlights the importance of early diagnosis, urgent surgery, and prompt antifungal therapy for the management of aspergillus spinal epidural abscesses. Associated morbidity and mortality can be substantially increased if physicians fail to recognise this condition and do not institute appropriate and timely surgical and medical treatment.
Evaluation of serological assays for the diagnosis of HIV infection in adults
(AOSIS, 2021-10) Bangalee, Avania; Bhoora, Sachin; Punchoo, Rivak; rivak.punchoo@up.ac.za
Serological tests based on the enzyme immunoassay (EIA) are the primary tool for the diagnosis of human immunodeficiency virus (HIV) in adults and have rapidly evolved to quicker, affordable and more accurate test formats to detect early HIV infection. Secondand third-generation HIV rapid tests detect the immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies to the HIV and are used at the point of care and in HIV self-testing. The tests are affordable and accessible in state and private diagnostic laboratories. The presentday fourth- and fifth-generation EIAs can detect both p24 antigen and IgG and IgM HIV antibodies and thereby diagnose early HIV infection at approximately 2 weeks. The fourthand fifth-generation EIAs also report sensitivity and specificity of more than 99%. The correct interpretation of HIV diagnosis of false-positive and false-negative EIA test results requires collaborative scrutiny of patient factors and laboratory test methodologies.
Structure‑based identification of SARS‑CoV‑2 main protease inhibitors from anti‑viral specific chemical libraries : an exhaustive computational screening approach
(Springer, 2021-08) Bhowmick, Shovonlal; Saha, Achintya; Osman, Sameh Mohamed; Alasmary, Fatmah Ali; Almutairi, Tahani Mazyad; Islam, Md Ataul
Worldwide coronavirus disease 2019 (COVID-19) outbreak is still threatening global health since its outbreak first reported in the late 2019. The causative novel virus has been designated as severe acute respiratory syndrome coronavirus 2 (SARSCoV- 2). Although COVID-19 emergent with significant mortality, there is no availability of definite treatment measures. It is now extremely desirable to identify potential chemical entities against SARS-CoV-2 for the treatment of COVID-19. In the present study, a state-of-art virtual screening protocol was implemented on three anti-viral specific chemical libraries against SARS-CoV-2 main protease ( Mpro). Particularly, viewing the large-scale biological role of Mpro in the viral replication process it has been considered as a prospective anti-viral drug target. Herein, on collected 79,892 compounds, hierarchical multistep docking followed by relative binding free energy estimation has been performed. Thereafter, implying a user-defined XP-dock and MM-GBSA cut-off scores as −8.00 and −45.00 kcal/mol, chemical space has been further reduced. Exhaustive molecular binding interactions analyses and various pharmacokinetics profiles assessment suggested four compounds (ChemDiv_D658-0159, ChemDiv_F431-0433, Enamine_Z3019991843 and Asinex_LAS_51389260) as potent inhibitors/modulators of SARS-CoV-2 Mpro. In-depth protein–ligand interactions stability in the dynamic state has been evaluated by 100 ns molecular dynamics (MD) simulation studies along with MM-GBSA-based binding free energy estimations of entire simulation trajectories that have revealed strong binding affinity of all identified compounds towards Mpro. Hence, all four identified compounds might be considered as promising candidates for future drug development specifically targeting the SARS-CoV-2 Mpro; however, they also need experimental assessment for a better understanding of molecular interaction mechanisms.
Flow cytometric analysis of apoptotic biomarkers in actinomycin D-treated SiHa cervical cancer cells
(MyJove Corporation, 2021-08-26) Punchoo, Rivak; Zhou, Esther; Bhoora, Sachin; rivak.punchoo@up.ac.za
Apoptosis biomarkers were investigated in actinomycin D-treated SiHa cervical cancer cells using a benchtop flow cytometer. Early biomarkers (Annexin V and mitochondrial membrane potential) and late biomarkers (caspases 3 and 7, and DNA damage) of apoptosis were measured in experimental and control cultures. Cultures were incubated for 24 hours in a humidified incubator at 37 °C with 5% CO2. The cells were then detached using trypsin and enumerated using a flow cytometric cell count assay. Cells were further analyzed for apoptosis using an Annexin V assay, a mitochondrial electrochemical transmembrane potential assay, a caspase 3/7 assay, and a DNA damage assay. This article provides an overview of apoptosis and traditional flow cytometry, and elaborates flow cytometric protocols for processing and analyzing SiHa cells. The results describe positive, negative, and sub-optimal experimental data. Also discussed are interpretation and caveats in performing flow cytometric analysis of apoptosis using this analytical platform. Flow cytometric analysis provides an accurate measurement of early and late biomarkers for apoptosis.
Pharmacoinformatics approach based identification of potential Nsp15 endoribonuclease modulators for SARS-CoV-2 inhibition
(Elsevier, 2021-03) Savale, Rutuja Umesh; Bhowmick, Shovonlal; Osman, Sameh Mohamed; Alasmary, Fatmah Ali; Almutairi, Tahani Mazyad; Abdullah, Dalal Saied; Patil, Pritee Chunarkar; Islam, Md Ataul
In the current study, a structure-based virtual screening paradigm was used to screen a small molecular database against the Non-structural protein 15 (Nsp15) endoribonuclease of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 is the causative agent of the recent outbreak of coronavirus disease 2019 (COVID-19) which left the entire world locked down inside the home. A multi-step molecular docking study was performed against antiviral specific compounds (~8722) collected from the Asinex antiviral database. The less or non-interacting molecules were wiped out sequentially in the molecular docking. Further, MM-GBSA based binding free energy was estimated for 26 compounds which shows a high affinity towards the Nsp15. The drug-likeness and pharmacokinetic parameters of all 26 compounds were explored, and five molecules were found to have an acceptable pharmacokinetic profile. Overall, the Glide-XP docking score and Prime-MM-GBSA binding free energy of the selected molecules were explained strong interaction potentiality towards the Nsp15 endoribonuclease. The dynamic behavior of each molecule with Nsp15 was assessed using conventional molecular dynamics (MD) simulation. The MD simulation information was strongly favors the Nsp15 and each identified ligand stability in dynamic condition. Finally, from the MD simulation trajectories, the binding free energy was estimated using the MM-PBSA method. Hence, the proposed final five molecules might be considered as potential Nsp15 modulators for SARS-CoV-2 inhibition.

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