COVID-19 Sample Pooling: From RNA Extraction to Quantitative Real-time RT-PCR.

The COVID-19 pandemic requires mass screening to identify those infected for isolation and quarantine. Individually screening large populations for the novel pathogen, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is costly and requires a lot of resources. Sample pooling methods improve the efficiency of mass screening and consume less reagents by increasing the capacity of testing and reducing the number of experiments performed, and are therefore especially suitable for under-developed countries with limited resources. Here, we propose a simple, reliable pooling strategy for COVID-19 testing using clinical nasopharyngeal (NP) and/or oropharyngeal (OP) swabs. The strategy includes the pooling of 10 NP/OP swabs for extraction and subsequent testing via quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR), and may also be applied to the screening of other pathogens.

Phosphoinositide-dependent Kinase-1 (PDPK1) regulates serum/glucocorticoid-regulated Kinase 3 (SGK3) for prostate cancer cell survival.

Prostate cancer (PCa) is the most common malignancy and is the second leading cause of cancer among men globally. Using a kinome-wide lentiviral small-hairpin RNA (shRNA) library screen, we identified phosphoinositide-dependent kinase-1 (PDPK1) as a potential mediator of cell survival in PCa cells. We showed that knock-down of endogenous human PDPK1 induced significant tumour-specific cell death in PCa cells (DU145 and PC3) but not in the normal prostate epithelial cells (RWPE-1). Further analyses revealed that PDPK1 mediates cancer cell survival predominantly via activation of serum/glucocorticoid-regulated kinase 3 (SGK3). Knock-down of endogenous PDPK1 in DU145 and PC3 cells significantly reduced SGK3 phosphorylation while ectopic expression of a constitutively active SGK3 completely abrogated the apoptosis induced by PDPK1. In contrast, no such effect was observed in SGK1 and AKT phosphorylation following PDPK1 knock-down. Importantly, PDPK1 inhibitors (GSK2334470 and BX-795) significantly reduced tumour-specific cell growth and synergized docetaxel sensitivity in PCa cells. In summary, our results demonstrated that PDPK1 mediates PCa cells' survival through SGK3 signalling and suggest that inactivation of this PDPK1-SGK3 axis may potentially serve as a novel therapeutic intervention for future treatment of PCa.

A novel strategy for community screening of SARS-CoV-2 (COVID-19): Sample pooling method.

The rapid global spread of the coronavirus disease (COVID-19) has inflicted significant health and socioeconomic burden on affected countries. As positive cases continued to rise in Malaysia, public health laboratories experienced an overwhelming demand for COVID-19 screening. The confirmation of positive cases of COVID-19 has solely been based on the detection of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) using real-time reverse transcription polymerase chain reaction (qRT-PCR). In efforts to increase the cost-effectiveness and efficiency of COVID-19 screening, we evaluated the feasibility of pooling clinical Nasopharyngeal/Oropharyngeal (NP/OP) swab specimens during nucleic acid extraction without a reduction in sensitivity of qRT-PCR. Pools of 10 specimens were extracted and subsequently tested by qRT-PCR according to the WHO-Charité protocol. We demonstrated that the sample pooling method showed no loss of sensitivity. The effectiveness of the pooled testing strategy was evaluated on both retrospective and prospective samples, and the results showed a similar detection sensitivity compared to testing individual sample alone. This study demonstrates the feasibility of using a pooled testing strategy to increase testing capacity and conserve resources, especially when there is a high demand for disease testing.

Bacillus thuringiensis parasporal proteins induce cell-cycle arrest and caspase-dependant apoptotic cell death in leukemic cells.

Bacillus thuringiensis (Bt) parasporal proteins with selective anticancer activity have recently garnered interest. This study determines the efficacy and mode of cell death of Bt 18 parasporal proteins against 3 leukemic cell lines (CEM-SS, CCRF-SB and CCRF-HSB-2).Cell-based biochemical analysis aimed to determine cell viability and the percentage of apoptotic cell death in treated cell lines; ultrastructural analysis to study apoptotic changes and Western blot to identify the parasporal proteins' binding site were performed. Bt 18 parasporal proteins moderately decreased viability of leukemic cells but not that of normal human T lymphocytes. Further purification of the proteins showed changes in inhibition selectivity. Phosphatidylserine externalization, active caspase-3, cell cycle, and ultrastructural analysis confirmed apoptotic activity and S-phase cell-cycle arrest. Western blot analysis demonstrated glyceraldehyde 3-phosphate dehydrogenase as a binding protein. We suggest that Bt 18 parasporal proteins inhibit leukemic cell viability by cell-cycle arrest and apoptosis and that glyceraldehyde 3-phosphate dehydrogenase binding initiates apoptosis.

Development of a purification method of pure primary lymphocytes for cell viability assays

The maintenance of pure primary lymphocytes culture for long periods may be difficult because of its inability to divide continuously. In addition, lymphocytes separation methods such as Ficoll-Paque, RBC lysis and immunomagnetic microbeads separation may have some affect on cell viability. The objective of this study is to determine various types of lymphocytes purification methods, in order to prolong primary lymphocytes culture to 72 hours. The second objective is to use these primary lymphocytes as targets for quantitative and qualitative cell viability assays when analysing the action of toxins isolated from natural products. Human blood was drawn and purified by using Ficoll-Paque, RBC lysis or immunomagnetic separation column method in various combinations. The purified lymphocytes were also grown with and without the growth enhancement factor, concanavalin-A. Cell viability assays were carried out for 72 hours at 24 hours interval. The lymphocytes purified using RBC lysis method, with or without concanavalin-A can prolong 100% cell viability for 72 hours whilst lymphocytes purified using Ficoll-Paque and supplemented with concanavalin-A showed an increase in cell viability of over 250% at 72 hours incubation. It was observed only lymphocytes purified using Ficoll-Paque followed by the immunomagnetic microbeads separation method and supplemented with concanavalin-A showed overall cell viability increase, reaching 300% at 72 hours incubation. This method was a reliable model to test the cytotoxicity of the Bacillus thuringiensis parasporal inclusion, suggesting that the method achieves the objectives of the study.