Management of the COVID-19 pandemic requires widespread SARS-CoV-2 testing. A main limitation for widespread SARS-CoV-2 testing is the global shortage of essential supplies, among these, RNA extraction kits. The need for commercial RNA extraction kits places a bottleneck on tests that detect SARS-CoV-2 genetic material, including PCR-based reference tests. Here we propose an alternative method we call PEARL (Precipitation Enhanced Analyte RetrievaL) that addresses this limitation. PEARL uses a lysis solution that disrupts cell membranes and viral envelopes while simultaneously providing conditions suitable for alcohol-based precipitation of RNA, DNA, and proteins. PEARL is a fast, low-cost, and simple method that uses common laboratory reagents and offers comparable performance to commercial RNA extraction kits. PEARL offers an alternative method to isolate host and pathogen nucleic acids and proteins to streamline the detection of DNA and RNA viruses, including SARS-CoV-2.

Whole blood fibrin clots capture platelets and release growth factors, and the addition of ASCs increases VEGF release for up to 2 weeks after clot formation. This suggests that whole blood fibrin clots may be a viable scaffold and delivery vehicle for future stem cell treatments.

The reversibility of chemotherapy-induced peripheral neuropathy (CIPN), a disabling and potentially permanent side effect of microtubule-targeting agents (MTAs), is becoming an increasingly important issue as treatment outcomes improve. The molecular mechanisms regulating the variability in time to onset, severity, and time to recovery from CIPN between the common MTAs paclitaxel and eribulin are unknown. Previously (Benbow et al. in Neurotox Res 29:299-313, 2016), we found that after 2 weeks of a maximum tolerated dose (MTD) in mice, paclitaxel treatment resulted in severe reductions in axon area density, higher frequency of myelin abnormalities, and increased numbers of Schwann cell nuclei in sciatic nerves. Biochemically, eribulin induced greater microtubule-stabilizing effects than paclitaxel. Here, we extended these comparative MTD studies to assess the recovery from these short-term effects of paclitaxel, eribulin, and a third MTA, ixabepilone, over the course of 6 months. Paclitaxel induced a persistent reduction in axon area density over the entire 6-month recovery period, unlike ixabepilone- or eribulin-treated animals. The abundance of myelin abnormalities rapidly declined after cessation of all drugs but recovered most slowly after paclitaxel treatment. Paclitaxel- and ixabepilone- but not eribulin-treated animals exhibited increased Schwann cell numbers during the recovery period. Tubulin composition and biochemistry rapidly returned from MTD-induced levels of α-tubulin, acetylated α-tubulin, and end-binding protein 1 to control levels following cessation of drug treatment. Taken together, sciatic nerve axons recovered more rapidly from morphological effects in eribulin- and ixabepilone-treated animals than in paclitaxel-treated animals and drug-induced increases in protein expression levels following paclitaxel and eribulin treatment were relatively transient.

Uncovering viral gene functions requires the modulation of gene expression through overexpression or loss-of-function. CRISPR interference (CRISPRi), a modification of the CRISPR-Cas9 gene editing technology, allows specific and efficient transcriptional silencing without genetic ablation. CRISPRi has been used to silence eukaryotic and prokaryotic genes at the single-gene and genome-wide levels. Here, we report the use of CRISPRi to silence latent and lytic viral genes, with an efficiency of ~80–90%, in epithelial and B-cells carrying multiple copies of the Kaposi’s sarcoma-associated herpesvirus (KSHV) genome. Our results validate CRISPRi for the analysis of KSHV viral elements, providing a functional genomics tool for studying virus–host interactions.

AbstractManagement of the COVID-19 pandemic requires widespread SARS-CoV-2 testing. A main limitation for widespread SARS-CoV-2 testing is the global shortage of essential supplies, among these, RNA extraction kits. The need for commercial RNA extraction kits places a bottleneck on tests that detect SARS-CoV-2 genetic material, including PCR-based reference tests. Here we propose an alternative method we call PEARL (Precipitation Enhanced Analyte RetrievaL) that addresses this limitation. PEARL uses a lysis solution that disrupts cell membranes and viral envelopes while simultaneously providing conditions suitable for alcohol-based precipitation of RNA, DNA, and proteins. PEARL is a fast, low-cost, and simple method that uses common laboratory reagents and offers comparable performance to commercial RNA extraction kits. PEARL offers an alternative method to isolate host and pathogen nucleic acids and proteins to streamline the detection of DNA and RNA viruses, including SARS-CoV-2.