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SARS coronavirus 2 (SARS-CoV-2) has caused an ongoing global pandemic with significant mortality and morbidity. At this time, the only FDA-approved therapeutic for COVID-19 is remdesivir, a broad-spectrum antiviral nucleoside analog. Efficacy is only moderate, and improved treatment strategies are urgently needed. To accomplish this goal, we devised a strategy to identify compounds that act synergistically with remdesivir in preventing SARS-CoV-2 replication. We conducted combinatorial high-throughput screening in the presence of submaximal remdesivir concentrations, using a human lung epithelial cell line infected with a clinical isolate of SARS-CoV-2. This identified 20 approved drugs that act synergistically with remdesivir, many with favorable pharmacokinetic and safety profiles. Strongest effects were observed with established antivirals, Hepatitis C virus nonstructural protein 5A (HCV NS5A) inhibitors velpatasvir and elbasvir. Combination with their partner drugs sofosbuvir and grazoprevir further increased efficacy, increasing remdesivir’s apparent potency > 25-fold. We report that HCV NS5A inhibitors act on the SARS-CoV-2 exonuclease proofreader, providing a possible explanation for the synergy observed with nucleoside analog remdesivir. FDA-approved Hepatitis C therapeutics Epclusa® (velpatasvir/sofosbuvir) and Zepatier® (elbasvir/grazoprevir) could be further optimized to achieve potency and pharmacokinetic properties that support clinical evaluation in combination with remdesivir.

Single-molecule imaging provides a powerful way to study biochemical processes in live cells, yet it remains challenging to track single molecules while simultaneously detecting their interactions. Here, we describe a novel property of rhodamine dyes, proximity-assisted photoactivation (PAPA), in which one fluorophore (the 'sender') can reactivate a second fluorophore (the 'receiver') from a dark state. PAPA requires proximity between the two fluorophores, yet it operates at a longer average intermolecular distance than Förster resonance energy transfer (FRET). We show that PAPA can be used in live cells both to detect protein-protein interactions and to highlight a subpopulation of labeled protein complexes in which two different labels are in proximity. In proof-of-concept experiments, PAPA detected the expected correlation between androgen receptor self-association and chromatin binding at the single-cell level. These results establish a new way in which a photophysical property of fluorophores can be harnessed to study molecular interactions in single-molecule imaging of live cells.A human body is made up of trillions of cells, each containing millions of proteins working to keep our bodies going. Since the invention of the microscope four hundred years ago, scientists have made large strides in visualizing cells and even single protein molecules within cells. To do this, proteins of interest are labeled with fluorescent dyes that absorb – or are ‘excited’ by – light of one color, and then give off light of a different color. The labeled proteins are excited by a powerful laser, and a sensitive camera detects the light emitted by single molecules of dye. This technique is called single-particle tracking (SPT), and it can reveal how proteins move around inside a cell. Because most proteins work together in teams or complexes, it would be useful to track the movement of proteins while at the same time observing their interactions. Unfortunately, SPT does not typically allow scientists to watch how proteins interact with each other. Graham et al. accidentally discovered how to do precisely this. First, they labeled proteins with two different colored dyes. Then, the dyes were excited using alternating red and green lasers. Repeated excitation destroys the fluorescent dye molecules, and sure enough, red-excited dye molecules went dark over time. Unexpectedly, however, molecules of the dye that had been excited with red light reappeared after exciting the second dye with green light. The fluorescent molecules were not dead, just sleeping. ‘Resuscitating’ one dye with the other required that they be close together, and therefore this process was called proximity-assisted photoactivation (PAPA for short). PAPA was able to detect interactions between proteins labeled with different dyes in live human cells, and combining PAPA with SPT allowed Graham et al. to distinguish protein molecules labeled with two different dyes from those labeled with a single dye. Finally, Graham et al. labeled molecules of the androgen receptor protein with two different dyes to monitor how they responded to testosterone. Combining PAPA and SPT measurements successfully detected the pairing of androgen receptor molecules, as well as increased binding of these paired androgen receptor molecules to DNA. This new way of observing how proteins interact will be useful for studying where and how fast these interactions happen in living cells. Understanding how teams of proteins work together under normal conditions will also shed light on how they misbehave in diseases.

The most common method for RNA detection involves reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR) analysis. Commercial one-step master mixes-which include both a reverse transcriptase and a thermostable polymerase and thus allow performing both the RT and qPCR steps consecutively in a sealed well-are key reagents for SARS-CoV-2 diagnostic testing; yet, these are typically expensive and have been affected by supply shortages in periods of high demand. As an alternative, we describe here how to express and purify Taq polymerase and M-MLV reverse transcriptase and assemble a homemade one-step RT-qPCR master mix. This mix can be easily assembled from scratch in any laboratory equipped for protein purification. We also describe two simple alternative methods to prepare clinical swab samples for SARS-CoV-2 RNA detection by RT-qPCR: heat-inactivation for direct addition, and concentration of RNA by isopropanol precipitation. Finally, we describe how to perform RT-qPCR using the homemade master mix, how to prepare in vitro-transcribed RNA standards, and how to use a fluorescence imager for endpoint detection of RT-PCR amplification in the absence of a qPCR machine In addition to being useful for diagnostics, these versatile protocols may be adapted for nucleic acid quantification in basic research. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of a one-step RT-qPCR master mix using homemade enzymes Basic Protocol 2: Preparation of swab samples for direct RT-PCR Alternate Protocol 1: Concentration of RNA from swab samples by isopropanol precipitation Basic Protocol 3: One-step RT-qPCR of RNA samples using a real-time thermocycler Support Protocol: Preparation of RNA concentration standards by in vitro transcription Alternate Protocol 2: One-step RT-PCR using endpoint fluorescence detection.

Clinicopathological data have been collected for 42 patients with cerebellar medulloblastoma diagnosed and treated in Western Australia between the years 1961 and 1984. Thirty-one patients were male and 12 were over 14 years of age. In 31 patients the neoplasms were localized to the midline, while in 10 they were placed laterally in the cerebellar hemispheres. The remaining patient had a diffusely spreading neoplasm in the meninges of the posterior fossa. Thirty-nine underwent surgical treatment followed by postoperative radiotherapy. Three patients died in the early postoperative period. Twenty-four patients survived for one year or longer, 15 for two or more years, 10 for five years, and five survived longer than 10 years. The survival proportions estimated by the life-table method were 66% at one year, 48% at two years and 32% at 5 and 10 years. In one patient who survived for 23 years a meningioma developed, possibly due to radiotherapy.

Paratuberculosis was studied for three years in a large goat herd. Post mortem lesions were seen mainly in the ileum and mesenteric lymph nodes where caseation was often present. In three successive years the annual faecal excretor rate was 15, 9 and 13 per cent despite removal of positives. Faecal culture and an agar gel immunodiffusion test proved useful for detecting the disease in live goats, but complement fixation and tuberculin tests were of less value. The prospects of controlling the disease are discussed.