Reproducibility and Sensitivity of Thirty-Six Methods to Quantify the SARS-CoV-2 Genetic Signal in Raw Wastewater: Findings from an Interlaboratory Methods Evaluation in the U.S.

In response to COVID-19, the international water community rapidly developed methods to quantify the SARS-CoV-2 genetic signal in untreated wastewater. Wastewater surveillance using such methods has the potential to complement clinical testing in assessing community health. This interlaboratory assessment evaluated the reproducibility and sensitivity of 36 standard operating procedures (SOPs), divided into eight method groups based on sample concentration approach and whether solids were removed. Two raw wastewater samples were collected in August 2020, amended with a matrix spike (betacoronavirus OC43), and distributed to 32 laboratories across the U.S. Replicate samples analyzed in accordance with the project\u27s quality assurance plan showed high reproducibility across the 36 SOPs: 80% of the recoverycorrected results fell within a band of ±1.15 log10 genome copies per L with higher reproducibility observed within a single SOP (standard deviation of 0.13 log10). The inclusion of a solids removal step and the selection of a concentration method did not show a clear, systematic impact on the recovery-corrected results. Other methodological variations (e.g., pasteurization, primer set selection, and use of RT-qPCR or RT-dPCR platforms) generally resulted in small differences compared to other sources of variability. These findings suggest that a variety of methods are capable of producing reproducible results, though the same SOP or laboratory should be selected to track SARS-CoV-2 trends at a given facility. The methods showed a 7 log10 range of recovery efficiency and limit of detection highlighting the importance of recovery correction and the need to consider method sensitivity when selecting methods for wastewater surveillance

Novel Retinoic Acid Receptor Alpha Agonists for Treatment of Kidney Disease

Development of pharmacologic agents that protect podocytes from injury is a critical strategy for the treatment of kidney glomerular diseases. Retinoic acid reduces proteinuria and glomerulosclerosis in multiple animal models of kidney diseases. However, clinical studies are limited because of significant side effects of retinoic acid. Animal studies suggest that all trans retinoic acid (ATRA) attenuates proteinuria by protecting podocytes from injury. The physiological actions of ATRA are mediated by binding to all three isoforms of the nuclear retinoic acid receptors (RARs): RARα, RARβ, and RARγ. We have previously shown that ATRA exerts its renal protective effects mainly through the agonism of RARα. Here, we designed and synthesized a novel boron-containing derivative of the RARα-specific agonist Am580. This new derivative, BD4, binds to RARα receptor specifically and is predicted to have less toxicity based on its structure. We confirmed experimentally that BD4 binds to RARα with a higher affinity and exhibits less cellular toxicity than Am580 and ATRA. BD4 induces the expression of podocyte differentiation markers (synaptopodin, nephrin, and WT-1) in cultured podocytes. Finally, we confirmed that BD4 reduces proteinuria and improves kidney injury in HIV-1 transgenic mice, a model for HIV-associated nephropathy (HIVAN). Mice treated with BD4 did not develop any obvious toxicity or side effect. Our data suggest that BD4 is a novel RARα agonist, which could be used as a potential therapy for patients with kidney disease such as HIVAN

Nitrone [2]rotaxanes: Simultaneous chemical protection and electrochemical activation of a functional group

We report on the use of the hydrogen bond accepting properties of neutral nitrone moieties to prepare benzylic-amide-macrocycle-containing [2]rotaxanes in yields as high as 70 %. X-Ray crystallography shows the presence of up to four intercomponent hydrogen bonds between the amide groups of the macrocycle and the two nitrone groups of the thread. Dynamic 1H NMR studies of the rates of macrocycle pirouetting in nonpolar solutions indicate that amide-nitrone hydrogen bonds are particularly strong, ~1.3 and ~0.2 kcal mol-1 stronger than similar amide-ester and amide-amide interactions, respectively. In addition to polarizing the N-O bond through hydrogen bonding, the rotaxane structure affects the chemistry of the nitrone groups in two significant ways: The intercomponent hydrogen bonding activates the nitrone groups to electrochemical reduction, a one electron reduction of the rotaxane being stablized by a remarkable 400 mV (8.1 kcal mol-1) with respect to the same process in the thread; encapsulation, however, protects the same functional groups from chemical reduction with an external reagent (and slows down electron transfer to and from the electroactive groups in cyclicvoltammetry experiments). Mechanical interlocking with a hydrogen bonding molecular sheath thus provides a route to an encapsulated polarized functional group and radical anions of significant kinetic and thermodynamic stability