Cov-MS: A Community-Based Template Assay for Mass-Spectrometry-Based Protein Detection in SARS-CoV-2 Patients.

Rising population density and global mobility are among the reasons why pathogens such as SARS-CoV-2, the virus that causes COVID-19, spread so rapidly across the globe. The policy response to such pandemics will always have to include accurate monitoring of the spread, as this provides one of the few alternatives to total lockdown. However, COVID-19 diagnosis is currently performed almost exclusively by reverse transcription polymerase chain reaction (RT-PCR). Although this is efficient, automatable, and acceptably cheap, reliance on one type of technology comes with serious caveats, as illustrated by recurring reagent and test shortages. We therefore developed an alternative diagnostic test that detects proteolytically digested SARS-CoV-2 proteins using mass spectrometry (MS). We established the Cov-MS consortium, consisting of 15 academic laboratories and several industrial partners to increase applicability, accessibility, sensitivity, and robustness of this kind of SARS-CoV-2 detection. This, in turn, gave rise to the Cov-MS Digital Incubator that allows other laboratories to join the effort, navigate, and share their optimizations and translate the assay into their clinic. As this test relies on viral proteins instead of RNA, it provides an orthogonal and complementary approach to RT-PCR using other reagents that are relatively inexpensive and widely available, as well as orthogonally skilled personnel and different instruments. Data are available via ProteomeXchange with identifier PXD022550.

Multiple co-occurring and persistently detected cyanotoxins and associated cyanobacteria in adjacent California lakes.

The global proliferation of toxin producing cyanobacterial blooms has been attributed to a wide variety of environmental factors with nutrient pollution, increased temperatures, and drought being three of the most significant. The current study is the first formal assessment of cyanotoxins in two impaired lakes, Canyon Lake and Lake Elsinore, in southern California that have a history of cyanobacterial blooms producing high biomass as measured by chl-a. Cyanotoxins in Lake Elsinore were detected at concentrations that persistently exceeded California recreational health thresholds, whereas Canyon Lake experienced persistent concentrations that only occasionally exceeded health thresholds. The study results are the highest recorded concentrations of microcystins, anatoxin-a, and cylindrospermopsin detected in southern California lakes. Concentrations exceeded health thresholds that caused both lakes to be closed for recreational activities. Cyanobacterial identifications indicated a high risk for the presence of potentially toxic genera and agreed with the cyanotoxin results that indicated frequent detection of multiple cyanotoxins simultaneously. A statistically significant correlation was observed between chlorophyll-a (chl-a) and microcystin concentrations for Lake Elsinore but not Canyon Lake, and chl-a was not a good indicator of cylindrospermopsin, anatoxin-a, or nodularin. Therefore, chl-a was not a viable screening indicator of cyanotoxin risk in these lakes. The study results indicate potential acute and chronic risk of exposure to cyanotoxins in these lakes and supports the need for future monitoring efforts to help minimize human and domestic pet exposure and to better understand potential effects to wildlife. The frequent co-occurrence of complex cyanotoxin mixtures further complicates the risk assessment process for these lakes given uncertainty in the toxicology of mixtures.

Mutations in the S-Adenosylmethionine Synthetase Genes SAM1 and SAM2 Differentially Affect Genome Stability in Saccharomyces cerevisiae.

Maintenance of genome integrity is a crucial cellular focus that involves a wide variety of proteins functioning in multiple processes. Defects in many different pathways can result in genome instability, a hallmark of cancer. Utilizing a diploid Saccharomyces cerevisiae model, we previously reported a collection of gene mutations that affect genome stability in a haploinsufficient state. In this work we explore the effect of gene dosage on genome instability for one of these genes and its paralog; SAM1 and SAM2 These genes encode S-Adenosylmethionine (AdoMet) synthetases, responsible for the creation of AdoMet from methionine and ATP. AdoMet is the universal methyl donor for methylation reactions and is essential for cell viability. It is the second most used cellular enzyme substrate and is exceptionally well-conserved through evolution. Mammalian cells express three genes, MAT1A, MAT2A, and MAT2B, with distinct expression profiles and functions. Alterations to these AdoMet synthetase genes, and AdoMet levels, are found in many cancers, making them a popular target for therapeutic intervention. However, significant variance in these alterations are found in different tumor types, with the cellular consequences of the variation still unknown. By studying this pathway in the yeast system, we demonstrate that losses of SAM1 and SAM2 have different effects on genome stability through distinctive effects on gene expression and AdoMet levels, and ultimately separate effects on the methyl cycle. Thus, this study provides insight into the mechanisms by which differential expression of the SAM genes have cellular consequences that affect genome instability.

Toxin composition of the 2016 Microcystis aeruginosa bloom in the St. Lucie Estuary, Florida.

A bloom of the cyanobacteria, Microcystis aeruginosa occurred in the St. Lucie Estuary during the summer of 2016, stimulated by the release of waters from Lake Okeechobee. This cyanobacterium produces the microcystins, a suite of heptapeptide hepatotoxins. The toxin composition of the bloom was analyzed and was compared to an archived bloom sample from 2005. Microcystin-LR was the most abundant toxin with lesser amounts of microcystin variants. Nodularin, cylindrospermopsin and anatoxin-a were not detected.

Microcystin production and ecological physiology of Caribbean black band disease cyanobacteria.

Molecular studies of black band disease (BBD), a coral disease found on tropical and subtropical reefs worldwide, have shown that one 16S rRNA gene sequence is ubiquitous. This sequence has been reported to be a member of the cyanobacterial genus Oscillatoria. In this study, extracts of two cultured laboratory strains of BBD Oscillatoria, and for comparison two strains of BBD Geitlerinema, all isolated from reefs of the wider Caribbean, were analysed using Ultra-Performance Liquid Chromatography-Tandem Quad Mass Spectrometry (UPLC-MS/MS). The cyanotoxin microcystin-LR (MC-LR) was found in all strains, and one Geitlerinema strain additionally produced MC-YR. Growth experiments that monitored toxin production using enzyme-linked immunosorbent assay (ELISA) showed that BBD Oscillatoria produced yields of MC-LR equivalent (0.02-0.04 mg g(-1)) independent of biomass and culture conditions (varying temperature, pH, light and organic carbon). This pattern is different from BBD Geitlerinema, which increased production of MC-LR equivalent in the presence of organic carbon in the light and dark and at a relatively lower temperature. These results indicate that different species and strains of BBD cyanobacteria, which can occur in the same BBD infection, may contribute to BBD pathobiology by producing different toxins and different amounts of toxin at different stages in the disease process. This is the first detailed study of laboratory cultures of the ubiquitous BBD cyanobacterium Oscillatoria sp. isolated from Caribbean reefs.

Microbial atrazine breakdown in a karst groundwater system and its effect on ecosystem energetics.

In the absence of sunlight energy, microbial community survival in subterranean aquifers depends on integrated mechanisms of energy and nutrient scavenging. Because karst aquifers are particularly sensitive to agricultural land use impacts due to rapid and direct hydrologic connections for pollutants to enter the groundwater, we examined the fate of an exogenous pesticide (atrazine) into such an aquifer and its impact on microbial ecosystem function. Atrazine and its degradation product deethylatrazine (DEA) were detected in a fast-flowing karst aquifer underlying atrazine-impacted agricultural land. By establishing microbial cultures with sediments from a cave conduit within this aquifer, we observed two distinct pathways of microbial atrazine degradation: (i) in cave sediments previously affected by atrazine, apparent surface-derived catabolic genes allowed the microbial communities to rapidly degrade atrazine via hydroxyatrazine, to cyanuric acid, and (ii) in low-impact sediments not previously exposed to this pesticide, atrazine was also degraded by microbial activity at a much slower rate, with DEA as the primary degradation product. In sediments from both locations, atrazine affected nitrogen cycling by altering the abundance of nitrogen dissimulatory species able to use nitrogenous compounds for energy. The sum of these effects was that the presence of atrazine altered the natural microbial processes in these cave sediments, leading to an accumulation of nitrate. Such changes in microbial ecosystem dynamics can alter the ability of DEA to serve as a proxy for atrazine contamination and can negatively affect ecosystem health and water quality in karst aquifers.

Detection of various freshwater cyanobacterial toxins using ultra-performance liquid chromatography tandem mass spectrometry.

Several freshwater cyanobacteria species have the capability to produce toxic compounds, frequently referred to as cyanotoxins. The most prevalent of these cyanotoxins is microcystin LR. Recognizing the potential health risk, France, Italy, Poland, Australia, Canada, and Brazil have set either standards or guidelines for the amount of microcystin LR permissible in drinking water based on the World Health Organization guideline of one microg/L of microcystin LR. Recently, the United States Environmental Protection Agency has begun to evaluate the occurrence and health effects of cyanotoxins and their susceptibility to water treatment under the Safe Drinking Water Act through the Contaminant Candidate List (CCL). A recent update of the Contaminant Candidate List focuses research and data collection on the cyanotoxins microcystin LR, anatoxin-a, and cylindrospermopsin. Liquid Chromatography/Tandem-Mass Spectrometry (LC/MS/MS) is a powerful tool for the analysis of various analytes in a wide variety of matrices because of its sensitivity and selectivity. The use of smaller column media (sub 2 microm particles) was investigated to both improve the speed, sensitivity and resolution, and to quantify the CCL cyanotoxins, in a single analysis, using Ultra-Performance Liquid Chromatography (UPLC) combined with tandem mass spectrometry. Natural waters and spiked samples were analyzed to show proof-of-performance. The presented method was able to clearly resolve each of the cyanotoxins in less than eight minutes with specificity and high spike recoveries.

Use of attenuated total reflectance Fourier transform infrared spectroscopy to identify microbial metabolic products on carbonate mineral surfaces.

This paper demonstrates the use of attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy to detect microbial metabolic products on carbonate mineral surfaces. By creating an ATR-FTIR spectral database for specific organic acids using ATR-FTIR spectroscopy we were able to distinguish metabolic acids on calcite surfaces following Escherichia coli growth. The production of these acids by E. coli was verified using high-performance liquid chromatography with refractive index detection. The development of this technique has allowed us to identify microbial metabolic products on carbonate surfaces in nutrient-limited cave environments.

Separation of substituted fullerenes using non-aqueous reversed-phase liquid chromatography-mass spectrometry.

A simple non-aqueous reversed-phase separation HPLC-MS method has been developed to allow for the rapid screening and separation of fullerenes and substituted fullerenes. This procedure provides confirmation that the proposed substitution methodology for the fullerene is not only successful but that multiple substitution products are obtained. The methodology is being expanded to analyze other substituted fullerene product mixtures.

Synthesis of a new ligand for transition metal-fullerene supramolecular systems.

A new ligand has been designed that provides a relatively simple framework to build supramolecular systems containing both fullerene and transition-metal moieties. The modular framework of the ligand allows for the easy design of more complex systems. Analysis of absorption and emission spectra suggests significant photoinduced charge transfer between the two moieties. More complex systems and the excited-state photophysics of the presented systems are being studied.