Insights from a workplace SARS-CoV-2 specimen collection program, with genomes placed into global sequence phylogeny.

In 2020, the Department of Energy established the National Virtual Biotechnology Laboratory (NVBL) to address key challenges associated with COVID-19. As part of that effort, Pacific Northwest National Laboratory (PNNL) established a capability to collect and analyze specimens from employees who self-reported symptoms consistent with the disease. During the spring and fall of 2021, 688 specimens were screened for SARS-CoV-2, with 64 (9.3%) testing positive using reverse-transcriptase quantitative PCR (RT-qPCR). Of these, 36 samples were released for research. All 36 positive samples released for research were sequenced and genotyped. Here, the relationship between patient age and viral load as measured by Ct values was measured and determined to be only weakly significant. Consensus sequences for each sample were placed into a global phylogeny and transmission dynamics were investigated, revealing that the closest relative for many samples was from outside of Washington state, indicating mixing of viral pools within geographic regions.

Evaluating Linear Ion Trap for MS3-Based Multiplexed Single-Cell Proteomics.

There is a growing demand to develop high-throughput and high-sensitivity mass spectrometry methods for single-cell proteomics. The commonly used isobaric labeling-based multiplexed single-cell proteomics approach suffers from distorted protein quantification due to co-isolated interfering ions during MS/MS fragmentation, also known as ratio compression. We reasoned that the use of MS3-based quantification could mitigate ratio compression and provide better quantification. However, previous studies indicated reduced proteome coverages in the MS3 method, likely due to long duty cycle time and ion losses during multilevel ion selection and fragmentation. Herein, we described an improved MS acquisition method for MS3-based single-cell proteomics by employing a linear ion trap to measure reporter ions. We demonstrated that linear ion trap can increase the proteome coverages for single-cell-level peptides with even higher gain obtained via the MS3 method. The optimized real-time search MS3 method was further applied to study the immune activation of single macrophages. Among a total of 126 single cells studied, over 1200 and 1000 proteins were quantifiable when at least 50 and 75% nonmissing data were required, respectively. Our evaluation also revealed several limitations of the low-resolution ion trap detector for multiplexed single-cell proteomics and suggested experimental solutions to minimize their impacts on single-cell analysis.

Detection of SARS-COV-2 by functionally imprinted micelles.

The near real-time detection of airborne particles-of-interest is needed for avoiding current/future threats. The incorporation of imprinted particles into a micelle-based electrochemical cell produced a signal when brought into contact with particle analytes (such as SARS-COV-2), previously imprinted onto the structure. Nanoamp scales of signals were generated from what may've been individual virus-micelle interactions. The system showed selectivity when tested against similar size and morphology particles. The technology was compatible with airborne aerosol sampling techniques. Overall, the application of imprinted micelle technology could provide near real-time detection methods to a host of possible analytes of interest in the field. Supplementary Information: The online version contains supplementary material available at 10.1557/s43579-022-00242-0.

Distinct Preflowering Drought Tolerance Strategies of Sorghum bicolor Genotype RTx430 Revealed by Subcellular Protein Profiling.

Drought is the largest stress affecting agricultural crops, resulting in substantial reductions in yield. Plant adaptation to water stress is a complex trait involving changes in hormone signaling, physiology, and morphology. Sorghum (Sorghum bicolor (L.) Moench) is a C4 cereal grass; it is an agricultural staple, and it is particularly drought-tolerant. To better understand drought adaptation strategies, we compared the cytosolic- and organelle-enriched protein profiles of leaves from two Sorghum bicolor genotypes, RTx430 and BTx642, with differing preflowering drought tolerances after 8 weeks of growth under water limitation in the field. In agreement with previous findings, we observed significant drought-induced changes in the abundance of multiple heat shock proteins and dehydrins in both genotypes. Interestingly, our data suggest a larger genotype-specific drought response in protein profiles of organelles, while cytosolic responses are largely similar between genotypes. Organelle-enriched proteins whose abundance significantly changed exclusively in the preflowering drought-tolerant genotype RTx430 upon drought stress suggest multiple mechanisms of drought tolerance. These include an RTx430-specific change in proteins associated with ABA metabolism and signal transduction, Rubisco activation, reactive oxygen species scavenging, flowering time regulation, and epicuticular wax production. We discuss the current understanding of these processes in relation to drought tolerance and their potential implications.

Amylases in the Human Vagina.

Dominance of Lactobacillus species in vaginal communities is a hallmark of healthy conditions in the female genital tract. Key nutrients for lactobacilli include sugars produced when glycogen is degraded by α-amylase in the vagina. While α-amylase activity has been demonstrated in vaginal fluids, it is unclear whether α-amylases are produced solely by the host, bacteria in the vagina, or both. We screened cervicovaginal mucus from 23 reproductive-age women, characterized the species composition of vaginal communities, measured vaginal pH, and determined levels of amylase activity, glycogen, and lactic acid. Based on differences in these measured variables, one sample from each of four individual donors was selected for metagenomic and proteomic analyses. Of eight putative bacterial amylases identified in the assembled bacterial metagenomes, we detected four in vaginal fluids. These amylases were produced by various bacteria in different vaginal communities. Moreover, no two communities were the same in terms of which bacteria were producing amylases. Although we detected bacterial amylases in vaginal fluids, there was no clear association between the bacterial species that was dominant in a community and the level of amylase activity. This association was likely masked by the presence of human α-amylase, which was also detected in vaginal fluids. Finally, the levels of amylase activity and glycogen were only weakly associated. Our findings show, for the first time, that multiple amylases from both bacterial and human origins can be present simultaneously in the vagina. This work also suggests that the link between glycogen, amylase, and Lactobacillus in the vagina is complex.IMPORTANCE In this study, we show that multiple bacteria in the vaginal community produce amylases that hydrolyze glycogen into simpler sugars (i.e., maltose and maltotriose). These sugars serve as "common goods" that sustain bacterial populations in vaginal communities. Given the temporal changes that are observed in the human vaginal microbiome, we expect the kinds of bacterial amylases produced will also vary over time. These differences influence the pool of resources that are broadly shared and shape the species composition of the vaginal bacterial community.

Top-down mass spectrometry of histone modifications in sorghum reveals potential epigenetic markers for drought acclimation.

Sorghum [Sorghum bicolor (L.) Moench] is an important cereal crop noted for its ability to survive water-limiting conditions. Herein, we present an analytical workflow to explore the changes in histone modifications through plant developmental stages and two drought stresses in two sorghum genotypes that differ in their response to drought. Top-down mass spectrometry (MS) is an ideal method to profile histone modifications and distinguish closely related histone proteoforms. We analyzed leaves of 48 plants and identified 26 unique histone proteins and 677 unique histone proteoforms (124 full-length and 553 truncated proteoforms). We detected trimethylation on nearly all H2B N-termini where acetylation is commonly expected. In addition, an unexpected modification on H2A histones was assigned to N-pyruvic acid 2-iminylation based on its unique neutral loss of CO2. Interestingly, some of the truncated histones, in particular H4 and H3.2, showed significant changes that correlated with the growth and water conditions. The histone proteoforms could serve as targets in search of chromatin modifiers and ultimately have important ramifications in future attempts of studying plant epigenetic reprogramming under stress.

Endocrine Regulation of Epimorphic Regeneration.

Studies aiming to uncover primary mechanisms of regeneration have predominantly focused on genetic pathways regulating specific stages in the regeneration process: wound healing, blastema formation, and pattern formation. However, studies across organisms show that environmental conditions and the physiological state of the animal can affect the rate or quality of regeneration, and endocrine signals are likely the mediators of these effects. Endocrine signals acting directly on receptors expressed in the tissue or via neuroendocrine pathways can affect regeneration by regulating the immune response to injury, allocation of energetic resources, or by enhancing or inhibiting proliferation and differentiation pathways involved in regeneration. This review discusses the cumulative knowledge in the literature about endocrine regulation of regeneration and its importance in future research to advance biomedical research.

Endocrine regulation of regeneration: Linking global signals to local processes.

Regeneration in amphibians and reptiles has been explored since the early 18th century, giving us a working in vivo model to study epimorphic regeneration in vertebrates. Studies aiming to uncover primary mechanisms of regeneration have predominantly focused on genetic pathways regulating specific stages of the regeneration process: wound healing, blastema formation and growth, and pattern formation. However, studies across organisms show that environmental conditions and physiological state of the animal can affect the rate or quality of regeneration, and endocrine signals are likely the mediators of these effects. Endocrine signals working/acting directly on receptors expressed in the structure or via neuroendocrine pathways can affect regeneration by modulating immune response to injury, allocation of energetic resources, or by enhancing or inhibiting proliferation and differentiation pathways in regenerating tissue. This review discusses the cumulative knowledge known about endocrine regulation of regeneration and important future research directions of interest to both ecological and biomedical research.

Drought delays development of the sorghum root microbiome and enriches for monoderm bacteria.

Drought stress is a major obstacle to crop productivity, and the severity and frequency of drought are expected to increase in the coming century. Certain root-associated bacteria have been shown to mitigate the negative effects of drought stress on plant growth, and manipulation of the crop microbiome is an emerging strategy for overcoming drought stress in agricultural systems, yet the effect of drought on the development of the root microbiome is poorly understood. Through 16S rRNA amplicon and metatranscriptome sequencing, as well as root metabolomics, we demonstrate that drought delays the development of the early sorghum root microbiome and causes increased abundance and activity of monoderm bacteria, which lack an outer cell membrane and contain thick cell walls. Our data suggest that altered plant metabolism and increased activity of bacterial ATP-binding cassette (ABC) transporter genes are correlated with these shifts in community composition. Finally, inoculation experiments with monoderm isolates indicate that increased colonization of the root during drought can positively impact plant growth. Collectively, these results demonstrate the role that drought plays in restructuring the root microbiome and highlight the importance of temporal sampling when studying plant-associated microbiomes.

Geophysiology of Wood Frogs: Landscape Patterns of Prevalence of Disease and Circulating Hormone Concentrations across the Eastern Range.

One of the major challenges for conservation physiologists is to determine how current or future environmental conditions relate to the health of animals at the population level. In this study, we measured prevalence of disease, mean condition of the body, and mean resting levels of corticosterone and testosterone in a total of 28 populations across the years 2011 and 2012, and correlated these measures of health to climatic suitability of habitat, using estimates from a model of the ecological niche of the wood frog's geographic range. Using the core-periphery hypothesis as a theoretical framework, we predicted a higher prevalence and intensity of infection of Batrachochytrium dendrobatidis (Bd) and ranaviruses, two major amphibian pathogens causing disease, and higher resting levels of circulating corticosterone, an indicator of allostatic load incurred from living in marginal habitats. We found that Bd infections were rare (2% of individuals tested), while infections with ranavirus were much more common: ranavirus-infected individuals were found in 92% of ponds tested over the 2 years. Contrary to our predictions, rates of infection with ranaviruses were positively correlated with quality of the habitat with the highest prevalence at the core of the range, and plasma corticosterone concentrations measured when frogs were at rest were not correlated with quality of the habitat, the prevalence of ranavirus, or the intensity of infection. Prevalence and mean viral titers of ranavirus infection were higher in 2012 than in 2011, which coincided with lower levels of circulating corticosterone and testosterone and an extremely early time of breeding due to relatively higher temperatures during the winter. In addition, the odds of having a ranavirus infection increased with decreased body condition, and if animals had an infection, viral titers were positively correlated to levels of circulating testosterone concentration. By resolving these patterns, experiments can be designed to test hypotheses about the mechanisms that produce them, such as whether transmission of the ranavirus and tolerance of the host are greater or whether virulence is lower in populations within core habitats. While there is debate about which metrics serve as the best bioindicators of population health, the findings of this study demonstrate the importance of long-term monitoring of multiple physiological parameters to better understand the dynamic relationship between the environment and the health of wildlife populations over space and time.