Background Physicians treating COVID-19 patients increasingly believe that the hyperinflammatory acute stage of COVID-19 results in a cytokine storm. The circulating biomarkers seen across the spectrum of COVID-19 have not been characterized compared to healthy controls, but such analyses are likely to yield insights into the pursuit of interventions that adequately reduce the burden of these cytokine storms. Objective To identify and characterize the host inflammatory response to SARS-CoV-2 infection, we assessed levels of proteins related to immune responses and cardiovascular disease, in patients stratified as mild, moderate, and severe, versus matched healthy controls. Methods Blood samples from adult patients hospitalized with COVID-19 were analyzed using high-throughput and ultrasensitive proteomic platforms and compared with age- and sex-matched healthy controls to provide insights into differential regulation of 185 markers. Results Results indicate a dominant hyperinflammatory milieu in the circulation and vascular endothelial damage markers within COVID-19 patients, and strong biomarker association with patient response as measured by Ordinal scale. As patients progress, we observe statistically significant dysregulation of IFNγ, IL-1RA, IL-6, IL-10, IL-19, MCP-1, -2, -3, CXCL9, CXCL10, CXCL5, ENRAGE and PARP-1. Furthermore, in a limited series of patients who were sampled frequently confirming reliability and reproducibility of our assays, we demonstrate that intervention with baricitinib attenuates these circulating biomarkers associated with the cytokine storm. Conclusion These wide-ranging circulating biomarkers show an association with increased disease severity and may help stratify patients and selection of therapeutic options. They also provide insights into mechanisms of SARS-CoV-2 pathogenesis and the host response.

Plant growth and development are constantly influenced by temperature fluctuations. To respond to temperature changes, different levels of gene regulation are modulated in the cell. Alternative splicing (AS) is a widespread mechanism increasing transcriptome complexity and proteome diversity. Although genome-wide studies have revealed complex AS patterns in plants, whether AS impacts the stress defense of plants is not known. We used heat shock (HS) treatments at nondamaging temperature and messenger RNA sequencing to obtain HS transcriptomes in the moss Physcomitrella patens. Data analysis identified a significant number of novel AS events in the moss protonema. Nearly 50% of genes are alternatively spliced. Intron retention (IR) is markedly repressed under elevated temperature but alternative donor/acceptor site and exon skipping are mainly induced, indicating differential regulation of AS in response to heat stress. Transcripts undergoing heat-sensitive IR are mostly involved in specific functions, which suggests that plants regulate AS with transcript specificity under elevated temperature. An exonic GAG-repeat motif in these IR regions may function as a regulatory cis-element in heat-mediated AS regulation. A conserved AS pattern for HS transcription factors in P. patens and Arabidopsis (Arabidopsis thaliana) reveals that heat regulation for AS evolved early during land colonization of green plants. Our results support that AS of specific genes, including key HS regulators, is fine-tuned under elevated temperature to modulate gene regulation and reorganize metabolic processes.Global warming in recent decades has caused annual temperature extremes that are becoming harmful for all living organisms. Although all living cells show rapid responses to changes of ambient temperature, unlike animals, plants are sessile and cannot escape adverse temperature conditions. Challenged by temperature changes, plants have evolved rapid and complex systems to sense the temperature signal and translate it into cellular defenses for acquired tolerance, such as enhancing protein folding/unfolding activities and maintaining membrane fluidity (Sung et al., 2003). Understanding how plants adapt to temperature stresses has been an important topic in improving thermotolerance in crops.The heat shock response (HSR) is conserved in eukaryotes in response to elevated temperature and induces the activity of heat shock transcription factors (HSFs) to promote the expression of HSR-related genes. Different mechanisms for temperature sensing and signal transduction have been proposed. The general model suggests that constitutively expressed chaperones in the cytoplasm form inactive complexes with HSFs. Upon heat shock (HS), cytosolic chaperones are recruited by misfolded proteins, thus allowing the release of HSFs for phosphorylation, oligomerization, and nuclear localization to regulate gene expression (Mosser et al

Mitochondria play an important role in maintaining metabolic and energy homeostasis in the cell. In plants, impairment in mitochondrial functions usually has detrimental effects on growth and development. To study genes that are important for plant growth, we have isolated a collection of slow growth (slo) mutants in Arabidopsis (Arabidopsis thaliana). One of the slo mutants, slo3, has a significant reduction in mitochondrial complex I activity. The slo3 mutant has a four-nucleotide deletion in At3g61360 that encodes a pentatricopeptide repeat (PPR) protein. The SLO3 protein contains nine classic PPR domains belonging to the P subfamily. The small deletion in the slo3 mutant changes the reading frame and creates a premature stop codon in the first PPR domain. We demonstrated that the SLO3-GFP is localized to the mitochondrion. Further analysis of mitochondrial RNA metabolism revealed that the slo3 mutant was defective in splicing of NADH dehydrogenase subunit7 (nad7) intron 2. This specific splicing defect led to a dramatic reduction in complex I activity in the mutant as revealed by blue native gel analysis. Complementation of slo3 by 35S:SLO3 or 35S:SLO3-GFP restored the splicing of nad7 intron 2, the complex I activity, and the growth defects of the mutant. Together, these results indicate that the SLO3 PPR protein is a splicing factor of nad7 intron 2 in Arabidopsis mitochondria.

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Non-invasive detection of colorectal cancer with blood-based markers is a critical clinical need. Here we describe a phased mass spectrometry-based approach for the discovery, screening, and validation of circulating protein biomarkers with diagnostic value. Initially, we profiled human primary tumor tissue epithelia and characterized about 300 secreted and cell surface candidate glycoproteins. These candidates were then screened in patient systemic circulation to identify detectable candidates in blood plasma. An 88-plex targeting method was established to systematically monitor these proteins in two large and independent cohorts of plasma samples, which generated quantitative clinical datasets at an unprecedented scale. The data were deployed to develop and evaluate a five-protein biomarker signature for colorectal cancer detection.

Plant isoprenoids are derived from two independent pathways, the cytosolic mevalonate pathway and the plastid methylerythritol 4-phosphate (MEP) pathway. We used green fluorescent fusion protein assays to demonstrate that the Arabidopsis MEP pathway enzymes are localized to the chloroplast. We have also characterized three Arabidopsis albino mutants, ispD-1, ispD-2 and ispE-1, which have T-DNA insertions in the IspD and IspE genes of the MEP pathway. Levels of photosynthetic pigments are almost undetectable in these albino mutants. Instead of thylakoids, the ispD and ispE mutant chloroplasts are filled with large vesicles. Impairments in chloroplast development and functions may signal changes in the expression of nuclear, chloroplast and mitochondrial genes. We used northern blot analysis to examine the expression of photosynthetic and respiratory genes in the ispD and ispE albino mutants. Steady-state mRNA levels of nucleus- and chloroplast-encoded photosynthetic genes are significantly decreased in the albino mutants. In contrast, transcript levels of nuclear and mitochondrial genes encoding subunits of the mitochondrial electron transport chain are increased or not affected in these mutants. Genomic Southern blot analysis revealed that the DNA amounts of mitochondrial genes are not enhanced in the ispD and ispE albino mutants. These results support the notion that the functional state of chloroplasts may affect the expression of nuclear and mitochondrial genes. The up-regulation of mitochondrial genes in the albino mutants is not caused by changes of mitochondrial DNA copy number in Arabidopsis.

Statins are widely used to prevent cardiovascular disease. In addition to their inhibitory effects on cholesterol synthesis, statins have beneficial effects in patients with sepsis and pneumonia, although molecular mechanisms have mostly remained unclear. Using human airway epithelial cells as a proper in vitro model, we show that prior exposure to physiological nanomolar serum concentrations of simvastatin (ranging from 10-1,000 nM) confers significant cellular resistance to the cytotoxicity of pneumolysin, a pore-forming toxin and the main virulence factor of Streptococcus pneumoniae. This protection could be demonstrated with a different statin, pravastatin, or on a different toxin, a-hemolysin. Furthermore, through the use of gene silencing, pharmacological inhibitors, immunofluorescence microscopy, and biochemical and metabolic rescue approaches, we demonstrate that the mechanism of protection conferred by simvastatin at physiological nanomolar concentrations could be different from the canonical mevalonate pathways seen in most other mechanistic studies conducted with statins at micromolar levels. All of these data are integrated into a protein synthesis-dependent, calcium-dependent model showing the interconnected pathways used by statins in airway epithelial cells to elicit an increased resistance to pore-forming toxins. This research fills large gaps in our understanding of how statins may confer host cellular protection against bacterial infections in the context of airway epithelial cells without the confounding effect from the presence of immune cells. In addition, our discovery could be potentially developed into a host-centric strategy for the adjuvant treatment of pore-forming toxin associated bacterial infections.Keywords: airway epithelium; pneumolysin; simvastatin; cholesterol; pneumonia Clinical RelevanceOur study dissects the cellular and molecular protection mechanisms of statins in an isolated airway epithelial cell-bacterial pore-forming toxin context. This research can fill large gaps in our understanding of how statins contribute to the reduced pathology observed during pneumonia and other bacterial infections. The better understanding of the molecular mechanisms in the statin-mediated protection will further affect the therapeutic development of host-centric approach therapies in combating microbial infections.

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