Mass spectrometry-based multiple reaction monitoring (MRM) quantitation of proteins can dramatically impact the discovery and quantitation of biomarkers via rapid, targeted, multiplexed protein ...expression profiling of clinical samples. A mixture of 45 peptide standards, easily adaptable to common plasma proteomics work flows, was created to permit absolute quantitation of 45 endogenous proteins in human plasma trypsin digests. All experiments were performed on simple tryptic digests of human EDTA-plasma without prior affinity depletion or enrichment. Stable isotope-labeled standard peptides were added immediately following tryptic digestion because addition of stable isotope-labeled standard peptides prior to trypsin digestion was found to generate elevated and unpredictable results. Proteotypic tryptic peptides containing isotopically coded amino acids (13C6Arg or 13C6Lys) were synthesized for all 45 proteins. Peptide purity was assessed by capillary zone electrophoresis, and the peptide quantity was determined by amino acid analysis. For maximum sensitivity and specificity, instrumental parameters were empirically determined to generate the most abundant precursor ions and y ion fragments. Concentrations of individual peptide standards in the mixture were optimized to approximate endogenous concentrations of analytes and to ensure the maximum linear dynamic range of the MRM assays. Excellent linear responses (r > 0.99) were obtained for 43 of the 45 proteins with attomole level limits of quantitation (<20% coefficient of variation) for 27 of the 45 proteins. Analytical precision for 44 of the 45 assays varied by <10%. LC-MRM/MS analyses performed on 3 different days on different batches of plasma trypsin digests resulted in coefficients of variation of <20% for 42 of the 45 assays. Concentrations for 39 of the 45 proteins are within a factor of 2 of reported literature values. This mixture of internal standards has many uses and can be applied to the characterization of trypsin digestion kinetics and plasma protein expression profiling because 31 of the 45 proteins are putative biomarkers of cardiovascular disease.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Asthma continues to disproportionately affect minority and low-income groups, with African American and Latino children who live in low-socioeconomic-status urban environments experiencing higher ...asthma morbidity and mortality than white children. This uneven burden in asthma morbidity has been ever increasing despite medical advancement. Many factors have contributed to these disparities in the areas of health care inequities, which result in inadequate treatment; poor housing, which leads to increased exposure to asthma allergens; and social and psychosocial stressors, which are often unappreciated. Interventions to reduce individual areas of disparities have had varying successes. Because asthma is a complex disease that affects millions of persons, multifaceted comprehensive interventions that combine all evidence-based successful strategies are essential to finally closing the gap in asthma morbidity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
The 2020 Focused Updates to the Asthma Management Guidelines: A Report from the National Asthma Education and Prevention Program Coordinating Committee Expert Panel Working Group was coordinated and ...supported by the National Heart, Lung, and Blood Institute (NHLBI) of the National Institutes of Health. It is designed to improve patient care and support informed decision making about asthma management in the clinical setting. This update addresses six priority topic areas as determined by the state of the science at the time of a needs assessment, and input from multiple stakeholders:•Fractional Exhaled Nitric Oxide Testing•Indoor Allergen Mitigation•Intermittent Inhaled Corticosteroids•Long-Acting Muscarinic Antagonists•Immunotherapy in the Treatment of Allergic Asthma•Bronchial ThermoplastyA rigorous process was undertaken to develop these evidence-based guidelines. The Agency for Healthcare Research and Quality's (AHRQ) Evidence-Based Practice Centers conducted systematic reviews on these topics, which were used by the Expert Panel Working Group as a basis for developing recommendations and guidance. The Expert Panel used GRADE (Grading of Recommendations, Assessment, Development and Evaluation), an internationally accepted framework, in consultation with an experienced methodology team for determining the certainty of evidence and the direction and strength of recommendations based on the evidence. Practical implementation guidance for each recommendation incorporates findings from NHLBI-led patient, caregiver, and clinician focus groups. To assist clincians in implementing these recommendations into patient care, the new recommendations have been integrated into the existing Expert Panel Report-3 (EPR-3) asthma management step diagram format.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
For over a thousand years, the common goldfish (
) was raised throughout Asia for food and as an ornamental pet. As a very close relative of the common carp (
), goldfish share the recent genome ...duplication that occurred approximately 14 million years ago in their common ancestor. The combination of centuries of breeding and a wide array of interesting body morphologies provides an exciting opportunity to link genotype to phenotype and to understand the dynamics of genome evolution and speciation. We generated a high-quality draft sequence and gene annotations of a "Wakin" goldfish using 71X PacBio long reads. The two subgenomes in goldfish retained extensive synteny and collinearity between goldfish and zebrafish. However, genes were lost quickly after the carp whole-genome duplication, and the expression of 30% of the retained duplicated gene diverged substantially across seven tissues sampled. Loss of sequence identity and/or exons determined the divergence of the expression levels across all tissues, while loss of conserved noncoding elements determined expression variance between different tissues. This assembly provides an important resource for comparative genomics and understanding the causes of goldfish variants.
In late July, 2023, SARS-CoV-2 surveillance programmes identified a new, highly mutated omicron subvariant, BA.2.86.1 Initial estimates place the effective reproduction number of BA.2.86 at ...1·29-times greater than that of XBB.1.5 and near equivalent to that of EG.5.1, one of the most rapidly expanding XBB subvariants.2 Since the emergence of BA.2.86, multiple countries have reported its presence, including an outbreak in a British care home, with an attack rate above 85%, where the majority of infected residents had received a SARS-CoV-2 Beta variant-based subunit vaccine 4 months earlier.3 Relative to the SARS-CoV-2 index strain, the BA.2.86 spike protein has 60 amino acid changes, located predominantly in the N-terminal and receptor binding domains (figure A). ...this study addresses the paucity of BA.2.86 and EG.5.1 virus characterisation data on clinical isolates, which represent the pathogens circulating among individuals. PJ is the science officer of the European Society of Clinical Microbiology and Infectious Diseases Study Group in Public Health Microbiology, and a member of the European Society of Clinical Microbiology and Infectious Diseases Emerging Infections Task Force.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Summary
INCREASED SIZE EXCLUSION LIMIT2 (ISE2) is a chloroplast‐localized RNA helicase that is indispensable for proper plant development. Chloroplasts in leaves with reduced ISE2 expression have ...previously been shown to exhibit reduced thylakoid contents and increased stromal volume, indicative of defective development. It has recently been reported that ISE2 is required for the splicing of group II introns from chloroplast transcripts. The current study extends these findings, and presents evidence for ISE2's role in multiple aspects of chloroplast RNA processing beyond group II intron splicing. Loss of ISE2 from Arabidopsis thaliana leaves resulted in defects in C‐to‐U RNA editing, altered accumulation of chloroplast transcripts and chloroplast‐encoded proteins, and defective processing of chloroplast ribosomal RNAs. Potential ISE2 substrates were identified by RNA immunoprecipitation followed by next‐generation sequencing (RIP‐seq), and the diversity of RNA species identified supports ISE2's involvement in multiple aspects of chloroplast RNA metabolism. Comprehensive phylogenetic analyses revealed that ISE2 is a non‐canonical Ski2‐like RNA helicase that represents a separate sub‐clade unique to green photosynthetic organisms, consistent with its function as an essential protein. Thus ISE2's evolutionary conservation may be explained by its numerous roles in regulating chloroplast gene expression.
Significance Statement
RNA processing is a critical part of modulating gene expression in chloroplasts and many of the nucleus‐encoded protein factors involved are unknown. In this study, we show that the evolutionarily conserved RNA helicase ISE2 functions in multiple aspects of chloroplast RNA processing including C‐to‐U RNA editing and ribosome biogenesis, and may also associate with a wide assortment of chloroplast RNA species to mediate RNA processing.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
The unfolded protein response (UPR) senses stress in the endoplasmic reticulum (ER) and initiates signal transduction cascades that culminate in changes to gene regulation. Long recognized as a means ...for improving ER protein folding through up-regulation of ER chaperones, the UPR is increasingly recognized to play a role in the regulation of metabolic pathways. ER stress is clearly connected to altered metabolism in tissues such as the liver, but the mechanisms underlying this connection are only beginning to be elucidated. Here, working exclusively in vivo, we tested the hypothesis that the UPR-regulated CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) participates in the transcriptional regulation of metabolism during hepatic ER stress. We found that metabolic dysregulation was associated with induction of eIF2α signaling and CHOP up-regulation during challenge with tunicamycin or Velcade. CHOP was necessary for suppression of genes encoding the transcriptional master regulators of lipid metabolism: Cebpa, Ppara, and Srebf1. This action of CHOP required a contemporaneous CHOP-independent stress signal. CHOP bound directly to C/EBP-binding regions in the promoters of target genes, whereas binding of C/EBPα and C/EBPβ to the same regions was diminished during ER stress. Our results thus highlight a role for CHOP in the transcriptional regulation of metabolism.
Background: ER stress regulates metabolic gene expression in liver.
Results: The ER stress-regulated pro-apoptotic transcription factor CHOP binds to the promoters of metabolic genes and is necessary for their suppression.
Conclusion: CHOP contributes to the metabolic alterations that accompany ER stress in vivo.
Significance: These findings suggest that CHOP has a non-apoptotic role in regulating metabolic physiology.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
In plants, cytidine-to-uridine (C-to-U) editing is a crucial step in processing mitochondria- and chloroplast-encoded transcripts. This editing requires nuclear-encoded proteins including members of ...the pentatricopeptide (PPR) family, especially PLS-type proteins carrying the DYW domain.
IPI1/emb175/PPR103
is a nuclear gene encoding a PLS-type PPR protein essential for survival in
Arabidopsis thaliana
and maize. Arabidopsis IPI1 was identified as likely interacting with ISE2, a chloroplast-localized RNA helicase associated with C-to-U RNA editing in Arabidopsis and maize. Notably, while the Arabidopsis and
Nicotiana
IPI1 orthologs possess complete DYW motifs at their C-termini, the maize homolog, ZmPPR103, lacks this triplet of residues which are essential for editing. In this study we examined the function of IPI1 in chloroplast RNA processing in
N. benthamiana
to gain insight into the importance of the DYW domain to the function of the EMB175/PPR103/ IPI1 proteins. Structural predictions suggest that evolutionary loss of residues identified as critical for catalyzing C-to-U editing in other members of this class of proteins, were likely to lead to reduced or absent editing activity in the
Nicotiana
and Arabidopsis IPI1 orthologs. Virus-induced gene silencing of
NbIPI1
led to defects in chloroplast ribosomal RNA processing and changes to stability of
rpl16
transcripts, revealing conserved function with its maize ortholog.
NbIPI1
-silenced plants also had defective C-to-U RNA editing in several chloroplast transcripts, a contrast from the finding that maize PPR103 had no role in editing. The results indicate that in addition to its role in transcript stability, NbIPI1 may contribute to C-to-U editing in
N. benthamiana
chloroplasts
.
Key message
The
Nicotiana benthamiana
DYW PPR protein NbIPI1 possess an intact C-terminal DYW domain and stabilizes the
rpl16-rpl14
transcript like its maize ortholog PPR103, and may also contribute to C-to-U RNA editing of some chloroplast transcripts.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Retroviruses integrate into the host genome in patterns specific to each virus. Understanding the causes of these patterns can provide insight into viral integration mechanisms, pathology and genome ...evolution, and is critical to the development of safe gene therapy vectors. We generated murine leukemia virus integrations in human HepG2 and K562 cells and subjected them to second-generation sequencing, using a DNA barcoding technique that allowed us to quantify independent integration events. We characterized >3,700,000 unique integration events in two ENCODE-characterized cell lines. We find that integrations were most highly enriched in a subset of strong enhancers and active promoters. In both cell types, approximately half the integrations were found in <2% of the genome, demonstrating genomic influences even narrower than previously believed. The integration pattern of murine leukemia virus appears to be largely driven by regions that have high enrichment for multiple marks of active chromatin; the combination of histone marks present was sufficient to explain why some strong enhancers were more prone to integration than others. The approach we used is applicable to analyzing the integration pattern of any exogenous element and could be a valuable preclinical screen to evaluate the safety of gene therapy vectors.
Both the USA and Europe experienced substantial excess mortality in 2020 and 2021 related to the COVID-19 pandemic. Methods used to estimate excess mortality vary, making comparisons difficult. This ...retrospective observational study included data on deaths from all causes occurring in the USA and 25 European countries or subnational areas participating in the network for European monitoring of excess mortality for public health action (EuroMOMO). We applied the EuroMOMO algorithm to estimate excess all-cause mortality in the USA and Europe during the first two years of the COVID-19 pandemic, 2020-2021, and compared excess mortality by age group and time periods reflecting three primary waves. During 2020-2021, the USA experienced 154.5 (95% Uncertainty Interval UI: 154.2-154.9) cumulative age-standardized excess all-cause deaths per 100,000 person years, compared with 110.4 (95% UI: 109.9-111.0) for the European countries. Excess all-cause mortality in the USA was higher than in Europe for nearly all age groups, with an additional 44.1 excess deaths per 100,000 person years overall from 2020-2021. If the USA had experienced an excess mortality rate similar to Europe, there would have been approximately 391 thousand (36%) fewer excess deaths in the USA.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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