Assigning Backbone NMR Resonances for Full Length Tau Isoforms: Efficient Compromise between Manual Assignments and Reduced Dimensionality

Tau protein is the longest disordered protein for which nearly complete backbone NMR resonance assignments have been reported. Full-length tau protein was initially assigned using a laborious combination of bootstrapping assignments from shorter tau fragments and conventional triple resonance NMR experiments. Subsequently it was reported that assignments of comparable quality could be obtained in a fully automated fashion from data obtained using reduced dimensionality NMR (RDNMR) experiments employing a large number of indirect dimensions. Although the latter strategy offers many advantages, it presents some difficulties if manual intervention, confirmation, or correction of the assignments is desirable, as may often be the case for long disordered and degenerate polypeptide sequences. Here we demonstrate that nearly complete backbone resonance assignments for full-length tau isoforms can be obtained without resorting either to bootstrapping from smaller fragments or to very high dimensionality experiments and automation. Instead, a set of RDNMR triple resonance experiments of modest dimensionality lend themselves readily to efficient and unambiguous manual assignments. An analysis of the backbone chemical shifts obtained in this fashion indicates several regions in full length tau with a notable propensity for helical or strand-like structure that are in good agreement with previous observations

Galectin-4 Controls Intestinal Inflammation by Selective Regulation of Peripheral and Mucosal T Cell Apoptosis and Cell Cycle

Galectin-4 is a carbohydrate-binding protein belonging to the galectin family. Here we provide novel evidence that galectin-4 is selectively expressed and secreted by intestinal epithelial cells and binds potently to activated peripheral and mucosal lamina propria T-cells at the CD3 epitope. The carbohydrate-dependent binding of galectin-4 at the CD3 epitope is fully functional and inhibited T cell activation, cycling and expansion. Galectin-4 induced apoptosis of activated peripheral and mucosal lamina propria T cells via calpain-, but not caspase-dependent, pathways. Providing further evidence for its important role in regulating T cell function, galectin-4 blockade by antisense oligonucleotides reduced TNF-alpha inhibitor induced T cell death. Furthermore, in T cells, galectin-4 reduced pro-inflammatory cytokine secretion including IL-17. In a model of experimental colitis, galectin-4 ameliorated mucosal inflammation, induced apoptosis of mucosal T-cells and decreased the secretion of pro-inflammatory cytokines. Our results show that galectin-4 plays a unique role in the intestine and assign a novel role of this protein in controlling intestinal inflammation by a selective induction of T cell apoptosis and cell cycle restriction. Conclusively, after defining its biological role, we propose Galectin-4 is a novel anti-inflammatory agent that could be therapeutically effective in diseases with a disturbed T cell expansion and apoptosis such as inflammatory bowel disease

Automated protein resonance assignments of magic angle spinning solid-state NMR spectra of β1 immunoglobulin binding domain of protein G (GB1)

Magic-angle spinning solid-state NMR (MAS SSNMR) represents a fast developing experimental technique with great potential to provide structural and dynamics information for proteins not amenable to other methods. However, few automated analysis tools are currently available for MAS SSNMR. We present a methodology for automating protein resonance assignments of MAS SSNMR spectral data and its application to experimental peak lists of the β1 immunoglobulin binding domain of protein G (GB1) derived from a uniformly 13C- and 15N-labeled sample. This application to the 56 amino acid GB1 produced an overall 84.1% assignment of the N, CO, CA, and CB resonances with no errors using peak lists from NCACX 3D, CANcoCA 3D, and CANCOCX 4D experiments. This proof of concept demonstrates the tractability of this problem

Probabilistic Interaction Network of Evidence Algorithm and its Application to Complete Labeling of Peak Lists from Protein NMR Spectroscopy

The process of assigning a finite set of tags or labels to a collection of observations, subject to side conditions, is notable for its computational complexity. This labeling paradigm is of theoretical and practical relevance to a wide range of biological applications, including the analysis of data from DNA microarrays, metabolomics experiments, and biomolecular nuclear magnetic resonance (NMR) spectroscopy. We present a novel algorithm, called Probabilistic Interaction Network of Evidence (PINE), that achieves robust, unsupervised probabilistic labeling of data. The computational core of PINE uses estimates of evidence derived from empirical distributions of previously observed data, along with consistency measures, to drive a fictitious system M with Hamiltonian H to a quasi-stationary state that produces probabilistic label assignments for relevant subsets of the data. We demonstrate the successful application of PINE to a key task in protein NMR spectroscopy: that of converting peak lists extracted from various NMR experiments into assignments associated with probabilities for their correctness. This application, called PINE-NMR, is available from a freely accessible computer server (http://pine.nmrfam.wisc.edu). The PINE-NMR server accepts as input the sequence of the protein plus user-specified combinations of data corresponding to an extensive list of NMR experiments; it provides as output a probabilistic assignment of NMR signals (chemical shifts) to sequence-specific backbone and aliphatic side chain atoms plus a probabilistic determination of the protein secondary structure. PINE-NMR can accommodate prior information about assignments or stable isotope labeling schemes. As part of the analysis, PINE-NMR identifies, verifies, and rectifies problems related to chemical shift referencing or erroneous input data. PINE-NMR achieves robust and consistent results that have been shown to be effective in subsequent steps of NMR structure determination

NMR Structure of Lipoprotein YxeF from Bacillus subtilis Reveals a Calycin Fold and Distant Homology with the Lipocalin Blc from Escherichia coli

The soluble monomeric domain of lipoprotein YxeF from the Gram positive bacterium B. subtilis was selected by the Northeast Structural Genomics Consortium (NESG) as a target of a biomedical theme project focusing on the structure determination of the soluble domains of bacterial lipoproteins. The solution NMR structure of YxeF reveals a calycin fold and distant homology with the lipocalin Blc from the Gram-negative bacterium E.coli. In particular, the characteristic β-barrel, which is open to the solvent at one end, is extremely well conserved in YxeF with respect to Blc. The identification of YxeF as the first lipocalin homologue occurring in a Gram-positive bacterium suggests that lipocalins emerged before the evolutionary divergence of Gram positive and Gram negative bacteria. Since YxeF is devoid of the α-helix that packs in all lipocalins with known structure against the β-barrel to form a second hydrophobic core, we propose to introduce a new lipocalin sub-family named ‘slim lipocalins’, with YxeF and the other members of Pfam family PF11631 to which YxeF belongs constituting the first representatives. The results presented here exemplify the impact of structural genomics to enhance our understanding of biology and to generate new biological hypotheses

The burden of diseases, injuries, and risk factors by state in the USA, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021

Background: The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2021 provides a comprehensive assessment of health and risk factor trends at global, regional, national, and subnational levels. This study aims to examine the burden of diseases, injuries, and risk factors in the USA and highlight the disparities in health outcomes across different states. Methods: GBD 2021 analysed trends in mortality, morbidity, and disability for 371 diseases and injuries and 88 risk factors in the USA between 1990 and 2021. We used several metrics to report sources of health and health loss related to specific diseases, injuries, and risk factors. GBD 2021 methods accounted for differences in data sources and biases. The analysis of levels and trends for causes and risk factors within the same computational framework enabled comparisons across states, years, age groups, and sex. GBD 2021 estimated years lived with disability (YLDs) and disability-adjusted life-years (DALYs; the sum of years of life lost to premature mortality and YLDs) for 371 diseases and injuries, years of life lost (YLLs) and mortality for 288 causes of death, and life expectancy and healthy life expectancy (HALE). We provided estimates for 88 risk factors in relation to 155 health outcomes for 631 risk–outcome pairs and produced risk-specific estimates of summary exposure value, relative health risk, population attributable fraction, and risk-attributable burden measured in DALYs and deaths. Estimates were produced by sex (male and female), age (25 age groups from birth to ≥95 years), and year (annually between 1990 and 2021). 95% uncertainty intervals (UIs) were generated for all final estimates as the 2·5th and 97·5th percentiles values of 500 draws (ie, 500 random samples from the estimate's distribution). Uncertainty was propagated at each step of the estimation process. Findings: We found disparities in health outcomes and risk factors across US states. Our analysis of GBD 2021 highlighted the relative decline in life expectancy and HALE compared with other countries, as well as the impact of COVID-19 during the first 2 years of the pandemic. We found a decline in the USA's ranking of life expectancy from 1990 to 2021: in 1990, the USA ranked 35th of 204 countries and territories for males and 19th for females, but dropped to 46th for males and 47th for females in 2021. When comparing life expectancy in the best-performing and worst-performing US states against all 203 other countries and territories (excluding the USA as a whole), Hawaii (the best-ranked state in 1990 and 2021) dropped from sixth-highest life expectancy in the world for males and fourth for females in 1990 to 28th for males and 22nd for females in 2021. The worst-ranked state in 2021 ranked 107th for males (Mississippi) and 99th for females (West Virginia). 14 US states lost life expectancy over the study period, with West Virginia experiencing the greatest loss (2·7 years between 1990 and 2021). HALE ranking declines were even greater; in 1990, the USA was ranked 42nd for males and 32nd for females but dropped to 69th for males and 76th for females in 2021. When comparing HALE in the best-performing and worst-performing US states against all 203 other countries and territories, Hawaii ranked 14th highest HALE for males and fifth for females in 1990, dropping to 39th for males and 34th for females in 2021. In 2021, West Virginia—the lowest-ranked state that year—ranked 141st for males and 137th for females. Nationally, age-standardised mortality rates declined between 1990 and 2021 for many leading causes of death, most notably for ischaemic heart disease (56·1% [95% UI 55·1–57·2] decline), lung cancer (41·9% [39·7–44·6]), and breast cancer (40·9% [38·7–43·7]). Over the same period, age-standardised mortality rates increased for other causes, particularly drug use disorders (878·0% [770·1–1015·5]), chronic kidney disease (158·3% [149·6–167·9]), and falls (89·7% [79·8–95·8]). We found substantial variation in mortality rates between states, with Hawaii having the lowest age-standardised mortality rate (433·2 per 100 000 [380·6–493·4]) in 2021 and Mississippi having the highest (867·5 per 100 000 [772·6–975·7]). Hawaii had the lowest age-standardised mortality rates throughout the study period, whereas Washington, DC, experienced the most improvement (a 40·7% decline [33·2–47·3]). Only six countries had age-standardised rates of YLDs higher than the USA in 2021: Afghanistan, Lesotho, Liberia, Mozambique, South Africa, and the Central African Republic, largely because the impact of musculoskeletal disorders, mental disorders, and substance use disorders on age-standardised disability rates in the USA is so large. At the state level, eight US states had higher age-standardised YLD rates than any country in the world: West Virginia, Kentucky, Oklahoma, Pennsylvania, New Mexico, Ohio, Tennessee, and Arizona. Low back pain was the leading cause of YLDs in the USA in 1990 and 2021, although the age-standardised rate declined by 7·9% (1·8–13·0) from 1990. Depressive disorders (56·0% increase [48·2–64·3]) and drug use disorders (287·6% [247·9–329·8]) were the second-leading and third-leading causes of age-standardised YLDs in 2021. For females, mental health disorders had the highest age-standardised YLD rate, with an increase of 59·8% (50·6–68·5) between 1990 and 2021. Hawaii had the lowest age-standardised rates of YLDs for all sexes combined (12 085·3 per 100 000 [9090·8–15 557·1]), whereas West Virginia had the highest (14 832·9 per 100 000 [11 226·9–18 882·5]). At the national level, the leading GBD Level 2 risk factors for death for all sexes combined in 2021 were high systolic blood pressure, high fasting plasma glucose, and tobacco use. From 1990 to 2021, the age-standardised mortality rates attributable to high systolic blood pressure decreased by 47·8% (43·4–52·5) and for tobacco use by 5·1% (48·3%–54·1%), but rates increased for high fasting plasma glucose by 9·3% (0·4–18·7). The burden attributable to risk factors varied by age and sex. For example, for ages 15–49 years, the leading risk factors for death were drug use, high alcohol use, and dietary risks. By comparison, for ages 50–69 years, tobacco was the leading risk factor for death, followed by dietary risks and high BMI. Interpretation: GBD 2021 provides valuable information for policy makers, health-care professionals, and researchers in the USA at the national and state levels to prioritise interventions, allocate resources effectively, and assess the effects of health policies and programmes. By addressing socioeconomic determinants, risk behaviours, environmental influences, and health disparities among minority populations, the USA can work towards improving health outcomes so that people can live longer and healthier lives. Funding: Bill & Melinda Gates Foundation

A target-protection mechanism of antibiotic resistance at atomic resolution: insights into FusB-type fusidic acid resistance

Antibiotic resistance in clinically important bacteria can be mediated by proteins that physically associate with the drug target and act to protect it from the inhibitory effects of an antibiotic. We present here the first detailed structural characterization of such a target protection mechanism mediated through a protein-protein interaction, revealing the architecture of the complex formed between the FusB fusidic acid resistance protein and the drug target (EF-G) it acts to protect. Binding of FusB to EF G induces conformational and dynamic changes in the latter, shedding light on the molecular mechanism of fusidic acid resistance

Interrogating host immunity to predict treatment response in inflammatory bowel disease

IBD treatment is undergoing a transformation with an expanding repertoire of drugs targeting different aspects of the immune response. Three novel classes of drugs have emerged in the past decade that target leukocyte trafficking to the gut (vedolizumab), neutralize key cytokines with antibodies (ustekinumab) and inhibit cytokine signalling pathways (tofacitinib). In advanced development are other drugs for IBD, including therapies targeting other cytokines such as IL-23 and IL-6. However, all agents tested so far are hampered by primary and secondary loss of response, so it is desirable to develop personalized strategies to identify which patients should be treated with which drugs. Stratification of patients with IBD by clinical parameters alone lacks sensitivity, and alternative modalities are now needed to deliver precision medicine in IBD. High-resolution profiling of immune response networks in individual patients is a promising approach and different technical platforms, including in vivo real-time molecular endoscopy, tissue transcriptomics and germline genetics, are promising tools to help predict responses to specific therapies. However, important challenges remain regarding the clinical utility of these technologies, including their scalability and accessibility. This Review focuses on unravelling some of the complexity of mucosal immune responses in IBD pathogenesis and how current and emerging analytical platforms might be harnessed to effectively stratify and individualise IBD therapy