Viperin triggers ribosome collision-dependent translation inhibition to restrict viral replication.

Innate immune responses induce hundreds of interferon-stimulated genes (ISGs). Viperin, a member of the radical S-adenosyl methionine (SAM) superfamily of enzymes, is the product of one such ISG that restricts the replication of a broad spectrum of viruses. Here, we report a previously unknown antiviral mechanism in which viperin activates a ribosome collision-dependent pathway that inhibits both cellular and viral RNA translation. We found that the radical SAM activity of viperin is required for translation inhibition and that this is mediated by viperin's enzymatic product, 3'-deoxy-3',4'-didehydro-CTP (ddhCTP). Viperin triggers ribosome collisions and activates the MAPKKK ZAK pathway that in turn activates the GCN2 arm of the integrated stress response pathway to inhibit translation. The study illustrates the importance of translational repression in the antiviral response and identifies viperin as a translation regulator in innate immunity.

Integrative Molecular Structure Elucidation and Construction of an Extended Metabolic Pathway Associated with an Ancient Innate Immune Response in COVID-19 Patients.

We present compelling evidence for the existence of an extended innate viperin-dependent pathway, which provides crucial evidence for an adaptive response to viral agents, such as SARS-CoV-2. We show the in vivo biosynthesis of a family of novel endogenous cytosine metabolites with potential antiviral activities. Two-dimensional nuclear magnetic resonance (NMR) spectroscopy revealed a characteristic spin-system motif, indicating the presence of an extended panel of urinary metabolites during the acute viral replication phase. Mass spectrometry additionally enabled the characterization and quantification of the most abundant serum metabolites, showing the potential diagnostic value of the compounds for viral infections. In total, we unveiled ten nucleoside (cytosine- and uracil-based) analogue structures, eight of which were previously unknown in humans allowing us to propose a new extended viperin pathway for the innate production of antiviral compounds. The molecular structures of the nucleoside analogues and their correlation with an array of serum cytokines, including IFN-α2, IFN-γ, and IL-10, suggest an association with the viperin enzyme contributing to an ancient endogenous innate immune defense mechanism against viral infection.

Quorum Sensing Controls Adaptive Immunity through the Regulation of Multiple CRISPR-Cas Systems.

Bacteria commonly exist in high cell density populations, making them prone to viral predation and horizontal gene transfer (HGT) through transformation and conjugation. To combat these invaders, bacteria possess an arsenal of defenses, such as CRISPR-Cas adaptive immunity. Many bacterial populations coordinate their behavior as cell density increases, using quorum sensing (QS) signaling. In this study, we demonstrate that QS regulation results in increased expression of the type I-E, I-F, and III-A CRISPR-Cas systems in Serratia cells in high-density populations. Strains unable to communicate via QS were less effective at defending against invaders targeted by any of the three CRISPR-Cas systems. Additionally, the acquisition of immunity by the type I-E and I-F systems was impaired in the absence of QS signaling. We propose that bacteria can use chemical communication to modulate the balance between community-level defense requirements in high cell density populations and host fitness costs of basal CRISPR-Cas activity.

Cumulative Neurotoxicological Air Pollution Exposure Is Associated with Lower Reading Improvement and Diminished Benefits of Literacy Interventions for Urban Elementary Students of Color.

The cognitive and behavioral deficits associated with air pollution exposure may have far-reaching negative effects on children's scholastic achievement. Moreover, air pollution may be conditioning the success of educational investments that support students who face greatest levels of societal adversity. This study examined the direct main effects of cumulative neurotoxicological exposure on annual reading improvement. We also tested the statistical interaction (i.e., moderation) of neurotoxicological exposure and academic intervention sessions on annual reading improvement for a large sample of ethnic minority (95%) elementary school children (n = 6080, k-6th grade) enrolled in a standard literacy enrichment program. These children were all behind grade level in reading and attended predominantly low-income schools (n = 85) in urban settings across the state of California. Multi-level modeling assessments accounted for random effects associated with school and neighborhood environments, and incorporated extensive individual, school, and community level covariates. Findings show individual elementary students of color to progress less in reading when exposed to greater accumulations of neurotoxin air pollution in their home and school environments, with the average deficit equivalent to 1.5 weeks of learning delay per year. Findings also show neurotoxicological exposure to diminish the efficacy of literacy intervention sessions received on reading improvement throughout the school year. Results suggest that pollution abatement can be a salient strategy to help bridge the child educational achievement gap. In addition to several methodological strengths, this study is one of the first to show that ambient pollution can undermine program efficacy of a literacy enrichment program.

Childhood poverty and psychological well-being: The mediating role of cumulative risk exposure.

The current study assessed whether the proportion of childhood (age 0-9 years) in poverty altered the developmental trajectories (ages 9-24) of multimethodological indicators of psychological well-being. In addition, we tested whether exposure to cumulative risk over time mediated the association between poverty exposure and psychological well-being. Measures of psychological well-being included internalizing and externalizing symptoms, a behavioral index of learned helplessness (task persistence), and chronic physiological stress (allostatic load). Exposure to poverty during childhood predicted the trajectory of each development outcome: individuals with more poverty exposure during childhood showed (a) relatively high levels of internalizing symptoms that diminished more slowly with maturation, (b) relatively high levels of externalizing symptoms that increased faster over time, (c) less task persistence indicative of greater learned helplessness, and (d) higher levels of chronic physiological stress which increased faster over time relative to persons with less childhood poverty exposure. Trajectories of cumulative risk exposure from physical and psychosocial surroundings from 9-24 years accounted for the association between childhood poverty and the growth curves of internalizing and externalizing symptoms but not for learned helplessness or chronic physiological stress. Additional sensitivity analyses indicate that early childhood disadvantage is particularly problematic for each outcome, except for internalizing symptoms which seem sensitive to the combination of early and lifetime poverty exposure. We also explored whether domains of cumulative risk as well as two alternatives, maternal sensitivity or family cohesion, functioned as mediators. Little evidence emerged for any of these alternative mediating constructs.

Measuring Repositioning in Home Care for Pressure Injury Prevention and Management.

Despite the widespread agreement on the need for the regular repositioning of at-risk individuals for pressure injury prevention and management, adherence to repositioning schedules remains poor in the clinical environment. The situation in the home environment is likely even worse. Our team has developed a non-contact system that can determine an individual's position in bed (left-side lying, supine, or right-side lying) using data from a set of inexpensive load cells placed under the bed. This system was able to detect whether healthy participants were left-side lying, supine, or right-side lying with 94.2% accuracy in the lab environment. The objective of the present work was to deploy and test our system in the home environment for use with individuals who were sleeping in their own beds. Our system was able to detect the position of our nine participants with an F1 score of 0.982. Future work will include improving generalizability by training our classifier on more participants as well as using this system to evaluate adherence to two-hour repositioning schedules for pressure injury prevention or management. We plan to deploy this technology as part of a prompting system to alert a caregiver when a patient requires repositioning.

Chemical Synthesis of the Antiviral Nucleotide Analogue ddhCTP.

3'-Deoxy-3',4'-didehydro-cytidine triphosphate (ddhCTP) is a novel antiviral molecule produced by the enzyme viperin as part of the innate immune response. ddhCTP has been shown to act as an obligate chain terminator of flavivirus and SARS-CoV-2 RNA-dependent RNA polymerases; however, further biophysical studies have been precluded by limited access to this promising antiviral. Herein, we report a robust and scalable synthesis of ddhCTP as well as the mono- and diphosphates ddhCMP and ddhCDP, respectively. Identification of a 2'-silyl ether protection strategy enabled selective synthesis and facile purification of the 5'-triphosphate, culminating in the preparation of ddhCTP on a gram scale.

Early childhood poverty and adult executive functioning: Distinct, mediating pathways for different domains of executive functioning.

Executive functioning in adulthood is associated with early-in-life disadvantage. Furthermore, distinct and independent underlying processes account for differences in specific domains of adult executive functioning. The duration of poverty from birth to age 9 is associated with reduced adult inhibitory control assessed by the Flanker task (n = 233, M = 23.52 years). This effect is largely explained by lower levels of maternal responsiveness in adolescence. Early poverty also related to worse working memory in adulthood, and this effect is partially explained by elevated allostatic load during adolescence, an index of chronic physiological stress.

A single point mutation converts a glutaryl-7-aminocephalosporanic acid acylase into an N-acyl-homoserine lactone acylase.

OBJECTIVE: To change the specificity of a glutaryl-7-aminocephalosporanic acid acylase (GCA) towards N-acyl homoserine lactones (AHLs; quorum sensing signalling molecules) by site-directed mutagenesis. RESULTS: Seven residues were identified by analysis of existing crystal structures as potential determinants of substrate specificity. Site-saturation mutagenesis libraries were created for each of the seven selected positions. High-throughput activity screening of each library identified two variants-Arg255Ala, Arg255Gly-with new activities towards N-acyl homoserine lactone substrates. Structural modelling of the Arg255Gly mutation suggests that the smaller side-chain of glycine (as compared to arginine in the wild-type enzyme) avoids a key clash with the acyl group of the N-acyl homoserine lactone substrate. CONCLUSIONS: Mutation of a single amino acid residue successfully converted a GCA (with no detectable activity against AHLs) into an AHL acylase. This approach may be useful for further engineering of 'quorum quenching' enzymes.

Childhood socioeconomic status is prospectively associated with surface morphometry in adulthood.

Childhood socioeconomic status (SES) has been associated with brain cortex surface area in children. However, the extent to which childhood SES is prospectively associated with brain morphometry in adulthood is unclear. We tested whether childhood SES (income-to-needs ratio averaged across ages 9, 13, and 17) is prospectively associated with cortical surface morphometry in adulthood. Average childhood income-to-needs ratio had a positive, prospective association with cortical thickness in adulthood in the precentral gyrus, postcentral gyrus, and caudal middle frontal gyrus (p < .05, FWE corrected). Childhood income-to-needs ratio also had a positive, prospective association with cortical surface area in adulthood in multiple regions, including the rostral and caudal middle frontal gyri and superior frontal gyrus (p < .05, FWE corrected). Concurrent income-to-needs ratio (measured at age 24) was not associated with cortical thickness or surface area in adulthood. The results underscore the importance of addressing poverty in childhood for brain morphological development.

Narrow-Spectrum Antibiotic Targeting of the Radical SAM Enzyme MqnE in Menaquinone Biosynthesis.

Antibiotic resistance continues to spread at an alarming rate, outpacing the introduction of new therapeutics and threatening to globally undermine health care. There is a crucial need for new strategies that selectively target specific pathogens while leaving the majority of the microbiome untouched, thus averting the debilitating and sometimes fatal occurrences of opportunistic infections. To address these challenges, we have adopted a unique strategy that focuses on oxygen-sensitive proteins, an untapped set of therapeutic targets. MqnE is a member of the radical S-adenosyl-l-methionine (RS) superfamily, all of which rely on an oxygen-sensitive [4Fe-4S] cluster for catalytic activity. MqnE catalyzes the conversion of didehydrochorismate to aminofutalosine in the essential menaquinone biosynthetic pathway present in a limited set of species, including the gut pathogen Helicobacter pylori (Hp), making it an attractive target for narrow-spectrum antibiotic development. Indeed, we show that MqnE is inhibited by the mechanism-derived 2-fluoro analogue of didehydrochorismate (2F-DHC) due to accumulation of a radical intermediate under turnover conditions. Structures of MqnE in the apo and product-bound states afford insight into its catalytic mechanism, and electron paramagnetic resonance approaches provide direct spectroscopic evidence consistent with the predicted structure of the radical intermediate. In addition, we demonstrate the essentiality of the menaquinone biosynthetic pathway and unambiguously validate 2F-DHC as a selective inhibitor of Hp growth that exclusively targets MqnE. These data provide the foundation for designing effective Hp therapies and demonstrate proof of principle that radical SAM proteins can be effectively leveraged as therapeutic targets.

Transition State Analogues Enhanced by Fragment-Based Structural Analysis: Bacterial Methylthioadenosine Nucleosidases.

Transition state analogue inhibitor design (TSID) and fragment-based drug design (FBDD) are drug design approaches typically used independently. Methylthio-DADMe-Immucillin-A (MTDIA) is a tight-binding transition state analogue of bacterial 5'-methylthioadenosine nucleosidases (MTANs). Previously, Salmonella enterica MTAN structures were found to bind MTDIA and ethylene glycol fragments, but MTDIA modified to contain similar fragments did not enhance affinity. Seventy-five published MTAN structures were analyzed, and co-crystallization fragments were found that might enhance the binding of MTDIA to other bacterial MTANs through contacts external to MTDIA binding. The fragment-modified MTDIAs were tested with Helicobacter pylori MTAN and Staphylococcus aureus MTANs (HpMTAN and SaMTAN) as test cases to explore inhibitor optimization by potential contacts beyond the transition state contacts. Replacement of a methyl group with a 2'-ethoxyethanol group in MTDIA improved the dissociation constant 14-fold (0.09 nM vs 1.25 nM) for HpMTAN and 81-fold for SaMTAN (0.096 nM vs 7.8 nM). TSID combined with FBDD can be useful in enhancing already powerful inhibitors.

Prospective associations, longitudinal patterns of childhood socioeconomic status, and white matter organization in adulthood.

The association between childhood socioeconomic status (SES) and brain development is an emerging area of research. The primary focus to date has been on SES and variations in gray matter structure with much less known about the relation between childhood SES and white matter structure. Using a longitudinal study of SES, with measures of income-to-needs ratio (INR) at age 9, 13, 17, and 24, we examined the prospective relationship between childhood SES (age 9 INR) and white matter organization in adulthood using diffusion tensor imaging. We also examined how changes in INR from childhood through young adulthood are associated with white matter organization in adult using a latent growth mixture model. Using tract-based spatial statistics (TBSS) we found that there is a significant prospective positive association between childhood INR and white matter organization in the bilateral uncinate fasciculus, bilateral cingulum bundle, bilateral superior longitudinal fasciculus, and corpus callosum (p < .05, FWE corrected). The probability that an individual was in the high-increasing INR profile across development compared with the low-increasing INR profile was positively associated with white matter organization in the bilateral uncinate fasciculus, left cingulum, and bilateral superior longitudinal fasciculus. The results of the current study have potential implications for interventions given that early childhood poverty may have long-lasting associations with white matter structure. Furthermore, trajectories of socioeconomic status during childhood are important-with individuals that belong to the latent profile that had high increases in INR having greater regional white matter organization in adulthood.

Projected Behavioral Impacts of Global Climate Change.

The projected behavioral impacts of global climate change emanate from environmental changes including temperature elevation, extreme weather events, and rising air pollution. Negative affect, interpersonal and intergroup conflict, and possibly psychological distress increase with rising temperature. Droughts, floods, and severe storms diminish quality of life, elevate stress, produce psychological distress, and may elevate interpersonal and intergroup conflict. Recreational opportunities are compromised by extreme weather, and children may suffer delayed cognitive development. Elevated pollutants concern citizens and may accentuate psychological distress. Outdoor recreational activity is curtailed by ambient pollutants. Limitations and issues in need of further investigation include the following: lack of data on direct experience with climate change rather than indirect assessments related to projected changes; poor spatial resolution in environmental exposures and behavioral assessments; few rigorous quasi-experimental studies; overreliance on self-reports of behavioral outcomes; little consideration of moderator effects; and scant investigation of underlying psychosocial processes to explain projected behavioral impacts.

E-cadherin-deficient cells have synthetic lethal vulnerabilities in plasma membrane organisation, dynamics and function.

BACKGROUND: The E-cadherin gene (CDH1) is frequently mutated in diffuse gastric cancer and lobular breast cancer, and germline mutations predispose to the cancer syndrome Hereditary Diffuse Gastric Cancer. We are taking a synthetic lethal approach to identify druggable vulnerabilities in CDH1-mutant cancers. METHODS: Density distributions of cell viability data from a genome-wide RNAi screen of isogenic MCF10A and MCF10A-CDH1-/- cells were used to identify protein classes affected by CDH1 mutation. The synthetic lethal relationship between selected protein classes and E-cadherin was characterised by drug sensitivity assays in both the isogenic breast MCF10A cells and CDH1-isogenic gastric NCI-N87. Endocytosis efficiency was quantified using cholera toxin B uptake. Pathway metagene expression of 415 TCGA gastric tumours was statistically correlated with CDH1 expression. RESULTS: MCF10A-CDH1-/- cells showed significantly altered sensitivity to RNAi inhibition of groups of genes including the PI3K/AKT pathway, GPCRs, ion channels, proteosomal subunit proteins and ubiquitinylation enzymes. Both MCF10A-CDH1-/- and NCI-N87-CDH1-/- cells were more sensitive than wild-type cells to compounds that disrupted plasma membrane composition and trafficking, but showed contrasting sensitivities to inhibitors of actin polymerisation and the chloride channel inhibitor NS3728. The MCF10A-CDH1-/- cell lines showed reduced capacity to endocytose cholera toxin B. Pathway metagene analysis identified 20 Reactome pathways that were potentially synthetic lethal in tumours. Genes involved in GPCR signalling, vesicle transport and the metabolism of PI3K and membrane lipids were strongly represented amongst the candidate synthetic lethal genes. CONCLUSIONS: E-cadherin loss leads to disturbances in receptor signalling and plasma membrane trafficking and organisation, creating druggable vulnerabilities.

Childhood poverty and adult psychological well-being.

Childhood disadvantage has repeatedly been linked to adult physical morbidity and mortality. We show in a prospective, longitudinal design that childhood poverty predicts multimethodological indices of adult (24 y of age) psychological well-being while holding constant similar childhood outcomes assessed at age 9. Adults from low-income families manifest more allostatic load, an index of chronic physiological stress, higher levels of externalizing symptoms (e.g., aggression) but not internalizing symptoms (e.g., depression), and more helplessness behaviors. In addition, childhood poverty predicts deficits in adult short-term spatial memory.

Neutron structures of the Helicobacter pylori 5'-methylthioadenosine nucleosidase highlight proton sharing and protonation states.

MTAN (5'-methylthioadenosine nucleosidase) catalyzes the hydrolysis of the N-ribosidic bond of a variety of adenosine-containing metabolites. The Helicobacter pylori MTAN (HpMTAN) hydrolyzes 6-amino-6-deoxyfutalosine in the second step of the alternative menaquinone biosynthetic pathway. Substrate binding of the adenine moiety is mediated almost exclusively by hydrogen bonds, and the proposed catalytic mechanism requires multiple proton-transfer events. Of particular interest is the protonation state of residue D198, which possesses a pKa above 8 and functions as a general acid to initiate the enzymatic reaction. In this study we present three corefined neutron/X-ray crystal structures of wild-type HpMTAN cocrystallized with S-adenosylhomocysteine (SAH), Formycin A (FMA), and (3R,4S)-4-(4-Chlorophenylthiomethyl)-1-[(9-deaza-adenin-9-yl)methyl]-3-hydroxypyrrolidine (p-ClPh-Thio-DADMe-ImmA) as well as one neutron/X-ray crystal structure of an inactive variant (HpMTAN-D198N) cocrystallized with SAH. These results support a mechanism of D198 pKa elevation through the unexpected sharing of a proton with atom N7 of the adenine moiety possessing unconventional hydrogen-bond geometry. Additionally, the neutron structures also highlight active site features that promote the stabilization of the transition state and slight variations in these interactions that result in 100-fold difference in binding affinities between the DADMe-ImmA and ImmA analogs.

Primordial-like enzymes from bacteria with reduced genomes.

The first cells probably possessed rudimentary metabolic networks, built using a handful of multifunctional enzymes. The promiscuous activities of modern enzymes are often assumed to be relics of this primordial era; however, by definition these activities are no longer physiological. There are many fewer examples of enzymes using a single active site to catalyze multiple physiologically-relevant reactions. Previously, we characterized the promiscuous alanine racemase (ALR) activity of Escherichia coli cystathionine ß-lyase (CBL). Now we have discovered that several bacteria with reduced genomes lack alr, but contain metC (encoding CBL). We characterized the CBL enzymes from three of these: Pelagibacter ubique, the Wolbachia endosymbiont of Drosophila melanogaster (wMel) and Thermotoga maritima. Each is a multifunctional CBL/ALR. However, we also show that CBL activity is no longer required in these bacteria. Instead, the wMel and T. maritima enzymes are physiologically bi-functional alanine/glutamate racemases. They are not highly active, but they are clearly sufficient. Given the abundance of the microorganisms using them, we suggest that much of the planet's biochemistry is carried out by enzymes that are quite different from the highly-active exemplars usually found in textbooks. Instead, primordial-like enzymes may be an essential part of the adaptive strategy associated with streamlining.

Childhood Origins of Young Adult Environmental Behavior.

Prospective, longitudinal analyses revealed that over a 12-year period from ages 6 to 18, individuals who grew up with mothers with more proenvironmental attitudes engaged in more proenvironmental behavior as young adults. A similar marginal association was uncovered between mothers' proenvironmental behaviors and the proenvironmental behavior of their young adult offspring. Maternal educational attainment, but not political ideology, was also associated with more proenvironmental behavior as children matured. Moreover, childhood time spent outdoors was positively associated with increased environmentally responsible behavior in young adulthood. Interestingly, one's own childhood proenvironmental behavior and attitude, at least as assessed at age 6, bear little on one's eventual proenvironmental behavior as a young adult. Finally, among this set of childhood factors, maternal education and childhood time spent outdoors were independent predictors of positive changes in environmental behavior from early childhood to young adulthood.

Transition State Analogue Inhibitors of 5'-Deoxyadenosine/5'-Methylthioadenosine Nucleosidase from Mycobacterium tuberculosis.

Mycobacterium tuberculosis 5'-deoxyadenosine/5'-methylthioadenosine nucleosidase (Rv0091) catalyzes the N-riboside hydrolysis of its substrates 5'-methylthioadenosine (MTA) and 5'-deoxyadenosine (5'-dAdo). 5'-dAdo is the preferred substrate, a product of radical S-adenosylmethionine-dependent enzyme reactions. Rv0091 is characterized by a ribocation-like transition state, with low N-ribosidic bond order, an N7-protonated adenine leaving group, and an activated but weakly bonded water nucleophile. DADMe-Immucillins incorporating 5'-substituents of the substrates 5'-dAdo and MTA were synthesized and characterized as inhibitors of Rv0091. 5'-Deoxy-DADMe-Immucillin-A was the most potent among the 5'-dAdo transition state analogues with a dissociation constant of 640 pM. Among the 5'-thio substituents, hexylthio-DADMe-Immucillin-A was the best inhibitor at 87 pM. The specificity of Rv0091 for the Immucillin transition state analogues differs from those of other bacterial homologues because of an altered hydrophobic tunnel accepting the 5'-substituents. Inhibitors of Rv0091 had weak cell growth effects on M. tuberculosis or Mycobacterium smegmatis but were lethal toward Helicobacter pylori, where the 5'-methylthioadenosine nucleosidase is essential in menaquinone biosynthesis. We propose that Rv0091 plays a role in 5'-deoxyadenosine recycling but is not essential for growth in these Mycobacteria.