A µ-opioid receptor modulator that works cooperatively with naloxone.

The µ-opioid receptor (µOR) is a well-established target for analgesia1, yet conventional opioid receptor agonists cause serious adverse effects, notably addiction and respiratory depression. These factors have contributed to the current opioid overdose epidemic driven by fentanyl2, a highly potent synthetic opioid. µOR negative allosteric modulators (NAMs) may serve as useful tools in preventing opioid overdose deaths, but promising chemical scaffolds remain elusive. Here we screened a large DNA-encoded chemical library against inactive µOR, counter-screening with active, G-protein and agonist-bound receptor to 'steer' hits towards conformationally selective modulators. We discovered a NAM compound with high and selective enrichment to inactive µOR that enhances the affinity of the key opioid overdose reversal molecule, naloxone. The NAM works cooperatively with naloxone to potently block opioid agonist signalling. Using cryogenic electron microscopy, we demonstrate that the NAM accomplishes this effect by binding a site on the extracellular vestibule in direct contact with naloxone while stabilizing a distinct inactive conformation of the extracellular portions of the second and seventh transmembrane helices. The NAM alters orthosteric ligand kinetics in therapeutically desirable ways and works cooperatively with low doses of naloxone to effectively inhibit various morphine-induced and fentanyl-induced behavioural effects in vivo while minimizing withdrawal behaviours. Our results provide detailed structural insights into the mechanism of negative allosteric modulation of the µOR and demonstrate how this can be exploited in vivo.

Structure-based design of bitopic ligands for the µ-opioid receptor.

Mu-opioid receptor (µOR) agonists such as fentanyl have long been used for pain management, but are considered a major public health concern owing to their adverse side effects, including lethal overdose1. Here, in an effort to design safer therapeutic agents, we report an approach targeting a conserved sodium ion-binding site2 found in µOR3 and many other class A G-protein-coupled receptors with bitopic fentanyl derivatives that are functionalized via a linker with a positively charged guanidino group. Cryo-electron microscopy structures of the most potent bitopic ligands in complex with µOR highlight the key interactions between the guanidine of the ligands and the key Asp2.50 residue in the Na+ site. Two bitopics (C5 and C6 guano) maintain nanomolar potency and high efficacy at Gi subtypes and show strongly reduced arrestin recruitment-one (C6 guano) also shows the lowest Gz efficacy among the panel of µOR agonists, including partial and biased morphinan and fentanyl analogues. In mice, C6 guano displayed µOR-dependent antinociception with attenuated adverse effects, supporting the µOR sodium ion-binding site as a potential target for the design of safer analgesics. In general, our study suggests that bitopic ligands that engage the sodium ion-binding pocket in class A G-protein-coupled receptors can be designed to control their efficacy and functional selectivity profiles for Gi, Go and Gz subtypes and arrestins, thus modulating their in vivo pharmacology.

Spinal cord extracts of amyotrophic lateral sclerosis spread TDP-43 pathology in cerebral organoids.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder caused by progressive loss of motor neurons and there is currently no effective therapy. Cytoplasmic mislocalization and aggregation of TAR DNA-binding protein 43 kDa (TDP-43) within the CNS is a pathological hallmark in sporadic ALS and prion-like propagation of pathogenic TDP-43 is thought to be implicated in disease progression. However, cell-to-cell transmission of pathogenic TDP-43 in the human CNS has not been confirmed experimentally. Here we used induced pluripotent stem cells (iPSCs)-derived cerebral organoids as recipient CNS tissue model that are anatomically relevant human brain. We injected postmortem spinal cord protein extracts individually from three non-ALS or five sporadic ALS patients containing pathogenic TDP-43 into the cerebral organoids to validate the templated propagation and spreading of TDP-43 pathology in human CNS tissue. We first demonstrated that the administration of spinal cord extracts from an ALS patient induced the formation of TDP-43 pathology that progressively spread in a time-dependent manner in cerebral organoids, suggesting that pathogenic TDP-43 from ALS functioned as seeds and propagated cell-to-cell to form de novo TDP-43 pathology. We also reported that the administration of ALS patient-derived protein extracts caused astrocyte proliferation to form astrogliosis in cerebral organoids, reproducing the pathological feature seen in ALS. Moreover, we showed pathogenic TDP-43 induced cellular apoptosis and that TDP-43 pathology correlated with genomic damage due to DNA double-strand breaks. Thus, our results provide evidence that patient-derived pathogenic TDP-43 can mimic the prion-like propagation of TDP-43 pathology in human CNS tissue. Our findings indicate that our assays with human cerebral organoids that replicate ALS pathophysiology have a promising strategy for creating readouts that could be used in future drug discovery efforts against ALS.

Surface-Area Enhanced Raman Spectroscopy of DNA in Porous Silica: A Quantitative and Reproducible Alternative to Plasmonic-Based SERS.

Despite the success of surface-enhanced Raman spectroscopy (SERS) for detecting DNA immobilized on plasmonic metal surfaces, its quantitative response is limited by the rapid falloff of enhancement with distance from the metal surface and variations in sensitivity that depend on orientation and proximity to plasmonic "hot spots". In this work, we assess an alternative approach for enhancing detection by immobilizing DNA on the interior surfaces of porous silica particles. These substrates provide over a 1000-fold greater surface area for detection compared to a planar support. The porous silica substrate is a purely dielectric material with randomly oriented internal surfaces, where scattering is independent of proximity and orientation of oligonucleotides relative to the silica surface. We characterize the quantitative response of Raman scattering from DNA in porous silica particles with sequences used in previous SERS investigations of DNA for comparison. The results show that Raman scattering of DNA in porous silica is independent of distance of nucleotides from the silica surface, allowing detection of longer DNA strands with constant sensitivity. The surface area enhancement within particles is reproducible (<4% particle-to-particle variation) owing to the uniform internal pore structure and surface chemistry of the silica support. DNA immobilization with a bis-thiosuccinimide linker provides a Raman-active internal standard for quantitative interpretation of Raman scattering results. Despite the high (30 mM) concentrations of immobilized DNA within porous silica particles, they can be used to measure nanomolar binding affinities of target molecules to DNA by equilibrating a very small number of particles with a sufficiently large volume of low-concentration solution of target molecules.

Rare-variant and polygenic analyses of amyotrophic lateral sclerosis in the French-Canadian genome.

PURPOSE: The genetic etiology of amyotrophic lateral sclerosis (ALS) includes few rare, large-effect variants and potentially many common, small-effect variants per case. The genetic risk liability for ALS might require a threshold comprised of a certain amount of variants. Here, we tested the degree to which risk for ALS was affected by rare variants in ALS genes, polygenic risk score, or both. METHODS: 335 ALS cases and 356 controls from Québec, Canada were concurrently tested by microarray genotyping and targeted sequencing of ALS genes known at the time of study inception. ALS genome-wide association studies summary statistics were used to estimate an ALS polygenic risk score (PRS). Cases and controls were subdivided into rare-variant heterozygotes and non-heterozygotes. RESULTS: Risk for ALS was significantly associated with PRS and rare variants independently in a logistic regression model. Although ALS PRS predicted a small amount of ALS risk overall, the effect was most pronounced between ALS cases and controls that were not heterozygous for a rare variant in the ALS genes surveyed. CONCLUSION: Both PRS and rare variants in ALS genes impact risk for ALS. PRS for ALS is most informative when rare variants are not observed in ALS genes.

Linking Ecological Specialization to Its Macroevolutionary Consequences: An Example with Passerine Nest Type.

A long-standing hypothesis in evolutionary biology is that the evolution of resource specialization can lead to an evolutionary dead end, where specialists have low diversification rates and limited ability to evolve into generalists. In recent years, advances in comparative methods investigating trait-based differences associated with diversification have enabled more robust tests of this idea and have found mixed support. We test the evolutionary dead end hypothesis by estimating net diversification rate differences associated with nest-type specialization among 3224 species of passerine birds. In particular, we test whether the adoption of hole-nesting, a nest-type specialization that decreases predation, results in reduced diversification rates relative to nesting outside of holes. Further, we examine whether evolutionary transitions to the specialist hole-nesting state have been more frequent than transitions out of hole-nesting. Using diversification models that accounted for background rate heterogeneity and different extinction rate scenarios, we found that hole-nesting specialization was not associated with diversification rate differences. Furthermore, contrary to the assumption that specialists rarely evolve into generalists, we found that transitions out of hole-nesting occur more frequently than transitions into hole-nesting. These results suggest that interspecific competition may limit adoption of hole-nesting, but that such competition does not result in limited diversification of hole-nesters. In conjunction with other recent studies using robust comparative methods, our results add to growing evidence that evolutionary dead ends are not a typical outcome of resource specialization. [Cavity nesting; diversification; hidden-state models; passerines; resource specialization.].

Reflective questioning to guide socially just global health reform: a narrative review and expert elicitation.

Recent research has highlighted the impacts of colonialism and racism in global health, yet few studies have presented concrete steps toward addressing the problems. We conducted a narrative review to identify published evidence that documented guiding frameworks for enhancing equity and inclusion in global health research and practice (GHRP). Based on this narrative review, we developed a questionnaire with a series of reflection questions related on commonly reported challenges related to diversity, inclusion, equity, and power imbalances. To reach consensus on a set of priority questions relevant to each theme, the questionnaire was sent to a sample of 18 global health experts virtually and two rounds of iterations were conducted. Results identified eight thematic areas and 19 reflective questions that can assist global health researchers and practitioners striving to implement socially just global health reforms. Key elements identified for improving GHRP include: (1) aiming to understand the historical context and power dynamics within the areas touched by the program; (2) promoting and mobilizing local stakeholders and leadership and ensuring measures for their participation in decision-making; (3) ensuring that knowledge products are co-produced and more equitably accessible; (4) establishing a more holistic feedback and accountability system to understand needed reforms based on local perspectives; and (5) applying systems thinking to addressing challenges and encouraging approaches that can be sustained long-term. GHRP professionals should reflect more deeply on how their goals align with those of their in-country collaborators. The consistent application of reflective processes has the potential to shift GHRP towards increased equity.

Obesity and metabolic syndrome in patients with heart failure with preserved ejection fraction: a cross-sectional analysis of the Veradigm Cardiology Registry.

BACKGROUND: The proportion of heart failure patients with preserved ejection fraction has been rising over the past decades and has coincided with increases in the prevalence of obesity and metabolic syndrome. The relationship between these interconnected comorbidities and heart failure with preserved ejection fraction (HFpEF) is still poorly understood. This study characterized obesity and metabolic syndrome among real-world patients with HFpEF. METHODS: We identified adults with heart failure in the Veradigm Cardiology Registry, previously the PINNACLE Registry, with a left ventricular ejection fraction measurement ≥ 50% between 01/01/2016 and 12/31/2019. Patients were stratified by obesity diagnosis and presence of metabolic syndrome (≥ 3 of the following: diabetes, hypertension, hyperlipidemia, and obesity). We captured baseline demographic and clinical characteristics and used multivariable logistic regression to examine the odds of having cardiac (atrial fibrillation, coronary artery disease, coronary artery bypass surgery, myocardial infarction, and stroke/transient ischemic attack) and non-cardiac (chronic kidney disease, chronic liver disease, and peripheral artery disease) comorbidities of interest. The models adjusted for age and sex, and the main covariates of interest were obesity and metabolic burden score (0-3 based on the presence of diabetes, hypertension, and hyperlipidemia). The models were run with and without an obesity*metabolic burden score interaction term. RESULTS: This study included 264,571 patients with HFpEF, of whom 55.7% had obesity, 52.5% had metabolic syndrome, 42.5% had both, and 34.3% had neither. After adjusting for age, sex, and burden of other metabolic syndrome-associated diagnoses, patients with HFpEF with obesity had lower odds of a diagnosis of other evaluated comorbidities relative to patients without obesity. The presence of metabolic syndrome in HFpEF appears to increase comorbidity burden as each additional metabolic syndrome-associated diagnosis was associated with higher odds of assessed comorbidities except atrial fibrillation. CONCLUSION: Obesity was common among patients with HFpEF and not always co-occurring with metabolic syndrome. Multivariable analysis suggested that patients with obesity may develop HFpEF in the absence of other driving factors such as cardiovascular disease or metabolic syndrome.

Utility of Thrombectomy in Nonagenarians: A Scoping Review.

BACKGROUND: Mechanical thrombectomy represents a mainstay of management for acute ischemic stroke in the setting of large vessel occlusion. However, there are no clinical practice guidelines defining the role of thrombectomy at the extremes of age. In this scoping review, we aimed to summarize the existing medical and neurosurgical literature pertaining to mechanical thrombectomy in nonagenarians. The PubMed database was queried using the following terms and relevant citations assessed: "thrombectomy nonagenarian," "thrombectomy age 90," "stroke nonagenarian," and "ischemic stroke thrombectomy." Common measurable outcomes, including mortality, modified Rankin scale (mRS) score, and Thrombolysis in Cerebral Infarction (TICI) scale score, were utilized to compare results. SUMMARY: Thrombectomy was shown to improve functional outcomes in all eight of the studies included in the analysis. Mortality was assessed in only two reported studies, and thrombectomy was shown to provide a mortality benefit in one study amongst patients for whom first-pass reperfusion was achieved. Other outcomes of reported interest included greater early neurologic recovery at discharge and improved functional outcomes at 90 days amongst nonagenarians who underwent thrombectomy as compared to those who received thrombolytic therapy alone. Nonagenarians with good functional status at baseline were the most likely to have favorable outcomes. KEY MESSAGES: Mechanical thrombectomy improves outcomes among nonagenarians presenting with acute ischemic stroke due to large vessel occlusion. Further large-scale prospective studies are warranted to optimize patient selection and develop clinical practice guidelines specific to this important patient demographic.

Positive allosteric modulation of the mu-opioid receptor produces analgesia with reduced side effects.

Positive allosteric modulators (PAMs) of the mu-opioid receptor (MOR) have been hypothesized as potentially safer analgesics than traditional opioid drugs. This is based on the idea that PAMs will promote the action of endogenous opioid peptides while preserving their temporal and spatial release patterns and so have an improved therapeutic index. However, this hypothesis has never been tested. Here, we show that a mu-PAM, BMS-986122, enhances the ability of the endogenous opioid Methionine-enkephalin (Met-Enk) to stimulate G protein activity in mouse brain homogenates without activity on its own and to enhance G protein activation to a greater extent than ß-arrestin recruitment in Chinese hamster ovary (CHO) cells expressing human mu-opioid receptors. Moreover, BMS-986122 increases the potency of Met-Enk to inhibit GABA release in the periaqueductal gray, an important site for antinociception. We describe in vivo experiments demonstrating that the mu-PAM produces antinociception in mouse models of acute noxious heat pain as well as inflammatory pain. These effects are blocked by MOR antagonists and are consistent with the hypothesis that in vivo mu-PAMs enhance the activity of endogenous opioid peptides. Because BMS-986122 does not bind to the orthosteric site and has no inherent agonist action at endogenously expressed levels of MOR, it produces a reduced level of morphine-like side effects of constipation, reward as measured by conditioned place preference, and respiratory depression. These data provide a rationale for the further exploration of the action and safety of mu-PAMs as an innovative approach to pain management.

Scalp microbiome composition changes and pathway evaluations due to effective treatment with Piroctone Olamine shampoo.

OBJECTIVE: To characterize the scalp microbial composition, function, and connection to dandruff severity using a metagenomics approach and to understand the impact of a Piroctone Olamine containing anti-dandruff shampoo on the scalp microbiome. METHODS: Shotgun metagenomics was used to characterize the composition of the scalp microbiomes from 94 subjects with and without clinically defined dandruff. Furthermore, the microbiome of the scalps of 100 dandruff sufferers before and after 3 weeks of treatment with either control or anti-dandruff shampoo containing 0.5% Piroctone Olamine (PO) was characterized and compared to identify microorganisms associated with the dandruff condition and the associated pathways and processes that may contribute to PO's effect on scalp microbiome. RESULTS: A higher relative abundance of Malassezia restricta and Staphylococcus capitis and a lower abundance of Cutibacterium acnes were associated with the dandruff scalps relative to the no-dandruff scalps. A 3-week PO shampoo treatment reduced the relative abundance of Malassezia species and Staphylococcus capitis and increased the relative abundance of Cutibacterium acnes. This change to the scalp microbiome composition is consistent with a return to a healthy no-dandruff microbiome and improved clinical signs and symptoms as measured by adherent scalp flaking score (ASFS) compared with the control shampoo. Functional genomics analysis showed that the PO shampoo treatment reduced oxidative stress-associated genes and decreased the abundance of protease, urease, and lipase genes. These changes correlated positively to improvements in dandruff severity. PO treatment favourably shifted scalp microbiomes in dandruff subjects toward the no-dandruff state. CONCLUSION: Our results suggest that part of the aetiology of dandruff can be attributed to dysbiosis of the scalp microbiome. PO treatment can restore a healthier microbiome, reducing oxidative stress and promoting better scalp health.

OBJECTIF: Caractériser la composition microbienne du cuir chevelu, sa fonction et son lien avec la sévérité des pellicules à l'aide d'une approche métagénomique. Comprendre l'impact d'un shampooing antipelliculaire à base de piroctone olamine sur le microbiome du cuir chevelu. MÉTHODES: La métagénomique shotgun a été utilisée pour caractériser la composition des microbiomes du cuir chevelu de 94 sujets avec et sans pellicules définies cliniquement. Par ailleurs, le microbiome des cuirs chevelus de 100 personnes ayant des pellicules avant et après trois semaines de traitement par un shampooing témoin ou un shampooing antipelliculaire contenant 0,5 % de piroctone olamine (PO) a été caractérisé et comparé pour identifier les micro­organismes associés à l'état pelliculaire, et les voies et processus associés pouvant contribuer à l'effet de la PO sur le microbiome du cuir chevelu. RÉSULTATS: Une abondance relative plus élevée de Malassezia restricta et de Staphylococcus capitis, et une abondance plus faible de Cutibacterium acnes étaient associées aux cuirs chevelus avec des pellicules par rapport aux cuirs chevelus sans pellicules. Un traitement avec un shampooing contenant de la PO de 3 semaines a réduit l'abondance relative des espèces Malassezia et Staphylococcus capitis, et a augmenté l'abondance relative de Cutibacterium acnes. Cette modification de la composition du microbiome du cuir chevelu est cohérente avec un retour à un microbiome sain sans pellicules, et une amélioration des signes et symptômes cliniques mesurés par le score de desquamation du cuir chevelu adhérent (Adherent Scalp Flaking Score, ASFS) par rapport au shampooing témoin. L'analyse génomique fonctionnelle a montré que le traitement avec un shampooing contenant de la PO réduisait les gènes associés au stress oxydatif et diminuait l'abondance des gènes de la protéase, de l'uréase et de la lipase. Ces modifications étaient corrélées positivement à des améliorations de la sévérité des pellicules. Le traitement avec la PO a favorisé l'évolution des microbiomes du cuir chevelu des sujets ayant des pellicules vers un état sans pellicules. CONCLUSION: Nos résultats suggèrent qu'une partie de l'étiologie des pellicules peut être attribuée à la dysbiose du microbiome du cuir chevelu. Le traitement avec la PO peut rétablir un microbiome plus sain, en réduisant le stress oxydatif et en favorisant une meilleure santé du cuir chevelu.

How Does Literacy Affect Speech Processing? Not by Enhancing Cortical Responses to Speech, But by Promoting Connectivity of Acoustic-Phonetic and Graphomotor Cortices.

Previous research suggests that literacy, specifically learning alphabetic letter-to-phoneme mappings, modifies online speech processing and enhances brain responses, as indexed by the BOLD, to speech in auditory areas associated with phonological processing (Dehaene et al., 2010). However, alphabets are not the only orthographic systems in use in the world, and hundreds of millions of individuals speak languages that are not written using alphabets. In order to make claims that literacy per se has broad and general consequences for brain responses to speech, one must seek confirmatory evidence from nonalphabetic literacy. To this end, we conducted a longitudinal fMRI study in India probing the effect of literacy in Devanagari, an abubgida, on functional connectivity and cerebral responses to speech in 91 variously literate Hindi-speaking male and female human participants. Twenty-two completely illiterate participants underwent 6 months of reading and writing training. Devanagari literacy increases functional connectivity between acoustic-phonetic and graphomotor brain areas, but we find no evidence that literacy changes brain responses to speech, either in cross-sectional or longitudinal analyses. These findings shows that a dramatic reconfiguration of the neurofunctional substrates of online speech processing may not be a universal result of learning to read, and suggest that the influence of writing on speech processing should also be investigated.SIGNIFICANCE STATEMENT It is widely claimed that a consequence of being able to read is enhanced auditory processing of speech, reflected by increased cortical responses in areas associated with phonological processing. Here we find no relationship between literacy and the magnitude of brain response to speech stimuli in individuals who speak Hindi, which is written using a nonalphabetic script, Devanagari, an abugida. We propose that the exact nature of the script under examination must be considered before making sweeping claims about the consequences of literacy for the brain. Further, we find evidence that literacy enhances functional connectivity between auditory processing areas and graphomotor areas, suggesting a mechanism whereby learning to write might influence speech perception.

Development of a Nickel-Catalyzed N-N Coupling for the Synthesis of Hydrazides.

A nickel-catalyzed N-N cross-coupling for the synthesis of hydrazides is reported. O-Benzoylated hydroxamates were efficiently coupled with a broad range of aryl and aliphatic amines via nickel catalysis to form hydrazides in an up to 81% yield. Experimental evidence implicates the intermediacy of electrophilic Ni-stabilized acyl nitrenoids and the formation of a Ni(I) catalyst via silane-mediated reduction. This report constitutes the first example of an intermolecular N-N coupling compatible with secondary aliphatic amines.

Risk factors for extended-spectrum beta-lactamase (ESBL)-producing E. coli carriage among children in a food animal-producing region of Ecuador: A repeated measures observational study.

BACKGROUND: The spread of antibiotic-resistant bacteria may be driven by human-animal-environment interactions, especially in regions with limited restrictions on antibiotic use, widespread food animal production, and free-roaming domestic animals. In this study, we aimed to identify risk factors related to commercial food animal production, small-scale or "backyard" food animal production, domestic animal ownership, and practices related to animal handling, waste disposal, and antibiotic use in Ecuadorian communities. METHODS AND FINDINGS: We conducted a repeated measures study from 2018 to 2021 in 7 semirural parishes of Quito, Ecuador to identify determinants of third-generation cephalosporin-resistant E. coli (3GCR-EC) and extended-spectrum beta-lactamase E. coli (ESBL-EC) in children. We collected 1,699 fecal samples from 600 children and 1,871 domestic animal fecal samples from 376 of the same households at up to 5 time points per household over the 3-year study period. We used multivariable log-binomial regression models to estimate relative risks (RR) of 3GCR-EC and ESBL-EC carriage, adjusting for child sex and age, caregiver education, household wealth, and recent child antibiotic use. Risk factors for 3GCR-EC included living within 5 km of more than 5 commercial food animal operations (RR: 1.26; 95% confidence interval (CI): 1.10, 1.45; p-value: 0.001), household pig ownership (RR: 1.23; 95% CI: 1.02, 1.48; p-value: 0.030) and child pet contact (RR: 1.23; 95% CI: 1.09, 1.39; p-value: 0.001). Risk factors for ESBL-EC were dog ownership (RR: 1.35; 95% CI: 1.00, 1.83; p-value: 0.053), child pet contact (RR: 1.54; 95% CI: 1.10, 2.16; p-value: 0.012), and placing animal feces on household land/crops (RR: 1.63; 95% CI: 1.09, 2.46; p-value: 0.019). The primary limitations of this study are the use of proxy and self-reported exposure measures and the use of a single beta-lactamase drug (ceftazidime with clavulanic acid) in combination disk diffusion tests for ESBL confirmation, potentially underestimating phenotypic ESBL production among cephalosporin-resistant E. coli isolates. To improve ESBL determination, it is recommended to use 2 combination disk diffusion tests (ceftazidime with clavulanic acid and cefotaxime with clavulanic acid) for ESBL confirmatory testing. Future studies should also characterize transmission pathways by assessing antibiotic resistance in commercial food animals and environmental reservoirs. CONCLUSIONS: In this study, we observed an increase in enteric colonization of antibiotic-resistant bacteria among children with exposures to domestic animals and their waste in the household environment and children living in areas with a higher density of commercial food animal production operations.

Evolution of a Human-Specific Tandem Repeat Associated with ALS.

Tandem repeats are proposed to contribute to human-specific traits, and more than 40 tandem repeat expansions are known to cause neurological disease. Here, we characterize a human-specific 69 bp variable number tandem repeat (VNTR) in the last intron of WDR7, which exhibits striking variability in both copy number and nucleotide composition, as revealed by long-read sequencing. In addition, greater repeat copy number is significantly enriched in three independent cohorts of individuals with sporadic amyotrophic lateral sclerosis (ALS). Each unit of the repeat forms a stem-loop structure with the potential to produce microRNAs, and the repeat RNA can aggregate when expressed in cells. We leveraged its remarkable sequence variability to align the repeat in 288 samples and uncover its mechanism of expansion. We found that the repeat expands in the 3'-5' direction, in groups of repeat units divisible by two. The expansion patterns we observed were consistent with duplication events, and a replication error called template switching. We also observed that the VNTR is expanded in both Denisovan and Neanderthal genomes but is fixed at one copy or fewer in non-human primates. Evaluating the repeat in 1000 Genomes Project samples reveals that some repeat segments are solely present or absent in certain geographic populations. The large size of the repeat unit in this VNTR, along with our multiplexed sequencing strategy, provides an unprecedented opportunity to study mechanisms of repeat expansion, and a framework for evaluating the roles of VNTRs in human evolution and disease.

Raman Scattering Reveals Ion-Dependent G-Quadruplex Formation in the 15-mer Thrombin-Binding Aptamer upon Association with α-Thrombin.

The discovery of DNA aptamers that bind biomolecular targets has enabled significant innovations in biosensing. Aptamers form secondary structures that exhibit selective high-affinity interactions with their binding partners. The binding of its target by an aptamer is often accompanied by conformational changes, and sensing by aptamers often relies on these changes to provide readout signals from extrinsic labels to detect target association. Many biosensing applications involve aptamers immobilized to surfaces, but methods to characterize conformations of immobilized aptamers and their in situ response have been lacking. To address this challenge, we have developed a structurally informative Raman spectroscopy method to determine conformations of the 15-mer thrombin-binding aptamer (TBA) immobilized on porous silica surfaces. The TBA is of interest because its binding of α-thrombin depends on the aptamer forming an antiparallel G-quadruplex, which is thought to drive signal changes that allow thrombin-binding to be detected. However, specific metal cations also stabilize the G-quadruplex conformation of the aptamer, even in the absence of its protein target. To develop a deeper understanding of the conformational response of the TBA, we utilize Raman spectroscopy to quantify the effects of the metal cations, K+ (stabilizing) and Li+ (nonstabilizing), on G-quadruplex versus unfolded populations of the TBA. In K+ or Li+ solutions, we then detect the association of α-thrombin with the immobilized aptamer, which can be observed in Raman scattering from the bound protein. The results show that the association of α-thrombin in K+ solutions produces no detectable change in aptamer conformation, which is found in the G-quadruplex form both before and after binding its target. In Li+ solutions, however, where the TBA is unfolded prior to α-thrombin association, protein binding occurs with the formation of a G-quadruplex by the aptamer.

The role of plasmids in carbapenem resistant E. coli in Alameda County, California.

BACKGROUND: Antimicrobial resistant infections continue to be a leading global public health crisis. Mobile genetic elements, such as plasmids, have been shown to play a major role in the dissemination of antimicrobial resistance (AMR) genes. Despite its ongoing threat to human health, surveillance of AMR in the United States is often limited to phenotypic resistance. Genomic analyses are important to better understand the underlying resistance mechanisms, assess risk, and implement appropriate prevention strategies. This study aimed to investigate the extent of plasmid mediated antimicrobial resistance that can be inferred from short read sequences of carbapenem resistant E. coli (CR-Ec) in Alameda County, California. E. coli isolates from healthcare locations in Alameda County were sequenced using an Illumina MiSeq and assembled with Unicycler. Genomes were categorized according to predefined multilocus sequence typing (MLST) and core genome multilocus sequence typing (cgMLST) schemes. Resistance genes were identified and corresponding contigs were predicted to be plasmid-borne or chromosome-borne using two bioinformatic tools (MOB-suite and mlplasmids). RESULTS: Among 82 of CR-Ec identified between 2017 and 2019, twenty-five sequence types (STs) were detected. ST131 was the most prominent (n = 17) followed closely by ST405 (n = 12). blaCTX-M were the most common ESBL genes and just over half (18/30) of these genes were predicted to be plasmid-borne by both MOB-suite and mlplasmids. Three genetically related groups of E. coli isolates were identified with cgMLST. One of the groups contained an isolate with a chromosome-borne blaCTX-M-15 gene and an isolate with a plasmid-borne blaCTX-M-15 gene. CONCLUSIONS: This study provides insights into the dominant clonal groups driving carbapenem resistant E. coli infections in Alameda County, CA, USA clinical sites and highlights the relevance of whole-genome sequencing in routine local genomic surveillance. The finding of multi-drug resistant plasmids harboring high-risk resistance genes is of concern as it indicates a risk of dissemination to previously susceptible clonal groups, potentially complicating clinical and public health intervention.

Structural and Functional Characteristics of Cerebral Arteries as an Explanation for Clinical Syndromes Limited to the Brain.

Vascular disease affects many different arterial beds throughout the body. Yet the brain is susceptible to several vascular disorders that either are not found in other parts of the body or when found are much less likely to cause clinical syndromes in other organs. This specific vulnerability of the brain may be explained by structural and functional differences between the vessels of the brain and those of vessels in other parts of the body. In this review, we focus on how cerebrovascular anatomy and physiology may make the brain and its vessels more susceptible to unique vascular pathologies. To highlight these differences, we use our knowledge of five diseases and syndromes that most commonly manifest in the intracranial vasculature. For each, we identify characteristics of the intracranial arteries that make them susceptible to these diseases, while noting areas of uncertainty requiring further research.

Diagnostic Performance in Low- and High-Contrast Tasks of an Image-Based Denoising Algorithm Applied to Radiation Dose-Reduced Multiphase Abdominal CT Examinations.

BACKGROUND. Anatomic redundancy between phases can be used to achieve denoising of multiphase CT examinations. A limitation of iterative reconstruction (IR) techniques is that they generally require use of CT projection data. A frequency-split multi-band-filtration algorithm applies denoising to the multiphase CT images themselves. This method does not require knowledge of the acquisition process or integration into the reconstruction system of the scanner, and it can be implemented as a supplement to commercially available IR algorithms. OBJECTIVE. The purpose of the present study is to compare radiologists' performance for low-contrast and high-contrast diagnostic tasks (i.e., tasks for which differences in CT attenuation between the imaging target and its anatomic background are subtle or large, respectively) evaluated on multiphase abdominal CT between routine-dose images and radiation dose-reduced images processed by a frequency-split multiband-filtration denoising algorithm. METHODS. This retrospective single-center study included 47 patients who underwent multiphase contrast-enhanced CT for known or suspected liver metastases (a low-contrast task) and 45 patients who underwent multiphase contrast-enhanced CT for pancreatic cancer staging (a high-contrast task). Radiation dose-reduced images corresponding to dose reduction of 50% or more were created using a validated noise insertion technique and then underwent denoising using the frequency-split multi-band-filtration algorithm. Images were independently evaluated in multiple sessions by different groups of abdominal radiologists for each task (three readers in the low-contrast arm and four readers in the high-contrast arm). The noninferiority of denoised radiation dose-reduced images to routine-dose images was assessed using the jackknife alternative free-response ROC (JAFROC) figure-of-merit (FOM; limit of noninferiority, -0.10) for liver metastases detection and using the Cohen kappa statistic and reader confidence scores (100-point scale) for pancreatic cancer vascular invasion. RESULTS. For liver metastases detection, the JAFROC FOM for denoised radiation dose-reduced images was 0.644 (95% CI, 0.510-0.778), and that for routine-dose images was 0.668 (95% CI, 0.543-0.792; estimated difference, -0.024 [95% CI, -0.084 to 0.037]). Intraobserver agreement for pancreatic cancer vascular invasion was substantial to near perfect when the two image sets were compared (κ = 0.53-1.00); the 95% CIs of all differences in confidence scores between image sets contained zero. CONCLUSION. Multiphase contrast-enhanced abdominal CT images with a radiation dose reduction of 50% or greater that undergo denoising by a frequency-split multiband-filtration algorithm yield performance similar to that of routine-dose images for detection of liver metastases and vascular staging of pancreatic cancer. CLINICAL IMPACT. The image-based denoising algorithm facilitates radiation dose reduction of multiphase examinations for both low- and high-contrast diagnostic tasks without requiring manufacturer-specific hardware or software.

Vestibular Vertigo and Disparities in Healthcare Access Among Adults in the United States.

OBJECTIVE: Vertigo and dizziness have a high lifetime prevalence with significant impacts on daily life. We sought to explore differences in access to and ability to afford care among adults with vestibular vertigo by race/ethnicity, income, and insurance type. DESIGN: This is a cross-sectional study using the 2016 National Health Interview Survey. A total of 32,047 adults who completed the 2016 National Health Interview Survey Balance Supplement were analyzed. We used a previously validated definition of vertigo defined as (1) positional vertigo, (2) rotational vertigo, or (3) recurrent dizziness with nausea and either oscillopsia or imbalance. We examined several self-reported measures of healthcare utilization and access. RESULTS: Among adults with vestibular vertigo, African Americans had significantly increased odds of delayed care due to lack of transportation; Hispanic ethnicity was associated with decreased odds of skipping medication doses and asking a doctor for a lower-cost medication. Adults with public insurance had significantly lower odds of reporting delayed care due to worry about cost, not receiving medical care due to cost, and delayed filling of a prescription, but had greater odds of reporting delayed care due to lack of transportation. Lack of insurance and lower income were associated with increased odds of delaying and not receiving care due to cost. CONCLUSION: These findings demonstrate significant differences in access to care among adults with vestibular vertigo in the United States based on race, income, and health insurance status.