bluuebunny/crossref_metadata_2025 huggingface dataset:
DistinctWolbachialocalization patterns in oocytes of diverse host species reveal multiple strategies of maternal transmission
Authors: Radousky Yonah A. , Hague Michael T.J. , Fowler Sommer , Paneru Eliza , Codina Adan , Rugamas Cecilia , Hartzog Grant , Cooper Brandon S. , Sullivan William
Published: October 2022

A broad array of endosymbionts radiate through host populations via vertical transmission, yet much remains unknown concerning the cellular basis, diversity and routes underlying this transmission strategy. Here we address these issues, by examining the cellular distributions ofWolbachiastrains that diverged up to 50 million years ago in the oocytes of 18 divergentDrosophilaspecies. This analysis revealed threeWolbachiadistribution patterns: 1) a tight clustering at the posterior pole plasm (the site of germline formation); 2) a concentration at the posterior pole plasm, but with a significant bacteria population distributed throughout the oocyte; 3) and a distribution throughout the oocyte, with none or very few located at the posterior pole plasm. Examination of this latter class indicatesWolbachiaaccesses the posterior pole plasm during the interval between late oogenesis and the blastoderm formation. We also find that oneWolbachiastrain in this class concentrates in the posterior somatic follicle cells that encompass the pole plasm of the developing oocyte. In contrast, strains in whichWolbachiaconcentrate at the posterior pole plasm generally exhibit no or fewWolbachiain the follicle cells associated with the pole plasm. Taken together, these studies suggest that for someDrosophilaspecies,Wolbachiainvade the germline from neighboring somatic follicle cells. Phylogenomic analysis indicates that closely relatedWolbachiastrains tend to exhibit similar patterns of posterior localization, suggesting that specific localization strategies are a function ofWolbachia-associated factors. Previous studies revealed that endosymbionts rely ononeof two distinct routes of vertical transmission: continuous maintenance in the germline (germline-to-germline) or a more circuitous route via the soma (germline-to-soma-to-germline). Here we provide compelling evidence thatWolbachiastrains infectingDrosophilaspecies maintain the diverse arrays of cellular mechanisms necessary forbothof these distinct transmission routes. This characteristic may account for its ability to infect and spread globally through a vast range of host insect species.
Single-cell RNA-seq reveals alterations in peripheralCX3CR1and nonclassical monocytes in familial tauopathy
Authors: Sirkis Daniel W. , Solsberg Caroline Warly , Johnson Taylor P. , Bonham Luke W. , Sturm Virginia E. , Lee Suzee E. , Rankin Katherine P. , Rosen Howard J. , Boxer Adam L. , Seeley William W. , Miller Bruce L. , Geier Ethan G. , Yokoyama Jennifer S.
Published: October 2022

Background

Emerging evidence from mouse models is beginning to elucidate the brain’s immune response to tau pathology, but little is known about the nature of this response in humans. In addition, it remains unclear to what extent tau pathology and the local inflammatory response within the brain influence the broader immune system.

Methods

To address these questions, we performed single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) from carriers of pathogenic variants inMAPT, the gene encoding tau.

Results

Analysis of ∼181,000 individual PBMC transcriptomes fromMAPTpathogenic variant carriers (n= 8) and healthy non-carrier controls (n= 8) demonstrated striking differential expression in monocytes and natural killer (NK) cells. We observed a marked reduction in the expression ofCX3CR1– the gene encoding the fractalkine receptor that is known to modulate tau pathology in mouse models – in monocytes and NK cells. We also observed a significant reduction in the abundance of nonclassical monocytes and dysregulated expression of nonclassical monocyte marker genes, includingFCGR3A. Finally, we identified reductions inTMEM176AandTMEM176B, genes thought to be involved in the inflammatory response in human microglia. We confirmed differential expression of select biologically relevant genes dysregulated in our scRNA-seq data using droplet digital PCR as an orthogonal technique for quantitative validation.

Conclusions

Our results suggest that human peripheral immune cell expression and abundance are modulated by tau-associated pathophysiologic changes.CX3CR1and nonclassical monocytes in particular will be a focus of future work exploring the role of these peripheral signals in additional tau-associated neurodegenerative diseases.
Role of mesolimbic cannabinoid receptor 1 in stress-driven increases in cocaine self-administration in male rats
Authors: McReynolds Jayme R. , Wolf Colten P. , Starck Dylan M. , Mathy Jacob C. , Schaps Rebecca , Krause Leslie A. , Hillard Cecilia J. , Mantsch John R.
Published: October 2022

Stress is prevalent in the lives of those with substance use disorders (SUDs) and influences SUD outcomes. Understanding the neurobiological mechanisms through which stress promotes drug use is important for the development of effective SUD interventions. We have developed a model wherein exposure to a stressor, uncontrollable electric footshock, daily at the time of cocaine self-administration (SA) escalates intake in male rats. Here we test the hypothesis that stress-induced escalation of cocaine SA requires CB1 cannabinoid receptor signaling. Male Sprague-Dawley rats self-administered cocaine (0.5 mg/kg/inf, i.v.) during 2-h sessions comprised of four 30-min SA components separated by 5-min shock sequences or 5-min shock-free periods for 14 days. Footshock produced an escalation of cocaine SA that persisted following shock removal. Systemic administration of the cannabinoid receptor type 1 (CB1R) antagonist, AM251, attenuated cocaine intake only in rats with a history of stress. This effect was localized to the mesolimbic system, as intra-nucleus accumbens (NAc) shell and intra-ventral tegmental area (VTA) micro-infusions of AM251 attenuated cocaine intake only in stress-escalated rats. Cocaine SA, regardless of stress history, increases CB1R binding site density in the VTA, but not NAc shell. Following extinction, cocaine-primed reinstatement (10 mg/kg, ip) was increased in rats with prior footshock during SA. AM251 attenuated reinstatement only in rats with a stress history. Altogether, these data demonstrate that mesolimbic CB1R signaling is required to escalate intake and heighten relapse susceptibility and suggest that repeated stress at the time of cocaine use enhances mesolimbic CB1R signaling through a currently unknown mechanism.
Allosteric mechanism of transcription inhibition by NusG-dependent pausing of RNA polymerase
Authors: Vishwakarma Rishi K. , Qayyum M. Zuhaib , Babitzke Paul , Murakami Katsuhiko S.
Published: October 2022

NusG is a transcription elongation factor that stimulates transcription pausing in Gram+ bacteria includingBacillus subtilisby sequence-specific interaction with a conserved pause-inducing-11TTNTTT-6motif found in the non-template DNA (ntDNA) strand within the transcription bubble. To reveal the structural basis of NusG-dependent pausing, we determined a cryo-EM structure of a paused transcription complex containing RNAP, NusG, and the TTNTTT motif in the ntDNA strand. Interaction of NusG with the ntDNA strand rearranges the transcription bubble by positioning three consecutive T residues in a cleft between NusG and the β-lobe domain of RNAP. We revealed that the RNAP swivel module rotation (swiveling), which widens (swiveled state) and narrows (non-swiveled state) a cleft between NusG and the β-lobe, is an intrinsic motion of RNAP and is directly linked to nucleotide binding at the active site and to trigger loop folding, an essential conformational change of all cellular RNAPs for the RNA synthesis reaction. We also determined cryo-EM structures of RNAP escaping from a paused transcription complex. These structures revealed the NusG-dependent pausing mechanism by which NusG-ntDNA interaction inhibits the transition from swiveled to non-swiveled states, thereby preventing trigger loop folding and RNA synthesis allosterically. This motion is also reduced by formation of an RNA hairpin within the RNA exit channel. Thus, the pause half-life can be modulated by the strength of the NusG-ntDNA interaction and/or the stability of the RNA hairpin. NusG residues that interact with the TTNTTT motif are widely conserved in bacteria, suggesting that NusG-dependent pausing of transcription is widespread.

Significance statement

Transcription pausing by RNA polymerase (RNAP) regulates gene expression where it controls co-transcriptional RNA folding, synchronizes transcription with translation, and provides time for binding of regulatory factors. Transcription elongation factor NusG stimulates pausing in Gram+ bacteria includingBacillus subtilisandMycobacterium tuberculosisby sequence-specific interaction with a conserved pause motif found in the non-template DNA (ntDNA) strand within the transcription bubble. Our structural and biochemical results revealed that part of the conserved TTNTTT motif in ntDNA is extruded and sandwiched between NusG and RNAP. Our results further demonstrate that an essential global conformational change in RNAP is directly linked to RNA synthesis, and that the NusG-ntDNA interaction pauses RNA synthesis by interfering with this conformational change.
Distribution and survival strategies of diazotrophs in the Arctic Ocean revealed by global-scale metagenomic analysis
Authors: Shiozaki Takuhei , Nishimura Yosuke , Yoshizawa Susumu , Takami Hideto , Hamasaki Koji , Fujiwara Amane , Nishino Shigeto , Harada Naomi
Published: October 2022

Nitrogen fixation is the major source of reactive nitrogen in the ocean and has been considered to occur specifically in low-latitude oligotrophic oceans. Recent studies have shown that nitrogen fixation also occurs in the polar regions and thus is a global process, although the physiological and ecological characteristics of polar diazotrophs are not yet known. Here, we successfully reconstructed genomes, including that of cyanobacterium UCYN-A (Candidatus‘Atelocyanobacterium thalassa’), from metagenome data corresponding to 111 samples isolated from the Arctic Ocean. These diazotrophs were highly abundant in the Arctic Ocean (max., 1.28% of the total microbial community), suggesting that they have important roles in the Arctic ecosystem and biogeochemical cycles. Diazotrophs in the Arctic Ocean were either Arctic-specific or universal species. Arctic-specific diazotrophs, including Arctic UCYN-A, had unique gene sets (e.g., aromatics degradation) and/or a very small cell size (<0.2 µm), suggesting adaptations to Arctic-specific conditions. Universal diazotrophs were generally heterotrophs and commonly had the gene that encodes the cold-inducible RNA chaperone, which presumably makes their survival possible even in deep, cold waters and polar regions. Thus both types of diazotroph have physiological traits adaptable to their environments, which allow nitrogen fixation on a global scale.
Vascular endothelial growth factor A contributes to the increasing of mammalian respiratory epithelial permeability induced byPasteurella multocidainfection
Authors: Lin Lin , Yang Jie , Zhang Dajun , Lv Qingjie , Wang Fei , Liu Peng , Wang Mixue , Shi Congcong , Huang Xi , Liang Wan , Tan Chen , Wang Xiangru , Chen Huanchun , Wilson Brenda A , Wu Bin , Peng Zhong
Published: October 2022

Infections withPasteurella multocidacan cause significant zoonotic respiratory problems in both humans and animals.In vivotests in mouse infection models were used to investigate the mechanisms of respiratory epithelial barrier dysfunction during respiratory bacterial infection with these pathogens. Results revealed thatP. multocidainfection significantly increased epithelial permeability and increased expression of vascular endothelial growth factor A (VEGFA) and endothelial nitric oxide synthase (eNOS) in murine tracheae and lungs. In murine lung epithelial cell (MLE-12) models,P. multocidainfection decreased the expression of tight junctions (ZO-1) and adherens junctions (β-catenin, E-cadherin), but induced the activation of the hypoxia-inducible factor-1α (HIF-1α) and VEGFA signaling. When expression of HIF-1α is suppressed, the induction of VEGFA and ZO-1expression byP. multocidainfection is decreased. We also found that intervention of HIF-1α and VEGFA signaling affected infection outcomes caused by respiratory bacteria in mouse models. Most importantly, we demonstrated thatP. multocidainfection increased permeability of human respiratory epithelial cells and this process was associated with the activation of the HIF-1α and VEGFA signaling and likely contributes to the pathogenesis ofP. multocidain humans.

Importance

Mammalian respiratory epithelium forms the first line of defense against infections withPasteurella multocida, an important zoonotic respiratory pathogen. In this study, we foundP. multocidainfection increased respiratory epithelial permeability and promoted the induction of the hypoxia-HIF-1α-VEGFA axis in both mouse and murine cell models. Similar findings were also demonstrated in human respiratory epithelial cells. The results from this study gain important knowledge about the pathogenesis ofP. multocidacausing infections in both animals and humans.
Behavioral interventions to improve sleep outcomes in people with multiple sclerosis: A systematic review
Authors: Turkowitch David , Donkers Sarah J. , Costa Silvana L. , Vaduvathiriyan Prasanna , Williams Joy , Siengsukon Catherine
Published: October 2022

Objective

To determine effective behavioral interventions to improve sleep in people with MS.

Methods

Systematic review following PRISMA guidelines.

Data Sources

Literature searches were performed in December 2021 in Ovid MEDLINE, CINAHL, and Web of Science along with hand searching for grey literature and cited references. Out of the 837 search results, 830 unique references were reviewed after duplicates were removed.

Study Selection

Four reviewers independently reviewed titles and abstracts (two reviewers for each article), and a fifth reviewer resolved discrepancies. The full-text articles (n = 81) were reviewed independently by four reviewers (two for each article) for eligibility, and consensus for inclusion was achieved by a fifth reviewer as needed. Thirty-seven articles were determined eligible for inclusion.

Data Extraction

Four reviewers extracted relevant data from each study (two reviewers for each article) using a standard data-extraction table. Consensus was achieved for completeness and accuracy of the data extraction table by a fifth reviewer. Four reviewers (two reviewers for each article) conducted a quality appraisal of each article to assess the risk for bias and quality of the articles and consensus was achieved by a fifth reviewer as needed.

Data Synthesis

Descriptions were used to describe types of interventions, sleep outcomes, results, and key components across interventions.

Conclusions

The variability in the intervention types, intervention dose, outcomes used, training/expertise of interventionist, specific sample included, and quality of the study made it difficult to compare and synthesize results. Overall, the CBT-I, CBT/psychotherapy, and education/self-management support interventions reported positive improvements in sleep outcomes. The quality appraisal scores ranged from low to high quality indicating potential for bias. Further research is necessary to demonstrate efficacy of most of the interventions.
Engagement with the HCV care cascade among high-risk groups: a population-based study
Authors: Erman Aysegul , Everett Karl , Wong William W. L. , Forouzannia Farinaz , Greenaway Christina , Janjua Naveed , Kwong Jeffrey C. , Sander Beate
Published: October 2022

Background

Hepatitis C virus (HCV) elimination requires a thorough understanding of the care cascade. A direct-acting-antiviral (DAA)-era description of the care cascade has not been undertaken in Ontario, Canada. Our primary objective was to describe the current population-level care cascade in the general Ontario population and among key risk-groups — baby-boomers, immigrants, and individuals experiencing residential instability. The secondary objective was to identify predictors of engagement.

Methods

We conducted a population-based cohort study of Ontario residents undergoing HCV testing between January 1, 1999, and December 31, 2018, and mapped the care cascade [antibody diagnosed, RNA tested, RNA positive, genotyped, treated, achieved sustained virologic response (SVR), reinfected/relapsed] as of December 31, 2018. The cascade was stratified by risk groups. Cause-specific hazard modeling was used to identify demographic, and socioeconomic predictors of engagement with key steps of the cascade.

Results

Among 108,428 Ontario resident living with an HCV antibody diagnosis, 88% received confirmatory RNA testing; of these, 62% tested positive and 94% of positive tests were genotyped. Of those with confirmed viremia, 53% initiated treatment and 76% of treated individuals achieved SVR, while ∼1% experienced reinfection or relapse. Males, older birth cohorts, long-term residents, those with a history of substance use disorder and social marginalisation (e.g., material deprivation, residential instability), and those initially diagnosed in the pre-DAA era exhibited lower rates of engagement with almost every step of HCV care.

Conclusions

Despite DAA-era improvements, treatment initiation remains a major gap. HCV screening and linkage-to-treatment, particularly for those with a history of substance use disorder and social marginalisation, will be needed to equitably close gaps in HCV care in Ontario.
Prevalence of comorbidities and their impact on survival among older adults with the five most common cancers in Taiwan: A population study
Authors: Chien Li-Hsin , Tseng Tzu-Jui , Chen Tzu-Yu , Chen Chung-Hsing , Chen Chia-Yu , Jiang Hsin-Fang , Tsai Fang-Yu , Ku Hsiu-Ying , Jiang Shih Sheng , Hsiung Chao A. , Liu Tsang-Wu , Chang I-Shou
Published: October 2022

Because of the cancer incidence increase and population aging in Taiwan, we aimed to assess the cancer prevalence, to summarize the comorbidities of older patients with the five most common cancers (i.e., breast, colorectal, liver, lung, and oral), and to develop a Taiwan comorbidity index (TCI) for studying their actual prognosis. The linkage of the Taiwan Cancer Registry, Cause of Death Database, and National Health Insurance Research Database was used. We followed the standard statistical learning steps to obtain a survival model with good discriminatory accuracy in predicting death due to noncancer causes, from which we obtained the TCI and defined comorbidity levels. We reported the actual prognosis by age, stage, and comorbidity level. In Taiwan, cancer prevalence nearly doubled in 2004–2014, and comorbidities were common among older patients. Stage was the major predictor of patients’ actual prognoses. For localized and regional breast, colorectal, and oral cancers, comorbidities correlated with noncancer-related deaths. Compared with the US, the chances of dying from comorbidities in Taiwan were lower and the chances of dying from cancer were higher for breast, colorectal, and male lung cancers. These actual prognoses could help clinicians and patients in treatment decision-making and help policymakers in resource planning.
Preferences and Acceptability for Long-Acting PrEP Agents Among Pregnant and Postpartum Women with Experience Using Daily Oral PrEP in South Africa and Kenya
Authors: Wara Nafisa J. , Mvududu Rufaro , Marwa Mary M. , Gómez Laurén , Mashele Nyiko , Orrell Catherine , Moucheraud Corrina , Kinuthia John , John-Stewart Grace , Myer Landon , Hoffman Risa , Pintye Jillian , Davey Dvora L. Joseph
Published: October 2022

Introduction

Long-acting pre-exposure prophylaxis (PrEP) options could overcome some barriers to oral PrEP persistence during pregnancy and postpartum. We evaluated long-acting PrEP preferences among oral PrEP-experienced pregnant and postpartum women in South Africa and Kenya, two countries with high coverage of oral PrEP and with pending regulatory approvals for long-acting injectable cabotegravir and the dapivirine vaginal ring (approved in South Africa, under review in Kenya).

Methods

From September 2021 to February 2022, we surveyed pregnant and postpartum women enrolled in oral PrEP studies in South Africa and Kenya. We evaluated oral PrEP attitudes and preferences for existing and future long-acting PrEP methods.

Results

We surveyed 190 women in South Africa (67% postpartum; median age 27 years [IQR 22-32]) and 204 women in Kenya (79% postpartum; median age 29 years [IQR 25-33]). 75% of participants reported oral PrEP use within the last 30 days. Overall, 49% of participants reported negative oral PrEP attributes, including side effects (21% South Africa, 30% Kenya) and pill burden (20% South Africa, 25% Kenya). Preferred PrEP attributes included long-acting method, effectiveness, safety while pregnant and breastfeeding, and free medication. Most participants (75%, South Africa and Kenya) preferred a potential long-acting injectable over oral PrEP, most frequently for longer duration of effectiveness in South Africa (87% South Africa, 42% Kenya) versus discretion in Kenya (5% South Africa, 49% Kenya). 87% of participants preferred oral PrEP over a potential long-acting vaginal ring, mostly due to concern about possible discomfort with vaginal insertion (82% South Africa, 48% Kenya). Significant predictors of long-acting PrEP preference included past use of injectable contraceptive (aOR 2.48, 95% CI: 1.34, 4.57), disliking at least one oral PrEP attribute (aOR 1.72, 95% CI: 1.05, 2.80), and preferring infrequent PrEP use (aOR 1.58, 95% CI: 0.94, 2.65).

Conclusions

Oral PrEP-experienced pregnant and postpartum women expressed a theoretical preference for long-acting injectable PrEP over other modalities, demonstrating potential acceptability among a key population who must be at the forefront of injectable PrEP rollout. Reasons for PrEP preferences differed by country, emphasizing the importance of increasing context-specific options and choice of PrEP modalities for pregnant and postpartum women.
Large effect loci have a prominent role in Darwin’s finch evolution
Authors: Enbody Erik D. , Sendell-Price Ashley T. , Sprehn C. Grace , Rubin Carl-Johan , Visscher Peter M. , Grant B. Rosemary , Grant Peter R. , Andersson Leif
Published: October 2022

A fundamental goal in evolutionary biology is to understand the genetic architecture of adaptive traits and its evolutionary relevance. Using whole-genome data of 3,958 Darwin’s finches on the Galápagos Island of Daphne Major we identify six loci of large effect that explain 46% of the variation in beak size ofGeospiza fortis, a key ecological trait. Allele frequency changes across 30 years at these loci affected beak morphology in two ways. An abrupt change in beak size occurred inGeospiza fortisas a result of natural selection associated with a drought, and a more gradual change occurred inG. scandensas a result of introgressive hybridization. This study demonstrates how large effect loci are a major contributor to the genetic architecture of rapid diversification during adaptive radiations.

One Sentence Summary

Allele frequency change at six loci of large effect causes evolutionary change in key ecological traits.
Cortical tracking of continuous speech under bimodal divided attention
Authors: Xie Zilong , Brodbeck Christian , Chandrasekaran Bharath
Published: October 2022

Speech processing often occurs amidst competing inputs from other modalities, e.g., listening to the radio while driving. We examined the extent to whichdividingattention between auditory and visual modalities (bimodal divided attention) impacts neural processing of natural continuous speech from acoustic to linguistic levels of representation. We recorded electroencephalographic (EEG) responses when human participants performed a challenging primary visual task, imposing low or high cognitive load while listening to audiobook stories as a secondary task. The two dual-task conditions were contrasted with an auditory single-task condition in which participants attended to stories while ignoring visual stimuli. Behaviorally, the high load dual-task condition was associated with lower speech comprehension accuracy relative to the other two conditions. We fitted multivariate temporal response function encoding models to predict EEG responses from acoustic and linguistic speech features at different representation levels, including auditory spectrograms and information-theoretic models of sublexical-, word-form-, and sentence-level representations. Neural tracking of most acoustic and linguistic features remained unchanged with increasing dual-task load, despite unambiguous behavioral and neural evidence of the high load dual-task condition being more demanding. Compared to the auditory single-task condition, dual-task conditions selectively reduced neural tracking of only some acoustic and linguistic features, mainly at latencies >200 ms, while earlier latencies were surprisingly unaffected. These findings indicate that behavioral effects of bimodal divided attention on continuous speech processing occur not due to impaired early sensory representations but likely at later cognitive processing stages. Crossmodal attention-related mechanisms may not be uniform across different speech processing levels.
Clustering laminar cytoarchitecture: in vivo parcellation based on cortical granularity
Authors: Shamir Ittai , Assaf Yaniv , Shamir Ron
Published: October 2022

The laminar microstructure of the cerebral cortex is considered a unique anatomical mark of the development, function, connectivity, and even various pathologies of the brain. In recent years, multiple neuroimaging studies have utilized magnetic resonance imaging (MRI) relaxometry to visualize and explore this intricate microstructure. By successfully delineating the cortical laminar components, the applicability of T1 relaxometry has been expanded beyond solely a direct measure of myeline content. However, validating its applicability as a measure of cortical cytoarchitecture demands analyzing the complex resulting laminar datasets. In this study, we adapt and implement an algorithm for clustering cell omics profiles to cluster these complex microstructural cortical datasets. The resulting clusters correspond with an established atlas of cytoarchitectonic features, providing robust validation of T1 imaging as a tool for exploring cortical laminar composition. Lastly, we demonstrate the applicability of this framework in the exploration of the cytoarchitectonic features behind various unique skillsets.
An expanded proteomic survey of the human parasiteLeishmania majorfocusing on changes in null mutants of the Golgi GDP-Mannose/Fucose/ArabinopyranosetransporterLPG2or the mitochondrial fucosyltransferaseFUT1
Authors: Polanco Gloria , Scott Nichollas E. , Lye Lon F. , Beverley Stephen M.
Published: October 2022

The trypanosomatid protozoan parasiteLeishmaniahas a significant impact on human health globally. Understanding the pathways associated with virulence within this significant pathogen is critical for identifying novel vaccination and chemotherapy targets. Within this study we leverage an ultradeep proteomic approach to improve our understanding of two virulence associated genes inLeishmania, encoding the Golgi Mannose/Arabinopyranose/Fucose nucleotide-sugar transporterLPG2, and the mitochondrial fucosyltransferaseFUT1. Using deep peptide fractionation followed by complementary fragmentation approaches with higher energy collisional dissociation (HCD) and Electron-transfer dissociation (ETD) allowed the identification of over 6500 proteins, nearly doubling the experimentally knownLeishmania majorproteome. This deep proteomic analysis revealed significant quantitative differences in bothΔlpg2-andΔfut1smutants withFUT1-dependent changes linked to marked alterations within mitochondrial associated proteins whileLPG2-dependent changes impacted many pathways including the secretory pathway. While the FUT1 enzyme has been shown to fucosylate peptidesin vitro, no evidence for protein fucosylation was identified within our ultradeep analysis nor did we observe fucosylated glycans withinLeishmaniaglycopeptides isolated using HILIC enrichment. Combined this work provides a critical resource for the community on the observableLeishmaniaproteome as well as highlights phenotypic changes associated withLPG2orFUT1ablation which may guide the development of future therapeutics.

Importance

Leishmaniais a widespread trypanosomatid protozoan parasite of humans with ∼12 million cases ranging from mild to fatal, and hundreds of millions asymptomatically infected. This work advances knowledge of the experimental proteome by nearly 2 fold, to more than 6500 proteins a great resource to investigators seeking to decode how this parasite is transmitted and causes disease, and new targets for therapeutic intervention. The ultradeep proteomics approach identified potential proteins underlying the ‘persistence without pathology’ phenotype of deletion mutants of the Golgi nucleotide transporter LPG2, showing many alterations and several candidates. Studies of a rare deletion mutant of the mitochondrial fucosyltransferase FUT1 revealed changes underlying its strong mitochondrial dysfunction, but did not reveal examples of fucosylation of either peptides or N-glycans. This suggests this vital protein’s elusive target(s) may be more complex than the methods used could detect, or may not be a protein, perhaps another glycoconjugate or glycolipid.
Computational and experimental analyses of alanine racemase suggest new avenues for developing allosteric small-molecule antibiotics
Authors: Van Wieren Arie , Durant Jacob D , Majumdar Sudipta
Published: October 2022

Given the ever-present threat of antibacterial resistance, there is an urgent need to identify new antibacterial drugs and targets. One such target is alanine racemase (Alr), an enzyme required for bacterial cell-wall biosynthesis. Alr is an attractive drug target because it is essential for bacterial survival but is absent in humans. Here, we investigate the Alr fromM. tuberculosis(MT), the pathogen responsible for human tuberculosis, as a model Alr enzyme. MT-Alr functions exclusively as an obligate homodimer formed by two identical monomers. Both monomers contribute to the overall composition of their active sites. Therefore, disrupting the dimer interface could inhibit MT-Alr activity. Using computational methods, we identified seven interfacial residues predicted to be responsible for MT-Alr dimerization. Mutating one of the seven residues, Lys261, to alanine resulted in a completely inactive enzyme. Further investigation suggested a potential drug-binding site near Lys261 that might be useful for allosteric drug discovery.

The bacterial protein alanine racemase (Alr) converts L-alanine to D-alanine, a critical component of the bacterial cell wall. Cycloserine, a known antibiotic, inhibits Alr by binding to the same pocket that alanine binds. Several human proteins have similar pockets, so cycloserine has severe side effects. We identified additional Alr pockets and discovered that altering one of them abolishes Alr activity. Molecules that bind this pocket may similarly impact Alr activity, helping to address the ongoing antibiotic resistance crisis.
Quantifying quantify 64 drugs, illicit substances, and D- and L- isomers in human oral fluid with liquid-liquid extraction
Authors: Robbins Brian , Carpenter Rob E. , Long Mary , Perry Jacob
Published: October 2022

Although human oral fluid has become more routine for quantitative drug detection in pain management, detecting a large scope of medications and substances is costly and technically challenging for laboratories. This paper presents a quantitative assay for 64 pain medications, illicit substances, and drug metabolites in human oral fluid. The novelty of this assay is that it was developed on an older model AB SCIEX 4000 instrument and renders obscure the need for more technical and expensive laboratory equipment. This method includes addition of internal standard and a 2-step liquid-liquid extraction and dry-down step to concentrate and clean the samples. The samples were suspended in 50% MeOH in water and separation and detection was accomplished using triple quadrupole mass spectrometry (LC-MS/MS). Separation was achieved using reverse-phase liquid chromatography with detection by LC-MS/MS. A second injection was done in negative mode to determine THC-COOH concentration as an indicator of THC. An aliquot of the (already) extracted samples was analyzed for D- and L- isomers of amphetamine and methamphetamine using a chiral column. The standard curve spanned from 5 to 2000 ng/mL for most of the analytes (1 to 2000 ng/mL for fentanyl and THC-COOH) and up to 1000 ng/mL for 13 analytes. Pregabalin and gabapentin ranged from 25 to 2000 ng/mL. The result is a low-cost method for the sensitive detection of a wide-ranging oral fluid menu for pain management. This assay has a high sensitivity, and good precision and accuracy for all analytes with an older model mass spectrometer.
Molecular mechanism of tRNA binding by theEscherichia coliN7 guanosine methyltransferase TrmB
Authors: Schultz Sarah K. , Meadows Kieran , Kothe Ute
Published: October 2022

Among the large and diverse collection of tRNA modifications, 7-methylguanosine is frequently found in the tRNA variable loop at position 46. This modification is introduced by the TrmB enzyme, which is conserved in bacteria and eukaryotes. Complementing the report of various phenotypes for different organisms lacking TrmB homologs, we report here hydrogen peroxide sensitivity for theEscherichia coli ΔtrmBknockout strain. To gain insight into the molecular mechanism of tRNA binding byE. coliTrmB in real-time, we developed a new assay based on introducing a 4-thiouridine modification at position 8 ofin vitrotranscribed tRNAPheenabling us to fluorescently labelled this unmodified tRNA. Using rapid kinetic stopped-flow measurements with this fluorescent tRNA, we examined the interaction of wild-type and single substitution variants of TrmB with tRNA. Our results reveal the role of SAM for rapid and stable tRNA binding, the rate-limiting nature of m7G46 catalysis for tRNA release, and the importance of residues R26, T127 and R155 across the entire surface of TrmB for tRNA binding.
Routing States Transition During Oscillatory Bursts and Attentional Selection
Authors: Boroujeni Kianoush Banaie , Womelsdorf Thilo
Published: October 2022

Neural information routing relies on spatiotemporal activity dynamics across interconnected brain areas. However, it remains unclear how routing states emerge at fast spiking timescales and interact with the slower activity dynamics of larger networks during cognitive processes.

Here, we show that localized neural spiking events generate long-range directional routing states with spiking activity in distant brain areas that dynamically switch or amplify during oscillatory bursts, selective attention, and decision-making. Computational modeling and neural recordings from lateral prefrontal cortex (LPFC), anterior cingulate cortex (ACC), and striatum of nonhuman primates revealed that cross-areal, directional routing states arise within ∼20 ms around spikes of single neurons. On average, LPFC spikes led activity in the ACC and striatum by few milliseconds. The routing state was amplified during LPFC beta bursts between the LPFC and striatum and switched direction during ACC theta/alpha bursts between ACC and LPFC. Selective attention amplified the lead of these theta/alpha-specific lead-ensembles in the ACC, while decision-making amplified the lead of ACC and LPFC spiking output over the striatum. Notably, the fast lead/lag relationships of cross-areal neuronal ensembles that were modulated by attention states or decision-making predicted firing rate dynamics of their neurons during those functional states at slower timescales. Overall, our findings demonstrate directional routing of spiking activity across nonhuman primate frontal and striatal areas, as well as the functional and network states that modulate the direction and magnitude of these interactions.

Fast spatio-temporal dynamics of brain activity subserves the routing of information across distant regions and is integral to flexible cognition, decision-making, and selective attention. This study demonstrates that routing dynamics emerge as 20 ms brief lead and lag relationships of spiking activities across distant brain areas. The direction and magnitude of the lead and lag relationships systematically switched during frequency-specific oscillatory bursts and when attention shifts to visual cues.
Whole Human-Brain Mapping of Single Cortical Neurons for Profiling Morphological Diversity and Stereotypy
Authors: Han Xiaofeng , Guo Shuxia , Ji Nan , Li Tian , Liu Jian , Ye Xiangqiao , Wang Yi , Yun Zhixi , Xiong Feng , Rong Jing , Liu Di , Ma Hui , Wang Yujin , Huang Yue , Zhang Peng , Wu Wenhao , Ding Liya , Hawrylycz Michael , Lein Ed , Ascoli Giorgio A. , Xie Wei , Liu Lijuan , Zhang Liwei , Peng Hanchuan
Published: October 2022

Quantification of individual cells’ morphology and their distribution at the whole brain scale is essential to understand the structure and diversity of cell types. Despite recent technological advances, especially single cell labeling and whole brain imaging, for many prevailing animal models, it is exceedingly challenging to reuse similar technologies to study human brains. Here we propose Adaptive Cell Tomography (ACTomography), a low-cost, high-throughput, high-efficacy tomography approach, based on adaptive targeting of individual cells suitable for human-brain scale modeling of single neurons to characterize their 3-D structures, statistical distributions, and extensible for other cellular features. Specifically, we established a platform to inject dyes into cortical neurons in surgical tissues of 18 patients with brain tumors or other conditions and 1 donated fresh postmortem brain. We collected 3-D images of 1746 cortical neurons, of which 852 neurons were subsequentially reconstructed to quantify their local dendritic morphology, and mapped to standard atlases both computationally and semantically. In our data, human neurons are more diverse across brain regions than by subject age or gender. The strong stereotypy within cohorts of brain regions allows generating a statistical tensor-field of neuron morphology to characterize 3-D anatomical modularity of a human brain.
Improving the sensitivity of in vivo CRISPR off-target detection with DISCOVER-Seq+
Authors: Zou Roger S. , Liu Yang , Gaido Oscar E. Reyes , Konig Maximilian F. , Mog Brian J. , Shen Leo L. , Aviles-Vazquez Franklin , Marin-Gonzalez Alberto , Ha Taekjip
Published: October 2022

Discovery of off-target CRISPR-Cas genome editing activity in patient-derived cells and animal models is crucial for therapeutic applications, but currently exhibits low sensitivity. We demonstrate that inhibition of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) accumulates repair protein MRE11 at CRISPR-targeted sites, enabling high-sensitivity mapping of off-target sites to positions of MRE11 binding using chromatin immunoprecipitation sequencing (ChIP-seq). This technique, termed DISCOVER-Seq+, discovered up to 5-fold more CRISPR off-target sites in immortalized cell lines, primary human cells, and mice compared to previous methods. We demonstrated applicability to ex vivo knock-in of a cancer-directed transgenic T-cell receptor in primary human T cells and in vivo adenovirus knock-out of cardiovascular risk genePCSK9in mice. DISCOVER-Seq+ is the most sensitive method to-date for discovering off-target genome editing in vivo.
G-PROTEIN COUPLED RECEPTOR 19 (GPR19) KNOCKOUT MICE DISPLAY SEX-DEPENDENT METABOLIC DYSFUNCTION
Authors: Mushala Bellina A.S. , Xie Bingxian , Sipula Ian J. , Stoner Michael W. , Thapa Dharendra , Manning Janet R. , Bugga Paramesha , Vandevender Amber M. , Jurczak Michael J. , Scott Iain
Published: October 2022

G-protein coupled receptors (GPCRs) mediate signal transduction from the cellular surface to intracellular metabolic pathways. While the function of many GPCRs has been delineated previously, a significant number require further characterization to elucidate their cellular function. G-protein coupled receptor 19 (GPR19) is a poorly characterized class A GPCR which has been implicated in the regulation of circadian rhythm, tumor metastasis, and mitochondrial homeostasis. In this report, we use a novel knockout (KO) mouse model to examine the role of GPR19 in whole-body metabolic regulation. We show that loss of GPR19 promotes increased energy expenditure and decreased activity in both male and female mice. However, only male GPR19 KO mice display glucose intolerance in response to a high fat diet (HFD). Loss of GPR19 expression in male mice, but not female mice, resulted in diet-induced hepatomegaly, which was associated with decreased expression of key fatty acid oxidation genes in male GPR19 KO livers. Overall, our data suggest that loss of GPR19 impacts whole-body energy metabolism in diet-induced obese mice in a sex-dependent manner.
“UNTARGETING” AUTOANTIBODIES USING GENOME EDITING, A PROOF-OF-CONCEPT STUDY
Authors: Keppeke Gerson Dierley , Diogenes Larissa , Gomes Kethellen , Andrade Luis Eduardo Coelho
Published: October 2022

Autoantibodies are useful biomarkers of autoimmune diseases and some have direct pathogenic role. Current standard therapies for elimination of specific B/plasma-cell clones are not fully efficient. In this proof-of-concept study, we used the CRISPR/Cas9 genome-editing system to knockout V(D)J rearrangements that produce pathogenic autoantibodiesin vitro.

HEK293T cell lines were established with stable expression of two monoclonal antibodies, a humanized anti-dsDNA (clone 3H9) and a human-derived anti-nAChR-α1-subunit (clone B12L). For each clone, five CRISPR/Cas9 guided-RNAs (T-gRNAs) were designed to target the heavy chain CDR2/3 variable regions. After CRISPR/Cas9 editing, levels of secreted immunoglobulins were evaluated, in addition to 3H9 anti-dsDNA reactivity by ELISA and B12L anti-AChR reactivity using cells overexpressing mouse genes of AChR-α1/β1/δ/γ/ε-subunits.

The T-gRNAs decreased the expression of the heavy chain to ∼50-60%, compared to >90% in Non-Target-gRNA. Levels of secreted IgG and reactivity to the respective target antigens decreased ∼90% and ∼95% after knockout with the T-gRNAs compared to Non-Target-gRNA for clones 3H9 and B12L, respectively. Sequencing indicated the presence ofindelsat the Cas9 cut-site, which could lead to codon jam, the likely cause of the knockout. Additionally, remaining secreted 3H9 antibodies presented variable reactivity to dsDNA among the five T-gRNA, suggesting that the exact Cas9 cut-site andindelsmay further interfere with antibody-antigen interaction.

CRISPR/Cas9 genome-editing was very effective to knockout the Heavy-Chain-IgG genes, considerably affecting the secretion and binding capacity of the autoantibodiesin vitro, warranting application of this concept toin vivomodels as a potential novel therapeutic approach for autoantibody-mediated diseases.

Highlights

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Autoantibodies can have a direct pathogenic role in some autoimmune diseases.

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Elimination of specific B/plasma-cell clones is not attainable with current therapies.

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CRISPR/Cas9 allows targeting of specific DNA sites, such as V(D)J rearrangements.

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CRISPR/Cas9 genome-editing was very effective in knocking out the heavy chain of autoantibodies.

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Indels introduced at Cas9 cut site interfered with autoantibody-antigen interaction.
Overweight and Obesity among Women at Reproductive Age in Cambodia: Data Analysis of Cambodia Demographic and Health Survey 2014
Authors: Um Samnang , Yom An , Muir Jonathan A. , Sopheab Heng
Published: October 2022

Introduction

Overweight and obesity increase the risk of cardiovascular disease, type 2 diabetes mellitus, hypertension, stroke, and some type of cancers, and maternal health globally. In Cambodia, the prevalence of overweight and obesity among women aged 15-49 years increased from 6% in 2000 to 18% in 2014, becoming a public health burden. We examined socio-demographic and behavioral factors associated with overweight and/or obesity among women of reproductive age in Cambodia.

Methods

We analyzed existing data from the 2014 Cambodia Demographic and Health Survey (CDHS) that used a two-stage stratified cluster sampling design. Data analysis was restricted to non-pregnant women, resulting in an analytic sample of 10,818 women. Multivariable logistic regressions that accounted for the CDHS sampling design were performed using STATA V16 to examine factors associated with overweight and obesity.

Results

Prevalence of overweight and obesity among non-pregnant women of reproductive age were 15.2% and 2.8% respectively. Factors independently associated with increased odds of overweight and/or obesity including women aged 20-29 years with adjusted odds ratio [AOR=2.4; 95% CI: 1.6-3.6], 30-39 years [AOR=4.6; 95% CI: 3.0-6.9], and 40-49 years [AOR=6.6; 95% CI: 4.3-10.1], married women [AOR=1.8; 95% CI: 1.3-2.7], urban residence [AOR=1.3; 95% CI: 1.1-1.5], and women having at least 4 children [AOR=1.7; 95% CI: 1.2-2.5]. The following factors were associated with decreased odds of overweight and obesity: completed at least secondary education [AOR=0.7; 95% CI: 0.6-0.8], agricultural work [AOR=0.7; 95% CI: 0.5-0.8], and manual labor work [AOR=0.7; 95% CI: 0.6-0.9].

Conclusions

Increased age, married women, living in urban residence, and having at least four children were the main risk factors associated with overweight and/or obesity. Conversely, higher education, working in agriculture, and working in manual labor were negatively associated with overweight and/or obesity. Cambodia’s non-communicable disease (NCD) public health programs should consider these characteristic for targeting interventions to further reduce overweight and/or obesity in the coming years.