bluuebunny/crossref_metadata_2025 huggingface dataset:
School-going learners are more likely to access HIV and contraceptive care at locations with friendly providers, Wi-Fi and other value-added services: Findings from a discrete choice experiment among learners in Gauteng, South Africa
Authors: Govathson Caroline , Long Lawrence , Moolla Aneesa , Mngadi-Ncube Sithabile , Ngcobo Nkosinathi , Mongwenyana Constance , Lince-Deroche Naomi , Pascoe Sophie
Published: July 2022

Background

Many Adolescents in Sub-Saharan Africa don’t access HIV and reproductive health services optimally. To improve uptake of these services, it is important to understand the students’ preferences for how services are delivered so that implementation strategies can reflect this.

Methods

A discrete choice experiment (DCE) was used to elicit preferences. The DCE was conducted in 10 high schools situated in neighbourhoods of varying socio-economic status (SES) in Gauteng (South Africa). Students aged ≥15 years (Grades 9-12) were consented and enrolled in the DCE. Conditional logistic regression was used to determine preferred attributes for HIV and contraceptive service delivery. Results were stratified by gender and neighbourhood SES quintile. (1=Lowest SES; 5=Highest SES).

Results

The DCE was completed between 07/2018-09/2019; 805 students were enrolled (67% female; 66% 15-17 years; 51% in grades 9-10). 54% of students in quintile 1 schools had no monthly income; 38% in quintile 5 schools had access to USD7 per month. Preferences were similar for male and female students. Students strongly preferred services provided by friendly, non-judgmental staff (Odds ratio 1.63; 95% Confidence Interval: 1.55-1.72) where confidentiality was ensured (1.33; 1.26-1.40). They preferred services offered after school (1.14; 1.04-1.25) with value-added services like free Wi-Fi (1.19; 1.07-1.32), food (1.23; 1.11-1.37) and youth-only waiting areas (1.18; 1.07-1.32). Students did not have a specific location preference, but preferred not to receive services within the community (0.82; 0.74-0.91) or school (0.88; 0.80-0.96). Students attending schools in high SES neighbourhoods expressed a preference for private care (1.15; 0.98-1.35). Costs to access services were a deterrent for most students irrespective of school neighbourhood; female students were deterred by costs ≥USD3 (0.79; 0.70-0.91); males by costs ≥USD7 (0.86; 0.74-1.00)

Conclusions

Preferences that encourage utilisation of services do not significantly differ by gender or school neighbourhood SES. Staff attitude and confidentiality are key issues affecting students’ decisions to access HIV and contraceptive services. Addressing how healthcare providers respond to young people seeking sexual and reproductive health services is critical for improving adolescents’ uptake of these services.
SARS-CoV-2 infections during Omicron (BA.1) dominant wave and subsequent population immunity in Gauteng, South Africa
Authors: Madhi Shabir A. , Kwatra Gaurav , Myers Jonathan E. , Jassat Waasila , Dhar Nisha , Mukendi Christian K. , Blumberg Lucille , Welch Richard , Izu Alane , Mutevedzi Portia C.
Published: July 2022

Background

The B.1.1.529 (Omicron BA.1) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a global resurgence of coronavirus disease 2019 (Covid-19). The contribution of BA.1 infection to population immunity and its effect on subsequent resurgence of B.1.1.529 sub-lineages warrant investigation.

Methods

We conducted an epidemiologic survey to determine the sero-prevalence of SARS-CoV-2 IgG from March 1 to April 11, 2022, after the BA.1-dominant wave had subsided in Gauteng (South Africa), and prior to a resurgence of Covid-19 dominated by the BA.4 and BA.5 (BA.4/BA.5) sub-lineages. Population-based sampling included households in an earlier survey from October 22 to December 9, 2021 preceding the BA.1 dominant wave. Dried-blood-spot samples were quantitatively tested for IgG against SARS-CoV-2 spike protein and nucleocapsid protein. Epidemiologic trends in Gauteng for cases, hospitalizations, recorded deaths, and excess deaths were evaluated from the inception of the pandemic to the onset of the BA.1 dominant wave (pre-BA.1), during the BA.1 dominant wave, and for the BA.4/BA.5 dominant wave through June 6, 2022.

Results

The 7510 participants included 2420 with paired samples from the earlier survey. Despite only 26.7% (1995/7470) of individuals having received a Covid-19 vaccine, the overall sero-prevalence was 90.9% (95% confidence interval [CI], 90.2 to 91.5), including 89.5% in Covid-19 unvaccinated individuals. Sixty-four percent (95%CI, 61.8-65.9) of individuals with paired samples had serological evidence of SARS-CoV-2 infection during the BA.1 dominant wave. Of all cumulative recorded hospitalisations and deaths, 14.1% and 5.9% were contributed by the BA.1 dominant wave, and 5.1% and 1.6% by the BA.4/BA.5 dominant wave. The SARS-CoV-2 infection fatality risk was lower in the BA.1 compared with pre-BA.1 waves for recorded deaths (0.02% vs. 0.33%) and Covid-19 attributable deaths based on excess mortality estimates (0.03% vs. 0.67%).

Conclusions

Gauteng province experienced high levels of infections in the BA.1 -dominant wave against a backdrop of high (73%) sero-prevalence. Covid-19 hospitalizations and deaths were further decoupled from infections during BA.4/BA.5 dominant wave than that observed during the BA.1 dominant wave.

(Funded by the Bill and Melinda Gates Foundation.)
Semantic and phonetic markers in schizophrenia-spectrum disorders; a combinatory machine learning approach
Authors: Voppel A.E. , de Boer J.N. , Brederoo S.G. , Schnack H.G. , Sommer I.E.C.
Published: July 2022

Introduction

Speech is a promising marker for schizophrenia-spectrum disorder diagnosis, as it closely reflects symptoms. Previous approaches have made use of different feature domains of speech in classification, including semantic and phonetic features. However, an examination of the relative contribution and accuracy per domain remains an area of active investigation. Here, we examine these domains (i.e. phonetic and semantic) separately and in combination.

Methods

Using a semi-structured interview with neutral topics, speech of 94 schizophrenia-spectrum subjects (SSD) and 73 healthy controls (HC) was recorded. Phonetic features were extracted using a standardized feature set, and transcribed interviews were used to assess word connectedness using a word2vec model. Separate cross-validated random forest classifiers were trained on each feature domain. A third, combinatory classifier was used to combine features from both domains.

Results

The phonetic domain random forest achieved 81% accuracy in classifying SSD from HC. For the semantic domain, the classifier reached an accuracy of 80% with a sparse set of features with 10-fold cross-validation. Joining features from the domains, the combined classifier reached 85% accuracy, significantly improving on models trained on separate domains. Top features were fragmented speech for phonetic and variance of connectedness for semantic, with both being the top features for the combined classifier.

Discussion

Both semantic and phonetic domains achieved similar results compared with previous research. Combining these features shows the relative value of each domain, as well as the increased classification performance from implementing features from multiple domains. Explainability of models and their feature importance is a requirement for future clinical applications.
COVID-19 pandemic surges can induce bias in trials using response adaptive randomization: A simulation study
Authors: Yarnell Christopher J , Fowler Robert A , Sung Lillian , Tomlinson George
Published: July 2022

Response-adaptive randomization is being used in COVID-19 trials, but it is unknown whether outcome rate changes during surges of COVID-19 will lead to bias in trial results. In response-adaptive randomization, allocation ratios are adjusted according to interim analyses to assign more patients to promising interventions. Although it is known that response-adaptive randomization may give biased estimates if outcome rates drift over time, observed mortality fluctuations in the COVID-19 pandemic are more extreme than any previously tested in simulation. We hypothesized that pandemic surges induce bias in trials using response-adaptive randomization, and that adjustment for time will alleviate that bias. Bayesian 4-arm superiority trials with a mortality outcome were simulated to investigate bias in treatment effect, comparing complete and response-adaptive randomization under different pandemic scenarios based on data from New York, Spain, and Italy. Relative bias in the odds ratio associated with treatment ranged from 0.3% to 11% and was largest in trials with a surge and an effective intervention that did not adjust for time. Bias was attenuated by adjustment for time without compromising the false-positive rate. Trials using response-adaptive randomization were more likely to identify effective interventions but were slower to drop ineffective interventions. Even with variation in outcome rates similar to observed pandemic surges, COVID-19 trials using response-adaptive randomization that adjust for time can provide accurate estimates of treatment effects.
Factors Associated with COVID-19 Breakthrough Infection in the Pre-Omicron Era Among Vaccinated Patients with Rheumatic Diseases: A Cohort Study
Authors: Patel Naomi J. , Wang Xiaosong , Fu Xiaoqing , Kawano Yumeko , Cook Claire , Vanni Kathleen M.M. , Qian Grace , Banasiak Emily , Kowalski Emily , Zhang Yuqing , Sparks Jeffrey A. , Wallace Zachary S.
Published: July 2022

Objective

Rheumatic disease patients on certain immunomodulators are at increased risk of impaired humoral response to SARS-CoV-2 vaccines. We aimed to identify factors associated with breakthrough infection among patients with rheumatic diseases.

Methods

We identified patients with rheumatic diseases being treated with immunomodulators in a large healthcare system who received at least two doses of either the mRNA-1273 (Moderna) or BNT162b2 (Pfizer-BioNTech) vaccines or one dose of the Johnson & Johnson-Janssen (J&J) vaccine. We followed patients until SARS-CoV-2 infection, death, or December 15, 2021, when the Omicron variant became dominant in our region. We estimated the association of baseline characteristics with the risk of breakthrough infection using multivariable Cox regression.

Results

We analyzed 11,468 patients (75% female, mean age 60 years). Compared to antimalarial monotherapy, multiple immunomodulators were associated with higher risk of infection: anti-CD20 monoclonal antibodies (aHR 5.20, 95% CI: 2.85, 9.48), CTLA-4 Ig (aHR 3.52, 95% CI: 1.90, 6.51), mycophenolate (aHR 2.31, 95% CI: 1.25, 4.27), IL-6 inhibitors (aHR 2.15, 95% CI: 1.09, 4.24), JAK inhibitors (aHR 2.02, 95% CI: 1.01, 4.06), and TNF inhibitors (aHR 1.70, 95% CI: 1.09, 2.66). mRNA-1273 recipients had a lower risk of breakthrough infection compared to BNT162b2 recipients (aHR 0.66, 95% CI: 0.50, 0.86). There was no association of sex, body mass index, smoking status, race, or ethnicity with risk of breakthrough infection.

Conclusion

Among patients with rheumatic diseases, multiple immunomodulators were associated with increased risk of breakthrough infection. These results highlight the need for additional mitigation strategies in this vulnerable population.
Intrinsic linking of chromatin fiber in human cells
Authors: Borodzik Maciej , Denkiewicz Michał , Spaliński Krzysztof , Winnicka-Sztachelska Kamila , Sengupta Kaustav , Pilipczuk Marcin , Pilipczuk Michał , Ruan Yijun , Plewczynski Dariusz
Published: July 2022

Motivation

We propose a practical algorithm based on graph theory, with the purpose of identifying CTCF-mediated chromatin loops that are linked in 3D space. Our method is based finding clique minors in graphs constructed from pairwise chromatin interaction data obtained from the ChIA-PET experiments. We show that such a graph structure, representing a particular arrangement of loops, mathematically necessitates linking, if co-occurring in an individual cell. The presence of these linked structures can advance our understanding of the principles of spatial organization of the genome.

Results

We apply our method to graphs created from in situ ChIA-PET data for GM12878, H1ESC, HFFC6 and WTC11 cell lines, and from long-read ChIA-PET data. We look at these datasets as divided into CCDs - closely interconnected regions defined based on CTCF loops. We find numerous candidate regions with minors, indicating the presence of links. The graph-theoretic characteristics of these linked regions, including betweenness and closeness centrality, differ from regions without, in which no minors were found, which supports their non-random nature. We also look at the position of the linked regions with respect to chromatin compartments.

Availability

The implementation of the algorithm is available at https://github.com/SFGLab/cKNOTs

Contact

Dariusz.Plewczynski@pw.edu.pl
Effect of Histidine Covalent Modification on Strigolactone Receptor Activation and Selectivity
Authors: Chen Jiming , Shukla Diwakar
Published: July 2022

The parasitic weed Striga has led to billions of dollars’ worth of agricultural productivity loss worldwide. Striga detects host plants using the plant hormone strigolactone. Early steps in the strigolactone signaling pathway involve substrate binding and hydrolysis followed by a conformational change to an “active” or “closed” state, after which it associates with a MAX2-family downstream signaling partner. The structures of the inactive and active states of strigolactone receptors are known through X-ray crystallography, and the transition pathway of from the inactive to active state in apo receptors has previously been characterized using molecular dynamics simulations. However, it also has been suggested that a covalent butenolide modification of the receptor on the catalytic histidine through substrate hydrolysis promotes formation of the active state. Using molecular dynamics simulations, we show that the presence of the covalent butenolide enhances activation in both At D14 and ShHTL7, but the enhancement is ∼50 times greater in ShHTL7. We also show that several conserved interactions with the covalent butenolide modification promote transition to the active state in both At D14 (non-parasite) and ShHTL7 (parasite). Finally, we demonstrate that the enhanced activation of ShHTL7 likely results from disruption of ShHTL7-specific histidine interactions that inhibited activation in the apo case.
Lokatt: A hybrid DNA nanopore basecaller with an explicit duration hidden Markov model and a residual LSTM network
Authors: Xu Xuechun , Bhalla Nayanika , Ståhl Patrik , Jaldén Joakim
Published: July 2022

Background

Basecalling long DNA sequences is a crucial step in nanopore-based DNA sequencing protocols. In recent years, the CTC-RNN model has become the leading basecalling model, supplanting preceding hidden Markov models (HMMs) that relied on pre-segmenting ion current measurements. However, the CTC-RNN model operates independently of prior biological and physical insights.

Results

We present a novel basecaller namedLokatt: explicit duration Markov model and residual-LSTM network. It leverages an explicit duration HMM (EDHMM) designed to model the nanopore sequencing processes. Trained on a newly generated library with methylation-free Ecoli samples and MinION R9.4.1chemistry, the Lokatt basecaller achieves basecalling performances with a median single read identity score of0.930, a genome coverage ratio of99.750%, on par with existing state-of-the-art structure when trained on the same datasets.

Conclusion

Our research underlines the potential of incorporating prior knowledge into the basecalling processes, particularly through integrating HMMs and recurrent neural networks. The Lokatt basecaller showcases the efficacy of a hybrid approach, emphasizing its capacity to achieve high-quality basecalling performance while accommodating the nuances of nanopore sequencing. These outcomes pave the way for advanced basecalling methodologies, with potential implications for enhancing the accuracy and efficiency of nanopore-based DNA sequencing protocols.

Supplementary information

Supplementary data are available online.
Heteroplasmic Mitochondrial Genetic Variation in the Human Heart
Authors: Xue Liying , Sun Xianbang , Moreira Jesse D. , Li Yi , Smith Karan Kacy , Padera Robert F. , Lenburg Marc E. , Levy Daniel , Benjamin Emelia J. , Gopal Deepa M. , Liu Chunyu , Fetterman Jessica L.
Published: July 2022

Whether heteroplasmic mitochondrial genetic variants in readily accessible tissues (blood, skeletal muscle) reflect those in the human heart (atrial appendage, left ventricle) is unknown. Using next generation sequencing data from paired tissue samples (n=233) collected postmortem in the Genotype-Tissue Expression project, we identified 558 unique heteroplasmic mitochondrial genetic variants across the four tissues, of which only 13% were shared across all four tissue sites. Between the two cardiac sites, 61% of heteroplasmic mitochondrial genetic variants were unique to one site. A greater proportion of the heteroplasmic variants were non-synonymous or frameshift variants in the muscle sites compared to blood or those variants shared across all four tissues. Compared to blood, the total number of heteroplasmic variants was higher in cardiac tissue, which was associated with advancing age. Our findings suggest that human cardiac tissue has unique heteroplasmic mtDNA variants and may be relevant to aging-related diseases.
The fitness landscape of TEM-1 β-lactamase is stratified and inverted by sublethal concentrations of cefotaxime
Authors: Farr Andrew D. , Pesce Diego , Zwart Mark P. , de Visser J. Arjan G. M.
Published: July 2022

Adaptive evolutionary processes are constrained by the availability of mutations which cause a fitness benefit – a concept that may be illustrated by ‘fitness landscapes’ which map the relationship of genotype space with fitness. Experimentally derived landscapes have demonstrated a predictability to evolution by identifying limited ‘mutational routes’ that evolution by natural selection may take between low and high-fitness genotypes. However, such studies often utilise indirect measures to determine fitness. We estimated the competitive fitness of each mutant relative to all of its single-mutation neighbours to describe the fitness landscape of three mutations in a β-lactamase enzyme at sub-lethal concentrations of the antibiotic cefotaxime in a structured and unstructured environment. We found that in the unstructured environment the antibiotic selected for higher-resistance types – but with an equivalent fitness for subsets of mutants, despite substantial variation in resistance – resulting in a stratified fitness landscape. In contrast, in a structured environment with low antibiotic concentration, antibiotic-susceptible genotypes had a relative fitness advantage, which was associated with antibiotic-induced filamentation. These results cast doubt that highly resistant genotypes have a unique selective advantage in environments with sub-inhibitory concentrations of antibiotics, and demonstrate that direct fitness measures are required for meaningful predictions of the accessibility of evolutionary routes.

Importance

The evolution of antibiotic resistant bacterial populations underpins the ongoing antibiotic-resistance crisis. We aim to understand how antibiotic-degrading enzymes can evolve to cause increased resistance, how this process is constrained and whether it can be predictable. To this end we performed competition experiments with a combinatorially-complete set of mutants of a β-lactamase gene subject to sub-inhibitory concentrations of the antibiotic cefotaxime. While some mutants confer their hosts with high resistance to cefotaxime, in competition these mutants do not always confer a selective advantage. Similarly, we identified conditions involving spatial structure where mutations causing high resistance result in a selective disadvantage. Together, this work suggests that the relationship between resistance level and fitness at sub-inhibitory concentrations is complex; predicting the evolution of antibiotic resistance requires knowledge of the conditions that select for resistant genotypes and the selective advantage evolved types have over their predecessors.
SARS-CoV-2 infection dynamics and genomic surveillance reveals early variant transmission in urban wastewater
Authors: Lamba Sanjay , Sutharsan G , Daroch Namrta , Paul Kiran , Joshi Soumya Gopal , Darshan S , Annamalai N , Vishwanath S , Mishra Rakesh , Ramakrishnan Uma , Ishtiaq Farah
Published: July 2022

Environmental surveillance (ES) of a pathogen is crucial for understanding the community load of disease. As an early warning system, ES for SARS-CoV-2 has complemented routine diagnostic surveillance by capturing near real-time virus circulation at a population level. In this longitudinal study in 28 sewershed sites in Bangalore city, we quantified SARS-CoV-2 RNA to track infection dynamics and provide evidence of change in the relative abundance of emerging variants. We describe an early warning system using the exponentially weighted moving average control chart and demonstrate how SARS-CoV-2 RNA concentrations in wastewater correlated with clinically diagnosed new COVID-19 cases, with the trends appearing 8–14 days earlier in wastewater than in clinical data. This was further corroborated by showing that the estimated number of infections is strongly correlated with SARS-CoV-2 RNA copies detected in the wastewater. Using a deconvolution matrix, we detected emerging variants of concern up to two months earlier in wastewater samples. In addition, we found a huge diversity in variants detected in wastewater compared to clinical samples. Our study highlights that quantifying viral titres, correlating it with a known number of cases in the area, and combined with genomic surveillance helps in tracking VOCs over time and space, enabling timely and making informed policy decisions.
Investigation of in-phase bilateral exercise effects on corticospinal plasticity in relapsing remitting multiple sclerosis: a multiple baseline design
Authors: Sokratous Dimitris , Charalambous Charalambos C. , Zamba-Papanicolaou Eleni , Michailidou Kyriaki , Konstantinou Nikos
Published: July 2022

Relapsing-remitting multiple sclerosis is the most common type of multiple sclerosis characterized by periods of relapses and generating various motor symptoms. These symptoms are associated with the corticospinal tract integrity, which is quantified by means of corticospinal plasticity which can be probed via transcranial magnetic stimulation and assessed with motor threshold, motor evoked potential and central motor conduction time. Several factors, such as exercise and interlimb coordination, can influence corticospinal plasticity. Previous work in healthy and in stroke patients showed that the greatest improvement in corticospinal plasticity occurred during in-phase bilateral arm exercises. Altered corticospinal plasticity due to bilateral cortical lesions is common in multiple sclerosis, yet, the impact of these type of exercises in this cohort is unclear. The aim of this concurrent multiple baseline design study is to investigate the effects of in-phase bilateral exercises on corticospinal plasticity and on clinical measures using transcranial magnetic stimulation and standardized clinical assessment, in five people with relapsing-remitting multiple sclerosis. The intervention protocol will last for 12 consecutive weeks (30-60 minutes /session x 3 sessions/week) and include in-phase bilateral movements of the upper limbs, adapted to different sports activities and to functional training. To define functional relation between the intervention and the results on corticospinal plasticity (i.e., resting motor threshold, motor evoked potential amplitude, latency) and on clinical measures (i.e., balance, gait, bilateral hand dexterity and strength, cognitive function), we will perform a visual analysis followed by multilevel modelling and the single case educational design-specific mean difference in order to estimate the magnitude of the effect size across cases. We assume that possible effects from our study, will introduce a type of exercise that will be effective during the disease progression.
Therapeutic potential of IL6R blockade for the treatment of sepsis and sepsis-related death: Findings from a Mendelian randomisation study
Authors: Hamilton Fergus , Thomas Matt , Arnold David , Palmer Tom , Moran Ed , Mentzer Alexander J , Maskell Nick , Baillie Kenneth , Summers Charlotte , Hingorani Aroon , MacGowan Alasdair , Khandakar Golam M , Mitchell Ruth , Smith George Davey , Ghazal Peter , Timpson Nicholas J
Published: July 2022

Introduction

Sepsis is characterised by dysregulated, life-threatening immune responses, which are thought to be driven by cytokines such as interleukin-6 (IL-6). Genetic variants in IL6R known to downregulate IL-6 signalling are associated with improved COVID-19 outcomes, a finding later confirmed in randomised trials of IL-6 receptor antagonists (IL6RA). We hypothesised that blockade of IL6R could also improve outcomes in sepsis.

Methods

We performed a Mendelian randomisation analysis using single nucleotide polymorphisms (SNPs) in and near IL6R to evaluate the likely causal effects of IL6R blockade on sepsis, sepsis severity, other infections, and COVID-19. We weighted SNPs by their effect on CRP and combined results across them in inverse variance weighted meta-analysis, proxying the effect of IL6RA. Our outcomes were measured in UK Biobank, FinnGen, the COVID-19 Host Genetics Initiative (HGI), and the GenOSept and GainS consortium. We performed several sensitivity analyses to test assumptions of our methods, including utilising variants around CRP in a similar analysis.

Results

In the UK Biobank cohort (N=485,825, including 11,643 with sepsis), IL6R blockade was associated with a decreased risk of sepsis (OR=0.80; 95% CI 0.66-0.96, per unit of natural log transformed CRP decrease). The size of this effect increased with severity, with larger effects on 28-day sepsis mortality (OR=0.74; 95% CI 0.38-0.70); critical care admission with sepsis (OR=0.48, 95% CI 0.30-0.78) and critical care death with sepsis (OR=0.37, 95% CI 0.14 - 0.98) Similar associations were seen with severe respiratory infection: OR for pneumonia in critical care 0.69 (95% CI 0.49 - 0.97) and for sepsis survival in critical care (OR=0.22; 95% CI 0.04- 1.31) in the GainS and GenOSept consortium. We also confirm the previously reported protective effect of IL6R blockade on severe COVID-19 (OR=0.69, 95% 0.57 - 0.84) in the COVID-19 HGI, which was of similar magnitude to that seen in sepsis. Sensitivity analyses did not alter our primary results.

Conclusions

IL6R blockade is causally associated with reduced incidence of sepsis, sepsis related critical care admission, and sepsis related mortality. These effects are comparable in size to the effect seen in severe COVID-19, where IL-6 receptor antagonists were shown to improve survival. This data suggests a randomised trial of IL-6 receptor antagonists in sepsis should be considered.
A custom library construction method for super-resolution ribosome profiling in Arabidopsis
Authors: Wu Hsin-Yen Larry , Hsu Polly Yingshan
Published: July 2022

Ribosome profiling (aka Ribo-seq) is the deep sequencing of ribosome footprints (RFs). It maps and quantifies ribosome occupancy on mRNA, which enables the identification of coding regions and the accurate quantification of translation efficiency. We previously optimized the Ribo-seq method in Arabidopsis and tomato (Hsu et al., 2016; Wu et al., 2019; Wu and Hsu, 2022) to obtain precise RFs with strong 3-nucleotide periodicity, a feature displayed by actively translating ribosomes and a benchmark of high-quality Ribo-seq (Brar and Weissman, 2015). This strong periodicity allowed us to confidently define numerous unannotated translation events across plants (Hsu et al., 2016; Wu et al., 2019; Wu and Hsu, 2022). Recently, several key commercial reagents used in our methods were discontinued; thus, there is an urgent need to develop a new protocol. Here, we report an updated protocol that adapts two custom library construction methods (McGlincy and Ingolia, 2017; Li et al., 2021) for plants. We applied this new protocol to Arabidopsis seedlings and obtained high-quality data. We describe our step-by-step method and discuss crucial considerations for Ribo-seq experiments. We also provide a bioinformatic pipeline to perform essential quality control analyses on Ribo-seq data. Our approach should be readily applicable to other plant species with minimal modifications.
Fc effector activity and neutralization against SARS-CoV-2 BA.4 is compromised in convalescent sera, regardless of the infecting variant
Authors: Richardson Simone I. , Kgagudi Prudence , Manamela Nelia P. , Kaldine Haajira , Venter Elizabeth M. , Pillay Thanusha , Lambson Bronwen E. , van der Mescht Mieke A. , Hermanus Tandile , de Beer Zelda , de Villiers Talita Roma , Bodenstein Annie , van den Berg Gretha , du Pisanie Marizane , Burgers Wendy A. , Ntusi Ntobeko A.B. , Ueckermann Veronica , Rossouw Theresa M. , Boswell Michael T. , Moore Penny L.
Published: July 2022

The SARS-CoV-2 Omicron BA.1 variant, which exhibits high level neutralization resistance, has since evolved into several sub-lineages including BA.4 and BA.5, which have dominated the fifth wave of infection in South Africa. Here we assessed the sensitivity of BA.4 to neutralization and antibody dependent cellular cytotoxicity (ADCC) in convalescent donors infected with four previous variants of SARS-CoV-2, as well as in post-vaccination breakthrough infections (BTIs) caused by Delta or BA.1. We confirm that BA.4 shows high level resistance to neutralization, regardless of the infecting variant. However, breakthrough infections, which trigger potent neutralization, retained activity against BA.4, albeit at reduced titers. Fold reduction of neutralization in BTIs was lower than that seen in unvaccinated convalescent donors, suggesting maturation of neutralizing responses to become more resilient against VOCs in hybrid immunity. BA.4 sensitivity to ADCC was reduced but remained detectable in both convalescent donors and in BTIs. Overall, the high neutralization resistance of BA.4, even to antibodies from BA.1 infections, provides an immunological mechanism for the rapid spread of BA.4 immediately after a BA.1-dominated wave. Furthermore, although ADCC activity against BA.4 was reduced, residual activity may nonetheless contribute to the protection from disease.
Mutagenesis Induced by Protonation of Single-Stranded DNA is Linked to Glycolytic Sugar Metabolism
Authors: Gelova Suzana P. , Chan Kin
Published: July 2022

Mutagenesis can be thought of as random, in the sense that the occurrence of each mutational event cannot be predicted with precision in space or time. However, when sufficiently large numbers of mutations are analyzed, recurrent patterns of base changes called mutational signatures can be identified. To date, some 60 single base substitution or SBS signatures have been derived from analysis of cancer genomics data. We recently reported that the ubiquitous signature SBS5 matches the pattern of single nucleotide polymorphisms (SNPs) in humans and has analogs in many species. Using a temperature-sensitive single-stranded DNA mutation reporter system, we also showed that a similar mutational pattern in yeast is dependent on error-prone translesion DNA synthesis and glycolytic sugar metabolism. Here, we further investigated mechanisms that are responsible for this form of mutagenesis in yeast. We first confirmed that excess sugar metabolism leads to increased mutation rate, which was detectable by fluctuation assay. We then ruled out a significant role for aerobic respiration in SBS5-like mutagenesis by observing that petite and wild-type cells did not exhibit statistical differences in mutation frequencies. Since glycolysis is known to produce excess protons, we then investigated the effects of experimental manipulations on pH and mutagenesis. We hypothesized that yeast metabolizing 8% glucose would produce more excess protons than cells metabolizing 2% glucose. Consistent with this, cells metabolizing 8% glucose had lower intracellular and extracellular pH values. Similarly, deletion ofvma3(encoding a vacuolar H+-ATPase subunit) increased mutagenesis. We also found that treating cells with edelfosine (which renders membranes more permeable, including to protons) or culturing in low pH media increased mutagenesis. Altogether, our results agree with multiple biochemical studies showing that protonation of nitrogenous bases can alter base pairing so as to stabilize some mispairs, and shed new light on a common form of intrinsic mutagenesis.

Graphical Abstract
Substrate binding and lipid-mediated allostery in the human organic anion transporter 1 at the atomic-scale
Authors: Janaszkiewicz Angelika , Tóth Ágota , Faucher Quentin , Arnion Hélène , Védrenne Nicolas , Guellec Chantal Barin-Le , Marquet Pierre , Meo Florent Di
Published: July 2022

The Organic Anion Transporter 1 is a membrane transporter known for its central role in drug elimination by the kidney.hOAT1 is an antiporter translocating substrate in exchange forα-ketoglutarate. The understanding ofhOAT1 structure and function remains limited due to the absence of resolved structure ofhOAT1. Benefiting from conserved structural and functional patterns shared with other Major Facilitator Superfamily transporters, the present study intended to investigate fragments ofhOAT1 transport function and modulation of its activity in order to make a step forward the understanding of its transport cycle.μs-long molecular dynamics simulation ofhOAT1 were carried out suggesting two plausible binding sites for a typical substrate, adefovir, in line with experimental observations. The well-known B-like motif binding site was observed in line with previous studies. However, we here propose a new inner binding cavity which is expected to be involved in substrate translocation event. Binding modes ofhOAT1 co-substrateα-ketoglutarate were also investigated suggesting that it may binds to highly conserved intracellular motifs. We here hypothesize thatα-ketoglutarate may disrupt the pseudo-symmetrical intracellular charge-relay system which in turn may participate to the destabilisation of OF conformation. Investigations regarding allosteric communications alonghOAT1 also suggest that substrate binding event might modulate the dynamics of intracellular charge relay system, assisted by surrounding lipids as active partners. We here proposed a structural rationalisation of transport impairments observed for two single nucleotide polymorphisms, p.Arg50His and p.Arg454Gln suggesting that the present model may be used to transport dysfunctions arising fromhOAT1 mutations.

Highlights

Adefovir has at least two binding pockets onhOAT1 in the outward-facing conformation.

The highly conserved B-motif within MFS is strongly involved in substrate binding.

α-Ketoglutarate binds to the intracellular domain ofhOAT1 and destabilizes its OF conformation.

The lipid membrane bilayer plays an active role in the allosteric communication between intracellular and extracellular domains ofhOAT1.

Graphical Abstract

The present work (from left): (i) reveals binding modes of adefovir (top) andα-ketoglutarate (bottom) tohOAT1; (ii) maps Single Nucleotide Polymorphisms on outward-facing (top) and inward-facing (bottom) conformation ofhOAT1; (iii) asses the allosteric effect of lipidic environment and presence of substrates.
How do tuna schools associate to dFADs? A study using echo-sounder buoys to identify global patterns
Authors: Navarro-García Manuel , Precioso Daniel , Gavira-O’Neill Kathryn , Torres-Barrán Alberto , Gordo David , Gallego Víctor , Gómez-Ullate David
Published: July 2022

Based on the data gathered by echo-sounder buoys attached to drifting Fish Aggregating Devices (dFADs) across tropical oceans, the current study applies a Machine Learning protocol to examine the temporal trends of tuna schools’ association to drifting objects. Using a binary output, metrics typically used in the literature were adapted to account for the fact that the entire tuna aggregation under the dFAD was considered. The median time it took tuna to colonize the dFADs for the first time varied between 25 and 43 days, depending on the ocean, and the longest soak and colonization times were registered in the Pacific Ocean. The tuna schools’ Continuous Residence Times were generally shorter than Continuous Absence Times (median values between 5 and 7 days, and 9 and 11 days, respectively), in line with the results found by previous studies. Using a regression output, two novel metrics, namely aggregation time and disaggregation time, were estimated to obtain further insight into the symmetry of the aggregation process. Across all oceans, the time it took for the tuna aggregation to depart from the dFADs was not significantly longer than the time it took for the aggregation to form. The value of these results in the context of the “ecological trap” hypothesis is discussed, and further analyses to enrich and make use of this data source are proposed.
Power and precision: Evaluation and recommendations of quantitative trait analysis methods for RNA expression levels in the Hybrid Rat Diversity Panel
Authors: Pattee Jack , Vanderlinden Lauren A. , Mahaffey Spencer , Hoffman Paula , Tabakoff Boris , Saba Laura M
Published: July 2022

The Hybrid Rat Diversity Panel (HRDP) is a stable and well-characterized set of more than 90 inbred rat strains that can be leveraged for systems genetics approaches to understanding the genetic and genomic variation associated with complex disease. The HRDP exhibits substantial between-strain diversity while retaining substantial within-strain isogenicity, allowing for the precise mapping of genetic variation associated with complex phenotypes and providing statistical power to identify associated variants. In order to robustly identify associated genetic variants, it is important to account for the population structure induced by inbreeding. To this end, we investigate the performance of various plausible approaches towards modeling quantitative traits in the HRDP and quantify their operating characteristics. Towards facilitating study planning and design, we conduct extensive simulations to investigate the power of genetic association analyses in the HRDP, and characterize the impressive attained power.
Epistasis reduces fitness costs of influenza A virus escape from stem-binding antibodies
Authors: Lee Chung-Young , Caceres C. Joaquin , Geiger Ginger , Seibert Brittany , Faccin Flavio Cargnin , Gay L. Claire , Ferreri Lucas M. , Kaul Drishti , Wrammert Jens , Tan Gene S. , Perez Daniel R. , Lowen Anice C.
Published: July 2022

The hemagglutinin (HA) stem region is a major target of universal influenza vaccine efforts owing to the presence of highly conserved epitopes across multiple influenza A virus strains and subtypes. To explore the potential impact of vaccine-induced immunity targeting the HA stem, we examined the fitness effects of viral escape from stem-binding broadly neutralizing antibodies (stem-bnAbs). Recombinant viruses containing each individual antibody escape substitution showed diminished replication compared to wild-type virus, indicating that stem-bnAb escape incurred fitness costs. A second-site mutation in the HA head domain (N133D) reduced the fitness effects observed in primary cell cultures and likely enabled the selection of escape mutations. This putative permissive mutation was not, however, sufficient to ease fitness costs in a ferret transmission model. Taken together, these data suggest that viral escape from stem-bnAbs is costly but highlight the potential for epistatic interactions to enable evolution within the functionally constrained HA stem domain.
Melanization ofCandida aurisis Associated with Alteration of Extracellular pH
Authors: Smith Daniel F. Q. , Mudrak Nathan J. , Zamith-Miranda Daniel , Honorato Leandro , Nimrichter Leonardo , Chrissian Christine , Smith Barbara , Gerfen Gary J , Stark Ruth E. , Nosanchuk Joshua D. , Casadevall Arturo
Published: July 2022

Candida aurisis a recently emerged global fungal pathogen, which causes life-threatening infections, often in healthcare settings.C. aurisinfections are worrisome because the fungus is often resistant to multiple antifungal drug classes. Furthermore,C. aurisforms durable and difficult to remove biofilms. Due to the relatively recent, resilient, and resistant nature ofC. auris, we investigated whether it produces the common fungal virulence factor melanin. Melanin is a black-brown pigment typically produced following enzymatic oxidation of aromatic precursors, which promotes fungal virulence through oxidative stress resistance, mammalian immune response evasion, and antifungal peptide and pharmaceutical inactivation. We found that certain strains ofC. aurisoxidized L-DOPA and catecholamines into melanin. Melanization occurred extracellularly in a process mediated by alkalinization of the extracellular environment, resulting in granule-like structures that adhere to the fungus external surface.C. aurishad relatively high cell surface hydrophobicity, but there was no correlation between hydrophobicity and melanization. Melanin protected the fungus from oxidative damage, but we did not observe a protective role during infection of macrophages orGalleria mellonellalarvae. In summary,C. aurisalkalinizes the extracellular medium, which promotes the non-enzymatic oxidation of L-DOPA to melanin that attaches to its surface, thus illustrating a novel mechanism for fungal melanization.
The Herpes Simplex Virus Tegument Protein pUL21 is Required for Viral Genome Retention Within Capsids
Authors: Thomas Ethan C.M. , Bossert Maike , Banfield Bruce W.
Published: July 2022

During virion morphogenesis herpes simplex virus nucleocapsids transit from the nucleoplasm to the cytoplasm, through a process called nuclear egress, where the final stages of virion assembly occur. Coupled to nuclear egress is a poorly understood quality-control mechanism that preferentially selects genome-containing C-capsids, rather than A- and B-capsids that lack genomes, for transit to the cytoplasm. We and others have reported that cells infected with HSV strains deleted for the tegument protein pUL21 accumulate both empty A-capsids and C-capsids in the cytoplasm of infected cells. Quantitative microscopy experiments indicated that C-capsids were preferentially selected for envelopment at the inner nuclear membrane and that nuclear integrity remained intact in cells infected with pUL21 mutants, prompting alternative explanations for the accumulation of A-capsids in the cytoplasm. More A-capsids were also found in the nuclei of cells infected with pUL21 mutants compared to their wild type (WT) counterparts, suggesting pUL21 might be required for optimal genome packaging or genome retention within capsids. In support of this, more viral genomes were prematurely released into the cytoplasm during pUL21 mutant infection compared to WT infection and led to enhanced activation of cellular cytoplasmic DNA sensors. Mass spectrometry and western blot analysis of WT and pUL21 mutant capsids revealed an increased association of the known pUL21 binding protein, pUL16, with pUL21 mutant capsids, suggesting that premature and/or enhanced association of pUL16 with capsids might result in capsid destabilization. Further supporting this idea, deletion of pUL16 from a pUL21 mutant strain rescued genome retention within capsids. Taken together, these findings suggest that pUL21 regulates pUL16 addition to nuclear capsids and that premature, and/or, over-addition of pUL16 impairs HSV genome retention within capsids.

Importance

pUL21 is a conserved, multifunctional alphaherpesvirus protein involved in cell-to-cell spread of infection, transport of capsids along microtubules, and regulation of the phosphorylation status of both viral and host proteins. pUL21 thereby controls diverse processes such as nuclear egress of capsids and cellular lipid trafficking. This study provides additional insight into HSV-1 and HSV-2 pUL21 activities and suggests that, by binding pUL16, pUL21 prevents pUL16 from interacting prematurely with nuclear capsids that would otherwise lead to capsid destabilization and premature ejection of viral genomes. Thus, prevention of pUL21 interaction with pUL16 may prove to be a useful strategy for interfering with virion assembly.
Lasting alterations in monocyte and dendritic cell subsets in individuals after hospitalization for COVID-19
Authors: Hopkins Francis R. , Govender Melissa , Svanberg Cecilia , Nordgren Johan , Waller Hjalmar , Nilsdotter-Augustinsson Åsa , Henningsson Anna J. , Hagbom Marie , Sjöwall Johanna , Nyström Sofia , Larsson Marie
Published: July 2022

After more than two years the COVID-19 pandemic continues to burden healthcare systems and economies worldwide, and it is evident that long-term effects of the disease can persist for months post-recovery in some individuals. The activity of myeloid cells such as monocytes and dendritic cells (DC) is essential for correct mobilization of the innate and adaptive responses to a pathogen. Impaired levels and responses of monocytes and DC to SARS-CoV-2 is likely to be a driving force behind the immune dysregulation that characterizes severe COVID-19. Here, we followed, for 6-7 months, a cohort of COVID-19 patients hospitalized during the early waves of the pandemic. The levels and phenotypes of circulating monocyte and DC subsets were assessed to determine both the early and long-term effects of the SARS-CoV-2 infection. We found increased monocyte levels that persisted for 6-7 months, mostly attributed to elevated levels of classical monocytes. While most DC subsets recovered from an initial decrease, we found elevated levels of cDC2/cDC3 at the 6-7 month timepoint. Analysis of functional markers on monocytes and DC revealed sustained reduction in PD-L1 expression but increased CD86 expression across almost all cell types examined. Finally, viral load and CRP correlated to the appearance of circulating antibodies and levels of circulating DC and monocyte subsets, respectively. By elucidating some of the long-term effects that SARS-CoV-2 infection has on these key innate myeloid cells, we have shed more light on how the immune landscape remains affected in the months following severe COVID-19.
Small-angle X-ray scattering: characterization of cubic Au nanoparticles using Debye's scattering formula
Authors: Deumer Jérôme , Pauw Brian R. , Marguet Sylvie , Skroblin Dieter , Taché Olivier , Krumrey Michael , Gollwitzer Christian
Published: July 2022

A versatile software package in the form of a Python extension, named CDEF (computing Debye's scattering formula for extraordinary form factors), is proposed to calculate approximate scattering profiles of arbitrarily shaped nanoparticles for small-angle X-ray scattering (SAXS). CDEF generates a quasi-randomly distributed point cloud in the desired particle shape and then applies the open-source software DEBYER for efficient evaluation of Debye's scattering formula to calculate the SAXS pattern (https://github.com/j-from-b/CDEF). If self-correlation of the scattering signal is not omitted, the quasi-random distribution provides faster convergence compared with a true-random distribution of the scatterers, especially at higher momentum transfer. The usage of the software is demonstrated for the evaluation of scattering data of Au nanocubes with rounded edges, which were measured at the four-crystal monochromator beamline of PTB at the synchrotron radiation facility BESSY II in Berlin. The implementation is fast enough to run on a single desktop computer and perform model fits within minutes. The accuracy of the method was analyzed by comparison with analytically known form factors and verified with another implementation, the SPONGE, based on a similar principle with fewer approximations. Additionally, the SPONGE coupled to McSAS3 allows one to retrieve information on the uncertainty of the size distribution using a Monte Carlo uncertainty estimation algorithm.