bluuebunny/crossref_metadata_2025 huggingface dataset:
A data driven approach to address missing data in the 1970 British birth cohort
Authors: Katsoulis Michail , Narayanan Martina , Dodgeon Brian , Ploubidis George , Silverwood Richard
Published: February 2024

Background

Missing data may induce bias when analysing longitudinal population surveys. We aimed to tackle this problem in the 1970 British Cohort Study (BCS70)

Methods

We utilised a data-driven approach to address missing data issues in BCS70. Our method consisted of a 3-step process to identify important predictors of non-response from a pool of ∼20,000 variables from 9 sweeps in 18037 individuals. We used parametric regression models to identify a moderate set of variables (predictors of non-response) that can be used as auxiliary variables in principled methods of missing data handling to restore baseline sample representativeness.

Results

Individuals from disadvantaged socio-economic backgrounds, increased number of older siblings, non-response at previous sweeps and ethnic minority background were consistently associated with non-response in BCS70 at both early (ages 5-16) and later sweeps (ages 26-46). Country of birth, parents not being married and higher father’s age at completion of education were additional consistent predictors of non-response only at early sweeps. Moreover, being male, greater number of household moves, low cognitive ability, and non-participation in the UK 1997 elections were additional consistent predictors of non-response only at later sweeps. Using this information, we were able to restore sample representativeness, as we could replicate the original sample distribution of father’s social class and cognitive ability and reduce the bias due to missing data in the relationship between father’s socioeconomic status and mortality.

Conclusions

We provide a set of variables that researchers can utilise as auxiliary variables to address missing data issues in BCS70 and restore sample representativeness.

Key Messages

We aimed to address the problem of missing data in the 1970 British Cohort Study (BCS70) caused by non-response at different sweeps

We identified a set of predictors of non-response that can successfully restore baseline sample representativeness across sweeps

The information from this study can be used from researchers in the future to utilise appropriate auxiliary variables to tackle problems due to missing data in BCS70
Epidemiological model can forecast COVID-19 outbreaks from wastewater-based surveillance in rural communities
Authors: Meadows Tyler , Coats Erik R. , Narum Solana , Top Eva , Ridenhour Benjamin J. , Stalder Thibault
Published: February 2024

Wastewater can play a vital role in infectious disease surveillance, especially in underserved communities where it can reduce the equity gap to larger municipalities. However, using wastewater surveillance in a predictive manner remains a challenge. We tested if detecting SARS-CoV-2 in wastewater can predict outbreaks in rural communities. Under the CDC National Wastewater Surveillance program, we monitored several rural communities in Idaho (USA). While high daily variations in wastewater viral load made real-time interpretation difficult, a SEIR model could factor out the data noise and forecast the start of the Omicron outbreak in five of the six cities that were sampled soon after SARS-CoV-2 quantities increased in wastewater. For one city, the model could predict an outbreak 11 days before reported clinical cases began to increase. An epidemiological modeling approach can transform how epidemiologists use wastewater data to provide public health guidance on infectious diseases in rural communities.
The Association Between Dietary Changes to Lose Weight and Mental Health Status in the National Health and Nutrition Examination Survey, 2005-2006
Authors: Min Jihyun Jane , Noh Keeyoon , Nam Sojeong , Ellison-Barnes Alejandra
Published: February 2024

Background

Weight loss is a common endeavor taken by millions of residents annually in the U.S.—a country with a 31% overweight and 42% obesity rate. Weight loss is associated with numerous physical health benefits, such as better cardiovascular health. However, unhealthy weight loss strategies can cause adverse mental health effects. Past research has focused on how weight loss strategies affect the mental health of a general population—rather than those who are trying to lose weight—or has investigated a diverse array of weight loss strategies. This paper explores how dietary changes for weight loss are associated with mental health, specifically through several variables used in the Patient Health Questionnaire-9 (PHQ9), which measures depression severity. We hypothesize that eating less and skipping meals will be associated with poorer mental health status, while eating fewer carbohydrates, eating less fat, and drinking more water will be associated with better mental health status.

Methods and Findings

This study uses the U.S. 2005-2006 National Health and Nutrition Examination Survey (NHANES) 2005. Univariate (descriptive statistics), bivariate (correlation coefficient), and multivariate (ordinal logistic regression) analyses were performed. The main results show that ‘skipped meals’ was positively associated with ‘feeling bad about yourself,’ ‘feeling down, depressed, or hopeless,’ and ‘little interest in doing things.’ ‘Ate fewer carbohydrates’ was negatively associated with the ‘little interest in doing things.’

Conclusion

Differing dietary changes used for weight loss, particularly skipping meals and eating fewer carbohydrates, are associated with differences in mental health status. Health care professionals providing weight loss guidance should be cognizant of patients’ baseline mental health and the potential for changes in mental health with different dietary strategies. Future research employing a longitudinal approach to determine whether there is evidence of a causal relationship between these and other dietary strategies and subsequent mental health outcomes.
Consent in Canadian-Led Critical Care Research During the COVID-19 Pandemic: A Scoping Review
Authors: Krewulak Karla , Albrecht Lisa , Cameron Saoirse , Gibson Jessica , Martin Dori-Ann , Porteous Rebecca , Sampson Margaret , O’Hearn Katie
Published: February 2024

Introduction

Despite the importance of critical care research during the SARS-CoV-2 pandemic, several pandemic-related factors made the process of obtaining prior written informed consent for research infeasible. To overcome these challenges, research studies utilized alternate informed consent models suggested by available guidance.

Objective

To describe the consent models used in Canadian intensive care unit (ICU) and pediatric ICU (PICU) studies during the COVID-19 pandemic.

Data Sources

We searched MEDLINE, EMBASE, CENTRAL,clinicaltrials.gov, and medRxiv from 01-Jan-2020 to 28-Apr-2023 using Medical Subject Headings and keywords related to the setting (ICU, PICU), study design (e.g., RCT) and study region (i.e., Canada). We included Canadian-led studies that were enrolling during the SARS-CoV-2 and reported on consent. Two independent reviewers reviewed titles/abstracts and full text articles for inclusion.

Results

We included 13 studies from adult (n=12, 92.3%) and pediatric (n=1, 7.7%) populations. Some study authors reported that informed (n=3/13, 23.1%) or a priori (n=2/13, 15.4%) consent was obtained, without further details. Study authors also reported using written informed (n=4/13, 30.8%), deferred (n=3/13, 23.1%), verbal/waived/assent (each n=2/13, 15.4%), or that ethics approval was not necessary which means consent was not required (n=1/13, 7.7%). Five studies (n=5/13, 38.5%) used multiple consent models: a priori/deferred (n=2/5, 40%), written/verbal (n=2/5, 40%), or waived/assent (n=1/5, 20%).

Conclusion

This scoping review underscores the importance of transparent reporting of or modifications to trial procedures during crises, such as the COVID-19 pandemic. Improved reporting practices and exploration of alternate consent models, including electronic consent, are crucial for advancing critical care trials beyond the pandemic and preparing for future health emergencies.
AutoCumulus: an Automated Mammographic Density Measure Created Using Artificial Intelligence
Authors: Al-qershi Osamah , Nguyen Tuong L , Elliott Michael S , Schmidt Daniel F , Makalic Enes , Li Shuai , Fox Samantha K , Dowty James G , Peña-Solorzano Carlos A , Kwok Chun Fung , Chen Yuanhong , Wang Chong , Lippey Jocelyn , Brotchie Peter , Carneiro Gustavo , McCarthy Davis J , Jeong Yeojin , Sung Joohon , Frazer Helen ML , Hopper John L
Published: February 2024

Background

Mammographic (or breast) density is an established risk factor for breast cancer. There are a variety of approaches to measurement including quantitative, semi-automated and automated approaches. We present a new automated measure, AutoCumulus, learnt from applying deep learning to semi-automated measures.

Methods

We used mammograms of 9,057 population-screened women in the BRAIx study for which semi-automated measurements of mammographic density had been made by experienced readers using the CUMULUS software. The dataset was split into training, testing, and validation sets (80%, 10%, 10%, respectively). We applied a deep learning regression model (fine-tuned ConvNeXtSmall) to estimate percentage density and assessed performance by the correlation between estimated and measured percent density and a Bland-Altman plot. The automated measure was tested on an independent CSAW-CC dataset in which density had been measured using the LIBRA software, comparing measures for left and right breasts, sensitivity for high sensitivity, and areas under the receiver operating characteristic curve (AUCs).

Results

Based on the testing dataset, the correlation in percent density between the automated and human measures was 0.95, and the differences were only slightly larger for women with higher density. Based on the CSAW-CC dataset, AltoCumulus outperformed LIBRA in correlation between left and right breast (0.95 versus 0.79; P<0.001), specificity for 95% sensitivity (13% versus 10% (P<0.001)), and AUC (0.638 cf. 0.597; P<0.001).

Conclusion

We have created an automated measure of mammographic density that is accurate and gives superior performance on repeatability within a woman, and for prediction of interval cancers, than another well-established automated measure.
Dietary Sodium and Potassium Patterns in Adults with Food Insecurity in the Context of Hypertension Risk
Authors: Onugha Elizabeth A. , Banerjee Ankona , Vimalajeewa Horahenage D. , Nobleza Kenneth J. , Nguyen Duc T. , Racette Susan B. , Dave Jayna M.
Published: February 2024

Background

Food insecurity (FIS), characterized by the lack of consistent access to nutritious food, is associated with hypertension and adverse health outcomes. Despite evidence of a higher prevalence of hypertension (HTN) in patients living with FIS, there is limited data exploring the underlying mechanism.

Methods

We conducted a cross-sectional analysis of 17,015 adults aged 18-65 years, using dietary recall data from the National Health and Nutrition Examination Survey (2011-2018). Univariate and multivariable analyses were used to examine the association between FIS, HTN, and dietary sodium and potassium levels.

Results

Individuals reporting FIS had a significantly lower mean intake of potassium (2.5±0.03 gm) compared to those in food-secure households (2.74±0.02 gm). No significant difference was found in the mean dietary sodium intake based on food security status. Non-Hispanic Black participants showed a high prevalence of HTN and FIS. While Non-Hispanic White and Hispanic participants had a high prevalence of FIS, it did not appear to influence their risk of HTN.

Conclusions

Adults with FIS and HTN were more likely to report a lower dietary potassium intake. Increasing access to healthy foods, particularly potassium-rich foods, for individuals facing FIS, may contribute to reducing the HTN prevalence and improving cardiovascular outcomes.
Scalable single-cell pooled CRISPR screens with conventional knockout vector libraries
Authors: Islam Mirazul , Yang Yilin , Simmons Alan J. , Xu Yanwen , Fisher Emilie L. , Deng Wentao , Grieb Brian C , Molina Paola , de Caestecker Christian , Ramirez-Solano Marisol A. , Liu Qi , Tansey William P. , Macara Ian G. , Rathmell Jeffrey C. , Coffey Robert J. , Lau Ken S.
Published: February 2024

Current methods for single-cell RNA profiling of pooled CRISPR screens are limited, either by indirect capture of single guide RNAs (sgRNAs) or by custom modification of plasmid libraries. Here, we present a direct sgRNA capture platform called Native sgRNA Capture and sequencing (NSC-seq) that enables single-cell CRISPR screens using common knockout plasmid libraries, facilitating genotype-phenotype mapping at multiple scalesin vitroandin vivo. Additionally, we characterize sgRNA expression in three whole-genome knockout libraries, revealing a substantial subset of truncated (isoform) spacer reads. We provide this dataset as a reference of expressed sgRNA isoforms that may potentially have compromised CRISPR gene editing efficacy and precision.
A hierarchical Bayesian interaction model to estimate cell-type-specific methylation quantitative trait loci incorporating priors from cell-sorted bisulfite sequencing data
Authors: Cheng Youshu , Cai Biao , Li Hongyu , Zhang Xinyu , D’Souza Gypsyamber , Shrestha Sadeep , Edmonds Andrew , Meyers Jacquelyn , Fischl Margaret , Kassaye Seble , Anastos Kathryn , Cohen Mardge , Aouizerat Bradley E , Xu Ke , Zhao Hongyu
Published: February 2024

Background

Methylation Quantitative Trait Loci (meQTLs) are chromosomal regions that harbor genetic variants affecting DNA methylation levels. The identification of meQTLs can be accomplished through quantifying the effects of single nucleotide polymorphisms (SNPs) on DNA methylation levels, and these inferred meQTLs can shed light on the complex interplay between the genome and methylome. However, most meQTL studies to date utilize bulk methylation datasets composed of different cell types that may have distinct methylation patterns in each cell type. Current technological challenges hinder the comprehensive collection of large-scale, cell-type-specific (CTS) methylation data, which limits our understanding of CTS methylation regulation. To address this challenge, we propose a hierarchical Bayesian interaction model (HBI) to infer CTS meQTLs from bulk methylation data.

Results

Our HBI method integrates bulk methylations data from a large number of samples and CTS methylation data from a small number of samples to estimate CTS meQTLs. Through simulations, we show that HBI improves the estimation (accuracy and power) of CTS genetic effects on DNA methylation. To systematically characterize genome-wide SNP-methylation level associations in multiple cell types, we apply HBI to bulk methylation data measured in peripheral blood mononuclear cells (PBMC) from a cohort of 431 individuals together with flow-sorted cell-derived methylation sequencing (MC-seq) data measured in isolated white blood cells (CD4+ T-cells, CD8+ T-cells, CD16+ monocytes) for 47 individuals. We demonstrate that HBI can identify CTS meQTLs and improve the functional annotation of SNPs.

Conclusions

HBI can incorporate strong and robust signals from MC-seq data to improve the estimation of CTS meQTLs. Applying HBI to link the methylome and genome data helps to identify biologically relevant cell types for complex traits.
The long noncoding RNA lnc-FANCI-2 intrinsically restricts RAS signaling and phosphorylation of Akt and Erk in HPV16-infected cervical cancer
Authors: Liu Haibin , Yu Lulu , Majerciak Vladimir , Meyer Thomas , Yi Ming , Johnson Peter F. , Cam Maggie , Lowy Douglas R. , Zheng Zhi-Ming
Published: February 2024

We recently discovered increased expression of a long noncoding RNA, lnc-FANCI-2, coinciding with cervical lesion progression from CIN1, CIN2-3 to cervical cancer. Viral E7 of high-risk HPVs and host transcription factor YY1 are two major factors promoting lnc-FANCI-2 expression. To explore possible roles of lnc-FANCI-2 in HPV-induced cervical carcinogenesis, we ablated the expression oflnc-FANCI-2in the HPV16-positive cervical cancer cell line, CaSki. Knock-out (KO) single cell clones expressed HPV16 oncogenes normally but displayed altered cell morphology when compared with the parental cells. Proteomic profiling of cytosolic and secreted proteins from the parental and KO cells showed that lnc-FANCI-2 regulates expression of a subset of cell surface and adhesion-related proteins, including inhibition of MCAM, PODXL2 and ECM1 and increased levels of ADAM8 and TIMP2. RNA-seq analyses revealed that, relative to the parental cells, KO cells exhibited significantly increased RAS signaling but decreased IFN pathways. In KO cells, phosphorylated Akt and Erk1/2, two important RAS pathway effectors, were increased more than 3-fold, accompanied by increase of IGFBP3, MCAM, VIM, and CCND2 (cyclin D2) and decrease of RAC3. Accordingly, high levels of lnc-FANCI-2 and lower levels of MCAM in cervical cancer patients are associated with improved survival. We found that lnc-FANCI-2 in CaSki cells interacts specifically with 32 host proteins, including H13, HNRH1, K1H1, MAP4K4, and RNPS1, and knockdown of MAP4K4 led to increase phosphorylation of Akt and Erk1/2. In summary, a key function of lnc-FANCI-2 is to intrinsically regulate RAS signaling, thereby affecting cervical cancer outcome.

Significance

Expression of lnc-FANCI-2 is related to cervical lesion progression. Knock-out (KO) or knock-down (KD) of lnc-FANCI-2 expression in HPV16-positive cervical cancer cells CaSki significantly increases RAS signaling, phosphorylation of Akt and Erk1/2, and increase of epithelial mesenchymal transition factors. lnc-FANCI-2 KO also regulates the expression of a subset of cell surface and adhesion-related proteins IGFBP3 and MCAM. A high level of lnc-FANCI-2 and lower level of MCAM in cervical cancer patients are associated with improved survival. lnc-FANCI-2 in CaSki cells interacts specifically with 32 host proteins, including MAP4K4. KD of MAP4K4 expression in CaSki cells led to increase phosphorylation of Akt and Erk1/2. Thus, one lnc-FANCI-2 function is to intrinsically regulate RAS signaling to impact cervical lesion progression.
Improved nuclease-based prime editing by DNA repair modulation and pegRNA engineering
Authors: Antoniou Panagiotis , Dacquay Louis , Selfjord Niklas , Madeyski-Bengtson Katja , Loyd Anna-Lena , Gordon Euan , Thom George , Hsieh Pei-Pei , Wimberger Sandra , Šviković Saša , Firth Mike , Akrap Nina , Maresca Marcello , Peterka Martin
Published: February 2024

Prime editing is a genome engineering tool that allows installation of small edits with high precision. However, prime editing efficiency and purity can vary widely across different edits, genomic targets, and cell types. Prime editing nuclease (PEn) utilizes a fully active Cas9 instead of the nickase employed in conventional prime editors. PEn is capable of editing sites resistant to nickase-based prime editors but induces more undesired editing events. In this work, we introduce two strategies to enhance PEn precision and efficiency. First, we apply a small molecule approach, selectively modulating DNA repair pathways, to improve PEn precision up to 9.8-fold and reduce off-target editing by 90%. Second, through pegRNA engineering, we devise a strategy that mitigates unintended pegRNA scaffold integration, which is a common prime editing by-product, enhancing precision up to 3.5-fold. We apply this approach to a specific type of PEn editing mediated through non-homologous end joining and use it to achieve efficient and precise prime editing in multiple human cell lines, primary human hepatocytes, and mouse embryos. Together, this work presents two general strategies to improve prime editing, overcomes the limitations of current PEn editors, and provides reliable and precise genome editing outcomes, a pivotal requirement for therapeutic applications.
Genomic and phenotypic studies amongClostridioides difficileisolates show a high prevalence of clade 2 and great diversity in clinical isolates from Mexican adults and children with healthcare-associated diarrhea
Authors: Meléndez Sánchez D , Hernández Laura , Ares Miguel , Méndez Tenorio A , Flores-Luna Lourdes , Torres Javier , Camorlinga-Ponce M
Published: February 2024

Clostridioides difficileis the most common cause of healthcare-associated infection worldwide. Its pathogenesis is mainly due to the production of toxins A, B, and CDT, whose genetic variants may be associated with disease severity. We studied genetic diversity inC. difficileisolates from hospitalized adults and children using different gene and genome typing methods and investigated their association with in vitro expression of toxins.

Whole-genome was sequenced in 39 toxigenicC. difficileisolates and used for multilocus sequence typing (MLST),tcdA, andtcdBtyping sequence type, and for phylogenetic analysis. Strains were grown in broth media, and expression of toxin genes was measured by real-time PCR and cytotoxicity was determined in culture assays.

We found that clustering after genome-wide phylogeny followed clade classification, although different clusters were identified within each clade. The toxin profiletcdA+/tcdB+/cdtA+/cdtB+ and clade 2/ST1 were the most prevalent among isolates from children and adults. Isolates presented two TcdA and three TcdB subtypes, of which A2 and B2 were dominant. Toxin gene expression or cytotoxicity were not associated with genotyping or toxin subtypes. In conclusion, genomic and phenotypic analysis shows high diversity amongC. difficileisolates from patients with healthcare-associated-diarrhea.

Importance

Clostridioides difficileis a toxin-producing bacterial pathogen associated with healthcare, and different genetic or phenotypic typing have been proposed for classification. We extensively studied cytotoxicity, expression of toxins, whole genome phylogeny, and toxin typing in clinicalC. difficileisolates. Most isolates presented atcdA+ tcdB+cdtA+ cdtB+pattern, with high diversity in cytotoxicity, and clade 2/ST1 was the most prevalent. However, they all had the same TcdA2/TcdB2 toxin subtype. Advances in genomics and bioinformatics tools offer the opportunity to understand the virulence ofC. difficilebetter and find markers with better clinical use.
Antagonizing the SAMD9 pathway is key to myxoma virus host shut-off and immune evasion
Authors: Raza Tahseen , Perterson Erich , Liem Jason , Liu Jia
Published: February 2024

Poxviruses have life cycles exclusively in the cytoplasm. However, these viruses can have profound impact to host transcription. One possible mechanism is through viral manipulation of host protein synthesis and such ability is critical for viral immune evasion. Many mammalian poxviruses encode more than one viral protein to interact with the host SAMD9 protein. In myxoma virus (MYXV), a rabbit specific poxvirus and non-pathogenic for other species, viral M062 protein is the lone inhibitor to SAMD9 with broad species specificity and loss ofM062Rin viral genome (ΔM062Rmutant) leads to profound infection defect. We previously found ΔM062Rremodeled transcriptomic landscape in monocytes/macrophages that is associated with the crosstalk between the SAMD9 pathway and cGAS/STING/IRF3 DNA sensing pathway. In this study we completed the characterization of ΔM062Rinfection. We observed that although this replication-defective virus preserved intact early protein synthesis, it failed to conduct host shutoff. Despite a defect in viral DNA replication, ΔM062Rinfection retained intact intermediate protein synthesis comparable to the wildtype virus. Using time course dual RNAseq analyses we found that the overall viral gene transcription profile was mostly indistinguishable from that of the wildtype MYXV. However, the slightly attenuated late RNA synthesis along with the block at viral protein synthesis led to its infection defect. Infection by ΔM062Rin macrophages potentiated the antiviral responses to new danger signals. We provided an initial characterization of such a state in which host antiviral protein synthesis may be promoted leading to the immunological consequence.

Importance

Poxviruses utilize multi-faceted strategies to evade and manipulate host immunity. Through targeted gene deletion, we generated useful tools of mutant poxviruses to investigate specific crosstalk between host defense mechanisms. Through studying MYXV M062 protein function, we previously identified SAMD9 as one host target of poxvirusC7Lsuperfamily, in which familyM062Ris one member. However, what kind of cellular outcome caused by ΔM062Rinfection remained unknown. The infection defect of ΔM062Rcaused the induction of host inflammation program is likely due to the activation of the host pathway governed by SAMD9. Because little is known about SAMD9 cellular function and the pathways it regulates, which are important for cellular homeostasis and immune regulation, this study on ΔM062Rinduced effect in host cells will provide new insight on how SAMD9 affects cellular protein synthesis and immunological responses.
αMI-domain of Integrin Mac-1 Binds the Cytokine Pleiotrophin Using Multiple Mechanisms
Authors: Nguyen Hoa , Podolnikova Nataly P. , Ugarova Tatiana P. , Wang Xu
Published: February 2024

The integrin Mac-1 (αMβ2, CD11b/CD18, CR3) is an important adhesion receptor expressed on macrophages and neutrophils. Mac-1 is also the most promiscuous member of the integrin family that binds a diverse set of ligands through its αMI-domain. However, the binding mechanism of most ligands is not clear. We have determined the interaction of αMI-domain with the cytokine pleiotrophin (PTN), a cationic protein known to bind αMI-domain and induce Mac-1-mediated cell adhesion and migration. Our data show that PTN’s N-terminal domain binds a unique site near the N- and C-termini of the αMI-domain using a metal-independent mechanism. However, stronger interaction is achieved when an acidic amino acid in a zwitterionic motif in PTN’s C-terminal domain chelates the divalent cation in the metal ion-dependent adhesion site of the active αMI-domain. These results indicate that αMI-domain can bind ligands using multiple mechanisms, and suggest that active αMI-domain prefers acidic amino acids in zwitterionic motifs.

HIGHLIGHTS

αMI-domain’s interaction with the cytokine pleiotrophin (PTN) was investigated with solution NMR.

αMI-domain binds PTN using multiple mechanisms.

PTN’s N-terminal domain binds both active and inactive αMI-domains using a unique site near αMI-domain’s termini.

PTN’s C-terminal domain binds only active αMI-domain through a metal-dependent interaction.
GSDMD pore formation regulates caspase-4 cleavage to limit IL-18 production in the intestinal epithelium
Authors: Bruce J.K. , Li L. , Tang Y. , Winsor N. , Krustev C. , Keely S. , Philpott D.J. , Girardin S.E.
Published: February 2024

Epithelial inflammasomes induce pyroptosis and release cytokines to defend against cytosolic pathogens. However, pyroptosis in epithelial barriers must be carefully regulated to facilitate elimination of infected cells while limiting widespread pyroptosis to preserve the single cell barrier. How epithelial cells achieve this is unknown. In this study, we describe a novel epithelial caspase regulation mechanism. By examining caspase-4 activation in human epithelial cells, we discovered that GSDMD pore formation serves as a signal to terminate caspase-4 activity thus facilitating epithelial cell expulsion while controlling cytokine secretion. Inhibition of epithelial pyroptosis led to IL-18 hyperproduction, likely as a mechanism to combat increased pathogen burden and initiate a wider immune response. Moreover, we demonstrate that full-length, rather than cleaved caspase-4 is active against IL-18 and propose that GSDMD pore formation facilitates cleavage of caspase-4 to terminate its catalytic activity. By comparing human cells and murine epithelial organoids to immune cells, we show that GSDMD pore mediated inhibition of caspase activity is largely specific to epithelial cells. Overall, these studies characterise a novel, epithelial-specific negative feedback loop that modulates inflammasome activity and challenge the dogma that autocatalytic caspase cleavage is required for caspase activity against substrates.

Graphical Abstract

In intestinal epithelial cells, caspase activation simultaneously leads to GSDMD pore formation and IL-18 release. GSDMD pore formation provides a signal to terminate caspase activity and limit cytokine production. In GSDMD deficient cells, lack of an inhibition signal leads to caspase mediated IL-18 hyperproduction. Upon cell death this leads to release of massive amounts of IL-18. Created with BioRender.com
A combination of two-enzyme system and enzyme engineering improved the activity of a new PET hydrolase identified from soil bacterial genome
Authors: Mabashi-Asazuma Hideaki , Hirai Makoto , Sakurai Shigeru , Ide Keigo , Kogawa Masato , Matsushita Ai , Hosokawa Masahito , Tsuda Soichiro
Published: February 2024

We here report a novel PET hydrolase originating from a soil microbial genome sequence. This enzyme, bbPET0069, exhibits characteristics resembling a cutinase-like Type I PET-degrading enzyme but lacks disulfide bonds. Notably, bbPET0069 displayed remarkable synergy withCandida antarcticalipase B (CALB), demonstrating rapid and efficient PET degradation. To improve the PET degradation activity of bbPET0069, we employed a three-dimensional (3D) structural modeling to identify mutation sites around its substrate binding domain combined with a protein language model for effective mutation prediction. Through three initial rounds of directed evolution, we achieved a significant enhancement in PET degradation with CALB, resulting in a 12.6-fold increase compared to wild-type bbPET0069 without CALB. We confirmed its PET degradation activity in PET nanoparticles and films, and our proposed approach enabled efficient PET degradation to terephthalic acid monomers up to 95.5%. Our approach, which integrates a two-enzyme system with protein engineering, demonstrates the potential for enhancing the activity of emerging PET-degradation enzymes, which may possess unique attributes.

Graphical Abstract

A novel PET hydrolase, bbPET0069, was identified from a soil microbial genome. bbPET0069 and CALB showed remarkable synergy in PET degradation. Using surface feature analysis, PET degradation activity of bbPET0069 was significantly improved. This combination of a two-enzyme system and surface feature analysis holds promise for enhancing emerging PET-degradation enzymes.
Characterization of severe fever with thrombocytopenia syndrome virus Japanese isolate YG1 strain quasispecies using reverse genetics approaches
Authors: Lokupathirage Sithumini M.W. , Muthusinghe Devinda S. , Sarii Rakiiya S. , Akanbi Olusola A. , Shimizu Kenta , Tsuda Yoshimi , Yoshimatsu Kumiko
Published: February 2024

Three amino acid mutations have been identified in the isolated YG1 strain of severe fever thrombocytopenia syndrome virus (SFTSV), Gn (Y328H) accounts for 26.9% of the virus in patients’ blood, Gc (R624W) and L (N1891K) those are minor. To investigate viral properties caused by each mutation, we rescued viruses with one–three mutations. Mutations Y328H and R624W in GP increased the cell fusion activity and plaque size. Theses enhancement was more pronounced for both Y328H and R624W. The pseudotyped vesicular stomatitis virus coated with the SFTSV GP Y328H mutant showed lower infectivity in Vero E6 cells, which was compensated for by the additional R624W mutation. In the process of adaptation for virus with Y328H, the R624W mutation may be acquired. Moreover, only the viruses with the N1891K mutation in L showed significant CPE and the CPE was inhibited by the pan-caspase inhibitor, suggesting that caspase-dependent cell death occurred. Programmed cell death associated molecules caspase-1 and caspase-3 were induced in both CPE inducing and wild-type virus-infected cells. Furthermore, infection with the wild-type virus suppressed actinomycin D-induced cell death. These results suggest that SFTSV infected cells initiate programmed cell death, whereas wildt-ype virus may inhibit cell death. Furthermore, the N1891K mutation in L virus was outcompeted by a 10-fold less wild-type virus in Vero E6 cells indicating that it was not advantageous for viral survival in Vero E6 cells. Thus the quasispecies composition of SFTSV appeared to be influenced by propagative environment.

Importance

This study shows information on viral pathogenesis by analyzing quasispecies derived from one fatal case of severe fever with thrombocytopenia syndrome virus (SFTSV) infection. Observation with recombinant SFTSV altered Gn and Gc suggests that combining mutations may increase the viability of mutant viruses, selecting viruses to create a suitable population for propagation. The N1891K mutation in L protein of SFTSV was related to CPE appearance. On the other hand, wild-type virus which is major population in patient infection was suppressive for cell death. It was suggested that SFTSV has a mechanism to escape cell death for the prolonged viral propagation in infected cells. Although the mechanism is still unknown, it has been suggested that RNA virus polymerase might be involved in the regulation of cell death. This study proposed the mechanism underlying the adaptation to the environment and survival of virus as quasispecies.
Fast and scalable off-target assessment for CRISPR guide RNAs using partial matches
Authors: Schmitz Carl , Bradford Jacob , Salomone Robert , Perrin Dimitri
Published: February 2024

The design of CRISPR-Cas9 guide RNAs is not trivial. In particular, it is crucial to evaluate the risk of unintended, off-target modifications, but this is computationally expensive. To avoid a brute-force approach where each guide RNA is compared against every possible CRISPR target site in the genome, we previously introduced Crackling, a guide RNA design tool that relies on exact matches over 4bp subsequences to approximate a neighbourhood and accelerate off-target scoring by greatly reducing the search space. While this was faster than other existing tools, it still generates large neighbourhoods. Here, we aim to further reduce the search space by requiring more, now non-contiguous, exact matches. The new implementation, called Crackling++, is benchmarked against our initial approach and other off-target evaluation tools. We show that it provides the fastest way to assess candidate guide RNAs. By using memorymapped files, it also scales to the largest genomes. Crackling++ is available athttps://github.com/bmds-lab/CracklingPlusPlusunder the Berkeley Software Distribution (BSD) 3-Clause license.
Reducing stomatal density by expression of a synthetic EPF increases leaf intrinsic water use efficiency and reduces plant water use in a C4crop
Authors: Ferguson John N. , Schmuker Peter , Dmitrieva Anna , Quach Truyen , Zhang Tieling , Ge Zhengxiang , Nersesian Natalya , Sato Shirley J , Clemente Tom E. , Leakey Andrew D.B.
Published: February 2024

Enhancing crop water use efficiency (WUE) is a key target trait for climatic resilience and expanding cultivation on marginal lands. Reducing stomatal conductance (gs) through manipulating stomatal density has been observed to translate to improved WUE in multiple C3crop species. However, reducinggsin C3species often reduces photosynthetic carbon gain. A different response is expected in C4plants because they possess specialized anatomy and biochemistry which concentrates CO2at the site of fixation. This modifies the photosynthesis (AN) relationship with intracellular CO2concentration (ci) so that photosynthesis is CO2-saturated and reductions ingsare unlikely to impairAN. To test this hypothesis, genetic strategies were investigated to reduce stomatal density in the C4crop sorghum. Constitutive expression of a synthetic epidermal patterning factor (EPF) transgenic allele in sorghum, lead to reduced stomatal densities. A moderate reduction in stomatal density did not strengthen stomatal limitation to AN, improved WUE, reduced water use, and avoided loss of carbon fixation during a period of water deprivation. However, these positive outcomes were associated with negative pleiotropic effects on reproductive development and photosynthetic capacity. Avoiding pleiotropy by targeting expression of the transgene to specific tissues provides a potential pathway to optimal agronomic outcomes.
Hospital Care Trajectories of Older Adults with Cancer and the Associated Clinical Profiles: linking a French Prospective Cohort Study and a Clinical Data Warehouse
Authors: Jean Charline , Paillaud Elena , Boudou-Rouquette Pascaline , Martinez-Tapia Claudia , Pamoukdjian Frédéric , Hagège Meoïn , Bréant Stéphane , Hassen-Khodja Claire , Natella Pierre-André , Cudennec Tristan , Laurent Marie , Caillet Philippe , Audureau Etienne , Canouï-Poitrine Florence
Published: February 2024

Background

The longitudinal hospital care experiences of older adults with cancer, from the treatment decision-making process until their end of life, remain unexplored. We examined the hospital care trajectories of these patients and identified associated clinical determinants.

Methods

We linked the ELCAPA multicenter cohort study (patients aged ≥70 with a solid tumor and having been referred for a geriatric assessment between 2012 and 2019) and the Greater Paris University Hospitals’ clinical data warehouse. Individual care trajectories, defined as series of consultations, hospital admissions (in day, acute or rehabilitation units) and emergency room visits, were clustered using multichannel sequence analysis. Cluster membership determinants were identified among socio-demographic, oncological and geriatric parameters by logistic regression analysis.

Results

707 patients (median age: 82; metastatic cancer: 45.2%; 10,998 care episodes) were included. Four trajectory clusters were identified: cluster A (n=149, 21.1%) with in-hospital surgical trajectories, cluster B (n=198, 28.0%) with outpatient care trajectories with chemotherapy and/or radiotherapy, cluster C (n=302, 42.7%) without any hospital cancer treatments, and cluster D (n=58, 8.2%) with mostly chemotherapy and high hospital care consumption. Cluster belonging determinants included metastatic status and cancer site (for cluster A), cognition, mobility and mood status (unimpaired parameters for cluster B and impaired for cluster C), and younger age (for cluster D).

Conclusion

While highlighting varied hospital care experiences among older patients with cancer, we found that age remains an independent determinant of chemotherapy-dominant care trajectories.
Dissecting quantitative trait nucleotides by saturation genome editing
Authors: Roy Kevin R. , Smith Justin D. , Li Shengdi , Vonesch Sibylle C. , Nguyen Michelle , Burnett Wallace T. , Orsley Kevin M. , Lee Cheng-Sheng , Haber James E. , St.Onge Robert P. , Steinmetz Lars M.
Published: February 2024

Genome editing technologies have the potential to transform our understanding of how genetic variation gives rise to complex traits through the systematic engineering and phenotypic characterization of genetic variants. However, there has yet to be a system with sufficient efficiency, fidelity, and throughput to comprehensively identify causal variants at the genome scale. Here we explored the ability of templated CRISPR editing systems to install natural variants genome-wide in budding yeast. We optimized several approaches to enhance homology-directed repair (HDR) with donor DNA templates, including donor recruitment to target sites, single-stranded donor production by bacterial retrons, and in vivo plasmid assembly. We uncovered unique advantages of each system that we integrated into a single superior system named MAGESTIC 3.0. We used MAGESTIC 3.0 to dissect causal variants residing in 112 quantitative trait loci across 32 environmental conditions, revealing an enrichment for missense variants and loci with multiple causal variants. MAGESTIC 3.0 will facilitate the functional analysis of the genome at single-nucleotide resolution and provides a roadmap for improving template-based genome editing systems in other organisms.
Temperature-induced changes in protein interactions control RNA recruitment to G3BP1 condensates
Authors: Fischer Charlotte M. , Ausserwöger Hannes , Sneideris Tomas , Qian Daoyuan , Scrutton Rob , Qamar Seema , St George-Hyslop Peter , Knowles Tuomas P. J.
Published: February 2024

Biomolecular condensates have emerged as prominent regulators of dynamic subcellular organisation and essential biological processes. Temperature, in particular, exerts a significant influence on the formation and behaviour of biomolecular condensation. For example, during cellular heat stress, stress granules (SGs) are formed from RNA-binding proteins (RBPs) and RNA, forming liquid condensates to protect the RNA from damage. However, the molecular mechanisms leading to changes in protein phase behaviour are not well understood. To answer how temperature modulates protein interactions and phase behaviour, we developed a high-throughput microfluidic platform, capable of mapping the phase space and quantifying protein interactions in a temperature-dependent manner. Specifically, our approach measures high-resolution protein phase diagrams as a function of temperature, while accurately quantifying changes in the binodal, condensate stoichiometry and free energy contribution of a solute, hence, providing information about the underlying mechanistic driving forces. We employ this approach to investigate the effect of temperature changes on the phase separation of the stress granule scaffold protein Ras GTPase-activating protein-binding protein 1 (G3BP1) with PolyA-RNA. Surprisingly, we find that the G3BP1/RNA phase boundary remains unaffected by the increasing temperature but the underlying stoichiometry and energetics shift, which can only be revealed with high-resolution phase diagrams. This indicates that temperature-induced dissolution is counteracted by entropic processes driving phase separation. With increasing temperature, the G3BP1 content in condensates decreases alongside with a reduction of the free energy of protein interactions, while the RNA content increases driven by entropically favoured hydrophobic interactions. In the context of cellular heat SG formation, these findings could indicate that during heat shock, elevated temperatures directly induce RNA recruitment to stress granules as a cytoprotective mechanism by finetuning the strength of protein and RNA interactions.
Dynamics of the membrane- and cell wall-associated proteome ofArabidopsis thalianaroots in response to uranium stress
Authors: Przybyla-Toscano Jonathan , Chetouhi Cherif , Balliau Thierry , Alban Claude , Bourguignon Jacques , Ravanel Stéphane
Published: February 2024

Uranium (U) is a non-essential and toxic metal for plants, which have the ability to take up uranyl ions from the soil and preferentially accumulate them in the roots. We showed that the bulk of the radionuclide accumulates in the root insoluble proteome of Arabidopsis plants challenged with U. Therefore, to elucidate new molecular mechanisms related to U stress response and tolerance, we used label-free quantitative proteomics to analyze the dynamics of the root membrane- and cell wall-enriched proteome under U stress. Of the 2,802 proteins identified, 458 showed differential accumulation in response to U. Biological processes affected by U include response to stress, amino acid metabolism, and previously unexplored functions associated with membranes and the cell wall. Indeed, our analysis supports a dynamic and complex reorganization of the cell wall in response to U stress, including lignin and suberin synthesis, pectin modifications, polysaccharide hydrolysis, and Casparian strips formation. Water flux through aquaporins and vesicular trafficking were also significantly perturbed by U stress. Finally, the abundance of metal transporters and iron, calcium, and other metal-binding proteins was affected by U. These proteins may play a role in controlling the fate and toxicity of U in plants.
Grapevine cell response to carbon deficiency requires transcriptome and methylome reprogramming
Authors: Berger Margot M J , Garcia Virginie , Lacrampe Nathalie , Rubio Bernadette , Decros Guillaume , Pétriacq Pierre , Flandin Amélie , Cassan Cédric , Hilbert-Masson Ghislaine , Colombié Sophie , Atanassova Rossitza , Gallusci Philippe
Published: February 2024

Sugar limitation has dramatic consequences on plant cells, which include a profound reorganization of the cell metabolism, transcriptional reprogramming, and the recycling of cellular components to maintain fundamental cell functions. There is so far no description of the possible contribution of epigenetic regulations in the adaptation of plant cells to limited carbon availability. We investigated this question using non-photosynthetic grapevine cells (Vitis vinifera, cv Cabernet Sauvignon) culturedin vitrowith contrasted glucose concentrations. As expected, limited sugar availability in the culture medium led to a rapid cell growth arrest. This was associated with a major metabolic shift characterized by depletion in soluble sugar and total amino acids, an increase in malate content and changes in the cell redox status. Consistently, flux modeling showed a dramatic slowdown of many pathways required for biomass accumulation such as cell wall polymers and total protein content. In contrast, anaplerotic fluxes, the synthesis of some amino acids, redox and polyamine metabolism were enhanced.

Carbon deprivation also resulted in a major transcriptional reprogramming characterized by the induction of genes involved in photosynthesis, and the repression of those related to sucrose mobilization or cell cycle control. Similarly, the epigenetic landscape was deeply modified. Glucose-depleted cells showed a higher global DNA methylation level than those grown with glucose. Changes in DNA methylation mainly occurred at transposable elements, but also at genes including differentially expressed genes, suggesting that DNA methylation could participate in the adaptation of cells to limited sugar availability. In addition, genes encoding histone modifiers were differentially expressed suggesting that additional epigenetic mechanisms may be at work during the response of plant cells to carbon shortage.
Translational arrest and mRNA decay are independent activities of alphaherpesvirus virion host shutoff proteins
Authors: Eke Lucy , Tweedie Alistair , Cutts Sophie , Wise Emma L , Elliott Gillian
Published: February 2024

The herpes simplex virus 1 (HSV1) virion host shutoff (vhs) protein is an endoribonuclease that regulates the translational environment of the infected cell, by inducing the degradation of host mRNA via cellular exonuclease activity. To further understand the relationship between translational shutoff and mRNA decay, we have used ectopic expression to compare HSV1 vhs (vhsH) to its homologues from four other alphaherpesviruses – varicella zoster virus (vhsV), bovine herpesvirus 1 (vhsB), equine herpesvirus 1 (vhsE) and Marek’s disease virus (vhsM). Only vhsH, vhsB and vhsE induced degradation of a reporter luciferase mRNA, with polyA+in situhybridisation indicating a global depletion of cytoplasmic polyA+ RNA and a concomitant increase in nuclear polyA+ RNA and the polyA tail binding protein PABPC1 in cells expressing these variants. By contrast, vhsV and vhsM failed to induce reporter mRNA decay and polyA+ depletion, but rather, induced cytoplasmic G3BP1 and polyA+ mRNA-containing granules and phosphorylation of the stress response proteins eIF2α and protein kinase R. Intriguingly, regardless of their apparent endoribonuclease activity, all vhs homologues induced an equivalent general blockade to translation as measured by single cell puromycin incorporation. Taken together, these data suggest that the activities of translational arrest and mRNA decay induced by vhs are separable and we propose that they represent sequential steps of the vhs host interaction pathway.