bluuebunny/crossref_metadata_2025_split huggingface dataset:
CRISPR-Cas9 screen of E3 ubiquitin ligases identifies TRAF2 and UHRF1 as regulators of HIV latency in primary human T cells
Authors: Rathore Ujjwal , Haas Paige , Easwar Kumar Vigneshwari , Hiatt Joseph , Haas Kelsey M. , Bouhaddou Mehdi , Swaney Danielle L. , Stevenson Erica , Zuliani-Alvarez Lorena , McGregor Michael J. , Turner-Groth Autumn , Ochieng' Olwal Charles , Bediako Yaw , Braberg Hannes , Soucheray Margaret , Ott Melanie , Eckhardt Manon , Hultquist Judd F. , Marson Alexander , Kaake Robyn M. , Krogan Nevan J. 
Published: February 2024

ABSTRACT

During HIV infection of CD4+ T cells, ubiquitin pathways are essential to viral replication and host innate immune response; however, the role of specific E3 ubiquitin ligases is not well understood. Proteomics analyses identified 116 single-subunit E3 ubiquitin ligases expressed in activated primary human CD4+ T cells. Using a CRISPR-based arrayed spreading infectivity assay, we systematically knocked out 116 E3s from activated primary CD4+ T cells and infected them with NL4-3 GFP reporter HIV-1. We found 10 E3s significantly positively or negatively affected HIV infection in activated primary CD4+ T cells, including UHRF1 (pro-viral) and TRAF2 (anti-viral). Furthermore, deletion of either TRAF2 or UHRF1 in three JLat models of latency spontaneously increased HIV transcription. To verify this effect, we developed a CRISPR-compatible resting primary human CD4+ T cell model of latency. Using this system, we found that deletion of TRAF2 or UHRF1 initiated latency reactivation and increased virus production from primary human resting CD4+ T cells, suggesting these two E3s represent promising targets for future HIV latency reversal strategies.

IMPORTANCE
HIV, the virus that causes AIDS, heavily relies on the machinery of human cells to infect and replicate. Our study focuses on the host cell’s ubiquitination system which is crucial for numerous cellular processes. Many pathogens, including HIV, exploit this system to enhance their own replication and survival. E3 proteins are part of the ubiquitination pathway that are useful drug targets for host-directed therapies. We interrogated the 116 E3s found in human immune cells known as CD4+ T cells, since these are the target cells infected by HIV. Using CRISPR, a gene-editing tool, we individually removed each of these enzymes and observed the impact on HIV infection in human CD4+ T cells isolated from healthy donors. We discovered that 10 of the E3 enzymes had a significant effect on HIV infection. Two of them, TRAF2 and UHRF1, modulated HIV activity within the cells and triggered an increased release of HIV from previously dormant or “latent” cells in a new primary T cell assay. This finding could guide strategies to perturb hidden HIV reservoirs, a major hurdle to curing HIV. Our study offers insights into HIV-host interactions, identifies new factors that influence HIV infection in immune cells, and introduces a novel methodology for studying HIV infection and latency in human immune cells.
The dCas9-based genome editing in
Plasmodium yoelii
Authors: Zhang Chao , Yang Shijie , Quansah Elvis , Zhang Ziyu , Da Weiran , Wang Bingjie 
Published: February 2024

ABSTRACT

Genetic editing is a powerful tool for functional characterization of genes in various organisms. With its simplicity and specificity, the CRISPR-Cas9 technology has become a popular editing tool, which introduces site-specific DNA double-strand breaks (DSBs), and then leverages the endogenous repair pathway for DSB repair
via
homology-directed repair (HDR) or the more error-prone non-homologous end joining (NHEJ) pathways. However, in the
Plasmodium
parasites, the lack of a typical NHEJ pathway selects for DSB repair through the HDR pathway when a homologous DNA template is available. The AT-rich nature of the
Plasmodium
genome exacerbates this drawback by making it difficult to clone longer homologous repair DNA templates. To circumvent these challenges, we adopted the hybrid catalytically inactive Cas9 (dCas9)-microbial single-stranded annealing proteins (SSAP) editor to the
Plasmodium
genome. In
Plasmodium yoelii
, we demonstrated the use of the dCas9-SSAP, as the cleavage-free gene editor, by targeted gene deletion and gene tagging, even using shorter homologous DNA templates. This dCas9-SSAP method with a shorter DNA template, which did not require DSBs, independent of HDR and NHEJ, would be a great addition to the existing genetic toolbox and could be deployed for the functional characterization of genes in
Plasmodium
, contributing to improving the ability of the malaria research community in characterizing more than half of genes with unknown functions.

IMPORTANCE

Malaria caused by
Plasmodium
parasites infection remains a serious threat to human health, with an estimated 249 million malaria cases and 608,000 deaths worldwide in 2022, according to the latest report from the World Health Organization (WHO). Here, we demonstrated the use of dCas9-single-stranded annealing protein, as the cleavage-free gene editor in
Plasmodium yoelii
, by targeted deletion and gene tagging, even using shorter homologous DNA templates. This method with a shorter DNA template, which did not require DSBs, independent of HDR and NHEJ, showing the potential significance in greatly improving our ability to elucidate gene functions, would contribute to assisting the malaria research community in deciphering more than half of genes with unknown functions to identify new drug and vaccine targets.
Heterogeneous lineage-specific arginine deiminase expression within dental microbiome species
Authors: Mann Allison E. , Chakraborty Brinta , O'Connell Lauren M. , Nascimento Marcelle M. , Burne Robert A. , Richards Vincent P. 
Published: February 2024

ABSTRACT

Arginine catabolism by the bacterial arginine deiminase system (ADS) has anticariogenic properties through the production of ammonia, which modulates the pH of the oral environment. Given the potential protective capacity of the ADS pathway, the exploitation of ADS-competent oral microbes through pre- or probiotic applications is a promising therapeutic target to prevent tooth decay. To date, most investigations of the ADS in the oral cavity and its relation to caries have focused on indirect measures of activity or on specific bacterial groups, yet the pervasiveness and rate of expression of the ADS operon in diverse mixed microbial communities in oral health and disease remain an open question. Here, we use a multivariate approach, combining ultra-deep metatranscriptomic sequencing with paired metataxonomic and
in vitro
citrulline quantification to characterize the microbial community and ADS operon expression in healthy and late-stage cavitated teeth. While ADS activity is higher in healthy teeth, we identify multiple bacterial lineages with upregulated ADS activity on cavitated teeth that are distinct from those found on healthy teeth using both reference-based mapping and
de novo
assembly methods. Our dual metataxonomic and metatranscriptomic approach demonstrates the importance of species abundance for gene expression data interpretation and that patterns of differential expression can be skewed by low-abundance groups. Finally, we identify several potential candidate probiotic bacterial lineages within species that may be useful therapeutic targets for the prevention of tooth decay and propose that the development of a strain-specific, mixed-microbial probiotic may be a beneficial approach given the heterogeneity of taxa identified here across health groups.

IMPORTANCE
Tooth decay is the most common preventable chronic disease, affecting more than two billion people globally. The development of caries on teeth is primarily a consequence of acid production by cariogenic bacteria that inhabit the plaque microbiome. Other bacterial strains in the oral cavity may suppress or prevent tooth decay by producing ammonia as a byproduct of the arginine deiminase metabolic pathway, increasing the pH of the plaque biofilm. While the benefits of arginine metabolism on oral health have been extensively documented in specific bacterial groups, the prevalence and consistency of arginine deiminase system (ADS) activity among oral bacteria in a community context remain an open question. In the current study, we use a multi-omics approach to document the pervasiveness of the expression of the ADS operon in both health and disease to better understand the conditions in which ADS activity may prevent tooth decay.
Distribution and genetic characterization of fluoroquinolone resistance gene
qnr
among
Salmonella
strains from chicken in China
Authors: Chen Yang , Liu Lihui , Guo Yali , Chu Jinhua , Wang Bangjuan , Sui Yuxin , Wei Hanqi , Hao Haihong , Huang Lingli , Cheng Guyue 
Published: February 2024

ABSTRACT

The prevalence and dissemination of the plasmid-mediated fluoroquinolone (FQ) resistance gene
qnr
in
Salmonella
are considered serious public health concerns worldwide. So far, no comprehensive large-scale studies have focused on the prevalence and genetic characteristics of the
qnr
gene in
Salmonella
isolated from chickens. Herein, this study aimed to investigate the prevalence, antimicrobial resistance (AMR) patterns, and molecular characteristics of chicken-originated
qnr
-positive
Salmonella
strains from chicken farms, slaughterhouses, and markets in 12 provinces of China in 2020–2021. The overall prevalence of the
qnr
gene was 21.13% (56/265), with the highest prevalence in markets (36.11%, 26/72), followed in farms (17.95%, 21/117), and slaughterhouses (10.53%, 9/76). Only the
qnrS
and
qnrB
genes were detected, and the prevalence rate of the
qnrS
gene (19.25%, 51/265) was higher than that of the
qnrB
gene (1.89%, 5/265). Whole genome sequencing identified 37 distinct AMR genes and 15 plasmid replicons, and the most frequent mutation in quinolone resistance determining regions was
parC
(T57S; 91.49%, 43/47). Meanwhile, four different
qnrS
and two
qnrB
genetic environments were discovered among 47
qnr
-positive
Salmonella
strains. In total, 21.28% (10/47) of the strains were capable of conjugative transfer, and all were
qnrS1
-positive strains, with the majority of transferable plasmids being IncHI2 types (
n
= 4). Overall, the prevalence of
qnr
-positive
Salmonella
strains from chickens in China and their carriage of multiple resistance and virulence genes and transferable plasmids is a major concern, which calls for continuous surveillance of
qnr
-positive
Salmonella
and the development of measures to control its prevalence and transmission.

IMPORTANCE

Salmonella
is a common foodborne pathogen responsible for 155,000 deaths annually worldwide. Fluoroquinolones (FQs) are used as first-line drugs for the treatment of
Salmonella
infections in several countries and regions. However, the emergence and increasing prevalence of the FQ-resistant gene
qnr
in
Salmonella
isolated from chickens have been widely reported. Gaining insight into the genetic mechanisms of AMR genes in chicken could lead to the development of preventive measures to control and reduce the risk of drug resistance. In this study, we identified
qnr
-positive
Salmonellae
isolated from chickens in different regions of China and their AMR patterns and genome-wide characteristics, providing a theoretical basis for further control of their prevalence and transmission.
Validation of a real-time polymerase chain reaction for the detection and quantification of the nucleic acid of
Histoplasma
from equine clinical samples
Authors: Fisher Lewis W. S. , Ceesay Abdou , Jallow Demba , Hawkes Sophie F. , Showering Alicia , Kane Yaghouba , Doumbia Amadou , Stringer Andrew P. , Scantlebury Claire E. 
Published: February 2024

ABSTRACT

Histoplasma capsulatum
var
. farciminosum
(HCF) is a dimorphic fungus that causes epizootic lymphangitis in equids. Current diagnostic approaches, including culture, microscopy, and clinical presentation, lack speed, sensitivity, and specificity when diagnosing clinical cases. In this study, equine blood and pus samples on Whatman FTA cards from Senegal (
n
= 3), The Gambia (
n
= 19), Ethiopia (
n
= 16), and Mali (
n
= 13) were tested using a real-time PCR (qPCR) protocol. The assay was optimized and tested for its suitability to detect and quantify HCF in blood and pus loaded onto Whatman FTA cards at sampling. Whatman FTA cards were tested for their suitability for use with qPCR and were found to recover DNA more efficiently than from direct extraction. Using TaqMan fluorescent probes and specific primers, the assay demonstrated 100% analytical specificity when detecting multiple strains of
Histoplasma
and no false positives with off-target organisms. The assay’s diagnostic performance was measured against an existing nested internal transcribed spacer PCR protocol using a receiver operating characteristic curve. The test was found to have a diagnostic specificity and sensitivity of 100% and 71.4%, respectively, when analyzing pus samples using a cycle threshold (Ct) cutoff determined by Youden’s index (27.75). Blood sample cutoff Ct value was proposed at 34.55. Further optimization is required to improve the performance of the protocol when applied to blood samples. This study has, for the first time, demonstrated the ability to detect and quantify the DNA of
Histoplasma
spp. in equine blood and pus samples with a high degree of accuracy, providing a platform to further investigate the pathogenesis and epidemiology of this disease.

IMPORTANCE

Histoplasmosis is a neglected yet major cause of morbidity and mortality in both equids and people in resource-scarce settings. One of the major hindrances to the control of histoplasmosis is a lack of readily available diagnostic tests. Tests are needed to support clinical decision-making and to be applied in population-based research to further understand this disease
in situ
. This paper reports, for the first time, the validation and application of a qPCR to detect
Histoplasma
directly from equine clinical samples, bypassing the need to culture this notoriously difficult organism. We report and comment on the performance of the qPCR in comparison with our previously developed nested PCR.
In-depth characterization of multidrug-resistant NDM-1 and KPC-3 co-producing
Klebsiella pneumoniae
bloodstream isolates from Italian hospital patients
Authors: Posteraro Brunella , De Maio Flavio , Motro Yair , Menchinelli Giulia , De Lorenzis Desy , Marano Roberto B. M. , Aljanazreh Bessan , Errico Federica Maria , Massaria Giuseppe , Spanu Teresa , Posteraro Patrizia , Moran-Gilad Jacob , Sanguinetti Maurizio 
Published: February 2024

ABSTRACT

Bloodstream infection (BSI) caused by carbapenem-resistant
Klebsiella pneumoniae
(KP) poses significant challenges, particularly when the infecting isolate carries multiple antimicrobial resistance (AMR) genes/determinants. This study, employing short- and long-read whole-genome sequencing, characterizes six New Delhi metallo-β-lactamase (NDM) 1 and KP carbapenemase (KPC) 3 co-producing KP isolates, the largest cohort investigated in Europe to date. Five [sequence type (ST) 512] and one (ST11) isolates were recovered from patients who developed BSI from February to August 2022 or February 2023 at two different hospitals in Rome, Italy. Phylogenetic analysis revealed two distinct clusters among ST512 isolates and a separate cluster for the ST11 isolate. Beyond
bla
NDM-1
and
bla
KPC-3
, various AMR genes, indicative of a multidrug resistance phenotype, including colistin resistance, were found. Each cluster-representative ST512 isolate harbored a
bla
NDM-1
plasmid (IncC) and a
bla
KPC-3
plasmid [IncFIB(pQil)/IncFII(K)], while the ST11 isolate harbored a
bla
NDM-1
plasmid [IncFII(pKPX1)] and a
bla
KPC-3
plasmid [IncFIB(K)/IncFII(K)]. The
bla
NDM-1
plasmids carried genes conferring resistance to clinically relevant antimicrobial agents, and the aminoglycoside resistance gene
aac
(
6
′)-
Ib
was found on different plasmids. Colistin resistance-associated
mgrB
/
pmrB
gene mutations were present in all isolates, and the yersiniabactin-encoding
ybt
gene was unique to the ST11 isolate. In conclusion, our findings provide insights into the genomic context of
bla
NDM-1
/
bla
KPC-3
carbapenemase-producing KP isolates.

IMPORTANCE

This study underscores the critical role of genomic surveillance as a proactive measure to restrict the spread of carbapenemase-producing KP isolates, especially when key antimicrobial resistance genes, such as
bla
NDM-1
/
bla
KPC-3
, are plasmid borne. In-depth characterization of these isolates may help identify plasmid similarities contributing to their intra-hospital/inter-hospital adaptation and transmission. Despite the lack of data on patient movements, it is possible that carbapenem-resistant isolates were selected to co-produce KP carbapenemase and New Delhi metallo-β-lactamase via plasmid acquisition. Studies employing long-read whole-genome sequencing should be encouraged to address the emergence of KP clones with converging phenotypes of virulence and resistance to last-resort antimicrobial agents.
Bacteroides fragilis
aggravates high-fat diet-induced non-alcoholic fatty liver disease by regulating lipid metabolism and remodeling gut microbiota
Authors: Huang Yumei , Cao Jiali , Zhu Mengpei , Wang Ziwen , Jin Ze , Xiong Zhifan 
Published: February 2024

ABSTRACT

Gut microbiota dysbiosis is a prominent determinant that significantly contributes to the disruption of lipid metabolism. Consequently, it is essential to the occurrence and development of non-alcoholic fatty liver disease (NAFLD). Nevertheless, the connection between diet and symbiotic gut microbiota in the progression of NAFLD remains uncertain. The purpose of this study was to explore the role of supplementing commensal
Bacteroides fragilis
(
B. fragilis
) on lipid metabolism, gut microbiota, and metabolites in high-fat diet (HFD)-fed mice, elucidating the impact of gut microbiota and metabolites on the development of NAFLD. Our study revealed that supplementation with
B. fragilis
exacerbated both weight gain and obesity in mice.
B. fragilis
exacerbated blood glucose levels and liver dysfunction in mice. Furthermore, an increase in liver lipid accumulation and the upregulation of genes correlated with lipid metabolism were observed in mice. Under an HFD, supplementation of commensal
B. fragilis
resulted in alterations in the gut microbiota, notably a significant increase in
Desulfovibrionaceae
, which led to elevated endotoxin levels and thereby influenced the progression of NAFLD. It was interesting that the simultaneous examination of gut microbiota metabolites revealed a more pronounced impact of diet on short-chain fatty acids. This study represented the pioneering investigation into the impact of
B. fragilis
on NAFLD. Our findings demonstrated that
B. fragilis
induced dysregulation in the intestinal microbiota, leading to elevated levels of lipopolysaccharide and dysfunction in glucose and lipid metabolism, thereby exacerbating NAFLD.

IMPORTANCE

Some intestinal symbiotic microbes are involved in the occurrence of the metabolic disorders. Our study investigated the impact of supplementing commensal
Bacteroides fragilis
on host metabolism in high-fat diet-fed mice. Research results indicated that adding a specific bacterial strain to the complex intestinal microecology can worsen metabolic conditions. This effect mainly affects the structural diversity of intestinal microorganisms, the increase in harmful bacteria in the gut, and the elevation of endotoxin levels, blood glucose, and lipid metabolism, thereby impacting the progression of non-alcoholic fatty liver disease (NAFLD). Understanding the principles that govern the establishment of microbial communities comprising multiple species is crucial for preventing or repairing dysfunctions in these communities, thereby enhancing host health and facilitating disease treatment. This study demonstrated that gut microbiota dysbiosis could contribute to metabolic dysfunction and provides new insights into how to promote gut microbiota in the prevention and therapy of NAFLD.
Pelvic floor muscle training in female functional fitness exercisers: an assessor-blinded randomised controlled trial
Authors: Skaug Kristina Lindquist , Engh Marie Ellström , Bø Kari 
Published: February 2024

ObjectiveStress urinary incontinence (SUI) is common among females during functional fitness training, such as CrossFit. The aim of this study was to assess the effect of pelvic floor muscle training (PFMT) on SUI in female functional fitness exercisers.MethodsThis was an assessor-blinded randomised controlled trial with a PFMT group (n=22) and a control group (n=25). The PFMT group followed a 16-week home-training programme with 3 sets of 8–12 maximum pelvic floor muscle (PFM) contractions daily and weekly follow-up/reminders by phone. The primary outcome was change in a total score of the International Consultation on Incontinence Questionnaire-Urinary Incontinence Short Form (ICIQ-UI-SF). The secondary outcomes were perceived change of symptoms of SUI, change of PFM strength measured by vaginal manometry and symptoms of anal incontinence (AI) and pelvic organ prolapse (POP).Results47 women, mean age of 33.5 years (SD: 8.1), participated. At 16 weeks, there was a mean difference between groups of −1.4 (95% CI: −2.6 to −0.2) in the change of the ICIQ-UI-SF score in favour of the PFMT group. The PFMT group completed a mean of 70% (SD: 23) of the prescribed protocol. 64% in the PFMT group versus 8% in the control group reported improved symptoms of SUI (p<0.001, relative risk: 7.96, 95% CI, 2.03 to 31.19). There were no group differences in the change of PFM strength or AI/POP symptoms.ConclusionA 16-week home-training programme of the PFM led to improvements in SUI in female functional fitness exercisers. However, PFM strength and AI and POP symptoms did not improve significantly in the PFMT group compared with the control group.