bluuebunny/crossref_metadata_2025_split huggingface dataset:
Flagellar interference with plasmid uptake in biofilms: a joint experimental and modeling study
Authors: Røder Henriette Lyng , Christidi Eleni , Amador Cristina I. , Music Samra , Olesen Asmus Kalckar , Svensson Birte , Madsen Jonas Stenløkke , Herschend Jakob , Kreft Jan-Ulrich , Burmølle Mette 
Published: December 2023

ABSTRACT

Plasmid conjugation is a key facilitator of horizontal gene transfer (HGT), and plasmids encoding antibiotic resistance drive the increasing prevalence of antibiotic resistance. In natural, engineered, and clinical environments, bacteria often grow in protective biofilms. Therefore, a better understanding of plasmid transfer in biofilms is needed. Our aim was to investigate plasmid transfer in a biofilm-adapted wrinkly colony mutant of
Xanthomonas retroflexus
(XRw) with enhanced matrix production and reduced motility. We found that XRw biofilms had an increased uptake of the broad host-range IncP-1ϵ plasmid pKJK5 compared to the wild type (WT). Proteomics revealed fewer flagellar-associated proteins in XRw, suggesting that flagella were responsible for reducing plasmid uptake. This was confirmed by the higher plasmid uptake of non-flagellated
fliM
mutants of the
X. retroflexus
wrinkly mutant as well as the wild type. Moreover, testing several flagellar mutants of
Pseudomonas putida
suggested that the flagellar effect was more general. We identified seven mechanisms with the potential to explain the flagellar effect and simulated them in an individual-based model. Two mechanisms could thus be eliminated (increased distances between cells and increased lag times due to flagella). Another mechanism identified as viable in the modeling was eliminated by further experiments. The possibility of steric hindrance of pilus movement and binding by flagella, reducing the frequency of contact and thus plasmid uptake, proved viable, and the three other viable mechanisms had a reduced probability of plasmid transfer in common. Our findings highlight the important yet complex effects of flagella during bacterial conjugation in biofilms.

IMPORTANCE

Biofilms are the dominant form of microbial life and bacteria living in biofilms are markedly different from their planktonic counterparts, yet the impact of the biofilm lifestyle on horizontal gene transfer (HGT) is still poorly understood. Horizontal gene transfer by conjugative plasmids is a major driver in bacterial evolution and adaptation, as exemplified by the troubling spread of antibiotic resistance. To either limit or promote plasmid prevalence and dissemination, we need a better understanding of plasmid transfer between bacterial cells, especially in biofilms. Here, we identified a new factor impacting the transfer of plasmids, flagella, which are required for many types of bacterial motility. We show that their absence or altered activity can lead to enhanced plasmid uptake in two bacterial species,
Xanthomonas retroflexus
and
Pseudomonas putida
. Moreover, we demonstrate the utility of mathematical modeling to eliminate hypothetical mechanisms.
Wastewater-based surveillance as a tool for public health action: SARS-CoV-2 and beyond
Authors: Parkins Michael D. , Lee Bonita E. , Acosta Nicole , Bautista Maria , Hubert Casey R. J. , Hrudey Steve E. , Frankowski Kevin , Pang Xiao-Li 
Published: December 2023

SUMMARY
Wastewater-based surveillance (WBS) has undergone dramatic advancement in the context of the coronavirus disease 2019 (COVID-19) pandemic. The power and potential of this platform technology were rapidly realized when it became evident that not only did WBS-measured SARS-CoV-2 RNA correlate strongly with COVID-19 clinical disease within monitored populations but also, in fact, it functioned as a leading indicator. Teams from across the globe rapidly innovated novel approaches by which wastewater could be collected from diverse sewersheds ranging from wastewater treatment plants (enabling community-level surveillance) to more granular locations including individual neighborhoods and high-risk buildings such as long-term care facilities (LTCF). Efficient processes enabled SARS-CoV-2 RNA extraction and concentration from the highly dilute wastewater matrix. Molecular and genomic tools to identify, quantify, and characterize SARS-CoV-2 and its various variants were adapted from clinical programs and applied to these mixed environmental systems. Novel data-sharing tools allowed this information to be mobilized and made immediately available to public health and government decision-makers and even the public, enabling evidence-informed decision-making based on local disease dynamics. WBS has since been recognized as a tool of transformative potential, providing near-real-time cost-effective, objective, comprehensive, and inclusive data on the changing prevalence of measured analytes across space and time in populations. However, as a consequence of rapid innovation from hundreds of teams simultaneously, tremendous heterogeneity currently exists in the SARS-CoV-2 WBS literature. This manuscript provides a state-of-the-art review of WBS as established with SARS-CoV-2 and details the current work underway expanding its scope to other infectious disease targets.
KSHV-encoded LANA bypasses transcriptional block through the stabilization of RNA Pol II in hypoxia
Authors: Bose Dipayan , Singh Rajnish Kumar , Robertson Erle S. 
Published: December 2023

ABSTRACT
Hypoxia results from an insufficient supply of oxygen, which results in physiological stress in biological systems. Cells respond to this unfavorable condition by reducing critical cellular functions, which include replication, transcription, and translation. Oncogenic Kaposi sarcoma-associated virus (KSHV) undergoes lytic reactivation in hypoxic conditions. Furthermore, reactivation requires the synthesis of specific viral proteins, both structural and non-structural. Therefore, the virus manipulates the cellular microenvironment to ensure efficient functioning of the cellular transcription machinery, including RNA polymerase II (Pol II), by protecting it from hypoxia-mediated degradation. In this study, we demonstrated that hypoxia induces degradation of RNA Pol II via ubiquitination mediated by the NEDD4 E3 ligase, which results in transcription inhibition. However, in KSHV-infected cells during hypoxia, the virus-encoded latency-associated nuclear antigen interacts with NEDD4, inhibiting its E3 ubiquitin ligase activity and requires complex formation with hypoxia inducible factor HIF1α. This activity inhibits the polyubiquitination of RNA Pol II to maintain steady transcription levels necessary for the synthesis of the essential viral genes needed for successful lytic reactivation.

IMPORTANCE
Hypoxia can induce the reactivation of Kaposi sarcoma-associated virus (KSHV), which necessitates the synthesis of critical structural proteins. Despite the unfavorable energetic conditions of hypoxia, KSHV utilizes mechanisms to prevent the degradation of essential cellular machinery required for successful reactivation. Our study provides new insights on strategies employed by KSHV-infected cells to maintain steady-state transcription by overcoming hypoxia-mediated metabolic stress to enable successful reactivation. Our discovery that the interaction of latency-associated nuclear antigen with HIF1α and NEDD4 inhibits its polyubiquitination activity, which blocks the degradation of RNA Pol II during hypoxia, is a significant contribution to our understanding of KSHV biology. This newfound knowledge provides new leads in the development of novel therapies for KSHV-associated diseases.
Microbiota-accessible fiber activates short-chain fatty acid and bile acid metabolism to improve intestinal mucus barrier in broiler chickens
Authors: Yang Jiantao , Qin Kailong , Sun Yanpeng , Yang Xiaojun 
Published: December 2023

ABSTRACT

Dietary polysaccharides are closely associated with gut microbiota and intestinal function homeostasis. This study aimed to investigate the mechanisms by which the β-glucan, arabinoxylan, and resistant starch selectively modulate gut microbiota and microbiota-derived metabolites to protect the intestinal mucus barrier in broiler chicken. In the present study, cecal microbiota samples from 21-day-old broilers were cultured
in vitro
with β-glucan, arabinoxylan, or resistant starch as the sole carbon source. We found that β-glucan, arabinoxylan and resistant starch alter community structure and selectively target the enrichment of
Bacteroides
,
Lactobacillus, Coprococcus
,
Butyricicoccus
,
Ruminococcus
and
Blautia
, respectively. Notably, supplementing fiber-deficient diets with arabinoxylan and resistant starch, but not with β-glucan, improved the intestinal mucus barrier by cecal microbiota enrichment and increase in the concentration of microbiota-derived short-chain fatty acids (SCFAs). In addition, we illustrated through bacterial cultures
in vitro
that the supplementation of arabinoxylan and resistant starch resulted in a change in bacterial biotransformation of secondary bile acids. Our findings provide insight into how arabinoxylan and resistant starch may selectively target gut microbiota and mediate the production of SCFAs and bile acid biotransformation to improve intestinal mucus barrier function.

IMPORTANCE
The intestinal mucus barrier, located at the interface of the intestinal epithelium and the microbiota, is the first line of defense against pathogenic microorganisms and environmental antigens. Dietary polysaccharides, which act as microbiota-accessible fiber, play a key role in the regulation of intestinal microbial communities. However, the mechanism via which dietary fiber affects the intestinal mucus barrier through targeted regulation of the gut microbiota is not clear. This study provides fundamental evidence for the benefits of dietary fiber supplementation in broiler chickens through improvement in the intestinal mucus barrier by targeted regulation of the gut ecosystem. Our findings suggest that the microbiota-accessible fiber-gut microbiota-short-chain fatty acid/bile acid axis plays a key role in regulating intestinal function.
Hierarchic regulation of a metabolic pathway: H-NS, CRP, and SsrB control
myo
-inositol utilization by
Salmonella enterica
Authors: Felsl Angela , Brokatzky Dominik , Kröger Carsten , Heermann Ralf , Fuchs Thilo M. 
Published: December 2023

ABSTRACT

The metabolic island GEI4417/4436 of
Salmonella enterica
serovar Typhimurium (
S
. Typhimurium) enables the degradation of
myo
-inositol (MI) as the sole carbon and energy source. The island encodes two regulatory factors, the autoregulated repressor IolR that inhibits the transcription of most
iol
genes in the absence of MI, and the activator ReiD that induces the expression of an
iol
gene operon that is not controlled by IolR. In this study, we investigated the putative role of global regulators in the control of MI utilization by
S
. Typhimurium. The histone-like nucleoid structuring protein H-NS is demonstrated here to interact with 16 regions of GEI4417/4436 and to silence the transcriptional activity of the promoters P

reiD

and P

iolE

, thus controlling the expression of genes initial for MI degradation. The cAMP-binding regulatory protein (CRP) is shown to bind numerous promoters in GEI4417/4436 and is required for the growth of
S
. Typhimurium strain 14028 in a minimal medium with MI. The binding kinetics of H-NS and CRP toward promoters of GEI4417/4436 were quantified by surface resonance spectroscopy, showing that H-NS weakly binds to promoters of initial genes and that CRP has a high affinity to promoters of all genes essential for MI utilization. Furthermore, three promoters of the metabolic island were identified here to belong to the virulon of the two-component system SsrA/SsrB of salmonellae as demonstrated by electrophoretic mobility shift assays and the use of chromosomal luciferase reporter fusions.

IMPORTANCE

The capacity to utilize myo-inositol (MI) as sole carbon and energy source is widespread among bacteria, among them the intestinal pathogen
S
. Typhimurium. This study elucidates the complex and hierarchical regulation that underlies the utilization of MI by
S
. Typhimurium under substrate limitation. A total of seven regulatory factors have been identified so far, allowing the pathogen an environment-dependent, efficient, and fine-tuned regulation of a metabolic property that provides growth advantages in different environments.
Analysis of antiviral drug properties of thymidine kinase of herpes B virus using recombinant herpes simplex virus 1
Authors: Nguyen Phu Hoang Anh , Fukushi Shuetsu , Yamada Souichi , Harada Shizuko , Yoshikawa Tomoki , Kinoshita Hitomi , Kawahara Madoka , Ogawa Takuma , Ebihara Hideki , Moi Meng Ling , Saijo Masayuki 
Published: December 2023

ABSTRACT
Zoonotic infection by herpes B virus (BV) causes severe neurological disease, which in the absence of appropriate treatment can have a mortality rate of 80%. Long-term administration of acyclovir or ganciclovir is the recommended prophylactic treatment option for BV infections. However, the emergence of acyclovir- and/or ganciclovir-resistant BV may become a major problem. Here, we examined the ability of BV thymidine kinase (TK) from monkeys (mBVTK) and humans (hBVTK) to acquire resistance to acyclovir and ganciclovir. The sequences of mBVTK and hBVTK differ by two amino acids. We constructed and expressed four HSV-1-based recombinant viruses (HSV-1_mBVTK, HSV-1_hBVTK, HSV-1_VZVTK, and HSV_HSV-1TK) in which the HSV-1TK gene was deleted and the TK gene of monkey BV, human BV, varicella-zoster virus, and HSV-1 was inserted, respectively. We then tested the sensitivity of these recombinant viruses to acyclovir, ganciclovir, penciclovir, and brivudine. HSV-1_HSV-1TK and wild-type HSV-1 were more susceptible to ganciclovir, acyclovir, and penciclovir than HSV-1_VZVTK, HSV-1_hBVTK, and HSV-1_mBVTK. Both HSV-1_hBVTK and HSV-1_mBVTK exhibited similar sensitivity profiles against all tested drugs: both were susceptible to acyclovir, ganciclovir, and penciclovir but resistant to brivudine. In addition, we forced HSV-1_hBVTK to replicate under selective pressure from acyclovir or ganciclovir and then examined the ability of the BV TK gene to develop drug resistance. Investigation of the drug-resistant HSV-1_hBVTK gene revealed the absence of amino acid substitutions in BV TK, suggesting that the development of acyclovir- or ganciclovir-resistant BV through mutations in the TK gene is a rare occurrence in this experimental system.

IMPORTANCE
Zoonotic infection of humans with herpes B virus (BV) causes severe neurological diseases. Acyclovir (ACV) and ganciclovir (GCV), most frequently used as anti-herpes drugs, are recommended for prophylaxis and therapy in human BV infection. In this study, we examined the property of BV thymidine kinase (TK) against anti-herpes drugs using a recombinant herpes simplex virus type 1 (HSV-1) carrying BV TK gene. We found that HSV-1 carrying BV TK was similarly sensitive to GCV as HSV-1 carrying varicella zoster virus TK. In addition, we demonstrated that BV TK was not mutated in the GCV- and ACV-resistant HSV-1 carrying BV TK, suggesting that ACV- or GCV-resistant BV might be rare during treatment with these antiviral drugs. These data can provide a new insight into the properties of BV TK in terms of the development of drug resistance.
Clinical impact of metagenomic next-generation sequencing of peripheral blood for the diagnosis of invasive mucormycosis: a single-center retrospective study
Authors: Wang Wei , Yao Yake , Li Xi , Zhang Shanshan , Zeng Zhu , Zhou Hua , Yang Qing 
Published: December 2023

ABSTRACT
This study aims to assess the usefulness of peripheral blood metagenomic next-generation sequencing (mNGS) in diagnosing invasive mucormycosis (IM). We conducted a retrospective analysis of clinical data from 73 patients with mucorales sequences detected by peripheral blood mNGS at the First Affiliated Hospital, Zhejiang University School of Medicine, between February 2021 and December 2022. We examined data on risk factors, clinical symptoms, imaging characteristics, pathogenic findings, clinical diagnoses, and treatment outcomes. Among the 73 patients with mucorales sequences detected by peripheral blood mNGS, 7 cases were confirmed, 12 were probable, 34 were possible, and 20 were false positives, based on the diagnostic criteria for IM. Among the 34 possible cases, patients exhibited risk factors, clinical symptoms, imaging features, and prognoses consistent with IM. In 7 out of the 12 probable cases, the same mucorale nucleic acid was found in peripheral blood mNGS samples collected at an average interval of 8.1 days. In the remaining 20 patients, mucorales nucleic acid was detected in peripheral blood mNGS, but they lacked risk factors or clinical features of mucorales infection and lacked other pathological evidence, leading to a judgment of false positive. The positive predictive value of peripheral blood mNGS for diagnosing invasive mucormycosis in the cases studied was 72.6%. Peripheral blood mNGS testing provides valuable insights for diagnosing invasive mucormycosis. However, it should be used in conjunction with risk factors, clinical features, and other pathogenic test results to make a comprehensive assessment.

IMPORTANCE
Given the high fatality rates, prompt and accurate identification of the fungal culprit is crucial, emphasizing the need for invasive mucormycosis. Unfortunately, mucormycosis lacks definitive biomarkers, depending primarily on smears, cultures, or pathology, all necessitating invasive specimen collection from the infection site. However, obtaining valid specimens early in critically ill patients poses substantial risks and challenges. Whether peripheral blood metagenomic next-generation sequencing (mNGS) can enhance early mucormycosis diagnosis, especially when direct specimen collection from the infection site is challenging, is warranted. This is a large-scale clinical study conducted to evaluate the utility and clinical impact of mNGS of peripheral blood for the diagnosis of invasive mucormycosis. We believe our study provided both novelty in translational medicine and a great value for the medical community to understand the strengths and limitations of mNGS of peripheral blood as a new diagnostic tool for the diagnosis and management of invasive mucormycosis.
Spermine enhances the activity of anti-tuberculosis drugs
Authors: Sao Emani Carine , Reiling Norbert 
Published: December 2023

ABSTRACT

The discovery of molecules that could shorten the treatment period of tuberculosis (TB) holds the promise of improving the current regimen. Polyamines are organic polycationic alkylamines found in millimolar concentrations in all living cells. Spermine (Spm) is a polyamine that was shown (more than 70 years ago) to be toxic to
Mycobacterium tuberculosis
(M.tb). In this study, we used various biochemical assays to assess the mechanism of action of Spm. Furthermore, using various drug susceptibility testing methods, we investigated the ability of Spm to enhance the activity of first-line TB drugs, isoniazid (INH) and rifampicin (RIF), as well as second-line TB drugs, para-aminosalicylic acid (PAS) and bedaquiline (BDQ). Our findings revealed that at toxic concentrations, Spm induced the formation of reactive oxygen species (ROS), a reaction that was influenced by the pH and the iron concentration of the media. Furthermore, the generation of ROS by Spm was associated with increased ferric iron and NADP levels. Moreover, the ROS produced by Spm synergized with the ROS generated by the lipophilic peroxide cumene hydroperoxide (CuOOH), as well as with the ROS generated by RIF and PAS, while enhancing their antimycobacterial activity. Additionally, we found that Spm enhanced the combined activity of INH and RIF against M.tb. Lastly, Spm was also able to enhance the activity of BDQ due to another mechanism of action. This study provides data and findings that would fuel future studies on the suitability of Spm as a potential adjunct TB therapy. The enhanced antimycobacterial activity of TB drugs in combination with Spm could offer promising new avenues for improving TB treatment outcomes.

IMPORTANCE

This is the first study that attempted to demonstrate the mechanisms of reactive oxygen species (ROS) generation by spermine (Spm) in
Mycobacterium tuberculosis
(M.tb). Furthermore, this is the first study to demonstrate that it is able to enhance the activity of currently available and World Health Organization (WHO)-approved tuberculosis (TB) drugs. Spermine can easily be obtained since it is already found in our diet. Moreover, as opposed to conventional antibiotics, it is less toxic to humans since it is found in millimolar concentrations in the body. Finally, with the difficulty of curing TB with conventional antibiotics, this study suggests that less toxic molecules, such as Spm, could in a long-term perspective be incorporated in a TB regimen to boost the treatment.
Effects of variation in sample storage conditions and swab order on 16S vaginal microbiome analyses
Authors: Kumar Tanya , Bryant MacKenzie , Cantrell Kalen , Song Se Jin , McDonald Daniel , Tubb Helena M. , Farmer Sawyer , Lewis Amanda , Lukacz Emily S. , Brubaker Linda , Knight Rob 
Published: December 2023

ABSTRACT
Technical bias is a pressing issue in microbiome research, and variability can be introduced at any stage from sample collection to figure generation. In this study, we aim to reduce biases in studying the human vaginal microbiome by examining the impact of sample storage buffer and multiple swabbing events using 16S rRNA gene amplicon sequencing data generated from vaginal swabs. We show that AssayAssure Genelock, a clinically relevant preservative for urine samples, is effective in preserving vaginal samples for microbiome studies. When comparing Genelock to 95% (vol/vol) ethanol and no preservative (air only), host variability explained more variance in both weighted and unweighted UniFrac measurements than the preservation method. We further examined the impact of three successive self-swabbing events, as the relatively low biomass nature of vaginal samples can inherently introduce bias. It is important to know if taking multiple swabs can provide replicable results and thus allow for additional technical replicates and an increased sample size. We found that up to three swabbing events do not introduce bias when examining the presence or absence of taxa but can explain 3% of the variability in the amount of taxa calculated. A study with more participants is warranted to provide further validation of these findings, but in producing this pilot study, we aim to continue laying the groundwork so that universally standardized and accessible studies can be created.

IMPORTANCE
The composition of the human vaginal microbiome has been linked to a variety of medical conditions including yeast infection, bacterial vaginosis, and sexually transmitted infection. The vaginal microbiome is becoming increasingly acknowledged as a key factor in personal health, and it is essential to establish methods to collect and process accurate samples with self-collection techniques to allow large, population-based studies. In this study, we investigate if using AssayAssure Genelock, a nucleic acid preservative, introduces microbial biases in self-collected vaginal samples. To our knowledge, we also contribute some of the first evidence regarding the impacts of multiple swabs taken at one time point. Vaginal samples have relatively low biomass, so the ability to collect multiple swabs from a unique participant at a single time would greatly improve the replicability and data available for future studies. This will hopefully lay the groundwork to gain a more complete and accurate understanding of the vaginal microbiome.