bluuebunny/crossref_metadata_2025_split huggingface dataset:
Insights into the mechanism of substrate specificity in a novel PL15_3 subfamily oligo-alginate lyase VBAly15A
Authors: Tang Yongqi , Song Ziyan , Xu Xiaodong , Li Yingjie , Wang Lushan 
Published: February 2025

ABSTRACT

Alginate is a major component of brown algae cell walls and can be degraded via β-elimination by alginate lyases. These enzymes are classified into polysaccharide lyases and oligo-alginate lyases (Oals), with Oals mainly represented by the PL15 and PL17 families. Unlike PL17 Oals, which are widely present in alginate-degrading microorganisms, PL15 enzymes are only identified in a limited number of microorganisms, and their biochemical characteristics remain poorly understood. In this research, a novel PL15 alginate lyase, VBAly15A, from the marine bacterium,
Vibrio
sp. B1Z05, was identified and characterized. It belongs to a new PL15_3 subfamily and exhibits high activity toward polyM substrates. VBAly15A is thermostable in medium temperatures, tolerant to alkaline up to 11.0, and polyM-specific Oal, and it can first degrade alginate polymers into disaccharides and subsequently catalyze disaccharides into monomers via an exolytic mode. Site-directed mutagenesis showed that Arg
114
, Tyr
470
, and Arg
110
in the active groove are essential for the stable binding of the substrate. In addition, the amino acid His
226
in VBAly15A, previously suggested to act as a catalytic base, is not essential for catalysis, whereas Tyr
280
, previously proposed to act as a catalytic acid, is required for enzyme activity. Structural bioinformatic and biochemical analyses revealed that His
226
functions as a catalytic base, specifically abstracting protons from G-type substrates, while Tyr
280
acts as both a catalytic acid and a base. This catalytic mechanism is likely conserved in PL15 family alginate lyases.

IMPORTANCE
Alginate, as a renewable resource for sustainability, has great application prospects. In addition to polysaccharide lyases, Oals are critical for the full degradation of alginate, a key prerequisite for biorefinery. So far, most identified and well-characterized Oals belong to the PL17 family. However, the catalytic mechanism of PL15 Oals is limited, and even the catalytic base and acid are not fully elucidated. The significance of this study lies in discovering and characterizing a novel Oal VBAly15A that divides into a new PL15 subfamily, PL15_3. Not only are key amino acid residues involved in enzyme activity identified, but residues acting as the catalytic base and acid are also demonstrated. The distance of the catalytic residues His and Tyr to the C5 proton of the sugar ring determines the substrate specificity. Therefore, this work provides new insights into the mechanism of substrate specificity in alginate lyases.
Rho-dependent termination and RNase E-mediated cleavage: dual pathways for RNA 3′ end processing in polycistronic mRNA
Authors: Jeon Heung Jin , N Monford Paul Abishek , Wang Xun , Lim Heon M. 
Published: February 2025

ABSTRACT

“Pre-full-length” transcripts are produced at the end of the polycistronic galactose (
gal
) operon, 5′
galE-galT-galK-galM 3′
, via Rho-dependent transcription termination (RDT) and -independent transcription termination. The 3′ end of the full-length
galETKM
mRNA is acquired by exonucleolytic processing of the 3′-OH ends of the pre-full-length transcripts. However, the
gal
operon produces an mRNA termed
galE
whose 3′ end forms approximately 120 nucleotides downstream of the
galE
stop codon, within the subsequent gene,
galT
, thereby establishing polarity in gene expression. In this study, we investigated the molecular processes that generate the 3′ end of
galE
mRNA. We discovered that the 3′ ends of pre-galE mRNA are produced in the middle of
galT
as a result of the combination of two separate molecular processes—one previously reported as RDT and the other as unreported RNase E-mediated transcript cleavage. The 3′ ends of pre-
galE
mRNA undergo exonucleolytic processing to the 3′ end of
galE
mRNA observed
in vivo
. A hairpin structure containing an 8 bp stem and a 4-nucleotide loop, located 5–10 nucleotides upstream of the 3′ ends of
galE
mRNA, blocks exoribonuclease digestion and renders transcript stability. These findings demonstrate that RNase E—contrary to its general role in mRNA degradation—produces RNA 3′ ends that regulate polarity in gene expression.

IMPORTANCE

This study reports the findings of two molecular mechanisms that generate the 3′ ends of pre-
galE
mRNA in the
gal
operon, viz., Rho-dependent transcription termination and RNase E-mediated cleavage. These 3′ ends are subsequently processed to produce stable
galE
mRNA with a hairpin structure that prevents exoribonuclease degradation. This mechanism establishes gene expression polarity by generating the 3′ end of
galE
mRNA within
galT
in contrast to the usual mRNA degradation role of RNase E. The study reveals a unique role of RNase E in mRNA processing and stability.
Germination of
Bacillus
spores by LiCl
Authors: Wicander James , Gorsuch John , Chen Longjiao , Caldbeck Rebecca , Korza George , Brul Stanley , Christie Graham , Setlow Peter 
Published: February 2025

ABSTRACT

Spores of
Bacillus subtilis
have been found to germinate when incubated with LiCl, but not with other monovalent or divalent metal cations.
Bacillus megaterium
spores also germinated with LiCl, but
B. cereus
spores did not. In
B. subtilis
, the LiCl germination was via the activation of spores’ GerA germinant receptor (GR), and in
B. megaterium,
it was the GerU GR. Notably, LiCl germination was much slower than normal physiological germinant triggered GR germination. In
B. subtilis
spores, rates of LiCl germination were increased in spores with a more fluid IM and decreased in spores with a less fluid IM. Analyses of the GerA germinant binding site suggested that Li
+
could bind in a specific site in the
B. subtilis
GerAB subunit where normally a Na
+
likely binds. Importantly, NaCl strongly inhibited LiCl germination of
B. subtilis
spores, much more so than the larger cation in KCl, although neither salt inhibited L-alanine germination via the GerA GR. These findings increase the understanding of features of mechanisms of germination of
Bacillus
spores.

IMPORTANCE

The ability of some bacteria to form spores upon nutrient starvation confers properties of metabolic dormancy and enhanced resistance to environmental stressors that would otherwise kill vegetative cells. Since spore-forming bacteria include several notable pathogens and economically significant spoilage organisms, insight into how spores are stimulated to germinate and form new vegetative cells is important. Here, we reveal that relatively high concentrations of the inorganic salt lithium chloride trigger the germination of
Bacillus subtilis
and
Bacillus megaterium
spores by stimulating one of the spores of each species cohort of nutrient germinant receptors. This is significant since novel germinants and increased knowledge of the germination process should provide opportunities for improved control of spores in healthcare, food, and environmental sectors.
The African swine fever virus MGF360-16R protein functions as a mitochondrial-dependent apoptosis inducer by competing with BAX to bind to the HSP60 protein
Authors: Xiang Zhiyong , Xu Zhen , Weng Wenlian , Wang Hua , Wu Jiajun , Jiang Fei , Qu Yajin , Li Quanlin , Gao Peng , Zhou Lei , Ge Xinna , Guo Xin , Han Jun , Zhang Yongning , Yang Hanchun 
Published: February 2025

ABSTRACT

Induction of cell apoptosis is a critical pathogenic feature of the African swine fever virus (ASFV), a devastating threat to the world pig industry, but the underlying mechanisms have remained unclear. Here, we report the genome-wide screening and identification of 27 ASFV-encoded apoptosis inducers. Of them, the viral protein MGF360-16R was found to be a mitochondria-targeting protein and exhibits strong activity to induce mitochondrial-dependent apoptosis. Interestingly, this protein exhibits a dynamic distribution pattern during viral infection of porcine macrophages; it is mainly localized to the viral factory in the early stage and then shifts a portion to mitochondria in the late stage, a timing that coincides with virus-induced apoptosis. Mechanistically, MGF360-16R interacts with and competes for the cellular heat shock protein 60 (HSP60), and this results in the release of BAX from the HSP60-BAX complex, leading to the activation of BAX and induction of apoptosis. The ASFV mutant lacking MGF360-16R showed a reduced ability to induce cellular apoptosis, highlighting the complex nature of ASFV-induced cell apoptosis. Overall, our study adds a new function to MGF360-16R and reveals a novel mechanism of ASFV-mediated cell apoptosis.

IMPORTANCE
ASFV is a complex virus with a huge genome and numerous encoded proteins. Induction of cell apoptosis is associated with pathological damage in pigs caused by ASFV, but its inducers and underlying mechanisms remain unclear. Through genome-wide screening, the viral protein MGF360-16R was identified as a potent apoptosis inducer with a unique localization transfer from the viral factory to mitochondria during ASFV infection of porcine macrophages. MGF360-16R induces apoptosis by interacting with the cellular HSP60 protein to release BAX from the HSP60-BAX complex in a competitive binding manner. Our research findings not only reveal a novel function of MGF360-16R but also provide clues for understanding the pathogenesis of ASFV and potentially developing new therapeutic strategies.
Virus-like particle vaccine with authentic quaternary epitopes protects against Zika virus-induced viremia and testicular damage
Authors: Abbo Sandra R. , Yan Kexin , Geertsema Corinne , Hick Tessy A. H. , Altenburg Jort J. , Nowee Gwen , van Toor Chris , van Lent Jan W. , Nakayama Eri , Tang Bing , Metz Stefan W. , Bhowmik Ryan , de Silva Aravinda M. , Prow Natalie A. , Correia Ricardo , Alves Paula M. , Roldão António , Martens Dirk E. , van Oers Monique M. , Suhrbier Andreas , Pijlman Gorben P. 
Published: February 2025

ABSTRACT

Zika virus (ZIKV) caused unprecedented outbreaks in South America and the Caribbean in 2015–2016, leading primarily to a series of abnormalities in neonates termed congenital Zika syndrome. The threat of ZIKV reemergence has seen the development of multiple ZIKV vaccines that are at the preclinical stage or in early-stage clinical trials. Herein, we describe a pathway to the development of ZIKV vaccines generated using a baculovirus-insect cell expression system, which is widely applied for the manufacture of biologics for human use. Virus-like particle (VLP) vaccines comprising CprME and subviral particle (SVP) vaccines comprising prME were evaluated for their ability to mediate protection against ZIKV challenge in
Ifnar1
−/−
mice. Initial attempts resulted in VLP and SVP vaccines that failed to present quaternary epitopes and did not provide effective protection. To improve the SVP vaccine, two modifications were introduced: firstly, an alanine to cysteine substitution (A264C) in the E domain II region to promote the formation of stabilized E homodimers and, secondly, the use of
Spodoptera frugiperda
Sf9 insect cells that had been adapted to grow and produce vaccine at a neutral pH of 7. E homodimers largely retain their pre-fusion conformation at pH 7, which is a requirement for the induction of effective neutralizing antibody responses. The stabilized SVP-A26C vaccine induced high levels of neutralizing antibodies and protected male
Ifnar1
−/−
mice against viremia and testicular damage. Our study reiterates the need to present the immune system with E dimers arranged in authentic quaternary conformations and provides a scalable production method for this novel ZIKV vaccine.

IMPORTANCE

We describe the generation of a subviral particle (SVP) vaccine comprising prME proteins of ZIKV, with an envelope protein substitution, A264C, that stabilizes E dimer formation. The SVP vaccine was produced in a novel Sf9 insect cell line adapted to grow in suspension at pH 7. The study highlights the importance of challenge experiments to ascertain whether the responses induced by an experimental vaccine actually mediate protection against virus infection and disease. The study also reiterates the contention that effective flavivirus vaccines need to present the immunogen in an authentic tertiary and quaternary structure with a pre-fusion conformation.
Association of tumor microbiome with survival in resected early-stage PDAC
Authors: Meng Yixuan , Wang Chan , Usyk Mykhaylo , Kwak Soyoung , Peng Chengwei , Hu Kenneth S. , Oberstein Paul E. , Krogsgaard Michelle , Li Huilin , Hayes Richard B. , Ahn Jiyoung 
Published: February 2025

ABSTRACT

The pancreas tumor microbiota may influence tumor microenvironment and influence survival in early-stage pancreatic ductal adenocarcinoma (PDAC); however, current studies are limited and small. We investigated the relationship of tumor microbiota to survival in 201 surgically resected patients with localized PDAC (Stages I–II), from The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) cohorts. We characterized the tumor microbiome using RNA-sequencing data. We examined the association of the tumor microbiome with overall survival (OS), via meta-analysis with the Cox PH model. A microbial risk score (MRS) was calculated from the OS-associated microbiota. We further explored whether the OS-associated microbiota is related to host tumor immune infiltration. PDAC tumor microbiome α- and β-diversities were not associated with OS; however, 11 bacterial species, including species of
Gammaproteobacteria
, confirmed by extensive resampling, were significantly associated with OS (all
Q
< 0.05). The MRS summarizing these bacteria was related to a threefold change in OS (hazard ratio = 2.96 per standard deviation change in the MRS, 95% confidence interval = 2.26–3.86). This result was consistent across the two cohorts and in stratified analyses by adjuvant therapy (chemotherapy/radiation). Identified microbiota and the MRS also exhibited association with memory B cells and naïve CD4
+
T cells, which may be related to the immune landscape through BCR and TCR signaling pathways. Our study shows that a unique tumor microbiome structure, potentially affecting the tumor immune microenvironment, is associated with poorer survival in resected early-stage PDAC. These findings suggest that microbial mechanisms may be involved in PDAC survival, potentially informing PDAC prognosis and guiding personalized treatment strategies.

IMPORTANCE
Much of the available data on the PDAC tumor microbiome and survival are derived from relatively small and heterogeneous studies, including those involving patients with advanced stages of pancreatic cancer. There is a critical knowledge gap in terms of the tumor microbiome and survival in early-stage patients treated by surgical resection; we expect that advancements in survival may initially be best achieved in these patients who are treated with curative intent.
Comparative metagenome-associated analysis of gut microbiota and antibiotic resistance genes in acute gastrointestinal injury patients with the risk of in-hospital mortality
Authors: Bai Yunpeng , Hu Yali , Chen Xiangyin , Hu Linhui , Wu Kunyong , Liang Silin , Zheng Jinshui , Gänzle Michael G. , Chen Chunbo 
Published: February 2025

ABSTRACT

Acute gastrointestinal injury (AGI) is known for its poor long-term prognosis and the associated increase in mortality among intensive care unit (ICU) patients. As the role of the gut microbiome and resistome in AGI remains unclear, the present study aimed to explore the possible associations between dysbacteriosis and in-hospital mortality in ICU patients with gastrointestinal dysfunction. Fecal samples were collected from a prospective cohort of 210 ICU patients with AGI, and shotgun metagenomic sequencing was used to determine the taxonomic composition of gut microbiota and the differences of antibiotic resistance genes (ARGs) between the Death and Survival groups. Compared to the Survival group, patients in the Death group shifted from strict anaerobes to facultative anaerobes in the fecal microbial community, with more
Klebsiella
but less
Prevotella
. The co-occurrence patterns revealed that more ARG subtypes were enriched in microbial taxa in the Death group, especially for
Clostridium
and
Methanobrevibacter
. Furthermore, the ARG type had large area under the curve (AUCs) in receiver operating characteristic for predicting the disease severity, and a combined gut microbiota-ARG subtype classifiers showed better performance than either of them. Thus, comparative metagenome-associated analysis can help to obtain valuable information about gut microbiota and gene coding for antibiotic resistance in AGI patients.

IMPORTANCE

A metagenomic-related strategy was conducted to obtain a highly valuable resource to improve understanding of intestinal microbiota dysbiosis and antibiotic resistance genes (ARGs) profiles. The results indicate that intestinal microbiota, including
Klebsiella
and
Prevotella
, changed dramatically in intensive care unit (ICU) patients with acute gastrointestinal injury (AGI). Due to longer ICU stays and receiving more antibiotic treatment, the types and correlations of ARGs in the Death group were significantly higher than those in the Survival group. The findings of this study are expected to expand our knowledge of gut microbiota and resistome profiles reflecting gastrointestinal status, accelerate the identification of disease biomarkers, and provide new insights into the prevention and treatment of AGI-related diseases.
Exploring oral microbiome in oral squamous cell carcinoma across environment-associated sample types
Authors: Wang Zizheng , Chen Yilong , Li Haoning , Yue Yuan , Yu Haopeng 
Published: February 2025

ABSTRACT

The relationship between the oral microbiome and oral squamous cell carcinoma (OSCC) has been extensively investigated. Nonetheless, most previous studies were single-center, resulting in the absence of systematic evaluations. To address this gap, we performed a comprehensive meta-analysis on 1,255 samples from OSCC-related 16S rRNA gene data sets, representing a diverse range of OSCC phenotypes. It is recognized that the progression of cancer is related to the alterations in the microbiome among different phenotypes. Our findings revealed distinct microbiome characteristics among different sample types, with Biopsy (Bios) and Swab samples exhibiting significant differences between phenotypes. In Bios samples, the microbiomes of the Cancer group and the normal tissue adjacent to the tumor (NAT) group display a higher similarity, while both differ from the microbiome of the Fibroepithelial polyp (FEP) group. Moreover, the identified differential genera and pathways corresponded with these observations. We developed a diagnostic model using the random forest algorithm on Swab samples, achieving an area under the receiver operating characteristic curve (AUC) of 0.918. Importantly, this model exhibited considerable effectiveness (AUC = 0.849) when applied to another sequencing platform. Taken together, our study provides a comprehensive overview of the oral microbiome during various OSCC progression stages, potentially enhancing early detection and treatment.

IMPORTANCE
This study answers key questions regarding the universal microbial characteristics and comprehensive oral microbiome dynamics during oral squamous cell carcinoma (OSCC) progression. By integrating multiple data sets, we examine the following critical aspects: (1) Do different sample types harbor distinct microbial communities within the oral cavity? (2) Which sample types offer greater potential for investigating OSCC progression? (3) How are the oral microbiomes of the Cancer group, normal tissue adjacent to the tumor group, and Fibroepithelial polyp group related, and what is their potential association with OSCC development? (4) Can a diagnostic model based on microbial signatures effectively distinguish between Cancer and Health groups using Swab samples?
Novel application of ribonucleoprotein-mediated CRISPR-Cas9 gene editing in plant pathogenic oomycete species
Authors: Dort Erika N. , Feau Nicolas , Hamelin Richard C. 
Published: February 2025

ABSTRACT

CRISPR-Cas9 gene editing has become an important tool for the study of plant pathogens, allowing researchers to functionally characterize specific genes involved in phytopathogenicity, virulence, and fungicide resistance. Protocols for CRISPR-Cas9 gene editing have already been developed for Phytophthoras, an important group of oomycete plant pathogens; however, these efforts have exclusively focused on agricultural pathosystems, with research lacking for forest pathosystems. We sought to develop CRISPR-Cas9 gene editing in two forest pathogenic Phytophthoras,
Phytophthora cactorum
and
P. ramorum
, using a plasmid-ribonucleoprotein (RNP) co-transformation approach. Our gene target in both species was the ortholog of
PcORP1
, which encodes an oxysterol-binding protein that is the target of the fungicide oxathiapiprolin in the agricultural pathogen
P. capsici
. We delivered liposome complexes, each containing plasmid DNA and CRISPR-Cas9 RNPs, to
Phytophthora
protoplasts using a polyethylene glycol-mediated transformation protocol. We obtained two
ORP1
mutants in
P. cactorum
but were unable to obtain any mutants in
P. ramorum
. The two
P. cactorum
mutants exhibited decreased resistance to oxathiapiprolin, as measured by their radial growth relative to wild-type cultures on oxathiapiprolin-supplemented medium. Our results demonstrate the potential for RNP-mediated CRISPR-Cas9 gene editing in
P. cactorum
and provide a foundation for future optimization of our protocol in other forest pathogenic
Phytophthora
species.

IMPORTANCE

CRISPR-Cas9 gene editing has become a valuable tool for characterizing the genetics driving virulence and pathogenicity in plant pathogens. CRISPR-Cas9 protocols are now well-established in several
Phytophthora
species, an oomycete genus with significant economic and ecological impact globally. These protocols, however, have been developed for agricultural
Phytophthora
pathogens only; CRISPR-Cas9 systems have not yet been developed for any forest pathogenic Phytophthoras. In this study, we sought to establish CRISPR-Cas9 gene editing in two forest
Phytophthora
pathogens that cause widespread tree mortality:
P. cactorum
and
P. ramorum
. We successfully obtained gene mutations in
P. cactorum
and demonstrated a decrease in fungicide resistance, a trait that could impact the pathogen’s ability to cause disease. However, the same protocol did not yield any mutants in
P. ramorum
. The results of our study will serve as a baseline for the development of CRISPR-Cas9 gene editing in forest Phytophthoras and other oomycetes.