bluuebunny/crossref_metadata_2025_split huggingface dataset:
Signatures of Mollicutes-related endobacteria in publicly available Mucoromycota genomes
Authors: Longley Reid , Robinson Aaron J. , Asher Olivia A. , Middlebrook Earl , Bonito Gregory , Chain Patrick S. G. 
Published: August 2024

ABSTRACT

Mucoromycota fungi and their Mollicutes-related endobacteria (MRE) are an ideal system for studying bacterial–fungal interactions and evolution due to the long-term and intimate nature of their interactions. However, methods for detecting MRE face specific challenges due to the poor representation of MRE in sequencing databases coupled with the high sequence divergence of their genomes, making traditional similarity searches unreliable. This has precluded estimations on the diversity of MRE associated with Mucoromycota. To determine the prevalence of previously undetected MRE in fungal genome sequences, we scanned 389 Mucoromycota genome assemblies available from the National Center for Biotechnology Information for the presence of MRE sequences using publicly available tools to map contigs from fungal assemblies to publicly available MRE genomes. We demonstrate a higher diversity of MRE genomes than previously described in Mucoromycota and a lack of cophylogeny between MRE and the majority of their fungal hosts. This supports the late invasion hypothesis regarding MRE acquisition across most of the examined fungal families. In contrast with other Mucoromycota lineages, MRE from the Gigasporaceae displayed some degree of cophylogeny with their hosts, which may indicate that horizontal transmission is restricted between members of this family or that transmission is strictly vertical. These results underscore the need for a refined process to capture sequencing data from potential fungal endosymbionts to discern their evolution and transmission. Screens of fungal genomes for MRE can help improve the quality of fungal genome assemblies while identifying new MRE lineages to further test hypotheses on their origin and evolution.

IMPORTANCE
Mollicutes-related endobacteria (MRE) are obligate intracellular bacteria found within Mucoromycota fungi. Despite their frequent detection, MRE roles in host functioning are still unknown. Comparative genomic investigations can improve our understanding of the impact of MRE on their fungal hosts by identifying similarities and differences in MRE genome evolution. However, MRE genomes have only been assembled from a small fraction of Mucoromycota hosts. Here, we demonstrate that MRE can be present yet undetected in publicly available Mucoromycota genome assemblies. We use these newfound sequences to assess the broader diversity of MRE and their phylogenetic relationships with respect to their hosts. We demonstrate that publicly available tools can be used to extract novel MRE sequences from assembled fungal genomes leading to insights on MRE evolution. This work contributes to a greater understanding of the fungal microbiome, which is crucial to improving knowledge on the dynamics and impacts of fungi in microbial ecosystems.
Enzymatically enhanced ultrastructure expansion microscopy unlocks expansion of
in vitro Toxoplasma gondii
cysts
Authors: Bondarenko Kseniia , Limoge Floriane , Pedram Kayvon , Gissot Mathieu , Young Joanna C. 
Published: August 2024

ABSTRACT

Expansion microscopy (ExM) is an innovative approach to achieve super-resolution images without using super-resolution microscopes, based on the physical expansion of the sample. The advent of ExM has unlocked the detail of super-resolution images for a broader scientific circle, lowering the cost and entry skill requirements for the field. One of its branches, ultrastructure expansion microscopy (U-ExM), has become popular among research groups studying apicomplexan parasites, including the acute stage of
Toxoplasma gondii
infection. Here, we show that the chronic cyst-forming stage of
Toxoplasma
, however, resists U-ExM expansion, impeding precise protein localization. We then solve the
in vitro
cyst’s resistance to denaturation required for successful U-ExM. As the cyst’s main structural protein CST1 contains a mucin domain, we added an enzymatic digestion step using the pan-mucinase StcE prior to the expansion protocol. This allowed full expansion of the cysts in fibroblasts and primary neuronal cell culture without disrupting immunofluorescence analysis of parasite proteins. Using StcE-enhanced U-ExM, we clarified the localization of the GRA2 protein, which is important for establishing a normal cyst, observing GRA2 granules spanning across the CST1 cyst wall. The StcE–U-ExM protocol allows accurate pinpointing of proteins in the bradyzoite cyst, which will greatly facilitate investigation of the underlying biology of cyst formation and its vulnerabilities.

IMPORTANCE

Toxoplasma gondii
is an intracellular parasite capable of establishing long-term chronic infection in nearly all warm-blooded animals. During the chronic stage, parasites encapsulate to form cysts predominantly in neurons and skeletal muscle. Current anti-
Toxoplasma
drugs do not eradicate chronic parasites, leaving a reservoir of infection. The cyst is critical for disease transmission and pathology, yet it is harder to study, with the function of many chronic-stage proteins still unknown. Ultrastructure expansion microscopy, a new method to overcome the light microscopy’s diffraction limit by physically expanding the sample, allowed in-depth studies of acute
Toxoplasma
infection. We show that
Toxoplasma
cysts resist expansion using standard protocol, but an additional enzymatic digestion with the mucinase StcE allows full expansion. This protocol offers new avenues for examining the chronic stage, including precise spatial organization of cyst-specific proteins, linking these locations to morphological structures, and detailed investigations of components of the durable cyst wall.
High prevalence of the recently identified clonal lineage ST1299/CT3109
vanA
among vancomycin-resistant
Enterococcus faecium
strains isolated from municipal wastewater
Authors: Valenza Giuseppe , Eisenberger David , Esse Jan , Held Jürgen , Lehner-Reindl Verena , Plaumann Peter-Louis , Ziegler Tobias , Knauer Max , Bogdan Christian , Dudler Patrick 
Published: August 2024

ABSTRACT

Previously, we demonstrated that the majority of vancomycin-resistant
Enterococcus faecium
(VREfm) strains from in-patients of the University Hospital Erlangen, Germany, belonged to only three clonal lineages, namely ST117/CT71
vanB
and two novel ST1299
vanA
lineages classified as CT3109 and CT1903. The goal of the current study was (i) to investigate whether VREfm is also detectable in wastewater of the city of Erlangen, (ii) to identify their molecular features, and (iii) to clarify whether VREfm could arise from the community of the city of Erlangen or can be (directly) connected to nosocomial infections in the hospital setting. From April to May 2023, a total of 244 VREfm strains from raw wastewater of the city of Erlangen were analyzed by core genome multilocus sequence typing (cgMLST). Moreover, 20 of them were further investigated for single nucleotide polymorphisms (SNPs). The molecular characterization of the wastewater VREfm strains revealed a high prevalence (27.9%) of the recently identified clonal lineage ST1299/CT3109
vanA,
which is mainly characterized by the presence of the tetracycline-resistance determinant
tet(M
) and the virulence genes
pilA
and
prpA
. The SNPs analysis revealed the presence of two major clusters, namely cluster I (≤65 SNPs), which included well-known hospital-adapted
vanB
clonal lineages such as ST117/CT71 and ST80/CT1065 and cluster II (≤70 SNPs), which were mainly characterized by the lineage ST1299/CT3109
vanA
. Based on the concomitant resistance to vancomycin and tetracycline, we propose that ST1299/CT3109
vanA
primarily originated and spread outside of hospital settings.

IMPORTANCE

This study provides a detailed genomic analysis of vancomycin-resistant
Enterococcus faecium
(VREfm) strains isolated from municipal wastewater with a particular focus on clonal lineages, antimicrobial resistance, and the presence of virulence genes. The high wastewater prevalence of the recently identified clonal lineage ST1299/CT3109
vanA
, which has been previously detected in hospitals, suggests an enormous potential for future spread in Germany.
Rapid and sensitive determination of residual prion infectivity from prion-decontaminated surfaces
Authors: Simmons Sara M. , Payne Vivianne L. , Hrdlicka Jay G. , Taylor Jack , Larsen Peter A. , Wolf Tiffany M. , Schwabenlander Marc D. , Yuan Qi , Bartz Jason C. 
Published: August 2024

ABSTRACT

Prion diseases are untreatable fatal transmissible neurodegenerative diseases that affect a wide range of mammals, including humans, and are caused by PrP
Sc
, the infectious self-templating conformation of the host-encoded protein, PrP
C
. Prion diseases can be transmitted via surfaces (e.g., forceps, EEG electrodes) in laboratory and clinical settings. Here, we use a combination of surface swabbing and real-time quaking-induced conversion (RT-QuIC) to test for residual surface-associated prions following prion disinfection. We found that treatment of several prion-contaminated laboratory and clinically relevant surfaces with either water or 70% EtOH resulted in robust detection of surface-associated prions. In contrast, treatment of surfaces with sodium hypochlorite resulted in a failure to detect surface-associated prions. RT-QuIC analysis of prion-contaminated stainless steel wires paralleled the findings of the surface swab studies. Importantly, animal bioassay and RT-QuIC analysis of the same swab extracts are in agreement. We report on conditions that may interfere with the assay that need to be taken into consideration before using this technique. Overall, this method can be used to survey laboratory and clinical surfaces for prion infectivity following prion decontamination protocols.

IMPORTANCE
Prion diseases can be accidentally transmitted in clinical and occupational settings. While effective means of prion decontamination exist, methods for determining the effectiveness are only beginning to be described. Here, we analyze surface swab extracts using real-time quaking-induced conversion (RT-QuIC) to test for residual prions following prion disinfection of relevant clinical and laboratory surfaces. We found that this method can rapidly determine the efficacy of surface prion decontamination. Importantly, examination of surface extracts with RT-QuIC and animal bioassay produced similar findings, suggesting that this method can accurately assess the reduction in prion titer. We identified surface contaminants that interfere with the assay, which may be found in clinical and laboratory settings. Overall, this method can enhance clinical and laboratory prion safety measures.
Diverse non-canonical electron bifurcating [FeFe]-hydrogenases of separate evolutionary origins in
Hydrogenedentota
Authors: Zheng Xiaowei , Huang Li 
Published: August 2024

ABSTRACT

Hydrogenedentota
, a globally distributed bacterial phylum-level lineage, is poorly understood. Here, we established a comprehensive genomic catalog of
Hydrogenedentota
, including a total of seven clades (or families) with 179 genomes, and explored the metabolic potential and evolutionary history of these organisms. We show that a single genome, especially those belonging to Clade 6, often encodes multiple hydrogenases with genomes in Clade 2, which rarely encode hydrogenases being the exception. Notably, most members of
Hydrogenedentota
contain a group A3 [FeFe]-hydrogenase (BfuABC) with a non-canonical electron bifurcation mechanism, in addition to substrate-level phosphorylation and electron transport-linked phosphorylation pathways, in energy conservation. Furthermore, we show that BfuABC from
Hydrogenedentota
fall into five sub-types. Phylogenetic analysis reveals five independent routes for the evolution of BfuABC homologs in
Hydrogenedentota
. We speculate that the five sub-types of BfuABC might be acquired from
Bacillota
(synonym
Firmicutes
) through separate horizontal gene transfer events. These data shed light on the diversity and evolution of bifurcating [FeFe]-hydrogenases and provide insight into the strategy of
Hydrogenedentota
to adapt to survival in various habitats.

IMPORTANCE

The phylum
Hydrogenedentota
is widely distributed in various environments. However, their physiology, ecology, and evolutionary history remain unknown, primarily due to the limited availability of the genomes and the lack of cultured representatives of the phylum. Our results have increased the knowledge of the genetic and metabolic diversity of these organisms and shed light on their diverse energy conservation strategies, especially those involving electron bifurcation with a non-canonical mechanism, which are likely responsible for their wide distribution. Besides, the organization and phylogenetic relationships of gene clusters coding for BfuABC in
Hydrogenedentota
provide valuable clues to the evolutionary history of group A3 electron bifurcating [FeFe]-hydrogenases.
Circulating Estrogen Metabolites and Risk of Breast Cancer among Postmenopausal Women in the Nurses’ Health Study
Authors: Brantley Kristen D. , Ziegler Regina G. , Craft Neal E. , Hankinson Susan E. , Eliassen A. Heather 
Published: August 2024

Abstract

Background:
Estradiol and estrone are well-established risk factors for postmenopausal breast cancer. Experimental evidence suggests that specific estrogen metabolites, produced via irreversible hydroxylation of estrone and estradiol at position 2 or 16, may independently influence carcinogenesis.

Methods:
We performed a nested case–control study of breast cancer (328 breast cancer cases; 639 controls) among postmenopausal women within the Nurses’ Health Study to examine the role of estrogens and estrogen metabolites (jointly referred to as EM). Plasma concentrations of each EM (unconjugated + conjugated forms) were measured by LC-MS/MS. Multivariable conditional logistic regression, adjusting for breast cancer risk factors, estimated relative risks (RR) and 95% confidence intervals of breast cancer across quintiles of individual EM, EM pathways, and pathway ratios. Associations by estrogen receptor (ER) and progesterone receptor (PR) status were analyzed by unconditional logistic regression.

Results:
Estradiol and estrone were strongly associated with increased breast cancer risk [estradiol: RRQ5 vs. Q1 (95% confidence interval) = 2.64 (1.64–4.26); estrone: 2.78 (1.74–4.45); both P-trends &lt;0.001]. The 2-hydroxylation pathway was strongly associated with risk [RRQ5 vs. Q1 = 3.09 (1.81–5.27); P-trend &lt;0.001] and remained so after adjusting for unconjugated estradiol [RRQ5 vs. Q1 = 2.23 (1.25–3.96); P-trend = 0.01]. Although the 16-hydroxylation pathway was modestly associated with risk [RRQ5 vs. Q1 = 1.62 (1.03–2.54); P-trend = 0.01], the association was attenuated after unconjugated estradiol adjustment [RRQ5 vs. Q1 = 1.24 (0.77–1.99); P-trend = 0.19]. Similar positive associations with the 2-pathway and 16-pathway were observed for ER+/PR+ and ER−/PR− tumors.

Conclusion:
In this cohort of postmenopausal women, 2-hydroxylation of estrone and estradiol was associated with increased breast cancer risk, independent of unconjugated estradiol.

Impact:
These results highlight the need to revisit the role of estrogen metabolism in breast cancer etiology and prevention.
Phase II Clinical Chemoprevention Trial of Weekly Erlotinib before Bladder Cancer Surgery
Authors: Downs Tracy M. , Bailey Howard H. , Lozar Taja , Schmitz Natalie S. , Green Heather , Scarlett Cameron O. , Havighurst Thomas C. , Twaroski Kyleigh , DeShong Katina , Wollmer Barbara , Bivalacqua Trinity J. , Saltzstein Daniel R. , Shore Neal , Kim KyungMann , Huang Wei , Ricke William A. , Barroilhet Lisa , House Margaret , Parnes Howard L. , Messing Edward 
Published: August 2024

Abstract
We performed a clinical trial in patients with non–muscle-invasive (NMI) urothelial cancer randomized (2:1) to the EGFR tyrosine kinase inhibitor erlotinib or placebo (either orally once weekly × 3 doses prior to scheduled surgery) to assess for a difference in EGFR phosphorylation in tumor-adjacent normal urothelium &lt;24 hours post–study dose and tolerance of weekly erlotinib therapy. Thirty-seven volunteers (6 female/31 male; mean age 70; 35 White/2 non-White) with confirmed or suspected NMI urothelial cancer were enrolled into either erlotinib (n = 24; 900 mg-13, 600 mg-11) or placebo (n = 13). IHC assessment of phosphorylated and total EGFR in tumor-adjacent normal urothelium (20 erlotinib and 9 placebo subjects) or tumor (21 erlotinib and 11 placebo subjects) at study end showed no significant difference between those receiving erlotinib or placebo. This was also true for other assessed tissue biomarkers (phosphorylated ERK, ERK, E-cadherin, p53, and Ki67). Adverse events were more common, in a dose-related fashion, in participants receiving erlotinib, e.g., 38% experienced grade 1 with rare grade 2 diarrhea and skin toxicity versus 8% in placebo. Clinically insignificant but statistically significant (P = 0.001) elevations in serum total bilirubin and creatinine were observed in participants receiving erlotinib. Serum erlotinib and metabolite concentrations (OSI-420) confirmed compliance in all subjects receiving erlotinib and did not significantly differ between the 600 and 900 mg doses. Despite compelling preclinical and clinical data for targeted EGFR inhibition in bladder cancer prevention, these data do not support the use of weekly erlotinib therapy to prevent progression of NMI bladder cancer.
Prevention Relevance: We evaluated the potential of erlotinib in preventing cancer by performing a randomized, double-blind, placebo-controlled trial of weekly erlotinib therapy in participants undergoing surgical removal of suspected noninvasive bladder neoplasia. Weekly erlotinib therapy was tolerated with common grade 1 to 2 toxicities but without evidence of beneficial effect upon urothelial tissue.
See related Spotlight, p. 7
Symmetric Dimethylarginine Predicts Previously Undetected Atrial Fibrillation in Patients With Ischemic Stroke
Authors: Hannemann Juliane , Wasser Katrin , Mileva Yoana , Kleinsang Fiona , Schubert Mario , Schwedhelm Edzard , Guan Kaomei , Wachter Rolf , Böger Rainer 
Published: August 2024

Background
Atrial fibrillation (AF) is a stroke risk factor that often remains undetected at hospital admission. Long‐term Holter monitoring helps to identify patients with previously unrecognized AF. Asymmetric (ADMA) and symmetric dimethylarginine (SDMA) are elevated in AF in cross‐sectional studies. We analyzed ADMA, SDMA, and other L‐arginine metabolites to assess their association with AF in the Find‐AF trial.

Methods and Results

We included 280 patients presenting with acute cerebral ischemia. Patients presenting in sinus rhythm received 7‐day Holter‐ECG. Biomarkers were quantified by ultra‐performance liquid chromatography–tandem mass spectrometry. We also analyzed protein methylation and L‐arginine‐related metabolites in human induced pluripotent stem cell‐derived cardiomyocytes in vitro. ADMA and SDMA were elevated in 44 patients who presented with AF. SDMA, but not ADMA, was significantly elevated in patients newly diagnosed with AF in Holter‐ECG as compared with those in sinus rhythm. SDMA plasma concentration >0.571 μmol/L significantly predicted presence of AF in Holter‐ECG (area under the curve=0.676 [0.530–0.822];
P
=0.029; sensitivity 0.786, specificity 0.572). SDMA levels further increased in patients with AF during the first 24 hours in hospital, and ADMA levels remained stable. In vitro, induced pluripotent stem cell‐derived cardiomyocytes showed increased symmetric protein methylation and elevated SDMA during rapid pacing (2.0 Hz versus 0.5 Hz), whereas asymmetric protein methylation and ADMA were unchanged.

Conclusions
SDMA at admission was significantly elevated in stroke patients presenting with AF and showed a moderate but significant prospective association with previously unrecognized AF. Thus, stroke patients with elevated SDMA concentration at admission may specifically benefit from extended Holter‐ECG monitoring.
Reduced Fetuin‐A Levels Are Associated With Exercise Intolerance and Predict the Risk of Adverse Outcomes in Patients With Heart Failure: The Role of Cardiac–Hepatic–Peripheral Interaction
Authors: Tomita Yusuke , Misaka Tomofumi , Sugawara Yukiko , Ichijo Yasuhiro , Anzai Fumiya , Sato Yu , Kimishima Yusuke , Yokokawa Tetsuro , Sato Takamasa , Oikawa Masayoshi , Kobayashi Atsushi , Yoshihisa Akiomi , Takeishi Yasuchika 
Published: August 2024

Background
Exercise intolerance in heart failure arises from multifactorial pathophysiological mechanisms. Hepatokines, liver‐synthesized molecules, regulate systemic metabolisms in peripheral tissues. We previously identified the hepatokine fetuin‐A as being linked to liver hypoperfusion in heart failure. Here, we investigated the role of fetuin‐A in connecting cardiac–hepatic–peripheral interaction.

Methods and Results
We conducted a prospective study involving 202 consecutive hospitalized patients (mean age, 56.8 years; 76.2% men) with heart failure who underwent cardiopulmonary exercise testing. We measured the serum concentration of fetuin‐A by ELISA. Correlation analysis revealed a negative association between fetuin‐A levels and the ratio of minimum minute ventilation to carbon dioxide production, its slope, and a tendency toward a positive correlation with peak oxygen uptake. Patients with impaired exercise tolerance exhibited lower fetuin‐A levels. During a median follow‐up of 1045 days, 18.3% experienced cardiac events, including 4 cardiac deaths and 33 cases of worsening heart failure. Classification and regression tree analysis identified a high‐risk subgroup with lower fetuin‐A (<24.3 mg/L) and impaired exercise tolerance (peak oxygen uptake<14.2 mL/kg per min). Kaplan–Meier analysis revealed that this subgroup had the highest risk of cardiac events. In a multivariable Cox proportional hazard model, the combination of lower fetuin‐A and exercise intolerance was independently associated with increased risks of cardiac events.

Conclusions
Reduced circulating fetuin‐A levels were associated with exercise intolerance in heart failure patients. Fetuin‐A could emerge as a target implicated in exercise capacity connecting cardiac–hepatic–peripheral interaction and as a valuable biomarker for predicting prognosis when combined with peak oxygen uptake.