bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of a novel NADPH generation reaction in the pentose phosphate pathway in
Escherichia coli
using mBFP
Authors: Ueno Koichiro , Sawada Shogo , Ishibashi Mai , Kanda Yoshiki , Shimizu Hiroshi , Toya Yoshihiro 
Published: October 2024

ABSTRACT

NADPH is a redox cofactor that drives the anabolic reactions. Although major NADPH generation reactions have been identified in
Escherichia coli
, some minor reactions have not been identified. In the present study, we explored novel NADPH generation reactions by monitoring the fluorescence dynamics after the addition of carbon sources to starved cells, using a metagenome-derived blue fluorescent protein (mBFP) as an intracellular NADPH reporter. Perturbation analyses were performed on a glucose-6-phosphate isomerase (PGI) deletion strain and its parental strain. Interestingly, mBFP fluorescence increased not only in the parental strain but also in the ΔPGI strain after the addition of xylose. Because the ΔPGI strain cannot metabolize xylose through the oxidative pentose phosphate pathway, this suggests that an unexpected NADPH generation reaction contributes to an increase in fluorescence. To unravel this mystery, we deleted the NADPH generation enzymes including transhydrogenase, isocitrate dehydrogenase, NADP
+
-dependent malic enzyme, glucose-6-phosphate dehydrogenase (G6PDH), and 6-phosphogluconate dehydrogenase (6PGDH) in the ΔPGI strain, and revealed that G6PDH and 6PGDH contribute to an increase in fluorescence under xylose conditions.
In vitro
assays using purified enzymes showed that G6PDH can produce NADPH using erythrose-4-phosphate (E4P) as a substitute for glucose-6-phosphate. Because the
Km
(0.65 mM) for E4P was much higher than the reported intracellular E4P concentrations in
E. coli
, little E4P must be metabolized through this bypass in the parental strain. However, the flux would increase when E4P accumulates in the cells owing to genetic modifications. This finding provides a metabolic engineering strategy for generating NADPH to produce useful compounds using xylose as a carbon source.

IMPORTANCE

Because NADPH is consumed during the synthesis of various useful compounds, enhancing NADPH regeneration is highly desirable in metabolic engineering. In this study, we explored novel NADPH generation reactions in
Escherichia coli
using a fluorescent NADPH reporter and found that glucose-6-phosphate dehydrogenase can produce NADPH using erythrose-4-phosphate as a substrate under xylose conditions. Xylose is an abundant sugar in nature and is an attractive carbon source for bioproduction. Therefore, this finding contributes to novel pathway engineering strategies using a xylose carbon source in
E. coli
to produce useful compounds that consume NADPH for their synthesis.
Lipophilic bisphosphonates reduced cyst burden and ameliorated hyperactivity of mice chronically infected with
Toxoplasma gondii
Authors: Sleda Melissa A. , Pitafi Zaid F. , Song WenZhan , Oldfield Eric , Moreno Silvia N. J. 
Published: October 2024

ABSTRACT

The current treatments for toxoplasmosis are only active against fast-growing tachyzoites, present in acute infections, with little effect on slow-growing bradyzoites within tissue cysts, present in latent chronic infections. The mitochondrion of
Toxoplasma gondii
is essential for its survival, and one of the major anti-parasitic drugs, atovaquone, inhibits the mitochondrial electron transport chain at the coenzyme Q:cytochrome c oxidoreductase site. Coenzyme Q (also known as ubiquinone [UQ]) consists of a quinone head and a lipophilic, isoprenoid tail that anchors UQ to membranes. The synthesis of the isoprenoid unit is essential for cell growth and is inhibited by lipophilic bisphosphonates, which inhibit the parasite growth. In this work, we investigated the effect of lipophilic bisphosphonates on the chronic stages of
T. gondii
. We discovered that three lipophilic bisphosphonates (BPH-1218, BPH-1236, and BPH-1238), effective for the acute infection, were also effective in controlling the development of chronic stages. We showed effectiveness by testing them against
in vitro
cysts and
in vivo
derived tissue cysts and, most importantly, these compounds reduced the cyst burden in the brains of chronically infected mice. We monitored the activity of infected mice non-invasively and continuously with a novel device termed the CageDot. A decrease in activity accompanied the acute phase, but mice recovered to normal activity and showed signs of hyperactivity when the chronic infection was established. Moreover, treatment with atovaquone or BPH-1218 ameliorated the hyperactivity observed during the chronic infection.

IMPORTANCE

Treatment for toxoplasmosis is challenged by a lack of effective drugs to eradicate the chronic stages. Most of the drugs currently used are poorly distributed to the central nervous system, and they trigger allergic reactions in a large number of patients. There is a compelling need for safe and effective treatments for toxoplasmosis. Bisphosphonates (BPs) are analogs of inorganic pyrophosphate and are used for the treatment of bone disorders. BPs target the isoprenoid pathway and are effective against several experimental parasitic infections. Some lipophilic BPs can specifically inhibit the mitochondrial activity of
Toxoplasma gondii
by interfering with the mechanism by which ubiquinone is inserted into the inner mitochondrial membrane. In this work, we present the effect of three lipophilic BPs against
T. gondii
chronic stages. We also present a new strategy for the monitoring of animal activity during disease and treatment that is non-invasive and continuous.
The putative type 4 secretion system effector BspD is involved in maintaining envelope integrity of the pathogen
Brucella
Authors: Ketterer Maren , Chiquet Petra , Esposito Mara , Sedzicki Jaroslaw , Québatte Maxime , Dehio Christoph 
Published: October 2024

ABSTRACT

Brucellosis is a debilitating disease caused by the Gram-negative, facultative intracellular zoonotic pathogen
Brucella.
En route to its intracellular replicative niche,
Brucella
encounters various stressful environments that may compromise envelope integrity. Here we show that the proposed type 4 secretion system (T4SS) effector BspD is a conserved protein of the Rhizobiales, which does not show signs of co-evolution with the presence of a T4SS or a certain lifestyle. We further present data indicating that BspD is critical for the envelope integrity of
Brucella abortus
in the stationary phase and in the presence of EDTA, a compound known to destabilize the outer membrane. Deletion of
bspD
resulted in abnormal bacterial morphologies, indicating its involvement in maintaining envelope integrity. Additionally, the absence of BspD led to the formation of fewer and smaller intracellular microcolonies in a macrophage infection model. From our observations, we propose that BspD of
B. abortus
is critical for preserving the integrity of the bacterial envelope, particularly under stressful conditions, which may enhance
Brucella
’s ability to survive within host cells.

IMPORTANCE

Brucellosis, caused by the intracellular pathogen
Brucella
, poses a significant health threat. Understanding how
Brucella
adapts to stressful environments is crucial. This study unveils BspD, a conserved protein within the Rhizobiales order, as a key player in maintaining
Brucella
's envelope integrity. Remarkably, BspD’s presence within the Rizobiales appears independent of the presence of a T4SS or a specific lifestyle. Deletion of
bspD
resulted in compromised envelope integrity, abnormal bacterial morphologies, and reduced intracellular microcolony formation. These findings underscore BspD’s critical role, particularly in stressful conditions like the stationary phase and EDTA exposure, and highlight its significance for the survival of
Brucella
within host cells. This elucidation deepens our understanding of
Brucella
pathogenesis and may inform future therapeutic strategies against brucellosis.
Development and evaluation of statistical and artificial intelligence approaches with microbial shotgun metagenomics data as an untargeted screening tool for use in food production
Authors: Beck Kristen L. , Haiminen Niina , Agarwal Akshay , Carrieri Anna Paola , Madgwick Matthew , Kelly Jennifer , Pylro Victor , Kawas Ban , Wiedmann Martin , Ganda Erika 
Published: October 2024

ABSTRACT

The increasing knowledge of microbial ecology in food products relating to quality and safety and the established usefulness of machine learning algorithms for anomaly detection in multiple scenarios suggests that the application of microbiome data in food production systems for anomaly detection could be a valuable approach to be used in food systems. These methods could be used to identify ingredients that deviate from their typical microbial composition, which could indicate food fraud or safety issues. The objective of this study was to assess the feasibility of using shotgun sequencing data as input into anomaly detection algorithms using fluid milk as a model system. Contrastive principal component analysis (PCA), cluster-based methods, and explainable artificial intelligence (AI) were evaluated for the detection of two anomalous sample classes using longitudinal metagenomic profiling of fluid milk compared to baseline (BL) samples collected under comparable circumstances. Traditional methods (alpha and beta diversity, clustering-based contrastive PCA, multidimensional scaling, and dendrograms) failed to differentiate anomalous sample classes; however, explainable AI was able to classify anomalous vs baseline samples and indicate microbial drivers in association with antibiotic use. We validated the potential for explainable AI to classify different milk sources using larger publicly available fluid milk 16S rDNA sequencing data sets and demonstrated that explainable AI is able to differentiate between milk storage methods, processing stages, and seasons. Our results indicate that the application of artificial intelligence continues to hold promise in the realm of microbiome data analysis and could present further opportunities for downstream analytic automation to aid in food safety and quality.

IMPORTANCE
We evaluated the feasibility of using untargeted metagenomic sequencing of raw milk for detecting anomalous food ingredient content with artificial intelligence methods in a study specifically designed to test this hypothesis. We also show through analysis of publicly available fluid milk microbial data that our artificial intelligence approach is able to successfully predict milk in different stages of processing. The approach could potentially be applied in the food industry for safety and quality control.
Novel motif associated with carbon catabolite repression in two major Gram-positive pathogen virulence regulatory proteins
Authors: Woo Jerry K. K. , Zimnicka Adriana M. , Federle Michael J. , Freitag Nancy E. 
Published: October 2024

ABSTRACT

Carbon catabolite repression (CCR) is a widely conserved regulatory process that ensures enzymes and transporters of less-preferred carbohydrates are transcriptionally repressed in the presence of a preferred carbohydrate. This phenomenon can be regulated via a CcpA-dependent or CcpA-independent mechanism. The CcpA-independent mechanism typically requires a transcriptional regulator harboring a phosphotransferase regulatory domain (PRD) that interacts with phospho
t
ransferase
s
ystem (PTS) components. PRDs contain a conserved histidine residue that is phosphorylated by the PTS-associated HPr-His15~P protein. PRD-containing regulators often harbor additional domains that resemble PTS-associated EIIB protein domains with a conserved cysteine residue that can be phosphorylated by cognate PTS components. We noted that Mga, the PRD-containing central virulence regulator of
Streptococcus pyogenes
, has an EIIB
Gat
domain containing a cysteine that, based on the presence of a similar motif in glycerol kinase, could be a target for phosphorylation. Using site-directed mutagenesis, we constructed phospho-ablative and phospho-mimetic substitutions of this cysteine and found that these substitutions modify the CCR of the Rgg2/3 quorum-sensing system. Moreover, we provide genetic evidence that the phospho-donor of this cysteine residue is likely to be ManL, the EIIA/B subunit of the mannose PTS system. Interestingly, a structurally distinct virulence gene regulator, PrfA of
Listeria monocytogenes
, harbors a similar cysteine-containing motif, and phospho-ablative and phospho-mimetic substitutions of the cysteine-altered CCR of PrfA-dependent virulence gene expression. Collectively, our data suggest that phosphorylation of a cysteine within the shared novel motif in Mga and PrfA may be a heretofore missing link between cellular metabolism and virulence.

IMPORTANCE
In this study, we identified a novel cysteine-containing motif within the amino acid sequence of two structurally distinct transcriptional regulators of virulence in two Gram-positive pathogens that appears to link carbon metabolism with virulence gene expression. The results also highlight the potential post-translational modification of cysteine in bacterial species, a rare and understudied modification.
Characterizing the urobiome in geriatric males with chronic indwelling urinary catheters: an exploratory longitudinal study
Authors: Stewart Emma , Hochstedler-Kramer Baylie R. , Khemmani Mark , Clark Nina M. , Parada Jorge P. , Farooq Ahmer , Doshi Chirag , Wolfe Alan J. , Albarillo Fritzie S. 
Published: October 2024

ABSTRACT

The impact of chronic indwelling urinary catheters (IUCs) on the composition and stability of the urinary microbiota remains unknown. The primary aim of this study was to describe the urinary microbiomes of geriatric males with chronic IUCs. A secondary aim was to explore clinical catheter-associated urinary tract infection (CAUTI) courses of the participants. Geriatric male patients with chronic IUCs were followed longitudinally. Catheterized urine, catheter tips, and both urethral and periurethral swabs were collected from participants at monthly intervals. Microbes were isolated and identified from each specimen using an enhanced culture method called expanded quantitative urine culture (EQUC) and targeted 16S rRNA gene DNA sequencing. Microbial outcomes were examined both in the absence of urinary symptoms and in the context of clinical diagnosis of CAUTI. Ten male participants (mean age 86 years) were enrolled. Urinary microbiomes differed for each participant. However, within each individual, microbiomes were similar over time and across niches (bladder, catheter, urethra, and periurethra). Within-niche microbiomes differed across individuals, and this was observed over time. The most abundant bacteria isolated from all niches were known uropathogens. Six of 10 individuals met diagnostic criteria for CAUTI at least once during the 12-month observation period, but no evidence of this or antibiotic treatment/response was discernable in our monthly samples. The microbiomes of each participant were unique and remained similar over time and across niches. Longitudinal EQUC or 16S rRNA gene sequencing data could be useful to clinicians when diagnosing or treating possible CAUTI.

IMPORTANCE
Catheter-associated urinary tract infections (CAUTIs) are serious but preventable nosocomial infections. The most common risk factor for developing CAUTI is prolonged use of indwelling urinary catheters (IUCs). This study provides the first longitudinal description of the urinary microbiomes of geriatric males with chronic IUCs, in the absence of urinary signs and symptoms, as a first step toward enhancing our knowledge of the impact of chronic IUCs on the composition and stability of the urinary microbiota. This is an understudied area, particularly for males.
Template plasmids optimized for deletion of multiple genes in yeast
Saccharomyces cerevisiae
Authors: Feng Yi-He , Song Jing-Zhen , Zhu Jing , Xie Zhiping 
Published: October 2024

ABSTRACT

For
Saccharomyces cerevisiae
, gene knockout is routinely performed by transformation with a linear DNA cassette consisting of a selection marker gene flanked by upstream and downstream sequences homologous to a target gene. Over the years, several plasmid sets containing a variety of selection marker genes have been developed. Targeting fidelity under this strategy was high when performing the first gene knockout in a strain. However, we found that targeting fidelity decreased substantially when performing subsequent gene knockouts. The majority of the transformants were “incorrect,” in which the new selection marker gene replaced a pre-existing selection marker gene instead of its intended target. This was caused by the presence of shared regions in the knockout DNA cassettes. To minimize shared regions among knockout cassettes, we developed a set of template plasmids, in which each selection marker open reading frame is flanked by a unique promoter/terminator combination. Our SJZ series templates cover eight selection markers, namely,
URA3
(
C. a
.),
TRP1
(
K.l
.),
his5
(
S.p
.),
LEU2
(
K.l
.),
nat
,
hph
,
kan
, and
amdS
. When using our templates, targeting fidelity in subsequent gene knockouts was restored to as high as that of the first knockout, with essentially all the transformants being correct. Our templates can therefore bring efficiency improvements in future research projects involving multi-gene knockouts.

IMPORTANCE
When knocking out multiple genes in yeast, recombination among selection markers produces a large portion of false-positive transformants. We developed a new set of templates designed to minimize shared regions among selection markers. The use of this new template set resulted in essentially all transformants being correct knockouts.
Method to apply temporal graph analysis on electronic patient record data to explore healthcare professional–patient interaction intensity: a cohort study
Authors: Booth John , Eriksson Maria H , Marks Stephen D , Bryant William A , Denaxas Spiros , Pope Rebecca , Sebire Neil J 
Published: October 2024

AimInteractions between patients and healthcare professionals (HCP) during hospital admissions are complex and difficult to interrogate using traditional analysis of electronic patient record (EPR) data. The objective of this study was to determine the feasibility of applying temporal network analytics to EPR data, focusing on HCP–patient interactions over time.MethodNetwork (graph) analysis was applied to routinely collected structured data from an EPR for HCP interactions with individual patients during admissions for patients undergoing renal transplantation between May 2019 and June 2023. Networks were constructed per day of admission within a session, defined by whether the patient was in the intensive care unit (ICU) or standard hospital ward. Connections between HCP were defined using a 60 min period. Reports were generated visualising daily interaction network structures, across individual admissions.Results2300 individual networks were constructed from 127 hospital admissions for renal transplantation. The number of nodes or HCP per network varied from 2 to 45, and network metrics provided detail regarding variation in the density and transitivity, changes in structure with different diameters and radii, and variations in centralisation. Each network analysis metric has a contribution to play in describing the dynamics of a daily HCP network and the composite findings provide insights that cannot be determined with standard approaches.ConclusionsNetwork analysis provides a novel approach to investigate and visualise patterns of HCP–patient interactions which allow for a deeper understanding of the complex nature of hospital patient care and could have numerous practical operational applications.
Paradoxical intention as a treatment for insomnia disorder: study protocol for a mixed-methods pilot trial
Authors: Salim Osame , Jansson-Fröjmark Markus , Sandlund Christina , Norell Annika 
Published: October 2024

IntroductionParadoxical intention (PI) is an insomnia treatment developed in the 1970s, which instructs patients to gently attempt to remain awake while in bed at night with the lights off. Previous research indicates PI’s potential in improving insomnia, although no study has been conducted in the last few decades during which the insomnia diagnostic criteria have changed. Additionally, there are knowledge gaps regarding outcomes related to wake after sleep onset, the treatment mechanisms as well as the acceptability and feasibility of the treatment. This study therefore aims to address these gaps by assessing the potential mechanisms, preliminary efficacy, and patient experience and acceptability of PI.Methods and analysisWe aim to include 40 adult participants with insomnia, aged 18 and above, from the Swedish general population. In this uncontrolled pilot study using a mixed-methods approach, both qualitative and quantitative data will be collected. The trial will be conducted on a self-help online platform, accessible from participants’ homes, with weekly phone call support by therapists. Process and outcome measures will be assessed weekly across a 4-week intervention period and at a 3-month follow-up. A subset of participants will be asked to participate in qualitative semistructured interviews regarding the treatment.Ethics and disseminationEthical approval for this project has been granted by the Swedish Ethical Review Authority (Dnr: 2023-06594-01). All participants will sign informed consent forms on a web service application prior to enrolment. From this mixed-methods study, we anticipate insights into the preliminary efficacy and mechanisms of paradoxical intention for treating insomnia, enriched by patient experience data. Results will be disseminated through peer-reviewed publications. The findings will inform adaptations to the treatment protocol and serve as groundwork for a possible larger scale randomised controlled trial.Trial registration numberNCT06259682.
Longitudinal frailty assessment in the prediction of survival among patients with advanced chronic kidney disease: a prospective observational single-centre cohort study
Authors: Kennard Alice , Richardson Alice , Rainsford Suzanne , Hamilton Kelly , Glasgow Nicholas , Pumpa Kate , Douglas Angela , Talaulikar Girish Scricant 
Published: October 2024

ObjectivesThis study aims to describe the prevalence, characteristics and longitudinal changes in frailty among outpatient chronic kidney disease (CKD) and haemodialysis (HD) populations and their impact on survival.DesignProspective observational cohort study.SettingSingle-centre ambulatory tertiary care setting, metropolitan Australian teaching hospital.ParticipantsAdult patients with advanced CKD (defined as estimated glomerular filtration rate <20 mL/min) or undergoing maintenance HD. Consent model was informed opt-out consent.InterventionsFried frailty assessment at baseline, 6 months and 12 months of longitudinal follow-up.Primary outcomesAll-cause mortality and kidney transplantation events.ResultsFrailty was identified in 36.3% of the 256 participants, while an additional 46.5% exhibited prefrailty. Frailty was equally common among CKD and HD cohorts. Frailty outperformed age, comorbidity and laboratory parameters in predicting mortality risk with HR 2.83 (95% CI 1.44 to 5.56, p<0.001). Frailty also substantially reduced access to transplantation. While most participants exhibited static Fried phenotype over longitudinal assessment, improvements in frailty were observed as frequently as frailty progression. Female gender and symptom burden predicted frailty progression.ConclusionsFrailty is highly prevalent and closely aligned with survival outcomes. Frailty among patients attending routine outpatient care may demonstrate responsiveness to intervention with subsequent improvements in mortality and other patient-level outcomes.