Pasa: leveraging population pangenome graph to scaffold prokaryote genome assemblies.

Whole genome sequencing has increasingly become the essential method for studying the genetic mechanisms of antimicrobial resistance and for surveillance of drug-resistant bacterial pathogens. The majority of bacterial genomes sequenced to date have been sequenced with Illumina sequencing technology, owing to its high-throughput, excellent sequence accuracy, and low cost. However, because of the short-read nature of the technology, these assemblies are fragmented into large numbers of contigs, hindering the obtaining of full information of the genome. We develop Pasa, a graph-based algorithm that utilizes the pangenome graph and the assembly graph information to improve scaffolding quality. By leveraging the population information of the bacteria species, Pasa is able to utilize the linkage information of the gene families of the species to resolve the contig graph of the assembly. We show that our method outperforms the current state of the arts in terms of accuracy, and at the same time, is computationally efficient to be applied to a large number of existing draft assemblies.

AMRViz enables seamless genomics analysis and visualization of antimicrobial resistance.

We have developed AMRViz, a toolkit for analyzing, visualizing, and managing bacterial genomics samples. The toolkit is bundled with the current best practice analysis pipeline allowing researchers to perform comprehensive analysis of a collection of samples directly from raw sequencing data with a single command line. The analysis results in a report showing the genome structure, genome annotations, antibiotic resistance and virulence profile for each sample. The pan-genome of all samples of the collection is analyzed to identify core- and accessory-genes. Phylogenies of the whole genome as well as all gene clusters are also generated. The toolkit provides a web-based visualization dashboard allowing researchers to interactively examine various aspects of the analysis results. Availability: AMRViz is implemented in Python and NodeJS, and is publicly available under open source MIT license at https://github.com/amromics/amrviz .

AMRomics: a scalable workflow to analyze large microbial genome collections.

Whole genome analysis for microbial genomics is critical to studying and monitoring antimicrobial resistance strains. The exponential growth of microbial sequencing data necessitates a fast and scalable computational pipeline to generate the desired outputs in a timely and cost-effective manner. Recent methods have been implemented to integrate individual genomes into large collections of specific bacterial populations and are widely employed for systematic genomic surveillance. However, they do not scale well when the population expands and turnaround time remains the main issue for this type of analysis. Here, we introduce AMRomics, an optimized microbial genomics pipeline that can work efficiently with big datasets. We use different bacterial data collections to compare AMRomics against competitive tools and show that our pipeline can generate similar results of interest but with better performance. The software is open source and is publicly available at https://github.com/amromics/amromics under an MIT license.

Lifestyle interventions with dietary strategies after breast cancer diagnosis: a systematic review.

PURPOSE: Obesity can increase mortality and morbidity in breast cancer survivors. Healthy lifestyle factors such as diet can help manage weight in this population. This systematic review examined lifestyle interventions with dietary strategies for breast cancer survivors and their effect on diet and/or weight-related outcomes. METHODS: Searches were conducted in Ovid MEDLINE® ALL (1946-February 14, 2022), Embase (Elsevier), CINAHL Complete (EBSCO), and APA PsycArticles (EBSCO), using keywords for diet, breast cancer, and intervention. The search was limited to human studies, English language, and publication processing date 2016-2023. RESULTS: The search yielded 3427 articles. After title and abstract review, 225 full-text articles were screened, and 67 articles with 61 distinct samples and interventions met inclusion criteria. Of these 61 lifestyle interventions with dietary strategies, 43 interventions also addressed physical activity. Most studies were randomized controlled trials (n = 41) and conducted post-treatment (n = 45). Mean participant age was 54 years. Of 29 studies that reported race/ethnicity, 20 (69%) reported ≥50% White participants. Of 36 that reported dietary outcomes, 29 (81%) reported significant findings. Of 57 that reported weight-related outcomes, 51 (89%) reported significant findings. CONCLUSION: This review demonstrated promising evidence for the efficacy of lifestyle interventions with dietary strategies in breast cancer survivors. However, culturally tailored interventions and interventions conducted before and during treatment are lacking.

Advanced patient-specific microglia cell models for pre-clinical studies in Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is an incurable neurodegenerative disorder with a rapidly increasing prevalence worldwide. Current approaches targeting hallmark pathological features of AD have had no consistent clinical benefit. Neuroinflammation is a major contributor to neurodegeneration and hence, microglia, the brain's resident immune cells, are an attractive target for potentially more effective therapeutic strategies. However, there is no current in vitro model system that captures AD patient-specific microglial characteristics using physiologically relevant and experimentally flexible culture conditions. METHODS: To address this shortcoming, we developed novel 3D Matrigel-based monocyte-derived microglia-like cell (MDMi) mono-cultures and co-cultures with neuro-glial cells (ReNcell VM). We used single-cell RNA sequencing (scRNAseq) analysis to compare the transcriptomic signatures of MDMi between model systems (2D, 3D and 3D co-culture) and against published human microglia datasets. To demonstrate the potential of MDMi for use in personalized pre-clinical strategies, we generated and characterized MDMi models from sixteen AD patients and matched healthy controls, and profiled cytokine responses upon treatment with anti-inflammatory drugs (dasatinib and spiperone). RESULTS: MDMi in 3D exhibited a more branched morphology and longer survival in culture compared to 2D. scRNAseq uncovered distinct MDMi subpopulations that exhibit higher functional heterogeneity and best resemble human microglia in 3D co-culture. AD MDMi in 3D co-culture showed altered cell-to-cell interactions, growth factor and cytokine secretion profiles and responses to amyloid-ß. Drug testing assays revealed patient- and model-specific cytokine responses. CONCLUSION: Our study presents a novel, physiologically relevant and AD patient-specific 3D microglia cell model that opens avenues towards improving personalized drug development strategies in AD.

Advanced Electrolyte Formula for Robust Operation of Vanadium Redox Flow Batteries at Elevated Temperatures.

Insufficient thermal stability of vanadium redox flow battery (VRFB) electrolytes at elevated temperatures (>40 °C) remains a challenge in the development and commercialization of this technology, which otherwise presents a broad range of technological advantages for the long-term storage of intermittent renewable energy. Herein, a new concept of combined additives is presented, which significantly increases thermal stability of the battery, enabling safe operation to the highest temperature (50 °C) tested to date. This is achieved by combining two chemically distinct additives-inorganic ammonium phosphate and polyvinylpyrrolidone (PVP) surfactant, which collectively decelerate both protonation and agglomeration of the oxo-vanadium species in solution and thereby significantly suppress detrimental formation of precipitates. Specifically, the precipitation rate is reduced by nearly 75% under static conditions at 50° C. This improvement is reflected in the robust operation of a complete VRFB device for over 300 h of continuous operation at 50 °C, achieving an impressive 83% voltage efficiency at 100 mA cm‒2 current density, with no precipitation detected in either the electrode/flow-frame or electrolyte tank.

Inhibition of the master regulator of Listeria monocytogenes virulence enables bacterial clearance from spacious replication vacuoles in infected macrophages.

A hallmark of Listeria (L.) monocytogenes pathogenesis is bacterial escape from maturing entry vacuoles, which is required for rapid bacterial replication in the host cell cytoplasm and cell-to-cell spread. The bacterial transcriptional activator PrfA controls expression of key virulence factors that enable exploitation of this intracellular niche. The transcriptional activity of PrfA within infected host cells is controlled by allosteric coactivation. Inhibitory occupation of the coactivator site has been shown to impair PrfA functions, but consequences of PrfA inhibition for L. monocytogenes infection and pathogenesis are unknown. Here we report the crystal structure of PrfA with a small molecule inhibitor occupying the coactivator site at 2.0 Å resolution. Using molecular imaging and infection studies in macrophages, we demonstrate that PrfA inhibition prevents the vacuolar escape of L. monocytogenes and enables extensive bacterial replication inside spacious vacuoles. In contrast to previously described spacious Listeria-containing vacuoles, which have been implicated in supporting chronic infection, PrfA inhibition facilitated progressive clearance of intracellular L. monocytogenes from spacious vacuoles through lysosomal degradation. Thus, inhibitory occupation of the PrfA coactivator site facilitates formation of a transient intravacuolar L. monocytogenes replication niche that licenses macrophages to effectively eliminate intracellular bacteria. Our findings encourage further exploration of PrfA as a potential target for antimicrobials and highlight that intra-vacuolar residence of L. monocytogenes in macrophages is not inevitably tied to bacterial persistence.

Blood volume reduction due to rapid plasma loss after birth in preterm piglets.

BACKGROUND: Understanding changes in blood volume after preterm birth is critical to preventing cardiovascular deterioration in preterm infants. The aims were to determine if blood volume is higher in preterm than term piglets and if blood volume changes in the hours after birth. METHODS: Paired blood volume measurements were conducted in preterm piglets (98/115d gestation, ~28wk gestation infant) at 0.5-5 h (n = 12), 0.5-9 h (n = 44) and 5-11 h (n = 7) after birth, and in a term cohort at 0.5-9 h (n = 40) while under intensive care. RESULTS: At 30 min after birth, blood volume was significantly lower in preterm piglets compared to term piglets. By 9 h after birth, blood volume had reduced by 18% in preterm piglets and 13% in term piglets. By 5-9 h after birth, preterm piglets had significantly lower blood volumes than at term (61 ± 10 vs. 76 ± 11 mL/kg). CONCLUSIONS: In contrast to clinical resources, preterm piglets have a lower blood volume than at term. Substantial reductions in blood volume after birth leave some preterm piglets hypovolemic. If this also occurs in preterm infants, this may have important clinical consequences. Modern studies of blood volume changes after birth are essential for improving preterm outcomes. IMPACT: Preterm piglets do not have a higher blood volume than their term counterparts, in contrast to current clinical estimates. Rapid reduction in blood volume after birth leads to hypovolemia in some preterm piglets. There is a critical need to understand blood volume changes after birth in preterm infants in order to improve clinical management of blood volume.

In Situ Synthesis and Self-Assembly of Peptide-PEG Conjugates: A Facile Method for the Construction of Fibrous Hydrogels.

Peptide-based hydrogels have gained considerable attention as a compelling platform for various biomedical applications in recent years. Their attractiveness stems from their ability to seamlessly integrate diverse properties, such as biocompatibility, biodegradability, easily adjustable hydrophilicity/hydrophobicity, and other functionalities. However, a significant drawback is that most of the functional self-assembling peptides cannot form robust hydrogels suitable for biological applications. In this study, we present the synthesis of novel peptide-PEG conjugates and explore their comprehensive hydrogel properties. The hydrogel comprises double networks, with the first network formed through the self-assembly of peptides to create a ß-sheet secondary structure. The second network is established through covalent bond formation via N-hydroxysuccinimide chemistry between peptides and a 4-arm PEG to form a covalently linked network. Importantly, our findings reveal that this hydrogel formation method can be applied to other peptides containing lysine-rich sequences. Upon encapsulation of the hydrogel with antimicrobial peptides, the hydrogel retained high bacterial killing efficiency while showing minimum cytotoxicity toward mammalian cells. We hope that this method opens new avenues for the development of a novel class of peptide-polymer hydrogel materials with enhanced performance in biomedical contexts, particularly in reducing the potential for infection in applications of tissue regeneration and drug delivery.

Development and Validation of Liquid Chromatographic Method for Fast Determination of Lincomycin, Polymyxin and Vancomycin in Preservation Solution for Transplants.

In this study, a liquid chromatographic method was developed for the fast determination of lincomycin, polymyxin and vancomycin in a preservation solution for transplants. A Kinetex EVO C18 (150 × 4.6 mm, 2.6 µm) column was utilized at 45 °C. Gradient elution was applied using a mixture of mobile phases A and B, both including 30 mM phosphate buffer at pH 2.0 and acetonitrile, at a ratio of 95:5 (v/v) for A and 50:50 (v/v) for B. A flow rate of 1.0 mL/min, an injection volume of 20 µL and UV detection at 210 nm were used. A degradation study treating the three antibiotics with 0.5 M hydrochloric acid, 0.5 M sodium hydroxide and 3% H2O2 indicated that the developed method was selective toward lincomycin, polymyxin, vancomycin and their degradation products. Other ingredients of the preservation solution, like those from the cell culture medium, did not interfere. The method was validated with good sensitivity, linearity, precision and accuracy. Furthermore, lincomycin, polymyxin and vancomycin were found to be stable in this preservation solution for 4 weeks when stored at -20 °C.

Positive Effects of Physical Activity on Prescription on Glycemic Control, Fitness, and Quality of Life in Newly Diagnosed Type 2 Diabetic Patients.

The aim was to investigate the effects of physical activity on prescription (PAP) compared with standard care (SC) in adult drug-naïve T2D patients. A randomized control trial was conducted with drug-naïve T2D patients attending an out-patient clinic Vietnam. Participants were randomly assigned to the PAP group (n+=+44) or the SC group (n+=+43). The PAP group received individualized recommendations for PA, intensive face-to-face training every two weeks. The SC group received the standard recommendations according to WHO guidelines. The mean HbA1c level change was larger (-10.6±6.4 mmol/mol) in the PAP group than in the SC group (-2.4±5.8 mmol/mol) (p<0.001). A one thousand step counts per day increase was significantly associated with a decrease of -2.43 mmol/mol in HbA1c [ß=-2.43, 95%CI: (-2.94, -1.92]) in the PAP group. The fasting plasma glucose levels of the PAP group decreased significantly compared with the SC group. The VO2-max increased significantly more in the PAP group than in the SC group. PAP had clear positive effects on health-related Quality of Life [mean between group difference: 9.54 (95%CI 5.84,13.23)]. Insulin resistance, BMI, waist circumference, total cholesterol, LDL cholesterol and triglycerides were significantly more decreased in the PAP group than in the control group. In conclusion, the fact that even a small change in mean step counts over three months had a beneficial effect on health-related outcomes in drug-naïve T2D patients can have large implications for treatment and management practices, not least in a middle-income country like Vietnam.

Post-stroke depression in Vietnamese patients is associated with decreased sleep quality and increased fatigue: a one-institution cross-sectional analysis.

PURPOSE: This study aimed to determine the prevalence of post-stroke depression (PSD) during the first year and its associated factors, especially focusing on sleep quality and fatigue severity. METHODS: A cross-sectional study was conducted among stroke patients in Vietnam's National Geriatric Hospital. Data were collected by using standardized questionnaires for interviewing and evaluating patients at the research site. Several covariables were presented including demographics, stroke-related characteristics, activities of daily living, post-stroke fatigue, and sleep quality (Pittsburgh Sleep Quality Index [PSQI] scale). PSD was assessed as an outcome variable through the Patient Health Questionnaire-9 scale. To summarize sociodemographic and clinical variables, descriptive statistics were performed. A logistic regression model was used to explore the factors related to PSD. RESULTS: Of 157 patients with stroke, mean age 73.1 (± 9.6), PSD was present in 60 patients (38%). The global score and all PSQI components of participants with PSD showed worse levels than those without depression. Furthermore, the prevalence of PSD was higher in patients with low IADL scores and functional disability at high levels. In the multivariate logistic regression analysis, the patients with PSD showed higher Fatigue Severity Scale (FSS) scores (OR = 4.11; 95% CI = 1.39; 12.19) and higher scores in two domains of the PSQI scale including subjective sleep quality (OR = 3.03; 95% CI = 1.21; 7.58) and sleep disturbance (OR = 5.22; 95% CI = 1.33; 20.47). CONCLUSION: There is a significant prevalence of depression following stroke. Furthermore, post-stroke fatigue and two PSQI scale components (subjective sleep quality and sleep disturbance) were shown to be associated with PSD. This finding may guide early screening and intervention strategies to address depression following stroke.

Type IV pili facilitated natural competence in Fusobacterium nucleatum.

OBJECTIVES: Many bacterial species naturally take up DNA from their surroundings and recombine it into their chromosome through homologous gene transfer (HGT) to aid in survival and gain advantageous functions. Herein we present the first characterization of Type IV pili facilitated natural competence in Fusobacterium nucleatum, which is a Gram-negative, anaerobic bacterium that participates in a range of infections and diseases including periodontitis, preterm birth, and cancer. METHODS: Here we used bioinformatics on multiple Fusobacterium species, as well as molecular genetics to characterize natural competence in strain F. nucleatum subsp. nucleatum ATCC 23726. RESULTS: We bioinformatically identified components of the Type IV conjugal pilus machinery and show this is a conserved system within the Fusobacterium genus. We next validate Type IV pili in natural competence in F. nucleatum ATCC 23726 and show that gene deletions in key components of pilus deployment (pilQ) and cytoplasmic DNA import (comEC) abolish DNA uptake and chromosomal incorporation. We next show that natural competence may require native F. nucleatum DNA methylation to bypass restriction modification systems and allow subsequent genomic homologous recombination. CONCLUSIONS: In summary, this proof of principle study provides the first characterization of natural competence in Fusobacterium nucleatum and highlights the potential to exploit this DNA import mechanism as a genetic tool to characterize virulence mechanisms of an opportunistic oral pathogen.

Pott's Puffy: First Shot is the Best Shot.

OBJECTIVE: Low incidence of Pott's Puffy tumor (PPT) has caused studying risk factors and recurrences of the disease to be difficult. We used the comparatively increased incidence at our institution to evaluate potential risk factors for the disease process itself and prognostic factors for recurrence of the disease. METHODS: Single institutional retrospective chart review identified 31 patients from 2010 to 2022 with PPT compared with a control group of 20 patients with either chronic rhinosinusitis or recurrent sinusitis. Patient mean age of PPT was 42 (range of 5 to 90) with the majority of the patient population as male (74%) and Caucasian (68%) in the setting of rural West Texas. Patient mean age of the control group was 50.7 (range of 30-78) with majority of patient population as male (55%) and Caucasian (70%). Interventions studied were functional endoscopic sinus surgery (FESS), FESS with trephination, and cranialization with or without FESS to compare prognostic factors for recurrence rates of PPT. These patients' prognostic risk factors for recurrence and risk factors to develop PPT were analyzed using Analysis of Variance (ANOVA) χ 2 statistical analysis with Fischer exact testing. RESULTS: Mean age was 42 years (range of 5-90) with the majority of the PPT patient population as male (74%) and Caucasian (68%) with an overall incidence of about 1 in 300,000. Pott's Puffy tumor patients were significantly favored in the younger and male population compared with the control patients. Risk factors of no prior allergy diagnosis, previous trauma, medication allergy to penicillin class or cephalosporin class, and lower body mass index were significant in the PPT population compared with the control group. Significant prognostic factors for recurrence of PPT were prior history of sinus surgery and operative treatment choice. Fifty percent (3/6) of patients with prior sinus surgery had recurrence of PPT. Of our 4 treatment options (FESS, FESS with trephination, FESS with cranialization, or cranialization alone), ;FESS had a recurrence of PPT of 0% (0/13), FESS with trephination had a recurrence of PPT of 50% (3/6), FESS with cranialization had a recurrence of PPT of 11% (1/9), and cranizalization alone had a recurrence of PPT of 0% (0/3). Of note, postop chronic rhinosinusitis was seen in 46% (6/13) of FESS alone, 17% (1/6) with FESS with trephination, 0% (0/9) with FESS with cranialization, and 33% (1/3) with just cranialization alone. CONCLUSIONS: Pott's Puffy tumor patients were younger and predominately male when compared to the control patients. No prior allergy diagnosis, previous trauma history, medication allergy to penicillin class or cephalosporin class, and lower body mass index are risk factors for PPT. There are 2 prognostic factors that predict recurrence of PPT: first operative treatment choice and prior sinus surgery. History of prior sinus surgery tends to increase the recurrence of PPT. The first operative treatment plan is the best shot at definitively treating PPT. Correct management surgically can prevent recurrence of PPT as well as long-term recurrence of chronic rhinosinusitis. With early diagnosis and mild disease, FESS is sufficient to prevent recurrence of PPT but chronic sinusitis may continue to occur if frontal sinus outflow track is not well opened. If considering trephination, a definitive cranialization may be more suited for more advanced disease since our study showed 50% of recurrence of PPT with trephination and FESS along with 17% chronic sinusitis long term. More advanced diseases with higher WBCs and intracranial extension do better with more aggressive surgical management with a cranialization with or without FESS which shows to reduce rates of PPT recurrence significantly.

Potent IPTG-inducible integrative expression vectors for production of recombinant proteins in Bacillus subtilis.

The IPTG-inducible promoter family, Pgrac, allows high protein expression levels in an inducible manner. In this study, we constructed IPTG-inducible expression vectors containing strong Pgrac promoters that allow integration of the transgene at either the amyE or lacA locus or both loci in Bacillus subtilis. Our novel integrative expression vectors based on Pgrac promoters could control the repression of protein production in the absence and the induction in the presence of an inducer, IPTG. The ß-galactosidase (BgaB) protein levels were 9.0%, 15% and 30% of the total cellular protein in the B. subtilis strains carrying single cassettes with the Pgrac01, Pgrac100 or Pgrac212 promoters, respectively. The maximal induction ratio of Pgrac01-bgaB was 35.5 while that of Pgrac100-bgaB was 7.5 and that of Pgrac212-bgaB was 9. The inducible expression of GFP and BgaB protein was stably maintained for 24 h, with the highest yield of GFP being 24% of cell total protein while the maximum amount of BgaB was found to be 38%. A dual integration of two copies of the gfp+ gene into the B. subtilis genome at the lacA and amyE loci resulted in a yield of about 40% of total cellular protein and a 1.74-fold increase in GFP compared with single-integrated strains containing the same Pgrac212 promoter. The capability of protein production from low to high levels of these inducible integrative systems is useful for fundamental and applied research in B. subtilis.

Orofacial dysfunction screening examinations in children with sleep-disordered breathing symptoms.

Orofacial myofunctional disorders (OMD) and sleep-disordered breathing (SDB) may present as comorbidities. Orofacial characteristics might serve as a clinical marker of SDB, allowing early identification and management of OMD and improving treatment outcomes for sleep disorders. The study aims to characterize OMD in children with SDB symptoms and to investigate possible relationships between the presence of various components of OMD and symptoms of SDB. A cross-sectional study of healthy children aged 6-8 from primary schools was conducted in central Vietnam in 2019. SDB symptoms were collected using the parental Pediatric Sleep Questionnaire, Snoring Severity Scale, Epworth Daytime Sleepiness Scale, and lip-taping nasal breathing assessment. Orofacial myofunctional evaluation included assessment of tongue mobility, as well as of lip and tongue strength using the Iowa Oral Performance Instrument, and of orofacial characteristics by the protocol of Orofacial Myofunctional Evaluation with Scores. Statistical analysis was used to investigate the relationship between OMD components and SDB symptoms. 487 healthy children were evaluated, of whom 46.2% were female. There were 7.6% of children at high risk of SDB. Children with habitual snoring (10.3%) had an increased incidence of restricted tongue mobility and decreased lip and tongue strength. Abnormal breathing patterns (22.4%) demonstrated lower posterior tongue mobility and lower muscle strength. Daytime sleepiness symptoms were associated with changes in muscle strength, facial appearance, and impaired orofacial function. Lower strengths of lip and tongue or improper nasal breathing were more likely to be present in children with reported sleep apnea (6.6%). Neurobehavioral symptoms of inattention and hyperactivity were linked to anomalous appearance/posture, increases in tongue mobility and oral strength. This study demonstrates a prevalence of orofacial myofunctional anomalies in children exhibiting SDB symptoms. Children with prominent SDB symptoms should be considered as candidates for further orofacial myofunctional assessment.

Enhanced Fusobacterium nucleatum Genetics Using Host DNA Methyltransferases To Bypass Restriction-Modification Systems.

Bacterial restriction-modification (R-M) systems are a first-line immune defense against foreign DNA from viruses and other bacteria. While R-M systems are critical in maintaining genome integrity, R-M nucleases unfortunately present significant barriers to targeted genetic modification. Bacteria of the genus Fusobacterium are oral, Gram-negative, anaerobic, opportunistic pathogens that are implicated in the progression and severity of multiple cancers and tissue infections, yet our understanding of their direct roles in disease have been severely hindered by their genetic recalcitrance. Here, we demonstrate a path to overcome these barriers in Fusobacterium by using native DNA methylation as a host mimicry strategy to bypass R-M system cleavage of transformed plasmid DNA. We report the identification, characterization, and successful use of Fusobacterium nucleatum type II and III DNA methyltransferase (MTase) enzymes to produce a multifold increase in gene knockout efficiency in the strain Fusobacterium nucleatum subsp. nucleatum 23726, as well as the first system for efficient gene knockouts and complementations in F. nucleatum subsp. nucleatum 25586. We show plasmid protection can be accomplished in vitro with purified enzymes, as well as in vivo in an Escherichia coli host that constitutively expresses F. nucleatum subsp. nucleatum MTase enzymes. In summary, this proof-of-concept study characterizes specific MTases that are critical for bypassing R-M systems and has enhanced our understanding of enzyme combinations that could be used to genetically modify clinical isolates of Fusobacterium that have thus far been inaccessible to molecular characterization. IMPORTANCE Fusobacterium nucleatum is an oral opportunistic pathogen associated with diseases that include cancer and preterm birth. Our understanding of how this bacterium modulates human disease has been hindered by a lack of genetic systems. Here, we show that F. nucleatum DNA methyltransferase-modified plasmid DNA overcomes the transformation barrier and has allowed the development of a genetic system in a previously inaccessible strain. We present a strategy that could potentially be expanded to enable the genetic modification of highly recalcitrant strains, thereby fostering investigational studies to uncover novel host-pathogen interactions in Fusobacterium.

Neisseria gonorrhoeae FC428 Subclone, Vietnam, 2019-2020.

Among 114 clinical Neisseria gonorrhoeae isolates collected in Vietnam during 2019-2020, we detected 15 of subclone sequence type 13871 of the FC428 clonal complex. Fourteen sequence type 13871 isolates with mosaic penA allele 60.001 were ceftriaxone or cefixime nonsusceptible, and 3/14 were azithromycin nonsusceptible. Emergence of this subclone threatens treatment effectiveness.

ALS monocyte-derived microglia-like cells reveal cytoplasmic TDP-43 accumulation, DNA damage, and cell-specific impairment of phagocytosis associated with disease progression.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a multifactorial neurodegenerative disease characterised by the loss of upper and lower motor neurons. Increasing evidence indicates that neuroinflammation mediated by microglia contributes to ALS pathogenesis. This microglial activation is evident in post-mortem brain tissues and neuroimaging data from patients with ALS. However, the role of microglia in the pathogenesis and progression of amyotrophic lateral sclerosis remains unclear, partly due to the lack of a model system that is able to faithfully recapitulate the clinical pathology of ALS. To address this shortcoming, we describe an approach that generates monocyte-derived microglia-like cells that are capable of expressing molecular markers, and functional characteristics similar to in vivo human brain microglia. METHODS: In this study, we have established monocyte-derived microglia-like cells from 30 sporadic patients with ALS, including 15 patients with slow disease progression, 6 with intermediate progression, and 9 with rapid progression, together with 20 non-affected healthy controls. RESULTS: We demonstrate that patient monocyte-derived microglia-like cells recapitulate canonical pathological features of ALS including non-phosphorylated and phosphorylated-TDP-43-positive inclusions. Moreover, ALS microglia-like cells showed significantly impaired phagocytosis, altered cytokine profiles, and abnormal morphologies consistent with a neuroinflammatory phenotype. Interestingly, all ALS microglia-like cells showed abnormal phagocytosis consistent with the progression of the disease. In-depth analysis of ALS microglia-like cells from the rapid disease progression cohort revealed significantly altered cell-specific variation in phagocytic function. In addition, DNA damage and NOD-leucine rich repeat and pyrin containing protein 3 (NLRP3) inflammasome activity were also elevated in ALS patient monocyte-derived microglia-like cells, indicating a potential new pathway involved in driving disease progression. CONCLUSIONS: Taken together, our work demonstrates that the monocyte-derived microglia-like cell model recapitulates disease-specific hallmarks and characteristics that substantiate patient heterogeneity associated with disease subgroups. Thus, monocyte-derived microglia-like cells are highly applicable to monitor disease progression and can be applied as a functional readout in clinical trials for anti-neuroinflammatory agents, providing a basis for personalised treatment for patients with ALS.

Liquid chromatographic method to follow-up ceftazidime and pyridine in portable elastomeric infusion pumps over 24 h.

Portable infusion pumps are an interesting solution to continue outpatient parenteral antimicrobial therapy (OPAT) at the patient's home. However, the use of ceftazidime for such applications is challenging in view of its relatively poor stability in solution. In this study, elastomeric infusion pumps with 6 or 7 g of ceftazidime were deflated over 24 h in an oven at 33°C while ceftazidime and its degradation product, pyridine, were regularly monitored. Hereto, a fast and sensitive liquid chromatographic (LC) method has been developed using a Kinetex® C18 (150 × 3 mm, 2.6 µm) column with gradient elution. Ammonium formate 20 mM and acetonitrile (ACN) were mixed in a ratio of 98:2 v/v for mobile phase A and 85:15 v/v for mobile phase B. Both were adjusted to pH 4.5 with formic acid. The flow rate was set at 0.4 mL/min. The solution with a starting dose of 6 g ceftazidime was found to be degraded 10% after an average of 19 h 11 min so that an administration of 6 g to the patient was not reached. For the solution with a starting dose of 7 g of ceftazidime, 10% degradation was observed after an average of 18 h 42 min. However, by starting from a higher dose, an average of 6.56 g of ceftazidime could be administered over 24 h. In addition, 1.0% of pyridine versus ceftazidime pentahydrate with sodium carbonate (=mixture for injection) was formed over 24 h.