Biology subjects

Phan, J.

Publications and source records attributed to Phan, J..

9 recordsLinked to original sources

bioRxiv2026

Glutathione Protects Hematopoietic Stem Cells from Proteotoxic Stress

Glutathione is a redox buffer, best known for preventing oxidative stress and ferroptosis, but rarely studied in stem cells. We studied glutathione function in hematopoietic stem cells (HSCs) by deleting the glutamate-cysteine ligase catalytic subunit (GCLC), which synthesizes glutathione, or glutathione peroxidase 4 (GPX4), which employs glutathione to prevent ferroptosis. GCLC deficiency eliminated glutathione from hematopoietic cells, severely impairing HSC function and hematopoiesis. In contrast, GPX4 deficiency did not affect HSC function and had little effect on hematopoiesis, even after vitamin E depletion, suggesting glutathione has ferroptosis-independent functions in HSCs. Consistent with this, GCLC deficiency increased reactive oxygen species levels in hematopoietic cells. But treatment with antioxidants or ferroptosis inhibitors gave little or no rescue of HSC function despite reducing ROS levels. Rather, GCLC-deficient, but not GPX4-deficient, HSCs exhibited unfolded protein accumulation, proteostatic stress, and cell death marked by elevated p53 levels and Annexin V staining. Deficiency for p53 prolonged the function of GCLC-deficient HSCs after transplantation. Glutathione is thus necessary in HSCs for proteostasis but may be dispensable for ferroptosis inhibition in vivo.

cell biology↗

bioRxiv2024

Enhanced Suppression of Stenotrophomonas maltophilia by a Three-Phage Cocktail: Genomic Insights and Kinetic Profiling

In our era of rising antibiotic resistance, Stenotrophomonas maltophilia (STM) is an understudied, gram-negative, aerobic bacterium widespread in the environment and increasingly causing opportunistic infections. Treating STM infections remains difficult, leading to an increase in disease severity and higher hospitalization rates in people with Cystic Fibrosis (pwCF), cancer, and other immunocompromised health conditions. The lack of effective antibiotics has led to renewed interest in phage therapy; however, there is a need for well-characterized phages. In response to an oncology patient with a respiratory infection, we collected 18 phages from Southern California wastewater influent that exhibit different plaque morphology against STM host strain B28B, cultivated from a blood sample. Here, we characterize the genomes and life cycle kinetics of our STM phage collection. We hypothesize that genetically distinct phages give rise to unique lytic life cycles that can enhance bacterial killing when combined into a phage cocktail compared to the individual phages alone. We identified three genetically distinct clusters of phages, and a representative from each group was screened for potential therapeutic use and investigated for infection kinetics. The results demonstrated that the three-phage cocktail significantly suppressed bacterial growth compared to individual phages when observed for 48 hours. We also assessed the lytic impacts of our three-phage cocktail against a collection of 46 STM strains to determine if a multi-phage cocktail can expand the host range of individual phages. Our phages remained strain-specific and infect >50% of tested strains. The multi-phage cocktail maintains bacterial growth suppression and prevents the emergence of phage-resistant strains throughout our 40-hour assay. These findings suggest specialized phage cocktails may be an effective avenue of treatment for recalcitrant STM infections resistant to current antibiotics. IMPORTANCEPhage therapy could provide a vital strategy in the fight against antimicrobial resistance (AMR) bacterial infections; however, significant knowledge gaps remain. This study investigates phage cocktail development for the opportunistic pathogen Stenotrophomonas maltophilia (STM). Our findings contribute novel phages, their lytic characteristics, and limitations when exposed to an array of clinically relevant STM strains. Eighteen bacteriophages were isolated from wastewater influent from Escondido, California, and subjected to genomic analysis. We investigated genetically distinct phages to establish their infection kinetics and developed them into a phage cocktail. Our findings suggest that a genetically distinct STM phage cocktail provides an effective strategy for bacterial suppression of host strain B28B and five other clinically relevant STM strains. Phage therapy against STM remains poorly understood, as only 39 phages have been previously isolated. Future research into the underlying mechanism of how phage cocktails overwhelm the host bacteria will provide essential information that could aid in optimizing phage applications and impact alternative treatment options.

microbiology↗

bioRxiv2023

Finding Candida auris in public metagenomic repositories

Candida auris is a newly emerged multidrug-resistant fungus capable of causing invasive infections with high mortality. Despite intense efforts to understand how this pathogen rapidly emerged and spread worldwide, its environmental reservoirs are poorly understood. Here, we present a collaborative effort between the U.S. Centers for Disease Control and Prevention, the National Center for Biotechnology Information, and GridRepublic (a volunteer computing platform) to identify C. auris sequences in publicly available metagenomic datasets. We developed the MetaNISH pipeline that uses SRPRISM to align sequences to a set of reference genomes and computes a score for each reference genome. We used MetaNISH to scan [~]300,000 SRA metagenomic runs from 2010 onwards and identified five datasets containing C. auris reads. Finally, GridRepublic has implemented a prospective C. auris molecular monitoring system using MetaNISH and volunteer computing.

bioinformatics↗

bioRxiv2022

A Trem2*R47H mouse model without cryptic splicing drives age- and disease-dependent tissue damage and synaptic loss in response to plaques

Genome-Wide Association Studies revealed the TREM2 R47H variant as one of the strongest genetic risk factors for late-onset Alzheimers Disease (AD). Unfortunately, many current TREM2*R47H mouse models are associated with cryptic mRNA splicing of the mutant allele that produces a confounding reduction in protein product. We have developed the Trem2R47H NSS (Normal Splice Site) mouse model where the Trem2 allele is expressed at a similar level to the wild-type Trem2 allele, without evidence of cryptic splicing products, and appropriate inflammatory responses to cuprizone challenge. Utilizing the 5xFAD mouse model, we report age- and disease-dependent changes in response to pathology. At an early disease stage (4 mo), homozygous Trem2R47H NSS; hemizygous 5xFAD (Trem2R47H NSS ; 5xFAD) mice have reduced size and number of microglia plus impaired interaction with plaques, that is associated with increased dystrophic neurites and axonal damage detected through plasma neurofilament light chain (NfL) level and suppressed inflammation. However, homozygosity for Trem2R47H NSS suppressed LTP deficits and presynaptic puncta loss caused by the 5xFAD transgene array. At a more advanced disease stage (12 mo,) Trem2R47H NSS ; 5xFAD mice no longer display impaired plaque-microglia interaction or suppressed inflammatory gene expression, although NfL levels remain elevated, and a unique interferon-related gene expression signature is seen. Furthermore, Trem2R47H NSS ; 5xFAD mice also display robust LTP deficits and exacerbated presynaptic loss. Collectively, we provide a Trem2R47H variant mouse without cryptic splicing, and demonstrate it has disease stage dependent effects when combined with a plaque bearing model, with an initial loss of function that ultimately resolves, giving rise to a unique interferon signature and associated tissue damage.

neuroscience↗

bioRxiv2021

High-Plex Multiomic Analysis in FFPE Tissue at Single-Cellular and Subcellular Resolution by Spatial Molecular Imaging

The Spatial Molecular Imaging platform (CosMxTM SMI, NanoString Technologies, Seattle, WA) utilizes high-plex in-situ imaging chemistry for both RNA and protein detection. This automated instrument provides 1000s of plex, at high sensitivity (1 to 2 copies/cell), very low error rate (0.0092 false calls/cell) and background ([~]0.04 counts/cell). The imaging system generates three-dimensional super-resolution localization of analytes at [~]2 million cells per sample, four samples per run. Cell segmentation is morphology-based using antibodies, compatible with FFPE samples. Multiomic data (980 RNAs, 108 proteins) were measured at subcellular resolution using FFPE tissues (non-small cell lung (NSCLC) and breast cancer) and allowed identification of over 18 distinct cell types, 10 unique tumor microenvironments, and 100 pairwise ligand-receptor interactions. Over 800,000 single cells and [~]260 million transcripts data are released into the public domain allowing extended data analysis by the entire spatial biology research community.

genomics↗

bioRxiv2021

Systematic phenotyping and characterization of the 3xTg-AD mouse model of Alzheimer's Disease

Animal models of disease are valuable resources for investigating pathogenic mechanisms and potential therapeutic interventions. However, for complex disorders such as Alzheimers disease (AD), the generation and availability of innumerous distinct animal models present unique challenges to AD researchers and hinder the success of useful therapies. Here, we conducted an in-depth analysis of the 3xTg-AD mouse model of AD across its lifespan to better inform the field of the various pathologies that appear at specific ages, and comment on drift that has occurred in the development of pathology in this line since its development 20 years ago. This modern characterization of the 3xTg-AD model includes an assessment of impairments in behavior, cognition, and long-term potentiation followed by quantification of amyloid beta (A{beta}) plaque burden and neurofibrillary tau tangles, biochemical levels of A{beta} and tau protein, and neuropathological markers such as gliosis and accumulation of dystrophic neurites. We also present a novel comparison of the 3xTg-AD model with the 5xFAD model using the same deep-phenotyping characterization pipeline. The results from these analyses are freely available via the AD Knowledge Portal (https://admodelexplorer.synapse.org). Our work demonstrates the utility of a characterization pipeline that generates robust and standardized information relevant to investigating and comparing disease etiologies of current and future models of AD. Contribution to the Field StatementAlzheimers Disease (AD) is an age-related neurodegenerative disorder characterized by progressive memory impairments and affects more than 30 million individuals worldwide. Using animal models of AD, researchers have elucidated disease progression and hallmark pathologies that may underpin the memory impairments seen in patients. However, therapeutic targets have failed to translate successfully from animal studies to human clinical trials, prompting a reassessment of the development, use, and interpretation of data acquired using the innumerous AD animal models available to researchers. To address these shortcomings, we have developed a robust and reproducible modern characterization of pathologies within current and future animal models of AD to better assess distinct pathologies that arise at specific brain regions and ages of different models. Using the popular 3xTg-AD mouse, we demonstrate the utility of these deep-phenotyping analyses and highlight the drift that affected development of pathologies in this line over the past two decades. Utilizing this same systematic characterization, we also perform a direct comparison with 5xFAD mice, another popular animal model of AD. The robust and standardized data generated from these systematic deep-phenotyping analyses are available for broad use by the AD research community to assess, compare, and determine appropriate animal models of AD.

neuroscience↗

bioRxiv2021

Alterations in gut microbiome composition and function in irritable bowel syndrome and increased probiotic abundance with daily supplementation

BackgroundIrritable bowel syndrome (IBS) is characterized by abdominal discomfort and irregular bowel movements and stool consistency. Because there are different symptoms associated with IBS, it is difficult to diagnose the role of the microbiome in IBS. ObjectiveHere, we present a study that includes metagenomic sequencing of stool samples from subjects with the predominant subtypes of IBS and a healthy cohort. We collected longitudinal samples from individuals with IBS who took daily made-to-order precision probiotic and prebiotic supplementation throughout the study. Materials and MethodsThis study includes a population of 489 individuals with IBS and 122 healthy controls. All stool samples were subjected to shotgun metagenomic sequencing. Precision probiotics and prebiotics were formulated for all subjects with longitudinal timepoints. ResultsThere was significant variation explained in the microbiome between the healthy and IBS cohorts. Individuals with IBS had a lower gut microbiome diversity and reduced anti-inflammatory microbes compared to the healthy controls. Eubacterium rectale and Faecalibacterium prausnitzii were associated with healthy microbiomes while Shigella species were associated with IBS. Pathway analysis indicated a functional imbalance of short chain fatty acids, vitamins, and a microbial component of Gram-negative bacteria in IBS compared to healthy controls. In the longitudinal dataset, there was a significant difference in microbiome composition between timepoints 1 and 3. There was also a significant increase in the overall microbiome score and relative abundances of probiotic species used to target the symptoms associated with IBS. ConclusionsWe identified microbes and pathways that differentiate healthy and IBS microbiomes. In response to precision probiotic supplementation, we identified a significant improvement in the overall microbiome score in individuals with IBS. These results suggest an important role for probiotics in managing IBS symptoms and modulation of the microbiome as a potential management strategy. ImportanceAn estimated 35 million people in the United States and 11.5% of the population globally are affected by IBS. Immunity, genetics, environment, diet, small intestinal bacterial overgrowth (SIBO), and the gut microbiome are all factors that contribute to the onset or triggers of IBS. With strong supporting evidence that the gut microbiome may influence symptoms associated with IBS, elucidating the important microbes that contribute to the symptoms and severity is important to make decisions for targeted treatment. As probiotics have become more common in treating IBS symptoms, identifying effective probiotics may help inform future studies and treatment.

microbiology↗

bioRxiv2021

Systematic Phenotyping and Characterization of the 5xFAD mouse model of Alzheimer's Disease

Mouse models of human diseases are invaluable tools for studying pathogenic mechanisms and testing interventions and therapeutics. For disorders such as Alzheimers disease in which numerous models are being generated, a challenging first step is to identify the most appropriate model and age to effectively evaluate new therapeutic approaches. Here we conducted a detailed phenotypic characterization of the 5xFAD model on a congenic C57BL/6J strain background, across its lifespan - including a seldomly analyzed 18-month old time point to provide temporally correlated phenotyping of this model and a template for characterization of new models of LOAD as they are generated. This comprehensive analysis included quantification of plaque burden, A{beta} biochemical levels, and neuropathology, neurophysiological measurements and behavioral and cognitive assessments, and evaluation of microglia, astrocytes, and neurons. Analysis of transcriptional changes was conducted using bulk-tissue generated RNA-seq data from microdissected cortices and hippocampi as a function of aging, which can be explored at the UCI Mouse Explorer and AD Knowledge Portal. This deep-phenotyping pipeline identified novel aspects of age-related pathology in the 5xFAD model.

neuroscience↗

bioRxiv2021

Capturing actively produced microbial volatile organic compounds from human associated samples with vacuum assisted sorbent extraction

Volatile organic compounds (VOC) from biological samples have unknown origins. VOCs may originate from the host or different microbial organisms from a microbial community. In order to disentangle the origin of microbial VOCs, we performed volatile headspace analysis of bacterial mono- and co-cultures of Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii, and stable isotope probing in biological samples of feces, saliva, sewage, and sputum. We utilized mono- and co-cultures to identify volatile production from individual bacterial species or in combination with stable isotope probing to identify the active metabolism of microbes from the biological samples. To extract the VOCs, we employed vacuum assisted sorbent extraction (VASE). VASE is an easy-to-use commercialized solvent-free headspace extraction method for semi-volatile and volatile compounds. The lack of solvents and the near vacuum conditions used make developing a method relatively easy and fast when compared to other extraction options like tert-butylation and solid phase microextraction. However, VASE does not work on nonvolatile compounds, thus excluding many protein analyses and heavy stable isotope labelling experiments. Using the workflow described here, we identified volatile signatures from mono- and co-cultures indicating there were volatiles specific to certain microbes or co-cultures. Furthermore, analysis of the stable isotope probing of biological samples identified VOCs that were either commonly or uniquely produced from the different human derived biological samples. Here we present the general workflow and experimental considerations of VASE. SUMMARYFrom this protocol, readers will be able to extract volatile organic compounds from a biological sample with the vacuum assisted sorbent extraction method, run samples on a GC-MS with the use of the Entech autosampler, and analyze data.

biochemistry↗