Biology subjects

Schwartz, J.

Publications and source records attributed to Schwartz, J..

31 records · Page 2Linked to original sources

bioRxiv2024

A Bacteriophage Cocktail Targeting Yersinia pestis Provides Strong Post-Exposure Protection in a Rat Pneumonic Plague Model

Yersinia pestis, one of the deadliest bacterial pathogens ever known, is responsible for three plague pandemics and several epidemics, with over 200 million deaths during recorded history. Due to high genomic plasticity, Y. pestis is amenable to genetic mutations as well as genetic engineering that can lead to the emergence or intentional development of pan-drug resistant strains. The dissemination of such Y. pestis strains could be catastrophic, with public health consequences far more daunting than those caused by the recent COVID-19 pandemic. Thus, there is an urgent need to develop novel, safe, and effective treatment approaches for managing Y. pestis infections. This includes infections by antigenically distinct strains for which vaccines, none FDA approved yet, may not be effective, and those that cannot be controlled by approved antibiotics. Lytic bacteriophages provide one such alternative approach. In this study, we examined post-exposure efficacy of a bacteriophage cocktail, YPP-401, to combat pneumonic plague caused by Y. pestis CO92. YPP-401 is a four-phage preparation with a 100% lytic activity against a panel of 68 genetically diverse Y. pestis strains. Using a pneumonic plague aerosol challenge model in gender-balanced Brown Norway rats, YPP-401 demonstrated [~]88% protection when delivered 18 hours post-exposure for each of two administration routes (i.e., intraperitoneal and intranasal) in a dose-dependent manner. Our studies suggest that YPP-401 could provide an innovative, safe, and effective approach for managing Y. pestis infections, including those caused by naturally occurring or intentionally developed strains that cannot be managed by vaccines in development and antibiotics.

microbiology↗

bioRxiv2023

Air pollution from biomass burning disrupts early adolescent cortical microarchitecture development

Exposure to outdoor particulate matter (PM2.5) represents a ubiquitous threat to human health, and particularly the neurotoxic effects of PM2.5 from multiple sources may disrupt neurodevelopment. Studies addressing neurodevelopmental implications of PM exposure have been limited by small, geographically limited samples and largely focus either on macroscale cortical morphology or postmortem histological staining and total PM mass. Here, we leverage residentially assigned exposure to six, data-driven sources of PM2.5 and neuroimaging data from the longitudinal Adolescent Brain Cognitive Development Study (ABCD Study(R)), collected from 21 different recruitment sites across the United States. To contribute an interpretable and actionable assessment of the role of air pollution in the developing brain, we identified alterations in cortical microstructure development associated with exposure to specific sources of PM2.5 using multivariate, partial least squares analyses. Specifically, average annual exposure (i.e., at ages 8-10 years) to PM2.5 from biomass burning was related to differences in neurite development across the cortex between 9 and 13 years of age. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/563430v3_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@eed0c1org.highwire.dtl.DTLVardef@cb3dbeorg.highwire.dtl.DTLVardef@1428660org.highwire.dtl.DTLVardef@8440aa_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2023

AAV-mediated Delivery of Plakophilin-2a Arrests Progression of Arrhythmogenic Right Ventricular Cardiomyopathy in Murine Hearts: Preclinical Evidence Supporting Gene Therapy in Humans

BackgroundPathogenic variants in plakophilin-2 (PKP2) cause arrhythmogenic right ventricular cardiomyopathy (ARVC), a disease characterized by life-threatening arrhythmias and progressive cardiomyopathy leading to heart failure. No effective medical therapy is available to prevent and/or arrest the disease. We tested the hypothesis that AAV-mediated delivery of the human PKP2 gene to an adult mammalian heart deficient in PKP2 can arrest disease progression and significantly prolong survival. MethodsExperiments were carried out using a cardiac-specific, tamoxifen (TAM)-activated PKP2 knockout murine model (PKP2-cKO). The potential therapeutic, AAVrh.74-PKP2a (RP-A601), is a recombinant AAVrh.74 gene therapy viral vector encoding the human PKP2 variant A (PKP2a). AAVrh.74-PKP2a was delivered to adult mice by a single tail vein injection either before or after TAM-activated PKP2-cKO. PKP2 expression was confirmed by molecular and histopathologic analyses. Cardiac function and disease progression were monitored by survival analyses, echocardiography and electrocardiography. ResultsConsistent with prior findings, loss of PKP2 expression caused 100% mortality within 50 days after TAM injection. In contrast, AAVrh.74-PKP2a-mediated PKP2a expression resulted in 100% survival for more than 5 months (at study termination). Echocardiographic analysis revealed that AAVrh.74-PKP2a prevented right ventricle dilation, arrested left ventricle functional decline, and mitigated arrhythmia burden. Molecular and histological analysis showed AAVrh.74-PKP2a- mediated transgene mRNA and protein expression and appropriate PKP2 localization at the cardiomyocyte intercalated disc. Importantly, therapeutic benefit was shown in mice receiving AAVrh.74-PKP2a after disease onset. ConclusionThese preclinical data demonstrate the potential for AAVrh.74-PKP2a (RP-A601) as a therapeutic for PKP2-related ARVC in both early and more advanced stages of disease.

physiology↗

bioRxiv2023

A rapid, multiplex digital PCR assay for EGFR, KRAS, BRAF, ERBB2 variants and ALK, RET, ROS1, NTRK1 gene fusions in non-small cell lung cancer

Digital PCR (dPCR) is emerging as an ideal platform for the detection and tracking of genomic variants in cancer due to its high sensitivity and simple workflow. The growing number of clinically-actionable cancer biomarkers creates a need for fast, accessible methods that allow for dense information content and high accuracy. Here, we describe a proof-of-concept amplitude modulation based multiplex dPCR assay capable of detecting 12 single nucleotide and indel variants in EGFR, KRAS, BRAF, and ERBB2, 14 gene fusions in ALK, RET, ROS1, NTRK1, and MET exon 14 skipping present in non-small cell lung cancer (NSCLC). We also demonstrate the use of multi-spectral target signal encoding to improve the specificity of variant detection by reducing background noise up to 11-fold. The assay reported an overall 100% PPA and 98.5% NPA compared to a sequencing-based assay in a cohort of 62 human FFPE samples. In addition, the dPCR assay rescued actionable information in 10 samples that failed to sequence, highlighting the utility of a multiplexed digital assay as a potential reflex solution for challenging NSCLC samples.

cancer biology↗

bioRxiv2023

B cell repertoire in children with skin barrier dysfunction supports altered IgE maturation associated with allergic food sensitization

IgE B cells produce antibodies responsible for the inappropriate specificity seen in allergic disease. In this study, we sequence antibody gene repertoires from a large, well-characterized early life cohort at risk for developing food allergy. We found that food allergen sensitization was associated with lower IgE mutation frequencies that was abrogated in children living with a pet dog, suggesting potential molecular mechanisms underlying the hygiene hypothesis (i.e., protective effects of pet ownership and non-antiseptic environs reported for allergic disease). We also observed increased IgE diversity and increased isotype-switching to IgE, suggesting that B cell development is altered in those with food allergen sensitizations relative to those without. IgE diversity was even more extreme in subjects who were highly likely to be peanut allergic. Unlike food antigen sensitization, we detected no effect of aeroallergen sensitization on IgE mutation levels and diversity. Consistent patterns of antibody rearrangement were associated with food allergen sensitization. These results have important implications for the allergic response driven by mast cells and basophils and the underlying drivers of the atopic march.

immunology↗

bioRxiv2022

Structure of Phosphorylated-like ClpXP Adaptor RssB Reveals an Interface Switch for Activation

SUMMARYIn {gamma}-proteobacteria such as Escherichia coli, the general stress response is mediated by {sigma}s, the stationary phase dissociable promoter specificity subunit of RNA polymerase. {sigma}s is degraded by ClpXP during active growth in a process dependent on the RssB adaptor, which acts catalytically and is thought to be stimulated by phosphorylation of a conserved aspartate in its N-terminal receiver domain. Here we present the crystal structure of full-length RssB bound to a beryllofluoride phosphomimic. Compared to the inhibited IraD anti-adaptor-bound RssB structure, our study reveals movements and coil-to-helix transitions in the C-terminal region of the RssB receiver domain and in the inter-domain segmented helical linker, accompanied by packing of the C-terminal effector domain onto the 4-{beta}5-5 (4-5-5) "signaling" face of the RssB receiver domain. This face is often the locus of protein-protein interactions in unphosphorylated receiver domains, but its masking is unusual in their phosphorylated forms. Our structure emphasizes the remarkable plasticity that underpins regulatory strategies within the large family of response regulators.

biophysics↗

bioRxiv2022

Open spaced ridged hydrogel scaffolds containing TiSAMP surface chemistry promotes regeneration and recovery following spinal cord injury.

The spinal cord has poor ability to regenerate after injury, which may be due to cell loss, cyst formation, inflammation, and scarring. A promising approach to treat spinal cord injury (SCI) is the use of biomaterials. We have developed a novel hydrogel scaffold fabricated from oligo(poly(ethylene glycol) fumarate) (OPF) as a 0.08 mm thick sheet containing polymer ridges and a cell-attractive surface chemistry on the other side. When the cells are cultured on OPF with the chemical patterning, the cells attach, align, and deposit ECM along the direction of the pattern. Animals implanted with the rolled scaffold sheets had greater hindlimb recovery compared to the multichannel scaffold control, likely due to the greater number of axons growing across. Inflammation, scarring, and ECM deposits were equal across conditions. Overall, the results suggest that the scaffold sheets promote axon outgrowth that can be guided across the scaffold, thereby promoting hindlimb recovery.

neuroscience↗

bioRxiv2021

Virtual partition digital PCR for high precision chromosomal counting applications

Digital PCR (dPCR) is the gold standard analytical platform for rapid high precision quantification of genomic fragments. However, current dPCR assays are generally limited to monitoring 1-2 analytes per sample, thereby limiting the platforms ability to address some clinical applications that require the simultaneous monitoring of 20 - 50 analytes per sample. Here we present Virtual Partition dPCR (VPdPCR), a novel analysis methodology enabling the detection of 10 or more target regions per color channel using conventional dPCR hardware and workflow. Furthermore, VPdPCR enables dPCR instruments to overcome upper quantitation limits caused by partitioning error. While traditional dPCR analysis establishes a single threshold to separate negative and positive partitions, VPdPCR establishes multiple thresholds to identify the number of unique targets present in each positive droplet based on fluorescent intensity. Each physical partition is then divided into a series of virtual partitions, and the resulting increase in partition count substantially decreases partitioning error. We present both a theoretical analysis of the advantages of VPdPCR and an experimental demonstration in the form of a 20-plex assay for non-invasive fetal aneuploidy testing. This demonstration assay - tested on 432 samples contrived from sheared cell-line DNA at multiple input concentrations and simulated fractions of euploid or trisomy-21 "fetal" DNA - is analyzed using both traditional dPCR thresholding and VPdPCR. VPdPCR analysis significantly lowers variance of chromosome ratio across replicates and increases the accuracy of trisomy identification when compared to traditional dPCR, yielding >98% single-well sensitivity and specificity. VPdPCR has substantial promise for increasing the utility of dPCR in applications requiring ultra-high-precision quantitation.

molecular biology↗

bioRxiv2021

Phospho-dependent Signaling during the General Stress Response by the Atypical Response Regulator and ClpXP Adaptor RssB

In the model organism Escherichia coli and related species, the general stress response relies on tight regulation of the intracellular levels of the promoter specificity subunit RpoS. RpoS turnover is exclusively dependent on RssB, a two-domain response regulator that functions as an adaptor that delivers RpoS to ClpXP for proteolysis. Here we report crystal structures of the receiver domain of RssB both in its unphosphorylated form and bound to the phosphomimic BeF3-. Surprisingly, we find only modest differences between these two structures, suggesting that truncating RssB may partially activate the receiver domain to a "meta-active" state. Our structural and sequence analysis points to RssB proteins not conforming to either the Y-T coupling scheme for signaling seen in prototypical response regulators, such as CheY, or to the signaling model of the less understood FATGUY proteins.

biophysics↗

bioRxiv2020

Haloferax volcanii immersed liquid biofilms develop independently of known biofilm machineries and exhibit rapid honeycomb pattern formation

The ability to form biofilms is shared by many microorganisms, including archaea. Cells in a biofilm are encased in extracellular polymeric substances that typically include polysaccharides, proteins, and extracellular DNA, conferring protection while providing a structure that allows for optimal nutrient flow. In many bacteria, flagella and evolutionarily conserved type IV pili are required for the formation of biofilms on solid surfaces or floating at the air-liquid interface of liquid media. Similarly, in many archaea it has been demonstrated that type IV pili and, in a subset of these species, archaella are required for biofilm formation on solid surfaces. Additionally, in the model archaeon Haloferax volcanii, chemotaxis and AglB-dependent glycosylation play important roles in this process. H. volcanii also forms immersed biofilms in liquid cultures poured into Petri dishes. This study reveals that mutants of this haloarchaeon that interfere with the biosynthesis of type IV pili or archaella, as well as a chemotaxis-targeting transposon and aglB-deletion mutants, lack obvious defects in biofilms formed in liquid cultures. Strikingly, we have observed that these liquid-based biofilms are capable of rearrangement into honeycomb-like patterns that rapidly form upon removal of the Petri dish lid, a phenomenon that is not dependent on changes in light or oxygen concentration but can be induced by controlled reduction of humidity. Taken together, this study demonstrates that H. volcanii requires novel, as-yet-unidentified strategies for immersed liquid biofilm formation and also exhibits rapid structural rearrangements. ImportanceThis first molecular biological study of archaeal immersed liquid biofilms advances our basic-biological understanding of the model archaeon Haloferax volcanii. Data gleaned from this study also provide an invaluable foundation for future studies to uncover components required for immersed liquid biofilms in this haloarchaeon and also potentially for liquid biofilm formation in general, which is poorly understood compared to the formation of biofilms on surfaces. Moreover, this first description of rapid honeycomb pattern formation is likely to yield novel insights into the underlying structural architecture of extracellular polymeric substances and cells within immersed liquid biofilms.

microbiology↗

bioRxiv2019

Promoting neuronal outgrowth using ridged scaffolds coated with extracellular matrix proteins.

Spinal cord injury (SCI) results in cell death, demyelination, and axonal loss. The spinal cord has a limited ability to regenerate and current clinical therapies for SCI are not effective in helping promote neurologic recovery. We have developed a novel scaffold biomaterial that is fabricated from the biodegradable hydrogel oligo[poly(ethylene glycol)fumarate] (OPF). We have previously shown that positively charged OPF scaffolds (OPF+) in an open spaced, multichannel design can be loaded with Schwann cells to support axonal generation and functional recovery following SCI. We have now developed a hybrid OPF+ biomaterial that increases the surface area available for cell attachment and that contains an aligned microarchitecture and extracellular matrix (ECM) proteins to better support axonal regeneration. OPF+ was fabricated as 0.08 mm thick sheets containing 100 m high polymer ridges that self-assembles into a spiral shape when hydrated. Laminin, fibronectin, or collagen I coating promoted neuron attachment and axonal outgrowth on the scaffold surface. In addition, the ridges aligned axons in a longitudinal bipolar orientation. Decreasing the space between the ridges increased the number of cells and neurites aligned in the direction of the ridge. Schwann cells seeded on laminin coated OPF+ sheets aligned along the ridges over a 6-day period and could myelinate dorsal root ganglion neurons over 4 weeks. The OPF+ sheets support axonal regeneration when implanted into the transected spinal cord. This novel scaffold design, with closer spaced ridges and Schwann cells is a novel biomaterial construct to promote regeneration after SCI.\n\nGraphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC=\"FIGDIR/small/788539v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (42K):\norg.highwire.dtl.DTLVardef@1ea0ae3org.highwire.dtl.DTLVardef@70e1f7org.highwire.dtl.DTLVardef@1034b77org.highwire.dtl.DTLVardef@14f784d_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2018

Implementation of an antibody characterization process: Application to the major ALS/FTD disease gene C9ORF72

Rigorously characterized antibodies are a key resource in biomedical research, yet there are no community-accepted processes to characterize the quality of research grade antibodies. This has led to a proliferation of poorly characterized antibodies of suspect quality, which in turn has led to flaws in the literature that hamper research progress, including the study of human disease. We selected the human protein C9ORF72 as a test case to implement a more standardized antibody characterization process. Mutations in the gene C9ORF72 are the major genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia but much remains unknown regarding the function of the encoded protein. We used CRISPR/Cas9-based knockout cells and knockout mice as controls to characterize 14 C9ORF72 antibodies including 12 commercially available antibodies advertised as selective for C9ORF72. We found one monoclonal antibody that is specific for the protein in immunoblot and that also recognizes C9ORF72 specifically in immunohistochemical applications on brain sections. A second C9ORF72 monoclonal antibody is effective for immunoprecipitation and immunofluorescence. Some of the antibodies that do not recognize C9ORF72 have been used in highly cited papers, raising concern over conclusions regarding previously reported C9ORF72 properties.

molecular biology↗

bioRxiv2018

Crowdsourced study of children with autism and their typically developing siblings identifies differences in taxonomic and predicted function for stool-associated microbes using exact sequence variant analysis.

BackgroundThe existence of a link between the gut microbiome and autism spectrum disorder (ASD) is well established in mice, but in human populations efforts to identify microbial biomarkers have been limited due to problems stratifying participants within the broad phenotype of ASD and a lack of appropriately matched controls. To overcome these limitations and investigate the relationship between ASD and the gut microbiome, we ran a crowdsourced study of families 2-7 year old sibling pairs, where one child of the pair had a diagnosis of ASD and the other child did not.\n\nMethodsParents of age-matched sibling pairs electronically consented and completed study procedures via a secure web portal (microbiome.stanford.edu). Parents collected stool samples from each child, responded to behavioral questionnaires about the ASD childs typical behavior, and whenever possible provided a home video of their ASD childs natural social behavior. We performed DNA extraction and 16S rRNA amplicon sequencing on 117 stool samples (60 ASD and 57 NT) that met all study design eligibility criteria. Using DADA2, Exact Sequence Variants (ESVs) were identified as taxonomic units, and three statistical tests were performed on ESV abundance counts: (1) permutation test to determine differences between sibling pairs, (2) differential abundance test using a zero-inflated gaussian mixture model to account for the sparse abundance matrix, and (3) differential abundance test after modeling under a negative binomial distribution. The potential functional gene abundance for each sample was also inferred from the 16S rRNA data, providing KEGG Ortholog (KO), which were analyzed for differential abundance.\n\nResultsIn total, 21 ESVs had significantly differentially proportions in stool of children with ASD and their neurotypical siblings. Of these 21 ESVs, 11 were enriched in neurotypical children and ten were enriched in children with ASD. ESVs enriched in the ASD cohort were predominantly associated with Ruminococcaceae and Bacteroidaceae; while those enriched in controls were more diverse including taxa associated with Bifidobacterium, Porphyromonas, Slackia, Desulfovibrio, Acinetobacter johnsonii, and Lachnospiraceae. Exact Variant Analysis suggested that Lachnospiraceae was specific to the control cohort, while Ruminococcaceae, Tissierellaceae and Bacteroidaceae were significantly enriched in children with ASD. Metabolic gene predictions determined that while both cohorts harbor the butyrogenic pathway, the ASD cohort was more likely to use the 4-aminobutanoate (4Ab) pathway, while the control cohort was more likely to use the pyruvate pathway. The 4Ab pathway releases harmful by-products like ammonia and can shunt glutamate, affecting its availability as an excitatory neurotransmitter. Finally, we observed differences in the carbohydrate uptake capabilities of various ESVs identified between the two cohorts.

microbiology↗