Publications by authors named "David J Gonzalez"

78 Publications

Case Series of Successful Intravenous Immunoglobulin (IVIG) Treatment in 4 Pregnant Patients with Severe COVID-19-Induced Hypoxia.

Am J Case Rep 2022 May 14;23:e936734. Epub 2022 May 14.

Department of Research, Sharp Healthcare, San Diego, CA, USA.

BACKGROUND Despite unprecedented speed in the execution of the COVID-19 vaccine and therapeutic clinical trials, pregnant patients have been largely excluded from initial studies. In addition, pregnant patients who are unvaccinated against SARS-CoV-2 have greater morbidity risk with severe COVID-19 disease as compared to patients of similar age and comorbidity status. Intravenous immunoglobulin (IVIG) has been deemed safe in pregnancy in other diseases. Prior data demonstrate the possible benefit of utilizing IVIG for the treatment in hospitalized patients with severe respiratory symptoms associated with COVID-19 active infections when administered within 14 days of COVID symptom onset. CASE REPORT We administered IVIG (Privigen®, CSL Behring) 0.5 g/kg daily for 3 consecutive days to 4 pregnant patients (ages 24-34 years of age) who were hospitalized with moderate-to-severe COVID-19 and not vaccinated against SARS-CoV-2. All patients received concomitant glucocorticoid therapy. Gestational ages were 26, 17, 35, and 35 weeks. All patients were discharged home breathing room air after a mean hospital stay of 15 days. Two patients had uncomplicated cesarean section at 35 weeks during the hospitalization. The pre-term pregnancies at 17 and 26 weeks were intact at hospital discharge and resulted in normal vaginal deliveries at term. All 4 patients consented to participate in this case series report. CONCLUSIONS IVIG may be a safe treatment consideration in pregnant women with severe COVID-19 to avoid pregnancy complications. Its use warrants further study in pregnancy acute respiratory distress syndrome (ARDS) due to SARS-CoV-2, influenza, and other respiratory viruses to which pregnant patients are vulnerable.
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http://dx.doi.org/10.12659/AJCR.936734DOI Listing
May 2022

Historic redlining and the siting of oil and gas wells in the United States.

J Expo Sci Environ Epidemiol 2022 Apr 13. Epub 2022 Apr 13.

Department of Environmental Health Sciences, Columbia University Mailman School of Public Health, New York, NY, USA.

Background: The presence of active or inactive (i.e., postproduction) oil and gas wells in neighborhoods may contribute to ongoing pollution. Racially discriminatory neighborhood security maps developed by the Home-Owners Loan Corporation (HOLC) in the 1930s may contribute to environmental exposure disparities.

Objective: To determine whether receiving worse HOLC grades was associated with exposure to more oil and gas wells.

Methods: We assessed exposure to oil and gas wells among HOLC-graded neighborhoods in 33 cities from 13 states where urban oil and gas wells were drilled and operated. Among the 17 cities for which 1940 census data were available, we used propensity score restriction and matching to compare well exposure neighborhoods that were similar on observed 1940 sociodemographic characteristics but that received different grades.

Results: Across all included cities, redlined D-graded neighborhoods had 12.2 ± 27.2 wells km, nearly twice the density in neighborhoods graded A (6.8 ± 8.9 wells km). In propensity score restricted and matched analyses, redlined neighborhoods had 2.0 (1.3, 2.7) more wells than comparable neighborhoods with a better grade.

Significance: Our study adds to the evidence that structural racism in federal policy is associated with the disproportionate siting of oil and gas wells in marginalized neighborhoods.
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http://dx.doi.org/10.1038/s41370-022-00434-9DOI Listing
April 2022

The Host-Microbiome Response to Hyperbaric Oxygen Therapy in Ulcerative Colitis Patients.

Cell Mol Gastroenterol Hepatol 2022 Apr 1;14(1):35-53. Epub 2022 Apr 1.

Department of Pathology, University of California San Diego, San Diego, California; Center for Microbiome Innovation, University of California San Diego, San Diego, California; Chiba University-UC San Diego Center for Mucosal Immunology, Allergy and Vaccines (cMAV), University of California, San Diego, La Jolla, California.

Background & Aims: Hyperbaric oxygen therapy (HBOT) is a promising treatment for moderate-to-severe ulcerative colitis. However, our current understanding of the host and microbial response to HBOT remains unclear. This study examined the molecular mechanisms underpinning HBOT using a multi-omic strategy.

Methods: Pre- and post-intervention mucosal biopsies, tissue, and fecal samples were collected from HBOT phase 2 clinical trials. Biopsies and fecal samples were subjected to shotgun metaproteomics, metabolomics, 16s rRNA sequencing, and metagenomics. Tissue was subjected to bulk RNA sequencing and digital spatial profiling (DSP) for single-cell RNA and protein analysis, and immunohistochemistry was performed. Fecal samples were also used for colonization experiments in IL10 germ-free UC mouse models.

Results: Proteomics identified negative associations between HBOT response and neutrophil azurophilic granule abundance. DSP identified an HBOT-specific reduction of neutrophil STAT3, which was confirmed by immunohistochemistry. HBOT decreased microbial diversity with a proportional increase in Firmicutes and a secondary bile acid lithocholic acid. A major source of the reduction in diversity was the loss of mucus-adherent taxa, resulting in increased MUC2 levels post-HBOT. Targeted database searching revealed strain-level associations between Akkermansia muciniphila and HBOT response status. Colonization of IL10 with stool obtained from HBOT responders resulted in lower colitis activity compared with non-responders, with no differences in STAT3 expression, suggesting complementary but independent host and microbial responses.

Conclusions: HBOT reduces host neutrophil STAT3 and azurophilic granule activity in UC patients and changes in microbial composition and metabolism in ways that improve colitis activity. Intestinal microbiota, especially strain level variations in A muciniphila, may contribute to HBOT non-response.
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http://dx.doi.org/10.1016/j.jcmgh.2022.03.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9117812PMC
April 2022

Phosphoproteomic analysis of thrombin- and p38 MAPK-regulated signaling networks in endothelial cells.

J Biol Chem 2022 04 4;298(4):101801. Epub 2022 Mar 4.

Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, California, USA. Electronic address:

Endothelial dysfunction is a hallmark of inflammation and is mediated by inflammatory factors that signal through G protein-coupled receptors including protease-activated receptor-1 (PAR1). PAR1, a receptor for thrombin, signals via the small GTPase RhoA and myosin light chain intermediates to facilitate endothelial barrier permeability. PAR1 also induces endothelial barrier disruption through a p38 mitogen-activated protein kinase-dependent pathway, which does not integrate into the RhoA/MLC pathway; however, the PAR1-p38 signaling pathways that promote endothelial dysfunction remain poorly defined. To identify effectors of this pathway, we performed a global phosphoproteome analysis of thrombin signaling regulated by p38 in human cultured endothelial cells using multiplexed quantitative mass spectrometry. We identified 5491 unique phosphopeptides and 2317 phosphoproteins, four distinct dynamic phosphoproteome profiles of thrombin-p38 signaling, and an enrichment of biological functions associated with endothelial dysfunction, including modulators of endothelial barrier disruption and a subset of kinases predicted to regulate p38-dependent thrombin signaling. Using available antibodies to detect identified phosphosites of key p38-regulated proteins, we discovered that inhibition of p38 activity and siRNA-targeted depletion of the p38α isoform increased basal phosphorylation of extracellular signal-regulated protein kinase 1/2, resulting in amplified thrombin-stimulated extracellular signal-regulated protein kinase 1/2 phosphorylation that was dependent on PAR1. We also discovered a role for p38 in the phosphorylation of α-catenin, a component of adherens junctions, suggesting that this phosphorylation may function as an important regulatory process. Taken together, these studies define a rich array of thrombin- and p38-regulated candidate proteins that may serve important roles in endothelial dysfunction.
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http://dx.doi.org/10.1016/j.jbc.2022.101801DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8987612PMC
April 2022

Multi-omics analyses of the ulcerative colitis gut microbiome link Bacteroides vulgatus proteases with disease severity.

Nat Microbiol 2022 02 27;7(2):262-276. Epub 2022 Jan 27.

Department of Pharmacology, University of California, San Diego, CA, USA.

Ulcerative colitis (UC) is driven by disruptions in host-microbiota homoeostasis, but current treatments exclusively target host inflammatory pathways. To understand how host-microbiota interactions become disrupted in UC, we collected and analysed six faecal- or serum-based omic datasets (metaproteomic, metabolomic, metagenomic, metapeptidomic and amplicon sequencing profiles of faecal samples and proteomic profiles of serum samples) from 40 UC patients at a single inflammatory bowel disease centre, as well as various clinical, endoscopic and histologic measures of disease activity. A validation cohort of 210 samples (73 UC, 117 Crohn's disease, 20 healthy controls) was collected and analysed separately and independently. Data integration across both cohorts showed that a subset of the clinically active UC patients had an overabundance of proteases that originated from the bacterium Bacteroides vulgatus. To test whether B. vulgatus proteases contribute to UC disease activity, we first profiled B. vulgatus proteases found in patients and bacterial cultures. Use of a broad-spectrum protease inhibitor improved B. vulgatus-induced barrier dysfunction in vitro, and prevented colitis in B. vulgatus monocolonized, IL10-deficient mice. Furthermore, transplantation of faeces from UC patients with a high abundance of B. vulgatus proteases into germfree mice induced colitis dependent on protease activity. These results, stemming from a multi-omics approach, improve understanding of functional microbiota alterations that drive UC and provide a resource for identifying other pathways that could be inhibited as a strategy to treat this disease.
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http://dx.doi.org/10.1038/s41564-021-01050-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8852248PMC
February 2022

Comparative Analysis of T-Cell Spatial Proteomics and the Influence of HIV Expression.

Mol Cell Proteomics 2022 Mar 8;21(3):100194. Epub 2022 Jan 8.

Biomedical Sciences Doctoral Program, University of California San Diego, La Jolla, California, USA; School of Medicine, University of California San Diego, La Jolla, California, USA; Veterans Medical Research Foundation, La Jolla, California, USA; VA San Diego Healthcare System, La Jolla, California, USA.

As systems biology approaches to virology have become more tractable, highly studied viruses such as HIV can now be analyzed in new unbiased ways, including spatial proteomics. We employed here a differential centrifugation protocol to fractionate Jurkat T cells for proteomic analysis by mass spectrometry; these cells contain inducible HIV-1 genomes, enabling us to look for changes in the spatial proteome induced by viral gene expression. Using these proteomics data, we evaluated the merits of several reported machine learning pipelines for classification of the spatial proteome and identification of protein translocations. From these analyses, we found that classifier performance in this system was organelle dependent, with Bayesian t-augmented Gaussian mixture modeling outperforming support vector machine learning for mitochondrial and endoplasmic reticulum proteins but underperforming on cytosolic, nuclear, and plasma membrane proteins by QSep analysis. We also observed a generally higher performance for protein translocation identification using a Bayesian model, Bayesian analysis of differential localization experiments, on row-normalized data. Comparative Bayesian analysis of differential localization experiment analysis of cells induced to express the WT viral genome versus cells induced to express a genome unable to express the accessory protein Nef identified known Nef-dependent interactors such as T-cell receptor signaling components and coatomer complex. Finally, we found that support vector machine classification showed higher consistency and was less sensitive to HIV-dependent noise. These findings illustrate important considerations for studies of the spatial proteome following viral infection or viral gene expression and provide a reference for future studies of HIV-gene-dropout viruses.
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http://dx.doi.org/10.1016/j.mcpro.2022.100194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8956815PMC
March 2022

Upstream oil and gas production and ambient air pollution in California.

Sci Total Environ 2022 Feb 11;806(Pt 1):150298. Epub 2021 Sep 11.

Department of Earth System Science, School of Earth, Energy and Environmental Sciences, Stanford University, Stanford, CA, USA.

Background: Prior studies have found that residential proximity to upstream oil and gas production is associated with increased risk of adverse health outcomes. Emissions of ambient air pollutants from oil and gas wells in the preproduction and production stages have been proposed as conferring risk of adverse health effects, but the extent of air pollutant emissions and resulting nearby pollution concentrations from wells is not clear.

Objectives: We examined the effects of upstream oil and gas preproduction (count of drilling sites) and production (total volume of oil and gas) activities on concentrations of five ambient air pollutants in California.

Methods: We obtained data on approximately 1 million daily observations from 314 monitors in the EPA Air Quality System, 2006-2019, including daily concentrations of five routinely monitored ambient air pollutants: PM, CO, NO, O, and VOCs. We obtained data on preproduction and production operations from Enverus and the California Geographic Energy Management Division (CalGEM) for all wells in the state. For each monitor and each day, we assessed exposure to upwind preproduction wells and total oil and gas production volume within 10 km. We used a panel regression approach in the analysis and fit adjusted fixed effects linear regression models for each pollutant, controlling for geographic, seasonal, temporal, and meteorological factors.

Results: We observed higher concentrations of PM and CO at monitors within 3 km of preproduction wells, NO at monitors at 1-2 km, and O at 2-4 km from the wells. Monitors with proximity to increased production volume observed higher concentrations of PM, NO, and VOCs within 1 km and higher O concentrations at 1-2 km. Results were robust to sensitivity analyses.

Conclusion: Adjusting for geographic, meteorological, seasonal, and time-trending factors, we observed higher concentrations of ambient air pollutants at air quality monitors in proximity to preproduction wells within 4 km and producing wells within 2 km.
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http://dx.doi.org/10.1016/j.scitotenv.2021.150298DOI Listing
February 2022

A combined EM and proteomic analysis places HIV-1 Vpu at the crossroads of retromer and ESCRT complexes: PTPN23 is a Vpu-cofactor.

PLoS Pathog 2021 11 29;17(11):e1009409. Epub 2021 Nov 29.

Department of Medicine, University of California, San Diego School of Medicine and Veterans Affairs San Diego Healthcare System, La Jolla, California, United States of America.

The HIV-1 accessory protein Vpu modulates membrane protein trafficking and degradation to provide evasion of immune surveillance. Targets of Vpu include CD4, HLAs, and BST-2. Several cellular pathways co-opted by Vpu have been identified, but the picture of Vpu's itinerary and activities within membrane systems remains incomplete. Here, we used fusion proteins of Vpu and the enzyme ascorbate peroxidase (APEX2) to compare the ultrastructural locations and the proximal proteomes of wild type Vpu and Vpu-mutants. The proximity-omes of the proteins correlated with their ultrastructural locations and placed wild type Vpu near both retromer and ESCRT-0 complexes. Hierarchical clustering of protein abundances across the mutants was essential to interpreting the data and identified Vpu degradation-targets including CD4, HLA-C, and SEC12 as well as Vpu-cofactors including HGS, STAM, clathrin, and PTPN23, an ALIX-like protein. The Vpu-directed degradation of BST-2 was supported by STAM and PTPN23 and to a much lesser extent by the retromer subunits Vps35 and SNX3. PTPN23 also supported the Vpu-directed decrease in CD4 at the cell surface. These data suggest that Vpu directs targets from sorting endosomes to degradation at multi-vesicular bodies via ESCRT-0 and PTPN23.
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http://dx.doi.org/10.1371/journal.ppat.1009409DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8659692PMC
November 2021

Multi-omics of human plasma reveals molecular features of dysregulated inflammation and accelerated aging in schizophrenia.

Mol Psychiatry 2022 Feb 5;27(2):1217-1225. Epub 2021 Nov 5.

Department of Pharmacology, University of California, San Diego, CA, 92093, USA.

Schizophrenia is a devastating psychiatric illness that detrimentally affects a significant portion of the worldwide population. Aging of schizophrenia patients is associated with reduced longevity, but the potential biological factors associated with aging in this population have not yet been investigated in a global manner. To address this gap in knowledge, the present study assesses proteomics and metabolomics profiles in the plasma of subjects afflicted with schizophrenia compared to non-psychiatric control patients over six decades of life. Global, unbiased analyses of circulating blood plasma can provide knowledge of prominently dysregulated molecular pathways and their association with schizophrenia, as well as features of aging and gender in this disease. The resulting data compiled in this study represent a compendium of molecular changes associated with schizophrenia over the human lifetime. Supporting the clinical finding of schizophrenia's association with more rapid aging, both schizophrenia diagnosis and age significantly influenced the plasma proteome in subjects assayed. Schizophrenia was broadly associated with prominent dysregulation of inflammatory and metabolic system components. Proteome changes demonstrated increased abundance of biomarkers for risk of physiologic comorbidities of schizophrenia, especially in younger individuals. These findings advance our understanding of the molecular etiology of schizophrenia and its associated comorbidities throughout the aging process.
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http://dx.doi.org/10.1038/s41380-021-01339-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9054664PMC
February 2022

The S Protein of Group B Is a Critical Virulence Determinant That Impacts the Cell Surface Virulome.

Front Microbiol 2021 14;12:729308. Epub 2021 Oct 14.

Department of Pharmacology, University of California San Diego, La Jolla, CA, United States.

Group B (GBS, ) is a human commensal and occasional pathogen that remains a leading cause of neonatal sepsis and meningitis with increasing disease burden in adult populations. Although programs for universal screening in pregnancy to guide intrapartum prophylaxis have reduced GBS invasive disease burden resulting from mother-to-newborn transfer during birth, better knowledge of disease mechanisms may elucidate new strategies to reduce antibiotic exposure. In our efforts to expand the knowledge base required for targeted anti-virulence therapies, we identified a GBS homolog for a recently identified virulence determinant of group A , S protein, and evaluated its role in GBS pathogenesis. A GBS S protein deletion mutant, Δ, showed altered cell-surface properties compared to the WT parent strain, including defective retention of its surface polysaccharide. Quantitative proteome analysis of enzymatically shaved surface epitopes of the GBS Δ mutant revealed a dysregulated cell surface virulome, with reduced abundance of several protein and glycoprotein components. The Δ mutant showed markedly attenuated virulence in a murine model of GBS systemic infection, with increased proteasome activity detected in the spleens of animals infected with the Δ mutant. These results expand the key roles S protein plays in streptococcal pathogenesis and introduces a new GBS virulence determinant and potential target for therapy development.
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http://dx.doi.org/10.3389/fmicb.2021.729308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8551713PMC
October 2021

Antimicrobials from a feline commensal bacterium inhibit skin infection by drug-resistant .

Elife 2021 10 19;10. Epub 2021 Oct 19.

Department of Dermatology, University of California, San Diego, San Diego, United States.

Methicillin-resistant (MRSP) is an important emerging zoonotic pathogen that causes severe skin infections. To combat infections from drug-resistant bacteria, the transplantation of commensal antimicrobial bacteria as a therapeutic has shown clinical promise. We screened a collection of diverse staphylococcus species from domestic dogs and cats for antimicrobial activity against MRSP. A unique strain ( C4) was isolated from feline skin that inhibited MRSP and multiple gram-positive pathogens. Whole genome sequencing and mass spectrometry revealed several secreted antimicrobials including a thiopeptide bacteriocin micrococcin P1 and phenol-soluble modulin beta (PSMβ) peptides that exhibited antimicrobial and anti-inflammatory activity. Fluorescence and electron microscopy revealed that antimicrobials inhibited translation and disrupted bacterial but not eukaryotic cell membranes. Competition experiments in mice showed that significantly reduced MRSP skin colonization and an antimicrobial extract from significantly reduced necrotic skin injury from MRSP infection. These findings indicate a feline commensal bacterium that could be utilized in bacteriotherapy against difficult-to-treat animal and human skin infections.
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http://dx.doi.org/10.7554/eLife.66793DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8592530PMC
October 2021

Unique virulence role of post-translocational chaperone PrsA in shaping secretome.

Virulence 2021 12;12(1):2633-2647

Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Taipei, Taiwan.

(group A , GAS) is a strict human pathogen causing a broad spectrum of diseases and a variety of autoimmune sequelae. The pathogenesis of GAS infection mostly relies on the production of an extensive network of cell wall-associated and secreted virulence proteins, such as adhesins, toxins, and exoenzymes. PrsA, the only extracellular parvulin-type peptidyl-prolyl isomerase expressed ubiquitously in Gram-positive bacteria, has been suggested to assist the folding and maturation of newly exported proteins to acquire their native conformation and activity. Two PrsA proteins, PrsA1 and PrsA2, have been identified in GAS, but the respective contribution of each PrsA in GAS pathogenesis remains largely unknown. By combining comparative proteomic and phenotypic analysis approaches, we demonstrate that both PrsA isoforms are required to maintain GAS proteome homeostasis and virulence-associated traits in a unique and overlapping manner. The inactivation of both PrsA in GAS caused remarkable impairment in biofilm formation, host adherence, infection-induced cytotoxicity, and virulence in a murine soft tissue infection model. The concordance of proteomic and phenotypic data clearly features the essential role of PrsA in GAS full virulence.
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http://dx.doi.org/10.1080/21505594.2021.1982501DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8489961PMC
December 2021

Heat shock protein 27 activity is linked to endothelial barrier recovery after proinflammatory GPCR-induced disruption.

Sci Signal 2021 Aug 31;14(698):eabc1044. Epub 2021 Aug 31.

Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093, USA.

Vascular inflammation causes endothelial barrier disruption and tissue edema. Several inflammatory mediators act through G protein–coupled receptors (GPCRs), including protease-activated receptor-1 (PAR1), to elicit inflammatory responses. The activation of PAR1 by its ligand thrombin stimulates proinflammatory, p38 mitogen-activated protein kinase (MAPK) signaling that promotes endothelial barrier disruption. Through mass spectrometry phosphoproteomics, we identified heat shock protein 27 (HSP27), which exists as a large oligomer that binds to actin, as a promising candidate for the p38-mediated regulation of barrier integrity. Depletion of HSP27 by siRNA enhanced endothelial cell barrier permeability and slowed recovery after thrombin stimulation. We further showed that two effector kinases of p38 MAPK, MAPKAPK2 (MK2) and MAPKAPK3 (MK3), differentially phosphorylated HSP27 at Ser, Ser, and Ser. Whereas inhibition of thrombin-stimulated p38 activation blocked HSP27 phosphorylation at all three sites, inhibition of MK2 reduced the phosphorylation of only Ser and Ser. Inhibition of both MK2 and MK3 was necessary to attenuate Ser phosphorylation. Thrombin-stimulated p38-MK2-MK3 signaling induced HSP27 oligomer disassembly. However, a phosphorylation-deficient mutant of HSP27 exhibited defective oligomer disassembly and altered the dynamics of barrier recovery after thrombin stimulation. Moreover, blocking HSP27 oligomer reassembly with the small-molecule inhibitor J2 enhanced endothelial barrier permeability in vitro and vascular leakage in vivo in response to PAR1 activation. These studies reveal the distinct regulation of HSP27 phosphorylation and function induced by the GPCR-stimulated p38-MK2-MK3 signaling axis that controls the dynamics of endothelial barrier recovery in vitro and vascular leakage in vivo.
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http://dx.doi.org/10.1126/scisignal.abc1044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8538426PMC
August 2021

The α-Arrestin ARRDC3 Is an Emerging Multifunctional Adaptor Protein in Cancer.

Antioxid Redox Signal 2022 May 4;36(13-15):1066-1079. Epub 2022 Jan 4.

Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, California, USA.

Adaptor proteins control the spatiotemporal dynamics of cellular signaling. Dysregulation of adaptor protein function can cause aberrant cell signaling and promote cancer. The arrestin family of adaptor proteins are known to regulate signaling by the superfamily of G protein-coupled receptors (GPCRs). The GPCRs are highly druggable and implicated in cancer progression. However, the molecular mechanisms responsible for arrestin dysregulation and the impact on GPCR function in cancer have yet to be fully elucidated. A new family of mammalian arrestins, termed the α-arrestins, was recently discovered. The α-arrestin, arrestin domain-containing protein 3 (ARRDC3), in particular, has been identified as a tumor suppressor and is reported to control cellular signaling of GPCRs in cancer. Compared with the extensively studied mammalian β-arrestins, there is limited information regarding the regulatory mechanisms that control α-arrestin activation and function. Here, we discuss the molecular mechanisms that regulate ARRDC3, which include post-translational modifications such as phosphorylation and ubiquitination. We also provide evidence that ARRDC3 can interact with a wide array of proteins that control diverse biological functions. ARRDC3 interacts with numerous proteins and is likely to display diverse functions in cancer, metabolic disease, and other syndromes. Thus, understanding the regulatory mechanisms of ARRDC3 activity in various cellular contexts is critically important. Recent studies suggest that α-arrestins may be regulated through post-translational modification, which is known to impact adaptor protein function. However, additional studies are needed to determine how these regulatory mechanisms affect ARRDC3 tumor suppressor function. 36, 1066-1079.
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http://dx.doi.org/10.1089/ars.2021.0193DOI Listing
May 2022

A Cdk4/6-dependent phosphorylation gradient regulates the early to late G1 phase transition.

Sci Rep 2021 07 19;11(1):14736. Epub 2021 Jul 19.

Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA.

During early G1 phase, Rb is exclusively mono-phosphorylated by cyclin D:Cdk4/6, generating 14 different isoforms with specific binding patterns to E2Fs and other cellular protein targets. While mono-phosphorylated Rb is dispensable for early G1 phase progression, interfering with cyclin D:Cdk4/6 kinase activity prevents G1 phase progression, questioning the role of cyclin D:Cdk4/6 in Rb inactivation. To dissect the molecular functions of cyclin D:Cdk4/6 during cell cycle entry, we generated a single cell reporter for Cdk2 activation, RB inactivation and cell cycle entry by CRISPR/Cas9 tagging endogenous p27 with mCherry. Through single cell tracing of Cdk4i cells, we identified a time-sensitive early G1 phase specific Cdk4/6-dependent phosphorylation gradient that regulates cell cycle entry timing and resides between serum-sensing and cyclin E:Cdk2 activation. To reveal the substrate identity of the Cdk4/6 phosphorylation gradient, we performed whole proteomic and phospho-proteomic mass spectrometry, and identified 147 proteins and 82 phospho-peptides that significantly changed due to Cdk4 inhibition in early G1 phase. In summary, we identified novel (non-Rb) cyclin D:Cdk4/6 substrates that connects early G1 phase functions with cyclin E:Cdk2 activation and Rb inactivation by hyper-phosphorylation.
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http://dx.doi.org/10.1038/s41598-021-94200-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8290049PMC
July 2021

mTORC2 controls the activity of PKC and Akt by phosphorylating a conserved TOR interaction motif.

Sci Signal 2021 04 13;14(678). Epub 2021 Apr 13.

Department of Pharmacology, University of California at San Diego, La Jolla, CA 92093, USA.

The complex mTORC2 is accepted to be the kinase that controls the phosphorylation of the hydrophobic motif, a key regulatory switch for AGC kinases, although whether mTOR directly phosphorylates this motif remains controversial. Here, we identified an mTOR-mediated phosphorylation site that we termed the TOR interaction motif (TIM; F-x-F-pT), which controls the phosphorylation of the hydrophobic motif of PKC and Akt and the activity of these kinases. The TIM is invariant in mTORC2-dependent AGC kinases, is evolutionarily conserved, and coevolved with mTORC2 components. Mutation of this motif in Akt1 and PKCβII abolished cellular kinase activity by impairing activation loop and hydrophobic motif phosphorylation. mTORC2 directly phosphorylated the PKC TIM in vitro, and this phosphorylation event was detected in mouse brain. Overexpression of PDK1 in mTORC2-deficient cells rescued hydrophobic motif phosphorylation of PKC and Akt by a mechanism dependent on their intrinsic catalytic activity, revealing that mTORC2 facilitates the PDK1 phosphorylation step, which, in turn, enables autophosphorylation. Structural analysis revealed that PKC homodimerization is driven by a TIM-containing helix, and biophysical proximity assays showed that newly synthesized, unphosphorylated PKC dimerizes in cells. Furthermore, disruption of the dimer interface by stapled peptides promoted hydrophobic motif phosphorylation. Our data support a model in which mTORC2 relieves nascent PKC dimerization through TIM phosphorylation, recruiting PDK1 to phosphorylate the activation loop and triggering intramolecular hydrophobic motif autophosphorylation. Identification of TIM phosphorylation and its role in the regulation of PKC provides the basis for AGC kinase regulation by mTORC2.
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http://dx.doi.org/10.1126/scisignal.abe4509DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8208635PMC
April 2021

CLK1 reorganizes the splicing factor U1-70K for early spliceosomal protein assembly.

Proc Natl Acad Sci U S A 2021 04;118(14)

Department of Pharmacology, University of California San Diego, La Jolla, CA 92093;

Early spliceosome assembly requires phosphorylation of U1-70K, a constituent of the U1 small nuclear ribonucleoprotein (snRNP), but it is unclear which sites are phosphorylated, and by what enzyme, and how such modification regulates function. By profiling the proteome, we found that the Cdc2-like kinase 1 (CLK1) phosphorylates Ser-226 in the C terminus of U1-70K. This releases U1-70K from subnuclear granules facilitating interaction with U1 snRNP and the serine-arginine (SR) protein SRSF1, critical steps in establishing the 5' splice site. CLK1 breaks contacts between the C terminus and the RNA recognition motif (RRM) in U1-70K releasing the RRM to bind SRSF1. This reorganization also permits stable interactions between U1-70K and several proteins associated with U1 snRNP. Nuclear induction of the SR protein kinase 1 (SRPK1) facilitates CLK1 dissociation from U1-70K, recycling the kinase for catalysis. These studies demonstrate that CLK1 plays a vital, signal-dependent role in early spliceosomal protein assembly by contouring U1-70K for protein-protein multitasking.
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http://dx.doi.org/10.1073/pnas.2018251118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8040622PMC
April 2021

Urinary Exosomes Identify Inflammatory Pathways in Vancomycin Associated Acute Kidney Injury.

Int J Mol Sci 2021 Mar 10;22(6). Epub 2021 Mar 10.

Department of Cellular and Molecular Medicine, University of Michigan Medical System, Ann Arbor, MI 48109, USA.

Background: Vancomycin is commonly used as a first line therapy for gram positive organisms such as methicillin resistant . Vancomycin-induced acute kidney injury (V-AKI) has been reported in up to 43% of patients, especially in those with higher targeted trough concentrations. The precise mechanism of injury in humans remains elusive, with recent evidence directed towards proximal tubule cell apoptosis. In this study, we investigated the protein contents of urinary exosomes in patients with V-AKI to further elucidate biomarkers of mechanisms of injury and potential responses.

Methods: Urine samples from patients with V-AKI who were enrolled in the DIRECT study and matched healthy controls from the UAB-UCSD O'Brien Center Biorepository were included in the analysis. Exosomes were extracted using solvent exclusion principle and polyethylene glycol induced precipitation. Protein identity and quantification was determined by label-free liquid chromatography mass spectrometry (LC/MS). The mean peak serum creatinine was 3.7 ± 1.4 mg/dL and time to kidney injury was 4.0 ± 3.0 days. At discharge, 90% of patients demonstrated partial recovery; 33% experienced full recovery by day 28. Proteomic analyses on five V-AKI and 7 control samples revealed 2009 proteins in all samples and 251 proteins significantly associated with V-AKI (Pi-score > 1). The top discriminatory proteins were complement C3, complement C4, galectin-3-binding protein, fibrinogen, alpha-2 macroglobulin, immunoglobulin heavy constant mu and serotransferrin.

Conclusion: Urinary exosomes reveal up-regulation of inflammatory proteins after nephrotoxic injury in V-AKI. Further studies are necessary in a large patient sample to confirm these findings for elucidation of pathophysiologic mechanisms and validation of potential injury biomarkers.
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http://dx.doi.org/10.3390/ijms22062784DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7999309PMC
March 2021

The tumor suppressor kinase DAPK3 drives tumor-intrinsic immunity through the STING-IFN-β pathway.

Nat Immunol 2021 04 25;22(4):485-496. Epub 2021 Mar 25.

La Jolla Institute for Immunology, La Jolla, CA, USA.

Evasion of host immunity is a hallmark of cancer; however, mechanisms linking oncogenic mutations and immune escape are incompletely understood. Through loss-of-function screening of 1,001 tumor suppressor genes, we identified death-associated protein kinase 3 (DAPK3) as a previously unrecognized driver of anti-tumor immunity through the stimulator of interferon genes (STING) pathway of cytosolic DNA sensing. Loss of DAPK3 expression or kinase activity impaired STING activation and interferon (IFN)-β-stimulated gene induction. DAPK3 deficiency in IFN-β-producing tumors drove rapid growth and reduced infiltration of CD103CD8α dendritic cells and cytotoxic lymphocytes, attenuating the response to cancer chemo-immunotherapy. Mechanistically, DAPK3 coordinated post-translational modification of STING. In unstimulated cells, DAPK3 inhibited STING K48-linked poly-ubiquitination and proteasome-mediated degradation. After cGAMP stimulation, DAPK3 was required for STING K63-linked poly-ubiquitination and STING-TANK-binding kinase 1 interaction. Comprehensive phospho-proteomics uncovered a DAPK3-specific phospho-site on the E3 ligase LMO7, critical for LMO7-STING interaction and STING K63-linked poly-ubiquitination. Thus, DAPK3 is an essential kinase for STING activation that drives tumor-intrinsic innate immunity and tumor immune surveillance.
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http://dx.doi.org/10.1038/s41590-021-00896-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8300883PMC
April 2021

Dissociation of DNA damage sensing by endoglycosidase HPSE.

iScience 2021 Mar 27;24(3):102242. Epub 2021 Feb 27.

Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL 60612, USA.

Balance between cell proliferation and elimination is critical in handling threats both exogenous and of internal dysfunction. Recent work has implicated a conserved but poorly understood endoglycosidase heparanase (HPSE) in the restriction of innate defense responses, yet biochemical mediators of these key functions remained unclear. Here, an unbiased immunopurification proteomics strategy is employed to identify and rank uncharacterized interactions between HPSE and mediators of canonical signaling pathways linking cell cycle and stress responses. We demonstrate with models of genotoxic stress including herpes simplex virus infection and chemotherapeutic treatment that HPSE dampens innate responses to double-stranded DNA breakage by interfering with signal transduction between initial sensors and downstream mediators. Given the long-standing recognition of HPSE in driving late-stage inflammatory disease exemplified by tissue destruction and cancer metastasis, modulation of this protein with control over the DNA damage response imparts a unique strategy in the development of unconventional multivalent therapy.
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http://dx.doi.org/10.1016/j.isci.2021.102242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7957091PMC
March 2021

Disruption of innate defense responses by endoglycosidase HPSE promotes cell survival.

JCI Insight 2021 04 8;6(7). Epub 2021 Apr 8.

Department of Microbiology and Immunology.

The drive to withstand environmental stresses and defend against invasion is a universal trait extant in all forms of life. While numerous canonical signaling cascades have been characterized in detail, it remains unclear how these pathways interface to generate coordinated responses to diverse stimuli. To dissect these connections, we followed heparanase (HPSE), a protein best known for its endoglycosidic activity at the extracellular matrix but recently recognized to drive various forms of late-stage disease through unknown mechanisms. Using herpes simplex virus-1 (HSV-1) infection as a model cellular perturbation, we demonstrate that HPSE acts beyond its established enzymatic role to restrict multiple forms of cell-intrinsic defense and facilitate host cell reprogramming by the invading pathogen. We reveal that cells devoid of HPSE are innately resistant to infection and counteract viral takeover through multiple amplified defense mechanisms. With a unique grasp of the fundamental processes of transcriptional regulation and cell death, HPSE represents a potent cellular intersection with broad therapeutic potential.
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http://dx.doi.org/10.1172/jci.insight.144255DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8119219PMC
April 2021

A Hetero-Multimeric Chitinase-Containing and Ookinete-Secreted Protein Complex Involved in Mosquito Midgut Invasion.

Front Cell Infect Microbiol 2020 8;10:615343. Epub 2021 Jan 8.

Section of Infectious Diseases, Department of Internal Medicine, Yale School of Medicine, New Haven, CT, United States.

Malaria parasites are transmitted by  mosquitoes. During its life cycle in the mosquito vector the ookinete escapes the proteolytic milieu of the post-blood meal midgut by traversing the midgut wall. This process requires penetration of the chitin-containing peritrophic matrix lining the midgut epithelium, which depends in part on ookinete-secreted chitinases. ookinetes have one chitinase (PfCHT1), whereas ookinetes of the avian-infecting parasite, , have two, a long and a short form, PgCHT1 and PgCHT2, respectively. Published data indicates that PgCHT2 forms a high molecular weight (HMW) reduction-sensitive complex; and one binding partner is the ookinete-produced von Willebrand A-domain-containing protein, WARP. Size exclusion chromatography data reported here show that PgCHT2 and its ortholog, PfCHT1 are covalently-linked components of a HMW chitinase-containing complex (> 1,300 kDa). Mass spectrometry of ookinete-secreted proteins isolated using a new chitin bead pull-down method identified chitinase-associated proteins in and ookinete-conditioned culture media. Mass spectrometry of this complex showed the presence of several micronemal proteins including von Willebrand factor A domain-related protein (WARP), ookinete surface enolase, and secreted ookinete adhesive protein (SOAP). To test the hypothesis that ookinete-produced PfCHT1 can form a high molecular homo-multimer or, alternatively, interacts with ookinete-produced proteins to produce an HMW hetero-multimer, we created chimeric parasites expressing to replace 1, enabling the production of large numbers of PfCHT1-expressing ookinetes. We show that chimeric ookinetes express monomeric PfCHT1, but a HMW complex containing is not present. A better understanding of the chitinase-containing HMW complex may enhance development of next-generation vaccines or drugs that target malaria transmission stages.
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http://dx.doi.org/10.3389/fcimb.2020.615343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7821095PMC
June 2021

Evaluating Organism-Wide Changes in the Metabolome and Microbiome following a Single Dose of Antibiotic.

mSystems 2020 Oct 6;5(5). Epub 2020 Oct 6.

Department of Pediatrics, UC San Diego, La Jolla, California, USA

Antibiotics are a mainstay of modern medicine, but as they kill their target pathogen(s), they often affect the commensal microbiota. Antibiotic-induced microbiome dysbiosis is a growing research focus and health concern, often assessed via analysis of fecal samples. However, such analysis does not inform how antibiotics influence the microbiome across the whole host or how such changes subsequently alter host chemistry. In this study, we investigated the acute (1 day postadministration) and delayed (6 days postadministration) effects of a single parenteral dose of two common antibiotics, ampicillin or vancomycin, on the global metabolome and microbiome of mice across 77 different body sites from 25 different organs. The broader-spectrum agent ampicillin had the greatest impact on the microbiota in the lower gastrointestinal tract (cecum and colon), where microbial diversity is highest. In the metabolome, the greatest effects were seen 1 day posttreatment, and changes in metabolite abundances were not confined to the gut. The local abundance of ampicillin and its metabolites correlated with increased metabolome effect size and a loss of alpha diversity versus control mice. Additionally, small peptides were elevated in the lower gastrointestinal tract of mice 1 day after antibiotic treatment. While a single parenteral dose of antibiotic did not drastically alter the microbiome, nevertheless, changes in the metabolome were observed both within and outside the gut. This study provides a framework for how whole-organism -omics approaches can be employed to understand the impact of antibiotics on the entire host. We are just beginning to understand the unintended effects of antibiotics on our microbiomes and health. In this study, we aimed to define an approach by which one could obtain a comprehensive picture of (i) how antibiotics spatiotemporally impact commensal microbes throughout the gut and (ii) how these changes influence host chemistry throughout the body. We found that just a single dose of antibiotic altered host chemistry in a variety of organs and that microbiome alterations were not uniform throughout the gut. As technological advances increase the feasibility of whole-organism studies, we argue that using these approaches can provide further insight on both the wide-ranging effects of antibiotics on health and how to restore microbial communities to mitigate these effects.
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http://dx.doi.org/10.1128/mSystems.00340-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7542558PMC
October 2020

Mortality Risk Profiling of Staphylococcus aureus Bacteremia by Multi-omic Serum Analysis Reveals Early Predictive and Pathogenic Signatures.

Cell 2020 09;182(5):1311-1327.e14

Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093, USA; Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA 92093, USA; Collaborative to Halt Antibiotic-Resistant Microbes, University of California, San Diego, La Jolla, CA 92093, USA; Center for Microbiome Innovation, University of California, San Diego, La Jolla, CA 92093, USA. Electronic address:

Staphylococcus aureus bacteremia (SaB) causes significant disease in humans, carrying mortality rates of ∼25%. The ability to rapidly predict SaB patient responses and guide personalized treatment regimens could reduce mortality. Here, we present a resource of SaB prognostic biomarkers. Integrating proteomic and metabolomic techniques enabled the identification of >10,000 features from >200 serum samples collected upon clinical presentation. We interrogated the complexity of serum using multiple computational strategies, which provided a comprehensive view of the early host response to infection. Our biomarkers exceed the predictive capabilities of those previously reported, particularly when used in combination. Last, we validated the biological contribution of mortality-associated pathways using a murine model of SaB. Our findings represent a starting point for the development of a prognostic test for identifying high-risk patients at a time early enough to trigger intensive monitoring and interventions.
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http://dx.doi.org/10.1016/j.cell.2020.07.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7494005PMC
September 2020

Kinetic profiling of metabolic specialists demonstrates stability and consistency of in vivo enzyme turnover numbers.

Proc Natl Acad Sci U S A 2020 09 1;117(37):23182-23190. Epub 2020 Sep 1.

Department of Bioengineering, University of California San Diego, La Jolla, CA 92093;

Enzyme turnover numbers ( s) are essential for a quantitative understanding of cells. Because s are traditionally measured in low-throughput assays, they can be inconsistent, labor-intensive to obtain, and can miss in vivo effects. We use a data-driven approach to estimate in vivo s using metabolic specialist strains that resulted from gene knockouts in central metabolism followed by metabolic optimization via laboratory evolution. By combining absolute proteomics with fluxomics data, we find that in vivo s are robust against genetic perturbations, suggesting that metabolic adaptation to gene loss is mostly achieved through other mechanisms, like gene-regulatory changes. Combining machine learning and genome-scale metabolic models, we show that the obtained in vivo s predict unseen proteomics data with much higher precision than in vitro s. The results demonstrate that in vivo s can solve the problem of inconsistent and low-coverage parameterizations of genome-scale cellular models.
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http://dx.doi.org/10.1073/pnas.2001562117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7502767PMC
September 2020

Multidimensional Proteome Profiling of Blood-Brain Barrier Perturbation by Group B .

mSystems 2020 Aug 25;5(4). Epub 2020 Aug 25.

Department of Pharmacology, University of California, San Diego, La Jolla, California, USA

Group B (GBS) remains the leading cause of neonatal meningitis, a disease associated with high rates of adverse neurological sequelae. The relationship between GBS and brain tissues remains poorly characterized, partly because past studies had focused on microbial rather than host processes. Additionally, the field has not capitalized on systems-level technologies to probe the host-pathogen relationship. Here, we use multiplexed quantitative proteomics to investigate the effect of GBS infection in the murine brain at various levels of tissue complexity, beginning with the whole organ and moving to brain vascular substructures. Infected whole brains showed classical signatures associated with the acute-phase response. In isolated brain microvessels, classical blood-brain barrier proteins were unaltered, but interferon signaling and leukocyte recruitment proteins were upregulated. The choroid plexus showed increases in peripheral immune cell proteins. Proteins that increased in abundance in the vasculature during GBS invasion were associated with major histocompatibility complex (MHC) class I antigen processing and endoplasmic reticulum dysfunction, a finding which correlated with altered host protein glycosylation profiles. Globally, there was low concordance between the infection proteome of whole brains and isolated vascular tissues. This report underscores the utility of unbiased, systems-scale analyses of functional tissue substructures for understanding disease. Group B (GBS) meningitis remains a major cause of poor health outcomes very early in life. Both the host-pathogen relationship leading to disease and the massive host response to infection contributing to these poor outcomes are orchestrated at the tissue and cell type levels. GBS meningitis is thought to result when bacteria present in the blood circumvent the selectively permeable vascular barriers that feed the brain. Additionally, tissue damage subsequent to bacterial invasion is mediated by inflammation and by immune cells from the periphery crossing the blood-brain barrier. Indeed, the vasculature plays a central role in disease processes occurring during GBS infection of the brain. Here, we employed quantitative proteomic analysis of brain vascular substructures during invasive GBS disease. We used the generated data to map molecular alterations associated with tissue perturbation, finding widespread intracellular dysfunction and punctuating the importance of investigations relegated to tissue type over the whole organ.
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http://dx.doi.org/10.1128/mSystems.00368-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7449606PMC
August 2020

Oil and gas production and spontaneous preterm birth in the San Joaquin Valley, CA: A case-control study.

Environ Epidemiol 2020 Aug 5;4(4):e099. Epub 2020 Jun 5.

Department of Pediatrics, School of Medicine, Stanford University, Stanford, California.

Background: Recent studies report an association between preterm birth and exposure to unconventional oil and gas wells. There has been limited previous study on exposure to conventional wells, which are common in California. Our objective was to determine whether exposure to well sites was associated with increased odds of spontaneous preterm birth (delivery at <37 weeks).

Methods: We conducted a case-control study using data on 27,913 preterm birth cases and 197,461 term birth controls. All births were without maternal comorbidities and were located in the San Joaquin Valley, CA, between 1998 and 2011. We obtained data for 83,559 wells in preproduction or production during the study period. We assessed exposure using inverse distance-squared weighting and, for each birth and trimester, we assigned an exposure tertile. Using logistic regression, we estimated adjusted odds ratios (ORs) for the association between exposure to well sites and preterm birth at 20-27, 28-31, and 32-36 weeks.

Results: We observed increased ORs for preterm birth with high exposure to wells in the first and second trimesters for births delivered at ≤31 weeks (adjusted ORs, 1.08-1.14). In stratified analyses, the associations were confined to births to Hispanic and non-Hispanic Black women and to women with ≤12 years of educational attainment. In a secondary analysis, we found evidence that exposure to wells in preproduction is associated with higher concentrations of particulate matter.

Conclusions: We found evidence that exposure to oil and gas well sites is associated with increased risk of spontaneous preterm birth.
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http://dx.doi.org/10.1097/EE9.0000000000000099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7423522PMC
August 2020

Fermentable fiber-induced hepatocellular carcinoma in mice recapitulates gene signatures found in human liver cancer.

PLoS One 2020 19;15(6):e0234726. Epub 2020 Jun 19.

Department of Physiology and Pharmacology, University of Toledo College of Medicine and Life Sciences, Toledo, OH, United States of America.

Hepatocellular carcinoma (HCC), the most malignant form of primary liver cancer, is the fourth most prevalent cause of cancer mortality globally. It was recently discovered that the dietary fermentable fiber, inulin, can reprogram the murine liver to favor HCC development in a gut microbiota-dependent manner. Determining the molecular pathways that are either over expressed or repressed during inulin-induced HCC would provide a platform of potential therapeutic targets. In the present study, we have combined analysis of the novel inulin-induced HCC murine model and human HCC samples to identify differentially expressed genes (DEGs) in hepatocarcinogenesis. Hepatic transcriptome profiling revealed that there were 674 DEGs in HCC mice compared to mice safeguarded from HCC. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis uncovered enrichment in ECM-receptor interaction, steroid hormone biosynthesis, PPAR signaling pathway, focal adhesion and protein digestion and absorption during inulin-induced HCC. Tandem mass tag based quantitative, multiplexed proteomic analysis delineated 57 differentially expressed proteins, where the over-expressed proteins were associated with cell adhesion molecules, valine, leucine and isoleucine degradation and ECM-receptor interaction. After obtaining the human orthologs of the mouse genes, we did a comparison analysis to level 3 RNA-seq data found in the Cancer Genome Atlas (TCGA) database, corresponding to human HCC (n = 361) and healthy liver (n = 50) samples. Out of the 549 up-regulated and 68 down-regulated human orthologs identified, 142 genes (137 significantly over-expressed and 5 significantly under-expressed) were associated with human HCC. Using univariate survival analysis, we found 27 over-expressed genes involved in cell-cell adhesion and cell division that were associated with poor HCC patient survival. Overall, the genetic and proteomics signatures highlight potential underlying mechanisms in inulin-induced HCC and support that this murine HCC model is human relevant.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0234726PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7304627PMC
September 2020

Molecular dissection of Chagas induced cardiomyopathy reveals central disease associated and druggable signaling pathways.

PLoS Negl Trop Dis 2020 05 20;14(5):e0007980. Epub 2020 May 20.

Skaggs School of Pharmacy and Pharmaceutical Sciences; University of California San Diego; La Jolla, CA, United States of America.

Chagas disease, the clinical presentation of T. cruzi infection, is a major human health concern. While the acute phase of Chagas disease is typically asymptomatic and self-resolving, chronically infected individuals suffer numerous sequelae later in life. Cardiomyopathies in particular are the most severe consequence of chronic Chagas disease and cannot be reversed solely by parasite load reduction. To prioritize new therapeutic targets, we unbiasedly interrogated the host signaling events in heart tissues isolated from a Chagas disease mouse model using quantitative, multiplexed proteomics. We defined the host response to infection at both the proteome and phospho-proteome levels. The proteome showed an increase in the immune response and a strong repression of several mitochondrial proteins. Complementing the proteome studies, the phospho-proteomic survey found an abundance of phospho-site alterations in plasma membrane and cytoskeletal proteins. Bioinformatic analysis of kinase activity provided substantial evidence for the activation of NDRG2 and JNK/p38 kinases during Chagas disease. A significant activation of DYRK2 and AMPKA2 and the inhibition of casein family kinases were also predicted. We concluded our analyses by linking the diseased heart proteome profile to known therapeutic interventions, uncovering a potential to target mitochondrial proteins, secreted immune effectors and core kinases for the treatment of chronic Chagas disease. Together, this study provides molecular insight into host proteome and phospho-proteome responses to T. cruzi infection in the heart for the first time, highlighting pathways that can be further validated for functional contributions to disease and suitability as drug targets.
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http://dx.doi.org/10.1371/journal.pntd.0007980DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7279607PMC
May 2020

Exposure of to Environmental Stress and Clinically Used Antibiotics Reveals Common Proteome Response among Pathogenic Mycobacteria.

Microorganisms 2020 May 9;8(5). Epub 2020 May 9.

Department of Biomedical Sciences, Carlson College of Veterinary Medicine, Oregon State University, Corvallis, OR 97331, USA.

subsp. (MAB) is a clinically important nontuberculous mycobacterium (NTM) causing pulmonary infection in patients such as cystic fibrosis and bronchiectasis. MAB is naturally resistant to the majority of available antibiotics. In attempts to identify the fundamental response of MAB to aerobic, anaerobic, and biofilm conditions (as it is encountered in patients) and during exposure to antibiotics, we studied bacterial proteome using tandem mass tag mass spectrometry sequencing. Numerous de novo synthesized proteins belonging to diverse metabolic pathways were found in anaerobic and biofilm conditions, including glycolysis/gluconeogenesis, tricarboxylic acid (TCA) cycle, oxidative phosphorylation, nitrogen metabolism, and glyoxylate and dicarboxylate metabolism. Upon exposure to amikacin and linezolid under stress environments, MAB displayed metabolic enrichment for glycerophospholipid metabolism and oxidative phosphorylation. By comparing proteomes of two significant NTMs, MAB and subsp. , we found highly synthesized shared enzymes of oxidative phosphorylation, TCA cycle, glycolysis/gluconeogenesis, glyoxylate/dicarboxylate, nitrogen metabolism, peptidoglycan biosynthesis, and glycerophospholipid/glycerolipid metabolism. The activation of peptidoglycan and fatty acid biosynthesis pathways indicates the attempt of bacteria to modify the cell wall, influencing the susceptibility to antibiotics. This study establishes global changes in the synthesis of enzymes promoting the metabolic shift and enhancing the pathogen resistance to antibiotics within different environments.
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http://dx.doi.org/10.3390/microorganisms8050698DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7285101PMC
May 2020
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