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The Vanderbilt University School of Medicine administers degree programs that provide students with the knowledge, skills, and attitudes they will need to practice safe, effective, ethical, evidence-based, and patient-centered health care in the 21st century, and to contribute to the knowledge base supporting it.

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Myeloid Cell-Derived HB-EGF Drives Tissue Recovery After Pancreatitis
(Cellular and Molecular Gastroenterology and Hepatology, 2019) Wen, Hui-Ju; Gao, Shan; Wang, Yin; Ray, Michael; Magnuson, Mark A.; Wright, Christopher V. E.; Di Magliano, Marina Pasca; Frankel, Timothy L.; Crawford, Howard C.
BACKGROUND & AIMS: Pancreatitis is a major cause of morbidity and mortality and is a risk factor for pancreatic tumorigenesis. Upon tissue damage, an inflammatory response, made up largely of macrophages, provides multiple growth factors that promote repair. Here, we examine the molecular pathways initiated by macrophages to promote pancreas recovery from pancreatitis. METHODS: To induce organ damage, mice were subjected to cerulein-induced experimental pancreatitis and analyzed at various times of recovery. CD11b-DTR mice were used to deplete myeloid cells. Hbeg(f/f);LysM-Cre mice were used to ablate myeloid cell-derived heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF). To ablate EGFR specifically during recovery, pancreatitis was induced in Egfr(f/f);Ptf1a(Flp0/+);FSF-Rosa26(CAG-CreERT2 )mice followed by tamoxifen treatment. RESULTS: Macrophages infiltrating the pancreas in experimental pancreatitis make high levels of HB-EGF. Both depletion of myeloid cells and ablation of myeloid cell HB-EGF delayed recovery from experimental pancreatitis, resulting from a decrease in cell proliferation and an increase in apoptosis. Mechanistically, ablation of myeloid cell HB-EGF impaired epithelial cell DNA repair, ultimately leading to cell death. Soluble HB-EGF induced EGFR nuclear translocation and methylation of histone H4, facilitating resolution of DNA damage in pancreatic acinar cells in vitro. Consistent with its role as the primary receptor of HB-EGF, in vivo ablation of EGFR from pancreatic epithelium during recovery from pancreatitis resulted in accumulation of DNA damage. CONCLUSIONS: By using novel conditional knockout mouse models, we determined that HB-EGF derived exclusively from myeloid cells induces epithelial cell proliferation and EGFR-dependent DNA repair, facilitating pancreas healing after injury.
Regulation of the Pancreatic Exocrine Differentiation Program and Morphogenesis by Onecut 1/Hnf6
(Cellular and Molecular Gastroenterology and Hepatology, 2019) Kropp, Peter A.; Zhu, Xiaodong; Gannon, Maureen
BACKGROUND & AIMS: The Onecut 1 transcription factor (Oc1, a.k.a. HNF6) promotes differentiation of endocrine and duct cells of the pancreas; however, it has no known role in acinar cell differentiation. We sought to better understand the role of Oc1 in exocrine pancreas development and to identify its direct transcriptional targets. METHODS: Pancreata from Oc1(Delta panc) (Oc1(fl/fl); Pdx1-Cre) mouse embryos and neonates were analyzed morphologically. High-throughput RNA-sequencing was performed on control and Oc1-deficient pancreas; chromatin immunoprecipitation sequencing was performed on wild-type embryonic mouse pancreata to identify direct Oc1 transcriptional targets. Immunofluorescence labeling was used to confirm the RNA-sequencing/chromatin immuno precipitation sequencing results and to further investigate the effects of Oc1 loss on acinar cells. RESULTS: Loss of Oc1 from the developing pancreatic epithelium resulted in disrupted duct and acinar cell development. RNA-sequencing revealed decreased expression of acinar cell regulatory factors (Nr5a2, Ptf1a, Gata4, Mist1) and functional genes (Amylase, Cpa1, Prss1, Spink1) at embryonic day (e) 18.5 in Oc1(Delta panc) samples. Approximately 1000 of the altered genes were also identified as direct Oc1 targets by chromatin immunoprecipitation sequencing, including most of the previously noted genes. By immunolabeling, we confirmed that Amylase, Mist1, and GATA4 protein levels are significantly decreased by P2, and Spink1 protein levels were significantly reduced and mislocalized. The pancreatic duct regulatory factors Hnf1 beta and FoxA2 were also identified as direct Oc1 targets. CONCLUSIONS: These findings confirm that Oc1 is an important regulator of both duct and acinar cell development in the embryonic pancreas. Novel transcriptional targets of Oc1 have now been identified and provide clarity into the mechanisms of Oc1 transcriptional regulation in the developing exocrine pancreas. Oc1 can now be included in the gene-regulatory network of acinar cell regulatory genes. Oc1 regulates other acinar cell regulatory factors and acinar cell functional genes directly, and it can also regulate some acinar cell regulatory factors (eg, Mist1) indirectly. Oc1 therefore plays an important role in acinar cell development.
Diaspora linkages benefit both sides: a single partnership experience
(Global Health Action, 2019-01) Kramer, Beverley; Zent, Roy
The emigration of physicians and scientists from resource-constrained countries decreases the country's ability to undertake research. Re-establishing research environments and increasing capacity reduced by these losses are important, particularly in the health sciences. One mechanism for re-establishing strong health sciences research is the introduction of an Alumni Diaspora Fellowship Programme. We define the beneficial effects of a successful single partnership in an Alumni Diaspora Programme. This Host/Alumnus collaboration demonstrates that bi-directional advantages have accrued for both the Host Institution situated in a resource-constrained country and the Alumni's Institution, located in a high-income country. In addition to expanding research in the resource-constrained country, collaborations expanded to other faculty beyond the Alumnus in the sending Institution, in multiple fields including those not readily available in the high-income country (HIV, TB, malaria). The environment at the host Institution in the resource-constrained country has been enriched by increased research publications, training of young scholars (over 200 trained in manuscript and grant application writing), and substantial advances in biomedical informatics. There has been considerable knowledge exchange and development between both Institutions, showing that 'brain circulation' and Diaspora Programmes are valuable strategies for expanding research.
Isotopic tagging of oxidized and reduced cysteines (iTORC) for detecting and quantifying sulfenic acids, disulfides, and free thiols in cells
(Journal of Biological Chemistry, 2019-04-19) Albertolle, Matthew E.; Glass, Sarah M.; Trefts, Elijah; Guengerich, F. Peter
Oxidative modifications of cysteine residues are an important component in signaling pathways, enzymatic regulation, and redox homeostasis. Current direct and indirect methods detect specific modifications and a general binary population of free or oxidized cysteines, respectively. In an effort to combine both direct and indirect detection strategies, here we developed a method that we designate isotopic tagging of oxidized and reduced cysteines (iTORC). This method uses synthetic molecules for rapid isotopic coding of sulfenic acids, reduced cysteines, and disulfides in cells. Our approach utilizes isotopically distinct benzothiazine and halogenated benzothiazine probes to sequentially alkylate sulfenic acids and then free thiols and, finally, after a reduction step, cysteines oxidized to disulfides or other phosphine-reducible states. We ascertained that the iodinated benzothiazine probe has reduced cross-reactivity toward primary amines and is highly reactive with the cysteine of GSH, with a calculated rate constant of 2 x 10(5) m(-1) s(-1) (pH 8.0, 23 degrees C) (i.e. 10-20 times faster than N-ethylmaleimide). We applied iTORC to a mouse hepatocyte lysate to identify known sulfenylated and disulfide-bonded proteins, including elongation factor 1-1 and mouse serum albumin, and found that iTORC reliably detected their expected oxidation status. This method can be easily employed to study the effects of oxidants on recombinant proteins and cell and tissue extracts, and the efficiencies of the alkylating agents enable completion of all three labeling steps within 2 h. In summary, we demonstrate here that halogenated benzothiazine-based alkylating agents can be utilized to rapidly measure the cellular thiol status in cells.
In Situ Molecular Architecture of the Helicobacter pylori Cag Type IV Secretion System
(MBIO, 2019-05) Hu, Bo; Khara, Pratick; Song, Liqiang; Lin, Aung Soe; Frick-Cheng, Arwen E.; Harvey, M. Lorena; Cover, Timothy L.; Christie, Peter J.
Helicobacter pylori colonizes about half of humans worldwide, and its presence in the gastric mucosa is associated with an increased risk of gastric adenocarcinoma, gastric lymphoma, and peptic ulcer disease. H. pylori strains carrying the cag pathogenicity island (cagPAI) are associated with increased risk of disease progression. The cagPAI encodes the Cag type IV secretion system (Cag(T4SS)), which delivers the CagA oncoprotein and other effector molecules into human gastric epithelial cells. We visualized structures of native and mutant Cag(T4SS) machines on the H. pylori cell envelope by cryoelectron tomography. Individual H. pylori cells contain multiple Cag(T4SS) nanomachines, each composed of a wheel-shaped outer membrane complex (OMC) with 14-fold symmetry and an inner membrane complex (IMC) with 6-fold symmetry. CagX, CagY, and CagM are required for assembly of the OMC, whereas strains lacking Cag3 and CagT produce outer membrane complexes lacking peripheral components. The IMC, which has never been visualized in detail, is configured as six tiers in cross-section view and three concentric rings surrounding a central channel in end-on view. The IMC contains three T4SS ATPases: (i) VirB4-like CagE, arranged as a hexamer of dimers at the channel entrance; (ii) a hexamer of VirB11-like Cag alpha, docked at the base of the CagE hexamer; and (iii) VirD4-like Cag beta and other unspecified Cag subunits, associated with the stacked CagE/Cag alpha complex and forming the outermost rings. The Cag(T4SS) and recently solved Legionella pneumophila Dot/Icm system comprise new structural prototypes for the T4SS superfamily. IMPORTANCE Bacterial type IV secretion systems (T4SSs) have been phylogenetically grouped into two subfamilies. The T4ASSs, represented by the Agrobacterium tumefaciens VirB/VirD4(T4SS), include "minimized" machines assembled from 12 VirB-and VirD4-like subunits and compositionally larger systems such as the Helicobacter pylori Cag(T4SS). T4BSSs encompass systems closely related in subunit composition to the Legionella pneumophila Dot/Icm(T4SS). Here, we present structures of native and mutant H. pylori Cag machines determined by in situ cryoelectron tomography. We identify distinct outer and inner membrane complexes and, for the first time, visualize structural contributions of all three "signature" ATPases of T4SSs at the cytoplasmic entrance of the translocation channel. Despite their evolutionary divergence, the Cag(T4SS) aligns structurally much more closely to the Dot/Icm(T4SS) than an available VirB/VirD4 subcomplex. Our findings highlight the diversity of T4SSs and suggest a structural classification scheme in which T4SSs are grouped as minimized VirB/VirD4-like or larger Cag-like and Dot/Icm-like systems.
Inhibition of MYC by the SMARCB1 tumor suppressor
(Nature Communications, 2019-05-01) Weissmiller, April M.; Wang, Jing; Lorey, Shelly L.; Howard, Gregory C.; Martinez, Ernest; Liu, Qi; Tansey, William P.
SMARCB1 encodes the SNF5 subunit of the SWI/SNF chromatin remodeler. SNF5 also interacts with the oncoprotein transcription factor MYC and is proposed to stimulate MYC activity. The concept that SNF5 is a coactivator for MYC, however, is at odds with its role as a tumor-suppressor, and with observations that loss of SNF5 leads to activation of MYC target genes. Here, we reexamine the relationship between MYC and SNF5 using biochemical and genome-wide approaches. We show that SNF5 inhibits the DNA-binding ability of MYC and impedes target gene recognition by MYC in cells. We further show that MYC regulation by SNF5 is separable from its role in chromatin remodeling, and that reintroduction of SNF5 into SMARCB1-null cells mimics the primary transcriptional effects of MYC inhibition. These observations reveal that SNF5 antagonizes MYC and provide a mechanism to explain how loss of SNF5 can drive malignancy.
Erenumab in chronic migraine with medication overuse Subgroup analysis of a randomized trial
(2019-05-14) Brandes, Jan Lewis
Objective To determine the effect of erenumab, a human anti-calcitonin gene-related peptide receptor monoclonal antibody, in patients with chronic migraine and medication overuse. Methods In this double-blind, placebo-controlled study, 667 adults with chronic migraine were randomized (3: 2: 2) to placebo or erenumab (70 or 140 mg), stratified by region and medication overuse status. Data from patients with baseline medication overuse at baseline were used to assess changes in monthly migraine days, acute migraine-specific medication treatment days, and proportion of patients achieving >= 50% reduction from baseline in monthly migraine days. Results Of 667 patients randomized, 41% (n = 274) met medication overuse criteria. In the medication overuse subgroup, erenumab 70 or 140 mg groups had greater reductions than the placebo group at month 3 in monthly migraine days (mean [95% confidence interval]-6.6 [-8.0 to -5.3] and-6.6 [-8.0 to -5.3] vs-3.5 [-4.6 to -2.4]) and acute migraine-specific medication treatment days (-5.4 [-6.5 to-4.4] and-4.9 [-6.0 to-3.8] vs-2.1 [-3.0 to -1.2]). In the placebo and 70 and 140 mg groups, = 50% reductions in monthly migraine days were achieved by 18%, 36% (odds ratio [95% confidence interval] 2.67 [1.36-5.22]) and 35% (odds ratio 2.51 [1.28-4.94]). These clinical responses paralleled improvements in patient-reported outcomes with a consistent benefit of erenumab across multiple measures of impact, disability, and healthrelated quality of life. The observed treatment effects were similar in the non-medication overuse subgroup. Conclusions Erenumab reduced migraine frequency and acute migraine-specific medication treatment days in patients with chronic migraine and medication overuse, improving disability and quality of life.
Enteropathogenic Escherichia coli remodels host endosomes to promote endocytic turnover and breakdown of surface polarity
(PLOS Pathogens, 2019-06) Kassa, Ephrem G.; Zlotkin-Rivkin, Efrat; Friedman, Gil; Ramachandran, Rachana P.; Melamed-Book, Naomi; Weiss, Aryeh M.; Belenky, Michael; Reichmann, Dana; Breuer, William; Pal, Ritesh Ranjan; Rosenshine, Ilan; Lapierre, Lynne A.; Goldenring, James R.; Aroeti, Benjamin
Enteropathogenic E. coli (EPEC) is an extracellular diarrheagenic human pathogen which infects the apical plasma membrane of the small intestinal enterocytes. EPEC utilizes a type III secretion system to translocate bacterial effector proteins into its epithelial hosts. This activity, which subverts numerous signaling and membrane trafficking pathways in the infected cells, is thought to contribute to pathogen virulence. The molecular and cellular mechanisms underlying these events are not well understood. We investigated the mode by which EPEC effectors hijack endosomes to modulate endocytosis, recycling and transcytosis in epithelial host cells. To this end, we developed a flow cytometry-based assay and imaging techniques to track endosomal dynamics and membrane cargo trafficking in the infected cells. We show that type-III secreted components prompt the recruitment of clathrin (clathrin and AP2), early (Rab5a and EEA1) and recycling (Rab4a, Rab11a, Rab11b, FIP2, Myo5b) endocytic machineries to peripheral plasma membrane infection sites. Protein cargoes, e.g. transferrin receptors, beta 1 integrins and aquaporins, which exploit the endocytic pathways mediated by these machineries, were also found to be recruited to these sites. Moreover, the endosomes and cargo recruitment to infection sites correlated with an increase in cargo endocytic turnover (i.e. endocytosis and recycling) and transcytosis to the infected plasma membrane. The hijacking of endosomes and associated endocytic activities depended on the translocated EspF and Map effectors in non-polarized epithelial cells, and mostly on EspF in polarized epithelial cells. These data suggest a model whereby EPEC effectors hijack endosomal recycling machineries to mislocalize and concentrate host plasma membrane proteins in endosomes and in the apically infected plasma membrane. We hypothesize that these activities contribute to bacterial colonization and virulence. Author summary Enteropathogenic Escherichia coli (EPEC) are pathogenic bacteria that cause infantile diarrhea. Upon ingestion, EPEC reaches the small intestine, where an injection device termed the type III secretion system is utilized to inject a set of effector proteins from the bacteria into the host cell. These proteins manipulate the localization and functions of host proteins, lipids and organelles and contribute to the emergence of the EPEC disease. The molecular mechanisms underlying the functions of the EPEC effector proteins are not completely understood. Here we show that early upon infection, two such effector proteins, EspF and Map, hijack host endosomes at bacterial adherence sites to facilitate endocytosis and recycling of plasma membrane proteins at these sites. The consequence of this event is the enrichment and mislocalization of host plasma membrane proteins at infection sites. One such protein is the transferrin receptor, which is a carrier for transferrin, whose function is to mediate cellular uptake of iron. Iron is a critical nutrient for bacterial growth and survival. We postulate that the unique manipulation of transferrin receptor endocytic membrane trafficking by EPEC plays an important role in its survival on the luminal surface of the intestinal epithelium.
Recognition of specific sialoglycan structures by oral streptococci impacts the severity of endocardial infection
(PLOS Pathogens, 2019-06) Bensing, Barbara A.; Li, Liang; Yakovenko, Olga; Wong, Maurice; Barnard, Karen N.; Iverson, T. M.; Lebrilla, Carlito B.; Parrish, Colin R.; Thomas, Wendy E.; Xiong, Yan; Sullam, Paul M.
Streptococcus gordonii and Streptococcus sanguinis are primary colonizers of the tooth surface. Although generally non-pathogenic in the oral environment, they are a frequent cause of infective endocarditis. Both streptococcal species express a serine-rich repeat surface adhesin that mediates attachment to sialylated glycans on mucin-like glycoproteins, but the specific sialoglycan structures recognized can vary from strain to strain. Previous studies have shown that sialoglycan binding is clearly important for aortic valve infections caused by some S. gordonii, but this process did not contribute to the virulence of a strain of S. sanguinis. However, these streptococci can bind to different subsets of sialoglycan structures. Here we generated isogenic strains of S. gordonii that differ only in the type and range of sialoglycan structures to which they adhere and examined whether this rendered them more or less virulent in a rat model of endocarditis. The findings indicate that the recognition of specific sialoglycans can either enhance or diminish pathogenicity. Binding to sialyllactosamine reduces the initial colonization of mechanically-damaged aortic valves, whereas binding to the closely-related trisaccharide sialyl T-antigen promotes higher bacterial densities in valve tissue 72 hours later. A surprising finding was that the initial attachment of streptococci to aortic valves was inversely proportional to the affinity of each strain for platelets, suggesting that binding to platelets circulating in the blood may divert bacteria away from the endocardial surface. Importantly, we found that human and rat platelet GPIb alpha (the major receptor for S. gordonii and S. sanguinis on platelets) display similar O-glycan structures, comprised mainly of a di-sialylated core 2 hexasaccharide, although the rat GPIb alpha has a more heterogenous composition of modified sialic acids. The combined results suggest that streptococcal interaction with a minor O-glycan on GPIb alpha may be more important than the over-all affinity for GPIb alpha for pathogenic effects. Author summary Infective endocarditis (IE) is a life-threatening infection of heart valves, and streptococci that normally reside in the mouth are a leading cause of this disease. Some oral streptococcal species express a protein on their surface that enables attachment to glycan (sugar) modifications on saliva proteins, an interaction that may be important for colonization of the tooth and other oral surfaces. These "Siglec-like adhesins" are hypervariable in the type and number of glycan structures they bind, ranging from just one to more than six of the structures displayed on the saliva proteins. If streptococci enter into the bloodstream, the Siglec-like adhesin can mediate attachment to similar glycans that decorate platelet or plasma proteins, which can impact the overall virulence of the organism. This study highlights how recognition of a specific type of glycan structure can cause a generally beneficial or neutral microbe to create damage to specific tissues-in this case the heart valves, illustrating one means by which commensal bacteria can become opportunistic or accidental pathogens. The findings further indicate that certain glycan-binding streptococci among the oral microbiota may be predisposed to produce infective endocarditis.
Changes in rapid HIV treatment initiation after national "treat all" policy adoption in 6 sub-Saharan African countries: Regression discontinuity analysis
(PLOS MEDICINE, 2019-06) Rebeiro, Peter F.; Tymejczyk, Olga; Brazier, Ellen; Yiannoutsos, Constantin T.; Vinikoor, Michael; van Lettow, Monique
Background Most countries have formally adopted the World Health Organization's 2015 recommendation of universal HIV treatment ("treat all"). However, there are few rigorous assessments of the real-world impact of treat all policies on antiretroviral treatment (ART) uptake across different contexts. Methods and findings We used longitudinal data for 814,603 patients enrolling in HIV care between 1 January 2004 and 10 July 2018 in 6 countries participating in the global International epidemiology Databases to Evaluate AIDS (IeDEA) consortium: Burundi (N = 11,176), Kenya (N = 179,941), Malawi (N = 84,558), Rwanda (N = 17,396), Uganda (N = 96,286), and Zambia (N = 425,246). Using a quasi-experimental regression discontinuity design, we assessed the change in the proportion initiating ART within 30 days of enrollment in HIV care (rapid ART initiation) after country-level adoption of the treat all policy. A modified Poisson model was used to identify factors associated with failure to initiate ART rapidly under treat all. In each of the 6 countries, over 60% of included patients were female, and median age at enrollment ranged from 32 to 36 years. In all countries studied, national adoption of treat all was associated with large increases in rapid ART initiation. Significant increases in rapid ART initiation immediately after treat all policy adoption were observed in Rwanda, from 44.4% to 78.9% of patients (34.5 percentage points [pp], 95% CI 27.2 to 41.7; p < 0.001), Kenya (25.7 pp, 95% CI 21.8 to 29.5; p < 0.001), Burundi (17.7 pp, 95% CI 6.5 to 28.9; p = 0.002), and Malawi (12.5 pp, 95% CI 7.5 to 17.5; p < 0.001), while no immediate increase was observed in Zambia (0.4 pp, 95% CI -2.9 to 3.8; p = 0.804) and Uganda (-4.2 pp, 95% CI -9.0 to 0.7; p = 0.090). The rate of rapid ART initiation accelerated sharply following treat all policy adoption in Malawi, Uganda, and Zambia; slowed in Kenya; and did not change in Rwanda and Burundi. In post hoc analyses restricted to patients enrolling under treat all, young adults (16-24 years) and men were at increased risk of not rapidly initiating ART (compared to older patients and women, respectively). However, rapid ART initiation following enrollment increased for all groups as more time elapsed since treat all policy adoption. Study limitations include incomplete data on potential ART eligibility criteria, such as clinical status, pregnancy, and enrollment CD4 count, which precluded the assessment of rapid ART initiation specifically among patients known to be eligible for ART before treat all. Conclusions Our analysis indicates that adoption of treat all policies had a strong effect on increasing rates of rapid ART initiation, and that these increases followed different trajectories across the 6 countries. Young adults and men still require additional attention to further improve rapid ART initiation.
Point mutations in the PDX1 transactivation domain impair human beta-cell development and function
(Molecular Metabolism, 2019-06) Wright, Christopher V. E.
Objective: Hundreds of missense mutations in the coding region of PDX1 exist; however, if these mutations predispose to diabetes mellitus is unknown. Methods: In this study, we screened a large cohort of subjects with increased risk for diabetes and identified two subjects with impaired glucose tolerance carrying common, heterozygous, missense mutations in the PDX1 coding region leading to single amino acid exchanges (P33T, C18R) in its transactivation domain. We generated iPSCs from patients with heterozygous PDX1(P33T/)(+), PDX1(C18R/+) mutations and engineered isogenic cell lines carrying homozygous PDX1(P33T/P33T), PDX1(C18R/C18R) mutations and a heterozygous PDX1 loss-of-function mutation (PDX1(+/-)). Results: Using an in vitro beta-cell differentiation protocol, we demonstrated that both, heterozygous PDXP33T/+, PDX1(C18R/+)and homozygous PDX1(P33T/P33T), PDX1(C18R/C18R) mutations impair beta-cell differentiation and function. Furthermore, PDX1(+/-) and PDX1(P33T/P33T )mutations reduced differentiation efficiency of pancreatic progenitors (PPs), due to downregulation of PDX1 -bound genes, including transcription factors MNX1 and PDX1 as well as insulin resistance gene CES1. Additionally, both PDX1(P33T/+ )and PDX1(P33T/P33T) mutations in PPs reduced the expression of PDX1-bound genes including the long-noncoding RNA, MEG3 and the imprinted gene NNAT, both involved in insulin synthesis and secretion. Conclusions: Our results reveal mechanistic details of how common coding mutations in PDX1 impair human pancreatic endocrine lineage formation and beta-cell function and contribute to the predisposition for diabetes. (C) 2019 The Authors. Published by Elsevier GmbH.
Loss of Function of Colgant1 Disrupts Collagen Post-translationa Modification and Causes Musculoskeletal Defects
(DISEASE MODELS & MECHANISMS, 2019-06) Geister, Krista A.; Lopez-Jimenez, Alberto Jose; Houghtaling, Scott; Ho, Tzu-Hua; Vanacore, Roberto; Beier, David R.
In a screen for organogenesis defects in N-ethyl-N-nitrosourea (ENU)-induced mutant mice, we discovered a line carrying a mutation in Colgaft1 [collagen beta(1-O)galactosyltransferase type 1], which is required for proper galactosylation of hydroxylysine residues in a number of collagens. Colgaft1 mutant embryos have not been previously characterized; here, we show that they exhibit skeletal and muscular defects. Analysis of mutant-derived embryonic fibroblasts reveals that COLGALT1 acts on collagen IV and VI, and, while collagen VI appears stable and its secretion is not affected, collagen IV accumulates inside of cells and within the extracellular matrix, possibly due to instability and increased degradation. We also generated mutant zebrafish that do not express the duplicated orthologs of mammalian Colgaft1. The double-homozygote mutants have muscle defects; they are viable through the larvae stage but do not survive to 10 days post-fertilization. We hypothesize that the Colgaft1 mutant could serve as a model of a human connective tissue disorder and/or congenital muscular dystrophy or myopathy.
The landscape of transcription initiation across latent and lytic KSHV genomes
(PLOS Pathogens, 2019-06) Ye, Xiang; Zhao, Yang; Karijolich, John
Precise promoter annotation is required for understanding the mechanistic basis of transcription initiation. In the context of complex genomes, such as herpesviruses where there is extensive genic overlap, identification of transcription start sites (TSSs) is particularly problematic and cannot be comprehensively accessed by standard RNA sequencing approaches. Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic gammaherpesvirus and the etiological agent of Kaposi's sarcoma and the B cell lymphoma primary effusion lymphoma (PEL). Here, we leverage RNA annotation and mapping of promoters for analysis of gene expression (RAMPAGE) and define KSHV TSSs transcriptome-wide and at nucleotide resolution in two widely used models of KSHV infection, namely iSLK.219 cells and the PEL cell line TREx-BCBL1-RTA. By mapping TSSs over a 96 h time course of reactivation we confirm 48 of 50 previously identified TSSs. Moreover, we identify over 100 novel transcription start site clusters (TSCs) in each cell line. Our analyses identified cell-type specific differences in TSC positions as well as promoter strength, and defined motifs within viral core promoters. Collectively, by defining TSSs at high resolution we have greatly expanded the transcriptional landscape of the KSHV genome and identified transcriptional control mechanisms at play during KSHV lytic reactivation. Author summary Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic gammaherpesvirus and the etiological agent of Kaposi's sarcoma and the B cell lymphoma primary effusion lymphoma (PEL). Despite identification of the virus over 20 years ago there is still an incomplete understanding of how many RNAs are transcribed from the viral genome and the location these RNAs are derived from. To fill this gap in knowledge we determined the landscape of transcription initiation on the KSHV genome. Our analyses more than tripled the number of known TSCs and thus viral-expressed RNAs. Furthermore, we identified key sequence features associated with the regulation of viral transcription start sites. This study provides the first transcriptome-wide characterization of KSHV transcription initiation sites as well as a framework for future studies to define functions of novel viral transcripts and viral gene regulatory elements.
Essentialism and Enhancing Healthcare Experiences: The Strategic Pursuit of "Less"
(Journal of Patient Experience, 2019-06) Cooley, Laura
Akt Signaling in Macrophage Polarization, Survival, and Atherosclerosis
(International Journal of Molecular Sciences, 2019-06-01) Linton, MacRae F.; Moslehi, Javid J.; Babaev, Vladimir R.
The PI3K/Akt pathway plays a crucial role in the survival, proliferation, and migration of macrophages, which may impact the development of atherosclerosis. Changes in Akt isoforms or modulation of the Akt activity levels in macrophages significantly affect their polarization phenotype and consequently atherosclerosis in mice. Moreover, the activity levels of Akt signaling determine the viability of monocytes/macrophages and their resistance to pro-apoptotic stimuli in atherosclerotic lesions. Therefore, elimination of pro-apoptotic factors as well as factors that antagonize or suppress Akt signaling in macrophages increases cell viability, protecting them from apoptosis, and this markedly accelerates atherosclerosis in mice. In contrast, inhibition of Akt signaling by the ablation of Rictor in myeloid cells, which disrupts mTORC2 assembly, significantly decreases the viability and proliferation of blood monocytes and macrophages with the suppression of atherosclerosis. In addition, monocytes and macrophages exhibit a threshold effect for Akt protein levels in their ability to survive. Ablation of two Akt isoforms, preserving only a single Akt isoform in myeloid cells, markedly compromises monocyte and macrophage viability, inducing monocytopenia and diminishing early atherosclerosis. These recent advances in our understanding of Akt signaling in macrophages in atherosclerosis may have significant relevance in the burgeoning field of cardio-oncology, where PI3K/Akt inhibitors being tested in cancer patients can have significant cardiovascular and metabolic ramifications.
Akt Signaling in Macrophage Polarization, Survival, and Atherosclerosis
(International Journal of Moledular Sciences, 2019-06-01) Linton, MacRae F.; Moslehi, Javid J.; Babaev, Vladimir R.
The PI3K/Akt pathway plays a crucial role in the survival, proliferation, and migration of macrophages, which may impact the development of atherosclerosis. Changes in Akt isoforms or modulation of the Akt activity levels in macrophages significantly affect their polarization phenotype and consequently atherosclerosis in mice. Moreover, the activity levels of Akt signaling determine the viability of monocytes/macrophages and their resistance to pro-apoptotic stimuli in atherosclerotic lesions. Therefore, elimination of pro-apoptotic factors as well as factors that antagonize or suppress Akt signaling in macrophages increases cell viability, protecting them from apoptosis, and this markedly accelerates atherosclerosis in mice. In contrast, inhibition of Akt signaling by the ablation of Rictor in myeloid cells, which disrupts mTORC2 assembly, significantly decreases the viability and proliferation of blood monocytes and macrophages with the suppression of atherosclerosis. In addition, monocytes and macrophages exhibit a threshold effect for Akt protein levels in their ability to survive. Ablation of two Akt isoforms, preserving only a single Akt isoform in myeloid cells, markedly compromises monocyte and macrophage viability, inducing monocytopenia and diminishing early atherosclerosis. These recent advances in our understanding of Akt signaling in macrophages in atherosclerosis may have significant relevance in the burgeoning field of cardio-oncology, where PI3K/Akt inhibitors being tested in cancer patients can have significant cardiovascular and metabolic ramifications.
Structure of the Helicobacter pylori Cag type IV Secretion System
(ELIFE, 2019-06-18) Campbell, Anne M.; Chung, Jeong Min; Sheedlo, Michael J.; Sawhney, Neha; Frick-Cheng, Arwen E.; Lacy, Dana Borden; Cover, Timothy L.; Ohi, Melanie D.
Bacterial type IV secretion systems (T4SSs) are molecular machines that can mediate interbacterial DNA transfer through conjugation and delivery of effector molecules into host cells. The Helicobacter pylori Cag T4SS translocates CagA, a bacterial oncoprotein, into gastric cells, contributing to gastric cancer pathogenesis. We report the structure of a membrane-spanning Cag T4SS assembly, which we describe as three sub-assemblies: a 14-fold symmetric outer membrane core complex (OMCC), 17-fold symmetric periplasmic ring complex (PRC), and central stalk. Features that differ markedly from those of prototypical T4SSs include an expanded OMCC and unexpected symmetry mismatch between the OMCC and PRC. This structure is one of the largest bacterial secretion system assemblies ever reported and illustrates the remarkable structural diversity that exists among bacterial T4SSs.
p73 regulates epidermal wound healing and induced keratinocyte programming
(Plos One, 2019-06-19) Beeler, J. Scott; Marshall, Clayton B.; Gonzalez-Ericsson, Paula I.; Shaver, Timothy M.; Guasch, Gabriela L. Santos; Lea, Spencer T.; Johnson, Kimberly N.; Jin, Hailing; Venters, Bryan J.; Sanders, Melinda E.; Pietenpol, Jennifer A.
p63 is a transcriptional regulator of ectodermal development that is required for basal cell proliferation and stem cell maintenance. p73 is a closely related p53 family member that is expressed in select p63-positive basal cells and can heterodimerize with p63. p73-/- mice lack multiciliated cells and have reduced numbers of basal epithelial cells in select tissues; however, the role of p73 in basal epithelial cells is unknown. Herein, we show that p73-deficient mice exhibit delayed wound healing despite morphologically normal-appearing skin. The delay in wound healing is accompanied by decreased proliferation and increased levels of biomarkers of the DNA damage response in basal keratinocytes at the epidermal wound edge. In wild-type mice, this same cell population exhibited increased p73 expression after wounding. Analyzing single-cell transcriptomic data, we found that p73 was expressed by epidermal and hair follicle stem cells, cell types required for wound healing. Moreover, we discovered that p73 isoforms expressed in the skin (Delta Np73) enhance p63-mediated expression of keratinocyte genes during cellular reprogramming from a mesenchymal to basal keratinocyte-like cell. We identified a set of 44 genes directly or indirectly regulated by Delta Np73 that are involved in skin development, cell junctions, cornification, proliferation, and wound healing. Our results establish a role for p73 in cutaneous wound healing through regulation of basal keratinocyte function.
Low plasma adropin concentrations increase risks of weight gain and metabolic dysregulation in response to a high-sugar diet in male nonhuman primates
(JOURNAL OF BIOLOGICAL CHEMISTRY, 2019-06-21) Butler, Andrew A.; Zhang, Jinsong; Price, Candice A.; Stevens, Joseph R.; Graham, James L.; Stanhope, Kimber L.; King, Sarah; Krauss, Ronald M.; Bremer, Andrew A.; Havel, Peter J.
Mouse studies linking adropin, a peptide hormone encoded by the energy homeostasis-associated (ENHO) gene, to biological clocks and to glucose and lipid metabolism suggest a potential therapeutic target for managing diseases of metabolism. However, adropin's roles in human metabolism are unclear. In silico expression profiling in a nonhuman primate diurnal transcriptome atlas (GSE98965) revealed a dynamic and diurnal pattern of ENHO expression. ENHO expression is abundant in brain, including ventromedial and lateral hypothalamic nuclei regulating appetite and autonomic function. Lower ENHO expression is present in liver, lung, kidney, ileum, and some endocrine glands. Hepatic ENHO expression associates with genes involved in glucose and lipid metabolism. Unsupervised hierarchical clustering identified 426 genes co-regulated with ENHO in liver, ileum, kidney medulla, and lung. Gene Ontology analysis of this cluster revealed enrichment for epigenetic silencing by histone H3K27 trimethylation and biological processes related to neural function. Dietary intervention experiments with 59 adult male rhesus macaques indicated low plasma adropin concentrations were positively correlated with fasting glucose, plasma leptin, and apolipoprotein C3 (APOC3) concentrations. During consumption of a high-sugar (fructose) diet, which induced 10% weight gain, animals with low adropin had larger increases of plasma leptin and more severe hyperglycemia. Declining adropin concentrations were correlated with increases of plasma APOC3 and triglycerides. In summary, peripheral ENHO expression associates with pathways related to epigenetic and neural functions, and carbohydrate and lipid metabolism, suggesting co-regulation in nonhuman primates. Low circulating adropin predicts increased weight gain and metabolic dysregulation during consumption of a high-sugar diet.
The Establishment of the Household Air Pollution Consortium (HAPCO)
(Atmosphere, 2019-07) Shu, Xiao-Ou; Yang, Gong
Household air pollution (HAP) is of public health concern, with 3 billion people worldwide (including >15 million in the US) exposed. HAP from coal use is a human lung carcinogen, yet the epidemiological evidence on carcinogenicity of HAP from biomass use, primarily wood, is not conclusive. To robustly assess biomass's carcinogenic potential, prospective studies of individuals experiencing a variety of HAP exposures are needed. We have built a global consortium of 13 prospective cohorts (HAPCO: Household Air Pollution Consortium) that have site- and disease-specific mortality and solid fuel use data, for a combined sample size of 587,257 participants and 57,483 deaths. HAPCO provides a novel opportunity to assess the association of HAP with lung cancer death while controlling for important confounders such as tobacco and outdoor air pollution exposures. HAPCO is also uniquely positioned to determine the risks associated with cancers other than lung as well as nonmalignant respiratory and cardiometabolic outcomes, for which prospective epidemiologic research is limited. HAPCO will facilitate research to address public health concerns associated with HAP-attributed exposures by enabling investigators to evaluate sex-specific and smoking status-specific effects under various exposure scenarios.

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