Reconstruction of 60 Years of Chikungunya Epidemiology in the Philippines Demonstrates Episodic and Focal Transmission
Proper understanding of the long-term epidemiology of chikungunya has been hampered by poor surveillance. Outbreak years are unpredictable and cases often misdiagnosed. Here we analyzed age-specific data from 2 serological studies (from 1973 and 2012) in Cebu, Philippines, to reconstruct both the annual probability of infection and population-level immunity over a 60-year period (1952-2012). We also explored whether seroconversions during 2012-2013 were spatially clustered. Our models identified 4 discrete outbreaks separated by an average delay of 17 years. On average, 23% (95% confidence interval [CI], 16%-37%) of the susceptible population was infected per outbreak, with > 50% of the entire population remaining susceptible at any point. Participants who seroconverted during 2012-2013 were clustered at distances of < 230 m, suggesting focal transmission. Large-scale outbreaks of chikungunya did not result in sustained multiyear transmission. Nevertheless, we estimate that > 350,000 infections were missed by surveillance systems. Serological studies could supplement surveillance to provide important insights on pathogen circulation.
Transcriptional activation of follistatin by Nrf2 protects pulmonary epithelial cells against silica nanoparticle-induced oxidative stress
Silica nanoparticles (SiO2 NPs) cause oxidative stress in respiratory system. Meanwhile, human cells launch adaptive responses to overcome SiO2 NP toxicity. However, besides a few examples, the regulation of SiO2 NP-responsive proteins and their functions in SiO2 NP response remain largely unknown. In this study, we demonstrated that SiO2 NP induced the expression of follistatin (FST), a stress responsive gene, in mouse lung tissue as well as in human lung epithelial cells (A549). The levels of Ac-H3(K9/18) and H3K4me2, two active gene markers, at FST promoter region were significantly increased during SiO2 NP treatment. The induction of FST transcription was mediated by the nuclear factor erythroid 2-related factor 2 (Nrf2), as evidenced by the decreased FST expression in Nrf2-deficient cells and the direct binding of Nrf2 to FST promoter region. Down-regulation of FST promoted SiO2 NP-induced apoptosis both in cultured cells and in mouse lung tissue. Furthermore, knockdown of FST increased while overexpression of FST decreased the expression level of NADPH oxidase 1 (NOX1) and NOX5 as well as the production of cellular reactive oxygen species (ROS). Taken together, these findings demonstrated a protective role of FST in SiO2 NP-induced oxidative stress and shed light on the interaction between SiO2 NPs and biological systems.
Manipulation of IL-1beta and IL-18 production by Yersinia pestis effectors YopJ and YopM and redundant impact on virulence
Innate immunity plays a central role in resolving infections by pathogens. Host survival during plague, caused by the Gram-negative bacterium Yersinia pestis, is favored by a robust early innate immune response initiated by Interleukin-1beta (IL-1beta) and IL-18. These cytokines are produced by a two-step mechanism involving NF-kB mediated pro-cytokine production and inflammasome-driven maturation into bioactive inflammatory mediators. Because of the anti-microbial effects induced by IL-1beta/IL-18, it may be desirable for pathogens to manipulate their production. Y. pestis type III secretion system effectors YopJ and YopM can interfere with different parts of this process. Both effectors have been reported to influence inflammasome caspase-1 activity; YopJ promotes caspase-8 dependent cell death and caspase-1 cleavage, while YopM inhibits caspase-1 activity via an incompletely understood mechanism. Yet neither effector appears essential for full virulence in vivo. Here we report that the sum of influences by YopJ and YopM on IL-1beta/IL-18 release is suppressive. In the absence of YopM, YopJ minimally affects caspase-1 cleavage, but suppresses IL-1beta, IL-18 and other cytokines and chemokines. Importantly, we find that Y. pestis containing combined deletions of YopJ and YopM induces elevated levels of IL-1beta/IL-18 in vitro and in vivo, and is significantly attenuated in a mouse model of bubonic plague. The reduced virulence of the YopJ-YopM mutant is dependent on the presence of IL-1beta, IL-18 and caspase-1. Thus, we conclude that Y. pestis YopJ and YopM can both exert a tight control of host IL-1beta/IL-18 production to benefit the bacteria, resulting in a redundant impact on virulence.
Humans that are heterozygous for the common S180L polymorphism in the Toll-like receptor (TLR) adaptor Mal (encoded by TIRAP) are protected from a number of infectious diseases, including tuberculosis (TB), whereas those homozygous for the allele are at increased risk. The reason for this difference in susceptibility is not clear. We report that Mal has a TLR-independent role in interferon-gamma (IFN-gamma) receptor signaling. Mal-dependent IFN-gamma receptor (IFNGR) signaling led to mitogen-activated protein kinase (MAPK) p38 phosphorylation and autophagy. IFN-gamma signaling via Mal was required for phagosome maturation and killing of intracellular Mycobacterium tuberculosis (Mtb). The S180L polymorphism, and its murine equivalent S200L, reduced the affinity of Mal for the IFNGR, thereby compromising IFNGR signaling in macrophages and impairing responses to TB. Our findings highlight a role for Mal outside the TLR system and imply that genetic variation in TIRAP may be linked to other IFN-gamma-related diseases including autoimmunity and cancer.
Traditional cloning methods have limitations on the number of DNA fragments that can be simultaneously manipulated, which dramatically slows the pace of molecular assembly. Here we describe GMAP, a Gibson assembly-based modular assembly platform consisting of a collection of promoters and genes, which allows for one-step production of DNA constructs. GMAP facilitates rapid assembly of expression and viral constructs using modular genetic components, as well as increasingly complicated genetic tools using contextually relevant genomic elements. Our data demonstrate the applicability of GMAP toward the validation of synthetic promoters, identification of potent RNAi constructs, establishment of inducible lentiviral systems, tumor initiation in genetically engineered mouse models, and gene-targeting for the generation of knock-in mice. GMAP represents a recombinant DNA technology designed for widespread circulation and easy adaptation for other uses, such as synthetic biology, genetic screens, and CRISPR-Cas9.
Alcoholic hepatitis accelerates early hepatobiliary cancer by increasing stemness and miR-122-mediated HIF-1alpha activation
Alcohol-related hepatocellular carcinoma (HCC) develops with advanced alcoholic liver disease and liver fibrosis. Using adult mice, we evaluate the effect of alcoholic steatohepatitis on early hepatobiliary carcinoma after initiation by diethyl-nitrosamine (DEN). Here we show that alcohol-fed DEN-injected mice have higher ALT and liver-to-body weight ratio compared to pair-fed DEN-injected mice. Alcohol feeding results in steatohepatitis indicated by increased pro-inflammatory cytokines and fibrotic genes. MRI and liver histology of alcohol+DEN mice shows hepatobiliary cysts, early hepatic neoplasia and increase in serum alpha-fetoprotein. Proliferation makers (BrdU, cyclin D1, p53) and cancer stem cell markers (CD133 and nanog) are significantly up-regulated in livers of alcohol-fed DEN-injected mice compared to controls. In livers with tumors, loss of miR-122 expression with a significant up-regulation of miR-122 target HIF-1alpha is seen. We conclude that alcoholic steatohepatitis accelerates hepatobiliary tumors with characteristic molecular features of HCC by up-regulating inflammation, cell proliferation, stemness, and miR-122 loss.
Autocrine VEGF signaling is critical for sustaining prostate and other cancer stem cells (CSCs), and it is a potential therapeutic target, but we observed that CSCs isolated from prostate tumors are resistant to anti-VEGF (bevacizumab) and anti-VEGFR (sunitinib) therapy. Intriguingly, resistance is mediated by VEGF/neuropilin signaling, which is not inhibited by bevacizumab and sunitinib, and it involves the induction of P-Rex1, a Rac GEF, and consequent Rac1-mediated ERK activation. This induction of P-Rex1 is dependent on Myc. CSCs isolated from the PTEN(pc-/-) transgenic model of prostate cancer exhibit Rac1-dependent resistance to bevacizumab. Rac1 inhibition or P-Rex1 downregulation increases the sensitivity of prostate tumors to bevacizumab. These data reveal that prostate tumors harbor cells with stem cell properties that are resistant to inhibitors of VEGF/VEGFR signaling. Combining the use of available VEGF/VEGFR-targeted therapies with P-Rex1 or Rac1 inhibition should improve the efficacy of these therapies significantly.
BACKGROUND: Emergence of drug-resistant Plasmodium falciparum has created an urgent need for new drug targets. DNA polymerase delta is an essential enzyme required for chromosomal DNA replication and repair, and therefore may be a potential target for anti-malarial drug development. However, little is known of the characteristics and function of this P. falciparum enzyme. METHODS: The coding sequences of DNA polymerase delta catalytic subunit (PfPoldelta-cat), DNA polymerase delta small subunit (PfPoldeltaS) and proliferating cell nuclear antigen (PfPCNA) from chloroquine- and pyrimethamine-resistant P. falciparum strain K1 were amplified, cloned into an expression vector and expressed in Escherichia coli. The recombinant proteins were analysed by SDS-PAGE and identified by LC-MS/MS. PfPoldelta-cat was biochemically characterized. The roles of PfPoldeltaS and PfPCNA in PfPoldelta-cat function were investigated. In addition, inhibitory effects of 11 compounds were tested on PfPoldelta-cat activity and on in vitro parasite growth using SYBR Green I assay. RESULTS: The purified recombinant protein PfPoldelta-cat, PfPoldeltaS and PfPCNA showed on SDS-PAGE the expected size of 143, 57 and 34 kDa, respectively. Predicted amino acid sequence of the PfPoldelta-cat and PfPoldeltaS had 59.2 and 24.7 % similarity respectively to that of the human counterpart. The PfPoldelta-cat possessed both DNA polymerase and 3'-5' exonuclease activities. It used both Mg(2+) and Mn(2+) as cofactors and was inhibited by high KCl salt ( > 200 mM). PfPoldeltaS stimulated PfPoldelta-cat activity threefolds and up to fourfolds when PfPCNA was included in the assay. Only two compounds were potent inhibitors of PfPoldelta-cat, namely, butylphenyl-dGTP (BuPdGTP; IC50 of 38 microM) and 7-acetoxypentyl-(3, 4 dichlorobenzyl) guanine (7-acetoxypentyl-DCBG; IC50 of 55 microM). The latter compound showed higher inhibition on parasite growth (IC50 of 4.1 microM). CONCLUSIONS: Recombinant PfPoldelta-cat, PfPoldeltaS and PfPCNA were successfully expressed and purified. PfPolS and PfPCNA increased DNA polymerase activity of PfPoldelta-cat. The high sensitivity of PfPoldelta to BuPdGTP can be used to differentiate parasite enzyme from mammalian and human counterparts. Interestingly, 7-acetoxypentyl-DCBG showed inhibitory effects on both enzyme activity and parasite growth. Thus, 7-acetoxypentyl-DCBG is a potential candidate for future development of a new class of anti-malarial agents targeting parasite replicative DNA polymerase.
Recycling of neurotransmitters is essential for sustained neuronal signaling, yet recycling pathways for various transmitters, including histamine, remain poorly understood. In the first visual ganglion (lamina) of Drosophila, photoreceptor-released histamine is taken up into perisynaptic glia, converted to carcinine, and delivered back to the photoreceptor for histamine regeneration. Here, we identify an organic cation transporter, CarT (carcinine transporter), that transports carcinine into photoreceptors during histamine recycling. CarT mediated in vitro uptake of carcinine. Deletion of the CarT gene caused an accumulation of carcinine in laminar glia accompanied by a reduction in histamine, resulting in abolished photoreceptor signal transmission and blindness in behavioral assays. These defects were rescued by expression of CarT cDNA in photoreceptors, and they were reproduced by photoreceptor-specific CarT knockdown. Our findings suggest a common role for the conserved family of CarT-like transporters in maintaining histamine homeostasis in both mammalian and fly brains.
Cross-Sectional Associations of Computed Tomography (CT)-Derived Adipose Tissue Density and Adipokines: The Framingham Heart Study
BACKGROUND: Excess accumulation of abdominal subcutaneous (SAT) and visceral adipose tissue (VAT) is associated with adverse levels of adipokines and cardiovascular disease risk. Whether fat quality is associated with adipokines has not been firmly established. This study examined the association between abdominal SAT and VAT density, an indirect measure of fat quality, with a panel of metabolic regulatory biomarkers secreted by adipose tissue or the liver independently of absolute fat volumes. METHODS AND RESULTS: We evaluated 1829 Framingham Heart Study participants (44.9% women). Abdominal SAT and VAT density was estimated indirectly by adipose tissue attenuation using computed tomography. Adipokines included adiponectin, leptin receptor, leptin, fatty acid-binding protein 4 (FABP-4), retinol-binding protein 4 (RBP-4), and fetuin-A. Fat density was associated with all the biomarkers evaluated, except fetuin-A. Lower fat density (ie, more-negative fat attenuation) was associated with lower adiponectin and leptin receptor, but higher leptin and FABP-4 levels (all P < 0.0001). SAT density was inversely associated with RPB-4 in both sexes, whereas the association between VAT density and RPB-4 was only observed in men (P < 0.0001). In women, after additional adjustment for respective fat volume, SAT density retained the significant associations with adiponectin, leptin, FABP-4, and RBP-4; and VAT density with adiponectin only (all P<0.0001). In men, significant associations were maintained upon additional adjustment for respective fat volume (P < 0.005). CONCLUSIONS: Lower abdominal fat density was associated with a profile of biomarkers suggestive of greater cardiometabolic risk. These observations support that fat density may be a valid biomarker of cardiometabolic risk.
Promoter-enhancer looping at the PPARgamma2 locus during adipogenic differentiation requires the Prmt5 methyltransferase
PPARgamma2 is a critical lineage-determining transcription factor that is essential for adipogenic differentiation. Here we report characterization of the three-dimensional structure of the PPARgamma2 locus after the onset of adipogenic differentiation and the mechanisms by which it forms. We identified a differentiation-dependent loop between the PPARgamma2 promoter and an enhancer sequence 10 kb upstream that forms at the onset of PPARgamma2 expression. The arginine methyltransferase Prmt5 was required for loop formation, and overexpression of Prmt5 resulted in premature loop formation and earlier onset of PPARgamma2 expression. Kinetic studies of regulatory factor interactions at the PPARgamma2 promoter and enhancer revealed enhanced interaction of Prmt5 with the promoter that preceded stable association of Prmt5 with enhancer sequences. Prmt5 knockdown prevented binding of both MED1, a subunit of Mediator complex that facilitates enhancer-promoter interactions, and Brg1, the ATPase of the mammalian SWI/SNF chromatin remodeling enzyme required for PPARgamma2 activation and adipogenic differentiation. The data indicate a dynamic association of Prmt5 with the regulatory sequences of the PPARgamma2 gene that facilitates differentiation-dependent, three-dimensional organization of the locus. In addition, other differentiation-specific, long-range chromatin interactions showed Prmt5-dependence, indicating a more general role for Prmt5 in mediating higher-order chromatin connections in differentiating adipocytes.
Results of a Culturally Adapted Internet-Enhanced Physical Activity Pilot Intervention for Overweight and Obese Young Adult African American Women
PURPOSE: This study evaluated a culturally relevant, social cognitive theory-based, Internet-enhanced physical activity (PA) pilot intervention developed for overweight/obese African American (AA) female college students.
DESIGN: Using a 3-month, single group, pretest-posttest design, participants accessed a culturally relevant PA promotion website and engaged in four moderate-intensity PA sessions each week.
RESULTS: Study completers (n = 25, mean age = 21.9 years) reported a decrease in sedentary screen time (p < .0001); however, no changes in moderate-to-vigorous PA were reported (p = .150). A significant increase in self-regulation for PA (p < .0001) and marginally significant increases in social support (p = .052) and outcome expectations (p = .057) for PA were observed. No changes in body mass index (p = .162), PA enjoyment (p = .151), or exercise self-efficacy (p = .086) were reported.
CONCLUSIONS: Findings of this exploratory study show some preliminary support for Internet-enhanced approaches to promote PA among overweight/obese AA women.
IMPLICATIONS FOR PRACTICE: Future studies with larger samples are needed to further explore culturally relevant Internet-enhanced PA programs in this underserved population.
The carboxyl terminal mutational hotspot of the ciliary disease protein RPGRORF15 (retinitis pigmentosa GTPase regulator) is glutamylated in vivo
Mutations inRPGR(ORF15)(retinitis pigmentosa GTPase regulator) are a major cause of inherited retinal degenerative diseases. RPGR(ORF15)(1152 residues) is a ciliary protein involved in regulating the composition and function of photoreceptor cilia. The mutational hotspot in RPGR(ORF15)is an unusual C-terminal domain encoded by exon ORF15, which is rich in polyglutamates and glycine residues (Glu-Gly domain) followed by a short stretch of basic amino acid residues (RPGR(C2)domain; residues 1072-1152). However, the properties of the ORF15-encoded domain and its involvement in the pathogenesis of the disease are unclear. Here we show that RPGR(ORF15)is glutamylated at the C-terminus, as determined by binding to GT335, which recognizes glutamylated substrates. This reactivity is lost in two mouse mutants ofRpgr, which do not express RPGR(ORF15)due to disease-causing mutations in exon ORF15. Our results indicate that RPGR(ORF15)is posttranslationally glutamylated in the Glu-Gly domain and that the GT335 antibody predominantly recognizes RPGR(ORF15)in photoreceptor cilia.
Black and Latino men in Central Massachusetts are an underserved population suffering significant health disparities. This presentation was part of training provided by the National Network of Libraries of Medicine/New England Region grant, Accessing Health Information for African American, African, and Latino Men: MedlinePlus training for the Barbershop Health Network and the Hector Reyes House, Worcester, MA. Train-the-trainer sessions were presented June 22, June 25, and July 8, 2015 to the staff from both organizations to learn how and where to access reliable consumer health information and, in turn, share these resources with their clients.