The eastern North American monarch butterfly, Danaus plexippus, is an emerging model system to study the neural, molecular, and genetic basis of animal long-distance migration and animal clockwork mechanisms. While genomic studies have provided new insight into migration-associated and circadian clock genes, the general lack of simple and versatile reverse-genetic methods has limited in vivo functional analysis of candidate genes in this species. Here, we report the establishment of highly efficient and heritable gene mutagenesis methods in the monarch butterfly using transcriptional activator-like effector nucleases (TALENs) and CRISPR-associated RNA-guided nuclease Cas9 (CRISPR/Cas9). Using two clock gene loci, cryptochrome 2 and clock (clk), as candidates, we show that both TALENs and CRISPR/Cas9 generate high-frequency nonhomologous end-joining (NHEJ)-mediated mutations at targeted sites (up to 100%), and that injecting fewer than 100 eggs is sufficient to recover mutant progeny and generate monarch knockout lines in about 3 months. Our study also genetically defines monarch CLK as an essential component of the transcriptional activation complex of the circadian clock. The methods presented should not only greatly accelerate functional analyses of many aspects of monarch biology, but are also anticipated to facilitate the development of these tools in other nontraditional insect species as well as the development of homology-directed knock-ins.
Ticks transmit more pathogens to humans and animals than any other arthropod. We describe the 2.1 Gbp nuclear genome of the tick, Ixodes scapularis (Say), which vectors pathogens that cause Lyme disease, human granulocytic anaplasmosis, babesiosis and other diseases. The large genome reflects accumulation of repetitive DNA, new lineages of retro-transposons, and gene architecture patterns resembling ancient metazoans rather than pancrustaceans. Annotation of scaffolds representing approximately 57% of the genome, reveals 20,486 protein-coding genes and expansions of gene families associated with tick-host interactions. We report insights from genome analyses into parasitic processes unique to ticks, including host 'questing', prolonged feeding, cuticle synthesis, blood meal concentration, novel methods of haemoglobin digestion, haem detoxification, vitellogenesis and prolonged off-host survival. We identify proteins associated with the agent of human granulocytic anaplasmosis, an emerging disease, and the encephalitis-causing Langat virus, and a population structure correlated to life-history traits and transmission of the Lyme disease agent.
Improved cell metabolism prolongs photoreceptor survival upon retinal-pigmented epithelium loss in the sodium iodate induced model of geographic atrophy
Age-related macular degeneration (AMD) is characterized by malfunction and loss of retinal-pigmented epithelium (RPE) cells. Because the RPE transfers nutrients from the choriocapillaris to photoreceptor (PR), PRs are affected as well. Geographic atrophy (GA) is an advanced form of AMD characterized by severe vision impairment due to RPE loss over large areas. Currently there is no treatment to delay the degeneration of nutrient deprived PRs once RPE cells die. Here we show that cell-autonomous activation of the key regulator of cell metabolism, the kinase mammalian target of rapamycin complex 1 (mTORC1), delays PR death in the sodium iodate induced model of RPE atrophy. Consistent with this finding loss of mTORC1 in cones accelerates cone death as cones fail to balance demand with supply. Interestingly, promoting rod survival does not promote cone survival in this model of RPE atrophy as both, rods and cones suffer from a sick and dying RPE. The findings suggest that activation of metabolic genes downstream of mTORC1 can serve as a strategy to prolong PR survival when RPE cells malfunction or die.
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.
Zika virus has emerged as a severe health threat with a rapidly expanding range. The IFITM family of restriction factors inhibits the replication of a broad range of viruses, including the closely related flaviruses West Nile virus and dengue virus. Here, we show that IFITM1 and IFITM3 inhibit Zika virus infection early in the viral life cycle. Moreover, IFITM3 can prevent Zika-virus-induced cell death. These results suggest that strategies to boost the actions and/or levels of the IFITMs might be useful for inhibiting a broad range of emerging viruses.
Muscle tissues are classically divided into two major types, depending on the presence or absence of striations. In striated muscles, the actin filaments are anchored at Z-lines and the myosin and actin filaments are in register, whereas in smooth muscles, the actin filaments are attached to dense bodies and the myosin and actin filaments are out of register. The structure of the filaments in smooth muscles is also different from that in striated muscles. Here we have studied the structure of myosin filaments from the smooth muscles of the human parasite Schistosoma mansoni. We find, surprisingly, that they are indistinguishable from those in an arthropod striated muscle. This structural similarity is supported by sequence comparison between the schistosome myosin II heavy chain and known striated muscle myosins. In contrast, the actin filaments of schistosomes are similar to those of smooth muscles, lacking troponin-dependent regulation. We conclude that schistosome muscles are hybrids, containing striated muscle-like myosin filaments and smooth muscle-like actin filaments in a smooth muscle architecture. This surprising finding has broad significance for understanding how muscles are built and how they evolved, and challenges the paradigm that smooth and striated muscles always have distinctly different components.
Drosophila sperm are unusual in that they do not require the intraflagellar transport (IFT) system for assembly of their flagella. In the mouse, the IFT proteins are very abundant in testis, but we here show that mature sperm are completely devoid of them, making the importance of IFT to mammalian sperm development unclear. To address this question, we characterized spermiogenesis and fertility in the Ift88(Tg737Rpw) mouse. This mouse has a hypomorphic mutation in the gene encoding the IFT88 subunit of the IFT particle. This mutation is highly disruptive to ciliary assembly in other organs. Ift88(-/-) mice are completely sterile. They produce approximately 350-fold fewer sperm than wild-type mice, and the remaining sperm completely lack or have very short flagella. The short flagella rarely have axonemes but assemble ectopic microtubules and outer dense fibers and accumulate improperly assembled fibrous sheath proteins. Thus IFT is essential for the formation but not the maintenance of mammalian sperm flagella.