Involvement of circadian clock genes in reproduction of Drosophila melanogaster

Daily (circadian) rhythms exist at molecular, physiological, and behavioral levels and coordinate many life functions. This coordination is believed to contribute to an organism's fitness, however, such contributions have not been convincingly demonstrated in any animal. The most significant measure of fitness is the reproductive output of the individual and species. In this thesis I examine the consequences of loss of clock function on reproductive fitness in Drosophila melanogaster. I demonstrated that single mating among couples with mutated period (per⁰), timeless (tim⁰), cycle (cyc⁰), and Clock (Clk[superscript Jrk]) genes resulted in approximately 40% fewer progeny compared to wild-type flies. Male and female contribution to this phenotype was demonstrated by a decrease in reproductive capacity among per⁰ and tim⁰ flies mated with wild-type flies of the opposite sex. The important role of clock genes for reproductive fitness was confirmed by reversal of the low fertility phenotype in flies with rescued per or tim function. These results prompted an investigation to determine the relative contribution of each sex to the fertility phenotype. Males lacking a functional clock showed a significant decline in the quantity of sperm released from the testes to seminal vesicles (SV), suggesting that this peripheral oscillator is involved in sperm maturation. We found that clock genes are rhythmically expressed in these tissues and the cycling of per and tim expression continued in vitro, hence the testes and SV complex contained an autonomous circadian clock. In contrast to males, PER and TIM were constantly present in the cytoplasm of follicular cells in fly ovaries. Ovarian expression of per and tim is not disrupted by constant light and females lacking per and tim produced nearly 50% fewer mature oocytes then wild-type flies. These results suggest that per and tim are acting in a non-circadian pathway in the ovaries. Taken together, this data demonstrates that circadian clock genes significantly contribute to the fitness of Drosophila melanogaster by affecting the fecundity of both sexes

Phytochemicals from cruciferous vegetables, epigenetics, and prostate cancer prevention

Epidemiological evidence has demonstrated a reduced risk of prostate cancer associated with cruciferous vegetable intake. Follow-up studies have attributed this protective activity to the metabolic products of glucosinolates, a class of secondary metabolites produced by crucifers. The metabolic products of glucoraphanin and glucobrassicin, sulforaphane and indole-3-carbinol respectively, have been the subject of intense investigation by cancer researchers. Sulforaphane and indole-3-carbinol inhibit prostate cancer by both blocking initiation and suppressing prostate cancer progression in vitro and in vivo. Research has largely focused on the anti-initiation and cytoprotective effects of sulforaphane and indole-3-carbinol through induction of Phase I and Phase II detoxification pathways. With regards to suppressive activity, research has focused on the ability of sulforaphane and indole-3-carbinol to antagonize cell signaling pathways known to be dysregulated in prostate cancer. More recent investigations have characterized the ability of sulforaphane and indole-3-carbinol derivatives to modulate the activity of enzymes controlling the epigenetic status of prostate cancer cells. In this review we will summarize the well-established, “classic” non-epigenetic targets of sulforaphane and indole-3-carbinol, and highlight more recent evidence supporting these phytochemicals as epigenetic modulators for prostate cancer chemoprevention.Keywords: sulforaphane, prostate cancer, I3C, epigeneti

Lung Inflammation, Injury, and Proliferative Response after Repetitive Particulate Hexavalent Chromium Exposure

BACKGROUND: Chronic inflammation is implicated in the development of several human cancers, including lung cancer. Certain particulate hexavalent chromium [Cr(VI)] compounds are well-documented human respiratory carcinogens that release genotoxic soluble chromate and are associated with fibrosis, fibrosarcomas, adenocarcinomas, and squamous cell carcinomas of the lung. Despite this, little is known about the pathologic injury and immune responses after repetitive exposure to particulate chromates. OBJECTIVES: In this study we investigated the lung injury, inflammation, proliferation, and survival signaling responses after repetitive exposure to particulate chromate. METHODS: BALB/c mice were repetitively treated with particulate basic zinc chromate or saline using an intranasal exposure regimen. We assessed lungs for Cr(VI)-induced changes by bronchoalveolar lavage, histologic examination, and immunohistochemistry. RESULTS: Single exposure to Cr(VI) resulted in inflammation of lung tissue that persists for up to 21 days. Repetitive Cr(VI) exposure induced a neutrophilic inflammatory airway response 24 hr after each treatment. Neutrophils were subsequently replaced by increasing numbers of macrophages by 5 days after treatment. Repetitive Cr(VI) exposure induced chronic peribronchial inflammation with alveolar and interstitial pneumonitis dominated by lymphocytes and macrophages. Moreover, chronic toxic mucosal injury was observed and accompanied by increased airway pro-matrix metalloprotease-9. Injury and inflammation correlated with airways becoming immuno reactive for phosphorylation of the survival signaling protein Akt and the proliferation marker Ki-67. We observed a reactive proliferative response in epithelial cells lining airways of chromate-exposed animals. CONCLUSIONS: These data illustrate that repetitive exposure to particulate chromate induces chronic injury and an inflammatory microenvironment that may promote Cr(VI) carcinogenesis

Inheritance analysis and identification of SNP markers associated with ZYMV resistance in Cucurbita pepo

[EN] Cucurbit crops are economically important worldwide. One of the most serious threats to cucurbit production is Zucchini yellow mosaic virus (ZYMV). Several resistant accessions were identified in Cucurbita moschata and their resistance was introgressed into Cucurbita pepo. However, the mode of inheritance of ZYMV resistance in C. pepo presents a great challenge to attempts at introgressing resistance into elite germplasm. The main goal of this work was to analyze the inheritance of ZYMV resistance and to identify markers associated with genes conferring resistance. An Illumina GoldenGate assay allowed us to assess polymorphism among nine squash genotypes and to discover six polymorphic single-nucleotide polymorphisms (SNPs) between two near-isogenic lines, "True French" (susceptible to ZYMV) and Accession 381e (resistant to ZYMV). Two F-2 and three BC1 populations obtained from crossing the ZYMV-resistant Accession 381e with two susceptible ones, the zucchini True French and the cocozelle "San Pasquale," were assayed for ZYMV resistance. Molecular analysis revealed an approximately 90% association between SNP1 and resistance, which was confirmed using High Resolution Melt (HRM) and a CAPS marker. Co-segregation up to 72% in populations segregating for resistance was observed for two other SNP markers that could be potentially linked to genes involved in resistance expression. A functional prediction of proteins involved in the resistance response was performed on genome scaffolds containing the three SNPs of interest. Indeed, 16 full-length pathogen recognition genes (PRGs) were identified around the three SNP markers. In particular, we discovered that two nucleotide-binding site leucine-rich repeat (NBS-LRR) protein-encoding genes were located near the SNP1 marker. The investigation of ZYMV resistance in squash populations and the genomic analysis performed in this work could be useful for better directing the introgression of disease resistance into elite C. pepo germplasm.This work was supported by the Ministry of University and Research (GenHORT project).Capuozzo, C.; Formisano, G.; Iovieno, P.; Andolfo, G.; Tomassoli, L.; Barbella, M.; Picó Sirvent, MB.... (2017). Inheritance analysis and identification of SNP markers associated with ZYMV resistance in Cucurbita pepo. Molecular Breeding. 37(8). https://doi.org/10.1007/s11032-017-0698-5S378Addinsoft (2007) XLSTAT, Analyse de données et statistique avec MS Excel. Addinsoft, NYAndolfo G, Ercolano MR (2015) Plant innate immunity multicomponent model. Front Plant Sci 6:987Andolfo G, Sanseverino W, Rombauts S et al (2013) Overview of tomato (Solanum lycopersicum) candidate pathogen recognition genes reveals important Solanum R locus dynamics. New Phytol 197:223–237Andolfo G, Ferriello F, Tardella L et al (2014) Tomato genome-wide transcriptional responses to fusarium wilt, and tomato mosaic virus. PLoS One 9:e94963Blanca J, Cañizares J, Roig C, Ziarsolo P, Nuez F, Picó B (2011) Transcriptome characterization and high throughput SSRs and SNPs discovery in Cucurbita pepo (Cucurbitaceae). BMC Genomics 12:104Brown RN, Bolanos-Herrera A, Myers JR, Jahn MM (2003) Inheritance of resistance to four cucurbit viruses in Cucurbita moschata. Euphytica 129:253–258Burge CB, Karlin S (1998) Finding the genes in genomic DNA. Curr Opin Struct Biol 8:346–354Cipollini D (2008) Constitutive expression of methyl jasmonate-inducible responses delays reproduction and constrains fitness responses to nutrients in Arabidopsis thaliana. Evol Ecol 24:59–68Cohen R, Hanan A, Paris HS (2003) Single-gene resistance to powdery mildew in zucchini squash (Cucurbita pepo). Euphytica 130:433–441Collum TD, Padmanabhan MS, Hsieh YC, Culver JN (2016) Tobacco mosaic virus-directed reprogramming of auxin/indole acetic acid protein transcriptional responses enhances virus phloem loading. Proc Natl Acad Sci U S A 113:E2740–E2749Desbiez C, Lecoq H (1997) Zucchini yellow mosaic virus. Plant Pathol 46:809–829Ercolano MR, Sanseverino W, Carli P, Ferriello F, Frusciante L (2012) Genetic and genomic approaches for R-gene mediated disease resistance in tomato: retrospects and prospects. Plant Cell Rep 31:973–985Esteras C, Gómez P, Monforte AJ, Blanca J, Vicente-Dólera N, Roig C, Nuez F, Picó B (2012) High-throughput SNP genotyping in Cucurbita pepo for map construction and quantitative trait loci mapping. BMC Genomics 13:80Formisano G, Paris HS, Frusciante L, Ercolano MR (2010) Commercial Cucurbita pepo squash hybrids carrying disease resistance introgressed from Cucurbita moschata have high genetic similarity. Plant Genet Resour 8:198–203Fulton TM, Chunwongse J, Tanksley SD (1995) Microprep protocol for extraction of DNA from tomato and other herbaceous plants. Plant Mol Biol Report 13:207–209Gal-On A (2007) Zucchini yellow mosaic virus: insect transmission and pathogenicity—the tails of two proteins. Mol Plant Pathol 8:139–150Gilbert-Albertini F, Lecoq H, Pitrat M, Nicolet JL (1993) Resistance of Cucurbita moschata to watermelon mosaic virus type 2 and its genetic relation to resistance to zucchini yellow mosaic virus. Euphytica 69:231–237Gómez P, Rodríguez-Hernández AM, Moury B, Aranda MA (2009) Genetic resistance for the sustainable control of plant virus diseases: breeding, mechanisms and durability. Eur J Plant Pathol 125:1–22Gong L, Stift G, Kofler R, Pachner M, Lelley T (2008a) Microsatellites for the genus Cucurbita and an SSR-based genetic linkage map of Cucurbita pepo L. Theor Appl Genet 117:37–48Gong L, Pachner M, Kalai K, Lelley T (2008b) SSR-based genetic linkage map of Cucurbita moschata and its synteny with Cucurbita pepo. Genome 51:878–887Iovieno P, Andolfo G, Schiavulli A, Catalano D, Ricciardi L, Frusciante L et al. (2015) Structure, evolution and functional inference on the MildewLocusO (MLO) gene family in three cultivated Cucurbitaceae. BMC Genomics 16:1112. doi: 10.1186/s12864-015-2325-3Ishibashi K, Kezuka Y, Kobayashi C, Kato M, Inoue T, Nonaka T et al (2014) Structural basis for the recognition–evasion arms race between Tomato mosaic virus and the resistance gene Tm-1. PNAS 111:E3486–E3495Lecoq H, Pitrat M, Clément M (1981) Identification et caractérisation d’un potyvirus provoquant la maladie du rabougrissement jaune du melon. Agronomie 1:827–834Lefebvre V, Palloix A (1996) Both epistatic and additive effects of QTLs are involved in polygenic induced resistance to disease: a case study, the interaction pepper—Phytophthora capsici Leonian. Theor Appl Genet 93:503–511Levi A, Thomas CE, Newman M, Zhan X, Xu Y, Wehner TC (2003) Massive preferential segregation and nonrandom assortment of linkage-groups produce quasi-linkage in an F2 mapping population of watermelon. Hortscience 38:782Lisa V, Lecoq H (1984) Zucchini yellow mosaic virus. Descriptions of Plant Viruses, Commonwealth Mycological Institute and Association of Applied Biologists 282Lisa V, Boccardo G, D'Agostino G, Dellavalle G, d’Aquilio M (1981) Characterization of a potyvirus that causes zucchini yellow mosaic. Phytopathology 71:667–672MacQueen A, Bergelson J (2016) Modulation of R-gene expression across environments. J Exp Bot 67:2093–2105Mantel N (1967) The detection of disease clustering and a generalized regression approach. Cancer Res 27:209–220Munger HM, Provvidenti R (1987) Inheritance of resistance to zucchini yellow mosaic virus in Cucurbita moschata. Cucurbit Genet Coop Rep 10:8–81Nameth ST, Dodds JA, Paulus AO, Laemmlen FF (1986) Cucurbit viruses of California: an ever-changing problem. Plant Dis 70:8–12Ott J, Wang J, Leal SM (2015) Genetic linkage analysis in the age of whole-genome sequencing. Nat Rev Genet 16(5):275–284Pachner M, Lelley T (2004) Different genes for resistance to zucchini yellow mosaic virus (ZYMV) in Cucurbita moschata. In: Lebeda A, Paris HS (eds) Progress in cucurbit genetics and breeding research: Proceedings of Cucurbitaceae 2004. Palacky University, Olomouc (Czech Republic), pp 237–243Pachner M, Paris HS, Lelley T (2011) Genes for resistance to zucchini yellow mosaic in tropical pumpkin. J Hered 102:330–335Pachner M, Paris HS, Winkler J, Lelley T (2015) Phenotypic and marker-assisted pyramiding of genes for resistance to zucchini yellow mosaic virus in oilseed pumpkin (Cucurbita pepo). Plant Breed 134:121–128Paris HS (1986) A proposed subspecific classification for Cucurbita pepo. Phytologia 61:133–138Paris HS (2001) Characterization of the Cucurbita pepo collection at the Newe Ya‘ar Research Center, Israel. Plant Genet Resour Newsl 126:41–45Paris HS (2008) Summer squash. In: Prohens J, Nuez F (eds) Handbook of plant breeding, Vegetables I: 351–379Paris HS, Cohen S (2000) Oligogenic inheritance for resistance to zucchini yellow mosaic virus in Cucurbita pepo. Ann Appl Biol 136:209–214Paris HS, Cohen S, Burger Y, Joseph R (1988) Single-gene resistance to zucchini yellow mosaic virus in Cucurbita moschata. Euphytica 37:27–29Peakall PE, Smouse R (2012) GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research—an update. Bioinformatics 28:2537–2539Sakamoto T, Deguchi M, Brustolini OJ, Santos AA, Silva FF, Fontes EP (2012) The tomato RLK superfamily: phylogeny and functional predictions about the role of the LRRII-RLK subfamily in antiviral defense. BMC Plant Biol 12:229Sanseverino W, Ercolano MR (2012) In silico approach to predict candidate R proteins and to define their domain architecture. BMC Res Notes 5:678Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739Teare MD, Santibanez Koref MF (2014) Linkage analysis and the study of Mendelian disease in the era of whole exome and genome sequencing. Brief Funct Genomics 13(5):378–383Valkonen JPT, Wiegmann K, Hämäläinen JH, Marczewski W, Watanabe KN (2008) Evidence for utility of the same PCR-based markers for selection of extreme resistance to Potato virus Y controlled by Rysto of Solanum stoloniferum derived from different sources. Ann Appl Biol 152:121–130Wessel-Beaver L (2005) Cultivar and germplasm release. Release of ‘Soler’ tropical pumpkin. J Agric Univ P R 89:263–266Whitaker TW, Davis GN (1962) Cucurbits: botany, cultivation and utilization. Interscience, New York, pp 105–116Whitaker TW, Robinson RW (1986) Squash breeding. In: Bassett MJ (ed) Breeding vegetable crops. Avi, Westport, pp 209–242Xu Y, Crouch JH (2008) Marker-assisted selection in plant breeding: from publications to practice. Crop Sci 48:391–407Xu R, Zhang S, Huang J, Zheng C (2013) Genome-wide comparative in silico analysis of the RNA helicase gene family in Zea mays and Glycine max: a comparison with Arabidopsis and Oryza sativa. PLoS One 8:e78982Ye G, Smith KF (2008) Marker-assisted gene pyramiding for inbred line development: basic principles and practical guidelines. Int J Plant Breed 2:1–10Zdobnov EM, Apweiler R (2001) InterProScan—an integration platform for the signature-recognition methods in InterPro. Bioinformatics 17:847–848Zraidi A, Stift G, Pachner M, Shojaeiyan A, Gong L, Lelley T (2007) A consensus map for Cucurbita pepo. Mol Breed 20:375–38

Phenotypic spectrum and transcriptomic profile associated with germline variants in TRAF7

PURPOSE: Somatic variants in tumor necrosis factor receptor-associated factor 7 (TRAF7) cause meningioma, while germline variants have recently been identified in seven patients with developmental delay and cardiac, facial, and digital anomalies. We aimed to define the clinical and mutational spectrum associated with TRAF7 germline variants in a large series of patients, and to determine the molecular effects of the variants through transcriptomic analysis of patient fibroblasts. METHODS: We performed exome, targeted capture, and Sanger sequencing of patients with undiagnosed developmental disorders, in multiple independent diagnostic or research centers. Phenotypic and mutational comparisons were facilitated through data exchange platforms. Whole-transcriptome sequencing was performed on RNA from patient- and control-derived fibroblasts. RESULTS: We identified heterozygous missense variants in TRAF7 as the cause of a developmental delay-malformation syndrome in 45 patients. Major features include a recognizable facial gestalt (characterized in particular by blepharophimosis), short neck, pectus carinatum, digital deviations, and patent ductus arteriosus. Almost all variants occur in the WD40 repeats and most are recurrent. Several differentially expressed genes were identified in patient fibroblasts. CONCLUSION: We provide the first large-scale analysis of the clinical and mutational spectrum associated with the TRAF7 developmental syndrome, and we shed light on its molecular etiology through transcriptome studies

Evaluating the Effects of SARS-CoV-2 Spike Mutation D614G on Transmissibility and Pathogenicity.

Global dispersal and increasing frequency of the SARS-CoV-2 spike protein variant D614G are suggestive of a selective advantage but may also be due to a random founder effect. We investigate the hypothesis for positive selection of spike D614G in the United Kingdom using more than 25,000 whole genome SARS-CoV-2 sequences. Despite the availability of a large dataset, well represented by both spike 614 variants, not all approaches showed a conclusive signal of positive selection. Population genetic analysis indicates that 614G increases in frequency relative to 614D in a manner consistent with a selective advantage. We do not find any indication that patients infected with the spike 614G variant have higher COVID-19 mortality or clinical severity, but 614G is associated with higher viral load and younger age of patients. Significant differences in growth and size of 614G phylogenetic clusters indicate a need for continued study of this variant

Evaluating the Effects of SARS-CoV-2 Spike Mutation D614G on Transmissibility and Pathogenicity

Global dispersal and increasing frequency of the SARS-CoV-2 spike protein variant D614G are suggestive of a selective advantage but may also be due to a random founder effect. We investigate the hypothesis for positive selection of spike D614G in the United Kingdom using more than 25,000 whole genome SARS-CoV-2 sequences. Despite the availability of a large dataset, well represented by both spike 614 variants, not all approaches showed a conclusive signal of positive selection. Population genetic analysis indicates that 614G increases in frequency relative to 614D in a manner consistent with a selective advantage. We do not find any indication that patients infected with the spike 614G variant have higher COVID-19 mortality or clinical severity, but 614G is associated with higher viral load and younger age of patients. Significant differences in growth and size of 614G phylogenetic clusters indicate a need for continued study of this variant

Genomic assessment of quarantine measures to prevent SARS-CoV-2 importation and transmission

Mitigation of SARS-CoV-2 transmission from international travel is a priority. We evaluated the effectiveness of travellers being required to quarantine for 14-days on return to England in Summer 2020. We identified 4,207 travel-related SARS-CoV-2 cases and their contacts, and identified 827 associated SARS-CoV-2 genomes. Overall, quarantine was associated with a lower rate of contacts, and the impact of quarantine was greatest in the 16–20 age-group. 186 SARS-CoV-2 genomes were sufficiently unique to identify travel-related clusters. Fewer genomically-linked cases were observed for index cases who returned from countries with quarantine requirement compared to countries with no quarantine requirement. This difference was explained by fewer importation events per identified genome for these cases, as opposed to fewer onward contacts per case. Overall, our study demonstrates that a 14-day quarantine period reduces, but does not completely eliminate, the onward transmission of imported cases, mainly by dissuading travel to countries with a quarantine requirement