Atrazine induction of a family 4 cytochrome P450 gene in \u3ci\u3eChironomus tentans\u3c/i\u3e (Diptera: Chironomidae)

Cytochrome P450-dependent microsomal monooxygenase (P450) activity was measured in control and atrazine-exposed third instar midge larvae, Chironomus tentans. Significantly elevated O-demethylase activity was observed in gut homogenates taken from midges exposed to atrazine concentrations from 1 to 10 ppm for 90 h. No significant induction was observed at atrazine concentrations below 1 ppm. A region of a cytochrome P450 family 4 gene was amplified and sequenced from C. tentans larvae. Alignments of inferred amino acid sequences with other insect CYP4 gene homologues indicate a high degree of similarity. Northern blot analysis employing the CYP4 gene fragment as a probe showed an overexpression in C. tentans exposed to atrazine. The results support the previously identified inducibility of cytochrome P450-dependent activity and provide insight into the potential consequences of atrazine exposure to aquatic organisms

Dengue Virus Infection of Aedes aegypti Requires a Putative Cysteine Rich Venom Protein

Citation: Londono-Renteria, B., Troupin, A., Conway, M. J., Vesely, D., Ledizet, M., Roundy, C. M., . . . Colpitts, T. M. (2015). Dengue Virus Infection of Aedes aegypti Requires a Putative Cysteine Rich Venom Protein. Plos Pathogens, 11(10), 23. doi:10.1371/journal.ppat.1005202Dengue virus (DENV) is a mosquito-borne flavivirus that causes serious human disease and mortality worldwide. There is no specific antiviral therapy or vaccine for DENV infection. Alterations in gene expression during DENV infection of the mosquito and the impact of these changes on virus infection are important events to investigate in hopes of creating new treatments and vaccines. We previously identified 203 genes that were >= 5-fold differentially upregulated during flavivirus infection of the mosquito. Here, we examined the impact of silencing 100 of the most highly upregulated gene targets on DENV infection in its mosquito vector. We identified 20 genes that reduced DENV infection by at least 60% when silenced. We focused on one gene, a putative cysteine rich venom protein (SeqID AAEL000379; CRVP379), whose silencing significantly reduced DENV infection in Aedes aegypti cells. Here, we examine the requirement for CRVP379 during DENV infection of the mosquito and investigate the mechanisms surrounding this phenomenon. We also show that blocking CRVP379 protein with either RNAi or specific antisera inhibits DENV infection in Aedes aegypti. This work identifies a novel mosquito gene target for controlling DENV infection in mosquitoes that may also be used to develop broad preventative and therapeutic measures for multiple flaviviruses

Biochemical and molecular effects of atrazine exposure in Chironomus tentans and Pimephales promelas

Cytochrome P450\u27s represent the single most important enzyme family involved with detoxification of xenobiotics. Induction of specific P450 isozymes in insects is often observed after treatment with a variety of inorganic chemicals (Scott 1999), and induction of the P450 system can have important consequences to the ability of insects to tolerate exposure to pesticides. Studies performed in our lab have measured the effect of atrazine exposure on cytochrome P450-dependent monooxygenase activity and have found increased activity as a result of atrazine exposure (10 ppm) in midge larvae (Chironomus tentans). Identification of specific atrazine-inducible genes enhances sensitivity of detection and provides insight into potential consequences of exposure. Research results presented in this thesis identified a CYP4 gene with an open reading frame of 1,680 bp (accession number AY880065). Northern blot analysis employing a fragment of 1,200 bp from the CYP4 gene as a probe confirmed over-expression in C. tentans exposed to atrazine (10 ppm). Additionally, expression of CYP4 was present in all five insect stages but was highest in late instar larvae. In a second set of experiments, proteomics were used to identify a set of fourteen proteins from C. tentans third instar larvae exposed to atrazine that exhibited differences in expression. The cytochrome P450 previously identified was not identified by these studies. The effect of atrazine exposure on cytochrome P450-dependent monooxygenase activity was also measured in adults of the fathead minnow (Pimephales promelas) and increased activity was found in males exposed to atrazine (2000 ppb). Additionally, proteomics were used to identify a set of four proteins from microsomes prepared from adult livers of P. promelas that showed differences in expression in midges exposed to atrazine. These results provide important methods for understanding pesticide toxicity in non-target organisms using molecular and proteomics techniques. The study of the affected proteins by atrazine in these species will contribute to the identification of target genes and provide a means to better predict the effects of exposure

Cloning and expression of an atrazine inducible cytochrome P450, \u3ci\u3eCYP4G33\u3c/i\u3e, from \u3ci\u3eChironomus tentans\u3c/i\u3e (Diptera: Chironomidae)

Previous studies performed in our laboratory have measured the effect of atrazine exposure on cytochrome P450-dependent monooxygenase activity and have found increased activity in midge larvae (Chironomus tentans) as a result of atrazine exposure (1– 10 ppm). Here we report the cloning and expression of a specific C. tentans CYP4 gene that is responsive to atrazine induction with an open reading frame of 1678 bp which encodes a putative protein of 559 amino acid residues. Alignments of deduced amino acid sequences with other insect P450 genes and phylogenetic analysis indicated a high degree of similarity to other insect CYP4 genes. Northern blotting analysis employing a fragment of 1200 bp from the CYP4 gene as a probe indicated that the CYP4 gene was expressed in all developmental stages, but was expressed at highest levels in late instar larvae. Additionally, over-expression of CYP4 in C. tentans exposed to atrazine (10 mg/l) confirms the ability of atrazine to induce specific P450 genes and provides insight into potential consequences of atrazine exposure in aquatic organisms

Expanded phenotypic spectrum of neurodevelopmental and neurodegenerative disorder Bryant-Li-Bhoj syndrome with 38 additional individuals.

Bryant-Li-Bhoj syndrome (BLBS), which became OMIM-classified in 2022 (OMIM: 619720, 619721), is caused by germline variants in the two genes that encode histone H3.3 (H3-3A/H3F3A and H3-3B/H3F3B) [1-4]. This syndrome is characterized by developmental delay/intellectual disability, craniofacial anomalies, hyper/hypotonia, and abnormal neuroimaging [1, 5]. BLBS was initially categorized as a progressive neurodegenerative syndrome caused by de novo heterozygous variants in either H3-3A or H3-3B [1-4]. Here, we analyze the data of the 58 previously published individuals along 38 unpublished, unrelated individuals. In this larger cohort of 96 people, we identify causative missense, synonymous, and stop-loss variants. We also expand upon the phenotypic characterization by elaborating on the neurodevelopmental component of BLBS. Notably, phenotypic heterogeneity was present even amongst individuals harboring the same variant. To explore the complex phenotypic variation in this expanded cohort, the relationships between syndromic phenotypes with three variables of interest were interrogated: sex, gene containing the causative variant, and variant location in the H3.3 protein. While specific genotype-phenotype correlations have not been conclusively delineated, the results presented here suggest that the location of the variants within the H3.3 protein and the affected gene (H3-3A or H3-3B) contribute more to the severity of distinct phenotypes than sex. Since these variables do not account for all BLBS phenotypic variability, these findings suggest that additional factors may play a role in modifying the phenotypes of affected individuals. Histones are poised at the interface of genetics and epigenetics, highlighting the potential role for gene-environment interactions and the importance of future research

Cardiac myosin activation with omecamtiv mecarbil in systolic heart failure

BACKGROUND The selective cardiac myosin activator omecamtiv mecarbil has been shown to improve cardiac function in patients with heart failure with a reduced ejection fraction. Its effect on cardiovascular outcomes is unknown. METHODS We randomly assigned 8256 patients (inpatients and outpatients) with symptomatic chronic heart failure and an ejection fraction of 35% or less to receive omecamtiv mecarbil (using pharmacokinetic-guided doses of 25 mg, 37.5 mg, or 50 mg twice daily) or placebo, in addition to standard heart-failure therapy. The primary outcome was a composite of a first heart-failure event (hospitalization or urgent visit for heart failure) or death from cardiovascular causes. RESULTS During a median of 21.8 months, a primary-outcome event occurred in 1523 of 4120 patients (37.0%) in the omecamtiv mecarbil group and in 1607 of 4112 patients (39.1%) in the placebo group (hazard ratio, 0.92; 95% confidence interval [CI], 0.86 to 0.99; P = 0.03). A total of 808 patients (19.6%) and 798 patients (19.4%), respectively, died from cardiovascular causes (hazard ratio, 1.01; 95% CI, 0.92 to 1.11). There was no significant difference between groups in the change from baseline on the Kansas City Cardiomyopathy Questionnaire total symptom score. At week 24, the change from baseline for the median N-terminal pro-B-type natriuretic peptide level was 10% lower in the omecamtiv mecarbil group than in the placebo group; the median cardiac troponin I level was 4 ng per liter higher. The frequency of cardiac ischemic and ventricular arrhythmia events was similar in the two groups. CONCLUSIONS Among patients with heart failure and a reduced ejection, those who received omecamtiv mecarbil had a lower incidence of a composite of a heart-failure event or death from cardiovascular causes than those who received placebo. (Funded by Amgen and others; GALACTIC-HF ClinicalTrials.gov number, NCT02929329; EudraCT number, 2016 -002299-28.)