Molecular Impacts of Dietary Exposure to Nanoplastics Combined or Not with Arsenic in the Caribbean Mangrove Oysters (Isognomon alatus)

Nanoplastics (NPs) are anthropogenic contaminants that raise concern, as they cross biological barriers. Metals’ adsorption on NPs’ surface also carries ecotoxicological risks to aquatic organisms. This study focuses on the impacts of three distinct NPs on the Caribbean oyster Isognomon alatus through dietary exposure. As such, marine microalgae Tisochrysis lutea were exposed to environmentally weathered mixed NPs from Guadeloupe (NPG), crushed pristine polystyrene nanoparticles (PSC), and carboxylated polystyrene nanoparticles of latex (PSL). Oysters were fed with NP-T. lutea at 10 and 100 µg L−1, concentrations considered environmentally relevant, combined or not with 1 mg L−1 pentoxide arsenic (As) in water. We investigated key gene expression in I. alatus’ gills and visceral mass. NP treatments revealed significant induction of cat and sod1 in gills and gapdh and sod1 in visceral mass. As treatment significantly induced sod1 expression in gills, but once combined with any of the NPs at both concentrations, basal mRNA levels were observed. Similarly, PSL treatment at 100 µg L−1 that significantly induced cat expression in gills or sod1 in visceral mass showed repressed mRNA levels when combined with As (reduction of 2222% and 34%, respectively, compared to the control). This study suggested a protective effect of the interaction between NPs and As, possibly by decreasing both contaminants’ surface reactivity.

Large Crystal Toxin Formation in Chromosomally Engineered Bacillus thuringiensis subsp. aizawai Due to σEAccumulation

ABSTRACTSeven distinctBacillus thuringiensissubsp.aizawaiintegrants were constructed that carried the chitinase (chiBlA) gene fromB. licheniformisunder the control of thecry11Aapromoter and terminator with and withoutp19andp20genes. The toxicity ofB. thuringiensissubsp.aizawaiintegrants against second-instarSpodoptera lituralarvae was increased 1.8- to 4.6-fold compared to that of the wild-type strain (BTA1). Surprisingly, the enhanced toxicity in some strains ofB. thuringiensissubsp.aizawaiintegrants (BtaP19CS,BtaP19CSter, andBtaCAT) correlated with an increase in toxin formation. To investigate the role of these genes in toxin production, the expression profiles of the toxin genes,cry1AaandchiBlA, as well as their transcriptional regulators (sigKandsigE), were analyzed by quantitative real-time RT-PCR (qPCR) from BTA1,BtaP19CS, andBtaCAT. Expression levels ofcry1Aain these two integrants increased about 2- to 3-fold compared to those of BTA1. The expression of the transcription factorsigKalso was prolonged in the integrants compared to that of the wild type; however,sigEexpression was unchanged. Western blot analysis of σEand σKshowed the prolonged accumulation of σEin the integrants compared to that of BTA1, resulting in the increased synthesis of pro-σKup toT17after the onset of sporulation in bothBtaP19CS andBtaCAT compared to that ofT13in BTA1. The results from qPCR indicate clearly that thecry1Aapromoter activity was influenced most strongly by σE, whereascry11Aadepended mostly on σK. These results on large-crystal toxin formation with enhanced toxicity should provide useful information for the generation of strains with improved insecticidal activity.

The antioxidant effects of garlic saponins protect PC12 cells from hypoxia-induced damage

Hypoxia frequently occurs under several different cellular circumstances. Excess reactive oxygen species that are induced by hypoxia may result in cell injury and dysfunction. Recently, garlic has been found to possess some biological and pharmacological activities. The present study examined the effects of garlic saponins (GSP) on the survival of differentiated PC12 (dPC12) cells and the oxidative–antioxidant system. dPC12 cells were exposed to 2 % O2 in order to establish a neuronal insult model. Cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide reduction assay and lactate dehydrogenase (LDH) release assay. The expression of selected genes (catalase (CAT), p65 and neuron-specific class III β-tubulin) was evaluated by real-time PCR and immunoblot assays. CAT activity, malondialdehyde (MDA) and 8-hydroxy-deoxyguanosine (8-OH-dG) concentrations were also determined. The data showed that hypoxia dramatically damaged dPC12 cells, while treatment with approximately 5 × 10− 2–10 ng/ml GSP improved cell viability, decreased LDH leakage and caused the cells to maintain neuronal-like characteristics in hypoxia. The production of MDA and 8-OH-dG was attenuated by GSP. CAT activity in dPC12 cells pretreated with GSP was higher than that of the hypoxic control. Moreover, GSP up-regulated CAT expression and decreased the total protein expression as well as the nuclear expression of p65 in hypoxic cells. These data indicate that GSP has antioxidant properties that can protect dPC12 cells from hypoxia-induced damage, which may be related to the up-regulation of CAT expression and activity as well as a decrease in the expression and nucleus distribution of p65 through effects on redox-sensitive signalling pathways.

The C, V and W proteins of Nipah virus inhibit minigenome replication

Nipah virus (NiV) is a recently emergent, highly pathogenic, zoonotic paramyxovirus of the genus Henipavirus. Like the phosphoprotein (P) gene of other paramyxoviruses, the P gene of NiV is predicted to encode three additional proteins, C, V and W. When the C, V and W proteins of NiV were tested for their ability to inhibit expression of the chloramphenicol acetyltransferase (CAT) reporter gene in plasmid-based, minigenome replication assays, each protein inhibited CAT expression in a dose-dependent manner. The C, V and W proteins of NiV also inhibited expression of CAT from a measles virus (MV) minigenome, but not from a human parainfluenzavirus 3 (hPIV3) minigenome. Interestingly, the C and V proteins of MV, which have previously been shown to inhibit MV minigenome replication, also inhibited NiV minigenome replication; however, they were not able to inhibit hPIV3 minigenome replication. In contrast, the C protein of hPIV3 inhibited minigenome replication of hPIV3, NiV and MV. Although there is very limited amino acid sequence similarity between the C, V and W proteins within the paramyxoviruses, the heterotypic inhibition of replication suggests that these proteins may share functional properties.

Stimulation of Na,K-ATPase by low potassium requires reactive oxygen species

The signaling pathway that transduces the stimulatory effect of low K+ on the biosynthesis of Na,K-ATPase remains largely unknown. The present study was undertaken to examine whether reactive oxygen species (ROS) mediated the effect of low K+ in Madin-Darby canine kidney (MDCK) cells. Low K+ increased ROS activity in a time- and dose-dependent manner, and this effect was abrogated by catalase and N-acetylcysteine (NAC). To determine the role of ROS in low-K+-induced gene expression, the cells were first stably transfected with expression constructs in which the reporter gene chloramphenicol acetyl transferase (CAT) was under the control of the avian Na,K-ATPase α-subunit 1.9 kb and 900-bp 5′-flanking regions that have a negative regulatory element. Low K+ increased the CAT expression in both constructs. Catalase or NAC inhibited the effect of low K+. To determine whether the increased CAT activity was mediated through releasing the repressive effect or a direct stimulation of the promoter, the cells were transfected with a CAT expression construct directed by a 96-bp promoter fragment that has no negative regulatory element. Low K+ also augmented the CAT activity expressed by this construct. More importantly, both catalase and NAC abolished the effect of low K+. Moreover, catalase and NAC also inhibited low-K+-induced increases in the Na,K-ATPase α1- and β1-subunit protein abundance and ouabain binding sites. The antioxidants had no significant effect on the basal levels of CAT activity, protein abundance, or ouabain binding sites. In conclusion, low K+ enhances the Na,K-ATPase gene expression by a direct stimulation of the promoter activity, and ROS mediate this stimulation and also low-K+-induced increases in the Na,K-ATPase protein contents and cell surface molecules.

Antioxidant Gene Expression in Active and Sedentary House Mice (Mus domesticus) Selected for High Voluntary Wheel-Running Behavior

We present liver mRNA levels of the two antioxidant enzymes catalase (CAT) and Mn-superoxide dismutase (SOD2) in four treatment groups of house mice assayed by RNase protection at 20 months of age. These groups were mice from four replicate selection and four replicate control lines from the sixteenth generation of selective breeding for high voluntary wheel running, housed with or without running wheels from age 3 weeks through 20 months. Exercising control females had induced CAT expression; SOD2 exhibited a similar pattern in females from two of the four control lines. Exercising male mice had induced CAT expression, but not SOD2 expression, irrespective of genetic background. We discuss these results with respect to both evolutionary (genetic) and training (exercise-induced) adaptations and explore predictions of these results in relation to the oxidative-damage theory of senescence.

Scanning mutagenesis identifies critical residues in the rinderpest virus genome promoter

Short regions at the 3′ and 5′ ends of the genome of Rinderpest virus (RPV) contain signals that regulate transcription of the viral genome, known as the genome promoter and the (complement to the) antigenome promoter, respectively. An RPV minigenome construct carrying the CAT coding sequence was used as a reporter to investigate residues in the 3′-terminal region of the genome important for these functions. Single-base scanning mutagenesis showed that modifications to nucleotides 1, 3, 4, 10 and 19 of the RPV leader had an extremely inhibitory effect on transcription and/or encapsidation of the minigenome, with CAT expression reduced to 0–10% of control values. Changes in any of the other first 22 nucleotides reduced the efficiency of the minigenome to 20–80% of the wild-type control, with the exception of nucleotides 16, 17 and 20, where mutations did not affect CAT expression significantly. Mutagenesis in blocks identified critical residues in positions 23–26, but changes to leader residues 27–48 had no major effect on CAT expression. A region of about 16 nucleotides (49–65) located around the start of the nucleocapsid gene, including the intergenic triplet CTT, was identified as essential for minigenome function. Mutations further into the nucleocapsid gene (nt 66–89) had a moderate effect (CAT activity 20–60% of control), while at least one critical residue was found in positions 93–96. The importance of four highly conserved G residues at positions 79, 85, 91 and 97 was also investigated. G79 was found to be optimal, though not critical, while a purine was required at 85 and 91. Although G97 is conserved in morbilliviruses, all bases were equally effective at this position.

Identification and functional analysis of sequence rearrangements in the long control region of human papillomavirus type 16 Af-1 variants isolated from Ugandan penile carcinomas

Human papillomavirus type 16 (HPV-16) is the predominant HPV isolate found in malignancies of male and female lower genital tracts. However, only a small percentage of individuals infected with high-risk HPVs develop a genital neoplasia, suggesting that additional events at both the cellular and the virus level are necessary for the progression to cancer, including genetic mutations/rearrangements of viral sequences involved in the oncogenic process. In this study, the genetic stability of the long control region (LCR) (nt 7289–114), which regulates expression levels of oncoproteins E6 and E7, was analysed in HPV-16 isolates from penile carcinoma (PC) biopsies of patients recruited from Uganda, one of the countries with the highest incidence of genital cancers in both men and women. Nucleotide changes within the LCR region typical of the African-1 (Af-1) lineage were observed in all HPV-16 isolates. Two out of five samples showed further rearrangements of the enhancer region. The functional activity of LCR with Af-1 mutations and/or rearrangements was evaluated by cloning each LCR into CAT expression vectors, followed by transfection in several epithelial and non-epithelial cell lines. CAT expression levels driven by a rearranged LCR were significantly higher than those driven by Af-1 or European prototype LCRs. Furthermore, in the NIH3T3 focus formation assay, the transforming activity of E6 and E7 genes, driven by a mutated or rearranged LCR, was 1·4- to 3·0-fold higher, respectively. These results indicate that rearrangements within the LCR of HPV-16 isolated from African PCs are frequently found (2 out of 5, 40%). It is also shown that increased HPV LCR activity is associated with an increased E6/E7-mediated in vitro transforming activity, suggesting that natural variants can play a major role in the pathogenesis of genital carcinomas.