The assessment of DNA from marine organisms via a modified salting-out protocol

We developed a rapid, practical and non-toxic salting-out method for the extraction of DNA from marine organisms, and tested it on two representative species of Porifera and Cnidaria, both living in association with symbiotic zooxanthellae. We tested the efficiency of the protocol by comparing the output of the method for fresh tissue, frozen tissue and tissue stored in ethanol. It proved to be effective for extracting DNA in the case of the methods of preservation considered here, and for obtaining quantities of DNA comparable to those obtained via the traditional approach. The DNA from both species was of good quality. The DNA obtained was amplified by PCR using specific primers for the large ribosomal subunit, allowing the identification of the presence of both the host and symbiont genomes.     

Selective degradation of reverse gyrase and DNA fragmentation induced by alkylating agent in the archaeon Sulfolobus solfataricus

Reverse gyrase is a peculiar DNA topoisomerase, specific of hyperthermophilic Archaea and Bacteria, which has the unique ability of introducing positive supercoiling into DNA molecules. Although the function of the enzyme has not been established directly, it has been suggested to be involved in DNA protection and repair. We show here that the enzyme is degraded after treatment of Sulfolobus solfataricus cells with the alkylating agent MMS. MMS-induced reverse gyrase degradation is highly specific, since (i) neither hydroxyurea (HU) nor puromycin have a similar effect, and (ii) topoisomerase VI and two chromatin components are not degraded. Reverse gyrase degradation does not depend on protein synthesis. Experiments in vitro show that direct exposure of cell extracts to MMS does not induce reverse gyrase degradation; instead, extracts from MMS-treated cells contain some factor(s) able to degrade the enzyme in extracts from control cells. In vitro, degradation is blocked by incubation with divalent metal chelators, suggesting that reverse gyrase is selectively degraded by a metal-dependent protease in MMS-treated cells. In addition, we find a striking concurrence of extensive genomic DNA degradation and reverse gyrase loss in MMS-treated cells. These results support the hypothesis that reverse gyrase plays an essential role in DNA thermoprotection and repair in hyperthermophilic organisms.     

Development of tetravalent IgG1 dual targeting IGF-1R-EGFR antibodies with potent tumor inhibition

In this study we present novel bispecific antibodies that simultaneously target the insulin-like growth factor receptor type I (IGF-1R) and epidermal growth factor receptor (EGFR). For this purpose disulfide stabilized scFv domains of the EGFR/ADCC antibody GA201 were fused via serine-glycine connectors to the C-terminus of the heavy (XGFR2) or light chain (XGFR4), or the N-termini of the light (XGFR5) or heavy chain (XGFR3) of the IGF-1R antibody R1507 as parental IgG1 antibody. The resulting bispecific IGF-1R-EGFR antibodies XGFR2, XGFR3 and XGFR4 were successfully generated with yields and stability comparable to conventional IgG1 antibodies. They effectively inhibited IGF-1R and EGFR phosphorylation and 3D proliferation of H322M and H460M2 tumor cells, induced strong down-modulation of IGF-1R as well as enhanced EGFR down-modulation compared to the parental EGFR antibody GA201 and were ADCC competent. The bispecific XGFR derivatives showed a strong format dependent influence of N- or C-terminal heavy and light chain scFv attachment on ADCC activity and an increase in receptor downregulation over the parental combination in vitro. XGFR2 and XGFR4 were selected for in vivo evaluation and showed potent anti-tumoral efficacy comparable to the combination of monospecific IGF-1R and EGFR antibodies in subcutaneous BxPC3 and H322M xenograft models. In summary, we have managed to overcome issues of stability and productivity of bispecific antibodies, discovered important antibody fusion protein design related differences on ADCC activity and receptor downmodulation and show that IGF-1R-EGFR antibodies represent an attractive therapeutic strategy to simultaneously target two key components de-regulated in multiple cancer types, with the ultimate goal to avoid the formation of resistance to therapy.     

Variation in the bitter-taste receptor gene TAS2R38, and adiposity in a genetically isolated population in Southern Italy

Objective: Variation in the bitter‐taste receptor gene, TAS2R38 confers the ability to taste 6‐n‐propylthiouracil (PROP). The objective of this study was to relate TAS2R38 haplotypes and PROP‐tasting phenotypes to adiposity in a genetically isolated population. We hypothesized that the nontaster phenotype would be associated with higher BMI and waist circumference (WC) in females, and that dietary restraint would mediate this relationship. Methods and Procedures: Participants were 540 healthy inhabitants of the genetically isolated village of Carlantino in southern Italy who were 15–89 years of age at the time of the study. Haplotype analyses were performed and PROP tasting was assessed using a filter paper method. Height, weight, and WC were measured and restrained eating was assessed using a brief questionnaire. Results: Nontaster females had higher BMI and WC than females who were phenotypic tasters, and this relationship was specific to females with low dietary restraint. Regression analysis showed that BMI declined by 1.7 units across taster groups in females when the model included the PROP by restraint interaction. PROP phenotype was not significantly associated with WC in the regression models. Polymorphisms in TAS2R38 were not associated with BMI or WC in females. Neither TAS2R38 haplotype nor PROP phenotype was strongly related to BMI or WC in males. Discussion: These data support previous findings of a relation between the nontaster phenotype and higher BMI in females that is modified by dietary restraint. Assessment of PROP phenotypes might provide unique information about adiposity that is not captured by haplotype analysis alone.     

Characterization of endocrine gland-derived vascular endothelial growth factor signaling in adrenal cortex capillary endothelial cells.

Endocrine gland-derived vascular endothelial growth factor (EG-VEGF) has been recently identified as a mitogen specific for the endothelium of steroidogenic glands. Here we report a characterization of the signal transduction of EG-VEGF in a responsive cell type, bovine adrenal cortex-derived endothelial (ACE) cells. EG-VEGF led to a time- and dose-dependent phosphorylation of p44/42 MAPK. This effect was blocked by pretreatment with pertussis toxin, suggesting that G alpha(i) plays an important role in mediating EG-VEGF-induced activation of MAPK signaling. The inhibitor of p44/42 MAPK phosphorylation PD 98059 resulted in suppression of both proliferation and migration in response to EG-VEGF. EG-VEGF also increased the phosphorylation of Akt in a phosphatidylinositol 3-kinase-dependent manner. Consistent with such an effect, EG-VEGF was a potent survival factor for ACE cells. We also identified endothelial nitric-oxide synthase as one of the downstream targets of Akt activation. Phosphorylation of endothelial nitric-oxide synthase in ACE cells was stimulated by EG-VEGF with a time course correlated to the Akt phosphorylation. Our data demonstrate that EG-VEGF, possibly through binding to a G-protein coupled receptor, results in the activation of MAPK p44/42 and phosphatidylinositol 3-kinase signaling pathways, leading to proliferation, migration, and survival of responsive endothelial cells.     

Increased expression of the neuropeptide Y receptor Y1 gene in the medial amygdala of transgenic mice induced by long-term treatment with progesterone or allopregnanolone

The neurosteroid allopregnanolone, a reduced metabolite of progesterone, induces anxiolytic effects by enhancing GABA(A) receptor function. Neuropeptide Y (NPY) and GABA are thought to interact functionally in the amygdala, and this interaction may be important in the regulation of anxiety. By using Y(1)R/LacZ transgenic mice, which harbour a fusion construct comprising the promoter of the mouse gene for the Y(1) receptor for NPY linked to the lacZ gene, we previously showed that long-term treatment with benzodiazepine receptor ligands modulates Y(1) receptor gene expression in the medial amygdala. We have now investigated the effects of prolonged treatment with progesterone or allopregnanolone on Y(1)R/LacZ transgene expression, as determined by quantitative histochemical analysis of beta-galactosidase activity. Progesterone increased both the cerebrocortical concentration of allopregnanolone and beta-galactosidase expression in the medial amygdala. Finasteride, a 5alpha-reductase inhibitor, prevented both of these effects. Long-term administration of allopregnanolone also increased both the cortical concentration of this neurosteroid and transgene expression in the medial amygdala. Treatment with neither progesterone nor allopregnanolone affected beta-galactosidase activity in the medial habenula. These data suggest that allopregnanolone regulates Y(1) receptor gene expression through modulation of GABA(A) receptor function, and they provide further support for a functional interaction between GABA and neuropeptide Y in the amygdala.     

Measuring Emotional Contagion in Social Media

Social media are used as main discussion channels by millions of individuals every day. The content individuals produce in daily social-media-based micro-communications, and the emotions therein expressed, may impact the emotional states of others. A recent experiment performed on Facebook hypothesized that emotions spread online, even in absence of non-verbal cues typical of in-person interactions, and that individuals are more likely to adopt positive or negative emotions if these are over-expressed in their social network. Experiments of this type, however, raise ethical concerns, as they require massive-scale content manipulation with unknown consequences for the individuals therein involved. Here, we study the dynamics of emotional contagion using a random sample of Twitter users, whose activity (and the stimuli they were exposed to) was observed during a week of September 2014. Rather than manipulating content, we devise a null model that discounts some confounding factors (including the effect of emotional contagion). We measure the emotional valence of content the users are exposed to before posting their own tweets. We determine that on average a negative post follows an over-exposure to 4.34% more negative content than baseline, while positive posts occur after an average over-exposure to 4.50% more positive contents. We highlight the presence of a linear relationship between the average emotional valence of the stimuli users are exposed to, and that of the responses they produce. We also identify two different classes of individuals: highly and scarcely susceptible to emotional contagion. Highly susceptible users are significantly less inclined to adopt negative emotions than the scarcely susceptible ones, but equally likely to adopt positive emotions. In general, the likelihood of adopting positive emotions is much greater than that of negative emotions.