Developmental outcomes in children exposed to Zika virus in utero from a Brazilian urban slum cohort study

BackgroundThe prevalence of developmental alterations associated with in-utero Zika virus (ZIKV) exposure in children is not well understood. Furthermore, estimation of the Population Attributable Fraction (PAF) of developmental alterations attributed to ZIKV has not been performed due to lack of population-based cohorts with data on symptomatic and asymptomatic ZIKV exposures and an appropriate control group. The aim of this study was to characterize neurodevelopmental outcomes of children at 11 to 32 months of age with intrauterine ZIKV exposure and estimate the PAF of alterations secondary to ZIKV exposure.Methodology/Principal findingsWe performed a cohort of biannual community-based prospective serosurveys in a slum community in Salvador, Brazil. We recruited women participating in our cohort, with a documented pregnancy from January 2015 to December 2016 and children born to those mothers. Children were classified as ZIKV exposed in utero (born from women with ZIKV seroconversion during pregnancy) or unexposed (born from women without ZIKV seroconversion or that seroconverted before/after pregnancy) by using an IgG monoclonal antibody blockade-of-binding (BoB). We interviewed mothers and performed anthropometric, audiometric, ophthalmological, neurologic, and neurodevelopmental evaluations of their children at 11 to 32 months of age. Among the 655 women participating in the cohort, 66 (10%) were pregnant during the study period. 46 (70%) of them completed follow-up, of whom ZIKV seroconversion occurred before, during, and after pregnancy in 25 (54%), 13 (28%), and 1 (2%), respectively. The rest of women, 7 (21.2%), did not present ZIKV seroconversion. At 11 to 32 months of life, the 13 ZIKV-exposed children had increased risk of mild cognitive delay (RR 5.1; 95%CI 1.1–24.4) compared with the 33 children unexposed, with a PAF of 53.5%. Exposed children also had increased risk of altered auditory behavior (RR 6.0; 95%CI 1.3–26.9), with a PAF of 59.5%.ConclusionsA significant proportion of children exposed in utero to ZIKV developed mild cognitive delay and auditory behavioral abnormalities even in the absence of gross birth defects such as microcephaly and other neurodevelopmental domains. Furthermore, our findings suggest that over half of these abnormalities could be attributed to intrauterine ZIKV exposure.     

Nutrient availability and grazing influence the strength of priority effects during freshwater bacterial community coalescence

When bacterial communities mix, immigration history can fundamentally affect the community composition as a result of priority effects. Priority effects arise when an early immigrant exhausts resources and/or alters habitat conditions, thereby influencing the establishment success of the late arriver. The strength of priority effects is context-dependent and expected to be stronger if environmental conditions favour the growth of the first arriver. In this study, we conducted a two-factorial experiment testing the importance of nutrient availability and grazing on the strength of priority effects in complex aquatic bacterial communities. We did so by mixing two dissimilar communities, simultaneously, and with a 38 h time-delay. Priority effects were measured as the invasion resistance of the first community to the invading second community. We found stronger priority effects in treatments with high nutrient availability and absence of grazing, but in general, the arrival timing was less important than the selection by nutrients and grazing. At the population level, the results were complex, but priority effects may have been driven by bacteria belonging to for example, the genera Rhodoferax and Herbaspirillum. Our study highlights the importance of arrival timing in complex bacterial communities, especially if environmental conditions favour rapid community growth.     

TWO NEUTRALITY TESTS OF Y-LINKED RDNA VARIATION IN DROSOPHILA MELANOGASTER

The evolutionary significance of Y-chromosomal ribosomal DNA sequence variation was tested by two different means. A single sample of males and females was collected from a peach orchard in central Pennsylvania. Wild-caught males and sons of wild-caught, wild-inseminated females were crossed to virgin females having an X-linked rDNA deficiency. Genomic DNA from male progeny of these crosses was extracted and digested with the single restriction endonuclease, DraI. Southern blots of these digestions, when probed with the complete rDNA probe, revealed 10 distinct patterns of restriction fragments. A chisquare test for the homogeneity of the frequency distributions of the sample of wild males and sons of wild females failed to reject a neutral null hypothesis. The allele frequency configuration was tested with the Ewens-Watterson test, and the departure from the infinite alleles neutral model was not significant. Simulations were performed to test the sensitivity of the tests to misclassification and to quantify the power of the two tests.     

Synergistic effects of adenosine A1 and P2Y receptor stimulation on calcium mobilization and PKC translocation in DDT1 MF-2 cells

1. The effect of adenosine analogues and of nucleotides, alone or in combination, on intracellular calcium, accumulation of inositol (1,4,5) trisphosphate (InsP₃), and on activation of protein kinase C (PKC) was studied in DDT₁ MF2 cells derived from a Syrian hamster myosarcoma. These cells were found to express mRNA for A₁ and some as yet unidentified P2Y receptor(s).2. Activation of either receptor type stimulated the production of InsP₃ and raised intracellular calcium in DDT₁ MF2 cells. Similarly, the A₁ selective agonist N⁶-cyclopentylade- nosine (CPA) increased PKC-dependent phosphorylation of the substrate MBP_4–14 and induced a PKC translocation to the plasma membrane as determined using [³H]-phorbol dibutyrate (PDBu) binding in DDT₁ MF-2 cells. However, neither adenosine nor CPA induced a significant translocation of transiently transfected -PKC-GFP from the cytosol to the cell membrane. In contrast to adenosine analogues, ATP and UTP also caused a rapid but transient translocation of -PKC-GFP and activation of PKC.3. Doses of the A₁ agonist CPA and of ATP or UTP per se caused barely detectable increases in intracellular Ca²⁺ but when combined, they caused an almost maximal stimulation. Similarly, adenosine (0.6 M) and UTP (or ATP, 2.5 M), which per se caused no detectable translocation of either - or -PKC-GFP, caused when combined a very clear-cut translocation of both PKC subforms, albeit with different time courses. These results show that simultaneous activation of P2Y and adenosine A₁ receptors synergistically increases Ca²⁺ transients and translocation of PKC in DDT₁ MF-2 cells. Since adenosine is rapidly formed by breakdown of extracellular ATP, such interactions may be biologically important.     

Functional expression of the transient receptor potential channel TRPA1, a sensor for toxic lung inhalants,in pulmonary epithelial cells

The cation channel TRPA1 functions as a chemosensory protein and is directly activated by a number of noxious inhalants. A pulmonary expression of TRPA1 has been described in sensory nerve endings and its stimulation leads to the acceleration of inflammatory responses in the lung. Whereas the function of TRPA1 in neuronal cells is well defined, only few reports exist suggesting a role in epithelial cells. The aim of the present study was therefore (1) to evaluate the expression of TRPA1 in pulmonary epithelial cell lines, (2) to characterize TRPA1-promoted signaling in these cells, and (3) to study the extra-neuronal expression of this channel in lung tissue sections. Our results revealed that the widely used alveolar type II cell line A549 expresses TRPA1 at the mRNA and protein level. Furthermore, stimulating A549 cells with known TRPA1 activators (i.e., allyl isothiocyanate) led to an increase in intracellular calcium levels, which was sensitive to the TRPA1 blocker ruthenium red. Investigating TRPA1 coupled downstream signaling cascades it was found that TRPA1 activation elicited a stimulation of ERK1/2 whereas other MAP kinases were not affected. Finally, using epithelial as well as neuronal markers in immunohistochemical approaches, a non-neuronal TRPA1 protein expression was detected in distal parts of the porcine lung epithelium, which was also found examining human lung sections. TRPA1-positive staining co-localized with both epithelial and neuronal markers underlining the observed epithelial expression pattern. Our findings of a functional expression of TRPA1 in pulmonary epithelial cells provide causal evidence for a non-neuronal TRPA1-mediated control of inflammatory responses elicited upon TRPA1-mediated registration of toxic inhalants in vivo.     

Modular broad-host-range expression vectors for single-protein and protein complex purification

A set of modular broad-host-range expression vectors with various affinity tags (six-His-tag, FLAG-tag, Strep-tag II, T7-tag) was created. The complete nucleotide sequences of the vectors are known, and these small vectors can be mobilized by conjugation. They are useful in the purification of proteins and protein complexes from gram-negative bacterial species. The plasmids were easily customized for Thiocapsa roseopersicina, Rhodobacter capsulatus, and Methylococcus capsulatus by inserting an appropriate promoter. These examples demonstrate the versatility and flexibility of the vectors. The constructs harbor the T7 promoter for easy overproduction of the desired protein in an appropriate Escherichia coli host. The vectors were useful in purifying different proteins from T. roseopersicina. The FLAG-tag-Strep-tag II combination was utilized for isolation of the HynL-HypC2 protein complex involved in hydrogenase maturation. These tools should be useful for protein purification and for studying protein-protein interactions in a range of bacterial species.     

Ser-474 is the major target of insulin-mediated phosphorylation of protein kinase B beta in primary rat adipocytes.

The mechanism of activation for protein kinase B (PKB), an important target for insulin signaling, has been scarcely investigated in primary cells. In this study, we have characterized the insulin-induced phosphorylation and activation of PKB beta in primary rat adipocytes. Insulin stimulation resulted in a translocation of PKB beta from cytosol to membranes, and phosphorylation and activation of PKB beta. Phosphoamino acid analysis and phosphopeptide mapping demonstrated that the phosphorylation occurred mainly on serines, also when using calyculin A, and that these were localized within one major phosphopeptide. Radiosequencing showed that the radioactivity was released in Cycle No. 7. In addition, the peptide was specifically immunoprecipitated from a tryptic digest of PKB beta using the anti-phospho-PKB (Ser-473) antibody. Taken together, these results show that rat adipocyte PKB beta mainly is phosphorylated on Ser-474 in response to insulin stimulation, in contrast to previous studies in human embryonic kidney (HEK) 293 cells demonstrating, in addition, phosphorylation of Thr-309.     

DNA adduct formation from quaternary benzo[c]phenanthridine alkaloids sanguinarine and chelerythrine as revealed by the 32P-postlabeling technique

Using the 32P-postlabeling assay, we investigated the ability of quaternary benzo[c]phenanthridine alkaloids, sanguinarine, chelerythrine and fagaronine, to form DNA adducts in vitro. Two enhanced versions of the assay (enrichment by nuclease P1 and 1-butanol extraction) were utilized in the study. Hepatic microsomes of rats pre-treated with beta-naphthoflavone or those of uninduced rats, used as metabolic activators, were incubated in the presence of calf thymus DNA and the alkaloids, with NADPH used as a cofactor. Under these conditions sanguinarine and chelerythrine, but not fagaronine, formed DNA adducts detectable by 32P-postlabeling. DNA adduct formation by both alkaloids was found to be concentration dependent. When analyzing different atomic and bond indices of the C11-C12 bond (ring B) in alkaloid molecules we found that fagaronine behaved differently from sanguinarine and chelerythrine. While sanguinarine and chelerythrine showed a preference for electrophilic attack indicating higher potential to be activated by cytochrome P450, fagaronine exhibited a tendency for nucleophilic attack. Our results demonstrate that sanguinarine and chelerythrine are metabolized by hepatic microsomes to species, which generate DNA adducts.