Selection of Diazotrophic Bacterial Communities in Biological Sand Filter Mesocosms Used for the Treatment of Phenolic-Laden Wastewater

Agri effluents such as winery or olive mill wastewaters are characterized by high phenolic concentrations. These compounds are highly toxic and generally refractory to biodegradation. Biological sand filters (BSFs) represent inexpensive, environmentally friendly, and sustainable wastewater treatment systems which rely vastly on microbial catabolic processes. Using denaturing gradient gel electrophoresis and terminal-restriction fragment length polymorphism, this study aimed to assess the impact of increasing concentrations of synthetic phenolic-rich wastewater, ranging from 96 mg L^?1 gallic acid and 138 mg L^?1 vanillin (i.e., a total chemical oxygen demand (COD) of 234 mg L^?1) to 2,400 mg L^?1 gallic acid and 3,442 mg L^?1 vanillin (5,842 mg COD L^?1), on bacterial communities and the specific functional diazotrophic community from BSF mesocosms. This amendment procedure instigated efficient BSF phenolic removal, significant modifications of the bacterial communities, and notably led to the selection of a phenolic-resistant and less diverse diazotrophic community. This suggests that bioavailable N is crucial in the functioning of biological treatment processes involving microbial communities, and thus that functional alterations in the bacterial communities in BSFs ensure provision of sufficient bioavailable nitrogen for the degradation of wastewater with a high C/N ratio.     

PIK3R1 Mutations Cause Syndromic Insulin Resistance with Lipoatrophy

Short stature, hyperextensibility of joints and/or inguinal hernia, ocular depression, Rieger anomaly, and teething delay (SHORT) syndrome is a developmental disorder with an unknown genetic cause and hallmarks that include insulin resistance and lack of subcutaneous fat. We ascertained two unrelated individuals with SHORT syndrome, hypothesized that the observed phenotype was most likely due to de novo mutations in the same gene, and performed whole-exome sequencing in the two probands and their unaffected parents. We then confirmed our initial observations in four other subjects with SHORT syndrome from three families, as well as 14 unrelated subjects presenting with syndromic insulin resistance and/or generalized lipoatrophy associated with dysmorphic features and growth retardation. Overall, we identified in nine affected individuals from eight families de novo or inherited PIK3R1 mutations, including a mutational hotspot (c.1945C>T [p.Arg649Trp]) present in four families. PIK3R1 encodes the p85α, p55α, and p50α regulatory subunits of class IA phosphatidylinositol 3 kinases (PI3Ks), which are known to play a key role in insulin signaling. Functional data from fibroblasts derived from individuals with PIK3R1 mutations showed severe insulin resistance for both proximal and distal PI3K-dependent signaling. Our findings extend the genetic causes of severe insulin-resistance syndromes and provide important information with respect to the function of PIK3R1 in normal development and its role in human diseases, including growth delay, Rieger anomaly and other ocular affections, insulin resistance, diabetes, paucity of fat, and ovarian cysts.     

Maternal Antiretroviral Therapy for the Prevention of Mother-To-Child Transmission of HIV in Malawi: Maternal and Infant Outcomes Two Years after Delivery

Background

Optimized preventive strategies are needed to reach the objective of eliminating pediatric AIDS. This study aimed to define the determinants of residual HIV transmission in the context of maternal antiretroviral therapy (ART) administration to pregnant women, to assess infant safety of this strategy, and to evaluate its impact on maternal disease.

Methodology/Principal Findings

A total of 311 HIV-infected pregnant women were enrolled in Malawi in an observational study and received a nevirapine-based regimen from week 25 of gestation until 6 months after delivery (end of breastfeeding period) if their CD4+ count was > 350/mm³ at baseline (n = 147), or indefinitely if they met the criteria for treatment (n. 164). Mother/child pairs were followed until 2 years after delivery. The Kaplan-Meier method was used to estimate HIV transmission, maternal disease progression, and survival at 24 months. The rate of HIV infant infection was 3.2% [95% confidence intervals (CI) 1.0-5.4]. Six of the 8 transmissions occurred among mothers with baseline CD4+ count > 350/mm³. HIV-free survival of children was 85.8% (95% CI 81.4-90.1). Children born to mothers with baseline CD4+ count < 350/mm³ were at increased risk of death (hazard ratio 2.6, 95% CI 1.1-6.1). Among women who had stopped treatment the risk of progression to CD4+ count < 350/mm³ was 20.6% (95% CI 9.2-31.9) by 18 months of drug discontinuation.

Conclusions

HIV transmission in this cohort was rare however, it occurred in a significative proportion among women with high CD4+ counts. Strategies to improve treatment adherence should be implemented to further reduce HIV transmission. Mortality in the uninfected exposed children was the major determinant of HIV-free survival and was associated to maternal disease stage. Given the considerable proportion of women reaching the criteria for treatment within 18 months of drug discontinuation, life-long ART administration to HIV-infected women should be considered.     

The cost of tsetse control using ‘Tiny Targets’ in the sleeping sickness endemic forest area of Bonon in Côte d’Ivoire: Implications for comparing costs across different settings

BackgroundWork to control the gambiense form of human African trypanosomiasis (gHAT), or sleeping sickness, is now directed towards ending transmission of the parasite by 2030. In order to supplement gHAT case-finding and treatment, since 2011 tsetse control has been implemented using Tiny Targets in a number of gHAT foci. As this intervention is extended to new foci, it is vital to understand the costs involved. Costs have already been analysed for the foci of Arua in Uganda and Mandoul in Chad. This paper examines the costs of controlling Glossina palpalis palpalis in the focus of Bonon in Côte d’Ivoire from 2016 to 2017.Methodology/Principal findingsSome 2000 targets were placed throughout the main gHAT transmission area of 130 km² at a density of 14.9 per km². The average annual cost was USD 0.5 per person protected, USD 31.6 per target deployed of which 12% was the cost of the target itself, or USD 471.2 per km² protected. Broken down by activity, 54% was for deployment and maintenance of targets, 34% for tsetse surveys/monitoring and 12% for sensitising populations.Conclusions/SignificanceThe cost of tsetse control per km² of the gHAT focus protected in Bonon was more expensive than in Chad or Uganda, while the cost per km² treated, that is the area where the targets were actually deployed, was cheaper. Per person protected, the Bonon cost fell between the two, with Uganda cheaper and Chad more expensive. In Bonon, targets were deployed throughout the protected area, because G. p. palpalis was present everywhere, whereas in Chad and Uganda G. fuscipes fuscipes was found only the riverine fringing vegetation. Thus, differences between gHAT foci, in terms of tsetse ecology and human geography, impact on the cost-effectiveness of tsetse control. It also demonstrates the need to take into account both the area treated and protected alongside other impact indicators, such as the cost per person protected.     

Catalytic and DNA-binding properties of the human Ogg1 DNA N-glycosylase/AP lyase: biochemical exploration of H270, Q315 and F319, three amino acids of the 8-oxoguanine-binding pocket

The human Ogg1 protein (hOgg1) is an antimutator DNA glycosylase/AP lyase that catalyzes the excision of 8-oxo-7,8-dihydroguanine (8-oxoG) and the incision of apurinic and apyrimidinic (AP) sites in DNA. In this study, we have investigated the functional role of H270, Q315 and F319, three amino acids that are located in the 8-oxoG-binding pocket of hOgg1. Wild-type and mutant hOgg1 proteins (H270A, H270R, H270L, Q315A and F319A) were purified to apparent homogeneity. The catalytic activities and the DNA-binding properties of the various hOgg1 mutants were compared to those of the wild-type. The results show that hOgg1 mutated at H270 (H270A and H270L) or F319 (F319A) exhibits greatly reduced (50- to 1000-fold) DNA glycosylase activity, whereas the AP lyase activity is only moderately affected (<4-fold). The affinity of the hOgg1 mutants (H270A, H270L and F319A) for 8-oxoG.C-containing DNA is also greatly reduced (>30-fold), whereas their affinity for THF.C-containing DNA is only moderately reduced (<7-fold). The results also show that hOgg1 mutated at Q315 (Q315A) exhibits catalytic and DNA-binding properties similar to those of the wild-type. Therefore, H270 and F319 are essential to form the functional 8-oxoG-binding pocket, whereas Q315 is less crucial. In contrast, H270, Q315 and F319 are not required for efficient binding of THF.C and cleavage of AP sites. Finally, hOgg1 mutant proteins with a substitution of H270A or F319A are members of a new type of hOgg1 that is deficient in DNA glycosylase but proficient in AP lyase.     

IL-10 inhibits vascular smooth muscle cell activation in vitro and in vivo

The anti-inflammatory cytokine IL-10 inhibits intimal hyperplasia after stent implantation via a powerful inactivation of monocytes. We tested the hypothesis that IL-10 may also inhibit vascular smooth muscle cell (SMC) activation via the inhibition of the NF-kappaB/I-kappaB system. The IL-10 receptor was detected in rat SMCs in vitro and in vivo. In LPS-stimulated rat SMCs, 1 ng/ml recombinant murine IL-10 (mIL-10) reduced I-kappaBalpha and I-kappaBbeta degradation, NF-kappaB activation, as well as the expression of the NF-kappaB-dependent gene IL-6 by 32%, 31%, 75%, and 19%, respectively (P < 0.05 for all). Similar results were obtained in vivo 6 h and 4 days after balloon abrasion of the rat aorta, a model in which intimal hyperplasia results essentially from SMC activation. Moreover, mIL-10 reduced SMC proliferation and migration in vitro (by 60% for both, P < 0.0001), resulting in reduced SMC proliferation and intimal growth 14 days after balloon abrasion of the rat aorta (by 76% and 75%, respectively; P < 0.005). In conclusion, mIL-10 has a direct inhibitory effect on SMCs in vitro and in vivo. This effect is mediated in part by NF-kappaB inactivation and may participate in the overall protective effect of IL-10 on postangioplasty restenosis.     

The amino-terminal extensions of the longer Sendai virus C proteins modulate pY701-Stat1 and bulk Stat1 levels independently of interferon signaling

The Sendai virus (SeV) C proteins are known to interact with Stat1 to prevent interferon (IFN)-induced pY701-Stat1 formation and IFN signaling. Nevertheless, pY701-Stat1 levels paradoxically increase during SeV infection. The C proteins also induce bulk Stat1 instability in some cells, similar to rubulavirus V proteins. We have found that SeV infection increases pY701-Stat1 levels even in cells in which bulk Stat1 levels strongly decrease. Remarkably, both the decrease in bulk Stat1 levels and the increase in pY701-Stat1 levels were found to be independent of the IFN signaling system, i.e., these events occur in mutant cells in which various components of the IFN signaling system have been disabled. Consistent with this, the C-induced decrease in Stat1 levels does not require Y701 of Stat1. We present evidence that C interacts with Stat1 in two different ways, one that prevents IFN-induced pY701-Stat1 formation and IFN signaling that has already been documented, and another that induces pY701-Stat1 formation (while decreasing bulk Stat1 levels) in a manner that does not require IFN signaling. These two types of Stat1 interaction are also distinguishable by C gene mutations. In particular, the IFN signaling-independent Stat1 interactions specifically require the amino-terminal extensions of the longer C proteins. The actions of the SeV C proteins in counteracting the cellular antiviral response are clearly more extensive than previously appreciated.     

A novel nuclear export signal and a REF interaction domain both promote mRNA export by the Epstein-Barr virus EB2 protein

A striking characteristic of mRNA export factors is that they shuttle continuously between the cytoplasm and the nucleus. This shuttling is mediated by specific factors interacting with peptide motifs called nuclear export signals (NES) and nuclear localization signals. We have identified a novel CRM-1-independent transferable NES and two nuclear localization signals in the Epstein-Barr virus mRNA export factor EB2 (also called BMLF1, Mta, or SM) localized at the N terminus of the protein between amino acids 61 and 146. We have also found that a previously described double NES (amino acids 213-236) does not mediate the nuclear shuttling of EB2, but is an interaction domain with the cellular export factor REF in vitro. This newly characterized REF interaction domain is essential for EB2-mediated mRNA export. Accordingly, in vivo, EB2 is found in complexes containing REF as well as the cellular factor TAP. However, these interactions are RNase-sensitive, suggesting that the RNA is an essential component of these complexes.     

Src homology-2 domain binding assays by scintillation proximity and surface plasmon resonance

Various SH2 competitive binding assays, based on different techniques, have been described in the literature to identify and characterize SH2 ligands. The consideration that most reported methods show experimental limitations associated with assay parameters has prompted us to base our Src-SH2 inhibitor discovery program on the use of two different assays. In this study, two conceptually different biochemical methods designed to discover Src-SH2 inhibitors, respectively scintillation proximity assay (SPA) and surface plasmon resonance (SPR), have been evaluated and compared. For its high sensitivity and adaptability to automation SPA was chosen for high capacity screening (primary screen), whereas SPR was used for hits confirmation (secondary screening). However with the drastic improvement of inhibitor affinities, the limit of sensitivity was rapidly reached for the SPR assay based on the canonical pYEEI ligand. The substitution of the natural, monophosphorylated peptide ligand with a triphosphorylated peptide has allowed us to remarkably increase its sensitivity, so that molecules with nanomolar affinities could be easily differentiated in terms of IC(50) ranking. Such a new, improved SPR assay can be of great interest for the study of high affinity ligands of different SH2-based drug targets.     

Physical and functional interaction of the Arabidopsis K(+) channel AKT2 and phosphatase AtPP2CA

The AKT2 K(+) channel is endowed with unique functional properties, being the only weak inward rectifier characterized to date in Arabidopsis. The gene is expressed widely, mainly in the phloem but also at lower levels in leaf epiderm, mesophyll, and guard cells. The AKT2 mRNA level is upregulated by abscisic acid. By screening a two-hybrid cDNA library, we isolated a protein phosphatase 2C (AtPP2CA) involved in abscisic acid signaling as a putative partner of AKT2. We further confirmed the interaction by in vitro binding studies. The expression of AtPP2CA (beta-glucuronidase reporter gene) displayed a pattern largely overlapping that of AKT2 and was upregulated by abscisic acid. Coexpression of AtPP2CA with AKT2 in COS cells and Xenopus laevis oocytes was found to induce both an inhibition of the AKT2 current and an increase of the channel inward rectification. Site-directed mutagenesis and pharmacological analysis revealed that this functional interaction involves AtPP2CA phosphatase activity. Regulation of AKT2 activity by AtPP2CA in planta could allow the control of K(+) transport and membrane polarization during stress situations.