Neurofibromatosis Type 1 Alternative Splicing Is a Key Regulator of Ras Signaling in Neurons

Neurofibromatosis type I (Nf1) is a GTPase-activating protein (GAP) that inactivates the oncoprotein Ras and plays important roles in nervous system development and learning. Alternative exon 23a falls within the Nf1 GAP domain coding sequence and is tightly regulated in favor of skipping in neurons; however, its biological function is not fully understood. Here we generated mouse embryonic stem (ES) cells with a constitutive endogenous Nf1 exon 23a inclusion, termed Nf1 23aIN/23aIN cells, by mutating the splicing signals surrounding the exon to better match consensus sequences. We also made Nf1 23aΔ/23aΔ cells lacking the exon. Active Ras levels are high in wild-type (WT) and Nf1 23aIN/23aIN ES cells, where the Nf1 exon 23a inclusion level is high, and low in Nf1 23aΔ/23aΔ cells. Upon neuronal differentiation, active Ras levels are high in Nf1 23aIN/23aIN cells, where the exon inclusion level remains high, but Ras activation is low in the other two genotypes, where the exon is skipped. Signaling downstream of Ras is significantly elevated in Nf1 23aIN/23aIN neurons. These results suggest that exon 23a suppresses the Ras-GAP activity of Nf1. Therefore, regulation of Nf1 exon 23a inclusion serves as a mechanism for providing appropriate levels of Ras signaling and may be important in modulating Ras-related neuronal functions.     

Prediction and Analysis of Canonical EF Hand Loop and Qualitative Estimation of Ca2+ Binding Affinity

The diversity of functions carried out by EF hand-containing calcium-binding proteins is due to various interactions made by these proteins as well as the range of affinity levels for Ca²⁺ displayed by them. However, accurate methods are not available for prediction of binding affinities. Here, amino acid patterns of canonical EF hand sequences obtained from available crystal structures were used to develop a classifier that distinguishes Ca²⁺-binding loops and non Ca²⁺-binding regions with 100% accuracy. To investigate further, we performed a proteome-wide prediction for E. histolytica, and classified known EF-hand proteins. We compared our results with published methods on the E. histolytica proteome scan, and demonstrated our method to be more specific and accurate for predicting potential canonical Ca²⁺-binding loops. Furthermore, we annotated canonical EF-hand motifs and classified them based on their Ca²⁺-binding affinities using support vector machines. Using a novel method generated from position-specific scoring metrics and then tested against three different experimentally derived EF-hand-motif datasets, predictions of Ca²⁺-binding affinities were between 87 and 90% accurate. Our results show that the tool described here is capable of predicting Ca²⁺-binding affinity constants of EF-hand proteins. The web server is freely available at http://202.41.10.46/calb/index.html.     

Characterization of Programmed Death-1 Homologue-1 (PD-1H) Expression and Function in Normal and HIV Infected Individuals

Chronic immune activation that persists despite anti-retroviral therapy (ART) is the strongest predictor of disease progression in HIV infection. Monocyte/macrophages in HIV-infected individuals are known to spontaneously secrete cytokines, although neither the mechanism nor the molecules involved are known. Here we show that overexpression of the newly described co-stimulatory molecule, PD1 homologue (PD-1H) in human monocyte/macrophages is sufficient to induce spontaneous secretion of multiple cytokines. The process requires signaling via PD-1H as cytokine secretion could be abrogated by deletion of the cytoplasmic domain. Such overexpression of PD-1H, associated with spontaneous cytokine expression is seen in monocytes from chronically HIV-infected individuals and this correlates with immune activation and CD4 depletion, but not viral load. Moreover, antigen presentation by PD-1H-overexpressing monocytes results in enhanced cytokine secretion by HIV-specific T cells. These results suggest that PD-1H might play a crucial role in modulating immune activation and immune response in HIV infection.     

Effect of sunflower seed oil emollient therapy on newborn infant survival in Uttar Pradesh,India: A community-based,cluster randomized,open-label controlled trial

BackgroundHospitalized preterm infants with compromised skin barrier function treated topically with sunflower seed oil (SSO) have shown reductions in sepsis and neonatal mortality rate (NMR). Mustard oil and products commonly used in high-mortality settings may possibly harm skin barrier integrity and enhance risk of infection and mortality in newborn infants. We hypothesized that SSO therapy may reduce NMR in such settings.Methods and findingsThis was a population-based, cluster randomized, controlled trial in 276 clusters in rural Uttar Pradesh, India. All newborn infants identified through population-based surveillance in the study clusters within 7 days of delivery were enrolled from November 2014 to October 2016. Exclusive, 3 times daily, gentle applications of 10 ml of SSO to newborn infants by families throughout the neonatal period were recommended in intervention clusters (n = 138 clusters); infants in comparison clusters (n = 138 clusters) received usual care, such as massage practice typically with mustard oil. Primary analysis was by intention-to-treat with NMR and post-24-hour NMR as the primary outcomes. Secondary analysis included per-protocol analysis and subgroup analyses for NMR. Regression analysis was adjusted for caste, first-visit weight, delivery attendant, gravidity, maternal age, maternal education, sex of the infant, and multiple births. We enrolled 13,478 (52.2% male, mean weight: 2,575.0 grams ± standard deviation [SD] 521.0) and 13,109 (52.0% male, mean weight: 2,607.0 grams ± SD 509.0) newborn infants in the intervention and comparison clusters, respectively. We found no overall difference in NMR in the intervention versus the comparison clusters [adjusted odds ratio (aOR) 0.96, 95% confidence interval (CI) 0.84 to 1.11, p = 0.61]. Acceptance of SSO in the intervention arm was high at 89.3%, but adherence to exclusive applications of SSO was 30.4%. Per-protocol analysis showed a significant 58% (95% CI 42% to 69%, p < 0.01) reduction in mortality among infants in the intervention group who were treated exclusively with SSO as intended versus infants in the comparison group who received exclusive applications of mustard oil. A significant 52% (95% CI 12% to 74%, p = 0.02) reduction in NMR was observed in the subgroup of infants weighing ≤1,500 g (n = 589); there were no statistically significant differences in other prespecified subgroup comparisons by low birth weight (LBW), birthplace, and wealth. No severe adverse events (SAEs) were attributable to the intervention. The study was limited by inability to mask allocation to study workers or participants and by measurement of emollient use based on caregiver responses and not actual observation.ConclusionsIn this trial, we observed that promotion of SSO therapy universally for all newborn infants was not effective in reducing NMR. However, this result may not necessarily establish equivalence between SSO and mustard oil massage in light of our secondary findings. Mortality reduction in the subgroup of infants ≤1,500 g was consistent with previous hospital-based efficacy studies, potentially extending the applicability of emollient therapy in very low-birth-weight (VLBW) infants along the facility–community continuum. Further research is recommended to develop and evaluate therapeutic regimens and continuum of care delivery strategies for emollient therapy for newborn infants at highest risk of compromised skin barrier function.Trial registrationISRCTN Registry ISRCTN38965585 and Clinical Trials Registry—India (CTRI/2014/12/005282) with WHO UTN # U1111-1158-4665.

In a cluster randomized trial, Aarti Kumar and colleagues study the effectiveness of a topical sunflower seed oil therapy in reducing neonatal mortality in Uttar Pradesh, India.     

Genetic incorporation of human metallothionein into the adenovirus protein IX for non-invasive SPECT imaging

As the limits of existing treatments for cancer are recognized, clearly novel therapies must be considered for successful treatment; cancer therapy using adenovirus vectors is a promising strategy. However tracking the biodistribution of adenovirus vectors in vivo is limited to invasive procedures such as biopsies, which are error prone, non-quantitative, and do not give a full representation of the pharmacokinetics involved. Current non-invasive imaging strategies using reporter gene expression have been applied to analyze adenoviral vectors. The major drawback to approaches that tag viruses with reporter genes is that these systems require initial viral infection and subsequent cellular expression of a reporter gene to allow non-invasive imaging. As an alternative to conventional vector detection techniques, we developed a specific genetic labeling system whereby an adenoviral vector incorporates a fusion between capsid protein IX and human metallothionein. Our study herein clearly demonstrates our ability to rescue viable adenoviral particles that display functional metallothionein (MT) as a component of their capsid surface. We demonstrate the feasibility of (99m)Tc binding in vitro to the pIX-MT fusion on the capsid of adenovirus virions using a simple transchelation reaction. SPECT imaging of a mouse after administration of a (99m)Tc-radiolabeled virus showed clear localization of radioactivity to the liver. This result strongly supports imaging using pIX-MT, visualizing the normal biodistribution of Ad primarily to the liver upon injection into mice. The ability we have developed to view real-time biodistribution in their physiological milieu represents a significant tool to study adenovirus biology in vivo.     

Enhanced production of ligninolytic enzymes and decolorization of molasses distillery wastewater by fungi under solid state fermentation

Selected isolates of fungi were grown on wheat straw and corncob in the presence of different moistening agents such as water, molasses, potato dextrose broth and distillery effluent. All the fungal isolates responded differently with respect to growth and ligninolytic enzyme production. Fungal growth on different substrates was checked by calculating ergosterol content, which varied widely within a single species when grown on different substrates. The maximum laccase production was obtained for Aspergillus flavus TERI DB9 grown on wheat straw with molasses. For manganese peroxidase, highest production was in Aspergillus niger TERI DB20 grown on corncob with effluent. Among the two isolates positive for lignin peroxidase, the highest production was in Fusarium verticillioides ITCC 6140. This immobilized fungal biomass was then used for decolorization of effluent from a cane molasses based distillery. Maximum decolorization (86.33%) was achieved in Pleurotus ostreatus (Florida) Eger EM 1303 immobilized on corncob with molasses in a period of 28 days.     

Characterisation of the yeast Pichia membranifaciens and its possible use in the biological control of Botrytis cinerea, causing the grey mould disease of grapevine

Pichia membranifaciens strain FY-101, isolated from grape skins, was found to be antagonistic to Botrytis cinerea, the causal organism of the grey mould disease of the grapevine. When grown together on solid as well as liquid media, the yeast brings about the inhibition of this parasitic fungus, coagulation and leakage of its cytoplasm, and suppression of its ability to produce the characteristic grey mould symptoms on the grapevine plantlets. In vitro experiments confirm that this yeast can be used as a biological control organism against B. cinerea. An account of the molecular characterisation of P. membranifaciens (complete sequence of the ITS region of its ribosomal DNA, GenBank accession No. AF 270935), as well as the interaction between B. cinerea and the yeast, are given here.     

Effect of bioaugmentation and nitrogen supplementation on composting of paddy straw

A composting experiment was conducted to evaluate the effect of a hyperlignocellulolytic fungal consortium and different nitrogen amendments on paddy straw composting in terms of changes in physicochemical and biological parameters. A fungal consortium comprising four lignocellulolytic mesophilic fungal cultures was used as inoculum for bioaugmentation of paddy straw in perforated pits. The comparative effect of farmyard manure (FYM), soybean trash, poultry litter and urea on the composting process was evaluated at monthly intervals in terms of physicochemical (pH, EC, available P, C:N ratio and humus content) and biological (enzymatic and microbial activity) parameters. The compost prepared from bioaugmented paddy straw composting mixture, with poultry manure as nitrogen supplement attained desirable C:N ratio in 1 month and displayed least phytotoxicity levels along with higher production of β-1,4-Exoglucanase. The combined activity of the autochthonous composting microbiota as well as the externally applied fungal inoculum accelerated the composting process of paddy straw. Supplementation of paddy straw with poultry manure in 8:1 ratio was identified as the best treatment to hasten the composting process. This study highlights the importance of application of fungal inoculum and an appropriate N-amendment such as poultry manure for preparation of compost using a substrate having high C:N ratio, such as paddy straw.