Proteomics and Metabolomics Analyses to Elucidate the Desulfurization Pathway of Chelatococcus sp.

Desulfurization of dibenzothiophene (DBT) and alkylated DBT derivatives present in transport fuel through specific cleavage of carbon-sulfur (C-S) bonds by a newly isolated bacterium Chelatococcus sp. is reported for the first time. Gas chromatography-mass spectrometry (GC-MS) analysis of the products of DBT degradation by Chelatococcus sp. showed the transient formation of 2-hydroxybiphenyl (2-HBP) which was subsequently converted to 2-methoxybiphenyl (2-MBP) by methylation at the hydroxyl group of 2-HBP. The relative ratio of 2-HBP and 2-MBP formed after 96 h of bacterial growth was determined at 4:1 suggesting partial conversion of 2-HBP or rapid degradation of 2-MBP. Nevertheless, the enzyme involved in this conversion process remains to be identified. This production of 2-MBP rather than 2-HBP from DBT desulfurization has a significant metabolic advantage for enhancing the growth and sulfur utilization from DBT by Chelatococcus sp. and it also reduces the environmental pollution by 2-HBP. Furthermore, desulfurization of DBT derivatives such as 4-M-DBT and 4, 6-DM-DBT by Chelatococcus sp. resulted in formation of 2-hydroxy-3-methyl-biphenyl and 2-hydroxy –3, 3^/- dimethyl-biphenyl, respectively as end product. The GC and X-ray fluorescence studies revealed that Chelatococcus sp. after 24 h of treatment at 37°C reduced the total sulfur content of diesel fuel by 12% by per gram resting cells, without compromising the quality of fuel. The LC-MS/MS analysis of tryptic digested intracellular proteins of Chelatococcus sp. when grown in DBT demonstrated the biosynthesis of 4S pathway desulfurizing enzymes viz. monoxygenases (DszC, DszA), desulfinase (DszB), and an NADH-dependent flavin reductase (DszD). Besides, several other intracellular proteins of Chelatococcus sp. having diverse biological functions were also identified by LC-MS/MS analysis. Many of these enzymes are directly involved with desulfurization process whereas the other enzymes/proteins support growth of bacteria at an expense of DBT. These combined results suggest that Chelatococcus sp. prefers sulfur-specific extended 4S pathway for deep-desulphurization which may have an advantage for its intended future application as a promising biodesulfurizing agent.     

Modeling of Mesoscale Variability in Biofilm Shear Behavior

Formation of bacterial colonies as biofilm on the surface/interface of various objects has the potential to impact not only human health and disease but also energy and environmental considerations. Biofilms can be regarded as soft materials, and comprehension of their shear response to external forces is a key element to the fundamental understanding. A mesoscale model has been presented in this article based on digitization of a biofilm microstructure. Its response under externally applied shear load is analyzed. Strain stiffening type behavior is readily observed under high strain loads due to the unfolding of chains within soft polymeric substrate. Sustained shear loading of the biofilm network results in strain localization along the diagonal direction. Rupture of the soft polymeric matrix can potentially reduce the intercellular interaction between the bacterial cells. Evolution of stiffness within the biofilm network under shear reveals two regimes: a) initial increase in stiffness due to strain stiffening of polymer matrix, and b) eventual reduction in stiffness because of tear in polymeric substrate.     

Interspecific diversity reduces and functionally substitutes for intraspecific variation in biofilm communities

Diversity has a key role in the dynamics and resilience of communities and both interspecific (species) and intraspecific (genotypic) diversity can have important effects on community structure and function. However, a critical and unresolved question for understanding the ecology of a community is to what extent these two levels of diversity are functionally substitutable? Here we show, for a mixed-species biofilm community composed of Pseudomonas aeruginosa, P. protegens and Klebsiella pneumoniae, that increased interspecific diversity reduces and functionally substitutes for intraspecific diversity in mediating tolerance to stress. Biofilm populations generated high percentages of genotypic variants, which were largely absent in biofilm communities. Biofilms with either high intra- or interspecific diversity were more tolerant to SDS stress than biofilms with no or low diversity. Unexpectedly, genotypic variants decreased the tolerance of biofilm communities when experimentally introduced into the communities. For example, substituting P. protegens wild type with its genotypic variant within biofilm communities decreased SDS tolerance by twofold, apparently due to perturbation of interspecific interactions. A decrease in variant frequency was also observed when biofilm populations were exposed to cell-free effluents from another species, suggesting that extracellular factors have a role in selection against the appearance of intraspecific variants. This work demonstrates the functional substitution of inter- and intraspecific diversity for an emergent property of biofilms. It also provides a potential explanation for a long-standing paradox in microbiology, in which morphotypic variants are common in laboratory grown biofilm populations, but are rare in diverse, environmental biofilm communities.     

Zebrafish TARP Cacng2 is required for the expression and normal development of AMPA receptors at excitatory synapses

Fast excitatory synaptic transmission in the CNS is mediated by the neurotransmitter glutamate, binding to and activating AMPA receptors (AMPARs). AMPARs are known to interact with auxiliary proteins that modulate their behavior. One such family of proteins is the transmembrane AMPA receptor‐related proteins, known as TARPs. Little is known about the role of TARPs during development, or about their function in non‐mammalian organisms. Here we report the presence of TARPs, specifically the prototypical TARP, stargazin, in developing zebrafish. We find that zebrafish express two forms of stargazin, Cacng2a and Cacng2b from as early as 12‐h post fertilization (hpf). Knockdown of Cacng2a and Cacng2b via splice‐blocking morpholinos resulted in embryos that exhibited deficits in C‐start escape responses, showing reduced C‐bend angles, smaller tail velocities and aberrant C‐bend turning directions. Injection of the morphants with Cacng2a or 2b mRNA rescued the morphological phenotype and the synaptic deficits. To investigate the effect of reduced Cacng2a and 2b levels on synaptic physiology, we performed whole cell patch clamp recordings of AMPA mEPSCs from zebrafish Mauthner cells. Knockdown of Cacng2a results in reduced AMPA currents and lower mEPSC frequencies, whereas knockdown of Cacng2b displayed no significant change in mEPSC amplitude or frequency. Non‐stationary fluctuation analysis confirmed a reduction in the number of active synaptic receptors in the Cacng2a but not in the Cacng2b morphants. Together, these results suggest that Cacng2a is required for normal trafficking and function of synaptic AMPARs, while Cacng2b is largely non‐functional with respect to the development of AMPA synaptic transmission. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 487–506, 2016     

Activation of the PI3K/mTOR Pathway following PARP Inhibition in Small Cell Lung Cancer

Small cell lung cancer (SCLC) is an aggressive malignancy with limited treatment options. We previously found that PARP is overexpressed in SCLC and that targeting PARP reduces cell line and tumor growth in preclinical models. However, SCLC cell lines with PI3K/mTOR pathway activation were relatively less sensitive to PARP inhibition. In this study, we investigated the proteomic changes in PI3K/mTOR and other pathways that occur following PAPR inhibition and/or knockdown in vitro and in vivo. Using reverse-phase protein array, we found the proteins most significantly upregulated following treatment with the PARP inhibitors olaparib and rucaparib were in the PI3K/mTOR pathway (p-mTOR, p-AKT, and pS6) (p≤0.02). Furthermore, amongst the most significantly down-regulated proteins were LKB1 and its targets AMPK and TSC, which negatively regulate the PI3K pathway (p≤0.042). Following PARP knockdown in cell lines, phosphorylated mTOR, AKT and S6 were elevated and LKB1 signaling was diminished. Global ATP concentrations increased following PARP inhibition (p≤0.02) leading us to hypothesize that the observed increased PI3K/mTOR pathway activation following PARP inhibition results from decreased ATP usage and a subsequent decrease in stress response signaling via LKB1. Based on these results, we then investigated whether co-targeting with a PARP and PI3K inhibitor (BKM-120) would work better than either single agent alone. A majority of SCLC cell lines were sensitive to BKM-120 at clinically achievable doses, and cMYC expression was the strongest biomarker of response. At clinically achievable doses of talazoparib (the most potent PARP inhibitor in SCLC clinical testing) and BKM-120, an additive effect was observed in vitro. When tested in two SCLC animal models, a greater than additive interaction was seen (p≤0.008). The data presented here suggest that combining PARP and PI3K inhibitors enhances the effect of either agent alone in preclinical models of SCLC, warranting further investigation of such combinations in SCLC patients.     

Chemical Aspects of Santalin as a Histological Stain

Recent research on the chemical nature of the red dyes isolated from Pterocarpus santalinus and certain West African plants, viz., Baphia nitida, Pterocarpus osun and Pterocarpus soyauxii, have been reviewed. P. santalinus contains santalins A, B and C., but no santarubin. Santalins and santarubins have been found in P. osun, P. soyauxii and B. nitida. The structural formulae of the santalins are presented and their differences from santarubins indicated. Santalins A and B have some similarities in structure with hematein. This is probably responsible for their staining properties; the possible mechanism of staining is discussed.     

Effect of desiccation on mosquito oviposition site selection in Mediterranean temporary habitats

1. Hydroperiod duration has been identified as the main factor determining the faunal composition and structure of aquatic communities in temporary habitats. It is hypothesised that desiccation will positively affect mosquito oviposition habitat selection during the post-drought period due to the lack of antagonists. 2. An experiment was carried out in outdoor mesocosms to assess whether desiccation events have post-drought effects on community richness and mosquito oviposition. Three different treatments were randomly assigned to the mesocosms: (i) eight mesocosms were left dried for a week and then reflooded; (ii) eight mesocosms stayed dry for 4 weeks before they were reflooded; (iii) eight mesocosms were maintained at a constant water volume of 30 litres during the entire experiment as controls. Mosquito oviposition and invertebrate community richness were monitored in every mesocosm, along with environmental parameters [water temperature, pH, conductivity, dissolved oxygen, total suspended solids (TSS) and chlorophyll a concentration]. 3. Post-drought mosquito oviposition and larval abundance were higher in the short-drought and long-drought pools than in the control. Desiccation negatively affected the biomass of the filter feeder invertebrates in both desiccation treatments. Chlorophyll a concentrations were higher in the long-drought pools than in controls. The negative impact of desiccation on zooplankton led to a post-drought increase in algae, associated with an increase in mosquito oviposition. 4. Despite immediate negative effect on mosquitoes, pulsed disturbances can benefit mosquitoes as they favour oviposition during the post-disturbance recovery period due to a lower abundance of mosquito antagonists and higher food resources for their offspring.     

Embryonic characteristics in superovulated mouse strains. Comparative analyses of the incidence of chromosomal aberrations, morphological malformations, and mortality of embryos from two strains of superovulated mice

Superovulation was induced in an inbred (C3HeB/FeJ) and a random-bred (CD-1) strain of mice and pregnant females were serially sacrificed from 6-15 days of gestation. All embryos were examined chromosomally and by external morphology. A G-banding technique was used to detect specific chromosomal constitutions. The common types of chromosomal aberrations were monosomies and trisomies. Our results showed that the proportion of chromosomally abnormal embryos was significantly greater in the superovulated group of inbred mice, than in the spontaneously ovulating inbred group, an observation not made in the random-bred mice. In each strain, superovulation yielded a significantly higher proportion of morphologically malformed embryos, but no significant difference between strains with respect to this criterion was noted after superovulation. There was a significant increase in the post-implantation embryonic mortality in the random-bred strain but not in the inbred strain following superovulation. This study indicates that each strain has its intrinsic pattern with regard to the fate of embryos following superovulation.