Studies on fermentative production of squalene

Fermentative production of squalene under anaerobic conditions using commercially available compressed baker's yeast (Saccharomyces cerevisiae), and a strain of Torulaspora delbrueckii isolated from molasses was studied. Yield of squalene from S. cerevisiae and T. delbrueckii were found to be 41.16 and 237.25 g g^–1 respectively, dry weight of yeast cells. Isolation and purification of squalene from the lipid extracts obtained by cell lysis of either strain were achieved chromatographically. The purified squalene was characterized spectroscopically against an authentic standard.     

Pharmacological targeting of the ephrin receptor kinase signalling by GLPG1790 in vitro and in vivo reverts oncophenotype,induces myogenic differentiation and radiosensitizes embryonal rhabdomyosarcoma cells

Background

EPH (erythropoietin-producing hepatocellular) receptors are clinically relevant targets in several malignancies. This report describes the effects of GLPG1790, a new potent pan-EPH inhibitor, in human embryonal rhabdomyosarcoma (ERMS) cell lines.

Methods

EPH-A2 and Ephrin-A1 mRNA expression was quantified by real-time PCR in 14 ERMS tumour samples and in normal skeletal muscle (NSM). GLPG1790 effects were tested in RD and TE671 cell lines, two in vitro models of ERMS, by performing flow cytometry analysis, Western blotting and immunofluorescence experiments. RNA interfering experiments were performed to assess the role of specific EPH receptors. Radiations were delivered using an x-6 MV photon linear accelerator. GLPG1790 (30 mg/kg) in vivo activity alone or in combination with irradiation (2 Gy) was determined in murine xenografts.

Results

Our study showed, for the first time, a significant upregulation of EPH-A2 receptor and Ephrin-A1 ligand in ERMS primary biopsies in comparison to NSM. GLPG1790 in vitro induced G1-growth arrest as demonstrated by Rb, Cyclin A and Cyclin B1 decrease, as well as by p21 and p27 increment. GLPG1790 reduced migratory capacity and clonogenic potential of ERMS cells, prevented rhabdosphere formation and downregulated CD133, CXCR4 and Nanog stem cell markers. Drug treatment committed ERMS cells towards skeletal muscle differentiation by inducing a myogenic-like phenotype and increasing MYOD1, Myogenin and MyHC levels. Furthermore, GLPG1790 significantly radiosensitized ERMS cells by impairing the DNA double-strand break repair pathway. Silencing of both EPH-A2 and EPH-B2, two receptors preferentially targeted by GLPG1790, closely matched the effects of the EPH pharmacological inhibition. GLPG1790 and radiation combined treatments reduced tumour mass by 83% in mouse TE671 xenografts.

Conclusions

Taken together, our data suggest that altered EPH signalling plays a key role in ERMS development and that its pharmacological inhibition might represent a potential therapeutic strategy to impair stemness and to rescue myogenic program in ERMS cells.

     

Phenotypic Switching in Biofilm-Forming Marine Bacterium Paenibacillus lautus NE3B01

Biofilm-forming marine bacterium Paenibacillus lautus NE3B01 was isolated from a mangrove ecosystem, Odisha, India. This isolate formed a swarming type of colony pattern on the solid culture medium with 0.5–2 % agar. Phase contrast microscopy study of a growing colony of P. lautus on solid media and swarming pattern revealed the existence of two phenotypically distinct cells (i.e. cocci and rods) across the colonies. However, in actively growing planktonic culture, only rod-shaped cells were observed. Biofilm growth studies (crystal violet assay) with the isolate showed significant biofilm formation by 6 h, and the detachment phase was observed after 18 h. Biofilm parameters (such as total biomass, roughness coefficient, biofilm thickness, etc.) of 24-h-old P. lautus biofilm were studied by confocal scanning laser microscopy (CSLM). The CSLM study showed that P. lautus formed a biofilm with an average thickness of 14.8 ± 2.6 μm, a high roughness coefficient (0.379 ± 0.103) and surface to bio-volume ratio (4.59 ± 1.12 μm²/μm³), indicating a highly uneven topography of the biofilm. This also indicates that the 24-h-old biofilm is in dispersal phase. Scanning electron microphotographs of P. lautus also supported the existence of two distinct phenotypes of P. lautus. The current findings suggest that P. lautus has two vegetative phenotypes and to decongest the overcrowded biofilm the bacterium can switch over to motile rods from nonmotile cocci and vice versa.     

Plasmodium berghei: influence of infection on the oxidant and antioxidants levels in pregnant BALB/c mice

Malarial infection during pregnancy has been associated with maternal anemia and death, abortion, still-birth and is a major cause of low birth weight, an important risk factor for infant morbidity and mortality in endemic areas. The present study was designed to delineate the oxidative stress in various organs (liver, spleen, kidney, brain and placenta) of pregnant Plasmodium berghei infected BALB/c mice. It was observed that pregnant-infected mice had higher parasitaemia than nonpregnant-infected mice. Most notably, levels of malondialdehyde (MDA), a measure of lipid peroxidation, reduced glutathione (GSH) and superoxide dismutase (SOD) levels were significantly higher in the liver, spleen, kidney and brain of pregnant-infected mice compared with pregnant mice. Although MDA levels were significantly higher, GSH and SOD levels remained unaltered in the placenta of pregnant-infected mice compared with pregnant mice. Furthermore, catalase activity was significantly lower in all the organs of pregnant-infected mice compared with pregnant mice. Histopathological observations in the organs clearly show the cellular and morphological alterations that may be occurring due to increased lipid peroxidation. Taken together, the data suggest that the increased severity of malarial infection during pregnancy may be due to accentuated oxidative stress.     

Comprehensive Analysis of Herpes Simplex Virus 1 (HSV-1) Entry Mediated by Zebrafish 3-O-Sulfotransferase Isoforms: Implications for the Development of a Zebrafish Model of HSV-1 Infection

Binding of herpes simplex virus 1 (HSV-1) envelope glycoprotein D (gD) to the receptor 3-O-sulfated heparan sulfate (3-OS HS) mediates viral entry. 3-O-Sulfation of HS is catalyzed by the 3-O-sulfotransferase (3-OST) enzyme. Multiple isoforms of 3-OST are differentially expressed in tissues of zebrafish (ZF) embryos. Here, we performed a comprehensive analysis of the role of ZF 3-OST isoforms (3-OST-1, 3-OST-5, 3-OST-6, and 3-OST-7) in HSV-1 entry. We found that a group of 3-OST gene family isoforms (3-OST-2, -3, -4, and -6) with conserved catalytic and substrate-binding residues of the enzyme mediates HSV-1 entry and spread, while the other group (3-OST-1, -5, and -7) lacks these properties. These results demonstrate that HSV-1 entry can be recapitulated by certain ZF 3-OST enzymes, a significant step toward the establishment of a ZF model of HSV-1 infection and tissue-specific tropism.     

Isolation of Enteric Nervous System Progenitor Cells from the Aganglionic Gut of Patients with Hirschsprung’s Disease

Enteric nervous system progenitor cells isolated from postnatal human gut and cultured as neurospheres can then be transplanted into aganglionic gut to restore normal patterns of contractility. These progenitor cells may be of future use to treat patients with Hirschprung’s disease, a congenital condition characterized by hindgut dysmotility due to the lack of enteric nervous system ganglia. Here we demonstrate that progenitor cells can also be isolated from aganglionic gut removed during corrective surgery for Hirschsprung’s disease. Although the enteric nervous system marker calretinin is not expressed in the aganglionic gut region, de novo expression is initiated in cultured neurosphere cells isolated from aganglionic Hirschsprung bowel. Furthermore, expression of the neural markers NOS, VIP and GFAP also increased during culture of aganglionic gut neurospheres which we show can be transplantation into cultured embryonic mouse gut explants to restore a normal frequency of contractility. To determine the origin of the progenitor cells in aganglionic region, we used fluorescence-activated cell sorting to demonstrate that only p75-positive neural crest-derived cells present in the thickened nerve trunks characteristic of the aganglionic region of Hirschsprung gut gave rise to neurons in culture. The derivation of enteric nervous system progenitors in the aganglionic gut region of Hirschprung’s patients not only means that this tissue is a potential source of cells for future autologous transplantation, but it also raises the possibility of inducing the differentiation of these endogenous cells in situ to compensate for the aganglionosis.     

An Efficiently Cleaved HIV-1 Clade C Env Selectively Binds to Neutralizing Antibodies

An ideal HIV-1 Env immunogen is expected to mimic the native trimeric conformation for inducing broadly neutralizing antibody responses. The native conformation is dependent on efficient cleavage of HIV-1 Env. The clade B isolate, JRFL Env is efficiently cleaved when expressed on the cell surface. Here, for the first time, we report the identification of a native clade C Env, 4-2.J41 that is naturally and efficiently cleaved on the cell surface as confirmed by its biochemical and antigenic characteristics. In addition to binding to several conformation-dependent neutralizing antibodies, 4-2.J41 Env binds efficiently to the cleavage-dependent antibody PGT151; thus validating its native cleaved conformation. In contrast, 4-2.J41 Env occludes non-neutralizing epitopes. The cytoplasmic-tail of 4-2.J41 Env plays an important role in maintaining its conformation. Furthermore, codon optimization of 4-2.J41 Env sequence significantly increases its expression while retaining its native conformation. Since clade C of HIV-1 is the prevalent subtype, identification and characterization of this efficiently cleaved Env would provide a platform for rational immunogen design.     

Protective effect of curcumin against intracerebral streptozotocin induced impairment in memory and cerebral blood flow

AimsThe aim of the present study is to investigate the effect of curcumin on cerebral blood flow (CBF), memory impairment, oxidative stress and cholinergic dysfunction in intracerebral (IC) streptozotocin (STZ) induced memory impairment in mice.Main methodsMemory impairment was induced by STZ (0.5 mg/kg, IC) administered twice with an interval of 48 h in mice. Memory function was assessed by Morris water maze and passive avoidance test. CBF was measured by Laser Doppler Flowmetry (LDF). To study the preventive effect, curcumin (10, 20 and 50 mg/kg, PO) was administered for 21 days starting from the first dose of STZ. In another set of experiment, curcumin was administered for 7 days from 19th day after confirming STZ induced dementia to observe its therapeutic effect. Biochemical parameters of oxidative stress and cholinergic function were estimated in brain on day 21.Key findingsThe major finding of this study is that STZ (IC) caused a significant reduction in CBF along with memory impairment, cholinergic dysfunction and enhanced oxidative stress. Curcumin dose dependently improved CBF in STZ treated mice together with amelioration of memory impairment both in preventive and therapeutic manner.SignificanceThe present study clearly demonstrates the beneficial effects of curcumin, the dietary staple of India, on CBF, memory and oxidative stress which can be exploited for dementia associated with age related vascular and neurodegenerative disorders.