Growth,pigments, and biochemical composition of marine red alga Gracilaria crassa

The marine red alga Gracilaria crassa was investigated for its proximate composition, minerals, fatty acids, amino acids, and agar content to decipher its nutritional implications. The growth performance and pigments were studied under different combinations of temperature and salinity. On a dry weight basis the total lipid content was 1.30?±?0.05 %, protein was 5.18?±?0.64 %, carbohydrate was 42.0?±?1.2 %, ash was 43.18?±?1.15 %, and agar content was 21.52?±?0.73 %. Appreciable amounts of macro-, micro-nutrients (K?>?Na, Ca, Mg, and Fe), and essential amino acids (Ileu, His, Thr, Leu, and Lys) were found. Palmitic, stearic acid, and arachidonic acid were major fatty acids detected. The alga showed maximum daily growth rate (DGR %) 5.8?±?0.09 % at 25 °C, 35 ‰ salinity. The highest content of pigment R-phycoerythrin (444.7?±?1.9 μg g^?1 fresh weight (FW) basis) was obtained at 25 ‰ salinity at 35 °C while that of R-phycocyanin (476.3?±?2.3 μg g^?1 DW) at 30 ‰ salinity at 30 °C. This study revealed that this alga can be utilized as a potential source for food and feed. The data generated on best growth conditions will be very useful for farming of G. crassa in open sea. This alga could be used for production of natural colorants at defined control condition.     

Order-disorder-order transitions mediate the activation of cholera toxin

Cholera toxin (CT) holotoxin must be activated to intoxicate host cells. This process requires the intracellular dissociation of the enzymatic CTA1 domain from the holotoxin components CTA2 and B5, followed by subsequent interaction with the host factor ADP ribosylation factor 6 (ARF6)-GTP. We report the first NMR-based solution structural data for the CT enzymatic domain (CTA1). We show that this free enzymatic domain partially unfolds at the C-terminus and binds its protein partners at both the beginning and the end of this activation process. Deviations from random coil chemical shifts (Δδ_coil) indicate helix formation in the activation loop, which is essential to open the toxin's active site and occurs prior to its association with human protein ARF6. We performed NMR titrations of both free CTA1 and an active CTA1:ARF6-GTP complex with NAD⁺, which revealed that the formation of the complex does not significantly enhance NAD⁺ binding. Partial unfolding of CTA1 is further illustrated by using 4,4′-bis(1-anilinonaphthalene 8-sulfonate) fluorescence as an indicator of the exposed hydrophobic character of the free enzyme, which is substantially reduced when bound to ARF6-GTP. We propose that the primary role of ARF6's allostery is to induce refolding of the C-terminus of CTA1. Thus, as a folded globular toxin complex, CTA1 escapes the chaperone and proteasomal components of the endoplasmic reticulum associated degradation pathway in the cytosol and then proceeds to ADP ribosylate its target G_sα, triggering the downstream events associated with the pathophysiology of cholera.     

Lead identification to generate isoquinolinedione inhibitors of insulin-like growth factor receptor (IGF-1R) for potential use in cancer treatment

Insulin-like growth factor receptor (IGF-1R) is a growth factor receptor tyrosine kinase that acts as a critical mediator of cell proliferation and survival. This receptor is over-expressed or activated in tumor cells and is emerging as a novel target in cancer therapy. Efforts in our "Hit to Lead" group have generated a novel series of submicromolar IGF-1R inhibitors based on a isoquinolinedione template originating from a Lance enzyme HTS screen. Chemical triage and parallel synthesis incorporating focused library arrays were instrumental in moving these investigations through the Wyeth exploratory medicinal chemistry process. The strategies, synthesis, and SAR behind this interesting kinase scaffold will be described.     

Searching Biomarkers in the Sequenced Genomes of <Emphasis Type="Italic">Staphylococcus</Emphasis> for their Rapid Identification

Bacterial identification using rrs (16S rRNA) gene is widely reported. Bacteria possessing multiple copies of rrs lead to overestimation of its diversity. Staphylococcus genomes carries 5–6 copies of rrs showing high similarity in their nucleotide sequences, which lead to ambiguous results. The genomes of 31 strains of Staphylococcus representing 7 species were searched for the presence of common genes. In silico digestion of 34 common genes using 10 restriction endonucleases (REs) lead to select gene-RE combinations, which could be used as biomarkers. RE digestion of recA allowed unambiguous identification of 13 genomes representing all the 7 species. In addition, a few more genes (argH, argR, cysS, gyrB, purH, and pyrE) and RE combinations permitted further identification of 12 strains. By employing additional RE and genes unique to a particular strain, it was possible to identify the rest 6 Staphylococcus aureus strains. This approach has the potential to be utilized for rapid detection of Staphylococcus strains.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-016-0565-9) contains supplementary material, which is available to authorized users.     

Polyelectrolyte complexes of gum kondagogu and chitosan,as diclofenac carriers

Polyelectrolyte complexes (PEC) of gum kondagogu (GKG) and chitosan were prepared by mixing polymeric solutions of different concentrations (0.02–0.18% w/v). The complex formed were loaded with diclofenac sodium, and the release of the drug was measured in vitro and in vivo, along with the measurement of particle size, zeta potential, complex formation, flow properties, and loading efficiency. Maximum yield of PEC was observed at gum kondagogu concentrations above 80%. The PEC showed lower release of diclofenac sodium in 0.1 N HCl as compared to phosphate buffer (pH 6.8). Increasing the concentration of gum kondagogu in PEC led to an increase in drug release. However, PEC 1:3 (gum kondagogu: chitosan) with higher concentration of chitosan showed 98% release with in 4.5 h, owing to the fact that chitosan has a higher degree of swelling in acidic medium. PEC 5:1 and 3:1 showed a 5.3- and 5.8-fold increase in relative bioavailability compared to the free drug when administered orally to the rats.     

CD44 regulates hepatocyte growth factor-mediated vascular integrity. Role of c-Met, Tiam1/Rac1, dynamin 2, and cortactin

The preservation of vascular endothelial cell (EC) barrier integrity is critical to normal vessel homeostasis, with barrier dysfunction being a feature of inflammation, tumor angiogenesis, atherosclerosis, and acute lung injury. Therefore, agents that preserve or restore vascular integrity have important therapeutic implications. In this study, we explored the regulation of hepatocyte growth factor (HGF)-mediated enhancement of EC barrier function via CD44 isoforms. We observed that HGF promoted c-Met association with CD44v10 and recruitment of c-Met into caveolin-enriched microdomains (CEM) containing CD44s (standard form). Treatment of EC with CD44v10-blocking antibodies inhibited HGF-mediated c-Met phosphorylation and c-Met recruitment to CEM. Silencing CD44 expression (small interfering RNA) attenuated HGF-induced recruitment of c-Met, Tiam1 (a Rac1 exchange factor), cortactin (an actin cytoskeletal regulator), and dynamin 2 (a vesicular regulator) to CEM as well as HGF-induced trans-EC electrical resistance. In addition, silencing Tiam1 or dynamin 2 reduced HGF-induced Rac1 activation, cortactin recruitment to CEM, and EC barrier regulation. We observed that both HGF- and high molecular weight hyaluronan (CD44 ligand)-mediated protection from lipopolysaccharide-induced pulmonary vascular hyperpermeability was significantly reduced in CD44 knock-out mice, thus validating these in vitro findings in an in vivo murine model of inflammatory lung injury. Taken together, these results suggest that CD44 is an important regulator of HGF/c-Met-mediated in vitro and in vivo barrier enhancement, a process with essential involvement of Tiam1, Rac1, dynamin 2, and cortactin.     

New slow-rusting leaf rust and stripe rust resistance genes <Emphasis Type="Italic">Lr67</Emphasis> and <Emphasis Type="Italic">Yr46</Emphasis> in wheat are pleiotropic or closely linked

The common wheat genotype ‘RL6077’ was believed to carry the gene Lr34/Yr18 that confers slow-rusting adult plant resistance (APR) to leaf rust and stripe rust but located to a different chromosome through inter-chromosomal reciprocal translocation. However, haplotyping using the cloned Lr34/Yr18 diagnostic marker and the complete sequencing of the gene indicated Lr34/Yr18 is absent in RL6077. We crossed RL6077 with the susceptible parent ‘Avocet’ and developed F₃, F₄ and F₆ populations from photoperiod-insensitive F₃ lines that were segregating for resistance to leaf rust and stripe rust. The populations were characterized for leaf rust resistance at two Mexican sites, Cd. Obregon during the 2008–2009 and 2009–2010 crop seasons, and El Batan during 2009, and for stripe rust resistance at Toluca, a third Mexican site, during 2009. The F₃ population was also evaluated for stripe rust resistance at Cobbitty, Australia, during 2009. Most lines had correlated responses to leaf rust and stripe rust, indicating that either the same gene, or closely linked genes, confers resistance to both diseases. Molecular mapping using microsatellites led to the identification of five markers (Xgwm165, Xgwm192, Xcfd71, Xbarc98 and Xcfd23) on chromosome 4DL that are associated with this gene(s), with the closest markers being located at 0.4 cM. In a parallel study in Canada using a Thatcher × RL6077 F₃ population, the same leaf rust resistance gene was designated as Lr67 and mapped to the same chromosomal region. The pleiotropic, or closely linked, gene derived from RL6077 that conferred stripe rust resistance in this study was designated as Yr46. The slow-rusting gene(s) Lr67/Yr46 can be utilized in combination with other slow-rusting genes to develop high levels of durable APR to leaf rust and stripe rust in wheat.     

Human CD8⁺ and CD4⁺ T cell memory to lymphocytic choriomeningitis virus infection

Although cellular immunity to acute lymphocytic choriomeningitis virus (LCMV) infection has been well characterized in experimental studies in mice, the T cell response to this virus in humans is incompletely understood. Thus, we analyzed the breadths, magnitudes, and differentiation phenotypes of memory LCMV-specific CD8(+) and CD4(+) T cells in three human donors displaying a variety of disease outcomes after accidental needle stick injury or exposure to LCMV. Although only a small cohort of donors was analyzed at a single time point postinfection, several interesting observations were made. First, we were able to detect LCMV-specific CD8(+) and CD4(+) T cell responses directly ex vivo at 4 to 8 years after exposure, demonstrating the longevity of T cell memory in humans. Second, unlike in murine models of LCMV infection, we found that the breadths of memory CD8(+) and CD4(+) T cell responses were not significantly different from one another. Third, it seemed that the overall CD8(+) T cell response was augmented with increasing severity of disease, while the LCMV-specific CD4(+) T cell response magnitude was highly variable between the three different donors. Next, we found that LCMV-specific CD8(+) T cells in the three donors analyzed seemed to undergo an effector memory differentiation program distinct from that of CD4(+) T cells. Finally, the levels of expression of memory, costimulatory, and inhibitory receptors on CD8(+) and CD4(+) T cell subsets, in some instances, correlated with disease outcome. These data demonstrate for the first time LCMV-specific CD8(+) and CD4(+) T cells in infected humans and begin to provide new insights into memory T cell responses following an acute virus infection.     

Huntington's Disease and its therapeutic target genes: a global functional profile based on the HD Research Crossroads database

ABSTRACT: BACKGROUND: Huntington's disease (HD) is a fatal progressive neurodegenerative disorder caused by the expansion of the polyglutamine repeat region in the huntingtin gene. Although the disease is triggered by the mutation of a single gene, intensive research has linked numerous other genes to its pathogenesis. To obtain a systematic overview of these genes, which may serve therapeutic targets, the Cure Huntington's Disease Initiative (CHDI) has recently established the HD Research Crossroads database. With currently over 800 cataloged genes, this web-based resource constitutes the most extensive curation of genes relevant to HD. It provides us with an unprecedented opportunity to survey molecular mechanisms involved in HD in a holistic manner. METHODS: To gain a synoptic view of therapeutic targets for HD, we have carried out a variety of bioinformatical and statistical analyses to scrutinize the functional association of genes curated in the HD Research Crossroads database. In particular, enrichment analyses were performed with respect to Gene Ontology categories, KEGG signaling pathways, and Pfam protein families. For selected processes, we also analyzed differential expression, using published microarray data. Additionally, we generated a candidate set of novel genetic modifiers of HD by combining information from the HD Research Crossroads database with previous genome-wide linkage studies. RESULTS: Our analyses led to a comprehensive identification of molecular mechanisms associated with HD. Remarkably, we not only recovered processes and pathways, which have frequently been linked to HD (such as cytotoxicity, apoptosis, and calcium signaling), but also found strong indications for other potentially disease-relevant mechanisms that have been less intensively studied in the context of HD (such as the cell cycle and RNA splicing, as well as Wnt and ErbB signaling). For follow-up studies, we provide a compendium of molecular mechanisms that are associated with HD. Additionally, we derived a candidate set of 24 novel genetic modifiers, including histone deacetylase 3 (HDAC3), metabotropic glutamate receptor 1 (GRM1), CDK5 regulatory subunit 2 (CDK5R2), and coactivator 1beta of the peroxisome proliferator-activated receptor gamma (PPARGC1B). CONCLUSIONS: The results of our study give us an intriguing picture of the molecular complexity of HD. Our analyses can be seen as a first step towards a comprehensive list of biological processes, molecular functions, and pathways involved in HD, and may provide a basis for the development of more holistic disease models and new therapeutics.