Derivation and characterization of cell cultures from the skin of the Indo-Pacific humpback dolphin Sousa chinensis

The marine mammalian Indo-Pacific humpback dolphin, once widely lived in waters of the Indian to western Pacific oceans, has become an endangered species. The individual number of this dolphin has significantly declined in recent decades, which raises the concern of extinction. Direct concentration on laboratorial conservation of the genetic and cell resources should be paid to this marine species. Here, we report the successful derivation of cell lines form the skin of Indo-Pacific humpback dolphin. The cell cultures displayed the characteristics of fibroblast in morphology and grew rapidly at early passages, but showed obvious growth arrest at higher passages. The karyotype of the cells consisted of 42 autosomes and sex chromosomes X and Y. The immortalized cell lines obtained by forced expression of the SV40 large T-antigen were capable of proliferation at high rate in long-term culture. Immortalization and long-term culture did not cause cytogenetically observable abnormality in the karyotype. The cell type of the primary cultures and immortalized cell lines were further characterized as fibroblasts by the specific expression of vimentin. Gene transfer experiments showed that exogenetic genes could be efficiently delivered into the cells by both plasmid transfection and lentivirus infection. The cells derived from the skin of the Indo-Pacific humpback dolphin may serve as a useful in vitro system for studies on the effects of environmental pollutants and pathogens in habitats on the dolphin animals. More importantly, because of their high proliferation rate and susceptibility to lentivirus, these cells are potential ideal materials for generation of induced pluripotent stem cells.     

Novel cytotoxic exhibition mode of antimicrobial peptide anoplin in MEL cells,the cell line of murine Friend leukemia virus‐induced leukemic cells

Anoplin is a recently discovered antimicrobial peptide (AMP) isolated from the venom sac of the spider wasp Anoplius samariensis, and it is one of the shortest α‐helical AMP found naturally to date consisting of only ten amino acids. Previous results showed that anoplin exhibits potent antimicrobial activity but little hemolytic activity. In this study, we synthesized anoplin, studied its cytotoxicity in Friend virus‐induced leukemia cells [murine erythroleukemia (MEL) cells], and proposed its possible mechanism. Our results showed that anoplin could inhibit the proliferation of MEL cells in a dose‐dependent and time‐dependent manner via disrupting the integrity of cell membrane, which indicated that anoplin exerts its cytotoxicity efficacy. In addition, the cell cycle distribution of MEL cells was arrested in the G₀/G₁ phase significantly. However, anoplin could not induce obvious apoptosis in MEL cells, as well as anoplin could not induce visible changes on morphology and quantity in the bone marrow cells isolated from normal mice. All of these results indicate that anoplin, as generally believed, is a selective AMP, a value characteristic in the design of safe therapeutic agents. The cytotoxicity of anoplin on MEL cells was mainly attributable to the plasma membrane perturbation and also to the intracellular events such as the arrest of cell cycle. Although this is an initial study that explored the activity of anoplin in vitro rather than in vivo, with the increasing resistance of conventional chemotherapy, there is no doubt that anoplin has desirable feature to be developed as a novel and selective anticancer agent. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesisof fructose laurate esters catalyzed by a CALB-displaying Pichia pastoris whole-cell biocatalyst in a non-aqueous system

Earlier studies on fructose laurate ester products have shown that recombinant Pichia pastoris displaying Candida antarctica lipase B (CALB) on the cell surface acts as an efficient whole-cell biocatalyst for sugar ester production from fructose and lauric acid in an organic solvent. The effects of various reaction factors, including solvent composition, substrate molar ratio, enzyme dose, temperature and water activity, on esterification catalyzed by the CALB-displaying P. pastoris whole-cell biocatalyst were examined in the present study. Under the preferred reaction conditions, specifically, 5 mL organic solvent mixture of 2-methyl-2-butanol/DMSO (20% v/v), 2 mmol fructose with a lauric acid to fructose molar ratio of 2:1, 0.3 g whole-cell biocatalyst (1,264 U/g dry cell) with an initial water activity of 0.11, 1.2 g 4Å molecular sieve, reaction temperature of 55oC and 200 rpm stirring speed, the fructose mono laurate ester yield was 78% (w/w). The CALBdisplaying P. pastoris whole-cell biocatalyst exhibited good operational stability, with an evident increase, rather than decrease, in relative activity after the continuous recover and reuse cycle. The relative activity of the biocatalyst remained 50% higher than that of the first batch, even following reuse for 15 batches. Our results collectively indicate that the CALB-displaying P. pastoris whole-cell biocatalyst may be potentially utilized in lieu of free or immobilized enzyme to effectively produce non-ionic surfactants such as fatty acid sugar esters, offering the significant advantages of cost-effectiveness, good operational stability and mild reaction conditions.

     

The anti-influenza virus effect of Phellinus igniarius extract

Herbal medicine has been used in the orient for thousands of years to treat large and small ailments, including microbial infections. Although there are treatments for influenza virus infection, there is no treatment for drug-resistant viruses. It is time that we explored and exploited the multi-component nature of herbal extracts as multi-drug combination therapies. Here, we present data on the anti-influenza virus effect of a medicinal mushroom, Phellinus igniarius. The P. igniarius water extract was effective against influenza A and B viruses, including 2009 pandemic H1N1, human H3N2, avian H9N2, and oseltamivir-resistant H1N1 viruses. Virological assays revealed that the extract may interfere with one or more early events in the influenza virus replication cycle, including viral attachment to the target cell. Therefore, our results provide new insights into the use of P. igniarius as an anti-influenza medicine.     

The effect of dietary intake of the acidic protein fraction of bovine colostrum on influenza A (H1N1) virus infection

Acidic protein levels in the milk decrease markedly as lactation progresses, suggesting that it is an important part of the colostrum. However, little attention has been paid to their biological function. In this study, we isolated the acidic protein fraction of bovine colostrum (AFC, isoelectric point <5) by anion-exchange chromatography, and investigated the effect of its dietary intake on influenza A (H1N1) virus infection. 100% of mice infected with 1 LD₅₀ of the virus survived when administered AFC for 14 days prior to infection, compared with 33% survival when administered phosphate buffered saline (PBS). Moreover, consumption of AFC reduced the weight loss associated with infection. We propose that dietary intake of AFC has a prophylactic effect on influenza A virus infection.     

Mathematical model for simultaneous nitrification and denitrification (SND) in membrane bioreactor (MBR) under Low Dissolved Oxygen (DO) concentrations

Activated Sludge Model no. 1 (ASM1) was modified and applied to Simultaneous Nitrification and Denitrification (SND) in oxygen-limited MBR. In order to calibrate the model correctly, the parametric sensitivity was performed using AQUASIM 2.0 to find the most important coefficients. The most sensitive coefficients in the model of oxygen-limited MBR were related to the growth of heterotrophic biomass. While the total autotrophic biomass concentration (X_BA) was decreased by decreasing DO concentration, there was an increase in the nitrite-oxidizing biomass concentration by a small amount. This model also showed that over 97% of permeate Soluble Chemical Oxygen Demand (S_COD) was the Soluble Inert (S_I). The model showed the change in the ammonia-oxidizing and nitrite-oxidizing biomass was decreased by decreasing DO concentration. However, there was an increase in the nitrite-oxidizing biomass concentration by a small amount due to the biomass retained in the bioreactor with membrane. It is contradictory to the reported observations for conventional activated sludge process.     

Molecular characterization of methicillin-resistant Panton-valentine leukocidin positive <Emphasis Type="Italic">staphylococcus aureus</Emphasis> clones disseminating in Tunisian hospitals and in the community

Background

The spread of MRSA strains at hospitals as well as in the community are of great concern worldwide. We characterized the MRSA clones isolated at Tunisian hospitals and in the community by comparing them to those isolated in other countries.

Results

We characterized 69 MRSA strains isolated from two Tunisian university hospitals between the years 2004-2008. Twenty-two of 28 (79%) community-associated MRSA (CA-MRSA) strains and 21 of 41 (51%) healthcare-associated MRSA (HA-MRSA) strains were PVL-positive. The PVL-positive strains belonged to predicted founder group (FG) 80 in MLST and carried either type IVc SCCmec or nontypeable SCCmec that harbours the class B mec gene complex. In contrast, very diverse clones were identified in PVL-negative strains: three FGs (5, 15, and 22) for HA-MRSA strains and four FGs (5, 15, 45, and 80) for CA-MRSA strains; and these strains carried the SCCmec element of either type I, III, IVc or was nontypeable. The nucleotide sequencing of phi7401PVL lysogenized in a CA-MRSA strain JCSC7401, revealed that the phage was highly homologous to phiSA2mw, with nucleotide identities of more than 95%. Furthermore, all PVL positive strains were found to carry the same PVL phage, since these strains were positive in two PCR studies, identifying gene linkage between lukS and mtp (major tail protein) and the lysogeny region, both of which are in common with phi7401PVL and phiSa2mw.

Conclusions

Our experiments suggest that FG80 S. aureus strains have changed to be more virulent by acquiring phi7401PVL, and to be resistant to β-lactams by acquiring SCCmec elements. These novel clones might have disseminated in the Tunisian community as well as at the Tunisian hospitals by taking over existing MRSA clones.

     

Knockout of 5‐lipoxygenase prevents dexamethasone‐induced tau pathology in 3xTg mice

Emerging evidence suggests that dysregulation stress hormones, such as glucocorticoids, in aged persons put them at a higher risk to develop Alzheimer's disease (AD). However, the mechanisms underlying such vulnerability remain to be unraveled. Pharmacologic inhibition of 5‐lipoxygenase (5LO), an active player in AD pathogenesis whose protein level increases with aging in the human, has been shown to blunt glucocorticoid‐mediated amyloid β (Ab) formation in vitro. In this article, we investigated the role of this pathway in modulating the development of the corticosteroid‐dependent AD‐like phenotype in the triple transgenic mice (3xTg). Dexamethasone was administered for 1 week to 3xTg or 3xTg genetically deficient for 5LO (3xTg/5LO^?/?) mice, and its effect on memory, amyloid‐β and tau levels, and metabolism assessed. At the end of the treatment, we observed that dexamethasone did not induce changes in behavior. Compared with controls, treated mice did not show significant alterations in brain soluble Aβ levels. While total tau protein levels were unmodified in all groups, we found that dexamethasone significantly increased tau phosphorylation at S396, as recognized by the antibody PHF‐13, which was specifically associated with an increase in the GSK3β activity. Additionally, dexamethasone‐treated mice had a significant increase in the tau insoluble fraction and reduction in the postsynaptic protein PDS‐95. By contrast, these modifications were blunted in the 3xTg/5LO^?/? mice. Our findings highlight the functional role that 5LO plays in stress‐induced AD tau pathology and support the hypothesis that pharmacologic inhibition of this enzyme could be a useful tool for individuals with this risk factor.