Differential expression of human basic fibroblast growth factor in Escherichia coli: potential role of promoter

Four different expression systems were developed for expression of the cDNA encoding human basic fibroblast growth factor (hbFGF), using Escherichia coli as host organism. The hbfgf structural gene was cloned into four expression vectors, pET-3a, pTrc99A, pPR37 and pKK223-3 differing only in their promoters, which were T7, trc, PR and tac respectively. Expression of the gene was induced by adding 0.5 mM (final concentration) of isopropyl--D-thio-galactopyranoside (IPTG) for the vectors carrying T7, trc and tac promoters or by a temperature shift from 30 to 42 °C for the vector carrying PR. The highest level of expression was observed in pET-1005 (a pET-3a derivative)/BL21 (DE3) system with 18.5 mg/l rhbFGF and the second high level expression was in pR37-1007 (pPR37 derivative) BL21 (DE3) system with 5 mg of rhbFGF/l. Since in the latter system a temperature shift was used for induction, 29% of the hbFGF was recovered as inclusion bodies in the insoluble cell fraction. The level of expression for the two other systems (pTrc-1006 and pKK-1008) was very low.     

<Emphasis Type="Italic">Piriformospora indica</Emphasis> Alleviates Salinity by Boosting Redox Poise and Antioxidative Potential of Tomato

More than 20% of irrigated land has been influenced by salt stress, decreasing crop production. In this research, we investigated the effect of different levels of salinity (0, 50, 100 and 150 mM NaCl) and the efficiency of Piriformospora indica on growth, biochemical traits, antioxidative defense system in tomato (Solanum lycopersicum L.). NaCl stress reduced chlorophyll content, height and biomass of plants. Higher level of salinity (150 mM) declined the plant height by 22.65%, total dry weight by 56.44% and total chlorophyll by 44.34%, however, P. indica inoculation raised plant height by 43.47%, dry weight by 69.23% and total chlorophyll content by 48.09%. Salinity stress increased H₂O₂, malondialdehyde (MDA), superoxide anion and 1,1-diphenyl-2-picrylhydrazyl (DPPH) level in leaves and roots tomato seedlings. However, P. indica inoculation reduced H₂O₂, MDA and superoxide anion and enhanced DPPH compared to non-inoculated plants at all NaCl levels. The total phenol and flavonoids increased with NaCl treatment. On the other hand, the total phenolic and flavonoid increased more in P. indica inoculated plants compared to non-inoculated ones. Moreover, inoculation of P. indica implicated noteworthy improvement of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR), and glutathione reductase (GR) activity in tomato upon salinity. Notably, colonization with P. indica significantly improved the content of reduced ascorbic acid (AsA), glutathione (GSH) and redox ratio in the tomato plants under salinity resulting in reduced redox state. Our findings confirmed that salinity had negative effect on tomato seedling; however, P. indica inoculation increased tolerance to salinity by improving the content of phenolic compounds, non-enzymatic antioxidants, and increasing the activity of antioxidant enzymes.     

Climate warming alters subsoil but not topsoil carbon dynamics in alpine grassland

Subsoil contains more than half of soil organic carbon (SOC) globally and is conventionally assumed to be relatively unresponsive to warming compared to the topsoil. Here, we show substantial changes in carbon allocation and dynamics of the subsoil but not topsoil in the Qinghai‐Tibetan alpine grasslands over 5 years of warming. Specifically, warming enhanced the accumulation of newly synthesized (¹⁴C‐enriched) carbon in the subsoil slow‐cycling pool (silt‐clay fraction) but promoted the decomposition of plant‐derived lignin in the fast‐cycling pool (macroaggregates). These changes mirrored an accumulation of lipids and sugars at the expense of lignin in the warmed bulk subsoil, likely associated with shortened soil freezing period and a deepening root system. As warming is accompanied by deepening roots in a wide range of ecosystems, root‐driven accrual of slow‐cycling pool may represent an important and overlooked mechanism for a potential long‐term carbon sink at depth. Moreover, given the contrasting sensitivity of SOC dynamics at varied depths, warming studies focusing only on surface soils may vastly misrepresent shifts in ecosystem carbon storage under climate change.     

Salt stress effects on growth,pigments, proteins and lipid peroxidation in <Emphasis Type="Italic">Salicornia persica</Emphasis> and <Emphasis Type="Italic">S. europaea</Emphasis>

The effects of NaCl stress on growth, water status, contents of protein, proline, malondialdehyde (MDA), various sugars and photosynthetic pigments were investigated in seedlings of Salicornia persica and S. europaea grown in vitro. Seeds were germinated under NaCl (0, 100, 200, 300, 400, 500 and 600 mM) on Murashige and Skoog medium for 45 d. The shoot growth of both species increased under low NaCl concentration (100 mM) and then decreased with increasing NaCl concentrations. In contrast to S. persica, root length in S. europaea reduced steadily with an increase in salinity. Proline content in S. persica was higher than in S. europaea at most NaCl concentrations. Proline, reducing saccharide, oligosaccharide and soluble saccharide contents increased under salinity in both species. In contrast, contents of proteins and polysaccharides reduced in both species under salt stress. MDA content remained close to control at moderate NaCl concentrations (100 and 200 mM) and increased at higher salinities. MDA content in S. europaea was significantly higher than S. persica at higher salinities. Salt treatments decreased K⁺ and P contents in seedlings of both species. Significant reduction in contents of chlorophylls and carotenoids due to NaCl stress was also observed in seedlings of both species. Some differences appeared between S. persica and S. europaea concerning proteins profile. On the basis of the data obtained, S. persica is more salt-tolerant than S. europaea.     

Measurement of opsonophagocytic activity of antibodies specific toNeisseria meningitidis serogroup A capsular polysaccharide-serogroup B outer membrane vesicle conjugate in animal model

Neisseria meningitidis is efficiently phagocytosed by polymorphonuclear leukocytes (PMNS) following opsonization with opsonic antibodies; opsonophagocytosis is the primary mechanism for clearance of meningococci from the host. Thus, in testing meningococcal vaccines, the level of opsonophagocytic antibodies appears to correlate with vaccine-induced protection. Our previous studies demonstrated that the conjugation ofN. meningitidis serogroup A capsular polysaccharide (CPSA) to serogroup B outer membrane vesicle (OMV) could induce a high level of bactericidal antibody response against serogroup A meningococci in animals. The purpose of this study was to evaluate opsonophagocytic activity of the conjugate of CPSA to OMV (CPSA-OMV). In order to evaluate the potential efficacy of CPSA-OMV a flow cytometric opsonophagocytic assay was used. The conjugate and controls were injected intramuscularly into four groups of rabbits with boosters on days 14, 28 and 42 following primary immunization. The rabbits were bled prior to injection and two weeks after each injection. Opsonophagocytic activity of antibodies in hyperimmune sera through rabbit PMNS were measured with flow cytometer, using dihydrorhodamine-123 as a probe. The results indicated that our conjugate could induce a highly significant level of opsonophagocytic activity against serogroup A meningococci after 56 days compared to the control groups (P<0.05). We conclude that this conjugate represents a vaccine candidate against serogroups A and B meningococci after further investigation.     

Fermentation of pomegranate juice by probiotic lactic acid bacteria

In this research, production of probiotic pomegranate juice through its fermentation by four strains of lactic acid bacteria: Lactobacillus plantarum, L. delbruekii, L. paracasei, L. acidophilus was examined. Fermentation was carried out at 30°C for 72 h under microaerophilic conditions. Microbial population, pH, titrable acidity, sugar and organic acid metabolism were measured during the fermentation period and the viability of all strains was also determined during the storage time at 4°C within 4 weeks. The results indicated that L. plantarum and L. delbruekii increased the pH sharply at the initial stages of fermentation and the sugar consumption was also higher in comparison with other strains, better microbial growth was also observed for these two strains during fermentation. Citric acid, as a major organic acid in pomegranate juice was significantly consumed by all probiotic lactic acid bacteria. L. plantarum and L. delbruekii showed higher viability during the storage time. Viable cells remained at their maximum level within 2 weeks but decreased dramatically after 4 weeks. Pomegranate juice was proved to be a suitable media for production of a fermented probiotic drink.     

Immunogenicity Evaluation of a Rationally Designed Polytope Construct Encoding HLA-A*0201 Restricted Epitopes Derived from Leishmania major Related Proteins in HLA-A2/DR1 Transgenic Mice: Steps toward Polytope Vaccine

Background

There are several reports demonstrating the role of CD8 T cells against Leishmania species. Therefore peptide vaccine might represent an effective approach to control the infection. We developed a rational polytope-DNA construct encoding immunogenic HLA-A2 restricted peptides and validated the processing and presentation of encoded epitopes in a preclinical mouse model humanized for the MHC-class-I and II.

Methods and Findings

HLA-A*0201 restricted epitopes from LPG-3, LmSTI-1, CPB and CPC along with H-2Kd restricted peptides, were lined-up together as a polytope string in a DNA construct. Polytope string was rationally designed by harnessing advantages of ubiquitin, spacers and HLA-DR restricted Th1 epitope. Endotoxin free pcDNA plasmid expressing the polytope was inoculated into humanized HLA-DRB1*0101/HLA-A*0201 transgenic mice intramuscularly 4 days after Cardiotoxin priming followed by 2 boosters at one week interval. Mice were sacrificed 10 days after the last booster, and splenocytes were subjected to ex-vivo and in-vitro evaluation of specific IFN-γ production and in-vitro cytotoxicity against individual peptides by ELISpot and standard chromium-51(⁵¹Cr) release assay respectively. 4 H-2Kd and 5 HLA-A*0201 restricted peptides were able to induce specific CD8 T cell responses in BALB/C and HLA-A2/DR1 mice respectively. IFN-γ and cytolytic activity together discriminated LPG-3-P1 as dominant, LmSTI-1-P3 and LmSTI-1-P6 as subdominant with both cytolytic activity and IFN-γ production, LmSTI-1-P4 and LPG-3-P5 as subdominant with only IFN-γ production potential.

Conclusions

Here we described a new DNA-polytope construct for Leishmania vaccination encompassing immunogenic HLA-A2 restricted peptides. Immunogenicity evaluation in HLA-transgenic model confirmed CD8 T cell induction with expected affinities and avidities showing almost efficient processing and presentation of the peptides in relevant preclinical model. Further evaluation will determine the efficacy of this polytope construct protecting against infectious challenge of Leishmania. Fortunately HLA transgenic mice are promising preclinical models helping to speed up immunogenicity analysis in a human related mouse model.     

TRPV1+ sensory neurons control beta cell stress and islet inflammation in autoimmune diabetes

In type 1 diabetes, T cell-mediated death of pancreatic beta cells produces insulin deficiency. However, what attracts or restricts broadly autoreactive lymphocyte pools to the pancreas remains unclear. We report that TRPV1(+) pancreatic sensory neurons control islet inflammation and insulin resistance. Eliminating these neurons in diabetes-prone NOD mice prevents insulitis and diabetes, despite systemic persistence of pathogenic T cell pools. Insulin resistance and beta cell stress of prediabetic NOD mice are prevented when TRPV1(+) neurons are eliminated. TRPV1(NOD), localized to the Idd4.1 diabetes-risk locus, is a hypofunctional mutant, mediating depressed neurogenic inflammation. Delivering the neuropeptide substance P by intra-arterial injection into the NOD pancreas reverses abnormal insulin resistance, insulitis, and diabetes for weeks. Concordantly, insulin sensitivity is enhanced in trpv1(-/-) mice, whereas insulitis/diabetes-resistant NODxB6Idd4-congenic mice, carrying wild-type TRPV1, show restored TRPV1 function and insulin sensitivity. Our data uncover a fundamental role for insulin-responsive TRPV1(+) sensory neurons in beta cell function and diabetes pathoetiology.     

Production of an extracellular alkaline metalloprotease from a newly isolated, moderately halophile, Salinivibrio sp. strain AF-2004

An extracellular protease was produced under stress conditions of high temperature and high salinity by a newly isolated moderate halophile, Salinivibrio sp. strain AF-2004 in a basal medium containing peptone, beef extract, glucose and NaCl. A modification of Kunitz method was used for protease assay. The isolate was capable of producing protease in the presence of sodium chloride, sodium sulfate, sodium nitrate, sodium nitrite, potassium chloride, sodium acetate and sodium citrate. The maximum protease was secreted in the presence of 7.5 to 10% (w/v) sodium sulfate or 3% (w/v) sodium acetate (4.6 U ml⁻¹). Various carbon sources including glucose, lactose, casein and peptone were capable of inducing enzyme production. The optimum pH, temperature and aeration for enzyme production were 9.0, 32 °C and 220 rpm, respectively. The enzyme production corresponded with growth and reached a maximum level during the mid-stationary phase. Maximum protease activity was exhibited in the medium containing 1% (w/v) NaCl at 60 °C, with 18% and 41% activity reductions at temperature 50 and 70 °C, respectively. The optimum pH for enzyme activity was 8.5, with 86% and 75% residual activities at pH 10 and 6, respectively. The activity of enzyme was inhibited by EDTA. These results suggest that the protease secreted by Salinivibrio sp. strain AF-2004 is industrially important from the perspectives of its activity at a broad pH ranges (5.0–10.0), its moderate thermoactivity in addition to its high tolerance to a wide range of salt concentration (0–10% NaCl).