Gene profiling and characterization of arginine kinase-1 (MrAK-1) from freshwater giant prawn (Macrobrachium rosenbergii)

Arginine kinase-1 (MrAK-1) was sequenced from the freshwater prawn Macrobrachium rosenbergii using Illumina Solexa Genome Analyzer Technique. MrAK-1 consisted of 1068 bp nucleotide encoded 355 polypeptide with an estimated molecular mass of 40 kDa. MrAK-1 sequence contains a potential ATP:guanido phosphotransferases active domain site. The deduced amino acid sequence of MrAK-1 was compared with other 7 homologous arginine kinase (AK) and showed the highest identity (96%) with AK-1 from cherry shrimp Neocaridina denticulate. The qRT-PCR analysis revealed a broad expression of MrAK-1 with the highest expression in the muscle and the lowest in the eyestalk. The expression of MrAK-1 after challenge with the infectious hypodermal and hematopoietic necrosis virus (IHHNV) was tested in muscle. In addition, MrAK-1 was expressed in Escherichia coli by prokaryotic expression plasmid pMAL-c2x. The optimum temperature (30 °C) and pH (8.5) was determined for the enzyme activity assay. MrAK-1 showed significant (P < 0.05) activity towards 10-50 mM ATP concentration. The enzyme activity was inhibited by α-ketoglutarate, glucose and ATP at the concentration of 10, 50 and 100 mM respectively. Conclusively, the findings of this study indicated that MrAK-1 might play an important role in the coupling of energy production and utilization and the immune response in shrimps.     

Leishmania major: Disruption of signal peptidase type I and its consequences on survival, growth and infectivity

Leishmania major (L. major) signal peptidase type I (SPase I) is an endopeptidase encoded by a single-copy gene. In all organisms, SPase I is responsible for removing the signal peptide from secretory pre-proteins and releasing mature proteins to cellular or extra-cellular space. In this study, the role of SPase I in L. major is investigated by gene deletion using homologous recombination (HR). The null mutant of SPase I was not possible to create, suggesting that SPase I is an essential gene for parasite survival.The obtained heterozygote mutant by disrupting one allele of SPase I in L. major showed significantly reduced level of infectivity in bone marrow-derived macrophages. In addition, the heterozygote mutants are unable to cause cutaneous lesion in susceptible BALB/c mice. This is the first report showing that SPase I may have an important role in Leishmania infectivity, e.g. in differentiation and survival of amastigotes. Apparently, the SPase I expression is not essential for in vitro growth of the parasite.     

Leishmania infantum: prime boost vaccination with C-terminal extension of cysteine proteinase type I displays both type 1 and 2 immune signatures in BALB/c mice

The aims of current study are to describe the immunogenicity and protective efficacy of prime boost vaccine using C-terminal extension (CTE) of cysteine proteinase type I of Leishmania infantum in BALB/c mice. Group I as vaccinated group primed with 100 microg of pcDNA-CTE and 3 weeks later boosted with combination of 30 microg rCTE, 50 microg of CpG and Montanide 720. Groups II and III were served as control groups. Although, this vaccination regimen did not protect mice against the infectious challenge but it was highly immunogenic. IgG2a has been raised strongly against rCTE in contrast to IgG1 and remains high at every time point under study. By analysis of CTE synthetic peptides (CTE100) before challenge, both IgG1 and IgG2a were produced and for all overlapping synthetic peptides (CTE 1-8) IgG1 raised significantly. This statue is changed at 7 weeks after challenge and only CTE2 and CTE3 have shown to induce considerable amount of IgG1. In all groups, the level of IL-5 started to increase with high concentration shortly passing only 3 weeks after infectious challenge. In compare with two control groups, the vaccinated group produced significantly higher level of IL-5 at 7 weeks post-infection. The parasite burden of all groups is similar at 4 weeks post-challenge in both liver and spleen. In contrast, at 8 weeks post challenge, the spleen of the vaccinated group showed significantly higher level of parasite load in compare with two control groups. This study demonstrated that immunization with CTE display both type 1 and 2 immune signatures in experimental murine model of L. infantum infection.     

Comparison of Glutathione S-transferase Activity and Concentration in Aflatoxin-Producing and their Non-Toxigenic Counterpart Isolates

In this study, two techniques were used to compare the specific activity and total concentration of mycelial glutathione S-transferase (GST) in fungal strains isolated from natural sources. The fungi identified as Aspergillus parasiticus and Aspergillus flavus have been divided into two groups based on their ability to produce aflatoxins. Altogether 26 fungi were isolated, among which 12 were capable of producing varying levels of aflatoxin and 14 were proved to be non-toxigenic. GST specific activity in mycelial preparation was measured spectrophotometrically using 2,1-chloro-2,4-dinitrobenzene as the substrate. The results showed that the mean GST activity in toxigenic isolates was 25.06 +/- 9.8 mumol/mg protein/min which was 2.8-fold greater than that measured in non-toxigenic isolates (8.84 +/- 5.5 mumol/mg protein/min). Moreover, the GST concentration was compared in toxigenic and non-toxigenic isolates using an Enzyme Linked Immunosorbent Assay based on antigen (fungal preparation) and antibody (antibody produced against fungal GST in rabbit). The results of ELISA showed that the mean GST level in toxigenic and non-toxigenic fungi was 1.17 +/- 0.55 and 0.40 +/- 0.24, respectively. These results further confirm that the aflatoxin production in the fungal strains is correlated with GST expression and using ELISA, it is possible to discriminate aflatoxin-producing fungi from their non-toxigenic counterparts.     

Host plant effects on the functional response of Stethorus gilvifrons to strawberry spider mites

The functional response of adult females of the coccinellid beetle Stethorus gilvifrons Mulsant to juveniles of strawberry spider mite, Tetranychus turkestani Ugarov & Nikolski was determined on cowpea, castor bean and cucumber leaves in the laboratory at 25°C and a 14 h L: 10 h D photoperiod. Beetles were isolated singly for 24 h in 9-cm Petri dishes with either 2, 4, 8, 16, 32, 64, or 128 nymphal stages of T. turkestani. Results showed a typical type II response on all plants tested, with up to 110.7, 100.8, and 53.0 prey attacked when 128 nymphal stages were provided on cowpea, castor bean, and cucumber leaves, respectively. Based on the Rogers random attack equation, the highest estimated attack rate and the lowest handling time were obtained on cowpea. It was therefore concluded that the host plant species can affect the predation rate and functional response components of S. gilvifrons, a specific and effective predator of spider mites.     

Heavy metals contamination and human health risk assessment in soils of an industrial area,Bandar Abbas – South Central Iran

The purpose of this study was to determine the contamination level, distribution, health risk and potential sources of Cr, Cd, Pb, Zn, Cu, Ni and As in 66 topsoil samples from industrial areas in Bandar Abbas County. The geoaccumulation index, pollution index and pollution load index were calculated to assess the pollution level in the industrial soils. The hazard index and carcinogenic risk were used to assess human health risk of heavy metals. Results showed that the contamination levels of heavy metals were in the descending order of Cu> Cd> Pb> Zn> As> Ni> Cr. Moreover, based on principal component analysis, Cd, Zn, Cu, and Pb originated mainly from anthropogenic sources, including power plants, oil and gas refinery, steel and zinc production factories and municipal waste landfills. For non-carcinogenic effects, hazard index of studied metals decreased in the order of Cr> As> Cd> Pb> Ni > Cu> Zn. Arsenic, chromium and cadmium were regarded as the priority pollutants. Carcinogenic risks due to Cd and As in suburban soils were within tolerable risk to human health; however, children faced more health risk in their daily life than adults via their unconscious ingestion and dermal contact pathway.     

Enhanced development of mouse single blastomeres into blastocysts via the simultaneous inhibition of TGF‐β and ERK pathways in microdroplet culture

Optimization of an in vitro culture that supports blastocyst (BL) development from single blastomeres (SBs) is essential to generate additional embryos for farm animals and humans and unravel the mechanisms that underlie totipotency. In this study, we have examined BL development from SBs that were derived from 2‐cell and 4‐cell mouse embryos in different media. Moreover, BLs were assessed for inner cell mass (ICM) by staining with Oct4. We found that BL development was improved in a lower volume of medium (1 µL) compared with a higher volume (5 µL). Furthermore, the supplementation of medium with the inhibitors of ERK1/2 and TGFβ (R2i) signaling pathways in 1 µL droplets of T6 medium improved BL development. The co‐culture of SBs with intact embryos in the presence of R2i showed more BL development and ICM to trophectoderm cell number ratio in comparison with SB culture and SB group culture. We also observed reduced total cell number, ICM, and trophectoderm cell numbers in all of the SB culture conditions versus intact embryo development. These findings might facilitate the successful generation of additional embryos for biomedical applications and elucidate the mechanisms that underlie totipotency.