Characterisation of lepidopteran-active Bacillus thuringiensis isolates recovered from infected larvae

Abstract

As a part of an ongoing nationwide programme focused on finding novel strains of Bacillus thuringiensis (Bt) that are toxic to some of the major pests that impact economically important crops, we initiated a search for Bt isolates native to Syria. We succeeded in assembling a collection of 40 Bt isolates recovered from infected larvae of Galleria mellonella, Helicoverpa armigera and Ephestia kuehniella. Light microscopy showed that all isolates produce bipyramidal and cuboidal crystal proteins. The 50% lethal concentration of the spore-crystal mixture of the 40 isolates against E. kuehniella larvae varied from 3 to more than 200 µg g^?1. A comparison of the LC₅₀ values of the tested isolates with the reference strain Bt kurstaki HD-1 (20.55 µg g^?1), showed that some of these isolates have a similar or up to six times higher toxicity potential. PCR screening revealed that all obtained isolates contain cry1 and cry2 genes, whereas only four contain cry9. Moreover, the proteins of 130 and 65/70 Kda encoded by these genes were detected in the SDS-PAGE of the purified parasporal bodies. Flagellar serotyping classified 30 as serovar kurstaki, six isolates serovar aizawai, one isolate cross-reacted with more than one H3 antisera and three were not typeable. Assays of toxicity of the aizawai isolates against third instar of G. mellonella showed that four, which contain cry9, have almost similar toxicity to the commercial strain Bt aizawai B401. Therefore, these isolates could be adopted for future applications to control G. mellonella. Moreover, this study contributes to our knowledge of Bt diversity in Syria where to date very few collections have been described.     

Omi, a recessive mutation on chromosome 10, is a novel allele of Ostm1

Large-scale N-ethyl-N-nitrosourea (ENU) mutagenesis has provided many rodent models for human disease. Here we describe the initial characterization and mapping of a recessive mutation that leads to degeneration of the incisors, failure of molars to erupt, a grey coat colour, and mild osteopetrosis. We mapped the omi mutation to chromosome 10 between D10Mit214 and D10Mit194. The Ostm1 gene is a likely candidate gene in this region and the grey-lethal allele, Ostm1 ^gl , and omi mutations fail to complement each other. We show that om/om mice have reduced levels of Ostm1 protein. To date we have not been able to identify the causative mutation. We propose that omi is a novel hypomorphic mutation affecting Ostm1 expression, potentially in a regulatory element.     

Study of Microflora in Malaysian Dried Fishes and Their Decontamination by Gamma-irradiation

The distribution of microorganisms in 10 samples of salted dried fish and the effects of irradiation of them were studied. The total aerobic bacteria in commercial dried fish were determined to be from 2 × 10⁴ to 3 × 10⁶ per gram. Mold counts were 1 × 10² to 7 × 10³ per gram with a lower amount of yeasts. In spoiled dried fish, total aerobic bacteria were determined to be 4× 10⁶ or 1 × 10⁷ per gram with a few yeasts. Coliforms were not isolated on MacConkey agar plates from any of the samples. The predominant bacteria occurring in spoiled dried fish were Pediococcus halophilus, Vibrio costicola and Planococcus sp. More than 50% of the molds consisted of the Aspergillus niger group, whereas lower amounts of the A. flavus, A. fumigatus and A. ochraceus groups, Penicillium chrysogenum series, etc. were also isolated from many samples of dried fish. All kinds of putrefactive microorganisms were radiation sensitive, and a dose of ca. 500 krad appears to be sufficient for extension of the shelf-life of dried fish from 2 to 4 times.     

Chitosan-Nanoconjugated Hormone Nanoparticles for Sustained Surge of Gonadotropins and Enhanced Reproductive Output in Female Fish

A controlled release delivery system helps to overcome the problem of short life of the leutinizing hormone releasing hormone (LHRH) in blood and avoids use of multiple injections to enhance reproductive efficacy. Chitosan- and chitosan-gold nanoconjugates of salmon LHRH of desired size, dispersity and zeta potential were synthesized and evaluated at half the dose rate against full dose of bare LHRH for their reproductive efficacy in the female fish, Cyprinus carpio. Whereas injections of both the nanoconjugates induced controlled and sustained surge of the hormones with peak (P<0.01) at 24 hrs, surge due to bare LHRH reached its peak at 7 hrs and either remained at plateau or sharply declined thereafter. While the percentage of relative total eggs produced by fish were 130 and 67 per cent higher, that of fertilised eggs were 171 and 88 per cent higher on chitosan- and chitosan-gold nanoconjugates than bare LHRH. Chitosan nanoconjugates had a 13 per cent higher and chitosan gold preparation had a 9 per cent higher fertilization rate than bare LHRH. Histology of the ovaries also attested the pronounced effect of nanoparticles on reproductive output. This is the first report on use of chitosan-conjugated nanodelivery of gonadotropic hormone in fish.     

<Emphasis Type="Italic">Bruguiera hainesii</Emphasis>, a critically endangered mangrove species,is a hybrid between <Emphasis Type="Italic">B. cylindrica</Emphasis> and <Emphasis Type="Italic">B. gymnorhiza</Emphasis> (Rhizophoraceae)

Bruguiera hainesii (Rhizophoraceae) is one of the two Critically Endangered mangrove species listed in the IUCN Red List of Threatened Species. Although the species is vulnerable to extinction, its genetic diversity and the evolutionary relationships with other Bruguiera species are not well understood. Also, intermediate morphological characters imply that the species might be of hybrid origin. To clarify the genetic relationship between B. hainesii and other Bruguiera species, we conducted molecular analyses including all six Bruguiera species using DNA sequences of two nuclear genes (CesA and UNK) and three chloroplast regions (intergenic spacer regions of trnL-trnF, trnS-trnG and atpB-rbcL). For nuclear DNA markers, all nine B. hainesii samples from five populations were heterozygous at both loci, with one allele was shared with B. cylindrica, and the other with B. gymnorhiza. For chloroplast DNA markers, the two haplotypes found in B. hainesii were shared only by B. cylindrica. These results suggested that B. hainesii is a hybrid between B. cylindrica as the maternal parent and B. gymnorhiza as the paternal one. Furthermore, chloroplast DNA haplotypes found in B. hainesii suggest that hybridization has occurred independently in regions where the distribution ranges of the parental species meet. As the IUCN Red List of Threatened Species currently excludes hybrids (except for apomictic plant hybrids), the conservation status of B. hainesii should be reconsidered.     

Interleukin 6 and/or Interleukin 17A Modulate the OPG/RANKL System of MC3T3-E1 Murine Osteoblast Cell Line

Cytokines such as interleukin-6 (IL-6) and IL-17 which act as key regulators of the immune response have been identified to have a potential role in the bone remodeling mechanism. Receptor activator of NF-κB ligand (RANKL) has been shown to regulate osteoclast differentiation and function while the osteoprotegerin (OPG) blocks the binding of RANKL and inhibits the differentiation of osteoclasts, thus favoring osteogenesis. Alkaline phosphatase (ALP) on the other hand works as early mineralization indicator in bone regulation. The current study aims to determine the potential role of IL-6 and IL-17A in regulating the OPG/RANKL system of the murine osteoblast cell line (MC3T3-E1). Gene expression analysis showed significant up-regulation of OPG and ALP by all the treated groups (rIL-6, rIL-17A and rIL-6 + rIL-17A). In contrast, treatment of cells with rIL-6 and/or rIL-17A showed down-regulation of RANKL expression. Interestingly, the osteoblast cells treated with combinations of rIL-6 + rIL17A showed marked increased in OPG/RANKL ratio. Similar pattern of protein expression was observed in the osteoblasts treated with rIL-6 and/or rIL-17A as detected by western blotting and ELISA. These findings suggest a new mechanism of regulation by these cytokines on the expression of OPG and RANKL, which could promote osteogenesis and diminish osteoclastogenesis.     

Production of cyclodextrin glycosyltransferase by immobilized <Emphasis Type="Italic">Bacillus</Emphasis> sp. on chitosan matrix

The whole-cell immobilization on chitosan matrix was evaluated. Bacillus sp., as producer of CGTase, was grown in solid-state and batch cultivation using three types of starches (cassava, potato and cornstarch). Biomass growth and substrate consumption were assessed by flow cytometry and modified phenol–sulfuric acid assays, respectively. Qualitative analysis of CGTase production was determined by colorless area formation on solid culture containing phenolphthalein. Scanning electron microscopy (SEM) analysis demonstrated that bacterial cells were immobilized on chitosan matrix efficiently. Free cells reached very high numbers during batch culture while immobilized cells maintained initial inoculum concentration. The maximum enzyme activity achieved by free cells was 58.15 U ml^?1 (36 h), 47.50 U ml^?1 (36 h) and 68.36 U ml^?1 (36 h) on cassava, potato and cornstarch, respectively. CGTase activities for immobilized cells were 82.15 U ml^?1 (18 h) on cassava, 79.17 U ml^?1 (12 h) on potato and 55.37 U ml^?1 (in 6 h and max 77.75 U ml^?1 in 36 h) on cornstarch. Application of immobilization technique increased CGTase activity significantly. The immobilized cells produced CGTase with higher activity in a shorter fermentation time comparing to free cells.     

Secretome analysis of virulent Pyrenophora teres f. teres isolates

Pyrenophora teres f. teres (Ptt) causes net form net blotch disease of barley, partially by producing necrosis‐inducing proteins. The protein profiles of the culture filtrates of 28 virulent isolates were compared by a combination of 2DE and 1D‐PAGE with 105 spots and 51 bands chosen for analysis by liquid chromatography electrospray ionization tandem mass spectrometry. A total of 259 individual proteins were identified with 63 of these proteins being common to the selected virulent isolates. Ptt secretes a broad spectrum of proteins including cell wall degrading enzymes; virulence factors and effectors; proteins associated with fungal pathogenesis and development; and proteins related to oxidation–reduction processes. Potential virulence factors and effectors identified included proteins with glucosidase activity, ricin B and concanavalin A‐like lectins, glucanases, spherulin, cutinase, pectin lyase, leucine‐rich repeat protein, and ceratoplatanin. Small proteins with unknown function but cysteine‐rich, common to effectors, were also identified. Differences in the secretion profile of the Ptt isolates have also provided important insight into the different mechanisms contributing to virulence and the development of net form net blotch symptoms.     

High Coke-Resistance Pt/Mg1-xNixO Catalyst for Dry Reforming of Methane

A highly active and stable nano structured Pt/Mg_1-xNi_xO catalysts was developed by a simple co-precipitation method. The obtained Pt/Mg_1-xNi_xO catalyst exhibited cubic structure nanocatalyst with a size of 50–80 nm and realized CH₄ and CO₂ conversions as high as 98% at 900°C with excellent stability in the dry reforming of methane. The characterization of catalyst was performed using various kinds of analytical techniques including XRD, BET, XRF, TPR-H₂, TGA, TEM, FESEM, FT-IR, and XPS analyses. Characterization of spent catalyst further confirms that Pt/Mg_1-xNi_xO catalyst has high coke-resistance for dry reforming. Thus, the catalyst demonstrated in this study, offers a promising catalyst for resolving the dilemma between dispersion and reducibility of supported metal, as well as activity and stability during high temperature reactions.     

Unmasking Heavily O-Glycosylated Serum Proteins Using Perchloric Acid: Identification of Serum Proteoglycan 4 and Protease C1 Inhibitor as Molecular Indicators for Screening of Breast Cancer

Heavily glycosylated mucin glycopeptides such as CA 27.29 and CA 15–3 are currently being used as biomarkers for detection and monitoring of breast cancer. However, they are not well detected at the early stages of the cancer. In the present study, perchloric acid (PCA) was used to enhance detection of mucin-type O-glycosylated proteins in the serum in an attempt to identify new biomarkers for early stage breast cancer. Sensitivity and specificity of an earlier developed sandwich enzyme-linked lectin assay were significantly improved with the use of serum PCA isolates. When a pilot case-control study was performed using the serum PCA isolates of normal participants (n = 105) and patients with stage 0 (n = 31) and stage I (n = 48) breast cancer, higher levels of total O-glycosylated proteins in sera of both groups of early stage breast cancer patients compared to the normal control women were demonstrated. Further analysis by gel-based proteomics detected significant inverse altered abundance of proteoglycan 4 and plasma protease C1 inhibitor in both the early stages of breast cancer patients compared to the controls. Our data suggests that the ratio of serum proteoglycan 4 to protease C1 inhibitor may be used for screening of early breast cancer although this requires further validation in clinically representative populations.