Detection of Bacillus cereus virulence factors in commercial products of Bacillus thuringiensis and expression of diarrheal enterotoxins in a target insect

We examined isolates from 4 commercial bioinsecticides based on different strains of Bacillus thuringiensis subspecies (kurstaki, israelensis, aizawai, and tenebrionis) for the presence of genes encoding proteins with known enterotoxigenicity (nhe, hbl, cytk, ces) and various other putative virulence genes (piplc, sph, bceT, entFM, entS, entT). The piplc and bceT sequences were present in all the isolates; sph was found in aizawai and israelensis; entFM only in israelensis; and entS in kurstaki, israelensis, and tenebrionis. Our results corroborate previous findings that isolates used in commercial products contain all nhe and hbl component genes but not the ces gene. We ascertained that the cytK gene present in the kurstaki-, israelensis-, and aizawai-based products belongs to the cytK-2 type and not the more toxigenic cytK-1 variant originally isolated from enterotoxic Bacillus cereus. We provide the first evidence that hemolytic (hblA) and nonhemolytic (nheA, nheB, nheC) enterotoxin genes are expressed during septicemia in a target insect. This opens the door for their possible participation in pathogenesis in target insects. If enterotoxins do not contribute to bacterial pathogenesis in target insects, their genes could be deleted from commercial production strains to pre-empt perceptions of public health risks.     

New Techniques for the Estimation of Naturally Occurring Brassinosteroids

We have developed enzyme-linked immunosorbent assays (ELISAs) for measuring 24-epicastasterone and related brassinolide analogs, with detection ranges of 0.005 to 50 pmoles. Polyclonal antibodies used in these assays were raised against 24-epicastasterone carboxymethyloxime-bovine serum albumin conjugates and were found to have high specificity for 24-epibrassinosteroids. Natural brassinosteroids (BRs), such as brassinolide and 24-epibrassinolide, exhibited relatively high cross-reactivities with the generated antibodies, whereas other BR analogs with β-oriented hydroxyl groups at C-2, C-3, C-22, and C23 lacked immunoreactivity. Through the use of internal standardization, dilution assays, recovery of authentic [³H]24-epicastasterone, and immunohistograms, the ELISAs have been shown to be applicable for estimating 24-epibrassinosteroid levels in crude plant extracts. To analyze brassinosteroids in tissues from young bean (Phaseolus vulgaris L., cv. Pinto), Daucus carota ssp.sativus plants and Arabidopsis thaliana L. Heynh. seedlings, and rape (Brassica napus L.) pollen, the extracts were fractionated by high performance liquid chromatography (HPLC) and the resulting fractions were analyzed by the ELISA method. Immunohistogram ELISA analysis of HPLC fractions indicated that major peaks of immunoreactivity co-chromatographed with the labeled and unlabeled 24-epibrassinolide. A highly sensitive electrospray ionization mass spectrometry (MS) technique (LOD: 50 fmol) was also developed and the results obtained by the HPLC-ELISA and HPLC-MS approaches were compared.     

Partial synchronization of cell division in root meristem induced by 5-aminouracil

5-aminouracil induces a partial synchronization of mitoses in barley, onion and garlic root tips. The highest degree of synchronization has been achieved in garlic where the mitotic index reached the value of about 36%, while in onion and barley the values equalled about 20%. The concentration causing the maximal synchronization in barley (400–750 ppm) was many times higher than in garlic (62.5 ppm) and onion (100 ppm). The occurrence of micronuclei was evaluated in garlic, under the conditions when synchronization was maximal. It was increased nearly tenfold as compared with the control.     

Molecular pathogen detection in biosolids with a focus on quantitative PCR using propidium monoazide for viable cell enumeration

Sewage sludge is the solid, organic material remaining after wastewater is treated and discharged from a wastewater treatment plant. Sludge is treated to stabilize the organic matter and reduce the amount of human pathogens. Once government regulations are met, including material quality standards (e.g., E. coli levels and heavy metal content) sludge is termed “biosolids”, which may be disposed of by land application according to regulations. Live-culture techniques have traditionally been used to enumerate select pathogens and/or indicator organisms to demonstrate compliance with regulatory requirements. However, these methods may result in underestimates of viable microorganisms due to several problems, including their inability to detect viable but non-culturable (VBNC) cells. Real-time quantitative polymerase chain reaction (qPCR) is currently under investigation as a fast, sensitive, and specific molecular tool for enumeration of pathogens in biosolids. Its main limitation is that it amplifies all target DNAs, including that from non-viable cells. This can be overcome by coupling qPCR with propidium monoazide (PMA), a microbial membrane-impermeant dye that binds to extracellular DNA and DNA in dead or membrane-compromised cells, inhibiting its amplification. PMA has successfully been used to monitor the presence of viable pathogens in several different matrices. In this review the use of PMA-qPCR is discussed as a suitable approach for viable microbial enumeration in biosolids. Recommendations for optimization of the method are made, with a focus on DNA extraction, dilution of sample turbidity, reagent concentration, and light exposure time.     

后路椎弓根螺钉内固定结合后外侧植骨融合治疗胸腰椎骨折的临床观察

目的：观察后路椎弓根螺钉内固定结合后外侧植骨融合治疗胸腰椎骨折的临床效果及安全性。方法：回顾性分析2010年6月-2012年4月在我院脊柱骨科住院治疗的71例胸腰脊椎骨折患者,所有患者随机被分成2组,治疗组38例接受采用后路椎弓根螺钉内固定结合后外侧植骨融合治疗,对照组33例接受传统椎弓根螺钉内固定。术后对患者椎体前缘高度、脊柱Cobb’s角、腰背痛、神经功能恢复情况、内固定并发症等方面进行手术效果的评价。结果：治疗组术后及随诊患者的椎体前、后缘高度的比值与对照组相比,均有统计学差异（P〈0．05）;治疗组术后及随诊患者的Cobb’s角较对照组明显减小,差异有统计学意义（P〈0．05）,治疗组手术时间与对照组相比差异有统计学意义（P〈0．05）;两组患者医疗费用、神经功能改善筝级相互比较,无统计学意义咿0．05）。结论：后路复位椎弓根螺钉内固定结合后外侧植骨融合治疗胸腰椎骨折的临床疗效确切。     

Riparian populations of minnesota reed canarygrass (Phalaris arundinacea) are most likely native,based on SNPs (DArTseqLD)

The native vs. exotic status of reed canarygrass (RCG), a major invasive species of Minnesota wetlands, is unknown. The aim of this study was to investigate this native vs. exotic status to enhance its management. Genetic comparison of wild RCG populations from six Minnesota and six Czech Republic rivers was performed. A total of 2521 polymorphic SNP markers (single nucleotide polymorphisms) were used to evaluate 478 RCG samples across all collections. In the PCoA, all (n = 256) tested extant wild, riparian RCG genotypes from six Minnesota Rivers and six Czech Republic Rivers were genetically distinct, although some SNPs were common in both populations since they are the same species. DAPC analysis also resulted in the formation of two primary clusters separating the Minnesota Rivers and Czech Republic Rivers riparian samples, with little overlap; STRUCTURE analysis also supported this clustering with k = 4 groups as it separated the Czech Republic Rivers populations into three groups, along with Minnesota Rivers. The uniformity of PCoA, DAPC, STRUCTURE, and Evanno results indicates the distinct separation of Minnesota Rivers and Czech Republic Rivers populations. Portions of the genome (specific SNPs) are preserved or in common across continents, as indicated by STRUCTURE similarities. Nonetheless, overall significant SNP differences between the continents indicate that the Minnesota riparian populations are distinct enough from the European (Czech) collections to be delineated as native N. American RCG. PCoA of all the Minnesota RCG collections clustered Minnesota Rivers, Herbarium, Extant Herbarium, Research Field and Native Field collections together. STRUCTURE analysis (k = 2; Evanno) divided these Minnesota collections from the Commercial Field and Cultivars collections. There are two genetically distinct groups of RCG in Minnesota and since the Minnesota Rivers, the Research Field, the Native Field and pre-1930 herbaria collections clustered together, they are most likely native N. American types. Analysis of molecular variance (AMOVA) indicated that the genetic variation was more significant within, rather than among, the RCG populations. Native, historic herbaria types cluster together with all wild RCG river populations in Minnesota, all of which were distinct from those in Central Europe, suggesting native RCG type persistence in N. America. Also, cultivated forage types of RCG are distinct from wild RCG Minnesota river populations. The SNP genetic data shows that riparian Minnesota RCG populations are native. These data will facilitate future management strategies to control RCG as a native, but invasive, species.

     

Diversity of Microsporidia,Cryptosporidium and Giardia in Mountain Gorillas (Gorilla beringei beringei) in Volcanoes National Park,Rwanda

Background

Infectious diseases represent the greatest threats to endangered species, and transmission from humans to wildlife under increased anthropogenic pressure has been always stated as a major risk of habituation.

Aims

To evaluate the impact of close contact with humans on the occurrence of potentially zoonotic protists in great apes, one hundred mountain gorillas (Gorilla beringei beringei) from seven groups habituated either for tourism or for research in Volcanoes National Park, Rwanda were screened for the presence of microsporidia, Cryptosporidium spp. and Giardia spp. using molecular diagnostics.

Results

The most frequently detected parasites were Enterocytozoon bieneusi found in 18 samples (including genotype EbpA, D, C, gorilla 2 and five novel genotypes gorilla 4–8) and Encephalitozoon cuniculi with genotype II being more prevalent (10 cases) compared to genotype I (1 case). Cryptosporidium muris (2 cases) and C. meleagridis (2 cases) were documented in great apes for the first time. Cryptosporidium sp. infections were identified only in research groups and occurrence of E. cuniculi in research groups was significantly higher in comparison to tourist groups. No difference in prevalence of E. bieneusi was observed between research and tourist groups.

Conclusion

Although our data showed the presence and diversity of important opportunistic protists in Volcanoes gorillas, the source and the routes of the circulation remain unknown. Repeated individual sampling, broad sampling of other hosts sharing the habitat with gorillas and quantification of studied protists would be necessary to acquire more complex data.     

Structural and Functional Study of the GlnB22-Insulin Mutant Responsible for Maturity-Onset Diabetes of the Young

The insulin gene mutation c.137G>A (R46Q), which changes an arginine at the B22 position of the mature hormone to glutamine, causes the monogenic diabetes variant maturity-onset diabetes of the young (MODY). In MODY patients, this mutation is heterozygous, and both mutant and wild-type (WT) human insulin are produced simultaneously. However, the patients often depend on administration of exogenous insulin. In this study, we chemically synthesized the MODY mutant [GlnB22]-insulin and characterized its biological and structural properties. The chemical synthesis of this insulin analogue revealed that its folding ability is severely impaired. In vitro and in vivo tests showed that its binding affinity and biological activity are reduced (both approximately 20% that of human insulin). Comparison of the solution structure of [GlnB22]-insulin with the solution structure of native human insulin revealed that the most significant structural effect of the mutation is distortion of the B20-B23 β-turn, leading to liberation of the B chain C-terminus from the protein core. The distortion of the B20-B23 β-turn is caused by the extended conformational freedom of the GlnB22 side chain, which is no longer anchored in a hydrogen bonding network like the native ArgB22. The partially disordered [GlnB22]-insulin structure appears to be one reason for the reduced binding potency of this mutant and may also be responsible for its low folding efficiency in vivo. The altered orientation and flexibility of the B20-B23 β-turn may interfere with the formation of disulfide bonds in proinsulin bearing the R46Q (GlnB22) mutation. This may also have a negative effect on the WT proinsulin simultaneously biosynthesized in β-cells and therefore play a major role in the development of MODY in patients producing [GlnB22]-insulin.     

Coevolution of Cryptosporidium tyzzeri and the house mouse (Mus musculus)

Two house mouse subspecies occur in Europe, eastern and northern Mus musculus musculus (Mmm) and western and southern Mus musculus domesticus (Mmd). A secondary hybrid zone occurs where their ranges meet, running from Scandinavia to the Black Sea. In this paper, we tested a hypothesis that the apicomplexan protozoan species Cryptosporidium tyzzeri has coevolved with the house mouse. More specifically, we assessed to what extent the evolution of this parasite mirrors divergence of the two subspecies. In order to test this hypothesis, we analysed sequence variation at five genes (ssrRNA, Cryptosporidium oocyst wall protein (COWP), thrombospondin-related adhesive protein of Cryptosporidium 1 (TRAP-C1), actin and gp60) in C. tyzzeri isolates from Mmd and Mmm sampled along a transect across the hybrid zone from the Czech Republic to Germany. Mmd samples were supplemented with mice from New Zealand. We found two distinct isolates of C. tyzzeri, each occurring exclusively in one of the mouse subspecies (C. tyzzeri-Mmm and C. tyzzeri-Mmd). In addition to genetic differentiation, oocysts of the C. tyzzeri-Mmd subtype (mean: 4.24 × 3.69 μm) were significantly smaller than oocysts of C. tyzzeri-Mmm (mean: 4.49 × 3.90 μm). Mmm and Mmd were susceptible to experimental infection with both C. tyzzeri subtypes; however, the subtypes were not infective for the rodent species Meriones unguiculatus, Mastomys coucha, Apodemus flavicollis or Cavia porcellus. Overall, our results support the hypothesis that C. tyzzeri is coevolving with Mmm and Mmd.