Identification and characterization of salt-responsive microRNAs in Populus tomentosa by high-throughput sequencing

Salt is one of the main environmental factors limiting plant growth and a better understanding of mechanisms of salt stress would aid efforts to bolster plant salt tolerance. MicroRNAs are well known for their important regulatory roles in response to abiotic stress in plants. In this study, high-throughput sequencing was employed to identify miRNAs in Populus tomentosa plantlets treated or not with salt (200 mM for 10 h). We found 141 conserved miRNAs belonging to 31 families, 29 non-conserved but previously-known miRNAs belonging to 26 families, and 17 novel miRNAs. Under salt stress, 19 miRNAs belonging to seven conserved miRNA families were significantly downregulated, and two miRNAs belonging to two conserved miRNA families were upregulated. Of seven non-conserved miRNAs with significantly altered expression, five were downregulated and two were upregulated. Furthermore, eight miRNAs were validated by qRT-PCR and their dynamic differential expressions were analyzed. In addition, 269 target genes of identified miRNAs were predicted and categorized by function. These results provide new insights into salt-responsive miRNAs in Populus.     

Structural characterization and biological implications of di-zinc binding in the ferroxidase center of Streptococcus pyogenes Dpr

Dps proteins contain a ferroxidase site that binds and oxidizes iron, thereby preventing hydroxyl radical formation by Fenton reaction. Although the involvement of a di-iron ferroxidase site has been suggested, X-ray crystal structures of various Dps members have shown either one or two iron cations with various occupancies despite the high structural conservation of the site. Similarly, structural studies with zinc, a redox-stable replacement for iron, have shown the binding of either one or two zinc ions. Here, the crystal structure of Streptococcus pyogenes Dpr in complex with zinc reveals the binding of two zinc cations in the ferroxidase center and an additional zinc-binding site at the surface of the protein. The results suggest a structural basis for the protection of Streptococcus pyogenes in zinc stress conditions and provide a clear evidence for a di-zinc and di-iron ferroxidase site in Streptococcus pyogenes Dpr protein.     

A proteomic analysis of Helicoverpa armigera adults after exposure to UV light irradiation

Ultraviolet (UV) light (blacklight), which emits UV in the range of 320-400 nm, has been used worldwide in light trapping of insect pests. To gain a better understanding of the response of Helicoverpa armigera adults to UV light irradiation, we carried out a comparative proteomic analysis. Three-day-old adults were exposed to UV light for 1 h. Total proteins were extracted and separated by two-dimensional gel electrophoresis. More than 1200 protein spots were reproducibly detected, including 12 that were more abundant and 21 less abundant. Mass spectrometry analysis and database searching helped us to identify 29 differentially abundant proteins. The identified proteins were categorized into several functional groups including signal transduction, RNA processing, protein processing, stress response, metabolisms, and cytoskeleton structure, etc. This study is the first analysis of differentially expressed proteins in phototactic insects under UV light irradiation conditions and gives new insights into the adaptation mechanisms responsive to UV light irradiation stress.     

The IspA protease's involvement in the regulation of the sporulation process of Bacillus thuringiensis is revealed by proteomic analysis

We have observed that the process of sporulation of the ispA-deficient mutant was delayed under phase-contrast microscopy. The protein profiles of the ispA-deficient mutant have been analyzed using two-dimensional gel electrophoresis. The results of a proteomic analysis using MALDI-TOF MS indicated that a sporulation-associated protein, pro- [Formula: see text], was upregulated, while two other sporulation-associated proteins, SpoVD and SpoVR, were downregulated in the ispA-deficient mutant. It has been known that pro- [Formula: see text] is a precursor of [Formula: see text] and is required for gene expression related to the late stage of sporulation. Moreover, SpoVD and SpoVR are known to be involved in the formation of the spore cortex. Based on these observations, we propose that the delay in the sporulation process observed in the ispA-deficient mutant may be due to a failure of [Formula: see text] to signal sporulation. This phenomenon may be further enhanced by insufficient amount of SpoVD and SpoVR for cortex formation. In this study, we have revealed for the first time a possible pathway for the regulation of sporulation-associated proteins via IspA.     

Analysis of Cry8Ka5-binding proteins from Anthonomus grandis (Coleoptera: Curculionidae) midgut

Biotech crops expressing Bacillus thuringiensis Cry toxins present a valuable approach for insect control. Cry8Ka5, which is highly toxic to the cotton boll weevil (Anthonomus grandis), was used as a model to study toxin-ligand interactions. Three Cry-binding proteins were detected after toxin overlay assays. Following de novo sequencing, a heat-shock cognate protein and a V-ATPase were identified, whilst a ∼120 kDa protein remained unknown. Additional Cry8Ka5-binding proteins were visualized by two-dimensional gel electrophoresis ligand blots.     

Structural and functional characterization of Streptococcus pyogenes Cas2 protein under different pH conditions

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins constitute an RNA-guided microbial defense system against invading foreign genetic materials. Cas2 is one of the core Cas proteins found universally in all the subtypes of CRISPR-Cas systems and is required for incorporating new spacers into CRISPR loci. Cas2 homologues from different CRISPR-Cas subtypes were characterized previously as metal-dependent nucleases with different substrate preferences, and it was proposed that a pH-dependent conformational change mediates metal binding and catalysis. Here, we report the crystal structures of Streptococcus pyogenes Cas2 at three different pHs (5.6, 6.5, and 7.5), as well as the results of its nuclease activity assay against double-stranded DNAs at varying pHs (6.0–9.0). Although S. pyogenes Cas2 exhibited strongly pH-dependent catalytic activity, there was no significant conformational difference among the three crystal structures. However, structural comparisons with other Cas2 homologues revealed structural variability and the flexible nature of its putative hinge regions, supporting the hypothesis that conformational switching is important for catalysis. Taken together, our results confirm that Cas2 proteins have pH-dependent nuclease activity against double-stranded DNAs, and provide indirect structural evidence for their conformational changes.     

Proteomic analysis of wild type and arsenite-resistant Leishmania donovani

Leishmania donovani, causative organism for visceral leishmaniasis, is responsible for considerable mortality and morbidity worldwide. Generation of drug-resistant variants continue to challenge the chemotherapy, the mainstay to fight the disease. The aim of current study was proteomic profiling of wild type (Ld-Wt) and arsenite-resistant (Ld-As20) L. donovani. Significant differences in protein profiles were observed between Ld-As20 and its parent Ld-Wt strain. Proteomic analysis of 158 spots from Ld-Wt and 144 spots from, Ld-As20 identified 77 and 74 protein entries, respectively, through MALDI-TOF/TOF based mass spectrometry and database search. A shift in the isoelectric point of few proteins was observed both in Ld-Wt and Ld-As20, which raises the possibility of continuous arsenite stress, resulting in the differences in the protein profiles of drug-resistant strain from its parent wild type strain. The comparative proteomic data holds the key for elucidation of the multifactorial and complex drug resistance mechanism, like arsenite resistance, in the parasite.     

长双歧杆菌BBMN68 可溶性蛋白质图谱的建立

为了建立长双歧杆菌BBMN68蛋白质图谱,采用双向电泳的方法建立了2-D参考图谱,通过MALDI-TOF/MS质谱鉴定和数据库搜索,鉴定到206个蛋白质(占长双歧杆菌BBMN68基因预测总蛋白的11.4%)。通过2-D胶分析,共有800±15(对数期)和800±20(稳定期)个蛋白质,其中282个蛋白点成功鉴定,代表206个不同的蛋白质。另外,分析了实验鉴定蛋白质的等电点和分子量,蛋白功能,密码子偏好性,蛋白质疏水性以及蛋白质细胞定位的分析。研究结果为长双歧杆菌的比较蛋白质组学研究提供了参考图谱和蛋白质基础信息数据。     

Nrc of Streptococcus pneumoniae suppresses capsule expression and enhances anti-phagocytosis

Streptococcus pneumoniae is a major pathogen of community-acquired pneumonia and one of its major virulence factors is pneumolysin, which functions as a cholesterol-dependent cytolytic pore-forming toxin. In this study, we identified the ply-like gene spd0729 in a BLAST search. Unexpectedly, hemolytic and cytotoxic assays showed no significant differences between a Δspd0729 mutant strain and the wild-type strain, whereas the mutant strain exhibited weaker anti-phagocytic activity in human peripheral blood. In addition, real-time RT-PCR analysis revealed that four capsular biosynthesis genes in the mutant strain had expressions 7- to 432-fold greater than those of the wild type, while an enzyme-linked immunoassay showed a mean 3-fold greater amount of total capsular polysaccharide in the mutant strain. These results suggest that Spd0729 is not a cytolysin, though it plays crucial roles in anti-phagocytosis and regulation of capsule expression. Thus, we named Spd0729 as a negative regulator of capsular polysaccharide synthesis (Nrc).     

Effects of quorum sensing on cell viability in Streptococcus mutans biofilm formation

Streptococcus mutans (S. mutans) uses a quorum sensing (QS) signaling system, which is dependent on competence stimulating peptide (CSP), to regulate diverse physiological activities including bacteriocin production, genetic transformation, and biofilm formation. However, the mechanism of the QS system-induced biofilm formation remains unclear. Here, we demonstrated that the late-stage biofilm formation was increased by the addition of exogenous CSP in S. mutans. The numbers of dead cells in biofilms formed in presence of CSP was 64.5% higher than that without CSP after 12 h (p < 0.05) and 76.3% higher after 24 h (p < 0.05), the numbers of live cells in biofilms formed in presence of CSP were 89.3% higher than that without CSP after 24 h (p < 0.01). The expression of QS-associated genes was increased 3.4-5.3-fold by CSP in biofilms. Our results revealed that cell viability of S. mutans grown in biofilms is affected by the CSP-dependent QS system.     

Down regulation of KMP-11 in Leishmania infantum axenic antimony resistant amastigotes as revealed by a proteomic screen

The therapeutic mainstay against the protozoan parasite Leishmania is still based on the antiquated pentavalent antimonials, but resistance is increasing in several parts of the world. Resistance is now partly understood in laboratory promastigote isolates, but the mechanism leading to drug resistance in amastigote isolates is lagging behind. Here we describe a comparative proteomic analysis of a genetically related pair of antimonial-sensitive and -resistant Leishmania infantum axenic amastigote strains. The proteomics screen has highlighted a number of proteins differentially expressed in the resistant parasite. The expression of the protein argininosuccinate synthetase (ARGG) was increased in the drug resistant mutant while a decrease in the expression of the kinetoplastid membrane protein (KMP-11) correlated with the drug resistance phenotype. This proteomic screen highlighted several novel proteins that are putatively involved in resistance to antimonials.     

造纸废水灌溉对毛白杨苗木生长及养分状况的影响

为了探讨工业造纸废水用于杨树人工林灌溉的可行性,以三倍体毛白杨(triploid Populus tomentosa)1年生盆栽苗木为对象,研究了不同浓度造纸废水(分别稀释到12.5％ (IF7Q)、16.7％ (1F5Q)、25％(1F3Q)、33.3％ (1F2Q)、50％ (1F1Q))灌溉对苗木生长及养分状况、土壤化学性质的影响.结果表明:造纸废水灌溉对土壤pH值、速效P含量无显著影响(P＞0.05),但能显著提高土壤有机质、全N及碱解N的含量(P＜0.05).适当稀释的废水灌溉能促进三倍体毛白杨的苗木生长,提高土壤和植株养分水平:灌溉后1F5Q地径、苗高生长量分别为10.5 mm和97.3 cm,较CK分别显著增加102％和47％ (P＜0.05);1F5Q和1F3Q处理苗木总生物量为247 g和230 g,分别较CK显著提高19％和11％(P＜0.05);废水灌溉可显著提高植株根、叶N含量和茎P含量(P＜0.05),但对植株叶、根P含量和茎N含量影响不大(P＞0.05).造纸废水通过一定处理后,可应用于苗木灌溉并促进其生长,提高地力.对于三倍体毛白杨,将废水稀释到16％-25％能起到较好的灌溉效果.     

Pili of oral Streptococcus sanguinis bind to fibronectin and contribute to cell adhesion

Streptococcus sanguinis is a predominant bacterium in the human oral cavity and occasionally causes infective endocarditis. We identified a unique cell surface polymeric structure named pili in this species and investigated its functions in regard to its potential virulence. Pili of S. sanguinis strain SK36 were shown to be composed of three distinctive pilus proteins (PilA, PilB, and PilC), and a pili-deficient mutant demonstrated reduced bacterial adherence to HeLa and human oral epithelial cells. PilC showed a binding ability to fibronectin, suggesting that pili are involved in colonization by this species. In addition, ATCC10556, a standard S. sanguinis strain, was unable to produce pili due to defective pilus genes, which indicates a diversity of pilus expression among various S. sanguinis strains.     

Relationships between Bambusa species (Poaceae,Bambusoideae) revealed by random amplified polymorphic DNA

RAPD (random amplified polymorphic DNA) molecular markers were used to investigate relationships between a sample of Bambusa species from South Eastern China that have been placed in Bambusa or in several segregate genera, Dendrocalamopsis, Leleba, Lingnania, Neosinocalamus and Sinocalamus by different authors. On the resultant neighbor-joining tree, a thorny core Bambusa cluster was distinguished, as was a Lingnania group, and a cluster of Dendrocalamus species with more capitate inflorescences. However, Leleba was found to be a polyphyletic group in the present study.     

Potential allelopatic effects of stilbenoids and flavonoids from leaves of Carex distachya Desf.

The potential allelopathic effects of 14 stilbenoids and five flavonoids, isolated from leaves of Carex distachya Desf., were evaluated on the seed germination and seedling growth of three coexisting Mediterranean species (Dactylis hispanica, Petrorhagia velutina, and Phleum subulatum). The structures of the metabolites have been elucidated on the basis of their spectroscopic features (1D and 2D NMR experiments and EI–MS and ESI–MS data). The bioassays showed species-specific effects of the metabolites from C. distachya, specially on the plant growth (root and shoot elongation) which resulted significantly stimulated or inhibited at 10⁻⁴ M concentration. The effects on root elongation is generally greater than the shoot growth at all the tested concentrations (10⁻⁴–10⁻⁸ M). Cluster of biological data showed interesting relationships between the chemical structures of the compounds and their biological effects.     

Characterisation of global protein expression by two-dimensional electrophoresis and mass spectrometry: proteomics of Toxoplasma gondii.

The development of tools for the analysis of global gene expression is vital for the optimal exploitation of the data on parasite genomes that are now being generated in abundance. Recent advances in two-dimensional electrophoresis (2-DE), mass spectrometry and bioinformatics have greatly enhanced the possibilities for mapping and characterisation of protein populations. We have employed these developments in a proteomics approach for the analysis of proteins expressed in the tachyzoite stage of Toxoplasma gondii. Over 1000 polypeptides were reproducibly separated by high-resolution 2-DE using the pH ranges 4-7 and 6-11. Further separations using narrow range gels suggest that at least 3000-4000 polypeptides should be resolvable by 2-DE using multiple single pH unit gels. Mass spectrometry was used to characterise a variety of protein spots on the 2-DE gels. Peptide mass fingerprints, acquired by matrix-assisted laser desorption/ionisation-(MALDI) mass spectrometry, enabled unambiguous protein identifications to be made where full gene sequence information was available. However, interpretation of peptide mass fingerprint data using the T. gondii expressed sequence tag (EST) database was less reliable. Peptide fragmentation data, acquired by post-source decay mass spectrometry, proved a more successful strategy for the putative identification of proteins using the T. gondii EST database and protein databases from other organisms. In some instances, several protein spots appeared to be encoded by the same gene, indicating that post-translational modification and/or alternative splicing events may be a common feature of functional gene expression in T. gondii. The data demonstrate that proteomic analyses are now viable for T. gondii and other protozoa for which there are good EST databases, even in the absence of complete genome sequence. Moreover, proteomics is of great value in interpreting and annotating EST databases.     

Population genetic differentiation of Schisandra chinensis and Schisandra sphenanthera as revealed by ISSR analysis

Schisandra chinensis (Turcz.) Baill. and Schisandra sphenanthera Rehd. et Wils. are well-known Chinese medicinal plants. The population genetic variation of the two species was studied using inter simple sequence repeat (ISSR) molecular markers. High levels of genetic diversity are revealed in both S. chinensis (P = 88.36%, h = 0.2894, I = 0.4396) and S. sphenanthera (P = 84.09%, H = 0.2782, I = 0.4280). However, the population genetic differentiation is significantly different between the two species. The S. sphenanthera harbors as high as 27% of the genetic variation among populations but 73% within populations, whereas in S. chinensis 17% of the genetic variation occurs among populations and 83% within populations. Both significant (P < 0.05) heterozygosity excess and shifted mode of allele frequency distribution are detected in four out of six populations of S. chinensis and one out of five populations of S. sphenanthera, suggesting the occurrence of recent population bottlenecks in the two species. The different patterns of genetic variation in S. chinensis and S. sphenanthera are discussed in relation to their differences in pollination mechanism, geographic distribution and historical events, and the level of gene flow and genetic drift.     

Diversity of Bacteria and Archaea from a landfill in Chandigarh,India as revealed by culture-dependent and culture-independent molecular approaches

The bacterial community structure of a municipal landfill in Chandigarh, India was analysed by culture-dependent as well as culture-independent molecular approaches, and archaeal structure by the latter method. Samples were collected in two phases from the surface and a depth of 0.91 m in June, 2004 and from 0.91 m, 1.52 m and 1.68 m in May, 2005. After serial dilutions, samples were plated onto tryptic soy agar (TSA), plate count agar (PCA), tryptic soy broth agar (TSBA) and TSBA100 (TSBA diluted 100 times and solidified with agarose), and incubated aerobically at 30 °C. The number of bacteria (CFU) on different media ranged between 9.4 × 10⁵ g⁻¹ (on PCA) and 1.9 × 10⁷ g⁻¹ (on TSA) (wet weight). The numbers of bacteria enumerated from plates incubated anaerobically (anaerobic agar and reinforced clostridial agar) were 2.1 × 10⁷ and 1.7 × 10⁶ g⁻¹, respectively. Of the 468 isolated and purified bacteria (183 in the first phase and 285 in the second phase), 135 were characterised using phenotypic characteristics as well as 16S rRNA gene sequence analysis. It was found that members of the phylum Firmicutes were overwhelmingly predominant (86.6%) in the landfill, followed by Actinobacteria (9.6%) and Proteobacteria (3.7%). Among the Firmicutes, at least 17 species from the single genus Bacillus were the most abundant inhabitants of the landfill. Detailed polyphasic characterisation of many of these isolates led to the discovery of a novel genus Paenisporosarcina (and the species P. quisquiliarum), a novel species of Microbacterium, M. immunditiarum, and reclassification of Sporosarcina macmurdoensis, Pelagibacillus goriensis, Bacillus silvestris, Bacillus insolitus, Bacillus psychrotolerans and Bacillus psychrodurans. Culture-independent analysis of two 16S rRNA gene libraries also revealed that the phylum Firmicutes was the predominant group in this community. The diversity of Archaea was found to be limited mainly to members of two orders: Methanosarcinales and Methanomicrobiales of the phylum Euryarchaeota. When these results were compared to those reported earlier on similar studies, it was found that irrespective of differences in composition of municipal solid waste (especially compostable organic matter and paper) and climate, the members of bacterial and archaeal communities in landfills of many countries remained broadly similar.     

Lavandula angustifolia essential oil as a novel and promising natural candidate for tick (Rhipicephalus (Boophilus) annulatus) control

Lavandula angustifolia is a well known herbal medicine with a variety of useful properties, including its acaricidal effect. This experiment was carried out to study the bioacaricidal activity of L. angustifolia essential oil (EO) against engorged Rhipicephalus (Boophilus) annulatus (Acari; Ixodidae) females. For this purpose six serial concentrations (0.5, 1.0, 2.0, 4.0, 6.0 and 8.0% w/v) of L. angustifolia EO were used. There was considerable mortality in concentrations more than 4.0% although there were no differences between 6.0 and 8.0% in all measured criteria. The mortality rate 24 h after inoculation was 73.26 and 100% in groups treated with 4.0 and 8.0% EO, respectively. Lavender EO also reduced tick egg weight in a concentration-dependent manner. The amount of eggs produced varied from 0.12 g (at 0.5% EO) to 0.00 g (at 8.0% EO) but did not differ statistically from the control. L. angustifolia EO caused 100% failure in egg laying at 6.0 and 8.0% whereas this value in the control group was zero. A positive correlation between L. angustifolia EO concentration and tick control, assessed by relative mortality rate and egg-laying weight, was observed by the EO LC/EC₅₀, which, when calculated using the Probit test, was 2.76-fold higher than the control. Lavender is a promising acaricidal against R. (B.) annulatusin vitro.