Diversity of Bacteria and Glycosyl Hydrolase Family 48 Genes in Cellulolytic Consortia Enriched from Thermophilic Biocompost

The enrichment from nature of novel microbial communities with high cellulolytic activity is useful in the identification of novel organisms and novel functions that enhance the fundamental understanding of microbial cellulose degradation. In this work we identify predominant organisms in three cellulolytic enrichment cultures with thermophilic compost as an inoculum. Community structure based on 16S rRNA gene clone libraries featured extensive representation of clostridia from cluster III, with minor representation of clostridial clusters I and XIV and a novel Lutispora species cluster. Our studies reveal different levels of 16S rRNA gene diversity, ranging from 3 to 18 operational taxonomic units (OTUs), as well as variability in community membership across the three enrichment cultures. By comparison, glycosyl hydrolase family 48 (GHF48) diversity analyses revealed a narrower breadth of novel clostridial genes associated with cultured and uncultured cellulose degraders. The novel GHF48 genes identified in this study were related to the novel clostridia Clostridium straminisolvens and Clostridium clariflavum, with one cluster sharing as little as 73% sequence similarity with the closest known relative. In all, 14 new GHF48 gene sequences were added to the known diversity of 35 genes from cultured species.The exploration and understanding of cellulose fermentation capabilities in nature could inform and enable industrial processes converting cellulosic biomass to fuels and other products. Enrichment of microbial communities that can utilize cellulose is useful in this context for the identification of novel organisms, novel metabolisms, and novel functions. Of particular interest are communities that can utilize cellulose at high temperatures and under anaerobic conditions, featuring high rates of solubilization under conditions where the energy and the reducing power of substrates are conserved in potentially useful fermentation products.Some evidence indicates that cocultures may be able to utilize cellulose more fully and produce higher concentrations of ethanol than pure cultures of model cellulolytic organisms such as Clostridium thermocellum and Clostridium straminisolvens (16, 20, 34). An initial step toward understanding the functional roles of community members in cooperative cellulose degradation is answering the question of what organisms are present in cellulolytic consortia obtained from nature. Currently, diversity estimation methods applied to cellulolytic communities range from traditional methods targeting the 16S rRNA gene (4, 12) to complex metagenomic analyses targeting the breadth of functional genes present in genomes of mixed cultures and the environment (3).From a functional gene standpoint, cellulase systems are complex assemblages of multifunctional glycosyl hydrolases. Even particularly relevant families, such as family 5 and family 9, tend to include hydrolases with multiple substrate specificities, deep evolutionary roots, and extensive sequence diversity within the same organism (19). However, family 48 glycosyl hydrolases include a select group of cellulosomal and unbound cellulases thought to play an essential role in cellulose solubilization by model cellulolytic clostridia (5, 7, 15), actinobacteria (6, 13), and anaerobic fungi (31). One key feature of this family of glycosyl hydrolases (mostly exoglucanases) is their ability to enhance cellulose solubilization in synergistic interactions with family 9 glycosyl hydrolases (2, 13). But unlike the latter, and with the notable exception of CelS and CelY in Clostridium thermocellum, family 48 hydrolases are present mostly in single copies in the genomes of cellulolytic microbes, making family 48 hydrolase genes a desirable target for primer design and molecular characterization.In this paper we describe the enrichment of microbial communities from a thermophilic compost pile and provide an assessment of diversity in stable cellulolytic enrichments by addressing total bacterial diversity using the 16S rRNA gene as well as introducing a novel method to assess functional diversity in cellulolytic consortia by targeting glycosyl hydrolase family 48 (GHF48) genes.     

From recommendation to action: psychosocial factors influencing physician intention to use Health Technology Assessment (HTA) recommendations

Background

The Evidence-Based Practice (EBP) Project has been investigating the implementation of evidence-based mental health practices (Assertive Community Treatment, Family Psychoeducation, Integrated Dual Diagnosis Treatment, Illness Management and Recovery, and Supported Employment) in state public mental health systems in the United States since 2001. To date, Project findings have yielded valuable insights into implementation strategy characteristics and effectiveness. This paper reports results of an effort to identify and classify state-level implementation activities and strategies employed across the eight states participating in the Project.

Methods

Content analysis and Greenhalgh et al's (2004) definition of innovation were used to identify and classify state-level activities employed during three phases of EBP implementation: Pre-Implementation, Initial Implementation and Sustainability Planning. Activities were coded from site visit reports created from documents and notes from key informant interviews conducted during two periods, Fall 2002 – Spring 2003, and Spring 2004. Frequency counts and rank-order analyses were used to examine patterns of implementation activities and strategies employed across the three phases of implementation.

Results

One hundred and six discreet implementation activities and strategies were identified as innovative and were classified into five categories: 1) state infrastructure building and commitment, 2) stakeholder relationship building and communications, 3) financing, 4) continuous quality management, and 5) service delivery practices and training. Implementation activities from different categories were employed at different phases of implementation.

Conclusion

Insights into effective strategies for implementing EBPs in mental health and other health sectors require qualitative and quantitative research that seeks to: a) empirically test the effects of tools and methods used to implement EBPs, and b) establish a stronger evidence-base from which to plan, implement and sustain such efforts. This paper offers a classification scheme and list of innovative implementation activities and strategies. The classification scheme offers potential value for future studies that seek to assess the effects of various implementation processes, and helps establish widely accepted standards and criteria that can be used to assess the value of innovative activities and strategies.     

Effect of ozone exposure on contractile activity and chemoreactivity of uterus horns longitudinal muscles of nonpregnant rats

With the purpose of studying the mechanism of ozone action on uterus smooth muscles it was investigated the influence of ozone-content (approximately 0.50 mkg/ml) Krebs' solution or its 10- and 100-fold dissolution on contractile activity and beta-adrenoreactivity of 56 longitudinal strips of uterus horns of 17 nonpregnant rats. Ozone at concentration approximately 0.50 mkg/ml (but not in concentration of approximately 0.05 and approximately 0.005 mkg/ml) reversibly raised frequency, amplitude and total contractile activity of intact myometrium strips, and also fast and reversibly reducel its beta-adrenoreactivity, i.e. decreased of inhibitory action of adrenaline (10(-8), 10(-7), 10(-6) g/ml), but did not change uterostimulatory effect of acetylcholine (10(-6) g/ml) and oxiyocin (5 x 10(-4) ME/ml), what evident about specificity of ozone beta-adrenoblokate effect. Ozone (approximately 0.50 and 0.05 mkg/ml) did not change ov value of potassium contracture of myometrium strips which was depolarized by hyperpotassium (60 mM KCL) Krebs' solution, but reduced inhibitory action of adrenaline (10(-8) g/ml). The question is being discussed about mechanisms of ozone beta-adrenoblocade actions, about clinical role of this phenomenon, and the possibility of using beta-adrenoreceptor sensibilizators direct action (histidine, tryptophan, tyrosine, trimetazidin and mildronat) at ozonotherapy with the purpose reduction of its negative effects.     

Streptomyces rimosus aminoglycoside-3'-phosphotransferase VIII: comparisons with aminoglycoside-3'-phosphotransferases of aminoglycoside-producing strains with eukaryotic protein kinases

The nucleotide sequence was established for the aphVIII aminoglycoside phosphotransferase gene of an oxytetracycline-producing Streptomyces rimosus strain. The gene is 804 bp in size and possibly codes for APHVIII of 267 residues. Heterologous expression of aphVIII was studied in Escherichia coli and Chlamydomonas reinhardtii. The deduced APHVIII sequence was compared with known sequences of aminoglycoside phosphotransferases of aminoglycoside-producing actinomycete strains and of eukaryotic protein kinases. A local homology of 38 residues was found between APHVIII and actinomycete serine-threonine protein kinases in the conserved region possibly involved in ATP binding. APHVIII differed from aminoglycoside 3'-phosphotransferases of aminoglycoside-producing actinomycete strains and of clinical isolates, and can be classed to a separate group.     

HIV infection in Saint Petersburg: the changing face of the epidemic

The specific features of the epidemiological situation in St. Petersburg at the first stage (1987-1995) and the second stage (1986-1999) of the development of HIV infection are described. During this period morbidity in HIV infection, starting from the insignificant rate of growth, sharply increased. For the first time injecting drug users were noted to come out to the foreground in the structure of the contingent of persons affected by HIV infection (57.5% and 72.6% in 1998 and 1999 respectively). Such change on the epidemic situation requires corrections in the strategy and tactics of preventive measures and medical assistance. To control HIV infection among drug users, only the combination of medical assistance given by narcologists with the realization of all elements of the program "Harm Reduction" can be effective.     

Use of glucose and carbon isotope fractionation by microbial cells immobilized on solid-phase surface

By the example of glucose uptake by the soil bacteria Pseudomonas aureofaciens BS1393(pBS216) and Rhodococcus sp. 3–30 immobilized on a solid-phase surface (quartz sand), their growth parameters were determined: growth rate (doubling time), total CO₂ production, CO₂ production per cell, lag period with respect to substrate uptake, respiratory quotient. The growth of P. aureofaciens and Rhodococcus sp. on glucose revealed (1) differences of the lag period with respect to substrate (lag time of ～4 h for P. aureofaciens and ～26 h for Rhodococcus sp.); (2) differences between the maximal rates of CO₂ production (～50 μg C-CO₂ g^?1 sand h^?1 for P. aureofaciens and ～8.5 μg C-CO₂ g^?1 sand h^?1 for Rhodococcus sp.); (3) differences in CO₂ production per cell (～1.94 × 10^?9 μM CO₂/CFU for P. aureofaciens and more than ～3.4 × 10^?9 μM CO₂/CFU for Rhodococcus sp.). The kinetics of the metabolic CO₂ isotopic composition was shown to be determined by the difference in the carbon isotopic characteristics of products in the cell. Upon introduction of glucose into the medium (the preparatory stage of the metabolism), the uptake of intracellular ¹³C-depleted products (lipids) is noted; at the stage of the maximal cell growth rate, introduced glucose is mainly metabolized; and at the final stage, upon exhaustion of substrate, the “stored” products—the lipid fraction—get involved in the metabolism. At the maximal rate of glucose uptake, the CO₂ carbon isotopic fractionation coefficient relative to organic products of microbial biosynthesis was determined to be α = 1.009 ± 0.002.     

Synthetic neoglycoconjugates of cell-surface phosphoglycans of Leishmania as potential anti-parasite carbohydrate vaccines

Leishmania are a genus of sandfly-transmitted protozoan parasites that cause a spectrum of debilitating and often fatal diseases in humans throughout the tropics and subtropics. During the parasite life cycle, Leishmania survive and proliferate in highly hostile environments. Their survival strategies involve the formation of an elaborate and dense cell-surface glycocalyx composed of diverse stage-specific glycoconjugates that form a protective barrier. Phosphoglycans constitute the variable structural and functional domain of major cell-surface lipophosphoglycan and secreted proteophosphoglycans. In this paper, we discuss structural aspects of various phosphoglycans from Leishmania with the major emphasis on the chemical preparation of neoglycoconjugates (neoglycoproteins and neoglycolipids) based on Leishmania lipophosphoglycan structures as well as the immunological evaluation for some of them as potential anti-leishmaniasis vaccines.