Development of a Universal Microarray Based on the Ligation Detection Reaction and 16S rRNA Gene Polymorphism To Target Diversity of Cyanobacteria

The cyanobacteria are photosynthetic prokaryotes of significant ecological and biotechnological interest, since they strongly contribute to primary production and are a rich source of bioactive compounds. In eutrophic fresh and brackish waters, their mass occurrences (water blooms) are often toxic and constitute a high potential risk for human health. Therefore, rapid and reliable identification of cyanobacterial species in complex environmental samples is important. Here we describe the development and validation of a microarray for the identification of cyanobacteria in aquatic environments. Our approach is based on the use of a ligation detection reaction coupled to a universal array. Probes were designed for detecting 19 cyanobacterial groups including Anabaena/Aphanizomenon, Calothrix, Cylindrospermopsis, Cylindrospermum, Gloeothece, halotolerants, Leptolyngbya, Palau Lyngbya, Microcystis, Nodularia, Nostoc, Planktothrix, Antarctic Phormidium, Prochlorococcus, Spirulina, Synechococcus, Synechocystis, Trichodesmium, and Woronichinia. These groups were identified based on an alignment of over 300 cyanobacterial 16S rRNA sequences. For validation of the microarrays, 95 samples (24 axenic strains from culture collections, 27 isolated strains, and 44 cloned fragments recovered from environmental samples) were tested. The results demonstrated a high discriminative power and sensitivity to 1 fmol of the PCR-amplified 16S rRNA gene. Accurate identification of target strains was also achieved with unbalanced mixes of PCR amplicons from different cyanobacteria and an environmental sample. Our universal array method shows great potential for rapid and reliable identification of cyanobacteria. It can be easily adapted to future development and could thus be applied both in research and environmental monitoring.     

Sequential anaerobic–aerobic degradation of munitions waste

A sequential anaerobic–aerobic biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) was studied. The results demonstrated that: (i) a complete degradation of RDX was achieved within 20 days using a consortium of bacteria from a wastewater activated sludge, (ii) RDX degradation did not occur under aerobic conditions alone, (iii) RDX-degrading bacterial strain that was isolated from the activated sludge completely degraded RDX within 2 days, and (iv) RDX- induced protein expressions were observed in the RDX-degrading bacterial strain. Based on fatty acid composition and a confirmation with a 16S rRNA analysis, the RDX-degrading bacterial strain was identified as a Bacillus pumilus—GC subgroup B.     

Phorbol 12, myristate 13, acetate potentiates the respiratory burst while inhibits phosphoinositide hydrolysis and calcium mobilization by formyl-methionyl-leucyl-phenylalanine in human neutrophils

It is widely believed that the transduction pathway in the activation of the NADPH oxidase by formyl-methionyl-leucyl-phenylalanine (FMLP) in neutrophils involves the stimulation of phosphoinositide hydrolysis, the increase in [Ca2+]i and the activity of the Ca2+ and phospholipid dependent protein kinase C. The results presented here show that the activation of the respiratory burst by FMLP can be dissociated by the stimulation of the hydrolysis of phosphatidylinositol 4,5-bisphosphate and Ca2+ changes. In fact, in neutrophils pretreated (primed) with non stimulatory doses of phorbol myristate acetate the respiratory burst by chemotactic peptide is greatly potentiated while the increase in [3H] inositol phosphates formation and in [Ca2+]i are depressed due to the inhibition of phospholipase C. This finding indicates that FMLP can trigger also a sequence of transduction reactions for the activation of the NADPH oxidase different from that involving the formation of the second messengers diacylglycerol and inositol phosphates and the increase in free Ca2+ concentration.     

Methane emission from feather moss stands

Data from remote sensing and Eddy towers indicate that forests are not always net sinks for atmospheric CH₄. However, studies describing specific sources within forests and functional analysis of microorganisms on sites with CH₄ turnover are scarce. Feather moss stands were considered to be net sinks for carbon dioxide, but received little attention to their role in CH₄ cycling. Therefore, we investigated methanogenic rates and pathways together with the methanogenic microbial community composition in feather moss stands from temperate and boreal forests. Potential rates of CH₄ emission from intact moss stands (n = 60) under aerobic conditions ranged between 19 and 133 pmol CH₄ h^?1 gdw^?1. Temperature and water content positively influenced CH₄ emission. Methanogenic potentials determined under N₂ atmosphere in darkness ranged between 22 and 157 pmol CH₄ h^?1 gdw^?1. Methane production was strongly inhibited by bromoethane sulfonate or chloroform, showing that CH₄ was of microbial origin. The moss samples tested contained fluorescent microbial cells and between 10⁴ and 10⁵ copies per gram dry weight moss of the mcrA gene coding for a subunit of the methyl CoM reductase. Archaeal 16S rRNA and mcrA gene sequences in the moss stands were characteristic for the archaeal families Methanobacteriaceae and Methanosarcinaceae. The potential methanogenic rates were similar in incubations with and without methyl fluoride, indicating that the CH₄ was produced by the hydrogenotrophic rather than aceticlastic pathway. Consistently, the CH₄ produced was depleted in ¹³C in comparison with the moss biomass carbon and acetate accumulated to rather high concentrations (3–62 mM). The δ¹³C of acetate was similar to that of the moss biomass, indicating acetate production by fermentation. Our study showed that the feather moss stands contained active methanogenic microbial communities producing CH₄ by hydrogenotrophic methanogenesis and causing net emission of CH₄ under ambient conditions, albeit at low rates.     

Aerobic sugar metabolism in the spoilage yeast Zygosaccharomyces bailii

Despite the importance of some Zygosaccharomyces species as agents causing spoilage of food, the carbon and energy metabolism of most of them is yet largely unknown. This is the case with Zygosaccharomyces bailii. In this study the occurrence of the Crabtree effect in the petite-negative yeast Z. bailii ATCC 36947 was investigated. In this yeast the aerobic ethanol production is strictly dependent on the carbon source utilised. In glucose-limited continuous cultures a very low level of ethanol was produced. In fructose-limited continuous cultures ethanol was produced at a higher level and its production increased with the dilution rate. As a consequence, on fructose the onset of respiro-fermentative metabolism caused a reduction in biomass yield. An immediate aerobic alcoholic fermentation in Z. bailii was observed during the transition from sugar limitation to sugar excess, both on glucose and on fructose. The analysis of some key enzymes of the fermentative metabolism showed a high level of acetyl-CoA synthetase in Z. bailii growing on fructose. At high dilution rates, the activities of glucose- and fructose-phosphorylating enzymes, as well as of pyruvate decarboxylase and alcohol dehydrogenase, were higher in cells during growth on fructose than on glucose.     

Concerted action of the new Genomic Peptide Finder and AUGUSTUS allows for automated proteogenomic annotation of the Chlamydomonas reinhardtii genome

The use and development of post-genomic tools naturally depends on large-scale genome sequencing projects. The usefulness of post-genomic applications is dependent on the accuracy of genome annotations, for which the correct identification of intron-exon borders in complex genomes of eukaryotic organisms is often an error-prone task. Although automated algorithms for predicting intron-exon structures are available, supporting exon evidence is necessary to achieve comprehensive genome annotation. Besides cDNA and EST support, peptides identified via MS/MS can be used as extrinsic evidence in a proteogenomic approach. We describe an improved version of the Genomic Peptide Finder (GPF), which aligns de novo predicted amino acid sequences to the genomic DNA sequence of an organism while correcting for peptide sequencing errors and accounting for the possibility of splicing. We have coupled GPF and the gene finding program AUGUSTUS in a way that provides automatic structural annotations of the Chlamydomonas reinhardtii genome, using highly unbiased GPF evidence. A comparison of the AUGUSTUS gene set incorporating GPF evidence to the standard JGI FM4 (Filtered Models 4) gene set reveals 932 GPF peptides that are not contained in the Filtered Models 4 gene set. Furthermore, the GPF evidence improved the AUGUSTUS gene models by altering 65 gene models and adding three previously unidentified genes.     

Antigenic drift in H5N1 avian influenza virus in poultry is driven by mutations in major antigenic sites of the hemagglutinin molecule analogous to those for human influenza virus

H5N1 highly pathogenic avian influenza virus has been endemic in poultry in Egypt since 2008, notwithstanding the implementation of mass vaccination and culling of infected birds. Extensive circulation of the virus has resulted in a progressive genetic evolution and an antigenic drift. In poultry, the occurrence of antigenic drift in avian influenza viruses is less well documented and the mechanisms remain to be clarified. To test the hypothesis that H5N1 antigenic drift is driven by mechanisms similar to type A influenza viruses in humans, we generated reassortant viruses, by reverse genetics, that harbored molecular changes identified in genetically divergent viruses circulating in the vaccinated population. Parental and reassortant phenotype viruses were antigenically analyzed by hemagglutination inhibition (HI) test and microneutralization (MN) assay. The results of the study indicate that the antigenic drift of H5N1 in poultry is driven by multiple mutations primarily occurring in major antigenic sites at the receptor binding subdomain, similarly to what has been described for human influenza H1 and H3 subtype viruses.     

Dominance of CD8 responses specific for epitopes bound by a single major histocompatibility complex class I molecule during the acute phase of viral infection.

Cytotoxic T-lymphocyte (CTL) responses are thought to control human immunodeficiency virus replication during the acute phase of infection. Understanding the CD8(+) T-cell immune responses early after infection may, therefore, be important to vaccine design. Analyzing these responses in humans is difficult since few patients are diagnosed during early infection. Additionally, patients are infected by a variety of viral subtypes, making it hard to design reagents to measure their acute-phase immune responses. Given the complexities in evaluating acute-phase CD8(+) responses in humans, we analyzed these important immune responses in rhesus macaques expressing a common rhesus macaque major histocompatibility complex class I molecule (Mamu-A*01) for which we had developed a variety of immunological assays. We infected eight Mamu-A*01-positive macaques and five Mamu-A*01-negative macaques with the molecularly cloned virus SIV(mac)239 and determined all of the simian immunodeficiency virus-specific CD8(+) T-cell responses against overlapping peptides spanning the entire virus. We also monitored the evolution of particular CD8(+) T-cell responses by tetramer staining of peripheral lymphocytes as well as lymph node cells in situ. In this first analysis of the entire CD8(+) immune response to autologous virus we show that between 2 and 12 responses are detected during the acute phase in each animal. CTL against the early proteins (Tat, Rev, and Nef) and against regulatory proteins Vif and Vpr dominated the acute phase. Interestingly, CD8(+) responses against Mamu-A*01-restricted epitopes Tat(28-35)SL8 and Gag(181-189)CM9 were immunodominant in the acute phase. After the acute phase, however, this pattern of reactivity changed, and the Mamu-A*01-restricted response against the Gag(181-189)CM9 epitope became dominant. In most of the Mamu-A*01-positive macaques tested, CTL responses against epitopes bound by Mamu-A*01 dominated the CD8(+) cellular immune response.     

The growing world of small heat shock proteins: from structure to functions

Small heat shock proteins (sHSPs) are present in all kingdoms of life and play fundamental roles in cell biology. sHSPs are key components of the cellular protein quality control system, acting as the first line of defense against conditions that affect protein homeostasis and proteome stability, from bacteria to plants to humans. sHSPs have the ability to bind to a large subset of substrates and to maintain them in a state competent for refolding or clearance with the assistance of the HSP70 machinery. sHSPs participate in a number of biological processes, from the cell cycle, to cell differentiation, from adaptation to stressful conditions, to apoptosis, and, even, to the transformation of a cell into a malignant state. As a consequence, sHSP malfunction has been implicated in abnormal placental development and preterm deliveries, in the prognosis of several types of cancer, and in the development of neurological diseases. Moreover, mutations in the genes encoding several mammalian sHSPs result in neurological, muscular, or cardiac age-related diseases in humans. Loss of protein homeostasis due to protein aggregation is typical of many age-related neurodegenerative and neuromuscular diseases. In light of the role of sHSPs in the clearance of un/misfolded aggregation-prone substrates, pharmacological modulation of sHSP expression or function and rescue of defective sHSPs represent possible routes to alleviate or cure protein conformation diseases. Here, we report the latest news and views on sHSPs discussed by many of the world’s experts in the sHSP field during a dedicated workshop organized in Italy (Bertinoro, CEUB, October 12–15, 2016).     

Expression of the major histocompatibility complex class I molecule Mamu-A*01 is associated with control of simian immunodeficiency virus SIVmac239 replication

Several HLA alleles are associated with attenuated human immunodeficiency virus disease progression. We explored the relationship between the expression of particular major histocompatibility complex (MHC) class I alleles and viremia in simian immunodeficiency virus SIV(mac)239-infected macaques. Of the common MHC class I alleles, animals that expressed Mamu-A*01 exhibited the best control of viral replication.