A new methodology combining PCR, cloning, and sequencing of clones discriminated by RFLP for the study of microbial populations: application to an UASB reactor sample

This work describes a methodology combining DNA extraction, polymerase chain reaction amplification with primers targeting 16S ribosomal RNA genes, cloning, and sequencing of clones previously analyzed by restriction fragment length polymorphism (RFLP), which can be applied to study the microbial diversity in a given habitat. The methodology allows the minimization of the sequencing effort, which is particularly relevant when analyzing large numbers of clones. The methodology does not require particularly skilled personnel and can easily be adaptable to the molecular characterization of virtually any particular microbial population, provided that both adequate primers and suitable restriction enzymes for RFLP analysis of the clone library have been chosen. An example of application is presented, in which a sample taken from a continuously operating upflow anaerobic sludge blanket reactor was analyzed. RFLP analysis of the initial 162 clones with HaeIII allowed the identification of only 28 distinct profiles. As expected, identical RFLP profiles corresponded to identical nucleotide sequences.     

Synthesis of Monomethoxypolyethyleneglycol—Cholesteryl Ester and Effect of its Incorporation in Liposomes

The objective of the present study was to synthesize monomethoxypolyethyleneglycol-5000 cholesteryl ester [PEG–CH] as a cost-effective substitute for polyethyleneglycol–phosphatidylethanolamine and to evaluate the influence of its incorporation in liposomal bilayers for surface modification. PEG–CH was synthesized and characterized by infrared (IR), proton nuclear magnetic resonance spectroscopy (¹H NMR), and differential scanning calorimetry (DSC) studies. Influence of incorporation of PEG–CH in liposomes was evaluated on various parameters such as zeta potential, DSC, and encapsulation efficiency of a hydrophilic drug pentoxyfylline. Conventional and PEG–CH containing pentoxyfylline liposomes were formulated and their stability was evaluated at 4°C for 3 months. PEG–CH could be successfully synthesized with good yields and the structure was confirmed by IR, DSC, and ¹H NMR. The incorporation of PEG–CH in liposomes resulted in reduction of the zeta potential and broadening of the DSC endotherm. Furthermore, incorporation of PEG–CH in liposomes decreased the encapsulation efficiency of pentoxifylline in liposomes when compared to conventional liposomes. Conventional and PEG–CH containing pentoxyfylline liposomes did not show any signs of pentoxyfylline degradation when stored at 4°C for 3 months.     

A new species of <Emphasis Type="Italic">Alcantarea</Emphasis> (Bromeliaceae,Tillandsioideae) from Serra dos Órgãos,Rio de Janeiro,Brazil

Alcantarea martinellii is described and illustrated as a new species endemic to Serra dos Órgãos, Petrópolis, Rio de Janeiro State, Brazil. The presence of a simple and erect inflorescence concomitant with a conspicuous water impounding rosette is an unreported combination of characters for Alcantarea. This new lithophytic taxon is unique, with no obvious close relative, but it is similar to A. benzingii, A. farneyi, and A. hatschbachii, which also have unbranched inflorescences.     

Genetic divergence within the monotypic tree genus <Emphasis Type="Italic">Platycarya</Emphasis> (Juglandaceae) and its implications for species’ past dynamics in subtropical China

Subtropical East Asia harbours a large plant diversity that is often attributed to allopatric speciation in this topographically complex region characterized by a relative climate stability. Here, we use observations of Platycarya, a widespread subtropical Asian tree genus, to explore the consequences of past climate stability on species’ evolutionary history in subtropical China. This genus has a controversial taxonomy: while it is now prevailingly treated as monotypic, two species have been originally described, Platycarya strobilacea and P. longipes. Previous information from species distribution models, fossil pollen data and genetic data based on chloroplast DNA (cpDNA) were integrated with newly obtained genetic data from the two putative species. We used both cpDNA (psbA-trnH and trnL-F intergenic spacers, including a partial trnL gene sequence) and nuclear markers. The latter included sequences of the internal transcribed spacer region (ITS1–5.8S–ITS2) of the nuclear ribosomal DNA and random genomic single nucleotide polymorphisms. Using these nuclear genetic markers, we found interspecific genetic divergence fitting with the ‘two species’ scenario and geographically structured intraspecific variation. Using cpDNA markers, we also found geographically structured intraspecific variation. Despite deep inter- and intraspecific genetic divergence, we detected genetic admixture in southwest China. Overall, our findings of genetic divergence within Platycarya support the hypothesis of allopatric speciation. However, episodes of population interconnection were identified, at least in southwest China, suggesting that the genus has had a dynamic population history.     

Production of rhamnolipids in solid-state cultivation using a mixture of sugarcane bagasse and corn bran supplemented with glycerol and soybean oil

Rhamnolipid biosurfactants are attracting attention due to their low toxicity, high biodegradability, and good ecological acceptability. However, production in submerged culture is made difficult by severe foaming problems. Solid-state cultivation (SSC) is a promising alternative production method. In the current work, we report the optimization of rhamnolipid production by Pseudomonas aeruginosa UFPEDA 614 on a solid substrate containing sugarcane bagasse and corn bran. The best rhamnolipid production, 45 g/l of impregnating solution used, was obtained with a 50:50 (m/m) mixture of sugarcane bagasse and corn bran supplemented with an impregnating solution containing 6% (v/v) of each of glycerol and soybean oil. This level is comparable with those of previous studies undertaken in solid-state cultivation; the composition of the biosurfactant is similar, but our medium is cheaper. Our work therefore provides a suitable basis for future studies of the development of an SSC-based process for rhamnolipid production.     

Proposed association between airborne pollen and pediatric asthma emergency room visits in the Caribbean Island of Trinidad and Tobago West Indies

There are a number of possible links between airborne pollen and respiratory disease that remain underappreciated by both the scientific community and health care providers within the Latin American and Caribbean region. A few regional studies have assimilated some preliminary information on this association, and most recently research on interactions between airborne pollen and pediatric asthma on the Caribbean island of Trinidad has shown critical areas that require further investigation. Pollen samples were collected over a 2-year period using a Burkard 7 Day Recording Volumetric Spore Sampler. Corresponding pediatric asthma data were recorded at a nearby medical hospital. Results indicated that the wet season showed higher pollen concentrations (82 pollen per cubic meter/day) than the dry season (53 pollen per cubic meter/day), and several pollen taxa were observed. An association between airborne pollen concentrations and pediatric asthma emergency room visits was suggested incorporating a threshold and lag period.     

Cloning of a Novel Omega-6 Desaturase from Flax (<Emphasis Type="Italic">Linum usitatissimum</Emphasis> L.) and Its Functional Analysis in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The Δ12 desaturase represents a diverse gene family in plants and is responsible for conversion of oleic acid (18:1) to linoleic acid (18:2). Several members of this family are known from plants like Arabidopsis and Soybean. Using primers from conserved C- and N-terminal regions, we have cloned a novel Δ12 desaturase gene amplified from flax genomic DNA, denoted as LuFAD2-2. This intron-less gene is 1,149-base pair long encoding 382 amino acids—putative membrane-bound Δ12 desaturase protein. Sequence comparisons show that the novel sequence has 85% similarity with previously reported flax Δ12 desaturase at amino acid level and shows typical features of membrane-bound desaturase such as three conserved histidine boxes along with four membrane-spanning regions that are universally present among plant desaturases. The signature amino acid sequence ‘YNNKL’ was also found to be present at the N terminus of the protein, which is necessary and sufficient for ER localization of enzyme. Neighbor-Joining tree generated from the sequence alignment grouped LuFAD2-2 among the other FAD2 sequences from Ricinus, Hevea, Jatropha, and Vernicia. When LuFAD2-2 and LuFAD2 were expressed in Saccharomyces cerevisiae, they could convert the oleic acid to linoleic acid, with an average conversion rate of 5.25 and 8.85%, respectively. However, exogenously supplied linoleic acid was feebly converted to linolenic acid suggesting that LuFAD2-2 encodes a functional FAD2 enzyme and has substrate specificity similar to LuFAD2.     

Assaying three new fungi against citrus mites in the laboratory,and a field trial

The Basidiomycotine fungi Meira geulakonigii, Meira argovae and Acaromyces ingoldii were assayed in the laboratory against five species of herbivorous mites: Phyllocoptruta oleivora (Eriophyidae), Panonychus citri, Eutetranychus orientalis, Tetranychus urticae and Tetranychus cinnabarinus (all four Tetranychidae). All fungi caused significantly high mortality rates (as compared to controls) after 14 days, some after 1 week. Phyllocoptruta oleivora was the most susceptible, showing >80% mortality even after 1 week. In a field trial, grapefruits sprayed either once a month or once a season with M. geulakonigii had significantly fewer P. oleivora and less damage than unsprayed fruit. These results suggest that M. geulakonigii may protect grapefruits against the injurious P. oleivora.