Diversity of fungi on rainforest litter in North Queensland, Australia

Ten leaves from each of 13 different tree types from two differentrainforest sites in North Queensland, Australia were examined in order toestablish the fungal diversity developing on these leaves. A total of 57microfungi were identified, most of which were mitosporic fungi. Speciesdiversity in terms of richness and evenness were compared and the Mt Lewis sitewas found to be richer as compared to the Butchers Creek site. Statisticalmeasurements of diversity indices, however, showed that the two forest siteswere of similar diversity. Thirty-six of the fungi identified occurred only onone leaf type, indicating possible host specificities or recurrences. The samplesize, however, is deemed to be insufficient, as a larger sample size may haveresulted in less of the fungi appearing to be host specific. It is recommendedthat future studies should include more leaf samples and less tree types. It isparticularly important that the same leaf species are collected within the samesite and at different sites in order to establish the effects of host on fungalcomposition.     

Carbohydrate-binding specificities of potential probiotic <Emphasis Type="Italic">Lactobacillus</Emphasis> strains in porcine jejunal (IPEC-J2) cells and porcine mucin

Bacterial lectins are carbohydrate-binding adhesins that recognize glycoreceptors in the gut mucus and epithelium of hosts. In this study, the contribution of lectin-like activities to adhesion of Lactobacillus mucosae LM1 and Lactobacillus johnsonii PF01, which were isolated from swine intestine, were compared to those of the commercial probiotic Lactobacillus rhamnosus GG. Both LM1 and PF01 strains have been reported to have good adhesion ability to crude intestinal mucus of pigs. To confirm this, we quantified their adhesion to porcine gastric mucin and intestinal porcine enterocytes isolated from the jejunum of piglets (IPEC-J2). In addition, we examined their carbohydrate-binding specificities by suspending bacterial cells in carbohydrate solutions prior to adhesion assays. We found that the selected carbohydrates affected the adherences of LM1 to IPEC-J2 cells and of LGG to mucin. In addition, compared to adhesion to IPEC-J2 cells, adhesion to mucin by both LM1 and LGG was characterized by enhanced specific recognition of glycoreceptor components such as galactose, mannose, and N-acetylglucosamine. Hydrophobic interactions might make a greater contribution to adhesion of PF01. A similar adhesin profile between a probiotic and a pathogen, suggest a correlation between shared pathogen–probiotic glycoreceptor recognition and the ability to exclude enteropathogens such as Escherichia coli K88 and Salmonella Typhimurium KCCM 40253. These findings extend our understanding of the mechanisms of the intestinal adhesion and pathogen-inhibition abilities of probiotic Lactobacillus strains.     

Next-generation sequencing revealed dominant fungal populations in collected dust from selected public school classrooms in Metro Manila

Fungal contaminants inside classrooms may increase the chance of health-related problems for school children and teachers, reducing their learning and productivity. Recent initiatives have utilized next-generation sequencing (NGS) technology in order to understand dust ecology and were used to significantly correlate some genera with health-related conditions. To our knowledge, this paper is the first report, describing the fungal community profile of collected dust using 454 pyrosequencing of the ITS region of the 18S rRNA gene from public school classrooms in Metro Manila, Philippines. Culture-dependent technique was done by gravimetric sampling on Sabouraud Dextrose Agar (SDA) to note the importance of existing viable spores present in the rooms. Composite samples of settled dust from each classroom were collected and pooled to represent one sample per school. The fungal ITS rRNA genes amplified from genomic DNA with barcoded primers were sent for pyrosequencing on a 454 GS FLX titanium platform, and sequences were analyzed using the ITScan pipeline. Fungal sequences from the collected dust samples clustered in 108 operational taxonomic units (OTUs), most of which occur as singleton. The number of OTUs that correspond to fungal species varied from 16 to 29 per sample. Rarefaction curves indicated that sampling coverage was partial and that the remaining fraction of the species diversity remains to be discovered. Genera that were detected by both NGS and by cultivation on SDA include Alternaria, Aspergillus, Cladosporium and Penicillium.     

Expression and characterisation of neopullulanase from <Emphasis Type="Italic">Lactobacillus mucosae</Emphasis>

Objectives

To clone and express a neopullulanase gene from Lactobacillus mucosae LM1 in Escherichia coli and characterise the resulting recombinant neopullulanase.

Results

An ORF in L. mucosae corresponding to a neopullulanase was cloned and expressed in E. coli. The predicted amino acid sequence of the neopullulanase contained catalytic sites and conserved motifs that are present in members of the neopullulanase subfamily. The resulting recombinant neopullulanase was efficiently purified by Ni–NTA affinity chromatography. The purified enzyme optimally hydrolyses pullulan at 37 °C and pH 6.0, producing panose as the major reaction product.

Conclusions

To the best of our knowledge, this is the first report of the cloning, expression and characterisation of a neopullulanase gene from a lactic acid bacterium.

     

Orthogonal HPLC methods for quantitating related substances and degradation products of pramlintide

Pramlintide is a 37-amino acid peptide that is being evaluated as a drug candidate for treating people with type 1 and insulin-using type 2 diabetes. Two high-performance liquid chromatography (HPLC) methods were developed for quantitating related substance impurities in pramlintide drug substance as well as degradation products of pramlintide formulated for parenteral administration. The methods differ with respect to separation mode and therefore provide orthogonal information concerning related substances and degradation products. One method uses a reverse phase (RP) separation mode, and the other involves a strong cation exchange (SCX) separation. Method performance testing showed that the RP- and SCX-HPLC methods both afford a high degree of selectivity, accuracy, precision, and sensitivity. The limit of quantitation for determining spiked authentic samples of degradation products was shown to be approximately 0.1% (relative to intact pramlintide) for both methods. Relative retention times for known pramlintide degradation products were determined for both the RP- and SCX-HPLC methods, demonstrating the selectivities of the 2 methods as well as the orthogonality of the information. The methods were also shown to be diastereospecific with respect to separating pramlintide from authentic samples of D-isomers at Ala⁵, Ala⁸, Ala⁵-Ala⁸, and Leu¹². The methods did not resolve pramlintide, however, from diastereomers with D-isomers near the C- and N-termini, namely Lys¹,Cys², and Tyr³⁷.     

Pyrosequencing-based analysis of fecal microbial communities in three purebred pig lines

This study examined the fecal bacterial diversity of 15-weekold pigs from three purebred lines: Duroc, Landrace, and Yorkshire. Taxon-dependent and -independent analyses were performed to evaluate differences in the fecal bacterial communities and to identify bacterial genera that can be used to discriminate breeds, following high-throughput pyrosequencing of 16S rRNA genes. Among the breeds evaluated, Landrace had the most diverse bacterial community composition. Prevotella, Blautia, Oscillibacter, and Clostridium were detected in all samples regardless of breed. On the other hand, Catenibacterium, Blautia, Dialister, and Sphaerochaeta were differentially detected among breeds, as demonstrated by the canonical loading plot. The discriminant analysis of principal components plot also showed clear separation of the three purebred pig lines, with a certain degree of similarity between Landrace and Yorkshire pigs and a distinct separation between Duroc pigs and the other two breeds. Other factors not related to breed, such as season or time of sampling and pen effects, may contribute to shaping the gut microbiota of pigs.