A rapid method for the detection of potentially viable Mycobacterium leprae in human biopsies: a novel application of PCR

Abstract A simple procedure based on the polymerase chain reaction has been developed to detect Mycobacterium leprae , rapidly and unambiguously, in biological samples. Its application to small numbers of M. leprae cells (∼ 10²) isolated from armadillo liver, mouse footpads or human biopsies is discussed.     

Temporal variations and pond age effect on plankton communities in semi-intensive freshwater marron (Cherax cainii,Austin and Ryan, 2002) earthen aquaculture ponds in Western Australia

The abundance and diversity of the plankton community represents the health of the aquatic ecosystem, and plays an important role in the growth of cultured animals under aquaculture conditions. The temporal variations of plankton abundance, taxonomic composition, diversity, evenness and species richness were studied in three old and three new semi-intensive marron (Cherax cainii, Austin and Ryan, 2002) ponds. Water parameters such as temperature, dissolved oxygen, pH, turbidity, TAN, nitrite, nitrate and reactive phosphate were recorded, and plankton samples were collected every two months, for one year of juvenile production cycle. A total of twenty-six phytoplankton and seven zooplankton genera were recorded. Chlorophyceae was the dominant class of phytoplankton throughout the year, followed by Trebouxiophyceae. Rotifera comprised 49.8% of the total zooplankton community (individuals L^?1), the largest proportion of any group. Temporal variations impacted the plankton abundance and community structure, and plankton abundance were more abundant during summer. The pond age did not influence the phytoplankton abundance, whereas zooplankton abundance was higher in older ponds.     

Discovery and initial optimization of 5,5′-disubstituted aminohydantoins as potent β-secretase (BACE1) inhibitors

8,8-Diphenyl-2,3,4,8-tetrahydroimidazo[1,5-a]pyrimidin-6-amine (1) was identified through HTS, as a weak (micromolar) inhibitor of BACE1. X-Ray crystallographic studies indicate the 2-aminoimidazole ring forms key H-bonding interactions with Asp32 and Asp228 in the catalytic site of BACE1. Lead optimization using structure-based focused libraries led to the identification of low nanomolar BACE1 inhibitors such as 20b with substituents which extend from the S₁ to the S₃ pocket.     

Resistance to human serum of gonococci in urethral exudates is reduced by neuraminidase

Gonococci examined directly from urethral exudates are resistant to killing by human serum, but most strains become susceptible on subculture. Previous work with gonococci grown in vitro indicates that resistance in vivo is due to sialylation of gonococcal lipopolysaccharide (LPS) by a host factor, cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NANA) or a related compound present in urogenital secretions and blood cells including phagocytes, which exude during inflammation. This sialylation inhibits the reaction between bactericidal IgM in serum and its target LPS sites. Here, we confirm the indication by using gonococci grown in vivo. Crucial to the above conclusions was the marked reduction of CMP-NANA-conferred serum resistance when gonococci were treated with neuraminidase to remove sialyl groups from their LPS. We now show that the serum resistance of gonococci in urethral exudates was reduced by treatment with neuraminidase from more than 95% (calculated in relation to controls incubated with heated serum) to 2-11% according to sample and incubation time. Subculture of the gonococci also reduced resistance to 9-11% but resistance was restored to more than 95% by incubation with CMP-NANA. This work is the culmination of an investigation that underlines the need to identify specific host factors and the virulence determinants they induce in vivo in future studies of pathogenicity.     

How many biological replicates are needed in an RNA-seq experiment and which differential expression tool should you use?

RNA-seq is now the technology of choice for genome-wide differential gene expression experiments, but it is not clear how many biological replicates are needed to ensure valid biological interpretation of the results or which statistical tools are best for analyzing the data. An RNA-seq experiment with 48 biological replicates in each of two conditions was performed to answer these questions and provide guidelines for experimental design. With three biological replicates, nine of the 11 tools evaluated found only 20%–40% of the significantly differentially expressed (SDE) genes identified with the full set of 42 clean replicates. This rises to >85% for the subset of SDE genes changing in expression by more than fourfold. To achieve >85% for all SDE genes regardless of fold change requires more than 20 biological replicates. The same nine tools successfully control their false discovery rate at ≲5% for all numbers of replicates, while the remaining two tools fail to control their FDR adequately, particularly for low numbers of replicates. For future RNA-seq experiments, these results suggest that at least six biological replicates should be used, rising to at least 12 when it is important to identify SDE genes for all fold changes. If fewer than 12 replicates are used, a superior combination of true positive and false positive performances makes edgeR and DESeq2 the leading tools. For higher replicate numbers, minimizing false positives is more important and DESeq marginally outperforms the other tools.     

Utilizing natural diversity to evolve protein function: applications towards thermostability

Protein evolution relies on designing a library of sequences that capture meaningful functional diversity in a limited number of protein variants. Several approaches take advantage of the sequence space already explored through natural selection by incorporating sequence diversity available from modern genomes (and their ancestors) when designing these libraries. The success of these approaches is, partly, owing to the fact that modern sequence diversity has already been subjected to evolutionary selective forces and thus the diversity has already been deemed 'fit to survive'. Five of these approaches will be discussed in this review to highlight how protein engineers can use evolutionary sequence history/diversity of homologous proteins in unique ways to design protein libraries.     

Reducing the impact of pesticides on biological control in Australian vineyards: pesticide mortality and fecundity effects on an indicator species, the predatory mite Euseius victoriensis (Acari: Phytoseiidae)

Laboratory bioassays on detached soybean, Glycine max (L.) Merr., leaves were used to test 23 fungicides, five insecticides, two acaricides, one herbicide, and two adjuvants on a key Australian predatory mite species Euseius victoriensis (Womersley) in "worst-case scenario" direct overspray assays. Zero- to 48-h-old juveniles, their initial food, and water supply were sprayed to runoff with a Potter tower; spinosad and wettable sulfur residues also were tested. Tests were standardized to deliver a pesticide dose comparable with commercial application of highest label rates at 1,000 liter/ha. Cumulative mortality was assessed 48 h, 4 d, and 7 d after spraying. Fecundity was assessed for 7 d from start of oviposition. No significant mortality or fecundity effects were detected for the following compounds at single-use application at 1,000 liter/ha: azoxystrobin, Bacillus thuringiensis (Bt) subsp. kurstaki, captan, chlorothalonil, copper hydroxide, fenarimol, glyphosate, hexaconazole, indoxacarb, metalaxyl/copper hydroxide, myclobutanil, nonyl phenol ethylene oxide, phosphorous acid, potassium bicarbonate, pyraclostrobin, quinoxyfen, spiroxamine, synthetic latex, tebufenozide, triadimenol, and trifloxystrobin. Iprodione and penconazole had some detrimental effect on fecundity. Canola oil as acaricide (2 liter/100 liter) and wettable sulfur (200 g/100 liter) had some detrimental effect on survival and fecundity and cyprodinil/fludioxonil on survivor. The following compounds were highly toxic (high 48-h mortality): benomyl, carbendazim, emamectin benzoate, mancozeb, spinosad (direct overspray and residue), wettable sulfur (> or = 400 g/100 liter), and pyrimethanil; pyrimethanil had no significant effect on fecundity of surviving females. Indoxacarb safety to E. victoriensis contrasts with its toxicity to key parasitoids and chrysopid predators. Potential impact of findings is discussed.