Method for flow cytometric detection of Listeria monocytogenes in milk.

This report describes a method for the detection of Listeria monocytogenes in raw milk by flow cytometric analysis of fluorescently labeled bacterial populations. The use of immunofluorescence in combination with measures of DNA content by propidium iodide labeling and size by light scattering enabled specific identification of L. monocytogenes from Streptococcus faecalis, Streptococcus agalactiae, Streptococcus uberis, Staphylococcus epidermidis, and Staphylococcus hyicus. Additional specific resolution of L. monocytogenes populations was achieved through selective enrichment of raw milk in Listeria enrichment broth. These procedures should permit the rapid screening of milk and other food samples for L. monocytogenes and eliminate many of the short-comings associated with conventional fluorescent-antibody procedures.     

Oligo targeting for profiling drug resistance mutations in the parasitic trypanosomatids

Trypanosomatids cause the neglected tropical diseases, sleeping sickness, Chagas disease and the leishmaniases. Studies on these lethal parasites would be further facilitated by new and improved genetic technologies. Scalable precision editing methods, for example, could be used to improve our understanding of potential mutations associated with drug resistance, a current priority given that several new anti-trypanosomal drugs, with known targets, are currently in clinical development. We report the development of a simple oligo targeting method for rapid and precise editing of priority drug targets in otherwise wild type trypanosomatids. In Trypanosoma brucei, approx. 50-b single-stranded oligodeoxynucleotides were optimal, multiple base edits could be incorporated, and editing efficiency was substantially increased when mismatch repair was suppressed. Resistance-associated edits were introduced in T. brucei cyclin dependent kinase 12 (CRK12, L⁴⁸²F) or cleavage and polyadenylation specificity factor 3 (N²³²H), in the Trypanosoma cruzi proteasome β5 subunit (G²⁰⁸S), or in Leishmania donovani CRK12 (G⁵⁷²D). We further implemented oligo targeting for site saturation mutagenesis, targeting codon G⁴⁹² in T. brucei CRK12. This approach, combined with amplicon sequencing for codon variant scoring, revealed fourteen resistance conferring G⁴⁹² edits encoding six distinct amino acids. The outputs confirm on-target drug activity, reveal a variety of resistance-associated mutations, and facilitate rapid assessment of potential impacts on drug efficacy.     

The probability that related individuals share some section of genome identical by descent

A formal mathematical framework is presented for the study of linkage in man and the concept of chromosome pedigree is defined for both autosomes and X chromosomes. It is shown that, assuming no interference, all the crossover processes in the pedigree may be viewed jointly as a continuous-time Markov random walk on the vertices of a hypercube, the time parameter being map distance along the chromosome. The event that two individuals have a segment of chromosome in common, thus proving them to be related, corresponds to the random walk hitting a particular set of vertices. The probability of this happening is calculated for various types of relationship, making use of the symmetry of the situation to partition the vertices into a very much smaller number of orbits and render the computation manageable. The probability that an individual with n children passes on all his or her genes to them is also calculated in this way.     

Heparin is not metabolized by the perfused rat liver

The role of the liver in metabolism of heparin was studied using the isolated rat liver perfused in vitro for 10 hr. Porcine intestinal heparin (1000 u) was added to the recirculating liver perfusate, and serial heparin measurements were performed on the liver perfusate every 2 hr, as well as on bile samples secreted by the perfused liver. Heparin concentration remained at a constant level throughout the 10 hr of perfusion, and there was no detectable heparin secreted into bile samples. The findings suggest that hepatic metabolism/clearance plays a minimal role in heparin kinetics in plasma.     

Formaldehyde Treated Gelatin as a Gel Exclusion Agent

A high molecular weight fraction of gelatin precipitated from a solution of Difco gelatin with polyethylene glycol has several favourable properties in gel exclusion chromatography. To manufacture the gel exclusion agent, the gelatin was dissolved in hot water, allowed to set in the cold and rendered insoluble by reaction with formaldehyde. When finely disrupted and used as bed material in columns this gel is capable of separating protein components from mixtures of proteins of widely different molecular weights. Favourable properties include elasticity at relatively low concentration, its availability and its inexpensive nature.     

Spatial and temporal patterns of non-colonial boring organisms (polychaetes,sipunculans and bivalve molluscs) in Porites at Lizard Island,Great Barrier Reef

A study of the spatial and temporal patterns of colonisation by non-colonial boring organisms to dead Porites coral substrate was conducted at Lizard Island, Great Barrier Reef over a 4 year period. These fluctuations were analysed for each group of borers, and most exhibited strong site preferences, with preferred sites being on the windward slope in 10 m and on the reef flat in 1 m. A lagoonal patch reef site exhibited consistently low colonisation. Most groups showed inter-year variations in colonisation with spring/early summer dominating. These variations are discussed in terms of what is known about their life histories. These results together with those of Kiene (in preparation) which document varying rates of bioerosion, at these sites over the same time period, demonstrate that variations in borer colonisation are responsible for the variations in rates of bioerosion calculated. Thus rates of bioerosion by borers will vary significantly between different reef environments.     

Studies of the growth-regulating effects of ivermectin on larval Onchocerca lienalis in vitro

At concentrations of 0.1-100 ng/ml ivermectin inhibited L3-L4 molting by Onchocerca lienalis in vitro. The degree of inhibition was dose-dependent with a significant effect apparent at 0.1 ng/ml and complete inhibition occurring at 100 ng/ml. The ED50 for molt inhibition was 0.19 ng/ml. Molt-inhibiting levels of the drug were not acutely toxic to the worms. In the presence of 10 ng/ml, a concentration giving 95% molt inhibition, motility at day 7 postinoculation was 71% of that seen in nontreated controls. A more pronounced effect on motility was apparent in larvae under long-term cultivation in the presence of ivermectin. Kinetic studies indicated that the majority of the larvae respond irreversibly to the drug within the first 2 hr of exposure. Twenty-four hours of exposure were required for a maximal response. The inhibitory effects of ivermectin were less pronounced if larvae were allowed to develop under normal culture conditions for 24 or more hours prior to the initiation of drug treatment.