Applications of flow cytometry to clinical microbiology

Classical microbiology techniques are relatively slow in comparison to other analytical techniques, in many cases due to the need to culture the microorganisms. Furthermore, classical approaches are difficult with unculturable microorganisms. More recently, the emergence of molecular biology techniques, particularly those on antibodies and nucleic acid probes combined with amplification techniques, has provided speediness and specificity to microbiological diagnosis. Flow cytometry (FCM) allows single- or multiple-microbe detection in clinical samples in an easy, reliable, and fast way. Microbes can be identified on the basis of their peculiar cytometric parameters or by means of certain fluorochromes that can be used either independently or bound to specific antibodies or oligonucleotides. FCM has permitted the development of quantitative procedures to assess antimicrobial susceptibility and drug cytotoxicity in a rapid, accurate, and highly reproducible way. Furthermore, this technique allows the monitoring of in vitro antimicrobial activity and of antimicrobial treatments ex vivo. The most outstanding contribution of FCM is the possibility of detecting the presence of heterogeneous populations with different responses to antimicrobial treatments. Despite these advantages, the application of FCM in clinical microbiology is not yet widespread, probably due to the lack of access to flow cytometers or the lack of knowledge about the potential of this technique. One of the goals of this review is to attempt to mitigate this latter circumstance. We are convinced that in the near future, the availability of commercial kits should increase the use of this technique in the clinical microbiology laboratory.     

Molecular and functional characterization of a mutant allele of the mitogen-activated protein-kinase geneSLT2(MPK1) rescued from yeast autolytic mutants

 We have further characterized the functionality of the Saccharomyces cerevisiae gene SLT2(MPK1), coding for a MAP-kinase homolog essential for cell integrity, which is involved in the Pkc1p signalling pathway. This gene was isolated on the basis of its capacity to complement the thermosensitive-autolytic, osmotic-remediable phenotype of lyt2 mutants. Both slt2Δ and lyt2 mutants displayed a caffeine-sensitive phenotype consisting of cell lysis that was not dependent on temperature. Caffeine concentrations affecting the growth of these mutant strains were dependent on the genetic background, the SSD1 allele being very significant in this regard. The SLT2 allele of several lyt2 strains was both rescued and amplified by PCR. The recovered allele was shown to be non-functional as it could not complement the lytic phenotype of both deletion (slt2Δ) and lyt2 strains. After nucleotide sequencing of the recovered allele, we found that the defect of lyt2 mutants consists in a substitution of an aspartic acid for a glycine at position 35 of the amino-acid sequence of Slt2p. Gly35 is the third glycine of a glycine cluster (Gly-X-Gly-X-X-Gly), a conserved region in protein kinases and other nucleotide-binding proteins. Received: 13 October/27 November 1995     

High Blood Pressure Induced by Audiovisual Stimulation in Young Rats: Effect of Antihypertensive Agents

ABSTRACT

High systolic blood pressure (BP) was induced in young Wistar rats by daily exposure for 30 mins to environmental stimuli consisting of intense (100 dB) sound of 7500 cps and flashing light (0.3 cps). Maximal BP enhancement was obtained after the application of both these stimuli for 3 consecutive days. Such a hypertensive response was detected 24 hrs, but not 1 hr after the trial. High BP backed to normal 72 hrs after discontinuation of the audiovisual stimulation trials. Clinically effective antihypertensive agents (clonidine, prazosin, propranolol, practolol and metoprolol) as well as a new compound under investigation (cicletanine) were proved active in this model. Normotensive nonstressed rats run in parallel did not show any variation in their BP after administration of the same dose of the drugs assayed. This experimental model of hypertension may be a useful tool not only to test new antihypertensive agents but also to study the intriguing question of the role of stress in the development and maintenance of essential hypertension.     

The disproportionate burden of HIV and STIs among male sex workers in Mexico City and the rationale for economic incentives to reduce risks

Introduction

The objective of this article is to present the rationale and baseline results for a randomized controlled pilot trial using economic incentives to reduce HIV and sexually transmitted infection (STI) risk among male sex workers (MSWs) in Mexico City.

Methods

Participants (n=267) were tested and treated for STIs (chlamydia, gonorrhoea, syphilis and HIV) and viral hepatitis (hepatitis B and C), received HIV and STI prevention education and were randomized into four groups: (1) control, (2) medium conditional incentive ($50/six months), (3) high conditional incentive ($75/six months) and (4) unconditional incentive ($50/six months). In the conditional arms, incentives were contingent upon testing free of new curable STIs (chlamydia, gonorrhoea and syphilis) at follow-up assessments.

Results

Participants’ mean age was 25 years; 8% were homeless or lived in a shelter, 16% were unemployed and 21% lived in Mexico City less than 5 years. At baseline, 38% were living with HIV, and 32% tested positive for viral hepatitis or at least one STI (other than HIV). Participants had a mean of five male clients in the previous week; 18% reported condomless sex with their last client. For 37%, sex work was their main occupation and was conducted mainly on the streets (51%) or in bars/discotheques (24%) and hotels (24%). The average price for a sex transaction was $25 with a 35% higher payment for condomless sex.

Conclusions

The findings suggest that economic incentives are a relevant approach for HIV prevention among MSWs, given the market-based inducements for unprotected sex. This type of targeted intervention seems to be justified and should continue to be explored in the context of combination prevention efforts.     

@Note: A workbench for Biomedical Text Mining

Biomedical Text Mining (BioTM) is providing valuable approaches to the automated curation of scientific literature. However, most efforts have addressed the benchmarking of new algorithms rather than user operational needs. Bridging the gap between BioTM researchers and biologists’ needs is crucial to solve real-world problems and promote further research.We present @Note, a platform for BioTM that aims at the effective translation of the advances between three distinct classes of users: biologists, text miners and software developers. Its main functional contributions are the ability to process abstracts and full-texts; an information retrieval module enabling PubMed search and journal crawling; a pre-processing module with PDF-to-text conversion, tokenisation and stopword removal; a semantic annotation schema; a lexicon-based annotator; a user-friendly annotation view that allows to correct annotations and a Text Mining Module supporting dataset preparation and algorithm evaluation.@Note improves the interoperability, modularity and flexibility when integrating in-home and open-source third-party components. Its component-based architecture allows the rapid development of new applications, emphasizing the principles of transparency and simplicity of use. Although it is still on-going, it has already allowed the development of applications that are currently being used.