Clinical application of VNTR-typing of Mycobacteria tuberculosis strains: monitoring of treatment quality and of laboratory service

An early diagnosis of super-infection and mixed infection of Mycobacteria tuberculosis is highly important for the correction of an assigned treatment and for a positive prognostication. An analysis of loci with varying tandem repeats (VNTR) is a simple in use and reproducible method of M. tuberculosis genotyping. Thirty-seven serial isolates of M. tuberculosis from 12 patients with pulmonary tuberculosis including changing resistance to TB drugs registered in follow-up and treatment underwent VNTR-typing for 6 loci: ETR-A, C, E, V1, V3 and V4. Both super-infection and mixed infection were shown in 33.3% of cases to cause changes in the profile of resistance to TB drugs. In 3 patients, changes in the drug resistance profile were accompanied by a substitution of a colonizing strain for an epidemic clonal variant of the Beijing family with the 445446 genotype. Whereas in one patient, there was a substitution of the Beijing strain for the genotype 222422 strain, which is typical of M. tuberculosis with the S42 spoligotype. One intermediate serial isolate from the above patient had a mixed culture of 2 polyresistant strains of M. tuberculosis (445476 + 222422). In one case, the substitution of the infecting strain for a strain with changed genotypes and profiles of resistance to antibiotics occurred twice within an interval of 5-6 months. The 343543 genotype strain changed for the 452562 genotype strain and then--for the 445466 genotype strain; the final genotype variant belonged to the Beijing family. Serial isolates from 8 patients retained their original genotype (Beijing). In such cases, the changing spectrum of resistance to TB drugs can be associated with the secondary drug resistance acquired by strains in the process of treatment or with errors of laboratory equipment. Finally, the VNTR analysis is a rapid, easy-in-use and effective tool for the systemic typing of M. tuberculosis isolates in clinical practice.     

Genetic control of embryo lethality in crosses between common wheat and rye

The phenotypic manifestation and genetic control of embryo lethality observed in crosses between common wheat and rye were studied. It was found that crosses between common wheat and inbred self-fertile rye lines L2 and 535 gave rise to ungerminating grains, in which the development and differentiation of the hybrid embryo are arrested. Study of the degree of embryo development in the hybrid grains obtained by crossing common wheat varieties with inbred rye lines L2 and 535 showed that genotypes of the parents affected the ratio between undifferentiated embryos of various sizes. Analysis of this trait was performed by test crosses according to a novel pedigree program with the use of interlinear hybrids and a set of fourth-generation hybrid recombinant inbred lines. Rye line L2 was shown to bear the Eml (Embryo lethality) gene, which terminates the development of the hybrid embryo in amphihaploids. The suggestion of complementary interaction between wheat and rye genes during formation of a "n ew" character in wheat-rye F1 hybrids is discussed. A method of detecting an allele not complementary to the rye Eml allele in wheat is proposed. The proposed test program allows appropriate study of the system of wheat and rye genes involved in complementary interaction in the genotype of a distant hybrid.     

Complete probabilistic analysis of RNA shapes

Background  

Soon after the first algorithms for RNA folding became available, it was recognised that the prediction of only one energetically optimal structure is insufficient to achieve reliable results. An in-depth analysis of the folding space as a whole appeared necessary to deduce the structural properties of a given RNA molecule reliably. Folding space analysis comprises various methods such as suboptimal folding, computation of base pair probabilities, sampling procedures and abstract shape analysis. Common to many approaches is the idea of partitioning the folding space into classes of structures, for which certain properties can be derived.     

Concomitant requirement for Notch and Jak/Stat signaling during neuro-epithelial differentiation in the Drosophila optic lobe

The optic lobe forms a prominent compartment of the Drosophila adult brain that processes visual input from the compound eye. Neurons of the optic lobe are produced during the larval period from two neuroepithelial layers called the outer and inner optic anlage (OOA, IOA). In the early larva, the optic anlagen grow as epithelia by symmetric cell division. Subsequently, neuroepithelial cells (NE) convert into neuroblasts (NB) in a tightly regulated spatio-temporal progression that starts at the edges of the epithelia and gradually move towards its centers. Neuroblasts divide at a much faster pace in an asymmetric mode, producing lineages of neurons that populate the different parts of the optic lobe. In this paper we have reconstructed the complex morphogenesis of the optic lobe during the larval period, and established a role for the Notch and Jak/Stat signaling pathways during the NE-NB conversion. After an early phase of complete overlap in the OOA, signaling activities sort out such that Jak/Stat is active in the lateral OOA which gives rise to the lamina, and Notch remains in the medial cells that form the medulla. During the third instar, a wave front of enhanced Notch activity progressing over the OOA from medial to lateral controls the gradual NE-NB conversion. Neuroepithelial cells at the medial edge of the OOA, shortly prior to becoming neuroblasts, express high levels of Delta, which activates the Notch pathway and thereby maintains the OOA in an epithelial state. Loss of Notch signaling, as well as Jak/Stat signaling, results in a premature NE-NB conversion of the OOA, which in turn has severe effects on optic lobe patterning. Our findings present the Drosophila optic lobe as a useful model to analyze the key signaling mechanisms controlling transitions of progenitor cells from symmetric (growth) to asymmetric (differentiative) divisions.     

Development of enzyme flow calorimeter system for monitoring of microbial glycerol conversion

Glycerokinase from Cellulomonas sp. was used to develop biosensor based on flow calorimetry for quantitative analysis of glycerol during bioconversion process. An automatic flow injection analysis device with the glycerol biosensor was built and tested during growth on glycerol of 1,3-propanediol-producing bacteria. The biosensor exhibited an extreme storage and operational stability enabling us to use it for more than 2 years without significant loss of sensitivity. No interference with 1,3-propanediol and fermentation medium was observed. The linear range of glycerol concentration up to 70 mM was extended by developed automatic dilution technique with the aim of automatic online monitoring of microbial process. The analytical system was able to monitor the bioconversion process in a fully automatic way during the whole run with sampling frequency of one sample per 10 min.     

Isotopomer analysis of myocardial substrate metabolism: a systems biology approach

The increasing accessibility of mass isotopomer data via GC-MS and NMR technology has necessitated the use of a systematic and reliable method to take advantage of such data for flux analysis. Here we applied a nonlinear, optimization-based method to study substrate metabolism in cardiomyocytes using (13)C data from perfused mouse hearts. The myocardial metabolic network used in this study accounts for 257 reactions and 240 metabolites, which are further compartmentalized into extracellular space, cytosol, and mitochondrial matrix. Analysis of the perfused mouse heart showed that the steady-state ATP production rate was 16.6 +/- 2.3 micromol/min . gww, with 30% of the ATP coming from glycolysis. Of the four substrates available in the perfusate (glucose, pyruvate, lactate, and oleate), exogenous glucose forms the majority of cytosolic pyruvate. Pyruvate decaboxylation is significantly higher than carboxylation, suggesting that anaplerosis is low in the perfused heart. Exchange fluxes were predicted to be high for reversible enzymes in the citric acid cycle (CAC), but low in the glycolytic pathway. Pseudoketogenesis amounted to approximately 50% of the net ketone body uptake. Sensitivity analysis showed that the estimated flux distributions were relatively insensitive to experimental errors. The application of isotopomer data drastically improved the estimation of reaction fluxes compared to results computed with respect to reaction stoichiometry alone. Further study of 12 commonly used (13)C glucose mixtures showed that the mixtures of 20% [U-(13)C(6)] glucose, 80% [3 (13)C] glucose and 20% [U-(13)C(6)] glucose, 80% [4 (13)C] were best for resolving fluxes in the current network.     

Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia

Avian influenza A (H5N1) viruses cause severe disease in humans, but the basis for their virulence remains unclear. In vitro and animal studies indicate that high and disseminated viral replication is important for disease pathogenesis. Laboratory experiments suggest that virus-induced cytokine dysregulation may contribute to disease severity. To assess the relevance of these findings for human disease, we performed virological and immunological studies in 18 individuals with H5N1 and 8 individuals infected with human influenza virus subtypes. Influenza H5N1 infection in humans is characterized by high pharyngeal virus loads and frequent detection of viral RNA in rectum and blood. Viral RNA in blood was present only in fatal H5N1 cases and was associated with higher pharyngeal viral loads. We observed low peripheral blood T-lymphocyte counts and high chemokine and cytokine levels in H5N1-infected individuals, particularly in those who died, and these correlated with pharyngeal viral loads. Genetic characterization of H5N1 viruses revealed mutations in the viral polymerase complex associated with mammalian adaptation and virulence. Our observations indicate that high viral load, and the resulting intense inflammatory responses, are central to influenza H5N1 pathogenesis. The focus of clinical management should be on preventing this intense cytokine response, by early diagnosis and effective antiviral treatment.     

Blood flow augmentation by intrinsic venular contraction in vivo

Venomotion, spontaneous cyclic contractions of venules, was first observed in the bat wing 160 years ago. Of all the functional roles proposed since then, propulsion of blood by venomotion remains the most controversial. Common animal models that require anesthesia and surgery have failed to provide evidence for venular pumping of blood. To determine whether venomotion actively pumps blood in a minimally invasive, unanesthetized animal model, we reintroduced the batwing model. We evaluated the temporal and functional relationship between the venous contraction cycle and blood flow and luminal pressure. Furthermore, we determined the effect of inhibiting venomotion on blood flow. We found that the active venous contractions produced an increase in the blood flow and exhibited temporal vessel diameter-blood velocity and pressure relationships characteristic of a peristaltic pump. The presence of valves, a characteristic of reciprocating pumps, enhances the efficiency of the venular peristaltic pump by preventing retrograde flow. Instead of increasing blood flow by decreasing passive resistance, venular dilation with locally applied sodium nitroprusside decreased blood flow. Taken together, these observations provide evidence for active venular pumping of blood. Although strong venomotion may be unique to bats, venomotion has also been inferred from venous pressure oscillations in other animal models. The conventional paradigm of microvascular pressure and flow regulation assumes venules only act as passive resistors, a proposition that must be reevaluated in the presence of significant venomotion.