Multi-cell type human liver microtissues for hepatotoxicity testing.

Current 2-dimensional hepatic model systems often fail to predict chemically induced hepatotoxicity due to the loss of a hepatocyte-specific phenotype in culture. For more predictive in vitro models, hepatocytes have to be maintained in a 3-dimensional environment that allows for polarization and cell-cell contacts. Preferably, the model will reflect an in vivo-like multi-cell type environment necessary for liver-like responses. Here, we report the characterization of a multi-cell type microtissue model, generated from primary human hepatocytes and liver-derived non-parenchymal cells. Liver microtissues were stable and functional for 5 weeks in culture enabling, for example, long-term toxicity testing of acetaminophen and diclofenac. In addition, Kupffer cells were responsive to inflammatory stimuli such as LPS demonstrating the possibility to detect inflammation-mediated toxicity as exemplified by the drug trovafloxacin. Herewith, we present a novel 3D liver model for routine testing in 96-well format capable of reducing the risk of unwanted toxic effects in the clinic.

Genetic diversity among Curtobacterium flaccumfaciens pv. flaccumfaciens populations in the American high plains.

Curtobacterium flaccumfaciens pv. flaccumfaciens is a Gram-positive bacterium and has reemerged as an incitant of bacterial wilt in common (dry, edible) beans in western Nebraska, eastern Colorado, and southeastern Wyoming. Curtobacterium flaccumfaciens pv. flaccumfaciens is diverse phenotypically and genotypically and is represented by several different pathogen color variants. The population structure of 67 strains collected between 1957 and 2009, including some isolated from alternate hosts, was determined with 3 molecular typing techniques: amplified fragment length polymorphism (AFLP), repetitive extragenic palindromic polymerase chain reaction (rep-PCR), and pulsed-field gel electrophoresis (PFGE). All 3 typing techniques showed a great degree of population heterogeneity, but they were not congruent in cluster analysis of the C. flaccumfaciens pv. flaccumfaciens populations. Cluster analysis of a composite data set (AFLP, PFGE, and rep-PCR) using averages from all experiments yielded 2 distinct groups: cluster A included strains with colonies of yellow, orange, and pink pigments, and cluster B had strains of only yellow pigment. Strains producing purple extracellular pigment were assigned to both clusters. Thus, C. flaccumfaciens pv. flaccumfaciens is diverse phenotypically and genotypically.

Genetic diversity and population structure of Clavibacter michiganensis subsp. nebraskensis.

Clavibacter michiganensis subsp. nebraskensis (CMN) is a gram-positive bacterium and an incitant of Goss's bacterial wilt and leaf blight or "leaf freckles" in corn. A population structure of a wide temporal and geographic collection of CMN strains (n = 131), originating between 1969 and 2009, was determined using amplified fragment length polymorphism (AFLP) analysis and repetitive DNA sequence-based BOX-PCR. Analysis of the composite data set of AFLP and BOX-PCR fingerprints revealed two groups with a 60% cutoff similarity: a major group A (n = 118 strains) and a minor group B (n = 13 strains). The clustering in both groups was not correlated with strain pathogenicity. Group A contained two clusters, A1 (n = 78) and A2 (n = 40), with a linkage of 75%. Group A strains did not show any correlation with historical, geographical, morphological, or physiological properties of the strains. Group B was very heterogeneous and eight out of nine clusters were represented by a single strain. The mean similarity between clusters in group B varied from 13% to 63%. All strains in group B were isolated after 1999. The percentage of group B strains among all strains isolated after 1999 (n = 69) was 18.8%. Implications of the findings are discussed.

[Age and gender features of tobacco smoking].

On the basis of the conducted research, prevalence of tobacco smoking and its intensity are described as well as dependence on gender and age in an urban population of the middle Volga region (on the example of the city of Samara). It is proved, that in a general selection among men and women smoke: 49,37 and 14,17%; smoked before: 22,80 and 8,86%; never smoked: 27,83 and 76,97% of inspected men and women, accordingly. The contribution of the education level and marriage status are set in epidemiology of tobacco smoking dependence. Authors consider the development of public and medical measures in a fight against tobacco smoking should be realized, taking into account the gender, age, social and mental features of population, which is a difficult, but necessary process.

Genetic Diversity in Populations of Xanthomonas campestris pv. campestris in Cruciferous Weeds in Central Coastal California.

ABSTRACT Xanthomonas campestris pv. campestris (X. campestris) infects a large number of cruciferous plants, including weeds. California has one of the largest and most diverse populations of wild cruciferous plants in the world. Although considerable information is available on the genetic diversity of X. campestris in commercial crop plants, nothing is known about the diversity in strains infecting weeds. To assess the genetic diversity among strains of X. campestris in weeds in noncultivated and cultivated areas, strains of the pathogen were isolated from populations of cruciferous weeds growing in coastal valley crop-production sites and from remote nonproduction sites along the California central coast. Results of fingerprinting over 68 strains using amplified fragment length polymorphism along with representative strains by sequence analysis showed the presence of seven genotypes. Genotypes A and B were limited to coastal sites; genotypes C, D, and E were from inland cultivated sites; and genotypes F and G were present in both coastal noncultivated and inland cultivated sites. Crop strains were grouped outside any weed strain group and were separated from the weed strains and other pathovars of X. campestris. These results revealed, for the first time, that strains of X. campestris present in noncultivated coastal weed populations generally were unique to a site and genetically distinct from strains present in populations of weeds in crop-production areas located nearby.

Genetic Characterization and Diversity of Rathayibacter toxicus.

ABSTRACT Rathayibacter toxicus is a nematode-vectored gram-positive bacterium responsible for a gumming disease of grasses and production of a highly potent animal and human toxin that is often fatal to livestock and has a history of occurring in unexpected circumstances. DNA of 22 strains of R. toxicus from Australia were characterized using amplified fragment length polymorphism (AFLP) and pulsed-field gel electrophoresis (PFGE). AFLP analysis grouped the 22 strains into three genetic clusters that correspond to their geographic origin. The mean similarity between the three clusters was 85 to 86%. PFGE analysis generated three different banding patterns that enabled typing the strains into three genotypic groups corresponding to the same AFLP clusters. The similarity coefficient was 63 to 81% for XbaI and 79 to 84% for SpeI. AFLP and PFGE analyses exhibited an analogous level of discriminatory power and produced congruent results. PFGE analysis indicated that the R. toxicus genome was represented by a single linear chromosome, estimated to be 2.214 to 2.301 Mb. No plasmids were detected.

Study of the mechanical properties of myomesin proteins using dynamic force spectroscopy.

Myomesin is the most prominent structural component of the sarcomeric M-Band that is expressed in mammalian heart and skeletal muscles. Like titin, this protein is an intracellular member of the Ig-fibronectin superfamily, which has a flexible filamentous structure and which is largely composed of two types of domain that are similar to immunoglobulin (Ig)-like and fibronectin type III (FNIII) domains. Several myomesin isoforms have been identified, and their expression patterns are highly regulated both spatially and temporally. Particularly, alternative splicing in the central part of the molecule gives rise to an isoform, EH (embryonic heart)-myomesin, containing a serine and proline-rich insertion with no well-defined secondary structure, the EH segment. EH-myomesin represents the major myomesin isoform at embryonic stages of mammalian heart and is rapidly down-regulated around birth, but it is re-expressed in the heart of patients suffering from dilated cardio-myopathy. Here, in order to facilitate a better understanding of the physiological, and possibly pathological, functions of myomesin proteins, we explore the mechanical stability, elasticity and force-driven structural changes of human myomesin's sub-molecular segments using single-molecule force spectroscopy and protein engineering. We find that human myomesin molecules are composed of modules (Ig and FNIII), that are designed to withstand force and we demonstrate that the human cardiac EH segment functions like an additional elastic stretch in the middle part of the EH-myomesin and behaves like a random coil. Consequently myomesin isoforms (proteins with or without the EH segment) have different elastic properties, the EH-myomesin being the more compliant one. These findings imply that the compliance of the M-band increases with the amount of EH-myomesin it contains. So, we provide the evidence that not only titin but also other sarcomeric proteins have complicated visco-elastic properties depending on the contractile parameters in different muscle types.

A novel marker for vertebrate embryonic heart, the EH-myomesin isoform.

Myomesin is a structural component of the M-band that is expressed in all types of striated muscle. Its primary function may be the maintenance of the thick filament lattice and its anchoring to the elastic filament system composed of titin. Different myomesin isoforms have been described in chicken and mice, but no particular function has been assigned to them. Here we investigate the spatio-temporal expression pattern of myomesin isoforms by means of reverse transcriptase-polymerase chain reaction and isoform-specific antibodies. We find that two alternative splicing events give rise to four myomesin isoforms in chicken contrary to only one splicing event with two possible isoforms in mice. A splicing event at the C terminus results in two splice variants termed H-myomesin and S-myomesin, which represent the major myomesin species in heart and skeletal muscle of avian species, respectively. In contrast, in mammalian heart and skeletal muscle only S-myomesin is expressed. In embryonic heart of birds and mammals, alternative splicing in the central part of the molecule gives rise to the isoform that we termed EH-myomesin. It represents the major myomesin isoform at early embryonic stages of heart but is rapidly down-regulated around birth. Thus, the strict developmental regulation of the EH-myomesin makes it an ideally suited marker for embryonic heart.

[Role of the vitamin factor in preventing phenol poisoning]. / Rol' vitaminnogo faktora v profilaktike intoksikatsii fenolom.

Experiments on rats were made to examine the effect of vitamin B1, pantothenic and ascorbic acids on the acetylation system and some characteristics of protein metabolism under chronic exposure to phenol. Inhibition of phenol vapours led to inhibition of the acetylation on the 105th day of the experiment, to accumulation of pyruvic acid by the blood and diurnal urine, to elevation of cholesterol content in the blood serum. The total content of protein and protein fractions in the blood serum remained unchanged. Additional vitaminization of the animals with thiamine (150 micrograms), calcium pantothenate (650 micrograms) or with their mixture containing ascorbic acid (2 mg) resulted in normalization of the test characteristics of carbohydrate and fat metabolism. The data obtained and the clinical trials carried out by the authors suggest introduction of the physiological doses of thiamine, calcium pantothenate and ascorbic acid into the diet of the workers in order to prevent phenol poisonings more effectively.