A PCR-based diagnostic tool for distinguishing grape skin color mutants

Berry skin color mutants are phenotypically different from their original cultivars, but they show identical molecular profile if analysed by using microsatellite markers. This work gives an easy, inexpensive and quick diagnostic tool to discriminate these somatic variants. We distinguished some grape (Vitis vinifera L.) skin color mutants from white to red or pink and from black to grey, pink or white and we investigated their molecular bases by single-strand conformational polymorphism (SSCP), single base primer extension and coding sequence analysis of anthocyanin biosynthetic enzyme genes and by polymerase chain reaction (PCR) analysis of VvmybA1 regulatory gene. Analyses of structural genes did not reveal polymorphisms between wild type and mutant cultivars but only among different varieties, whereas the study of VvmybA1 regulatory gene has given important outcomes for color mutants characterisation. The discrimination between white wild type and its derived colored mutant and between black wild type and white mutant has been obtained through a simple test of amplification for presence/absence. The discrimination between black wild type and less colored mutant has occurred through a quantitative result on agarose gel confirmed by real-time PCR analysis: the amount of functional allele in less colored somatic variants genome was about one-fourth of the correspondent quantity in original black cultivars genome.     

XHas2 activity is required during somitogenesis and precursor cell migration in Xenopus development

In vertebrates, hyaluronan biosynthesis is regulated by three transmembrane catalytic enzymes denoted Has1, Has2 and Has3. We have previously cloned the Xenopus orthologues of the corresponding genes and defined their spatiotemporal distribution during development. During mammalian embryogenesis, Has2 activity is known to be crucial, as its abrogation in mice leads to early embryonic lethality. Here, we show that, in Xenopus, morpholino-mediated loss-of-function of XHas2 alters somitogenesis by causing a disruption of the metameric somitic pattern and leads to a defective myogenesis. In the absence of XHas2, early myoblasts underwent apoptosis, failing to complete their muscle differentiation programme. XHas2 activity is also required for migration of hypaxial muscle cells and trunk neural crest cells (NCC). To approach the mechanism whereby loss of HA, following XHas2 knockdown, could influence somitogenesis and precursor cell migration, we cloned the orthologue of the primary HA signalling receptor CD44 and addressed its function through an analogous knockdown approach. Loss of XCD44 did not disturb somitogenesis, but strongly impaired hypaxial muscle precursor cell migration and the subsequent formation of the ventral body wall musculature. In contrast to XHas2, loss of function of XCD44 did not seem to be essential for trunk NCC migration, suggesting that the HA dependence of NCC movement was rather associated with an altered macromolecular composition of the ECM structuring the cells' migratory pathways. The presented results, extend our knowledge on Has2 function and, for the first time, demonstrate a developmental role for CD44 in vertebrates. On the whole, these data underlie and confirm the emerging importance of cell-ECM interactions and modulation during embryonic development.     

The effects of humic substances within the Ah horizon of a Calcic Luvisol on morphological changes related to invertase and peroxidase activities in wheat roots

The chemical and biological characteristics of humus within the Ah horizon (Calcic-Luvisol) have been studied. Attention was paid to variation in the NMR spectra of humic fractions and ¹³C values and to how these changes are related to different biological humic fraction activities.The chemical changes in particular involve the decrease of the aromatic component and the increase of the non-aromatic component within the horizon and the different ¹³C value not only within the horizon but also among the humic fractions distinctive of different molecular sizes.An attempt has been made to explain the vertical chemical changes in terms of processes affecting the biological characteristics of the high and low molecular size humic fractions. The main conclusions are that the low molecular size humic fractions, in the upper part of the horizon, are of greater importance with respect to the other humic fractions in influencing the enzyme activities linked to growth metabolism. The biological role of the high molecular size humic fractions characterised by a relevant content of peptidic- and carbohydratic-C is also presented.     

Influence of the tracer characteristics on hydrodynamic models of packed-bed bioreactors

A bench-scale horizontal-flow anaerobic immobilized sludge (HAIS) reactor filled with porous ceramic spheres (5?mm diameter) was used for evaluating the effects of the tracer characteristics on the residence time distribution (RTD) curves and on the parameters of the hydrodynamic mathematical models. Stimulus-response assays were carried out with bromophenol blue, dextran blue, eosin Y, mordant violet, rhodamine WT and bromocresol green as tracers. The reactor was operated at the hydraulic residence time (HRT) of approximately 2 hours and the flow characteristics were evaluated by fitting the single-parameter models of dispersion and N-continuous stirred tank reactors (CSTR) in series to the experimental data. Tracer characteristics were found to affect deeply the form of RTD curves and the apparent degree of mixing observed in the responses were attributed to the tracer diffusion into the porous media, except for dextran blue. The best adjustment was obtained for the N-CSTR in series model. Thereafter, dextran blue and rhodamine WT were used in hydrodynamic experiments in the HAIS reactor operating with additional residence times. Values of N ranging from 34 to 62 were obtained from the dextran blue experiments for HRT ranging from 2 to 7 hours. The application of the statistical analysis ANOVA one-way method indicated that there was no significant statistical difference in the flow-pattern within the range of hydraulic residence times applied. The mean N value of 9 was obtained from the experiments with rhodamine WT. Such disagreement in the responses was attributed to the diffusion of rhodamine WT into the porous media, interfering in the shape of the experimental curves.     

The state of the art of adeno-associated virus-based vectors in gene therapy

Background

Genotyping of HBV is generally used for determining the epidemiological relationship between various virus strains and origin of infection mostly in research studies. The utility of genotyping for clinical applications is only beginning to gain importance. Whether HBV genotyping will constitute part of the clinical evaluation of Hepatitis B patients depends largely on the availability of the relevance of the evidence based information. Since Pakistan has a HBV genotype distribution which has been considered less virulent as investigated by earlier studies from south East Asian countries, a study on correlation between HBV genotypes and risk of progression to further complex hepatic infection was much needed

Methods

A total of 295 patients with HBsAg positive were selected from the Pakistan Medical Research Council's (PMRC) out patient clinics. Two hundred and twenty six (77%) were males, sixty nine (23%) were females (M to F ratio 3.3:1).

Results

Out of 295 patients, 156 (53.2%) had Acute(CAH), 71 (24.2%) were HBV Carriers, 54 (18.4%) had Chronic liver disease (CLD) Hepatitis. 14 (4.7%) were Cirrhosis and HCC patients. Genotype D was the most prevalent genotype in all categories of HBV patients, Acute (108), Chronic (39), and Carrier (53). Cirrhosis/HCC (7) were HBV/D positive. Genotype A was the second most prevalent with 28 (13%) in acute cases, 12 (22.2%) in chronics, 14 (19.7%) in carriers and 5 (41.7) in Cirrhosis/HCC patients. Mixed genotype (A/D) was found in 20 (12.8%) of Acute patients, 3 (5.6%) of Chronic and 4 (5.6%) of carriers, none in case of severe liver conditions.

Conclusion

Mixed HBV genotypes A, D and A/D combination were present in all categories of patients except that no A/D combination was detected in severe conditions. Genotype D was the dominant genotype. However, genotype A was found to be more strongly associated with severe liver disease. Mixed genotype (A/D) did not significantly appear to influence the clinical outcome.     

New residuals for Cox regression and their application to outlier screening

The identification of individuals who 'died far too early' or 'lived far too long' as compared to their survival probabilities from a Cox regression can lead to the detection of new prognostic factors. Methods to identify outliers are generally based on residuals. For Cox regression, only deviance residuals have been considered for this purpose, but we show that these residuals are not very suitable. Instead, we develop and propose two new types of residuals: the suggested log-odds and normal deviate residuals are simple and intuitively appealing and their theoretical properties and empirical performance make them very suitable for outlier identification. Finally, various practical aspects of screening for individuals with outlying survival times are discussed by means of a cancer study example.     

The solution structure of the N-terminal proteinase domain of the hepatitis C virus (HCV) NS3 protein provides new insights into its activation and catalytic mechanism.

The solution structure of the hepatitis C virus (BK strain) NS3 protein N-terminal domain (186 residues) has been solved by NMR spectroscopy. The protein is a serine protease with a chymotrypsin-type fold, and is involved in the maturation of the viral polyprotein. Despite the knowledge that its activity is enhanced by the action of a viral protein cofactor, NS4A, the mechanism of activation is not yet clear. The analysis of the folding in solution and the differences from the crystallographic structures allow the formulation of a model in which, in addition to the NS4A cofactor, the substrate plays an important role in the activation of the catalytic mechanism. A unique structural feature is the presence of a zinc-binding site exposed on the surface, subject to a slow conformational exchange process.