Human Intestinal Allografts Contain Functional Hematopoietic Stem and Progenitor Cells that Are Maintained by a Circulating Pool

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Protein flexibility and intrinsic disorder

Comparisons were made among four categories of protein flexibility: (1) low-B-factor ordered regions, (2) high-B-factor ordered regions, (3) short disordered regions, and (4) long disordered regions. Amino acid compositions of the four categories were found to be significantly different from each other, with high-B-factor ordered and short disordered regions being the most similar pair. The high-B-factor (flexible) ordered regions are characterized by a higher average flexibility index, higher average hydrophilicity, higher average absolute net charge, and higher total charge than disordered regions. The low-B-factor regions are significantly enriched in hydrophobic residues and depleted in the total number of charged residues compared to the other three categories. We examined the predictability of the high-B-factor regions and developed a predictor that discriminates between regions of low and high B-factors. This predictor achieved an accuracy of 70% and a correlation of 0.43 with experimental data, outperforming the 64% accuracy and 0.32 correlation of predictors based solely on flexibility indices. To further clarify the differences between short disordered regions and ordered regions, a predictor of short disordered regions was developed. Its relatively high accuracy of 81% indicates considerable differences between ordered and disordered regions. The distinctive amino acid biases of high-B-factor ordered regions, short disordered regions, and long disordered regions indicate that the sequence determinants for these flexibility categories differ from one another, whereas the significantly-greater-than-chance predictability of these categories from sequence suggest that flexible ordered regions, short disorder, and long disorder are, to a significant degree, encoded at the primary structure level.     

Drug use among youths

The levels of drug use among youths under 14 have remained extremely low for the past few years, as well as in most European countries. Yet, patterns of early drug use are often related to high-risk behaviours that require specific public policy strategies. Over the last years, the Government has set the target of reducing levels of drug use among specific groups such as young people: an awareness-raising media campaign has been launched and a specific outpatients clinics setting has been implemented for cannabis users in particular. This paper examines the relationship between health service providers and criminal justice authorities underlying the effectiveness of the current system. It underlines the need for an early screening and early interventions so as to avoid the shift from simple use to misuse or dependence.     

Functional anthology of intrinsic disorder. 3. Ligands, post-translational modifications, and diseases associated with intrinsically disordered proteins

Currently, the understanding of the relationships between function, amino acid sequence, and protein structure continues to represent one of the major challenges of the modern protein science. As many as 50% of eukaryotic proteins are likely to contain functionally important long disordered regions. Many proteins are wholly disordered but still possess numerous biologically important functions. However, the number of experimentally confirmed disordered proteins with known biological functions is substantially smaller than their actual number in nature. Therefore, there is a crucial need for novel bionformatics approaches that allow projection of the current knowledge from a few experimentally verified examples to much larger groups of known and potential proteins. The elaboration of a bioinformatics tool for the analysis of functional diversity of intrinsically disordered proteins and application of this data mining tool to >200 000 proteins from the Swiss-Prot database, each annotated with at least one of the 875 functional keywords, was described in the first paper of this series (Xie, H.; Vucetic, S.; Iakoucheva, L. M.; Oldfield, C. J.; Dunker, A. K.; Obradovic, Z.; Uversky, V.N. Functional anthology of intrinsic disorder. 1. Biological processes and functions of proteins with long disordered regions. J. Proteome Res. 2007, 5, 1882-1898). Using this tool, we have found that out of the 710 Swiss-Prot functional keywords associated with at least 20 proteins, 262 were strongly positively correlated with long intrinsically disordered regions, and 302 were strongly negatively correlated. Illustrative examples of functional disorder or order were found for the vast majority of keywords showing strongest positive or negative correlation with intrinsic disorder, respectively. Some 80 Swiss-Prot keywords associated with disorder- and order-driven biological processes and protein functions were described in the first paper (see above). The second paper of the series was devoted to the presentation of 87 Swiss-Prot keywords attributed to the cellular components, domains, technical terms, developmental processes, and coding sequence diversities possessing strong positive and negative correlation with long disordered regions (Vucetic, S.; Xie, H.; Iakoucheva, L. M.; Oldfield, C. J.; Dunker, A. K.; Obradovic, Z.; Uversky, V. N. Functional anthology of intrinsic disorder. 2. Cellular components, domains, technical terms, developmental processes, and coding sequence diversities correlated with long disordered regions. J. Proteome Res. 2007, 5, 1899-1916). Protein structure and functionality can be modulated by various post-translational modifications or/and as a result of binding of specific ligands. Numerous human diseases are associated with protein misfolding/misassembly/misfunctioning. This work concludes the series of papers dedicated to the functional anthology of intrinsic disorder and describes approximately 80 Swiss-Prot functional keywords that are related to ligands, post-translational modifications, and diseases possessing strong positive or negative correlation with the predicted long disordered regions in proteins.     

Bioreactor studies of native and tissue engineered cartilage

Functional tissue engineering of cartilage involves the use of bioreactors designed to provide a controlled in vitro environment that embodies some of the biochemical and physical signals known to regulate chondrogenesis. Hydrodynamic conditions can affect in vitro tissue formation in at least two ways: by direct effects of hydrodynamic forces on cell morphology and function, and by indirect flow-induced changes in mass transfer of nutrients and metabolites. In the present work, we discuss the effects of three different in vitro environments: static flasks (tissues fixed in place, static medium), mixed flasks (tissues fixed in place, unidirectional turbulent flow) and rotating bioreactors (tissues dynamically suspended in laminar flow) on engineered cartilage constructs and native cartilage explants. As compared to static and mixed flasks, dynamic laminar flow in rotating bioreactors resulted in the most rapid tissue growth and the highest final fractions of glycosaminoglycans and total collagen in both tissues. Mechanical properties (equilibrium modulus, dynamic stiffness, hydraulic permeability) of engineered constructs and explanted cartilage correlated with the wet weight fractions of glycosaminoglycans and collagen. Current research needs in the area of cartilage tissue engineering include the utilization of additional physiologically relevant regulatory signals, and the development of predictive mathematical models that enable optimization of the conditions and duration of tissue culture.     

Optimization of PCR in application of hot start Taq DNA polymerase for detection of Erwinia amylovora with primers FER1-F and FER1-R

There are two approaches in detection of bacterium Erwinia amylovora by PCR. One is based on detection of plasmid pEA29 and the other is based on detection of a chromosomal DNA sequence, specific for E. amylovora, in a sample. Since pathogenic strains without pEA29 have been isolated from the environment, methods based on this plasmid have been compromised and PCR methods based on chromosomal DNA species specific sequences became only reliable methods. PCR method with chromosomal primers FER1-F and FER1-R is currently the most reliable method due to its high sensitivity and specificity. The goal of this research is to make a significant improvement of the method by optimization of PCR in application of hot start DNA Taq polymerase, instead of wax, to obtain a hot start reaction. This enzyme, which is currently widely applied, can provide simpler achievement of hot start, saving labor and time and decreasing possibility of cross contamination of samples. Experiments showed that simple replacement of a regular recombinant Taq DNA polymerase by a hot start Taq DNA polymerase leads to complete failure of the reaction. Many optimization experiments had to be carried out to obtain an operational and reliable PCR which simultaneously has high sensitivity and specificity. Content of the reaction mixture, as well as temperature and time parameters of PCR, were significantly changed to achieve proper optimization.     

Changes in cardiac Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase expression and activity in female rats fed a high-fat diet

The aim of this study was to investigate whether the presence of endogenous estradiol alters the effects of a high-fat (HF) diet on activity/expression of the cardiac Na⁺/K⁺-ATPase, via PI3K/IRS and RhoA/ROCK signalling cascades in female rats. For this study, female Wistar rats (8 weeks old, 150–200 g) were fed a standard diet or a HF diet (balanced diet for laboratory rats enriched with 42% fat) for 10 weeks. The results show that rats fed a HF diet exhibited a decrease in phosphorylation of the α₁ subunit of Na⁺/K⁺-ATPase by 30% (p < 0.05), expression of total α₁ subunit of Na⁺/K⁺-ATPase by 31% (p < 0.05), and association of IRS1 with p85 subunit of PI3K by 42% (p < 0.05), while the levels of cardiac RhoA and ROCK2 were significantly increased by 84% (p < 0.01) and 62% (p < 0.05), respectively. Our results suggest that a HF diet alters cardiac Na⁺/K⁺-ATPase expression via molecular mechanisms involving RhoA/ROCK and IRS-1/PI3K signalling in female rats.     

Gas exchange is essential for bioreactor cultivation of tissue engineered cartilage

Tissue engineered cartilage can be grown in vitro if the necessary physical and biochemical factors are present in the tissue culture environment. Cell metabolism and tissue composition were studied for engineered cartilage cultured for 5 weeks using bovine articular chondrocytes, polymer scaffolds (5 mm diameter x 2 mm thick fibrous discs), and rotating bioreactors. Medium pH and concentrations of oxygen, carbon dioxide, glucose, lactate, ammonia, and glycosoaminoglycan (GAG) were varied by altering the exchange rates of gas and medium in the bioreactors. Cell-polymer constructs were assessed with respect to histomorphology, biochemical composition and metabolic activity. Low oxygen tension ( approximately 40 mmHg) and low pH ( approximately 6.7) were associated with anaerobic cell metabolism (yield of lactate on glucose, YL/G, of 2.2 mol/mol) while higher oxygen tension ( approximately 80 mmHg) and higher pH ( approximately 7.0) were associated with more aerobic cell metabolism (YL/G of 1.65-1.79 mol/mol). Under conditions of infrequent medium replacement (50% once per week), cells utilized more economical pathways such that glucose consumption and lactate production both decreased, cell metabolism remained relatively aerobic (YL/G of 1.67 mol/mol) and the resulting constructs were cartilaginous. More aerobic conditions generally resulted in larger constructs containing higher amounts of cartilaginous tissue components, while anaerobic conditions suppressed chondrogenesis in 3D tissue constructs.     

Detection of <Emphasis Type="Italic">Erwinia amylovora</Emphasis> by novel chromosomal polymerase chain reaction primers

A new sensitive and specific method for the detection of Erwinia amylovora was developed. The method is based on the detection of a chromosomal DNA sequence specific for this bacterial species and enables detection of E. amylovora pathogenic strains, including recent isolates that lack plasmid pEA29 and thus cannot be detected by the previously popular PCR methods based on the detection of this plasmid. A species-specific random amplified polymorphic DNA (RAPD) marker was identified, cloned, and sequenced, and sequence characterized amplified region (SCAR) primers for specific PCR were developed. The E. amylovora specific sequence, 1269 bp long, was amplified in polymerase chain reaction and detected with electrophoresis in agarose gel stained with ethidium bromide. Amplification with other bacterial species did not produce any PCR product detectable by electrophoresis. Matching of the E. amylovora specific sequence to chromosomal DNA was confirmed by computer analysis of the E. amylovora genome. A consistent sensitivity limit of the method was 3 CFU/reaction, and in some cases it was possible to detect 0.6 CFU/reaction. Due to its high sensitivity and specificity, our method of E. amylovora detection is currently the most reliable, taking into account that the reliability of PCR methods based on plasmid pEA29 has been compromised by the isolation of pathogenic E. amylovora strains that lack this plasmid.     

Substring selection for biomedical document classification

MOTIVATION: Attribute selection is a critical step in development of document classification systems. As a standard practice, words are stemmed and the most informative ones are used as attributes in classification. Owing to high complexity of biomedical terminology, general-purpose stemming algorithms are often conservative and could also remove informative stems. This can lead to accuracy reduction, especially when the number of labeled documents is small. To address this issue, we propose an algorithm that omits stemming and, instead, uses the most discriminative substrings as attributes. RESULTS: The approach was tested on five annotated sets of abstracts from iProLINK that report on the experimental evidence about five types of protein post-translational modifications. The experiments showed that Naive Bayes and support vector machine classifiers perform consistently better [with area under the ROC curve (AUC) accuracy in range 0.92-0.97] when using the proposed attribute selection than when using attributes obtained by the Porter stemmer algorithm (AUC in 0.86-0.93 range). The proposed approach is particularly useful when labeled datasets are small.