Type 1 diabetes genetic risk score discriminates between monogenic and Type 1 diabetes in children diagnosed at the age of <5 years in the Iranian population.

AIM: To examine the extent to which discriminatory testing using antibodies and Type 1 diabetes genetic risk score, validated in European populations, is applicable in a non-European population. METHODS: We recruited 127 unrelated children with diabetes diagnosed between 9 months and 5 years from two centres in Iran. All children underwent targeted next-generation sequencing of 35 monogenic diabetes genes. We measured three islet autoantibodies (islet antigen 2, glutamic acid decarboxylase and zinc transporter 8) and generated a Type 1 diabetes genetic risk score in all children. RESULTS: We identified six children with monogenic diabetes, including four novel mutations: homozygous mutations in WFS1 (n=3), SLC19A2 and SLC29A3, and a heterozygous mutation in GCK. All clinical features were similar in children with monogenic diabetes (n=6) and in the rest of the cohort (n=121). The Type 1 diabetes genetic risk score discriminated children with monogenic from Type 1 diabetes [area under the receiver-operating characteristic curve 0.90 (95% CI 0.83-0.97)]. All children with monogenic diabetes were autoantibody-negative. In children with no mutation, 59 were positive to glutamic acid decarboxylase, 39 to islet antigen 2 and 31 to zinc transporter 8. Measuring zinc transporter 8 increased the number of autoantibody-positive individuals by eight. CONCLUSIONS: The present study provides the first evidence that Type 1 diabetes genetic risk score can be used to distinguish monogenic from Type 1 diabetes in an Iranian population with a large number of consanguineous unions. This test can be used to identify children with a higher probability of having monogenic diabetes who could then undergo genetic testing. Identification of these individuals would reduce the cost of treatment and improve the management of their clinical course.

Diversity and genetic stability in banana genotypes in a breeding program using inter simple sequence repeats (ISSR) markers.

Banana (Musa spp) is a fruit species frequently cultivated and consumed worldwide. Molecular markers are important for estimating genetic diversity in germplasm and between genotypes in breeding programs. The objective of this study was to analyze the genetic diversity of 21 banana genotypes (FHIA 23, PA42-44, Maçã, Pacovan Ken, Bucaneiro, YB42-47, Grand Naine, Tropical, FHIA 18, PA94-01, YB42-17, Enxerto, Japira, Pacovã, Prata-Anã, Maravilha, PV79-34, Caipira, Princesa, Garantida, and Thap Maeo), by using inter-simple sequence repeat (ISSR) markers. Material was generated from the banana breeding program of Embrapa Cassava & Fruits and evaluated at Embrapa Coastal Tablelands. The 12 primers used in this study generated 97.5% polymorphism. Four clusters were identified among the different genotypes studied, and the sum of the first two principal components was 48.91%. From the Unweighted Pair Group Method using Arithmetic averages (UPGMA) dendrogram, it was possible to identify two main clusters and subclusters. Two genotypes (Garantida and Thap Maeo) remained isolated from the others, both in the UPGMA clustering and in the principal cordinate analysis (PCoA). Using ISSR markers, we could analyze the genetic diversity of the studied material and state that these markers were efficient at detecting sufficient polymorphism to estimate the genetic variability in banana genotypes.

Genetic diversity of Saccharum complex using ISSR markers.

Sugarcane (Saccharum sp, Poaceae) is native to Southeast Asia, and due to growing demand as raw material, its cultivation recently expanded to new frontiers. The genetic diversity analysis is essential for targeting strategies in the formation and maintenance of a germplasm. This study aimed to assess the genetic diversity of 26 accessions of sugarcane from the Active Germplasm Bank of Embrapa Coastal Tablelands, using inter-simple sequence repeat (ISSR) molecular markers. Sixteen primers were used, resulting in 87 fragments with 91.13% of polymorphism. The similarity of the individuals ranged between 0.22 and 0.87. Individuals RB867515 and RB92579 were closer genetically, and the most distant ones were PI240785 and NSL 291970. Four distinct clusters were formed, using UPGMA. This information can be used to prioritize the selection of accessions for the conduction of hybridization in breeding and germplasm exchange actions.

Analysis of genetic diversity of Laeliinae (Orchidaceae) in the State of Sergipe using ISSR markers.

The Orchidaceae represent one of the largest and most diverse families on the planet. However, this family is constantly threatened by predators and by the advancement of urban centers over its natural habitats. The objective of this study was to use inter-simple sequence repeat markers to evaluate the genetic diversity between orchid accessions of the Laeliinae subtribe, which comprise part of the Orchidaceae study collection at the Department of Agronomic Engineering of the Federal University of Sergipe. DNA was extracted from each specimen by using an adapted 2% cetyltrimethyl ammonium bromide protocol. Similarity between individuals was calculated using the Jaccard method. Clustering was carried out by the unweighted pair group method with arithmetic mean method, with resampling and 10,000 bootstraps. Eighty-seven fragments were obtained, all of which were polymorphic, revealing high variability between accessions. The mean similarity was 35.77% between Encyclia sp individuals, and 35.90% between specimens of Cattleya tigrina. For Epidendrum secundum, a relationship between geographic and genetic distances was observed, and the accession collected in the southern part of the State of Sergipe (Serra de Itabaiana National Park) was more divergent than that of the other parts of the state. The data generated in this study will guide further research aimed at the ex situ conservation of these materials.

Genetic diversity in natural populations of mangaba in Sergipe, the largest producer State in Brazil.

Mangaba (Hancornia speciosa Gomes) is found in areas of coastal tablelands in the Brazilian Northeast and Cerrado regions. This species has been subjected to habitat fragmentation that is mainly due to human activity, and requires conservation strategies. The aim of this study was to analyze the structure and inter- and intrapopulation genetic diversity of natural populations of H. speciosa Gomes using inter-simple sequence repeat (ISSR) molecular markers. A total of 155 individuals were sampled in 10 natural populations (ITA, PAC, IND, EST, BC, PIR, JAP, BG, NEO, and SANT) in the State of Sergipe, Brazil. Fifteen primers were used to generate 162 fragments with 100% polymorphism. Genetic analysis showed that the variability between populations (77%) was higher than within populations (23%). It was possible to identify five different groups by the unweighted pair group method with arithmetic mean and principal coordinate analysis, and only one individual (E10) remained isolated. Using ISSR markers it was possible to obtain a molecular profile of the populations evaluated, showing that these markers were effective and exhibited sufficient polymorphism to estimate the genetic variability of natural populations of H. speciosa Gomes.

Estimation of genetic diversity in a natural population of cambui tree (Myrciaria tenella O. Berg) using ISSR markers.

Cambui (Myrciaria tenella O. Berg) is a native species from Brazil, which belongs to the family Myrtaceae. Molecular characterization is one of the most used tools for the study of the biotechnological potential of species because the diversity level between individuals can be inferred. Analysis of genetic diversity is fundamental to the direction of the strategies necessary to form and maintain a germplasm. This study aimed to evaluate the genetic diversity in a natural population of cambui using inter-simple sequence repeat (ISSR) molecular markers. The natural population, which provided the plant material, is found at the Private Reserve of Natural Heritage of Caju, which belongs to the experimental field of Embrapa Tabuleiros Costeiros, in the municipality of Itaporanga d'Ajuda, SE, Brazil. Young leaves of each individual were collected for DNA extraction and analysis of PCR-ISSR. Thirty primers were tested and the top 10 were selected. The use of these primers resulted in 71 fragments with 98.3% polymorphism. Similarity of individuals ranged between 0.30 and 0.92. The most similar individuals were C13 and C17 and the most distant were C1 and C41. Through UPGMA, six distinct groups were identified. This information may be used for conservation of these genetic resources, germplasm exchange, creation of germplasm bank and in future studies with this species.

Genetic diversity analysis of Varronia curassavica Jacq. accessions using ISSR markers.

Varronia curassavica Jacq. is a medicinal and aromatic plant from Brazil with significant economic importance. Studies on genetic diversity in active germplasm banks (AGB) are essential for conservation and breeding programs. The aim of this study was to analyze the genetic diversity of V. curassavica accessions of the AGB of Medicinal and Aromatic Plants of the Federal University of Sergipe (UFS), using inter-simple sequence repeat molecular markers. Twenty-four primers were tested, and 14 were polymorphic and informative, resulting in 149 bands with 97.98% polymorphism. The UPGMA dendrogram divided the accessions into Clusters I and II. Jaccard similarity coefficients for pair-wise comparisons of accessions ranged between 0.24 and 0.78. The pairs of accessions VCUR-001/VCUR-503, VCUR-001/VCUR-504, and VCUR-104/VCUR-501 showed relatively low similarity (0.24), and the pair of accessions VCUR-402/VCUR- 403 showed medium similarity (0.78). Twenty-eight accessions were divided into three distinct clusters, according to the STRUCTURE analysis. The genetic diversity of V. curassavica in the AGB of UFS is low to medium, and it requires expansion. Accession VCUR-802 is the most suitable for selection in breeding program of this species, since it clearly represents all of the diversity present in the AGB.

Populations of Erythrina velutina Willd. at risk of extinction.

The goal of this study was to characterize the structure of two natural populations of the coral tree using RAPD and ISSR markers. The study evaluated all individuals in two different areas in the northeastern region of Brazil: the first was in the riparian area, 10 km x 100 m along the edge of the lower São Francisco River, and the second was in the municipality of Pinhão, in a semiarid region between the municipalities of Neópolis and Santana do São Francisco. We used all the coral trees present in those two areas (37 individuals). The results of the RAPD and ISSR markers were highly congruent, supporting the reliability of the techniques used. Similarity was estimated using the Jaccard arithmetic complement index. A dendrogram was constructed using the unweighted pair group method with arithmetic mean cluster algorithm, and the robustness of the data was bootstrapped with 5000 replicates. A principal coordinate analysis was performed on the basis of Jaccard coefficients. The total genetic variation observed was 21%, corresponding to the variation between the populations, and 79% of the variation was observed within the populations.

Molecular characterization of bromeliads from northeast Brazil.

Bromeliaceae is an important botany family that includes many species with economic value; demand for members of this family is increasing. However, illegal collection frequently occurs, drastically reducing the species populations; thus, it is necessary to collect and store Bromeliaceae genetic material. In this study, we identified and quantified genetic variability of the Bromeliad family using dominant markers to create the first Germplasm Bank in the northeast region of Brazil. Molecular tools were used to characterize the collected accessions. The combination of 11 inter-simple sequence repeats and 13 random amplified polymorphic DNA markers were used to detect the genetic variability of wild bromeliad accessions.

Effect of shark cartilage derived protein on the NK cells activity.

CONTEXT: Shark cartilage has been used for its beneficial effects on various diseases. There are evidences, that shark cartilage stimulates cellular and humoral immune responses, which makes it an anti-tumor and immunomodulator candidate. OBJECTIVE: The immunostimulatory effect of shark cartilage derived proteins on the cytotoxic activity of natural killer (NK) cells from healthy human peripheral blood mononuclear cells was studied. MATERIAL AND METHODS: The shark cartilage was extracted and its bioactive proteins were purified using ion-exchange chromatography (DE-52) and sequential fractionation on Amicon ultrafiltration membranes. The effect of each protein fraction on the modulation of cytotoxic activity of NK cells, as effectors, against K562, as target cells, was assayed by enzymatic lactate dehydrogenase test. RESULTS: The most immunostimulatory effect on the cytotoxic activity of NK cells was observed for AR10 fraction, containing proteins with molecular weight of about 14.5 kDa on the reducible discontinuous sodium dodecyl sulfate polyacrylamide gel electrophoresis. DISCUSSION: Among the examined shark cartilage derived proteins, the most immunostimulatory effects on the NK cells cytotoxicity was found for AR10 fraction with molecular weight of about 14 kDa. We propose-the direct interactions of shark cartilage derived proteins with NK cells surface receptors may lead to the enhancing in the cytotoxic activity of NK cells. CONCLUSION: Thus AR10 fraction, proteins of about 14.5 kDa, has a novel immunostimulatory effect on the NK cells activity in vitro and if confirmed by in vivo trials, it may lead to its future clinical applications as, immunotherapy of cancer, HIV, and augmentation of host immune system related immunodeficiency disorders.

Understanding the context of male and transgender sex work using peer ethnography.

OBJECTIVES: To distinguish between three distinct groups of male and transgender sex workers in Pakistan and to demonstrate how members of these stigmatized groups need to be engaged in the research process to go beyond stated norms of behaviour. METHODS: A peer ethnography study was undertaken in a major city in Pakistan. 15 male and 15 transgender sex workers were trained as peer researchers to each interview three peers in their network. Analysis was based on interviews with peer researchers as well as observation of dynamics during training and analysis workshops. RESULTS: The research process revealed that, within the epidemiological category of biological males who sell sex, there are three sociologically different sexual identities: khusras (transgender), khotkis (feminized males) and banthas (mainstream male identity). Both khusras and khotkis are organised in strong social structures based on a shared identity. While these networks provide emotional and material support, they also come with rigid group norms based on expected "feminine" behaviours. In everyday reality, sex workers showed fluidity in both behaviour and identity according to the situational context, transgressing both wider societal and group norms. The informal observational component in peer ethnography was crucial for the accurate interpretation of interview data. Participant accounts of behaviour and relationships are shaped by the research contexts including who interviews them, at what stage of familiarity and who may overhear the conversation. CONCLUSIONS: To avoid imposing a "false clarity" on categorisation of identity and assumed behaviour, it is necessary to go beyond verbal accounts to document the fluidity of everyday reality.

Generation of soluble P- and E-selectins in vivo is dependent on expression of P-selectin glycoprotein ligand-1.

BACKGROUND: Factors contributing to the generation of soluble P- and E-selectins remain unclear. RESULTS: This work demonstrates that mice lacking P-selectin glycoprotein ligand-1 (Psgl-1(-/-)) are deficient in soluble P-selectin (sP-sel), which is due to a defective binding interaction between PSGL-1 and P-sel, because mice lacking alpha(1,3)-fucosyltransferase-VII are also deficient in sP-sel. Psgl-1(-/-) mice are also deficient in soluble E-selectin (sE-sel) indicating that leukocyte interactions with endothelial cells lead to the generation of sE-sel. The generation of sE-sel requires an interaction between PSGL-1 and P-sel, as deficiency of sE-sel is observed in both Psgl-1(-/-) and P-sel(-/-) mice. Bone marrow transplantation from Psgl-1(-/-) to Psgl-1(+/+) mice leads to deficiency of sP-sel and sE-sel in recipient mice, establishing the importance of bone marrow-derived PSGL-1 toward the generation of sP-sel and sE-sel. Bone marrow transplantation from P-sel(-/-) to P-sel1(+/+) mice does not lead to a significant reduction in sP-sel, confirming the importance of the endothelium toward the liberation of sP-sel. CONCLUSION: sP-sel and sE-sel reflect an interaction between leukocyte PSGL-1 and endothelial P-sel.

Effects of hyperthermia on the colony-stimulating factor production by the lung.

Hyperthermic treatment of the murine lung in the range of 40-46 degrees C inhibited the production of colony-stimulating factors by the lung in vitro. This inhibition was dose dependent. Thermodynamic analysis was used to determine the activation energies. The Arrhenius plot contained a transition at 43 degrees C. At temperatures below and above the transition temperature, the activation energies were 40.49 and 197 kcal/mole, respectively. Below the transition temperature, the effect of hyperthermia was characterized by a delayed response represented by the broad initial shoulder of the hyperthermic dose-response curves. To investigate the mechanism of hyperthermia-induced reduction of the colony-stimulating factor production, the effect of hyperthermia on the protein synthesis by the lung was also studied. The results indicated an immediate response to hyperthermia, characterized by the absence of the initial shoulder and the high slope of the hyperthermic dose response curves. The corresponding Arrhenius plot did not have any transition point. The single activation energy calculated was 97.25 kcal/mole. It is concluded that the hyperthermic depression of the colony-stimulating factor production by the lung cannot be explained solely on the basis of the effects of hyperthermia on the protein synthesis.

The effect of indomethacin on colony-stimulating-factor production by the lung.

In this study, indomethacin was used to investigate the production of colony-stimulating-factor by the lung tissue. Addition of various concentrations of indomethacin (10(-5)-1 mg/ml) to the lung culture showed that it enhances CSF production in a dose dependent manner. The number of colonies reached a maximum at 1 microgram/ml and gradually diminished at higher concentrations. Addition of exogenous E-series prostaglandins alone had no effect on the CSF activity of normal lung. However, in the presence of indomethacin, E-series prostaglandins reversed the enhancing effect produced by indomethacin. On the other hand, cAMP or its dibutyryl derivative also increased CSF production. Removal of alveolar macrophages from the lung by lavaging had no effect on the CSF production by the lung but reduced the enhancing effect of indomethacin by 50%. The results suggest that indomethacin stimulates CSF production and that this process is partially regulated by prostaglandins and cAMP.

Dose and time dependent effects of caffeine on superoxide release, cell survival and DNA fragmentation of alveolar macrophages from rat lung.

In this study, the effect of two concentration ranges of the cAMP phosphodiesterase inhibitor, caffeine, on alveolar macrophage function was investigated by measuring survival rate, superoxide anion production and DNA fragmentation. The results show caffeine induced apoptosis in alveolar macrophages in a dose dependent manner. The survival rate of the cells exposed to low concentrations of caffeine (<5 mM) increased remarkably with a peak at 2.5 mM. At this concentration, caffeine failed to affect superoxide anion production and DNA degradation. However, at higher concentrations (5-20 mM), at which the viability was higher than the control, a significant increase in both superoxide production and DNA degradation, as judged by agarose gel and diphenylamine reaction, was obtained for 3 and 24 h of culture. The effect of caffeine on survival rate was also time dependent. At low caffeine concentrations, macrophages survived with a viability of 90-97% after 3 days. At moderate concentrations, the cells maintained viability up to 24 h but at concentrations higher than 20 mM, caffeine inhibited cell survival and killed a fraction of the population. The results suggest that low concentrations of caffeine prevent apoptosis of macrophages, whereas at moderate concentrations caffeine induces apoptosis in these cells. The results are discussed in relation to the mechanism of cAMP.

Lead nitrate induced apoptosis in alveolar macrophages from rat lung.

In this work we have attempted to characterize the programmed cell death (apoptosis) in alveolar macrophages exposed to various concentrations of lead nitrate. It was found that after 3 h of exposure a significant increase in superoxide anion production was observed, i.e. the number of trypan blue - exculding cells, was unchanged (< or = 95%) with any dose of lead employed. Agarose gel electrophoresis and diphenylamin reaction analysis revealed the occurrence of internucleosomal DNA fragmentation evaluated using cytological analysis by fluorescence dyes, suggesting that lead nitrate at low concentrations and short periods of exposure leads macrophages into apoptosis. However, time course studies showed that beyond 3 h, toxicity occurs, which could be attenuated by phosphodiesterase inhibitors, such as caffeine, suggesting a possible mechanism involving cAMP.

Interaction of a low mobility group protein, LMG160, with deoxyribonucleic acid.

A fraction of low mobility group (LMG) nonhistone protein designated LMG(160) was isolated from rat liver chromatin by preparative gel electrophoresis and its interaction with DNA was studied using thermal denaturation and DNA-cellulose affinity chromatography techniques. The results showed that LMG(160) with an isoelecteric point of 5-5.5 was bound to DNA and decreased its melting temperature. Increasing ionic strengths decreased this effect. DNA-cellulose affinity chromatography showed the affinity of LMG(160) to double stranded DNA was higher than that to single stranded DNA, since it required 0.6 M NaCl for elution. The results suggest that LMG(160) protein preferentially binds to double stranded DNA destabilizes it and the binding is electrostatic.

The effects of daunomycin antibiotic on histone H(1): thermal denaturation and fluorescence spectroscopy studies.

Using thermal denaturation and fluorescence spectroscopy, we have investigated the interaction of antitumor antibiotic, daunomycin, with calf thymus histone H(1) under several ionic strengths. The results show that daunomycin binds to histone H(1) and increases its melting temperature. Increasing ionic strength elevates this effect. Fluorescence emission data show that the interaction of daunomycin with histone H(1) decreases the emission intensity at 325 nm and induces hyperchromicity in the emission spectrum of the drug. The results suggest that histone H(1) can be considered as a new target for drug action at the chromatin level.

Three quantitative approaches to the diagnosis of abdominal pain in children: practical applications of decision theory.

BACKGROUND/PURPOSE: The authors compared 3 quantitative methods for assisting clinicians in the differential diagnosis of abdominal pain in children, where the most common important endpoint is whether the patient has appendicitis. Pretest probability in different age and sex groups were determined to perform Bayesian analysis, binary logistic regression was used to determine which variables were statistically significantly likely to contribute to a diagnosis, and recursive partitioning was used to build decision trees with quantitative endpoints. METHODS: The records of all children (1,208) seen at a large urban emergency department (ED) with a chief complaint of abdominal pain were immediately reviewed retrospectively (24 to 72 hours after the encounter). Attempts were made to contact all the patients' families to determine an accurate final diagnosis. A total of 1,008 (83%) families were contacted. Data were analyzed by calculation of the posttest probability, recursive partitioning, and binary logistic regression. RESULTS: In all groups the most common diagnosis was abdominal pain (ICD-9 Code 789). After this, however, the order of the most common final diagnoses for abdominal pain varied significantly. The entire group had a pretest probability of appendicitis of 0.06. This varied with age and sex from 0.02 in boys 2 to 5 years old to 0.16 in boys older than 12 years. In boys age 5 to 12, recursive partitioning and binary logistic regression agreed on guarding and anorexia as important variables. Guarding and tenderness were important in girls age 5 to 12. In boys age greater than 12, both agreed on guarding and anorexia. Using sensitivities and specificities from the literature, computed tomography improved the posttest probability for the group from.06 to.33; ultrasound improved it from.06 to.48; and barium enema improved it from.06 to.58. CONCLUSIONS: Knowing the pretest probabilities in a specific population allows the physician to evaluate the likely diagnoses first. Other quantitative methods can help judge how much importance a certain criterion should have in the decision making and how much a particular test is likely to influence the probability of a correct diagnosis. It now should be possible to make these sophisticated quantitative methods readily available to clinicians via the computer.

Ovarian Cyst Enlargement in a 14 Year Old Female with Persistent Ascities, Severe Hypothyroidism and Elevated Serum CA-125 Level.

A 14 year old female complained of abdominal pain and distention with vomiting. The physical exam showed thyroid enlargement and ascites. The imaging evaluation demonstrated a large ovarian cyst. Laboratory tests depicted hypothyroidism and marked elevation of Carbohydrate antigen 125 (CA-125) levels. As the bone age was 10 years, more retarded than the chronological age, Van Wyk and Grumbach syndrome was suspected. Treatment with thyroid hormone was initiated and the condition improved dramatically with disappearance of symptoms and signs 5 weeks later.