Molecular Characterization of Sclerospora graminicola, the Incitant of Pearl Millet Downy Mildew Using ISSR Markers

The DNA polymorphism among 22 isolates of Sclerospora graminicola, the causal agent of downy mildew disease of pearl millet was assessed using 20 inter simple sequence repeats (ISSR) primers. The objective of the study was to examine the effectiveness of using ISSR markers for unravelling the extent and pattern of genetic diversity in 22 S. graminicola isolates collected from different host cultivars in different states of India. The 19 functional ISSR primers generated 410 polymorphic bands and revealed 89% polymorphism and were able to distinguish all the 22 isolates. Polymorphic bands used to construct an unweighted pair group method of averages (UPGMA) dendrogram based on Jaccard's co-efficient of similarity and principal coordinate analysis resulted in the formation of four major clusters of 22 isolates. The standardized Nei genetic distance among the 22 isolates ranged from 0.0050 to 0.0206. The UPGMA clustering using the standardized genetic distance matrix resulted in the identification of four clusters of the 22 isolates with bootstrap values ranging from 15 to 100. The 3D-scale data supported the UPGMA results, which resulted into four clusters amounting to 70% variation among each other. However, comparing the two methods show that sub clustering by dendrogram and multi dimensional scaling plot is slightly different. All the S. graminicola isolates had distinct ISSR genotypes and cluster analysis origin. The results of ISSR fingerprints revealed significant level of genetic diversity among the isolates and that ISSR markers could be a powerful tool for fingerprinting and diversity analysis in fungal pathogens.

Cell surface heparan sulfate proteoglycans as novel markers of human neural stem cell fate determination

Multipotent neural stem cells (NSCs) provide a model to investigate neurogenesis and develop mechanisms of cell transplantation. In order to define improved markers of stemness and lineage specificity, we examined self-renewal and multi-lineage markers during long-term expansion and under neuronal and astrocyte differentiating conditions in human ESC-derived NSCs (hNSC H9 cells). In addition, with proteoglycans ubiquitous to the neural niche, we also examined heparan sulfate proteoglycans (HSPGs) and their regulatory enzymes. Our results demonstrate that hNSC H9 cells maintain self-renewal and multipotent capacity during extended culture and express HS biosynthesis enzymes and several HSPG core protein syndecans (SDCs) and glypicans (GPCs) at a high level. In addition, hNSC H9 cells exhibit high neuronal and a restricted glial differentiative potential with lineage differentiation significantly increasing expression of many HS biosynthesis enzymes. Furthermore, neuronal differentiation of the cells upregulated SDC4, GPC1, GPC2, GPC3 and GPC6 expression with increased GPC4 expression observed under astrocyte culture conditions. Finally, downregulation of selected HSPG core proteins altered hNSC H9 cell lineage potential. These findings demonstrate an involvement for HSPGs in mediating hNSC maintenance and lineage commitment and their potential use as novel markers of hNSC and neural cell lineage specification.

Ibuprofen ingestion does not affect markers of post-exercise muscle inflammation

Purpose: We investigated if oral ingestion of ibuprofen influenced leucocyte recruitment and infiltration following an acute bout of traditional resistance exercise Methods: Sixteen male subjects were divided into two groups that received the maximum over-the-counter dose of ibuprofen (1200mg d−1) or a similarly administered placebo following lower body resistance exercise. Muscle biopsies were taken from m.vastus lateralis and blood serum samples were obtained before and immediately after exercise, and at 3 and 24 h after exercise. Muscle cross-sections were stained with antibodies against neutrophils (CD66b and MPO) and macrophages (CD68). Muscle damage was assessed via creatine kinase and myoglobin in blood serum samples, and muscle soreness was rated on a ten-point pain scale. Results: The resistance exercise protocol stimulated a significant increase in the number of CD66b+ and MPO+ cells when measured 3 h post exercise. Serum creatine kinase, myoglobin and subjective muscle soreness all increased post-exercise. Muscle leucocyte infiltration, creatine kinase, myoglobin and subjective muscle soreness were unaffected by ibuprofen treatment when compared to placebo. There was also no association between increases in inflammatory leucocytes and any other marker of cellular muscle damage. Conclusion: Ibuprofen administration had no effect on the accumulation of neutrophils, markers of muscle damage or muscle soreness during the first 24 h of post-exercise muscle recovery.

Mitochondrial and nuclear markers suggest Hanuman langur (Primates: Colobinae) polyphyly: Implications for their species status

Recent molecular studies on langurs of the Indian subcontinent suggest that the widely-distributed and morphologically variable Hanuman langurs (Semnopithecus entellus) are polyphyletic with respect to Nilgiri and urple-faced langurs. To further investigate this scenario, we have analyzed additional sequences of mitochondrial cytochrome b as well as nuclear protamine P1 genes from these species. The results confirm Hanuman langur polyphyly in the mitochondrial tree and the nuclear markers suggest that the Hanuman langurs share protamine P1 alleles with Nilgiri and purple-faced langurs. We recommend provisional splitting of the so-called Hanuman langurs into three species such that the taxonomy is consistent with their evolutionary relationships.

Tumour markers as prognostic factors of survival of gastric cancer patients

The incidence of gastric cancer in the last decades has declined rapidly in the industrialised countries. Worldwide, however, gastric cancer is still the second most common cause of cancer death. Although surgery is currently the most effective treatment, the rapid progress in adjuvant chemotherapy and radiation therapy requires a re-evaluation of prognosis assessment. The TNM staging system of the UICC is ubiquitously used; it groups patients by decreasing survival times from stage I to stage IV based on the spread of disease, i.e. depth of tumour penetration (T), extent of spread to lymph nodes (N), and the presence or absence of distant (M) metastases. This is by far the most consistent prognostic classification system today. However, even within the stage groups there are patients that follow a varying course of disease. Our knowledge of the molecular differences between tumours of the same stage and morphology has been accumulating over the years and methods for a more accurate assessment of the phenotype of neoplasias are of value when evaluating the prognosis of individual patients with gastric cancer. In this study, the immunohistochemical expression of tumour markers involved in different phases in tumourigenesis was examined. The aim was to find new markers which could provide prognostic information in addition to what is provided by the TNM variables. A total of 337 specimens from the primary tumour of patients who underwent surgery for gastric cancer were collected and the immunohistochemical expression of seven different biomarkers was analysed. DNA ploidy and S-phase fraction (SPF) was assessed by flow cytometry. Finally, all biomarkers and clinicopathological prognostic factors were combined and evaluated by a multivariate Cox regression model to elucidate which specific factors provide independent prognostic information. By univariate survival analysis the following variables were significant prognostic factors: epithelial and stromal syndecan-1 expression, stromal tenascin-C expression, expression of tumour-associated trypsin inhibitor (TATI) in cancer cells, nuclear p53 expression, nuclear p21 expression, DNA ploidy, and SPF. By multivariate survival analysis adjusted for all available clinicopathological and biomolecular variables, p53 expression, p21 expression, and DNA ploidy emerged as independent prognostic biomarkers, together with penetration depth of the tumour, presence of nodal metastases, surgical cure of the cancer, and age of the patient at the time of diagnosis.

Assessment of Risk for Type 1 Diabetes in Children of Affected Families and in the General Population : Role of Immunological and Metabolic Markers

Background and aims. Type 1 diabetes (T1D), an autoimmune disease in which the insulin producing beta cells are gradually destroyed, is preceded by a prodromal phase characterized by appearance of diabetes-associated autoantibodies in circulation. Both the timing of the appearance of autoantibodies and their quality have been used in the prediction of T1D among first-degree relatives of diabetic patients (FDRs). So far, no general strategies for identifying individuals at increased disease risk in the general population have been established, although the majority of new cases originate in this population. The current work aimed at assessing the predictive role of diabetes-associated immunologic and metabolic risk factors in the general population, and comparing these factors with data obtained from studies on FDRs. Subjects and methods. Study subjects in the current work were subcohorts of participants of the Childhood Diabetes in Finland Study (DiMe; n=755), the Cardiovascular Risk in Young Finns Study (LASERI; n=3475), and the Finnish Type 1 Diabetes Prediction and Prevention Study (DIPP) Study subjects (n=7410). These children were observed for signs of beta-cell autoimmunity and progression to T1D, and the results obtained were compared between the FDRs and the general population cohorts. --- Results and conclusions. By combining HLA and autoantibody screening, T1D risks similar to those reported for autoantibody-positive FDRs are observed in the pediatric general population. Progression rate to T1D is high in genetically susceptible children with persistent multipositivity. Measurement of IAA affinity failed in stratifying the risk assessment in young IAA-positive children with HLA-conferred disease susceptibility, among whom affinity of IAA did not increase during the prediabetic period. Young age at seroconversion, increased weight-for-height, decreased early insulin response, and increased IAA and IA-2A levels predict T1D in young children with genetic disease susceptibility and signs of advanced beta-cell autoimmunity. Since the incidence of T1D continues to increase, efforts aimed at preventing T1D are important, and reliable disease prediction is needed both for intervention trials and for effective and safe preventive therapies in the future. Our observations confirmed that combined HLA-based screening and regular autoantibody measurements reveal similar disease risks in pediatric general population as those seen in prediabetic FDRs, and that risk assessment can be stratified further by studying glucose metabolism of prediabetic subjects. As these screening efforts are feasible in practice, the knowledge now obtained can be exploited while designing intervention trials aimed at secondary prevention of T1D.

Prognostic markers in head and neck carcinoma

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Well-known risk factors include tobacco smoking and alcohol consumption. Overall survival has improved, but is still low especially in developing countries. One reason for this is the often advanced stage of the disease at the time of diagnosis, but also lack of reliable prognostic tools to enable individualized patient treatment to improve outcome. To date, the TNM classification still serves as the best disease evaluation criterion, although it does not take into account the molecular basis of the tumor. The need for surrogate molecular markers for more accurate disease prediction has increased research interests in this field. We investigated the prevalence, physical status, and viral load of human papillomavirus (HPV) in HNSCC to determine the impact of HPV on head and neck carcinogenesis. The prevalence and genotyping of HPV were assessed with an SPF10 PCR microtiter plate-based hybridization assay (DEIA), followed by a line probe-based genotyping assay. More than half of the patients had HPV DNA in their tumor specimens. Oncogenic HPV-16 was the most common type, and coinfections with other oncogenic and benign associated types also existed. HPV-16 viral load was unevenly distributed among different tumor sites; the tonsils harbored significantly greater amounts of virus than other sites. Episomal location of HPV-16 was associated with large tumors, and both integrated and mixed forms of viral DNA were detected. In this series, we could not show that the presence of HPV DNA correlated with survival. In addition, we investigated the prevalence and genotype of HPV in laryngeal carcinoma patients in a prospective Nordic multicenter study based on fresh-frozen laryngeal tumor samples to determine whether the tumors were HPV-associated. These patients were also examined and interviewed at diagnosis for known risk factors, such as tobacco smoking and alcohol consumption, and for several other habituations to elucidate their effects on patient survival. HPV analysis was performed with the same protocols as in the first study. Only 4% of the specimens harbored HPV DNA. Heavy drinking was associated with poor survival. Heavy drinking patients were also younger than nonheavy drinkers and had a more advanced stage of disease at diagnosis. Heavy drinkers had worse oral hygiene than nonheavy drinkers; however, poor oral hygiene did not have prognostic significance. History of chronic laryngitis, gastroesophageal reflux disease, and orogenital sex contacts were rare in this series. To clarify why vocal cord carcinomas seldom metastasize, we determined tumor lymph vessel (LVD) and blood vessel (BVD) densities in HNSCC patients. We used a novel lymphatic vessel endothelial marker (LYVE-1 antibody) to locate the lymphatic vessels in HNSCC samples and CD31 to detect the blood microvessels. We found carcinomas of the vocal cords to harbor less lymphatic and blood microvessels than carcinomas arising from sites other than vocal cords. The lymphatic and blood microvessel densities did not correlate with tumor size. High BVD was strongly correlated with high LVD. Neither BVD nor LVD showed any association with survival in our series. The immune system plays an important role in tumorigenesis, as neoplastic cells have to escape the cytotoxic lymphocytes in order to survive. Several candidate HLA class II alleles have been reported to be prognostic in cervical carcinomas, an epithelial malignancy resembling HNSCC. These alleles may have an impact on head and neck carcinomas as well. We determined HLA-DRB1* and -DQB1* alleles in HNSCC patients. Healthy organ donors served as controls. The Inno-LiPA reverse dot-blot kit was used to identify alleles in patient samples. No single haplotype was found to be predictive of either the risk for head and neck cancer, or the clinical course of the disease. However, alleles observed to be prognostic in cervical carcinomas showed a similar tendency in our series. DRB1*03 was associated with node-negative disease at diagnosis. DRB1*08 and DRB1*13 were associated with early-stage disease; DRB1*04 had a lower risk for tumor relapse; and DQB1*03 and DQB1*0502 were more frequent in controls than in patients. However, these associations reached only borderline significance in our HNSCC patients.

Long-term effects of fenofibrate on lipoproteins and surrogate markers of macrovascular and microvascular disease in people with type 2 diabetes

Background and aims. Diabetic dyslipidemia is a highly atherogenic triad of increased triglycerides, decreased HDL cholesterol, and small dense LDL. Fibrates have a beneficial effect on diabetic dyslipidemia, and they have reduced cardiovascular events in randomized trials. Fenofibrate has reduced albuminuria and markers of low-grade inflammation and endothelial dysfunction. The present studies were undertaken to characterize the alterations of VLDL and LDL subclasses and to investigate the binding of LDL to arterial wall in type 2 diabetes. Further purpose was to elucidate the effects of fenofibrate on several lipoprotein subclasses, augmentation index (AIx), carotid intima-media thickness (IMT), and renal function. Subjects. 239 type 2 diabetic subjects were recruited among participants of the FIELD (Fenofibrate Intervention and Event Lowering in Diabetes) study at the Helsinki centre. The patients were randomized to fenofibrate (200mg/d) or placebo for 5 years. Additionally, a healthy control group (N = 93) was recruited. Results. VLDL1 triglycerides increased in similar proportion to total triglycerides in type 2 diabetic patients and control subjects. Despite the increase in total apoCIII levels, VLDL apoCIII was decreased in diabetic patients. Enrichment of LDL with apoCIII induced a small increase in binding of LDL to arterial wall proteoglycan. Intrinsic characteristics of diabetic LDL, rather than levels of apoCIII, were responsible for increased proteoglycan binding of diabetic LDL with high apoCIII. Fenofibrate reduced triglycerides, increased LDL size, and shifted HDL subclasses towards smaller particles with no change in levels of HDL cholesterol. High levels of homocysteine were associated with lower increase of HDL cholesterol and apoA-I during fenofibrate treatment. Long-term fenofibrate treatment did not improve IMT, AIx, inflammation, or endothelial function. Fenofibrate decreased creatinine clearance and estimated glomerular filtration rate. No effect on albuminuria was seen with fenofibrate. Instead, Cystatin C was increased during fenofibrate treatment. Conclusions. 1) Elevation of VLDL 1 triglycerides was the major determinant of plasma triglyceride concentration in control subjects and type 2 diabetic patients. 2) LDL with high apoCIII showed multiple atherogenic properties, that were only partially mediated by apoCIII per se in type 2 diabetes 3) Fenofibrate demonstrated no effect on surrogate markers of atherosclerosis. 4) Fenofibrate had no effect on albuminuria and the observed decrease in markers of renal function could complicate the clinical surveillance of the patients. 5) Fenofibrate can be used to treat severe hypertriglyceridemia or in combination therapy with statins, but not to increase HDL levels.

Serial Changes in Markers Measuring Coagulation, Fibrinolysis, and Vasoactivity in Patients with Ischemic Stroke

Ischemic stroke (IS) is a heterogeneous disease in which outcome is influenced by many factors. The hemostatic system is activated in association with cerebral ischemia, and thus, markers measuring coagulation, fibrinolysis, and vasoactivity could be useful tools in clinical practice. We investigated whether repeated measurements of these markers reveal patterns that might help in evaluating IS patients, including the early diagnosis of stroke subtypes, in estimating prognosis and risk of recurrence, and in selecting a treatment for secondary prevention of stroke. Vasoconstrictor peptide endothelin-1 (ET-1), homocysteine (Hcy), indicators of thrombin formation and activation (prothrombin fragment 1+2/F1+2, thrombin-antithrombin complex/TAT), indicators of plasmin formation and fibrinolysis (tissue plasminogen activator/t-PA, plasminogen activator inhibitor-1/PAI-1, and D-dimer), and natural anticoagulants (antithrombin/AT, protein C/PC, and protein S/PS) were measured in 102 consecutive mild to moderate IS patients on four occasions: on admission and at 1 week, 1 month, and 3 months after stroke, and once in controls. All patients underwent neurological examination and blood sampling in the same session. Furthermore, 42 IS patients with heterozygous factor V Leiden mutation (FVLm) were selected from 740 IS patients without an obvious etiology, and evaluated in detail for specific clinical, laboratory, and radiological features. Measurements of ET-1 and Hcy levels did not disclose information that could aid in the diagnostic evaluation of IS patients. F1+2 level at 3 months after IS had a positive correlation with recurrence of thromboembolic events, and thus, may be used as a predictive marker of subsequent cerebral events. The D-dimer and AT levels on admission and 1 week after IS were strongly associated with stroke severity, outcome, and disability. The specific analysis of IS patients with FVLm more often revealed a positive family history of thrombosis, a higher prevalence of peripheral vascular disease, and multiple infarctions in brain images, most of which were `silent infarcts´. Results of this study support the view that IS patients with sustained activation of both the fibrinolytic and the coagulation systems and increased thrombin generation may have an unfavorable prognosis. The level of activation may reflect the ongoing thrombotic process and the extent of thrombosis. Changes in these markers could be useful in predicting prognosis of IS patients. A clear need exists for a randomized prospective study to determine whether a subgroup of IS patients with markers indicating activation of fibrinolytic and coagulation systems might benefit from more aggressive secondary prevention of IS.