Differentiation of Cucumber mosaic virus isolates by hybridization to oligonucleotides in a microarray format

A system for microarrays was developed to detect and differentiate Cucumber mosaic virus (CMV) serogroups and subgroups. The coat protein genes of 14 different isolates were amplified using cy3-labelled generic but species-specific primers. These amplicons were hybridized against a set of five different serotype and subgroup specific 24-mer oligonucleotides bound to an aldehyde-coated glass slide via an aminolinker. The results of the hybridization revealed that the method allowed a clear differentiation of the 14 different CMV isolates into the serogroupes 1 and 2, and in addition was able to assign 9 out of 10 different serogroup 1 isolates correctly into subgroups 1a and 1b. This differentiation was not possible by RFLP analysis with the restriction enzyme MspI. The use of amplicons larger than 700 base pairs and their successful differentiation by hybridization to specific oligonucleotides opens avenues to highly parallel, yet sensitive assays for plant viruses.     

F2-isoprostanes stimulate collagen synthesis in activated hepatic stellate cells: a link with liver fibrosis?

Carbon tetrachloride (CCl4)-induced hepatic fibrosis has been considered to be linked to oxidative stress and mediated by aldehydic lipid peroxidation products. In the present study, we investigated whether collagen synthesis is induced by F2-isoprostanes, the most proximal products of lipid peroxidation and known mediators of important biological effects. By contrast with aldehydes, F2-isoprostanes act through receptors able to elicit definite signal transduction pathways. In a rat model of CCl4-induced hepatic fibrosis, plasma F2-isoprostanes were markedly elevated for the entire experimental period; hepatic collagen content also increased. When hepatic stellate cells (HSCs) from normal liver were cultured with F2-isoprostanes in the concentration range found in the in vivo studies (10(-9)-10(-8) M), a striking increase in DNA synthesis (reversed by the thromboxane A2 antagonist SQ 29 548), in cell proliferation and in collagen synthesis was observed. Total collagen content was similarly increased. Moreover, F2-isoprostanes markedly increased the production of transforming growth factor-beta1 by U937 cells, considered a model of liver macrophages. The data provide evidence for the possibility that F2-isoprostanes generated by lipid peroxidation in hepatocytes mediate HSC proliferation and collagen production seen in hepatic fibrosis.     

Differential sensitivities of severe acute respiratory syndrome (SARS) coronavirus spike polypeptide enzyme-linked immunosorbent assay (ELISA) and SARS coronavirus nucleocapsid protein ELISA for serodiagnosis of SARS coronavirus pneumonia

The use of recombinant severe acute respiratory syndrome-coronavirus (SARS-CoV) nucleocapsid protein (N) enzyme-linked immunosorbent assay (ELISA)-based antibody and antigen tests for diagnosis of SARS-CoV infections have been widely reported. However, no recombinant SARS-CoV spike protein (S)-based ELISA is currently available. In this article, we describe the problems and solutions of setting up the recombinant SARS-CoV S-based ELISA for antibody detection. The SARS-CoV S-based immunoglobulin M (IgM) and IgG ELISAs were evaluated and compared with the corresponding N-based ELISA for serodiagnosis of SARS-CoV pneumonia, using sera from 148 healthy blood donors who donated blood 3 years ago as controls and 95 SARS-CoV pneumonia patients in Hong Kong. Results obtained by the recombinant S (rS)-based IgG ELISA using the regenerated S prepared by dialysis with decreasing concentrations of urea or direct addition of different coating buffers, followed by addition of different regeneration buffer, identified 4 M urea and 1 M sarcosine for plate coating and no regeneration buffer as the most optimal conditions for antibody detection. The specificities of the S-based ELISA for IgG and IgM detection were 98.6% and 93.9%, with corresponding sensitivities of 58.9% and 74.7%, respectively. The sensitivity of the rN IgG ELISA (94.7%) is significantly higher than that of the rS IgG ELISA (P < 0.001), whereas the sensitivity of the rS IgM ELISA is significantly higher than that of the rN IgM ELISA (55.2%) (P < 0.01). An ELISA for detection of IgM against S and N could be more sensitive than one that detects IgM against N alone for serodiagnosis of SARS-CoV pneumonia.     

Autonomie in der Offenen Kinder- und Jugendarbeit fördern – Ergebnisse eines partizipativen Evaluationsprojekts

Bundesgesundheitsblatt - Gesundheitsforschung - Gesundheitsschutz - Partizipation zu ermöglichen ist Teil des Auftrags der Offenen Kinder- und Jugendarbeit. Dazu gehört die Einbindung der...     

Infection by CagA-Positive Helicobacter pylori Strains and Bone Fragility: A Prospective Cohort Study

Helicobacter pylori (HP) infection is a common and persistent disorder acting as a major cofactor for the development of upper gastrointestinal diseases and several extraintestinal disorders including osteoporosis. However, no prospective study assessed the effects of HP on bone health and fracture risk. We performed a HP screening in a population-based cohort of 1149 adults followed prospectively for up to 11 years. The presence of HP infection was assessed by serologic testing for serum antibodies to HP and the cytotoxin associated gene-A (CagA). The prevalence of HP infection did not differ among individuals with normal bone mineral density (BMD), osteoporosis, and osteopenia. However, HP infection by CagA-positive strains was significantly increased in osteoporotic (30%) and osteopenic (26%) patients respect to subjects with normal BMD (21%). Moreover, anti-CagA antibody levels were significantly and negatively associated with lumbar and femoral BMD. Consistent with these associations, patients affected by CagA-positive strains had a more than fivefold increased risk to sustain a clinical vertebral fracture (HR 5.27; 95% CI, 2.23–12.63; p < .0001) and a double risk to sustain a nonvertebral incident fracture (HR 2.09; 95% CI, 1.27–2.46; p < .005). Reduced estrogen and ghrelin levels, together with an impaired bone turnover balance after the meal were also observed in carriers of CagA-positive HP infection. HP infection by strains expressing CagA may be considered a risk factor for osteoporosis and fractures. Further studies are required to clarify in more detail the underlying pathogenetic mechanisms of this association. © 2020 American Society for Bone and Mineral Research (ASBMR).     

Needs for Youth Substance Abuse and Violence Prevention in Schools and Communities

Substance abuse and violence among today's youth are at an all time high. Numerous prevention programs have been implemented to address these issues. Not all are successful. Research has found that when schools and communities are involved in the planning of prevention programs, youth are more cognizant of risk factors and of behaviors that strengthen risk factors. The purpose of this study was to assess the emerging needs for prevention and health education among youth, and to identify effective approaches for prevention program development and service delivery in schools and communities. This study surveyed 312 youth prevention specialists, school and community health educators and counselors, and parents in a regional sample of ten states. Results indicated that substance abuse and violence are the two most critical and priority issues in need of comprehensive prevention. Multiple prevention service delivery strategies appropriate to youth, including training, technical assistance, conferencing and networking, needs assessment and evaluation, and information requests are discussed in detail. Implications for prevention programming emphasized reducing risk factors and strengthening protective factors, reaching and motivating youth participation.     

Analyzing patterns of staining in immunohistochemical studies: application to a study of prostate cancer recurrence.

BACKGROUND: Immunohistochemical studies use antibodies to stain tissues with the goal of quantifying protein expression. However, protein expression is often heterogeneous resulting in variable degrees and patterns of staining. This problem is particularly acute in prostate cancer, where tumors are infiltrative and heterogeneous in nature. In this article, we introduce analytic approaches that explicitly consider both the frequency and intensity of tissue staining. METHODS: Compositional data analysis is a technique used to analyze vectors of unit-sum proportions, such as those obtained from soil sample studies or species abundance surveys. We summarized specimen staining patterns by the proportion of cells staining at mild, moderate, and intense levels and used compositional data analysis to summarize and compare the resulting staining profiles. RESULTS: In a study of Syndecan-1 staining patterns among 44 localized prostate cancer cases with Gleason score 7 disease, compositional data analysis did not detect a statistically significant difference between the staining patterns in recurrent (n = 22) versus nonrecurrent (n = 22) patients. Results indicated only modest increases in the proportion of cells staining at a moderate intensity in the recurrent group. In contrast, an analysis that compared quantitative scores across groups indicated a (borderline) significant increase in staining in the recurrent group (P = 0.05, t test). CONCLUSIONS: Compositional data analysis offers a novel analytic approach for immunohistochemical studies, providing greater insight into differences in staining patterns between groups, but possibly lower statistical power than existing, score-based methods. When appropriate, we recommend conducting a compositional data analysis in addition to a standard score-based analysis.     

Thymoquinone Alterations of the Apoptotic Gene Expressions and Cell Cycle Arrest in Genetically Distinct Triple-Negative Breast Cancer Cells

Breast cancer (BC) is the most common cancer in women worldwide, and it is one of the leading causes of cancer death in women. triple-negative breast Cancer (TNBC), a subtype of BC, is typically associated with the highest pathogenic grade and incidence in premenopausal and young African American (AA) women. Chemotherapy, the most common treatment for TNBC today, can lead to acquired resistance and ineffective treatment. Therefore, novel therapeutic approaches are needed to combat medication resistance and ineffectiveness in TNBC patients. Thymoquinone (TQ) is shown to have a cytotoxic effect on human cancer cells in vitro. However, TQ’s mode of action and precise mechanism in TNBC disease in vitro have not been adequately investigated. Therefore, TQ’s effects on the genetically different MDA-MB-468 and MDA-MB-231 human breast cancer cell lines were assessed. The data obtained show that TQ displayed cytotoxic effects on MDA-MB-468 and MDA-MB-231 cells in a time- and concentration-dependent manner after 24 h, with IC₅₀ values of 25.37 µM and 27.39 µM, respectively. Moreover, MDA-MB-231 and MDA-MB-468 cells in a scratched wound-healing assay displayed poor wound closure, inhibiting invasion and migration via cell cycle blocking after 24 h. TQ arrested the cell cycle phase in MDA-MB-231 and MDA-MB-468 cells. The three cell cycle stages in MDA-MB-468 cells were significantly affected at 15 and 20 µM for G0/G1 and S phases, as well as all TQ concentrations for G2/M phases. In MDA-MB-468 cells, there was a significant decrease in G0/G1 phases with a substantial increase in the S phase and G2/M phases. In contrast, MDA-MB-231 showed a significant effect only during the two cell cycle stages (S and G2/M), at concentrations of 15 and 20 µM for S phases and all TQ values for G2/M phases. The TQ effect on the apoptotic gene profiles indicated that TQ upregulated 15 apoptotic genes in MDA-MB-231 TNBC cells, including caspases, GADD45A, TP53, DFFA, DIABLO, BNIP3, TRAF2/3, and TNFRSF10A. In MDA-MB-468 cells, 16 apoptotic genes were upregulated, including TNFRSF10A, TNF, TNFRSF11B, FADD TNFRSF10B, CASP2, and TRAF2, all of which are important for the apoptotic pathway andsuppress the expression of one anti-apoptotic gene, BIRC5, in MDA-MB-231 cells. Compared to MDA-MB-231 cells, elevated levels of TNF and their receptor proteins may contribute to their increased sensitivity to TQ-induced apoptosis. It was concluded from this study that TQ targets the MDA-MB-231 and MDA-MB-468 cells differently. Additionally, due to the aggressive nature of TNBC and the lack of specific therapies in chemoresistant TNBC, our findings related to the identified apoptotic gene profile may point to TQ as a potential agent for TNBC therapy.     

Identification of RAD51–BRCA2 Inhibitors Using N-Acylhydrazone-Based Dynamic Combinatorial Chemistry

RAD51 is an ATP-dependent recombinase, recruited by BRCA2 to mediate DNA double-strand breaks repair through homologous recombination and represents an attractive cancer drug target. Herein, we applied for the first-time protein-templated dynamic combinatorial chemistry on RAD51 as a hit identification strategy. Upon design of N-acylhydrazone-based dynamic combinatorial libraries, RAD51 showed a clear templating effect, amplifying 19 N-acylhydrazones. Screening against the RAD51–BRCA2 protein–protein interaction via ELISA assay afforded 10 inhibitors in the micromolar range. Further ¹⁹F NMR experiments revealed that 7 could bind RAD51 and be displaced by BRC4, suggesting an interaction in the same binding pocket of BRCA2. These results proved not only that ptDCC could be successfully applied on full-length oligomeric RAD51, but also that it could address the need of alternative strategies toward the identification of small-molecule PPI inhibitors.     

Correlation of catheterized and clean catch urine protein/creatinine ratios in preeclampsia evaluation

OBJECTIVE: To examine whether clean catch urine specimens correlate with catheterized specimens for determination of protein/creatinine ratios in pregnant women being evaluated for preeclampsia. METHODS: Sixty pregnant women who were at least at 20 weeks of gestation were enrolled. Patients with ruptured membranes, vaginal bleeding, or urinary tract infections were excluded. Midstream clean catch urine specimens were collected. Catheterized specimens were then collected and used for clinical management. The specimens were analyzed for protein, creatinine, urinalysis, and culture. Based on sample size calculations, 60 participants were needed to detect a correlation of 0.90 with 80% power and alpha=0.05. RESULTS: Mean gestational age at enrollment was 35.9 weeks (range 23.1-41.7 weeks). Median (range) clean catch and catheterized protein/creatinine ratios were 0.204 (0.089-3.465) and 0.181 (0.067-3.335), respectively, with a correlation coefficient of 0.897 (P<.001). When results were categorized by degree of proteinuria using a cutoff of 0.3, sensitivity and specificity of the clean catch protein/creatinine ratios were 95.2% and 97.4%. When using a more conservative cutoff of 0.19, sensitivity and specificity of the clean catch protein/creatinine ratios were 96.4% and 75.0%. CONCLUSION: Clean catch and catheterized urine specimens correlate well in women with suspected preeclampsia. Routine catheterization of pregnant women is not necessary in the evaluation of preeclampsia.