Measurement of Converting Enzyme Activity by Antibody-Trapping of Generated Angiotensin II: Comparison With Two Other Methods

Activity of the angiotensin converting enzyme (ACE) is usually measured in vitro by estimation of products cleaved by the enzyme from synthetic substrates. These substrates have affinities for ACE different from the natural substrate angiotensin I, and insensitive detection systems necessitate milli-molar substrate concentrations while physiological angiotensin I concentrations are in the picomolar range. A new assay for ACE activity measurement was developed which reliably quantitates femtomoles of generated angiotensin II in plasma from angiotensin I added at a 17 pmol/mL concentration. The production of high affinity monoclonal antibodies against angiotensin II (Kd = 7 × 10-11 mol/L) allowed a quantitative trapping (and thus protection from degrading enzymes) of angiotensin II generated during the incubation step and subsequent ra-dioimmunoassay by simple dilution with labelled angiotensin II. Using 40 µL plasma, the detection limit was 20 fmol/mL/min. Normal human plasma has an ACE activity of 335 ± 83 fmol/mL/min (mean ± SD). Precision was characterized by coefficients of variation of ^ 11% both within-assay and between-assays. Accuracy of the new method was established by comparing ACE activity with the ratio of plasma angiotensin II/angiotensin I in plasma obtained from normal volunteers 0.5 to 24 h after oral administration of 20 mg enalapril. The percentage of ACE inhibition indicated by both methods was almost identical (r = 0.93, n = 60, P < .001). Since the latter ratio appears to reflect in vivo ACE activity, these results indicate that accurate measurement in vitro of ACE activity in vivo has been achieved. Am J Hypertens 1992;5:393-39

ANÁLISE COMPARADA DA EVOLUÇÃO DE SÍTIOS TURÍSTICOS: SANTOS (SP) E BALNEÁRIO CAMBORIÚ (SC)

Este trabalho originou-se a partir de um estudo comparado entre os sítios turísticos de Santos (SP) e Balneário Camboriú (SC). Foram analisados aspectos históricos, políticos, econômicos e geográficos, bem como a evolução urbana e turística. Implicando assim uma reflexão sobre a construção destes espaços, considerados como produtos sociais em permanente processo de transformação, a partir das especificidades de cada lugar, onde o turismo, enquanto fenômeno e atividade, gera esta dinâmica. Considerou-se a importância da paisagem como recurso turístico e como elemento fundamental para o desenvolvimento sustentável e sustentado. Fundamentado neste princípio, efetuou-se uma análise aprofundada destes sítios, que possuem histórias entrelaçadas, principalmente em relação à ocupação e urbanização que ocorreram de modo espontâneo, cujo processo de crescimento desencadeou impactos negativos no âmbito econômico, social e, sobretudo, ambiental. Palavras-chave: sítios turísticos; turismo; paisagem; urbanização; impactos ambientais; Santos e Balneário Camboriú

Molecular characterization of breast cancer cell pools with normal or reduced ability to respond to progesterone: a study based on RNA-seq.

BACKGROUND About one-third of patients with estrogen receptor alpha (ERα)-positive breast cancer have tumors which are progesterone receptor (PR) negative. PR is an important prognostic factor in breast cancer. Patients with ERα-positive/PR-negative tumors have shorter disease-free and overall survival than patients with ERα-positive/PR-positive tumors. New evidence has shown that progesterone (P4) has an anti-proliferative effect in ERα-positive breast cancer cells. However, the role of PR in breast cancer is only poorly understood. METHODS We disrupted the PR gene (PGR) in ERα-positive/PR-positive T-47D cells using the CRISPR/Cas9 system. This resulted in cell pools we termed PR-low as P4 mediated effects were inhibited or blocked compared to control T-47D cells. We analyzed the gene expression profiles of PR-low and control T-47D cells in the absence of hormone and upon treatment with P4 alone or P4 together with estradiol (E2). Differentially expressed (DE) genes between experimental groups were characterized based on RNA-seq and Gene Ontology (GO) enrichment analyses. RESULTS The overall gene expression pattern was very similar between untreated PR-low and untreated control T-47D cells. More than 6000 genes were DE in control T-47D cells upon stimulation with P4 or P4 plus E2. When PR-low pools were subjected to the same hormonal treatment, up- or downregulation was either blocked/absent or consistently lower. We identified more than 3000 genes that were DE between hormone-treated PR-low and control T-47D cells. GO analysis revealed seven significantly enriched biological processes affected by PR and associated with G protein-coupled receptor (GPCR) pathways which have been described to support growth, invasiveness, and metastasis in breast cancer cells. CONCLUSIONS The present study provides new insights into the complex role of PR in ERα-positive/PR-positive breast cancer cells. Many of the genes affected by PR are part of central biological processes of tumorigenesis

River fragmentation increases localized population genetic structure and enhances asymmetry of dispersal in bullhead ( Cottus gobio )

Man-made habitat fragmentation is a major concern in river ecology and is expected to have particularly detrimental effects on aquatic species with limited dispersal abilities, like the bullhead (Cottus gobio). We used ten microsatellite markers to investigate small-scale patterns of gene flow, current dispersal and neutral genetic diversity in a morphologically diverse river where fragmented and unfragmented sections could be compared. We found high genetic differentiation between sampling sites with a maximum F ST of 0.32 between sites separated by only 35 km. A significant increase of genetic differentiation with geographical distance was observed in the continuous river section as well as in the full dataset which included headwater populations isolated by anthropogenic barriers. Several lines of evidence are consistent with the hypothesis that such barriers completely block upstream movement while downstream dispersal may be little affected. In the unfragmented habitat, dispersal rates were also higher in the direction of water flow than against it. The resulting asymmetry in gene flow likely contributes to the decrease of genetic variation observed from the lower reaches towards the headwaters, which is particularly pronounced in physically isolated populations. Our findings suggest that headwater populations, due to their isolation and low genetic variation, may be particularly vulnerable to extinctio

Impact of 6 month conjugated equine estrogen versus estradiol-treatment on biomarkers and enriched gene sets in healthy mammary tissue of non-human primates.

OBJECTIVE To identify distinctly regulated gene markers and enriched gene sets in breast tissue of cynomolgus monkeys (Macaca fascicularis) treated for six months with either conjugated equine estrogens (CEE) or estradiol (E2) by analysis of corresponding mRNA levels of genes associated with breast development, carcinogenesis, apoptosis and immune regulation. Additionally, translation of three nuclear markers was analyzed. METHODS RNA from breast biopsies and necropsies was isolated from two independent study trials from Ethun et al. (CEE) and Foth et al. (E2) after 6 month of treatment duration. RNA was subjected to qRT-PCR and MicroArray analysis. Immunohistochemical stainings were performed for the estrogen receptor alpha subunit (ERa), the progesterone receptor (PGR) and the proliferation marker Ki67. RESULTS We identified a total of 36 distinctly enriched gene sets. Thirty-one were found in the CEE treatment group and five were found in the E2 treatment group, with no overlap. Furthermore, two individual genes IGFBP1 and SGK493 were upregulated in CEE treated animals. Additional targeted qRT-PCR analysis of ten specific estrogen-related genes showed upregulation of three genes (TFF1, PGR and GREB1) after CEE treatment, respectively one gene (TFF1) after E2 treatment. Immunohistochemical stains of breast biopsies showed a significant increase in expression of the PGR marker after CEE treatment. CONCLUSIONS In this study we identified enriched gene sets possibly induced by CEE or E2 treatment in various processes associated with cancer biology and immunology. This preliminary translational data supports the concept that different estrogen types have different effects on healthy breast tissue and may help generate hypotheses for future research

Transition-Transversion Bias Is Not Universal: A Counter Example from Grasshopper Pseudogenes

Comparisons of the DNA sequences of metazoa show an excess of transitional over transversional substitutions. Part of this bias is due to the relatively high rate of mutation of methylated cytosines to thymine. Postmutation processes also introduce a bias, particularly selection for codon-usage bias in coding regions. It is generally assumed, however, that there is a universal bias in favour of transitions over transversions, possibly as a result of the underlying chemistry of mutation. Surprisingly, this underlying trend has been evaluated only in two types of metazoan, namely Drosophila and the Mammalia. Here, we investigate a third group, and find no such bias. We characterize the point substitution spectrum in Podisma pedestris, a grasshopper species with a very large genome. The accumulation of mutations was surveyed in two pseudogene families, nuclear mitochondrial and ribosomal DNA sequences. The cytosine-guanine (CpG) dinucleotides exhibit the high transition frequencies expected of methylated sites. The transition rate at other cytosine residues is significantly lower. After accounting for this methylation effect, there is no significant difference between transition and transversion rates. These results contrast with reports from other taxa and lead us to reject the hypothesis of a universal transition/transversion bias. Instead we suggest fundamental interspecific differences in point substitution processes

Human Wharton’s jelly mesenchymal stromal cell-derived small extracellular vesicles drive oligodendroglial maturation by restraining MAPK/ERK and Notch signaling pathways.

Peripartum cerebral hypoxia and ischemia, and intrauterine infection and inflammation, are detrimental for the precursor cells of the myelin-forming oligodendrocytes in the prematurely newborn, potentially leading to white matter injury (WMI) with long-term neurodevelopmental sequelae. Previous data show that hypomyelination observed in WMI is caused by arrested oligodendroglial maturation rather than oligodendrocyte-specific cell death. In a rat model of premature WMI, we have recently shown that small extracellular vesicles (sEV) derived from Wharton's jelly mesenchymal stromal cells (WJ-MSC) protect from myelination deficits. Thus, we hypothesized that sEV derived from WJ-MSC directly promote oligodendroglial maturation in oligodendrocyte precursor cells. To test this assumption, sEV were isolated from culture supernatants of human WJ-MSC by ultracentrifugation and co-cultured with the human immortalized oligodendrocyte precursor cell line MO3.13. As many regulatory functions in WMI have been ascribed to microRNA (miR) and as sEV are carriers of functional miR which can be delivered to target cells, we characterized and quantified the miR content of WJ-MSC-derived sEV by next-generation sequencing. We found that WJ-MSC-derived sEV co-localized with MO3.13 cells within 4 h. After 5 days of co-culture, the expression of myelin basic protein (MBP), a marker for mature oligodendrocytes, was significantly increased, while the oligodendrocyte precursor marker platelet-derived growth factor alpha (PDGFRα) was decreased. Notch and MAPK/ERK pathways known to inhibit oligodendrocyte maturation and differentiation were significantly reduced. The pathway enrichment analysis showed that the miR present in WJ-MSC-derived sEV target genes having key roles in the MAPK pathway. Our data strongly suggest that sEV from WJ-MSC directly drive the maturation of oligodendrocyte precursor cells by repressing Notch and MAPK/ERK signaling

Trends and Risk Factors of In-Hospital Mortality of Patients with COVID-19 in Germany: Results of a Large Nationwide Inpatient Sample

Unselected data of nationwide studies of hospitalized patients with COVID-19 are still sparse, but these data are of outstanding interest to avoid exceeding hospital capacities and overloading national healthcare systems. Thus, we sought to analyze seasonal/regional trends, predictors of in-hospital case-fatality, and mechanical ventilation (MV) in patients with COVID-19 in Germany. We used the German nationwide inpatient samples to analyze all hospitalized patients with a confirmed COVID-19 diagnosis in Germany between 1 January and 31 December in 2020. We analyzed data of 176,137 hospitalizations of patients with confirmed COVID-19-infection. Among those, 31,607 (17.9%) died, whereby in-hospital case-fatality grew exponentially with age. Overall, age ≥ 70 years (OR 5.91, 95%CI 5.70-6.13, p < 0.001), pneumonia (OR 4.58, 95%CI 4.42-4.74, p < 0.001) and acute respiratory distress syndrome (OR 8.51, 95%CI 8.12-8.92, p < 0.001) were strong predictors of in-hospital death. Most COVID-19 patients were treated in hospitals in urban areas (n = 92,971) associated with the lowest case-fatality (17.5%), as compared to hospitals in suburban (18.3%) or rural areas (18.8%). MV demand was highest in November/December 2020 (32.3%, 20.3%) in patients between the 6th and 8th age decade. In the first age decade, 78 of 1861 children (4.2%) with COVID-19-infection were treated with MV, and five of them died (0.3%). The results of our study indicate seasonal and regional variations concerning the number of COVID-19 patients, necessity of MV, and case fatality in Germany. These findings may help to ensure the flexible allocation of intensive care (human) resources, which is essential for managing enormous societal challenges worldwide to avoid overloaded regional healthcare systems

Social policies in oral health : review

Sabe-se que as políticas sociais podem ter efeito importante sobre a pobreza e sobre as iniquidades socioeconômicas na saúde. Esta revisão de literatura tem o objetivo de descrever conceitos básicos sobre políticas sociais com enfoque em políticas de transferência de renda como direitos sociais. Por fim, discutem-se as influências de tais políticas na saúde bucal e seus potenciais mecanismosSocial policies can have an important effect on poverty and socioeconomic health inequities. This review has the objective of explaining basic concepts about social policies, with special attention to cash transfer programmes as social rights. Finally, it discussed the influences of social policies on oral health and its potential mechanism