Reducing rates of Agrotain® for seedrow applications

Non-Peer Reviewe

Rapid Site-Directed Mutagenesis Using Two-PCR-Generated DNA Fragments Reproducing the Plasmid Template

We describe a new rapid and efficient polymerase chain reaction (PCR)-based site-directed mutagenesis method. This procedure is effective with any plasmid and it employs four oligonucleotide primers. One primer contains the desired mutation, the second is oriented in the opposite direction (one of these two primers should be phosphorylated), and the third and fourth should be coding in complementary fashion for a unique restriction site to be introduced in a nonessential region. The method consists of two simultaneous PCR reactions; the PCR products are digested with the enzyme that recognizes the newly introduced unique restriction site and then ligased and used to transform competent bacteria. Additionally, the use of Dpn I facilitates the elimination of template DNA. The newly introduced restriction site is essential for ligation in the correct orientation of the two-PCR products and is further used for mutant screening. Resulting plasmids carry both the new restriction site and the desired mutation. Using this method, more than 20 mutants have already been generated (using two different kinds of templates); all these mutants were sequenced for the desired mutation and transfected into AtT-20 cells and the expressed mutant proteins encoded by the vector were assayed

OR 47: Renal endothelin receptor type B upregulation in rats with low or high renin hypertension

Endothelin-1 (ET-1) is a potent renal and systemic vasoactive peptide. It acts through ETA and ETB receptors. We investigated density and subtype distribution of ET-1 receptors in hearts and kidneys of normotensive and hypertensive rats. Five groups of uninephrectomized Wistar rats were put on a low salt diet for six weeks. During this period, three groups of rats drank tap water and two groups received saline. One group of each regimen received DOCA subcutaneousely (1.6 mg/day). The fifth group of rats had the left renal artery clipped to induce 1K1C hypertension. At 6 weeks, mean arterial pressure (MAP) was recorded in conscious rats via a femoral artery catheter. Binding assays using 125I-ET-1 were carried out on membrane preparations in the presence and absence of the ETA receptor antagonist FR139317. On tap water, MAP was at 121.8±3.3 mmHg and DOCA or saline did not raise this MAP. On DOCA-salt and in 1K1C rats, MAP was increased to 154.5±5.8 mmHg (p<0.001) and 218.4±10.5 (p<0.001) mmHg, respectively. ET receptor subtypes were not equally expressed in the heart and the kidney: ETA was predominantly expressed in the heart, whereas ETB was predominant in the kidney. Both hypertensive models, the DOCA-salt and the 1K1C rats showed further significant changes: i) Cardiac weight index compared to controls of 2.49±0.06 mg/g was higher (p<0.001) at 3.89±0.10 and 4.86±0.18 mg/g in DOCA-salt and 1K1C hypertension, respectively, and kidney weight index compared to controls of 4.78±0.22 mg/g was higher at 10.10±0.54 mg/g in DOCA-salt (p<0.001) but tended to be below controls in 1K1C rats. ii) In the kidneys, the density of the ETB receptor subtype was upregulated in DOCA-salt and 1K1C rats from 160±8 to 217±12 and 190±2 fmol/mg (p<0.05), respectively, and ETA tended to be downregulated. iii) Plasma renin activity was decrased in DOCA-salt rats from 17±3 to 0.17±0.01 ng/ml/h and increased in 1K1C rats on low salt diet to 30±5 ng/ml/h (p<0.001). We conclude that upregulation of the ETB receptor mediating vasodilation and downregulation of the ETA receptor mediating vasoconstriction is compatible with a mainly renal counterregulatory effect of endothelin-1 to hypertension. This counterregulation may occur in both low and high renin models of hypertension. Am J Hypertens (2004) 17, 20A-21A; doi: 10.1016/j.amjhyper.2004.03.04

Analyser l’impact d’un projet de micro-finance : l’exemple d’ADéFI à Madagascar.

Cette étude s’attache à analyser l’impact d’une institution de microfinance opérant auprès de micro- entrepreneurs à Antananarivo (Madagascar). Elle débute par un passage en revue de la littérature récente consacrée à l’impact socio-économique de la micro-finance. Elle décrit ensuite succinctement l’état de l’offre et de la demande de micro-crédit à Madagascar. Elle présente enfin les résultats de l’analyse de l’impact des financements accordés par ADéFI. La méthodologie retenue consiste à comparer la situation d’un échantillon de micro-entreprises clientes d’ADéFI représentatives de l’ensemble de la clientèle de l’institution à celle d’un groupe de contrôle construit de façon quasi- expérimentale par une technique standard d’appariement. Dans l’ensemble, les résultats des analyses concluent à un impact positif du projet. En statique, les évaluations conduites en 2001 et 2004 montrent que les micro-entreprises financées enregistrent de meilleures performances en moyenne que les UPI non financées. En dynamique, les analyses menées sont plus nuancées. Si l’impact positif du projet est clairement établi en phase de croissance, son effet en période de récession paraît plus incertain.This study aims at assessing the impact of a microfinance institution serving micro-entrepreneurs in Antananarivo (Madagascar). It starts by reviewing the recent literature on the socioeconomic impact of microfinance, then moves on to describe the current state of supply and demand of micro-credit in Madagascar. Finally, the results of the examination of the impact of ADéFI financing on micro- entreprises are presented. The methodology consists of comparing the situation of a representative sample of micro-enterprise ADéFI clients with a control group, constructed in an almost experimental way through a standard matching technique. Overall, the results indicate a positive impact of the project. Taken as a snapshot, the studies conducted in 2001 and 2004 indicate that the micro- enterprises financed recorded better average performance than informal production units without funding. With a dynamic perspective however, the results were more nuanced. If the positive effect of the project is clear during growth phases, its effect during contractions appears less certain.impact assessment; Madagascar; score de propension; évaluation d'impact; Microfinance; propensity score matching;

Contraceptive Implanon: Why do GPs get asked to remove it early?

Nineteen women aged 18 to 39 years of age from four Coast City Country GP Training practices were interviewed regarding their experiences with Implanon and the reasons for early removal. All participants were in relationships and approximately half had children

Influence of Different Evolutive Forces on GDF5 Gene Variability

The GDF5 gene is involved in the development of skeletal elements, synovial joint formation, tendons, ligaments, and cartilage. Several polymorphisms are present within the gene, and two of them, rs143384 and 143383, were reported to be correlated with osteoarticular disease or muscle flexibility. The aim of this research is to verify if the worldwide distribution of the rs143384 polymorphism among human populations was shaped by selective pressure, or if it was the result of random genetic drift events. Ninety-four individuals of both the male and female sexes, 18–28 years old, from Sardinia were analyzed. We observed the following genotype frequencies: 28.72% of AA homozygotes, 13.83% of GG homozygotes, and 57.45% of AG heterozygotes. The allele frequencies were 0.574 for allele A and 0.426 for allele G. The relationships between the populations were verified via Multidimensional Scaling (MDS). Our data show (i) a clear heterogeneity within the African populations; (ii) a strong differentiation between the African populations and the other populations; and that (iii) the Sardinian population is placed within the European cluster. To reveal possible traces of selective pressure, the Population Branch Statistic (PBS) was calculated; both the rs143384 and 143383 SNPs have low PBS values, suggesting that there are no signals of selective pressure in those areas of the gene

Interaction of Adenovirus E1A with the HHV8 Promoter of Latent Genes: E1A Proteins are Able to Activate the HHV-8 LANAp in MV3 Reporter Cells

Human herpesvirus 8 (HHV-8) is associated with Kaposi’s sarcoma, body cavity-based lymphoma, and Castleman’s disease. Adenoviral (Ad) E1A proteins regulate the activity of cellular and viral promoters/enhancers and transcription factors and can suppress tumorigenicity of human cancers. As (i) HHV-8 and Ad may co-exist in immunocompromised patients and (ii) E1A might be considered as therapeutic transgene for HHV-8-associated neoplasms we investigated whether the promoter of the latency-associated nuclear antigen (LANAp) controlling expression of vCyclin, vFLIP, and LANA proteins required for latent type infection is regulated by E1A. Transfection experiments in MV3 melanoma cells revealed activation of the LANAp by Ad5 E1A constructs containing an intact N terminus (aa 1-119). In particular, an Ad12 E1A mutant, Spm2, lacking six consecutive alanine residues in the “spacer” region activated the HHV-8 promoter about 15-fold compared to vector controls. In summary, we report the activation of the LANAp by E1A as a novel interaction of E1A with a viral promoter. These data may have relevance for the management of viral infections in immunocompromised patients. A role for E1A as a therapeutic in this context remains to be defined

EXPLORING STUDENTS’ ATTITUDES TOWARDS ONLINE-BASED LEARNING SYSTEM IN THE NEW NORMAL: AN EXPLORATORY FACTOR ANALYSIS

The implementation of online learning modality in the “New Normal Education” shifted the track of education institutions across the globe from conducting face-to-face classes to holding online-classes. The study presented in this paper aimed to explore students’ attitude towards online-based learning system in the “New Normal” education. Specifically, it investigated the factor structure and the level of attitudes of 200 students towards online-based learning system. This study utilized a mixed method of research utilizing in-depth interview and a dimension reduction technique through Principal Component Analysis. Results revealed that, attitudes toward online-based learning system is multidimensional exploring eight dimensions namely: Engagement, Convenience, Satisfaction, Technology Acceptance, Adaptability, Interaction, Self-Regulation and Control. Moreover, the level of attitudes of students revealed a high level of convenience, technology acceptance, adaptability, interaction, assessment satisfaction, self-regulation and control and a moderate level of student engagement. Thus, the researchers recommend a training proposal for teachers as well as recalibrating the result of the study utilizing Confirmatory Factor Analysis.   Article visualizations

Statins (HMG-CoA reductase inhibitors) reduce CD40 expression in human vascular cells

Objective: HMG-CoA reductase inhibitors (statins) possess anti-inflammatory and immunomodulatory properties that are independent of their lipid-lowering action. As the CD40-CD40L signaling pathway is implicated in the modulation of inflammatory responses between vascular cells, involving adhesion molecules, pro-inflammatory cytokines, chemokines, we sought to investigate the potential role of statins in regulating the expression of CD40. Methods and Results: Using Western blot, flow cytometry and immunohistochemistry analyses, we observed that four different statins reduced IFN-γ-induced CD40 expression in human vascular cells (endothelial cells, smooth muscle cells, macrophages and fibroblasts). This effect was dose-dependent (from 5 μM to 80 nM) and reversed by addition of l-mevalonate. Activation of vascular cells by human recombinant CD40L, as measured by ELISA for IL-6, IL-8 and MCP-1, was strongly reduced when cells were treated with statins. Immunostaining of human carotid atherosclerotic lesions of patients subjected to statin treatment revealed less CD40 expression on a ‘per vascular cell' basis compared to control patients. Although many pleiotropic effects of statins are mediated by nitric oxide synthase (NOS)- or peroxisome proliferator-activated receptor (PPAR)-dependent signaling pathways, we observed similar statin-induced reduction of CD40 expression using NOS inhibitors or different PPAR ligands. Conclusion: Statins decrease CD40 expression and CD40-related activation of vascular cells. These effects are partially reversed by the HMG-CoA reductase product l-mevalonate and are mediated by NOS- or PPAR-dependent pathways. Altogether, these findings provide mechanistic insight into the beneficial effects of statins on atherogenesis. They also provide a scientific rationale for the use of statins as immunomodulators after organ transplantatio

B016 Impact of a 14-night intermittent hypoxia (IH) exposure on metabolic and cardiopulmonary adaptations to exercise in healthy subjects

IntroductionModifications in exercise tolerance have been reported in obstructive sleep apnea (OSA) patients. Also specific mechanisms have been speculated related to intermittent hypoxia (IH), hypertension, obesity or metabolic disturbance associated to OSA may play a significant role in exercise limitation. In order to eliminate these confounding factors we aimed to evaluate the effects of IH exposure during 14 nights in healthy subjects on exercise capacity, cardio-respiratory response and substrate oxidation during exercise.Methods12 healthy subjects (BMI: 21.8 0.5kg.m-2) were exposed to repetitive sequences of hypoxia — re-oxygenation during sleep in a hypoxic tent with appropriate cyclic re-oxygenation (rate: 30 desaturations.h-1). Maximal and sub-maximal exercise tests were performed before and after exposure in order to investigate cardiorespiratory variables and substrate oxidation parameters.ResultsIH did not modify maximal exercise parameters (VO2, heart rate, power output) nor ventilatory threshold (VTh). But this was achieved with a significant PETCO2 reduction and a VE/VCO2 increase during both maximal (Pre IH vs Post IH at VTh and Max, p<0.05) and sub-maximal (Pre vs Post at 30 % and 60 % Pmax, p<0.05) exercise tests, indicating hyperventilation. At the 1st min recovery after submaximal exercise test, diastolic arterial blood pressure (DBP) was higher after IH exposure (Pre: 60±3 vs Post: 78±2mmHg) in favour of a delayed DBP recovery following acute exercise. During sub-maximal exercise, subjects reached maximal lipid oxidation at higher power output and presented a decreased blood lactate at the same percentage of relative power after IH exposure.ConclusionExposure to 14 days of nocturnal IH is associated with an increased ventilatory response to subsequent exercise at sea level. Furthermore, delayed DBP recovery after exercise is in favor of early IH-induced cardiovascular modifications. This observation related to muscular exercise adaptations confirms the efficacy of the model in reproducing early cardiovascular alterations occurring in OSAS. Moreover, this model induces metabolic adaptations as soon as 14 nights of exposure