The Two-Component Signal Transduction System CopRS of Corynebacterium glutamicum Is Required for Adaptation to Copper-Excess Stress

Copper is an essential cofactor for many enzymes but at high concentrations it is toxic for the cell. Copper ion concentrations ≥50 µM inhibited growth of Corynebacterium glutamicum. The transcriptional response to 20 µM Cu2+ was studied using DNA microarrays and revealed 20 genes that showed a ≥ 3-fold increased mRNA level, including cg3281-cg3289. Several genes in this genomic region code for proteins presumably involved in the adaption to copper-induced stress, e. g. a multicopper oxidase (CopO) and a copper-transport ATPase (CopB). In addition, this region includes the copRS genes (previously named cgtRS9) which encode a two-component signal transduction system composed of the histidine kinase CopS and the response regulator CopR. Deletion of the copRS genes increased the sensitivity of C. glutamicum towards copper ions, but not to other heavy metal ions. Using comparative transcriptome analysis of the ΔcopRS mutant and the wild type in combination with electrophoretic mobility shift assays and reporter gene studies the CopR regulon and the DNA-binding motif of CopR were identified. Evidence was obtained that CopR binds only to the intergenic region between cg3285 (copR) and cg3286 in the genome of C. glutamicum and activates expression of the divergently oriented gene clusters cg3285-cg3281 and cg3286-cg3289. Altogether, our data suggest that CopRS is the key regulatory system in C. glutamicum for the extracytoplasmic sensing of elevated copper ion concentrations and for induction of a set of genes capable of diminishing copper stress

Effect of education and attitude on health professionals’ knowledge on prenatal screening

INTRODUCTION Ongoing developments in prenatal anomaly screening necessitate continuous updating of counsellors’ knowledge. We explored the effect of a refresher counselling course on participants’ knowledge of prenatal screening. METHODS We investigated the association between knowledge and counsellors’ working experience. Also, the association between knowledge and counsellors’ attitude towards prenatal screening was determined. All counsellors in the North-West region of the Netherlands were invited to attend a refresher counselling course and fill in both a pre-course and a post-course questionnaire. The participants consisted of midwifes, sonographers and gynaecologists. A 55-item questionnaire assessed pre-course (T0) and post-course (T1) knowledge. At T0, counsellors’ attitude towards the prenatal screening program was assessed and its association with knowledge analysed. RESULTS Of 387 counsellors, 68 (18%) attended the course and completed both questionnaires. Knowledge increased significantly from 77.7% to 84.6% (p<0.01). Scores were lowest regarding congenital heart diseases. Participants with ultrasound experience scored higher on T0, but improvement was seen in participants with and without ultrasound experience. Participants with a positive attitude towards a free-of-charge first trimester combined test had higher knowledge scores than participants with a negative attitude (62% vs 46%; p=0.002). CONCLUSIONS A refresher course improved counsellors’ knowledge on prenatal screening. Ultrasound experience and a positive attitude towards free screening may be associated with higher knowledge levels. Participating in a mandatory refresher counselling course is useful for the continuous improvement of healthcare practitioners’ knowledge. More research on the effect of knowledge and attitude on the quality of prenatal screening is necessar

The effectiveness of multifetal pregnancy reduction in trichorionic triplet gestation

The objective of the study was to assess in trichorionic triplet pregnancies the effectiveness of elective reduction to twins. This was a nationwide retrospective cohort study. We compared the time to delivery and perinatal mortality in trichorionic triplet pregnancies electively reduced to twins with ongoing trichorionic triplets and primary dichorionic twins. We identified 86 women with reduced trichorionic triplet pregnancies, 44 with ongoing trichorionic triplets, and 824 with primary twins. Reduced triplets had a median gestational age at delivery of 36.1 weeks (interquartile range [IQR], 33.3-37.5 weeks) vs 33.3 (IQR, 28.1-35.2) weeks for ongoing triplets and 37.1 (IQR, 35.3-38.1) weeks for primary twins (P < .001). The total number of surviving children in the reduced group was 155 (90%) vs 114 (86%) in the ongoing triplet group. After reduction, 75 of women (87%) had all their fetuses surviving, compared with 36 (82%) (relative risk [RR], 1.3; 95% confidence interval [CI], 0.72-2.3) for ongoing triplets and 770 (93%) (RR, 0.91; 95% CI, 0.82-1) for primary twins. There were 6 women without any surviving children (7%) after reduction vs 5 (11.4%) (RR, 0.81; 95% CI, 0.47-1.4) among women with ongoing triplets and 32 (3.9%) (RR, 1.7; 95% CI, 0.8-3.7) in women with primary twins. In women with a triplet pregnancy, fetal reduction increases gestational age at birth with 3 weeks as compared with ongoing triplets. However, there the impact on neonatal survival is limite

Dual Wavelength Photoactivation of cAMP- and cGMP-Dependent Protein Kinase Signaling Pathways

The spatial and temporal organization of biological systems offers a level of complexity that is challenging to probe with conventional reagents. Photoactivatable (caged) compounds represent one strategy by which spatiotemporal organizational complexities can be addressed. However, since the vast majority of caged species are triggered by UV light, it is not feasible to orthogonally control two or more spatiotemporal elements of the phenomenon under investigation. For example, the cGMP- and cAMP-dependent protein kinases are highly homologous enzymes, separated in time and space, which mediate the phosphorylation of both distinct and common protein substrates. However, current technology is unable to discriminate, in a temporally or spatially selective fashion, between these enzymes and/or the pathways they influence. We describe herein the intracellular triggering of a cGMP-mediated pathway with 360 nm light and the corresponding cAMP-mediated pathway with 440 nm light. Dual wavelength photoactivation was assessed in A10 cells by monitoring the phosphorylation of vasodilator-stimulated phosphoprotein (VASP), a known substrate for both the cAMP- and cGMP-dependent protein kinases. Illumination at 440 nm elicits a cAMP-dependent phosphorylation of VASP at Ser157 whereas 360 nm exposure triggers the phosphorylation of both Ser157 and Ser239. This is the first example of wavelength-distinct activation of two separate nodes of a common signaling pathway

Naive and memory human B cells have distinct requirements for STAT3 activation to differentiate into antibody-secreting plasma cells

Long-lived antibody memory is mediated by the combined effects of long-lived plasma cells (PCs) and memory B cells generated in response to T cell–dependent antigens (Ags). IL-10 and IL-21 can activate multiple signaling pathways, including STAT1, STAT3, and STAT5; ERK; PI3K/Akt, and potently promote human B cell differentiation. We previously showed that loss-of-function mutations in STAT3, but not STAT1, abrogate IL-10– and IL-21–mediated differentiation of human naive B cells into plasmablasts. We report here that, in contrast to naive B cells, STAT3-deficient memory B cells responded to these STAT3-activating cytokines, differentiating into plasmablasts and secreting high levels of IgM, IgG, and IgA, as well as Ag-specific IgG. This was associated with the induction of the molecular machinery necessary for PC formation. Mutations in IL21R, however, abolished IL-21–induced responses of both naive and memory human B cells and compromised memory B cell formation in vivo. These findings reveal a key role for IL-21R/STAT3 signaling in regulating human B cell function. Furthermore, our results indicate that the threshold of STAT3 activation required for differentiation is lower in memory compared with naive B cells, thereby identifying an intrinsic difference in the mechanism underlying differentiation of naive versus memory B cells