High resolution ultrasonography of peripheral nerves: measurements on 14 nerve segments in 56 healthy subjects and reliability assessments.

Purpose: The aim of this study was to assess different aspects of reliability in high-resolution ultrasonography (HRUS) of the peripheral nerves and to establish reference values for the most frequently examined nerve segments. Materials and Methods: A nerve size parameter, the cross-sectional area (CSA) of the C5, C6 and C7 cervical roots, the median, ulnar, radial, superficial radial, peroneal, tibial, and the sural nerves was measured using HRUS at a total of 14 predefined anatomical sites in two different cohorts of Results: The mean CSA of the 14 nerve segments ranged from 2 to 10mm2. The intra-rater, interrater and inter-equipment reliability was high with intraclass correlation coefficients of 0.93, 0.98, and 0.86, respectively. The CSA values showed no consistent correlation with age, height, and body weight, but males had significantly larger values than females for nerve segments on the armafter correcting for age,weight and height in multivariate analysis. CSA values did not differ when two independent cohorts were compared. Conclusion: Peripheral nerve ultrasonography is a reliable and reproducible diagnostic method in the hands of experienced examiners. Normal values for several upper and lower extremity nerves are provided by our study. healthy subjects (n = 56), and the inter-rater, intra- rater and inter-equipment reliability of measurements was assessed

The extent of PXE skin changes is related to cardiovascular complications and visual loss: a cross-sectional study

Pseudoxanthoma elasticum (PXE) is a genetic multisystem disorder with cutaneous, ophthalmological and cardiovascular (CV) involvement(1, 2, 3) . Having the capacity to predict the natural progression of PXE within various systems would improve preventive care in those patients at elevated risk of CV or ophthalmological complications. Yet, the means required to predict PXE progression are still lacking today. It is yet to be established whether each body system evolves at its own pace, however skin is typically involved earlier than other systems(4) . PXE skin lesions appear mainly in the first two decades of life; the initial manifestations are usually located at the neck and progressively extend to other skin areas. This extension is mostly over around 30 years old (y/o).(5) CV and ophthalmological complications may develop later. This article is protected by copyright. All rights reserved

The transcriptional activity of hepatocyte nuclear factor 4 alpha is inhibited via phosphorylation by ERK1/2

Hepatocyte nuclear factor 4 alpha (HNF4alpha) nuclear receptor is a master regulator of hepatocyte development, nutrient transport and metabolism. HNF4alpha is regulated both at the transcriptional and post-transcriptional levels by different mechanisms. Several kinases (PKA, PKC, AMPK) were shown to phosphorylate and decrease the activity of HNF4alpha. Activation of the ERK1/2 signalling pathway, inducing proliferation and survival, inhibits the expression of HNF4alpha. However, based on our previous results we hypothesized that HNF4alpha is also regulated at the post-transcriptional level by ERK1/2. Here we show that ERK1/2 is capable of directly phosphorylating HNF4alpha in vitro at several phosphorylation sites including residues previously shown to be targeted by other kinases, as well. Furthermore, we also demonstrate that phosphorylation of HNF4alpha leads to a reduced trans-activational capacity of the nuclear receptor in luciferase reporter gene assay. We confirm the functional relevance of these findings by demonstrating with ChIP-qPCR experiments that 30-minute activation of ERK1/2 leads to reduced chromatin binding of HNF4alpha. Accordingly, we have observed decreasing but not disappearing binding of HNF4alpha to the target genes. In addition, 24-hour activation of the pathway further decreased HNF4alpha chromatin binding to specific loci in ChIP-qPCR experiments, which confirms the previous reports on the decreased expression of the HNF4a gene due to ERK1/2 activation. Our data suggest that the ERK1/2 pathway plays an important role in the regulation of HNF4alpha-dependent hepatic gene expression

Expression and In Vivo Rescue of Human ABCC6 Disease-Causing Mutants in Mouse Liver

Loss-of-function mutations in ABCC6 can cause chronic or acute forms of dystrophic mineralization described in disease models such as pseudoxanthoma elasticum (OMIM 26480) in human and dystrophic cardiac calcification in mice. The ABCC6 protein is a large membrane-embedded organic anion transporter primarily found in the plasma membrane of hepatocytes. We have established a complex experimental strategy to determine the structural and functional consequences of disease-causing mutations in the human ABCC6. The major aim of our study was to identify mutants with preserved transport activity but failure in intracellular targeting. Five missense mutations were investigated: R1138Q, V1298F, R1314W, G1321S and R1339C. Using in vitro assays, we have identified two variants; R1138Q and R1314W that retained significant transport activity. All mutants were transiently expressed in vivo, in mouse liver via hydrodynamic tail vein injections. The inactive V1298F was the only mutant that showed normal cellular localization in liver hepatocytes while the other mutants showed mostly intracellular accumulation indicating abnormal trafficking. As both R1138Q and R1314W displayed endoplasmic reticulum localization, we tested whether 4-phenylbutyrate (4-PBA), a drug approved for clinical use, could restore their intracellular trafficking to the plasma membrane in MDCKII and mouse liver. The cellular localization of R1314W was significantly improved by 4-PBA treatment, thus potentially rescuing its physiological function. Our work demonstrates the feasibility of the in vivo rescue of cellular maturation of some ABCC6 mutants in physiological conditions very similar to the biology of the fully differentiated human liver and could have future human therapeutic application

Sodium butyrate modifies the stabilizing complexes of tyrosine hydroxylase mRNA.

Multiple mechanisms regulate the expression of the tyrosine hydroxylase (Th) gene, which encodes the rate-limiting enzyme in the biosynthesis of catecholamines. Sodium butyrate (SOB), a physiological histone deacetylase (HDAC) inhibitor, was reported to stimulate the Th gene promoter activity in reporter gene assays. However, the expression of the endogenous Th gene in PC12 cells was reported to be either stimulated or inhibited by SOB. Here, we report that SOB and other HDAC inhibitors drastically (up to 90%) and reversibly decrease the level of TH mRNA in PC12 cells. We also show that SOB does not influence the transcription initiation rate of the Th gene but perturbs the formation of protein-RNA complexes at the 3'UTR of the gene. Our results suggest that SOB inhibits the expression of the Th gene by destabilizing TH mRNAs

ABCC6 does not transport vitamin K3-glutathione conjugate from the liver: relevance to pathomechanisms of pseudoxanthoma elasticum

Vitamin K is a cofactor required for gamma-glutamyl carboxylation of several proteins regulating blood clotting, bone formation and soft tissue mineralization. Vitamin K3 is an important intermediate during conversion of the dietary vitamin K1 to the most abundant vitamin K2 form. It has been suggested that ABCC6 may have a role in transporting vitamin K or its derivatives from the liver to the periphery. This activity is missing in pseudoxanthoma elasticum, a genetic disorder caused by mutations in ABCC6 characterized by abnormal soft tissue mineralization. Here we examined the efflux of the glutathione conjugate of vitamin K3 (VK3GS) from the liver in wild type and Abcc6(-/-) mice, and in transport assays in vitro. We found in liver perfusion experiments that VK3GS is secreted into the inferior vena cava, but we observed no significant difference between wild type and Abcc6(-/-) animals. We overexpressed the human ABCC6 transporter in Sf9 insect and MDCKII cells and assayed its vitamin K3-conjugate transport activity in vitro. We found no measurable transport of VK3GS by ABCC6, whereas ABCC1 transported this compound at high rate in these assays. These results show that VK3GS is not the essential metabolite transported by ABCC6 from the liver and preventing the symptoms of pseudoxanthoma elasticu

il-10 Gene Locus DNA Methylation in Regulatory B Cells

Epigenetic studies are becoming increasingly common in the immunology field thanks to the support of cutting edge technology and to their potential of providing a large amount of data at the single cell level. Moreover, epigenetic modifications were shown to play a role in autoimmune/inflammatory disorders, paving the way for the possibility of using the results of epigenetic studies for therapeutic purposes. In recent years, epigenetic marks such as DNA methylation, histone modifications and nucleosome positioning were shown to regulate B cell fate and function during an immune response, but very little has been done in the context of one of the most recently discovered B cell subsets, that is regulatory B cells. Although no consensus has yet been found on the identity of these immunosuppressive B cells, the role of the IL-10 cytokine is consolidated, both in the murine and human setting. In this chapter we will focus on the analysis of the methylation profile of a gene of interest and we will specifically describe cloning and pyrosequencing bisulphite sequencing PCR (BSP). Given the specific context, we will provide tips and tricks for the analysis of the il-10 gene locus. Nonetheless, the methods presented are valid for the study of any gene of interest