A Strategy for Studying Epigenetic Diversity in Natural Populations: Proof of Concept in Poplar and Oak.

These last 20 years, several techniques have been developed for quantifying DNA methylation, the most studied epigenetic marks in eukaryotes, including the gold standard method, whole-genome bisulphite sequencing (WGBS). WGBS quantifies genome-wide DNA methylation but has several inconveniences rendering it less suitable for population-scale epigenetic studies. The high cost of deep sequencing and the large amounts of data generated prompted us to seek an alternative approach. Restricting studies to parts of the genome would be a satisfactory alternative had there not been a major limitation: the need to select upstream targets corresponding to differentially methylated regions (DMRs) as targets. Given the need to study large numbers of samples, we propose a strategy for investigating DNA methylation variation in natural populations, considering the structural complexity of the genomes with their size and their content in unique as coding regions versus repeated regions as transposable elements. We first identified regions of highly variable DNA methylation in a representative subset of genotypes representative of the biological diversity in the population by WGBS. We then analysed the variations of DNA methylation in these targeted regions at the population level by Sequencing Capture Bisulphite (SeqCapBis). The entire strategy was then validated by applying it to another species. Our strategy was developed as a proof of concept on natural populations of two forest species: Populus nigra and Quercus petraea.

Novel KDM6A (UTX) mutations and a clinical and molecular review of the X-linked Kabuki syndrome (KS2).

We describe seven patients with KDM6A (located on Xp11.3 and encodes UTX) mutations, a rare cause of Kabuki syndrome (KS2, MIM 300867) and report, for the first time, germ-line missense and splice-site mutations in the gene. We demonstrate that less than 5% cases of Kabuki syndrome are due to KDM6A mutations. Our work shows that similar to the commoner Type 1 Kabuki syndrome (KS1, MIM 147920) caused by KMT2D (previously called MLL2) mutations, KS2 patients are characterized by hypotonia and feeding difficulties during infancy and poor postnatal growth and short stature. Unlike KS1, developmental delay and learning disability are generally moderate-severe in boys but mild-moderate in girls with KS2. Some girls may have a normal developmental profile. Speech and cognition tend to be more severely affected than motor development. Increased susceptibility to infections, join laxity, heart, dental and ophthalmological anomalies are common. Hypoglycaemia is more common in KS2 than in KS1. Facial dysmorphism with KDM6A mutations is variable and diagnosis on facial gestalt alone may be difficult in some patients. Hypertrichosis, long halluces and large central incisors may be useful clues to an underlying KDM6A mutation in some patients.

[New forms of health care coordination in geriatrics]. / Nouvelles formes de coordination paramédicale en gériatrie.

The global care of elderly patients leads to new forms of coordination between allied healthcare professionals. They are based on completely new ethical issues relating to the responsibility of all the healthcare professionals involved. These new practices call for the mobilisation of new skills, the development of new teaching and research into interprofessional collaboration.

Specification of a Foxj1-dependent lineage in the forebrain is required for embryonic-to-postnatal transition of neurogenesis in the olfactory bulb.

Establishment of a neural stem cell niche in the postnatal subependymal zone (SEZ) and the rostral migratory stream (RMS) is required for postnatal and adult neurogenesis in the olfactory bulbs (OB). We report the discovery of a cellular lineage in the SEZ-RMS-OB continuum, the specification of which is dependent on the expression of the forkhead transcription factor Foxj1 in mice. Spatially and temporally restricted Foxj1+ neuronal progenitors emerge during embryonic periods, surge during perinatal development, and are active only for the first few postnatal weeks. We show that the development of the unique Foxj1-derived lineage is dependent on Foxj1 expression and is required for overall postnatal neurogenesis in the OB. Strikingly, the production of neurons from Foxj1+ progenitors significantly declines after the early postnatal weeks, but Foxj1-derived neurons in the OB persist during adult periods. For the first time, our study identifies the time- and region-specific activity of a perinatal progenitor domain that is required for transition and progression of OB neurogenesis from the embryonic-to-postnatal periods.

FoxJ1-dependent gene expression is required for differentiation of radial glia into ependymal cells and a subset of astrocytes in the postnatal brain.

Neuronal specification occurs at the periventricular surface of the embryonic central nervous system. During early postnatal periods, radial glial cells in various ventricular zones of the brain differentiate into ependymal cells and astrocytes. However, mechanisms that drive this time- and cell-specific differentiation remain largely unknown. Here, we show that expression of the forkhead transcription factor FoxJ1 in mice is required for differentiation into ependymal cells and a small subset of FoxJ1(+) astrocytes in the lateral ventricles, where these cells form a postnatal neural stem cell niche. Moreover, we show that a subset of FoxJ1(+) cells harvested from the stem cell niche can self-renew and possess neurogenic potential. Using a transcriptome comparison of FoxJ1-null and wild-type microdissected tissue, we identified candidate genes regulated by FoxJ1 during early postnatal development. The list includes a significant number of microtubule-associated proteins, some of which form a protein complex that could regulate the transport of basal bodies to the ventricular surface of differentiating ependymal cells during FoxJ1-dependent ciliogenesis. Our results suggest that time- and cell-specific expression of FoxJ1 in the brain acts on an array of target genes to regulate the differentiation of ependymal cells and a small subset of astrocytes in the adult stem cell niche.

National institutions should improve the information made available to patients about cervical smears.

OBJECTIVE: To assess the quality of the content of leaflets tools and websites of national institutions in United Kingdom and France informing patients about cervical smears. METHODS: We collected and analyzed the data and information on these two websites and leaflets made for patients. We screened those tools with the UP TO DATE SCIENTIFIC EVIDENCE IPDAS grid. RESULTS: None of the tools specify the level of evidence of the studies on which cervix cancer screening is based. The risk of complication due to cancer is poor. The effectiveness of screening in absolute value is not available. The risks and side-effects due to cervical smears are specified without the frequency. CONCLUSION: Information is truncated and pushes readers towards taking part in screening. This is not in accordance with the quality criteria of shared decision making. PRACTICE IMPLICATIONS: Patients should take part in the creation of decision making tools, so that the information is the most suited to their representations and understanding. This is why the documents made available by institutions should be based on recognized scientific sources. Responsible of health programs should be independent and separated from those responsible of information tool creation.

Validity of the Good Practice Guidelines: The example of type 2 diabetes.

AIMS: To assess the methodological quality of the systematic reviews of the literature for Good Practice Guidelines (GPGs) for treatment of type 2 diabetes (T2D). METHODS: The GPGs on treatment of T2D from May 2012 onwards were searched on PubMed, the Guidelines International Network, the National Guidelines Clearing House and the Infobanque des guides de pratique clinique. Quality of the GPGs was assessed by means of grading of levels of evidence, strength of recommendations, statements pertaining to systematic reviews, description of their methods, search for Randomized Controlled Trials meta-analyses, and citations from three meta-analyses which contested the strategy of intensive glycemic control and metformin as first-line treatment. RESULTS: Fiflty-two GPGs were included; half of them had and applied a system of grading and strength of recommendation and 58% stated they had carried out a systematic review. Only one GPG cited the three meta-analyses. Three quarters of the GPGs failed to detail their bibliographic research methods. CONCLUSION: The GPGs for treatment of T2D were of poor quality and their methodological rigor was insufficient. Even though the meta-analyses had a higher level of evidence, they were seldom cited.

Expanding the reach of probiotics through social enterprises.

The rapid rise in microbiome and probiotic science has led to estimates of product creation and sales exceeding $50 billion within five years. However, many people do not have access to affordable products, and regulatory agencies have stifled progress. The objective of a discussion group at the 2017 meeting of the International Scientific Association for Probiotics and Prebiotics was to identify mechanisms to confer the benefits of probiotics to a larger portion of the world's population. Three initiatives, built around fermented food, were discussed with different methods of targeting populations that face enormous challenges of malnutrition, infectious disease, poverty and violent conflict. As new candidate probiotic strains emerge, and the market diversifies towards more personalised interventions, manufacturing processes will need to evolve. Information dissemination through scientific channels and social media is projected to provide consumers and healthcare providers with rapid access to clinical results, and to identify the nearest location of sites making new and affordable probiotic food and supplements. This rapid translation of science to individual well-being will not only expand the beneficiaries of probiotics, but also fuel new social enterprises and economic business models.

Transcriptional activation of cyclooxygenase-2 by tumor suppressor p53 requires nuclear factor-kappaB.

Overexpression of cyclooxygenase-2 (Cox-2) is thought to exert antiapoptotic effects in cancer. Here we show that the tumor suppressor p53 upregulated Cox-2 in esophageal and colon cancer cell lines by inducing the binding of nuclear factor-kappaB (NF-kappaB) to its response element in the COX-2 promoter. Inhibition of NF-kappaB prevented p53 induction of Cox-2 expression. Cooperation between p53 and NF-kappaB was required for activation of COX-2 promoter in response to daunomycin, a DNA-damaging agent. Pharmacological inhibition of Cox-2 enhanced apoptosis in response to daunomycin, in particular in cells containing active p53. In esophageal cancer, there was a correlation between Cox-2 expression and wild-type TP53 in Barrett's esophagus (BE) and in adenocarcinoma, but not in squamous cell carcinoma (P<0.01). These results suggest that p53 and NF-kappaB cooperate in upregulating Cox-2 expression, promoting cell survival in inflammatory precursor lesions such as BE.

Even if they are not aware of it, general practitioners improve well-being in their adolescent patients.

BACKGROUND: Most adolescents consult their general practitioner (GP) for common reasons, somatic or administrative but many of them have hidden feelings of distress. OBJECTIVES: To assess the immediate impact of 'ordinary' consultations on feelings of distress among adolescents and to compare adolescents experiencing difficulties (D) to those with no difficulties (N). To analyse how accurately GPs assess the impact of their consultation on adolescents' feelings. METHODS: GPs were randomly selected from two non-contiguous French administrative areas between April and June 2006. Fifty-three GPs gave two questionnaires to the first 10 to 15 adolescents aged 12 to 20 seen in consultation. One questionnaire was issued before the consultation and the other one afterwards. Adolescents had to position themselves about different aspects of well-being and say where they would seek help if they had problems. A GP questionnaire assessed how well they estimated their impact on the adolescent's feeling of well-being. RESULTS: Six hundred and sixty-five adolescents were assessed. They reported feeling better about their health, being able to talk, having someone to talk to or to confide in and on feeling understood. The D group (n = 147) felt significantly better compared to the N group (n = 518). GPs tended to underestimate this improvement, especially regarding adolescents in the D group feeling better about their health. CONCLUSIONS: Consulting a GP generates increased well-being among adolescents, especially for those experiencing difficulties. GPs tend to underestimate the positive impact they may have. Further studies are needed to explore if this benefit is permanent over time.

Identification of an NPHP1 deletion causing adult form of nephronophthisis.

AIMS: Nephronophthisis (NPHP) is an autosomal recessive cystic disease of the kidney with main characteristic features of polyuria/polydipsia, mild or absent proteinuria, interstitial fibrosis, and tubular cysts. NPHP is responsible for 5-10 % of inheritable end-stage renal disease (ESRD) cases. We investigated the clinical features and genetic cause of NPHP in a Persian family with three siblings affected by tubulointerstitial nephropathy reaching ESRD in adulthood. METHODS: Uromodulin (UMOD), known to be involved in adult medullary cystic kidney disease, and nephronophthisis 1 (NPHP1) were investigated in the genomic DNA of the probands using DNA sequencing, multiplex ligation-dependent probe amplification (MLPA) analysis and molecular karyotyping. RESULTS: No mutation was detected in UMOD. Copy number variation analysis of the NPHP1 gene using the commercially available MLPA kit identified a recurrent large homozygous deletion encompassing all NPHP1 exons. The parents were heterozygous for this deletion. Whole genome array-CGH analysis confirmed a homozygous deletion on chromosome 2q13, NPHP1 site, and revealed that the size of the copy number loss was approximately 102 Kbp. CONCLUSION: This is the first report of determination of an NPHP1 deletion size using routine diagnostic methods. The results of this study expand the knowledge about the genotype-phenotype correlations in NPHP1, and have implications for genetic counseling and family planning advice for other affected families. This is the first molecular analysis of NPHP1 in an Iranian kindred.

What do troubled adolescents expect from their GPs?

BACKGROUND: Adolescents often have emotional and behavioural problems that general practitioners are likely to miss. While nearly 80% of them consult their GP every year, it is usually for physical, not psychological reasons. Trust in their GPs in necessary for screening. OBJECTIVES: To identify the key quality desired by adolescents for them to feel free to confide in GPs. To determine whether this quality differed according to gender, level of at-risk behaviours or interlocutor: friend, parent or GP. METHODS: A descriptive cross-sectional study was conducted in 182 French educational institutions chosen by lot. Fifteen-year-olds completed a self-administered questionnaire under examination conditions. While the questions on behaviour were drawn from the cross-national survey entitled 'Health behaviour in school-aged children (HBSC),' the questions on conditions conducive to trust were drawn from previous studies. RESULTS: A total of 1817 (911 boys, 906 girls) questionnaires were analysed. Adolescents said they seldom confided. The main quality they expected from a GP to whom they could confide in was 'honesty', which meant ensuring secrecy, refraining from judgment, and putting forward the right questions. This priority was modified by neither gender nor experience with health-risk behaviour. The quality of 'reliability' was more closely associated with their parents or friends, while 'emotionality' was cited less often. CONCLUSION: To gain the trust of adolescents, GPs have to be sincere and non-manipulative and have the ability to ensure confidentiality and to put forward the right questions without passing judgment. Can this be verified during consultations? Prospective studies could shed light on this point.[Box: see text].

Additive effect between NF-kappaB subunits and p53 protein for transcriptional activation of human p53 promoter.

The tumor suppressor p53 plays a pivotal role in the cellular response to DNA damage as it controls DNA repair, cell cycle arrest and apoptosis. We studied the autoregulation of human p53 gene transcription in colon cancer cell lines. Wild-type p53 has been shown to autoregulate its own transcription either positively or negatively and probably in a cell-type-specific manner. Indeed, a p53 binding site has been described in the human and murine p53 promoters, but a direct binding of wild-type p53 protein to this site has never been reported. In this study, we demonstrated a transactivation of human p53 promoter by wild-type p53 in human colon cancer cells. We identified in the human p53 promoter a novel potential p53-responsive element that binds wild-type p53. Moreover, wild-type p53 protein transactivated a reporter plasmid containing a luciferase gene driven by a minimal promoter harboring this p53 binding site. Finally, as the p53 promoter contains an NF-kappaB binding site, we demonstrated an additive effect when NF-kappaB subunits and p53 protein combined to transactivate the human p53 promoter.

Deacylation and uptake of the fatty acyl chain from enantiomeric dialkylacylglycerol by intact rat fat cells.

Isolated rat fat cells hydrolyze the ester bond in position 3 of enantiomeric dialkyl[3H]acylglycerol added as triester-analogue substrate. This lipolytic system constitutes an integrated model which allows, in the absence of di- and monoesters, specific determinations of both the rate-limiting step in triacylglycerol breakdown and the uptake of acyl residues in cell lipids. Splitting of the ester bond was catalyzed by a cell-bound acylhydrolase which exhibited the characteristic properties of lipoprotein lipase. About 70% of the total amount of 3H-labelled fatty acid released during hydrolysis were concomitantly taken up by the cells and reesterified, mainly as triacylglycerol, in cell lipids. The acyl residues appeared to penetrate the cells directly after lipoprotein lipase hydrolysis, without entering the extracellular compartment. This, along with previous finding of a high positive correlation between rates of lipolytic activity and fatty acid uptake (Vérine, A., Salers, P. and Boyer, J. (1982) Am. J. Physiol. 243, E175-E181), suggests that the uptake process is directly related to enzyme action. These results are consistent with the concept that lipoprotein lipase, besides being an acylhydrolase, could also function as acyltransferase.

Plasma membranes from rat intestinal epithelial cells at different stages of maturation. I. Preparation and characterization of plasma membrane subfractions originating from crypt cells and from villous cells.

To determine the mechanism of the maturation of the brush border membrane in intestinal epithelial cells, purification of the plasma membrane from undifferentiated rat crypt cells and of the basal-lateral membrane from villous cells has been performed. The method is based on density perturbation of the mitochondria to selectively disrupt their association with the membrane. With both cell populations, two membrane subfractions displaying the same respective density on sucrose gradient have been obtained with an overall yield of 15--20% and a 10-fold enrichment of the plasma membrane markers 5'-nucleotidase and (Na+ + K+)-dependent, ouabain-sensitive ATPase chosen to follow their purification. The four fractions were constituted by sheets and apparently closed vesicles of various sizes. Each fraction was characterized by a distinct protein composition and different levels of enzyme activities. The cells, used for the preparation of the membranes, were isolated as a villus to crypt gradient. This separation and that of the membranes, led to the conclusion that the (Na+ + K+)-dependent ATPase is localized principally in the plasma membrane of all cells whatever their state of maturation, while 5'-nucleotidase is predominantly located in the basal-lateral membrane of the villous cells and may serve as a specific marker for the purification of this membrane. Finally it has been shown that aminopeptidase, dissacharidases and alkaline phosphatase do not appear simultaneously in the maturation process of the cells, alkaline phosphatase being absent from the crypt cells and aminopeptidase being the first to be synthesized. This enzyme seems to appear in the crypt cells membrane before being integrated into the mature brush border membrane.

Fortification of Yogurts with Vitamin D and Calcium Enhances the Inhibition of Serum Parathyroid Hormone and Bone Resorption Markers: A Double Blind Randomized Controlled Trial in Women over 60 Living in a Community Dwelling Home.

OBJECTIVE: To evaluate whether fortification of yogurts with vitamin D and calcium exerts an additional lowering effect on serum parathyroid hormone (PTH) and bone resorption markers (BRM) as compared to iso-caloric and iso-protein dairy products in aged white women at risk of fragility fractures. DESIGN: A randomized double-blind controlled trial. SETTING: A community dwelling home. PARTICIPANTS: Forty-eight women over 60 years (mean age 73.4). INTERVENTION: Consumption during 84 days of two 125 g servings of either vitamin D and calcium-fortified yogurts (FY) at supplemental levels of 10 µg vitamin D3/d and 520 mg/d of calcium (total=800 mg/d), or non fortified control yogurts (CY) providing 280 mg/d of calcium. MEASUREMENTS: Serum changes from baseline (D0) to D28, D56 and D84 in 25OHD, PTH and in two BRM: Tartrate-resistant-acid-phosphatase-isoform-5b (TRAP5b) and carboxy-terminal-cross-linked-telopeptide of type-I-collagen (CTX). RESULTS: The 10 years risk of major and hip fractures were 13.1 and 5.0%, and 12.9 and 4.2 %, in FY and CY groups, respectively. From D0 to D84, serum 25OHD increased (mean±SE) from 34.3±2.4 to 56.3±2.4 nmol/L in FY (n=24) and from 35.0±2.5 to 41.3±3.0 nmol/L in CY (n=24), (P=0.00001). The corresponding changes in PTH were from 64.1±5.1 to 47.4±3.8 ng/L in FY and from 63.5±4.6 to 60.7±4.2 ng/L in CY (P=0.0011). After D84, TRAP5b was reduced significantly (P=0.0228) and CTX fell though not significantly (P=0.0773) in FY compared to CY. CONCLUSION: This trial in aged white women living in a community dwelling home at risk for osteoporotic fractures confirms that fortification of dairy products with vitamin D3 and calcium should provide a greater prevention of secondary hyperparathyroidism and accelerated bone resorption as compared to non-fortified equivalent foods.

Flame hydrolysis deposition of glass on silicon for the integration of optical and microfluidic devices

Flame hydrolysis deposition (FHD) of glasses has previously found applications in the telecommunications industry. This paper shows how the technology can be used to deposit silica with different refractive indices and thereby produce low-loss planar waveguides for use in analytical applications. We also show that the glasses can be patterned using a new reactive ion etch and sealed using a modification of anodic bonding, such that the resulting microstructures can be readily incorporated within a lithographically defined "chip", integrating both optical and fluidic circuitry on the same device. In the example described in this paper, waveguides, analytical microtiter chambers and fluidic capillary channels, with the necessary high aspect ratio features (and with depths up to 40 microm) were all produced in glass, using the appropriate deposition and etching technologies. The performance of the chip was assessed in the framework of a low-volume fluorescence assay, using waveguides to address miniaturized microtiter chambers with volumes of 230 and 570 pL. Devices featuring different optical detection configurations, including both in-line and orthogonal waveguide geometries, were fabricated. In the optimal configuration, the experimental detection limit was determined as ca. 20 pM (equivalent to 10 zmol) of a cyanine fluorophore, Cy5. The applicability of the device as a biochip platform was further illustrated by analytical measurements on fluorescently labeled oligodeoxynucleotides.

The complex regulation of ferredoxin/thioredoxin-related genes by light and the circadian clock.

The biochemical properties of the ferredoxin/thioredoxin transduction pathway regulating the activity of key carbon-fixation enzymes through post-translational modifications are well characterized but little is known about the regulation of the different genes. In the present study, we investigated in Chlamydomonas reinhardtii the regulation of the expression of ferredoxin, thioredoxin m, ferredoxin-NADP reductase, phosphoribulokinase, as well as that of cytosolic thioredoxin h, the function of which is still largely unknown. The effects of light, the circadian clock and active cell division were investigated by northern blotting. The five genes were found to be regulated by light and the circadian clock but with different kinetics and amplitudes. This leads for the first time to the proposal that an extra-chloroplastic thioredoxin is possibly implicated in light and/or circadian-related processes. An interplay between several light-transduction pathways in controlling the expression of the genes is suggested by the expression studies and the theoretical analysis of the promoters.