Identification of Novel Craniofacial Regulatory Domains Located far Upstream of SOX9 and Disrupted in Pierre Robin Sequence.

Mutations in the coding sequence of SOX9 cause campomelic dysplasia (CD), a disorder of skeletal development associated with 46,XY disorders of sex development (DSDs). Translocations, deletions, and duplications within a ∼2 Mb region upstream of SOX9 can recapitulate the CD-DSD phenotype fully or partially, suggesting the existence of an unusually large cis-regulatory control region. Pierre Robin sequence (PRS) is a craniofacial disorder that is frequently an endophenotype of CD and a locus for isolated PRS at ∼1.2-1.5 Mb upstream of SOX9 has been previously reported. The craniofacial regulatory potential within this locus, and within the greater genomic domain surrounding SOX9, remains poorly defined. We report two novel deletions upstream of SOX9 in families with PRS, allowing refinement of the regions harboring candidate craniofacial regulatory elements. In parallel, ChIP-Seq for p300 binding sites in mouse craniofacial tissue led to the identification of several novel craniofacial enhancers at the SOX9 locus, which were validated in transgenic reporter mice and zebrafish. Notably, some of the functionally validated elements fall within the PRS deletions. These studies suggest that multiple noncoding elements contribute to the craniofacial regulation of SOX9 expression, and that their disruption results in PRS.

Intrauterine growth restriction is associated with structural alterations in human umbilical cord and decreased nitric oxide-induced relaxation of umbilical vein.

INTRODUCTION: Intrauterine growth restriction (IUGR) affects ∼8% of all pregnancies and is associated with major perinatal mortality and morbidity, and with an increased risk to develop cardiovascular diseases in adulthood. Despite identification of several risk factors, the mechanisms implicated in the development of IUGR remain poorly understood. In case of placental insufficiency, reduced delivery of oxygen and/or nutrients to the fetus could be associated with alterations in the umbilical circulation, contributing further to the impairment of maternal-fetal exchanges. We compared the structural and functional properties of umbilical cords from growth-restricted and appropriate for gestational age (AGA) term newborns, with particular attention to the umbilical vein (UV). METHODS: Human umbilical cords were collected at delivery. Morphological changes were investigated by histomorphometry, and UV's reactivity by pharmacological studies. RESULTS: Growth-restricted newborns displayed significantly lower growth parameters, placental weight and umbilical cord diameter than AGA controls. Total cross-section and smooth muscle areas were significantly smaller in UV of growth-restricted neonates than in controls. Maximal vasoconstriction achieved in isolated UV was lower in growth-restricted boys than in controls, whereas nitric oxide-induced relaxation was significantly reduced in UV of growth-restricted girls compared to controls. CONCLUSION: IUGR is associated with structural alterations of the UV in both genders, and with a decreased nitric oxide-induced relaxation in UV of newborn girls, whereas boys display impaired vasoconstriction. Further investigations will allow to better understand the regulation of umbilical circulation in growth-restricted neonates, which could contribute to devise potential novel therapeutic strategies to prevent or limit the development of IUGR.

Identification of secretory IgA, free secretory piece and serum IgA in the ovine and caprine species.

Caprine and ovine IgA were identified by cross-reaction with anti-human and anti-bovine IgA sera in colostrum, mature milk, saliva, urine and serum. Secretory component (SC) was shown in the free form and associated with polymeric serum IgA in secretions. Mean molecular weights were determined for the IgA and the free secretory components. The high IgA content of saliva suggested that it was a major secretory immunoglobulin in these species. Traces of secretory IgA were also found in normal sera but most of the serum IgA had no secretory determinant. Secretory IgA, serum IgA and free secretory component were purified. Levels of the sheep and goat immunoglobulins were measured in various fluids.

Simultaneous quantification of selective serotonin reuptake inhibitors and metabolites in human plasma by liquid chromatography-electrospray mass spectrometry for therapeutic drug monitoring.

A simple and sensitive liquid chromatography-electrospray ionization mass spectrometry method was developed for the simultaneous quantification in human plasma of all selective serotonin reuptake inhibitors (citalopram, fluoxetine, fluvoxamine, paroxetine and sertraline) and their main active metabolites (desmethyl-citalopram and norfluoxetine). A stable isotope-labeled internal standard was used for each analyte to compensate for the global method variability, including extraction and ionization variations. After sample (250μl) pre-treatment with acetonitrile (500μl) to precipitate proteins, a fast solid-phase extraction procedure was performed using mixed mode Oasis MCX 96-well plate. Chromatographic separation was achieved in less than 9.0min on a XBridge C18 column (2.1×100mm; 3.5μm) using a gradient of ammonium acetate (pH 8.1; 50mM) and acetonitrile as mobile phase at a flow rate of 0.3ml/min. The method was fully validated according to Société Française des Sciences et Techniques Pharmaceutiques protocols and the latest Food and Drug Administration guidelines. Six point calibration curves were used to cover a large concentration range of 1-500ng/ml for citalopram, desmethyl-citalopram, paroxetine and sertraline, 1-1000ng/ml for fluoxetine and fluvoxamine, and 2-1000ng/ml for norfluoxetine. Good quantitative performances were achieved in terms of trueness (84.2-109.6%), repeatability (0.9-14.6%) and intermediate precision (1.8-18.0%) in the entire assay range including the lower limit of quantification. Internal standard-normalized matrix effects were lower than 13%. The accuracy profiles (total error) were mainly included in the acceptance limits of ±30% for biological samples. The method was successfully applied for routine therapeutic drug monitoring of more than 1600 patient plasma samples over 9 months. The β-expectation tolerance intervals determined during the validation phase were coherent with the results of quality control samples analyzed during routine use. This method is therefore precise and suitable both for therapeutic drug monitoring and pharmacokinetic studies in most clinical laboratories.

Use of transgenic mice for the characterization of human alpha 1-acid glycoprotein (orosomucoid) variants.

Sera from transgenic mice (TM) carrying human genes of alpha 1-acid glycoprotein (orosomucoid or ORM) have been analyzed by isoelectrofocusing and subsequent immunoblotting with antihuman ORM antibodies. With this technique it is possible to reveal selectively the human protein secreted in the TM sera. Orosomucoid bands present in TM sera have been compared with those of the most common human ORM phenotypes to correlate the products of specific genes to previously identified genetic variants. In this paper, we report the identification of the genes encoding for variants ORM1 F1 and ORM2 A, which are genes AGP-A and AGP-B/B' respectively. The nucleotide sequences of these genes are known; therefore a direct correlation between variants and specific amino acid sequences can be established.

Tonotopic mapping of human auditory cortex.

Since the early days of functional magnetic resonance imaging (fMRI), retinotopic mapping emerged as a powerful and widely-accepted tool, allowing the identification of individual visual cortical fields and furthering the study of visual processing. In contrast, tonotopic mapping in auditory cortex proved more challenging primarily because of the smaller size of auditory cortical fields. The spatial resolution capabilities of fMRI have since advanced, and recent reports from our labs and several others demonstrate the reliability of tonotopic mapping in human auditory cortex. Here we review the wide range of stimulus procedures and analysis methods that have been used to successfully map tonotopy in human auditory cortex. We point out that recent studies provide a remarkably consistent view of human tonotopic organisation, although the interpretation of the maps continues to vary. In particular, there remains controversy over the exact orientation of the primary gradients with respect to Heschl's gyrus, which leads to different predictions about the location of human A1, R, and surrounding fields. We discuss the development of this debate and argue that literature is converging towards an interpretation that core fields A1 and R fold across the rostral and caudal banks of Heschl's gyrus, with tonotopic gradients laid out in a distinctive V-shaped manner. This suggests an organisation that is largely homologous with non-human primates. This article is part of a Special Issue entitled Human Auditory Neuroimaging.

Identification of muscle-specific calpain and beta-sarcoglycan genes in progressive autosomal recessive muscular dystrophies.

The autosomal recessive forms of limb-girdle muscular dystrophies are encoded by at least five distinct genes. The work performed towards the identification of two of these is summarized in this report. This success illustrates the growing importance of genetics in modern nosology.

Toward synthetic combinatorial peptide libraries in positional scanning format (PS-SCL)-based identification of CD8+ Tumor-reactive T-Cell Ligands: a comparative analysis of PS-SCL recognition by a single tumor-reactive CD8+ cytolytic T-lymphocyte clone.

The use of synthetic combinatorial peptide libraries in positional scanning format (PS-SCL) has emerged recently as an alternative approach for the identification of peptides recognized by T lymphocytes. The choice of both the PS-SCL used for screening experiments and the method used for data analysis are crucial for implementing this approach. With this aim, we tested the recognition of different PS-SCL by a tyrosinase 368-376-specific CTL clone and analyzed the data obtained with a recently developed biometric data analysis based on a model of independent and additive contribution of individual amino acids to peptide antigen recognition. Mixtures defined with amino acids present at the corresponding positions in the native sequence were among the most active for all of the libraries. Somewhat surprisingly, a higher number of native amino acids were identifiable by using amidated COOH-terminal rather than free COOH-terminal PS-SCL. Also, our data clearly indicate that when using PS-SCL longer than optimal, frame shifts occur frequently and should be taken into account. Biometric analysis of the data obtained with the amidated COOH-terminal nonapeptide library allowed the identification of the native ligand as the sequence with the highest score in a public human protein database. However, the adequacy of the PS-SCL data for the identification for the peptide ligand varied depending on the PS-SCL used. Altogether these results provide insight into the potential of PS-SCL for the identification of CTL-defined tumor-derived antigenic sequences and may significantly implement our ability to interpret the results of these analyses.

Identification by Real-time PCR of 13 mature microRNAs differentially expressed in colorectal cancer and non-tumoral tissues

MicroRNAs (miRNAs) are short non-coding RNA molecules playing regulatory roles by repressing translation or cleaving RNA transcripts. Although the number of verified human miRNA is still expanding, only few have been functionally described. However, emerging evidences suggest the potential involvement of altered regulation of miRNA in pathogenesis of cancers and these genes are thought to function as both tumours suppressor and oncogenes. In our study, we examined by Real-Time PCR the expression of 156 mature miRNA in colorectal cancer. The analysis by several bioinformatics algorithms of colorectal tumours and adjacent non-neoplastic tissues from patients and colorectal cancer cell lines allowed identifying a group of 13 miRNA whose expression is significantly altered in this tumor. The most significantly deregulated miRNA being miR-31, miR-96, miR-133b, miR-135b, miR-145, and miR-183. In addition, the expression level of miR-31 was correlated with the stage of CRC tumor. Our results suggest that miRNA expression profile could have relevance to the biological and clinical behavior of colorectal neoplasia.

Biosafety assessment of probiotics used for human consumption: recommendations from the EU-PROSAFE project

On June 26-27, 2006, 60 academic and industry scientists gathered during the PROSAFE workshop to discuss recommendations on taxonomy, antibiotic resistance, in vitro assessment of virulence and in vivo assessment of safety of probiotics used for human consumption. For identification of lactic acid bacteria (LAB) intended for probiotic use, it was recommended that conventional biochemical methods should be complemented with molecular methods and that these should be performed by an expert lab. Using the newly developed LAB Susceptibility test Medium (LSM), tentative epidemiological cut-off values were proposed. It was recommended that potentially probiotic strains not belonging to the wildtype distributions of relevant antimicrobials should not be developed as future products for human or animal consumption. Furthermore, it was recommended that the use of strains harbouring known and confirmed virulence genes should be avoided. Finally, for in vivo assessment of safety by investigating strain pathogenicity in animal models, the rat endocarditis model appeared to be the most reliable model tested in the PROSAFE project. Moreover, consensus was reached for approving the necessity of a human colonisation study in a randomised placebo-controlled double-blind design; however, further discussions are needed on the details of such as study.

Identification of SPLUNC1's ENaC-inhibitory domain yields novel strategies to treat sodium hyperabsorption in cystic fibrosis airways.

The epithelial sodium channel (ENaC) is responsible for Na+ and fluid absorption across colon, kidney, and airway epithelia. We have previously identified SPLUNC1 as an autocrine inhibitor of ENaC. We have now located the ENaC inhibitory domain of SPLUNC1 to SPLUNC1's N terminus, and a peptide corresponding to this domain, G22-A39, inhibited ENaC activity to a similar degree as full-length SPLUNC1 (∼2.5 fold). However, G22-A39 had no effect on the structurally related acid-sensing ion channels, indicating specificity for ENaC. G22-A39 preferentially bound to the β-ENaC subunit in a glycosylation-dependent manner. ENaC hyperactivity is contributory to cystic fibrosis (CF) lung disease. Addition of G22-A39 to CF human bronchial epithelial cultures (HBECs) resulted in an increase in airway surface liquid height from 4.2±0.6 to 7.9±0.6 μm, comparable to heights seen in normal HBECs, even in the presence of neutrophil elastase. Our data also indicate that the ENaC inhibitory domain of SPLUNC1 may be cleaved away from the main molecule by neutrophil elastase, which suggests that it may still be active during inflammation or neutrophilia. Furthermore, the robust inhibition of ENaC by the G22-A39 peptide suggests that this peptide may be suitable for treating CF lung disease.

Virtual screening and computational optimization for the discovery of covalent prolyl oligopeptidase inhibitors with activity in human cells.

Our docking program, Fitted, implemented in our computational platform, Forecaster, has been modified to carry out automated virtual screening of covalent inhibitors. With this modified version of the program, virtual screening and further docking-based optimization of a selected hit led to the identification of potential covalent reversible inhibitors of prolyl oligopeptidase activity. After visual inspection, a virtual hit molecule together with four analogues were selected for synthesis and made in one-five chemical steps. Biological evaluations on recombinant POP and FAPα enzymes, cell extracts, and living cells demonstrated high potency and selectivity for POP over FAPα and DPPIV. Three compounds even exhibited high nanomolar inhibitory activities in intact living human cells and acceptable metabolic stability. This small set of molecules also demonstrated that covalent binding and/or geometrical constraints to the ligand/protein complex may lead to an increase in bioactivity.

General unknown screening procedure for the characterization of human drug metabolites: application to loratadine phase I metabolism.

This article describes the application of a recently developed general unknown screening (GUS) strategy based on LC coupled to a hybrid linear IT-triple quadrupole mass spectrometer (LC-MS/MS-LIT) for the simultaneous detection and identification of drug metabolites following in vitro incubation with human liver microsomes. The histamine H1 receptor antagonist loratadine was chosen as a model compound to demonstrate the interest of such approach, because of its previously described complex and extensive metabolism. Detection and mass spectral characterization were based on data-dependent acquisition, switching between a survey scan acquired in the ion-trapping Q3 scan mode with dynamic subtraction of background noise, and a dependent scan in the ion-trapping product ion scan mode of automatically selected parent ions. In addition, the MS(3) mode was used in a second step to confirm the structure of a few fragment ions. The sensitivity of the ion-trapping modes combined with the selectivity of the triple quadrupole modes allowed, with only one injection, the detection and identification of 17 phase I metabolites of loratadine. The GUS procedure used in this study may be applicable as a generic technique for the characterization of drug metabolites after in vitro incubation, as well as probably in vivo experiments.

Human melanoma-specific CD8(+) T-cells from metastases are capable of antigen-specific degranulation and cytolysis directly ex vivo.

The relatively low frequencies of tumor Ag-specific T-cells in PBMC and metastases from cancer patients have long precluded the analysis of their direct ex vivo cytolytic capacity. Using a new composite technique that works well with low cell numbers, we aimed at determining the functional competence of melanoma-specific CD8(+) T-cells. A multiparameter flow cytometry based technique was applied to assess the cytolytic function, degranulation and IFNγ production by tumor Ag-specific CD8(+) T-cells from PBMC and tumor-infiltrated lymph nodes (TILN) of melanoma patients. We found strong cytotoxicity by T-cells not only when they were isolated from PBMC but also from TILN. Cytotoxicity was observed against peptide-pulsed target cells and melanoma cells presenting the naturally processed endogenous antigen. However, unlike their PBMC-derived counterparts, T-cells from TILN produced only minimal amounts of IFNγ, while exhibiting similar levels of degranulation, revealing a critical functional dichotomy in metastatic lesions. Our finding of partial functional impairment fits well with the current knowledge that T-cells from cancer metastases are so-called exhausted, a state of T-cell hyporesponsiveness also found in chronic viral infections. The identification of responsible mechanisms in the tumor microenvironment is important for improving cancer therapies.

Identification of haptoglobin as an angiogenic factor in sera from patients with systemic vasculitis.

Angiogenesis is an important process in chronic inflammatory diseases. We observed that sera from patients with systemic vasculitis stimulated angiogenesis in an in vitro model using human umbilical vein endothelial cells cultured on a basement membrane (Matrigel) substrate. After 40% ammonium sulfate precipitation, angiogenic activity remained in the low molecular weight fraction and could be inactivated by heat. SDS-page of serum FPLC fractions exhibiting maximal angiogenic activity demonstrated two prominent species of 45 and 16-20 kD in patients' sera. These bands were much less apparent in sera obtained from control subjects. Amino-terminal sequencing of the 45-kD protein demonstrated that it was haptoglobin. Purified haptoglobin stimulated angiogenesis in a dose-dependent manner. The angiogenic activity of vasculitis patients' sera was partially inhibited by an antihaptoglobin antibody. Furthermore, serum haptoglobin levels in vasculitis patients correlated both with disease and angiogenic activity. Haptoglobin angiogenic activity was confirmed in two in vivo models using an implanted disc and a subcutaneous injection of basement membrane. Stimulation of angiogenesis is a newly recognized biological function of haptoglobin. The increased levels of haptoglobin found in chronic inflammatory conditions may play an important role in tissue repair. In systemic vasculitis, haptoglobin might also compensate for ischemia by promoting development of collateral vessels.