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Pathogenic GFM1 variants have been linked to neurological phenotypes with or without liver involvement, but only a few cases have been reported in the literature. Here, we report clinical, biochemical, and neuroimaging findings from nine unrelated children carrying GFM1 variants, 10 of which were not previously reported. All patients presented with neurological involvement-mainly axial hypotonia and dystonia during the neonatal period-with five diagnosed with West syndrome; two children had liver involvement with cytolysis episodes or hepatic failure. While two patients died in infancy, six exhibited a stable clinical course. Brain magnetic resonance imaging showed the involvement of basal ganglia, brainstem, and periventricular white matter. Mutant EFG1 and OXPHOS proteins were decreased in patient's fibroblasts consistent with impaired mitochondrial translation. Thus, we expand the genetic spectrum of GFM1-linked disease and provide detailed clinical profiles of the patients that will improve the diagnostic success for other patients carrying GFM1 mutations.

International audience; Heparan sulfate (HS) proteoglycan chains are key components of the breast tumor microenvironment that critically influence the behavior of cancer cells. It is established that abnormal synthesis and processing of HS play a prominent role in tumorigenesis, albeit mechanisms remain mostly obscure. HS function is mainly controlled by sulfotransferases, and here we report a novel cellular and pathophysiological significance for the 3-O-sulfotransferase 3-OST3A (HS3ST3A), catalyzing the final maturation step of HS, in breast cancer. We show that 3-OST3A is epigenetically repressed in all breast cancer cell lines of a panel representative of distinct molecular subgroups, except in human epidermal growth factor receptor 2-positive (HER2+) sloan-kettering breast cancer (SKBR3) cells. Epigenetic mechanisms involved both DNA methylation and histone modifications, producing different repressive chromatin environments depending on the cell molecular signature. Gain and loss of function experiments by cDNA and siRNA transfection revealed profound effects of 3-OST3A expression on cell behavior including apoptosis, proliferation, response to trastuzumab in vitro and tumor growth in xenografted mice. 3-OST3A exerted dual activities acting as tumor-suppressor in lumA-michigan cancer foundation (MCF)-7 and triple negative-MD Anderson (MDA) metastatic breast (MB)-231 cells, or as an oncogenic factor in HER2+-SKBR3 cells. Mechanistically, fluorescence-resonance energy transfer-fluorescence-lifetime imaging microscopy experiments indicated that the effects of 3-OST3A in MCF-7 cells were mediated by altered interactions between HS and fibroblast growth factor-7 (FGF-7). Further, this interplay between HS and FGF-7 modulated downstream ERK, AKT and p38 cascades, suggesting that altering 3-O-sulfation affects FGFR2IIIb-mediated signaling. Corroborating our cellular data, a clinical study conducted in a cohort of breast cancer patients uncovered that, in HER2+ patients, high level expression of 3-OST3A in tumors was associated with reduced relapse-free survival. Our findings define 3-OST3A as a novel regulator of breast cancer pathogenicity, displaying tumor-suppressive or oncogenic activities in a cell-and tumor-dependent context, and demonstrate the clinical value of the HS-O-sulfotransferase 3-OST3A as a prognostic marker in HER2+ patients.

Flow cytometry was used to demonstrate the presence of beta-lactoglobulin (betaLG) receptors on living murine hybridoma MARK-3 cells using a fluorescein isothiocyanate-betaLG conjugate (FITC-betaLG: molar ratio of 5:1). A site occupation curve was produced using a shift in the mean channel fluorescence at various concentrations of FITC-betaLG. The binding of labelled ligand was concentration dependent and was inhibited by unlabelled betaLG. The on-rate constant was 3.2x10(2) M(-1) min(-1) and the off-rate constant was 0.002 min(-1). Scatchard plot analysis gave a dissociation constant (K(d)) of 44+/-21x10(-7) and 39+/-24x10(-5) M (n=3). Flow cytometry indicated that at least 15% of the FITC-betaLG were internalized for 5 min and that internalization was temperature- and time-dependent. The internalization was confirmed by 3-D fluorescence microscopy (CELLScan system).

Thrombin plays a central role in thrombosis. Because of the medical need for novel antithrombotic drugs, a search for structurally novel thrombin inhibitors was undertaken. In the absence of a crystal structure, a class was designed based on a modeling approach which involved placing the essential functional groups of the thrombin antagonist MD-805 [1] (Argatroban) into the benzodiazepine nucleus. The best superposition was obtained with a 1,4-benzodiazepin-2-one containing a 1,2,3,4-tetrahydro quinolylsulfonyl moiety in the 3-position, a guanidino-phenyl at the 5-position, and N 1 -substituted with an acetic acid. Synthesis of these molecules provided compounds with an inhibitory activity with K 1 in the range of 40–1000 μJ.M. A report on the crystal structure of thrombin * hirudin(55-65) * MD-805 complex [2] suggested subsequent molecular modeling investigations to rationalize the pharmacological results.

Background and Objectives Epidural catheters placed for analgesia during labor and delivery can be reused in the postpartum period for postpartum tubal ligation anesthesia. This study examined the success rate of such reactivation of epidural catheters. Methods We studied all patients who had received an epidural anesthetic for labor and delivery and who had the epidural catheter reactivated at a later time to provide anesthesia for a postpartum tubal ligation (PPTL) procedure between January, 1993 and June, 1995. The patients were divided into four groups based on the interval between delivery and PPTL incision: (a) less than 8 hours, (b) 8 hours to less than 16 hours, (c) 16 hours to less than 24 hours and (d) 24 hours or more. Results Overall, 92% (129/140) of the patients had their epidural catheters successfully reactivated. The success rate in all but the last group was over 90%, while only 80% of catheters could be reactivated if left in place for more than 24 hours. Conclusions Epidural catheters can be reactivated reliably for as long as 24 hours after they are placed.

To describe the radiographic features and potential causes of urethral disruption in pancreas transplant recipients.Eight episodes of urinary extravasation were depicted with retrograde urethrography in five male patients who had undergone pancreatic transplantation. The patients' medical records were reviewed to determine a cause of the extravasations.Four extravasations occurred at the proximal (deep) bulbar urethra, three at the bulbomembranous junction of the urethra, and one at the distal bulbar urethra. Four of eight cases of extravasation were preceded by recent cystoscopy or placement of a Foley catheter; one case was preceded by possible urethral injury due to a fall.Urethral disruption occurs as a complication of pancreatic transplantation. It has so far been seen only in male patients and occurs at the bulbar urethra or bulbomembranous junction. If the treating physician is unaware of this condition, diagnosis and institution of appropriate therapy may be delayed. Recent prior urinary tract instrumentation or trauma may be predisposing factors in urinary extravasation.

The aim of this study was to assess the value of the measurement of os-placenta distance by translabial ultrasonography in the evaluation of placenta previa. This method was used in 40 women with suspected placenta previa to measure the distance between the placenta and internal cervical os. Sonographic diagnoses were compared to placental location determined at delivery. Translabial ultrasonography proved superior to the transabdominal route in both diagnosis and exclusion of placenta previa. Measurement of the os-placenta distance can be used as an adjunct to clinical assessment to predict the likelihood of safe vaginal delivery in cases of suspected placenta previa.

Summary Because the structural requirements for the binding of nicotine to central nicotine receptors remain largely uninvestigated, we undertook a systematic investigation of pyrrolidine ring-opened analogs. This led to a subsequent investigation of related conformationally restricted derivatives of these analogs. The results are reported relative to the binding of several well-known and widely used nicotine receptor ligands. Although none of the ring-opened analogs binds with higher affinity than (−)nicotine (Ki = 2.3 nM), 3-(N-methyl-N-ethylaminomethyl)pyridine (12a; Ki = 28 nM) binds with significant affinity. A conformationally restricted analog of 12a, N-methyl [2,7]naphthyridine 30b (Ki = 18 nM), binds with similar affinity. 6-Substitution of 12a and racemic nicotine seems to be tolerated when the substituent is halogen or methyl. In functional studies (hypolocomotion and antinociception in mice; stimulus generalization in nicotine-trained rats) 30b retains nicotine-like properties. Several of the 6-substituted compounds were 2 to 20 times more potent than (±)nicotine. Although the intact pyrrolidine ring of nicotine appears important for optimal affinity, its presence is not an absolute requirement for activity, and 6-position substitution of the pyridine nucleus can influence both binding and functional activity.

Viral and bacterial pathogens are a significant economic concern to the US broiler industry and the ecological epicenter for poultry pathogens is the mixture of bedding material, chicken excrement and feathers that comprises the litter of a poultry house. This study used high-throughput sequencing to assess the richness and diversity of poultry litter bacterial communities, and to look for connections between these communities and the environmental characteristics of a poultry house including its history of gangrenous dermatitis (GD). Cluster analysis of 16S rRNA gene sequences revealed differences in the distribution of bacterial phylotypes between Wet and Dry litter samples and between houses. Wet litter contained greater diversity with 90% of total bacterial abundance occurring within the top 214 OTU clusters. In contrast, only 50 clusters accounted for 90% of Dry litter bacterial abundance. The sixth largest OTU cluster across all samples classified as an Arcobacter sp., an emerging human pathogen, occurring in only the Wet litter samples of a house with a modern evaporative cooling system. Ironically, the primary pathogenic clostridial and staphylococcal species associated with GD were not found in any house; however, there were thirteen 16S rRNA gene phylotypes of mostly Gram-positive phyla that were unique to GD-affected houses and primarily occurred in Wet litter samples. Overall, the poultry house environment appeared to substantially impact the composition of litter bacterial communities and may play a key role in the emergence of food-borne pathogens.

Plant peptides of a rapeseed peptone were labelled with a coumarin fluorescent probe. The free-probe does not incorporate cell cytoplasm and is not cytotoxic at a concentration below 10 μM. Observations by Confocal Microscopy of CHO cells incubated in presence of labelled peptides indicate no fixation of the peptides on cell membrane receptors, but their incorporation inside cell cytoplasm. This integration seems to be time-dependant while the fluorescence intensity reaches saturation after more than 12 hours of incubation.

In refluxing diglyme, the title α-hydroxy imines 7 and 8 rearrange cleanly to amino ketones 9 and 10 substituted on the carbon α to nitrogen by an allyl or a propargyl group. In the case of α-hydroxy imine 9 , the migration of the allyl group occurs with an allylic transposition.

On 11 June 1996, three suspected cases of legionnaires’ disease in a group of 42 Dutch tourists were reported to the local public health authority by Millau hospital in south west France. The group (group 1) had been touring with caravans and staying at d

Mechanical forces regulate the morphology and the metabolism of endothelial cells through mechano-sensitive structures existed on cell surface. In this work, the influence of a steady laminar or disturbed flow on cell cytoskeleton and its connection with differential adhesion molecules expression at the apical side of the endothelial cell were studied in vitro. A 3D cell scanner coupled with a fluorescence microscope allowed the authors to visualize the spatial organization or distribution of cytoskeleton and adhesion molecules marked by specific fluorescent probes. In static conditions, the actin fibers presented a random structure, but after exposure to shear stresses, it was observed a reorganization of these fibers. The distribution of adhesion molecules at the cell surface seems to be linked to these structural modifications. These studies help one to better understand the response of endothelium to complex flows near vascular singularities.

Herpes simplex virus type-1 (HSV) amplicon vectors containing neuroprotective genes can alter cell physiology and enhance survival following various insults. However, to date, little is known about effects of viral infection itself (independent of the gene delivered) on neuronal physiology. Electrically-evoked synaptic responses are routinely recorded to measure functional alterations in the nervous system and were used here to assess the potential capability of HSV vectors to disrupt physiology of the hippocampus (a forebrain structure involved in learning that is highly susceptible to necrotic insult, making it a frequent target in gene therapy research). Population excitatory post-synaptic potentials (EPSPs) were recorded in the dentate gyrus (DG) and in area CA3 in vivo 72 h after infusion of an HSV vector expressing a reporter gene (lacZ) or vehicle into the DG. Evoked perforant path (PP-DG) or mossy fiber (MF-CA3) EPSPs slope values measured across input/output (I/O) curves were not altered by infection. Paired-pulse facilitation at either recording site was also unaffected. X-gal-positive granule cells surrounded the recording electrode (PP-DG recording) and stimulating electrode tracts (MF-CA3 recording) in animals that received vector, suggesting that we had measured function, at least in part, in infected neurons. Because of the negative electrophysiological result, we sought to deliver a gene with an HSV amplicon which would affect the measured endpoints, as a positive control. Delivery of calbindin D28kpotentiated PP-DG synaptic strength, indicating that our recording system could detect alterations due to vector expression. Thus, the data indicate that HSV vectors are benign, in regard to effects on synaptic function, and support the use of these vectors as a safe method to deliver selected genes to the central nervous system.

It is speculated that nicotine receptor ligands might be of therapeutic benefit for the treatment of obesity, anxiety, and memory loss. With the exception of nicotine, few selective high-affinity agents exist. (-)Epibatidine (1), isolated from Ecuadoran frogs, shows structural resemblence to (-)nicotine (2). Molecular modeling studies reveal that the N to N distance in 1 (5.5 A) is greater than that found in 2 (4.9 A). This distance exceeds that previously considered optimal for the nicotine receptor pharmacophore (4.8. ± 0.3 A) by about half a bond length. Nevertheless 1 binds (Ki = 0.055 nM) at [3H]nicotine-labeled receptors with higher affinity than 2 (Ki = 1.5 nM). Although our studies are still in progress, molecular superimposition and preliminary results with various structurally modified analogs suggest that it is the conformationally constrained nature of 1 that accounts for its high affinity. For example, 6-chloronicotine binds only with slightly higher affinity (Ki = 0.6 nM) than nicotine suggesting, at least for nicotine, that the chloro substituent is tolerated but does not contribute significantly to binding. Excision of the ethano bridge of deschloroepibatidine, to yield isonornicotine (Ki = 12.5 nM), results in reduced affinity. Isonicotine binds with similar (Ki = 7.3 nM) affinity. The bridged azabicycloheptane system likely fixes the structure of epibatidine in a highly receptor-preferred conformation. The pharmacophore for nicotine receptor binding will require reconsideration and should take into account the longer N-N distance of epibatidine. The N-N distance in epibatidine may be optimal, whereas that in isonornicotine is longer, and that for nicotine shorter. (Supported in part by funding from TDC/CIT.)

Thyroid cell cyclic AMP synthesis is stimulated by β-adrenergic agonists. We have characterized this sensitivity on cultured porcine thyroid cells and have studied its modulation by chronic treatment with thyrotrophin. The synthesis of cyclic AMP in intact porcine thyroid cells in primary culture was stimulated by the β-adrenergic agonist, isoproterenol. This stimulation was dose-dependent and was inhibited by the β-adrenergic antagonists propranolol and alprenolol. The cell responsiveness (i.e. the response elicited by 5 μm-isoproterenol after 5-min stimulation) was increased when the cells were cultured in the absence of thyrotrophin. Thyrotrophin, when present in the culture medium at the onset of culturing, inhibited this increase. A concentration of 100 μu. thyrotrophin/ml was sufficient to reduce the cyclic AMP response to 15% of its control value. Prostaglandin E2 or dibutyryl cyclic AMP did not mimic the effect of thyrotrophin. The low sensitivity of thyrotrophin-treated cells to β-adrenergic agonists could be explained by a decreased number of β-adrenergic receptors. [125I]Iodohydroxybenzyl pindolol specific binding was ten times greater in membrane preparations of control cells than in membranes derived from thyrotrophin-treated cells. The β-adrenergic sensitivity of cultured thyroid cells was also decreased after long-term treatment by terbutaline. A time- and dose-dependent desensitization was observed.