Hereditary axonal neuropathy related to MME gene mutation in a family with fetomaternal alloimmune glomerulonephritis.

We report a consanguineous family with a homozygous and heterozygous membrane metallo-endopeptidase (MME) mutation (c.467delC) and two clinical conditions: fetomaternal alloimmune membranous glomerulopathy (FMG) and hereditary motor and sensory axonal neuropathy. The penetrance of both phenotypes was variable. Some individuals experienced unusually fast neurological degradation. Pain and vasomotor signs were frequent complaints, possibly due to a loss of the neutral endopeptidase (NEP, the MME gene product) function and its subsequent inability to degrade substance P and vasomotor peptides. Electrophysiological and nerve biopsy findings were consistent with predominantly axonal neuropathy. This specific clinical phenotype was attributed to a c.467delC MME gene mutation. Diagnosis of such a mutation is important but can be challenging, due to allele dropout. Heterozygous subjects who had already reached the expected age of disease onset had peripheral neuropathy, but also suffered from additional diseases. Neurologists should advise women of childbearing age with MME mutations to seek pre-pregnancy genetic advice and nephrologists should search for neuropathy in patients with FMG.

[Pyopneumothorax in rheumatoid arthritis]. / Pyopneumothorax au cours d'une polyarthrite rhumatoïde.

INTRODUCTION: Rheumatoid arthritis (RA) is a chronic inflammatory disease affecting the joints but which frequently includes extra articular effects, including pulmonary nodules, which grow faster under immunosuppressive treatment. CASE REPORT: A 74 years old man, with mild asbestosis, underwent treatment with methotrexate then leflunomide (LEF) for seropositive RA. In February 2014, during monitoring of his asbestosis, chest CT scan showed the appearance of thick-walled cavitating lung nodules, with a central and sub pleural distribution. The patient was asymptomatic. Bronchoalveolar lavage excluded infection and tumor. LEF was stopped but in May 2014, the patient was admitted with respiratory infection and a pyopneumothorax which required surgical management. The postoperative course was complicated with a persistent pneumothorax. CONCLUSIONS: We describe a case of RA complicated by a pyopneumothorax after treatment with LEF. The risk of this complication could be reduced by regular chest imaging.

[Feasibility of assessing EGFR mutation and others using samples obtained by EBUS transbronchial needle aspiration]. / Faisabilité de la recherche de mutations EGFR et KRAS sur des prélèvements obtenus par EBUS-PTBA.

INTRODUCTION: Assessment of mutation status in patients with non-small cell lung cancer (NSCLC) is often required. The aim of this study was to confirm the feasibility of molecular mutation analysis in cytologic specimens obtained by endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA). METHODS: Patients with an EBUS-TBNA positive for adenocarcinoma or NSCLC (squamous cell carcinoma excluded) were included retrospectively from January to December 2010, and prospectively from January to August 2011. Specimens were collected on liquid based preparation and processed on paraffin-embedded cell blocks after ThinPrepÒ procedure. Molecular analysis was performed by a SnaPshot assay for EGFR and by pyrosequencing for KRAS on suitable samples (>5% tumor cells). RESULTS: Eighty-two patients were included (63 adenocarcinoma). Molecular analysis for EGFR was feasible in 80 (97.6%) patients and for KRAS in 78 (95.1%) patients. Molecular analysis identified EGFR and KRAS mutations in tumor samples from four (5%) and 18 (23%) patients respectively. All EGFR mutations were found in women. CONCLUSIONS: Molecular analysis mutations can be performed routinely in cytologic specimens obtained by EBUS-TBNA.

[Growth and tolerance in infants fed an infant formula based on hydrolyzed rice proteins]. / Efficacité sur la croissance et tolérance d'une préparation pour nourrissons à base de protéines de riz hydrolysées.

UNLABELLED: The objective of this study was to assess the efficacy in terms of growth and tolerance of an infant formula based on hydrolyzed rice proteins. PATIENTS AND METHODS: Healthy infants, born at term, less than 1 month old, and exclusively fed an infant formula based on hydrolyzed rice proteins until their diet was diversified, were included in this open-label, multicenter study. The main outcome measure was daily weight gain during the study period. The infant's weight, height, body mass index (BMI), and the data concerning tolerance (digestive disorders, allergy manifestations) were collected at inclusion in the study, at 2 and 4 months, and before diversifying the infant's diet between 4 and 6 months. The growth parameters were compared to the WHO standards by calculating the Z-score. RESULTS: Seventy-eight infants were included. The mean daily weight gain over 5 months was 23.2 ± 4.3 g/day, identical to the WHO standards (22.2 ± 1.8 g/day, P = 0.09). During the study period, the Z-scores for weight, height, and BMI varied between +1.1 and -0.5 SD according to the WHO standards. Formula acceptance and tolerance were both good. CONCLUSION: The infant formula studied, based on hydrolyzed rice proteins, was well tolerated and led to normal growth over the first few months of life, comparable to the WHO standards.

Experimental treatment of Staphylococcus aureus bovine intramammary infection using a guanine riboswitch ligand analog.

Staphylococcus aureus is a leading cause of intramammary infections (IMI). We recently demonstrated that Staph. aureus strains express the gene guaA during bovine IMI. This gene codes for a guanosine monophosphate synthetase and its expression is regulated by a guanine riboswitch. The guanine analog 2,5,6-triaminopyrimidine-4-one (PC1) is a ligand of the guanine riboswitch. Interactions between PC1 and its target result in inhibition of guanosine monophosphate synthesis and subsequent death of the bacterium. The present study describes the investigational use of PC1 for therapy of Staph. aureus IMI in lactating cows. The in vitro minimal inhibitory concentration of PC1 ranged from 0.5 to 4 µg/mL for a variety of Staph. aureus and Staphylococcus epidermidis strains and required a reducing agent for stability and full potency. A safety assessment study was performed, whereby the healthy quarters of 4 cows were infused with increasing doses of PC1 (0, 150, 250, and 500 mg). Over the 44 h following infusions, no obvious adverse effect was observed. Ten Holstein multiparous cows in mid lactation were then experimentally infused into 3 of the quarters with approximately 50 cfu of Staph. aureus strain SHY97-3906 and infection was allowed to progress for 2 wk before starting PC1 treatment. Bacterial counts reached then about 10(3) to 10(4) cfu/mL of milk. Infected quarters were treated with 1 of 3 doses of PC1 (0, 250, or 500 mg) after each morning and evening milking for 7d (i.e., 14 intramammary infusions of PC1). During the treatment period, milk from PC1-treated quarters showed a significant reduction in bacterial concentrations. However, this reduction of Staph. aureus count in milk was not maintained during the 4 wk following the end of the treatment and only 15% of the PC1-treated quarters underwent bacteriological cure. The somatic cell count and the quarter milk production were not affected by treatments. Although bacterial clearance was not achieved following treatment with PC1, these results demonstrate that the Staph. aureus guanine riboswitch represents a relevant and promising drug target for a novel class of antibiotics for the animal food industry.

Quality assurance issues in radical prostatectomy.

AIMS: Quality of surgery is a controversial issue and no studies are reporting on the standard of surgical quality in the treatment of urological cancer. The question is whether quality can be evaluated and whether there is a standard for a qualitatively well performed radical retropubic prostatectomy. METHODS: We reviewed the literature on this topic. Data of four large studies based on Medicare claims and an EORTC report were analysed. RESULTS: Two studies reflect hospital-volume rather than surgeon-volume. Two compared hospital-volume and surgeon-volume and in both studies there was no clear relationship between surgeon-volume and the parameters reviewed. Similarly, the EORTC study concluded that there is a variation in outcome that is not related to the caseload and proposed minimal quality standards for radical prostatectomy. CONCLUSIONS: There is no clear relationship between surgeon-volume and surgical quality. Since radical prostatectomy is the standard treatment for the most frequent male malignancy and is offered to many patients that might never even suffer from the disease, the procedure must be performed with the highest guarantee of quality. Although, quality control of radical prostatectomy is feasible, its implementation will still require an enormous effort from the urological community.

Disruption of a new X linked gene highly expressed in brain in a family with two mentally retarded males.

BACKGROUND: Mental retardation (MR) affects 2-3% of the human population and some of these cases are genetically determined. Although several genes responsible for MR have been identified, many cases have still not been explained. METHODS: We have identified a pericentric inversion of the X chromosome inv(X)(p22.3;q13.2) segregating in a family where two male carriers have severe MR while female carriers are not affected. RESULTS: The molecular characterisation of this inversion led us to identify two new genes which are disrupted by the breakpoints: KIAA2022 in Xq13.2 and P2RY8 in Xp22.3. These genes were not previously fully characterised in humans. KIAA2022 encodes a protein which lacks significant homology to any other known protein and is highly expressed in the brain. P2RY8 is a member of the purine nucleotide G-protein coupled receptor gene family. It is located in the pseudo-autosomal region of the X chromosome and is not expressed in brain. CONCLUSIONS: Because the haploinsufficiency of P2RY8 in carrier mothers does not have a phenotypic consequence, we propose that the severe MR of the affected males in this family is due to the absence of the KIAA2022 gene product. However, screening 20 probands from X linked MR families did not reveal mutations in KIAA2022. Nonetheless, the high expression of this gene in fetal brain and in the adult cerebral cortex could be consistent with a role in brain development and/or cognitive function.

Pancreatic pseudocyst with biliary fistula: treatment with endoscopic internal drainage.

Surgical drainage is the standard treatment for pancreatic pseudocysts and their complications. However, acute symptomatic pancreatic pseudocysts are amenable to endoscopic internal drainage in select cases. We report a case of pancreatic pseudocyst with biliary fistula resulting from a recurrent pseudocyst treated with endoscopic stent drainage.

Molecular biology for the pediatric surgeon.

Molecular biology is leading a revolution in our understanding, diagnosis, and treatment of disease and will continue to do so. Medicine in the future will require a greater understanding of this field and its methods by medical practitioners. This report reviews the basic aspects of the field including recombinant DNA methods. Of particular importance is how molecular biology will impact pediatric surgeons. Accordingly, the final section of this report briefly reviews the molecular biology of three diseases commonly treated by pediatric surgeons.

Length changes in the joining segment between domains 5 and 6 of a group II intron inhibit self-splicing and alter 3' splice site selection.

Domain 5 (D5) and domain 6 (D6) are adjacent folded hairpin substructures of self-splicing group II introns that appear to interact within the active ribozyme. Here we describe the effects of changing the length of the 3-nucleotide segment joining D5 to D6 [called J(56)3] on the splicing reactions of intron 5 gamma of the COXI gene of yeast mitochondrial DNA. Shortened variants J(56)0 and J(56)1 were defective in vitro for branching, and the second splicing step was performed inefficiently and inaccurately. The lengthened variant J(56)5 had a milder defect-splicing occurred at a reduced rate but with correct branching and a mostly accurate 3' splice junction choice. Yeast mitochondria were transformed with the J(56)5 allele, and the resulting yeast strain was respiration deficient because of ineffective aI5 gamma splicing. Respiration-competent revertants were recovered, and in one type a single joiner nucleotide was deleted while in the other type a nucleotide of D6 was deleted. Although these revertants still showed partial splicing blocks in vivo and in vitro, including a substantial defect in the second step of splicing, both spliced accurately in vivo. These results establish that a 3-nucleotide J(56) is optimal for this intron, especially for the accuracy of 3' splice junction selection, and indicate that D5 and D6 are probably not coaxially stacked.

Studies of point mutants define three essential paired nucleotides in the domain 5 substructure of a group II intron.

Domain 5 (D5) is a highly conserved, largely helical substructure of group II introns that is essential for self-splicing. Only three of the 14 base pairs present in most D5 structures (A2.U33, G3.U32, and C4.G31) are nearly invariant. We have studied effects of point mutations of those six nucleotides on self-splicing and in vivo splicing of aI5 gamma, an intron of the COXI gene of Saccharomyces cerevisiae mitochondria. Though none of the point mutations blocked self-splicing under one commonly used in vitro reaction condition, the most debilitating mutations were at G3 and G4. Following mitochondrial Biolistic transformation, it was found that mutations at A2, G3, and C4 blocked respiratory growth and splicing while mutations at the other sites had little effect on either phenotype. Intra-D5 second-site suppressors showed that pairing between nucleotides at positions 2 and 33 and 4 and 31 is especially important for D5 function. At the G3.U32 wobble pair, the mutant A.U pair blocks splicing, but a revertant of that mutant that can form an A+.C base pair regains some splicing. A dominant nuclear suppressor restores some splicing to the G3A mutant but not the G3U mutant, suggesting that a purine is required at position 3. These findings are discussed in terms of the hypothesis of Madhani and Guthrie (H. D. Madhani and C. Guthrie, Cell 71:803-817, 1992) that helix 1 formed between yeast U2 and U6 small nuclear RNAs may be the spliceosomal cognate of D5.

The stereochemical course of group II intron self-splicing.

The stereochemical specificities and reaction courses for both self-splicing steps of a group II intron have been determined by phosphorothioate substitution at the 5' and 3' splice site phosphodiester bonds. Both steps of the splicing reaction proceeded with a phosphorothioate in the Sp configuration but were blocked by the Rp diastereomer. Both steps also proceeded with inversion of stereochemical configuration around phosphorus, consistent with a concerted transesterification reaction. These results are identical to those found for nuclear precursor mRNA (pre-mRNA) splicing and provide support for the hypothesis that group II introns and nuclear pre-mRNA introns share a common evolutionary history.

Domain 5 interacts with domain 6 and influences the second transesterification reaction of group II intron self-splicing.

The role of domain 5 (d5) from the self-splicing group II intron 5 gamma of the COXI gene of yeast mitochondrial DNA in branching and 3' splice site utilization has been studied using a substrate transcript lacking d5 (delta d5 RNA). This RNA is completely unreactive in vitro, but releases 5' exon by hydrolysis under various reaction conditions when d5 RNA is added in trans. Under an extreme reaction condition, some accurate branching and splicing occur. Much more efficient use of a 3' splice site is obtained when delta d5 RNA is complemented by a transcript containing the wild-type domains 5 and 6 plus the 3' exon. While most delta d5 RNA molecules in that protocol still react by hydrolysis at the 5' splice site, the branching that occurs uses only the d6 tethered to d5 that is provided in trans. The use of this d6 and the 3' splice site also linked to d5, along with the observed indifference to the other d6 and 3' splice site resident in the delta d5 RNA, indicates that d5 plays a key role in positioning d6 for the first reaction step as well as in 3' splice site use. Two models for the manner by which d5 interacts with d6 are discussed.

Group II introns deleted for multiple substructures retain self-splicing activity.

Group II introns can be folded into highly conserved secondary structures with six major substructures or domains. Domains 1 and 5 are known to play key roles in self-splicing, while the roles of domains 2, 3, 4, and 6 are less clear. A trans assay for domain 5 function has been developed which indicates that domain 5 has a binding site on the precursor RNA that is not predicted from any secondary structure element. In this study, the self-splicing group II intron 5 gamma of the coxI gene of yeast mitochondrial DNA was deleted for various intron domains, singly and in combinations. Those mutant introns were characterized for self-splicing reactions in vitro as a means of locating the domain 5 binding site. A single deletion of domain 2, 3, 4, or 6 does not block in vitro reactions at either splice junction, though the deletion of domain 6 reduces the fidelity of 3' splice site selection somewhat. Even the triple deletion lacking domains 2, 4, and 6 retains some self-splicing activity. The deletion of domains 2, 3, 4, and 6 blocks the reaction at the 3' splice junction but not at the 5' junction. From these results, we conclude that the binding site for domain 5 is within domain 1 and that the complex of 5' exon, domain 1, and domain 5 (plus short connecting sequences) constitutes the essential catalytic core of this intron.