Formal [4 + 2] cycloaddition of imines with alkoxyisocoumarins.

A new preparation of δ-lactams is reported. In the presence of a Lewis acid promoter, alkoxyisocoumarins engage a range of N-aryl and N-alkyl imines to form δ-lactams with a pendent carboalkoxy substituent. A sulfonamide-thiourea catalyst enables the synthesis of these products in moderate to good enantioselectivities.

Chryseobacterium indologenes central nervous system infection in infancy: an emergent pathogen?

The isolation of Chryseobacterium indologenes as a causative micro-organism in human diseases is rare. Risk factors for infections caused by this pathogen include very young and very old age, indwelling devices, immune suppression and recent use of broad-spectrum antibiotics. Most cases suffer from bacteraemia or nosocomial pneumonia, whilst infection of the central nervous system (CNS) is extremely rare. We present a term-born infant diagnosed prenatally with holoprosencephaly and obstructive hydrocephalus, requiring post-natal ventriculoperitoneal shunt insertion. At 6 weeks of age, he suffered from Escherichia coli meningitis, showing satisfactory clinical response with antimicrobial therapy. Aged 11 months, he suffered from hyper-drainage syndrome, resulting in the removal of the shunt system. He represented 11 days post-operatively, with low-grade fever, irritability and cerebrospinal fluid (CSF) leakage. C. indologenes from CSF was isolated and antimicrobial therapy with ceftazidime and trimethoprim-sulfamethoxazole for 3 weeks resulted in good clinical response. This is the first documented community-acquired CNS infection due to C. indologenes in an infant without concomitant indwelling device or previous antibiotic pressure.

Phylogenetic analyses of symbiotic genes and characterization of functional traits of Mesorhizobium spp. strains associated with the promiscuous species Acacia seyal Del.

AIMS: To assess the phenotypic, symbiotic and genotypic diversity scope of Mesorhizobium spp. strains associated with Acacia seyal (Del.) isolated from different agro-ecological zones in Senegal, and uses of susceptible microbial inoculum in a reafforestation process. METHODS AND RESULTS: A polyphasic approach including phenotypic and genotypic techniques was used to study the diversity and their relationships with other biovars and species of rhizobia. The geographical origins of the strains have limited effect on their phylogenetic and phenotypic classification. Nodulation tests indicated promiscuity of the strains studied, because they were capable of nodulating six woody legume species (Acacia auriculiformis, Acacia senegal, A. seyal, Acacia tortilis ssp. raddiana, Leucaena leucocephala and Prosopis juliflora). Sequencing and phylogenetic analyses of nodA, nodC and nifH genes pointed out that in contrast to nodA gene, the phylogenies of nodC and nifH genes were not consistent with that of 16S rRNA, indicating that these genes of the A. seyal-nodulating rhizobia might have different origins. Microbial inoculation on nonsterile soil had significant effect on the nodules number and the growth of the seedlings, indicating that these strains of rhizobia might be used as inoculum. CONCLUSIONS: The results indicated that A. seyal is a nonselective host that can establish effective symbiosis with Mesorhizobium spp. strains from diverse genomic backgrounds and that the selected A. seyal-nodulating rhizobia could enhance plant growth. SIGNIFICANCE AND IMPACT OF THE STUDY: These results showed the important role that A. seyal could play in the improvement of reafforestation process as a promiscuous host, which can establish effective symbiosis with rhizobia from diverse genomic backgrounds.

Phenotypic and genotypic characteristics of Acacia senegal (L.) Willd. root-nodulating bacteria isolated from soils in the dryland part of Senegal.

AIMS: We characterized phenotypically and genotypically root-nodulating bacteria associated with Acacia senegal (L.) Willd. isolated from the soils surrounding A. senegal trees in the dry land area of Senegal. METHODS AND RESULTS: The phenotypical and genotypical characterizations we carried out showed a high diversity of A. senegal root-nodulating bacteria. Phenotypic patterns showed adaptations of the rhizobial strains to many environmental stresses such as heat, drought, and salinity. Twelve molecular groups were distinguished by profiles obtained using polymerase chain reaction/restriction fragment length polymorphism techniques from intergenic spacer region rDNA. The highest genetic diversity was found around the A. senegal rhizosphere. Therefore, A. senegal seemed to have a positive influence on occurrence and genotypical diversity of rhizobial populations. Rhizobial isolates obtained in this study belonged phylogenetically to the genera Mesorhizobium and Rhizobium. CONCLUSIONS: Our results provided information about the genetic diversity of the rhizobial strains associated with A. senegal and suggested the adaptability of natural rhizobial populations to major ecological environmental stress within these soil environments. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggested a potential selection of compatible and well adapted strains under stress conditions as inoculants for successful A. senegal growth in arid lands.

[Educational scheme for patients on home peritoneal dialysis in Spain]. / Programa de educación para los pacientes de diálisis peritoneal domiciliaria en España.

The objective of the present study is to obtain information about the training programme for patients undergoing Domiciliary Peritoneal Dialysis (DPD) in Spain. For the purposes of the study we designed a questionnaire comprising 50 closed-ended items and one open response item. The questionnaire was sent to 104 hospitals and was completed by 78.84% of them (n > or = 82). The average of patients undergoing peritoneal dialysis (PD) in the hospitals under study was 27.6: 15.8 of them receiving Chronic Ambulatory Peritoneal Dialysis (CAPD) and 11.8 Automatic Peritoneal Dialysis (APD). The questionnaire also served to investigate into the training methodology used in the different units, the involvement of the family in the programme, the basic knowledge patients received about Chronic Renal Insufficiency, the procedures associated with the therapy and the preparation they obtained to solve small-scale contingencies and emergency situations as well as the improvement of their quality of life. We also evaluated the training programme of autonomous patients on DPD and at the end of the questionnaire a blank space was left for facilities to add any comments or suggestions they considered relevant. From the results obtained we may conclude that most Spanish hospitals have devised a training planning for patients undergoing PD which helps them or caregivers to perform domiciliary treatment safely, provides them with basic knowledge about the disease and the routine procedures associated with the treatment, enables them to cope with contingencies and emergency situations and improves their quality of life during the dialysis period.

Genetic diversity and phylogeny of rhizobia that nodulate acacia spp. in morocco assessed by analysis of rRNA genes

Forty rhizobia nodulating four Acacia species (A. gummifera, A. raddiana, A. cyanophylla, and A. horrida) were isolated from different sites in Morocco. These rhizobia were compared by analyzing both the 16S rRNA gene (rDNA) and the 16S-23S rRNA spacer by PCR with restriction fragment length polymorphism (RFLP) analysis. Analysis of the length of 16S-23S spacer showed a considerable diversity within these microsymbionts, but RFLP analysis of the amplified spacer revealed no additional heterogeneity. Three clusters were identified when 16S rDNA analysis was carried out. Two of these clusters include some isolates which nodulate, nonspecifically, the four Acacia species. These clusters, A and B, fit within the Sinorhizobium lineage and are closely related to S. meliloti and S. fredii, respectively. The third cluster appeared to belong to the Agrobacterium-Rhizobium galegae phylum and is more closely related to the Agrobacterium tumefaciens species. These relations were confirmed by sequencing a representative strain from each cluster.

In situ detection of human cytomegalovirus DNA in gastrointestinal biopsies from AIDS patients by means of various PCR-derived methods.

The development of new in situ assessment of HCMV disease on endoscopical gastrointestinal biopsies from AIDS patients is described and compared with the viral load measured by semiquantitative solution-phase PCR (SQ-PCR). Ten biopsies were examined by viral isolation, standard histology, in situ hybridization (ISH), in situ PCR-hybridization (PCR-ISH) and SQ-PCR, using the same target sequence. The methods developed for in situ HCMV detection were HCMV primers, the plasmid pCMV 406-S, a vector-free-digoxigenin-labelled HCMV-362 probe and the pSK + MCS nonsense probe. Paraffin-embedded MRC5 cells, either HCMV-infected or uninfected served as controls of specificity for ISH. beta-Actin primers were designed as markers of DNA integrity. Computerized models of the PCR, solution-phase and in situ PCR on formalin-fixed DNA indicated that HCMV and beta-actin primers were efficient and specific. Nine biopsies were negative for HCMV by histology and virus isolation. SQ-PCR revealed 80,000; 80 and < 80 HCMV genomic equivalents in 6, 2 and 2 biopsies, respectively. In 8 biopsies, both ISH and PCR-ISH identified positive nuclei in the intestinal epithelium, with sparing of the lamina propria. This indicates that an improvement in in situ methods can help the timely diagnosis of HCMV infection. Direct in situ PCR with beta-actin primers showed a positive signal in all the nuclei in the tissue sections, whereas omission of Taq polymerase resulted in an absence of signal, implying optimal in situ PCR. The data suggest an early-stage reactivation of HCMV, possibly harboured in the intestinal epithelium.

[Ileocolitis caused by Herpes simplex virus type 2]. / Iléo-colite herpétique à virus Herpes simplex de type 2.

The authors report the case of a 51-year-old woman who was hospitalized for severe acute colitis. Sub-total colectomy was performed, due to clinical deterioration and resistance to treatment. The examination of the surgery specimen revealed a herpes-virus type 2 as the responsible pathogen agent. Complementary aciclovir treatment cured the patient.

[Recurrent hemothorax due to pleural endometriosis]. / Hémothorax récidivant par endométriose pleurale.

We report a case of a 31 year old woman with recurrent hemothorax at the beginning of mens. Pleural biopsy confirmed the diagnosis of pleural endometriosis. Medical treatment with a Gn-RH analogue failed to cure and we realised surgical pleurodesis.

[Value of transparietal lung biopsy in the cytological diagnosis of bronchiolo-alveolar carcinomas of the lung. Apropos of 3 cases]. / Apport de la ponction pulmonaire transpariétale au diagnostic cytologique des carcinomes bronchiolo-alvéolaires du poumon. A propos de 3 cas.

Bronchiolo-alveolar carcinoma is a rare primary lung tumour, which is difficult to diagnose by cytological techniques. This peripheral tumour, which develops on pre-existing alveolar walls, is not visible on bronchial endoscopy and brushing is often negative. The cellular material derived from aspiration or expectoration is characterised by numerous papillae, without any cytonuclear criteria of malignancy. The differential diagnosis is very difficult with reactive papillary hyperplasia and various forms of chronic bronchial inflammation. Transparietal aspiration lung biopsy facilitates the diagnosis of glandular carcinoma: the material examined corresponds to pathological tissue, as the biopsy is performed under image intensifier control. An inflammatory lesion is excluded by the rich cellularity and by the presence of architectural features of malignancy: cohesive and three-dimensional clumps of papillary tumour cells. The early cytological diagnosis of bronchiolo-alveolar and primary bronchiolar carcinomas of the lung by transparietal aspiration biopsy can allow the surgical cure of certain localised forms.

[Pseudotumor amylosis of the bladder. Apropos of a case]. / Amylose pseudo-tumorale de la vessie. A propos d'une observation.

A 57 year-old patient with localized pseudo-tumoral amyloidosis of bladder is described. There was no past medical history. Hematuria was the main symptom. The treatment consisted in transurethral resection. Two recurrences occurred at 4 and at 6 years which were also treated by resections. Clinical and biological evaluation was normal. Localized pseudo-tumoral amyloidosis of the urinary tract is a rare affection of good prognosis. Lesions present as pseudo-tumoral masses which can be biopsied without any risk of hematuria. Investigations are required to eliminate a generalized amyloidosis or a malignant lymphoproliferation. Treatment should be as conservative as possible.

[Hospital admissions due to varicella in a tertiary hospital]. / Hospitalizaciones por varicela en un hospital de tercer nivel.

INTRODUCTION: Varicella (chickenpox) can cause serious complications and admission to hospital. Several countries included the varicella vaccine in their immunization schedules. MATERIAL AND METHODS: A descriptive and retrospective study of hospitalizations due to varicella and its complications was conducted in a referral center from 2005 to 2011. RESULTS: A total of 1192 children with varicella were seen in the emergency room, of which 99 (8.5%) required admission. The annual incidence of admissions due to varicella and varicella complications was, 19.4 and 15.3 cases per 100,000 children under 14 years, respectively. Complications were more common in children under 5 years (79.5%), and with no underlying disease (78.2%). Infection of skin and soft tissue was the most common complication (62%). The mean hospital stay was 4.5 days (SD 4). CONCLUSIONS: Varicella causes high morbidity, and is more frequent in absolute terms in healthy children under 5 years of age. Therefore, routine vaccination recommended by the Immunization Advisory Committee should be mandatory.

Successful management of Churg-Strauss syndrome using omalizumab as adjuvant immunomodulatory therapy: first documented pediatric case.

Churg-Strauss syndrome (CSS) is an anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitis; it is extremely rare in childhood and defined according to the Chapel-Hill Consensus as an eosinophil-rich and granulomatous inflammation involving the respiratory tract and necrotizing vasculitis affecting small to medium-sized vessels. Children commonly have a history of asthma and sinusitis whilst clinical presentation typically involves pulmonary tract and less frequently skin, heart, gastrointestinal tract, and peripheral nerves. Cardiopulmonary disease is higher in children and prognosis is worse. It is associated with significant eosinophilia and raised serum IgE-levels. ANCA are only found in 25% of childhood cases. Here we report the case of a 10-year-old girl who presented to us with vomiting, abdominal pain, and weight loss, paresthesias of lower extremities and breathlessness as well as a history of asthma, sinusitis and allergic rhinitis. She was treated with corticosteroids, cyclophosphamide, intravenous immunoglobulin, mycophenolate mofetil (MMF), and rituximab. However, remission was only achieved after initiation of omalizumab therapy, a recombinant humanized anti-IgE antibody. To the best of our knowledge this is the first pediatric patient suffering from CSS successfully managed with adjuvant anti-IgE therapy resulting in the control of respiratory as well as gastrointestinal symptoms.

Optimization of DNA isolation from legume nodules.

AIMS: The aim of this study was to optimize DNA extraction from legume nodules to obtain large amounts of high-quality genomic DNA. METHODS AND RESULTS: Nodules of different legume species were used. Varied concentrations of guanidine thiocyanate (from 6 mol l(-1) to 0.05 mmol l(-1)), a component of DNAzol, were tested. The quality of DNA extract was determined by PCR-RFLP. The best results were obtained with 0.5 mmol l(-1) guanidine thiocyanate, which resulted in greater DNA yield than with higher and lower concentrations or with DNAzol. CONCLUSION: The procedure using 0.5 mmol l(-1) guanidine thiocyanate yields the highest DNA amount when compared with previously described protocols and offers a reliable method to isolate DNA from nodules of different origins. SIGNIFICANCE AND IMPACT OF THE STUDY: Irrespective of nodule origin, DNA yield was increased significantly, by two (e.g., Vigna nodules) to seven (Acacia auricoliformis nodules) times. In addition, the proposed procedure's costs are lower than those using the DNAzol.

Genetic diversity of Acacia tortilis ssp. raddiana rhizobia in Tunisia assessed by 16S and 16S-23S rDNA genes analysis.

AIMS: In order to understand the genetic diversity of Acacia tortilis ssp. raddiana-rhizobia in Tunisia, isolates from nine geographical locations were obtained and analysed. METHODS AND RESULTS: Characterization using restriction fragment length polymorphism analysis (RFLP) of PCR-amplified 16S rRNA gene and the intergenic spacer (IGS) between the 16S and 23S rRNA genes was undertaken. Symbiotic efficiency of the strains was also estimated. Analysis of the 16S rRNA by PCR-RFLP showed that the isolates were phylogenetically related to Ensifer ssp., Rhizobium tropicii-IIA, and Rhizobium tumefaciens species. Analysis of 16S-23S spacer by PCR-RFLP showed a high diversity of these rhizobia and revealed eleven additional groups, which indicates that these strains are genetically very diverse. Full 16S rRNA gene-sequencing showed that the majority of strains form a new subdivion inside the genera Ensifer, with Ensifer meliloti being its nearest neighbour. Nodulation test performed on the plant host demonstrated differences in the infectivity among the strains. CONCLUSION: Rhizobial populations that nodulate specifically and efficiently Acacia tortilis ssp. raddiana in representative soils of Tunisia is dominated by E. meliloti-like genomospecies. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper provides the first clear characterization and symbiotic efficiency data of rhizobia strains nodulating A. tortilis in Tunisia.

Ectomycorrhizal symbiosis enhanced the efficiency of inoculation with two Bradyrhizobium strains and Acacia holosericea growth.

Two strains of Bradyrhizobium sp., Aust 13C and Aust 11C, were dually or singly inoculated with an ectomycorrhizal fungus, Pisolithus albus to assess the interactions between ectomycorrhizal symbiosis and the nodulation process in glasshouse conditions. Sequencing of strains Aust 13C and Aust 11C confirmed their previous placement in the genus Bradyrhizobium. After 4 months' culture, the ectomycorrhizal symbiosis promoted plant growth and the nodulation process of both Bradyrhizobium strains, singly or dually inoculated. PCR/RFLP analysis of the nodules randomly collected in each treatment with Aust 13C and/or Aust 11C: (1) showed that all the nodules exhibited the same patterns as those of the Bradyrhizobium strains, and (2) did not detect contaminant rhizobia. When both Bradyrhizobium isolates were inoculated together, but without P. albus IR100, Aust 11C was recorded in 13% of the treated nodules compared to 87% for Aust 13C, whereas Aust 11C and Aust 13C were represented in 20 and 80% of the treated nodules, respectively, in the ectomycorrhizal treatment. Therefore Aust 13C had a high competitive ability and a great persistence in soil. The presence of the fungus did not significantly influence the frequencies of each Bradyrhizobium sp. root nodules. Although the mechanisms remain unknown, these results showed that the ectomycorrhizal and biological nitrogen-fixing symbioses were very dependent on each other. From a practical point of view, the role of ectomycorrhizal symbiosis is of great importance to N2 fixation and, consequently, these kinds of symbiosis must be associated in any controlled inoculation.

Arbuscular mycorrhizal symbiosis changes the colonization pattern of Acacia tortilis spp. Raddiana rhizosphere by two strains of rhizobia.

The aim of the study was to assess the effect of the mycorrhizosphere of A. tortillis spp. raddiana mycorrhized with Glomus intraradices on the root nodulation by Sinorhizobium terangae (ORS 1009) and/or Mesorhizobium plurifarium (ORS 1096) in two different culture substrates (sandy soil and sand). The endomycorrhizal fungus only stimulated plant growth in the sandy soil. Moreover, arbuscular mycorrhizal infection enhanced the nodulation process in both culture substrates. Beside the stimulatory effects of the mycorrhizosphere on both rhizobia development, fungal symbiosis induces two different dynamics of each bacterial strains in the sand-grown plants. These results suggest specific relationships could occur during the development of the tripartite symbiosis, at physiological and molecular level. From a practical point of view, the role of arbuscular mycorrhizas in improving nodulation and N2 fixation is universally recognized. The fungal symbiosis could modify the development of bacterial inoculants along the root systems. This effect is of particular interest in the controlled inoculation of selected rhizobia.

High-resolution phylogenetic analysis of NO2--oxidizing Nitrobacter species using the rrs-rrl IGS sequence and rrl genes.

A high-resolution phylogenetic analysis of Nitrobacter strains and their neighbours was made using the rrs-rrl intergenic spacer sequence and the hypervariable part of the rrl gene. The phylogenetic tree obtained was consistent with that which was obtained previously but was much more discriminating, permitting the design of genus-specific primers.

RER(+) phenotype in prostate intra-epithelial neoplasia associated with human prostate-carcinoma development.

The mutator (RER(+)) phenotype has been shown to be a mutational mechanism for tumour-suppressor-gene inactivation in colorectal cancer. A group of 60 prostate-carcinoma patients was studied to determine the frequency, intratumour distribution and timing of mutator phenotype in this cancer. Ten microsatellite loci were analyzed in 172 carcinoma foci (CF) and in 57 associated non-cancerous prostate tissues, including 31 areas of prostate intra-epithelial neoplasia (PIN) and 26 non-dysplastic areas with glandular hyperplasia (HP). We detected lesions with the RER(+) phenotype in 42% (25/60) of the prostate tumours. Clonal foci with RER(+) phenotype were detected at similar frequencies in pre-cancereous PIN (16%, 5/31) as in associated carcinoma foci (22%, 37/172), but were detected in only one of the 26 non-dysplastic prostate tissues studied (4%). Thus, clonal RER(+) foci were significantly more frequent in CF than in HP (p < 0.05). MI itself was significantly more frequent in CF (53%, p < 0.0001) and in PIN (35%, p < 0.05) than in HP (12%). Furthermore, 5 PIN harboured microsatellite mutations also detected in the associated cancer. Our overall results therefore strongly suggest that the mutator phenotype may occur as an early event in prostate tumorigenesis.