Pleiotropic role of the RNA chaperone protein Hfq in the human pathogen Clostridium difficile.

Clostridium difficile is an emergent human pathogen and the most common cause of nosocomial diarrhea. Our recent data strongly suggest the importance of RNA-based mechanisms for the control of gene expression in C. difficile. In an effort to understand the function of the RNA chaperone protein Hfq, we constructed and characterized an Hfq-depleted strain in C. difficile. Hfq depletion led to a growth defect, morphological changes, an increased sensitivity to stresses, and a better ability to sporulate and to form biofilms. The transcriptome analysis revealed pleiotropic effects of Hfq depletion on gene expression in C. difficile, including genes encoding proteins involved in sporulation, stress response, metabolic pathways, cell wall-associated proteins, transporters, and transcriptional regulators and genes of unknown function. Remarkably, a great number of genes of the regulon dependent on sporulation-specific sigma factor, SigK, were upregulated in the Hfq-depleted strain. The altered accumulation of several sRNAs and interaction of Hfq with selected sRNAs suggest potential involvement of Hfq in these regulatory RNA functions. Altogether, these results suggest the pleiotropic role of Hfq protein in C. difficile physiology, including processes important for the C. difficile infection cycle, and expand our knowledge of Hfq-dependent regulation in Gram-positive bacteria.

The TcdE holin drives toxin secretion and virulence in Clostridioides difficile.

Clostridioides difficile is the leading cause of healthcare associated infections. The Pathogenicity Locus (PaLoc) toxins TcdA and TcdB promote host disease. These toxins lack canonical N-terminal signal sequences for translocation across the bacterial membrane, suggesting alternate mechanisms of release, which have included targeted secretion and passive release from cell lysis. While the holin TcdE has been implicated in TcdA and TcdB release, its role in vivo remains unknown. Here, we show profound reductions in toxin secretion in ΔtcdE mutants in the highly virulent strains UK1 (epidemic ribotype 027, Clade 3) and VPI10463 (ribotype 087, Clade 1). Notably, tcdE deletion in either strain rescued highly susceptible gnotobiotic mice from lethal infection by reducing acute extracellular toxin to undetectable levels, limiting mucosal damage, and enabling long-term survival, in spite of continued toxin gene expression in ΔtcdE mutants. Our findings confirm TcdE's critical functions in vivo for toxin secretion and C. difficile virulence.

Rapid diagnosis of Clostridium difficile infection by multiplex real-time PCR.

The gold standards for the diagnosis of Clostridium difficile infections (CDIs) are the cytotoxicity assay and the toxigenic culture. However, both methods are time-consuming and the results are not available before 24-48 h. We developed and evaluated a multiplex in-house real-time polymerase chain reaction (PCR) assay for the simultaneous detection of toxigenic strains of C. difficile and the presumptive identification of the epidemic NAP1/027/BI strain from stools. Amplifications were performed using specific primers for tcdB and tcdC on an ABI Prism 7300 (Applied Biosystems). The detection of amplicons was done using TaqMan probes. The analytical sensitivity of the multiplex real-time PCR for detecting tcdB was estimated to 10 CFU/g of stools. This assay was assessed from 881 consecutive unformed stools from patients suspected of having CDI. The gold standard was the toxigenic culture for the diagnosis of CDI and PCR ribotyping for the identification of the NAP1/027/BI strain. The prevalence of positive toxigenic culture was 9.31%. Compared to the toxigenic culture, the sensitivity, specificity, and positive and negative predictive values were 86.59%, 97.43%, 78.02%, and 98.57%, respectively, for the real-time PCR and 70.73%, 100%, 100%, and 97.08%, respectively, for the cytotoxicity assay.

Further evidence of an Amerindian contribution to the Polynesian gene pool on Easter Island.

Available evidence suggests a Polynesian origin of the Easter Island population. We recently found that some native Easter Islanders also carried some common American Indian (Amerindian) human leukocyte antigen (HLA) alleles, which probably were introduced before Europeans discovered the island in 1722. In this study, we report molecular genetic investigations of 21 other selected native Easter Islanders. Analysis of mitochondrial DNA and Y chromosome markers showed no traces of an Amerindian contribution. However, high-resolution genomic HLA typing showed that two individuals carried some other common Amerindian HLA alleles, different from those found in our previous investigations. The new data support our previous evidence of an Amerindian contribution to the gene pool on Easter Island.

Clostridium difficile toxin synthesis is negatively regulated by TcdC.

Clostridium difficile toxin synthesis is growth phase-dependent and is regulated by various environmental signals. The toxin genes tcdA and tcdB are located in a pathogenicity locus, which also includes three accessory genes, tcdR, tcdC and tcdE. TcdR has been shown to act as an alternative sigma factor that mediates positive regulation of both the toxin genes and its own gene. The tcdA, tcdB and tcdR genes are transcribed during the stationary growth phase. The tcdC gene, however, is expressed during exponential phase. This expression pattern suggested that TcdC may act as a negative regulator of toxin gene expression. TcdC is a small acidic protein without any conserved DNA-binding motif. It is able to form dimers and its N-terminal region includes a putative transmembrane domain. Genetic and biochemical evidence showed that TcdC negatively regulates C. difficile toxin synthesis by interfering with the ability of TcdR-containing RNA polymerase to recognize the tcdA and tcdB promoters. In addition, the C. difficile NAP1/027 epidemic strains that produce higher levels of toxins have mutations in tcdC. Interestingly, a frameshift mutation at position 117 of the tcdC coding sequence seems to be, at least in part, responsible for the hypertoxigenicity phenotype of these epidemic strains.

Plasma nanotextured polymeric lab-on-a-chip for highly efficient bacteria capture and lysis.

We describe the design, fabrication, and successful demonstration of a sample preparation module comprising bacteria cell capture and thermal lysis on-chip with potential applications in food sample pathogen analysis. Plasma nanotexturing of the polymeric substrate allows increase of the surface area of the chip and the antibody binding capacity. Three different anti-Salmonella antibodies were directly and covalently linked to plasma treated chips without any additional linker chemistry or other treatment. Then, the Ab-modified chips were tested for their capacity to bind bacteria in the concentration range of 10(2)-10(8) cells per mL; the module exhibited 100% efficiency in Salmonella enterica serovar Typhimurium bacteria capture for cell suspensions below 10(5) cells per mL (10(4) cells injected with a 100 µL sample volume) and efficiency higher than 50% for 10(7) cells per mL. Moreover, thermal lysis achieved on-chip from as low as 10 captured cells was demonstrated and shown to compare well with off-chip lysis. Excellent selectivity (over 1 : 300) was obtained in a sample containing, in addition to S. Typhimurium and E. coli bacteria.

Calcitonin-loaded liposomes: stability under acidic conditions and bile salts-induced disruption resulting in calcitonin-phospholipid complex formation.

Calcitonin-loading in liposomes composed of phosphatidylcholine, cholesterol and stearylamine or dipalmitoyl phosphatidylglycerol was studied at low pH values and in the presence of bile salts to check whether liposomal entrapment could be a possible means of protecting the peptide against the aggressive conditions present in the gastrointestinal tract. The association of calcitonin with the lipidic vesicles was monitored using radioactive labelling of the peptide and gel-filtration separation of the free and liposome-associated fractions. The results show that for all phospholipid compositions tested, loading was preserved in light acidic or basic buffers, and that only a slight disruption was observed at pH 2.5. Cholate caused a significant but only partial release of calcitonin even when the cholate-to-phospholipid ratio was increased. To understand the mode of calcitonin entrapment in the vesicles, the release of liposome-entrapped calcein was monitored concomitantly and taken as a stability criterion. Liposome integrity appears to be resistant at low pHs but to be totally destroyed by 4 mM cholate in a manner quasi-independent of the phospholipid concentration. These results strongly suggest that bile salts induce a disruption of the liposomes which results in the formation of new lipidic structures involving calcitonin and probably cholate.

Use of the glucose clamp technique for confirmation of insulinoma autonomous hyperinsulinism.

The diagnosis of insulinoma on the basis of persistent hypoglycemia requires further confirmation. The insulin suppression test has been used to support this diagnosis prior to surgical intervention. In this study the euglycemic clamp technique was used to compare five control volunteers with four hypoglycemic patients with suspected insulinoma. Insulin was infused over successive two-hour periods at 2, 4, and 8 mU/kg/min. Plasma glucose levels were clamped at 80 mg/dL (4.4 mmol/L) using an artificial pancreas. High insulin levels were measured in all subjects, ranging from 225 +/- 30 microU/mL (1614 +/- 215 pmol/L) to 1018 +/- 239 microU/mL (7304 +/- 1714 pmol/L). Levels of C peptide fell to 0.1 ng/mL (0.028 nmol/L) in control subjects but remained at high levels in the patients. Insulinoma was confirmed on laparotomy in all four patients. In two patients tested after removal of the tumor the results were found to have returned to normal.

Y-chromosomal microsatellite mutation rates: differences in mutation rate between and within loci.

Precise estimates of mutation rates at Y-chromosomal microsatellite STR (short tandem repeat) loci make an important basis for paternity diagnostics and dating of Y chromosome lineage origins. There are indications of considerable locus mutation rate variability between (inter-) and within (intra-) loci. We have studied nine Y-STR loci-DYS19, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS385, and DYS388-in 1,766 father-son pairs of confirmed paternity (a total of 15,894 meioses). Five biallelic markers were also analyzed in the fathers-Tat, YAP, 12f2, SRY1532, and 92R7-defining haplogroups 1, 2, 3, 4, 9, and 16, respectively. A total of 36 fragment length mutations were observed: 24 gains (22 single-step, two double-step) and 12 single-step losses. Thus, there was a significant surplus of gains (p=0.045). Overall, the mutation rate was positively correlated to STR repeat length and there was a significant relative excess of losses in long alleles and gains in short alleles (p=0.043). In contrast to the situation in autosomal STR loci and in MSY-1, no noteworthy correlation between mutation rate and the father's age at the child's birth was observed. We observed significant interlocus differences in Y-STR mutation rates (p<0.01). The number of observed mutations ranged from zero in DYS392 to eight in DYS391 and DYS390. We have also demonstrated obvious differences in mutation rates between the haplogroups studied (p=0.024), a phenomenon that is a reflection of the dependence of mutation rate on allele size. Our study has thus demonstrated the necessity of not only locus-specific, but even allele-specific, mutation rate estimates for forensic and population genetic purposes, and provides a considerable basis for such estimates.

Nitrous oxide anesthesia-associated myelopathy.

BACKGROUND: The role of nitrous oxide exposure in neurologic complications of subclinical cobalamin deficiency has been reported, but few cases are well documented. OBSERVATION: Two weeks after surgery for prosthetic adenoma, a 69-year-old man developed ascending paresthesia of the limbs, severe ataxia of gait, tactile sensory loss on the 4 limbs and trunk, and absent tendon reflexes. After a second surgical intervention, the patient became confused. Four months after onset, the patient had paraplegia, severe weakness of the upper limbs, cutaneous anesthesia sparing the head, and confusion. Moderate macrocytosis, low serum B12 levels, and a positive Schilling test result led to the diagnosis of pernicious anemia. Results of electrophysiologic examinations showed a diffuse demyelinating neuropathy. Magnetic resonance imaging of the spinal cord disclosed hyperintensities of the dorsal columns on T2-weighted images. CONCLUSIONS: Pernicious anemia can result in severe neurologic symptoms with only mild hematologic changes. The role of nitrous oxide anesthesia in revealing subclinical B12 deficiency must be emphazised. Magnetic resonance imaging of the spinal cord might be helpful in making the diagnosis.

Microencapsulation of isolated pituitary cells by polyacrylamide microlatex coagulation on agarose beads.

Microlatex beads of homogenous size were made by polymerization of a mixture of acrylamide/bisacrylamide dispersed in a microemulsion. The microlatex was aggregated by dilution of the microemulsion in acrylamide solutions. The aggregates were then coagulated by polymerization at the interfaces of agarose beads circulating in a capillary tube containing paraffin oil. Biocompatibility was tested on isolated pituitary cells microencapsulated by this procedure.

Cellular biocompatibility and resistance to compression of macroporous beta-tricalcium phosphate ceramics.

The main problem for macroporous structures used as bone substitutes is their lower resistances when compared to that of cancellous bone. The present investigation aimed to improve the strength of ceramics with 65% porosities based on beta-TCP. The initial mixtures were rendered plastic by addition of non-ionic carbohydrate binders. Macropores were created using substances which were eliminated by heat. Mechanical tests indicated that the resistance of the ceramics depended more on the quantity than the nature of the binders. Porosity measurements were done with a mercury porosimeter, and cellular biocompatibility was evaluated by performing cellular attachment tests and observing the proliferation of differentiated cells.

Study of a (trimethylenecarbonate-co-epsilon-caprolactone) polymer--part 2: in vitro cytocompatibility analysis and in vivo ED1 cell response of a new nerve guide.

Future surgical strategies to restore neurological function in peripheral nerve loss may involve replacement of nerve tissue with cultured Schwann cells using biodegradable guiding implants. Random copolymers of trimethylene carbonate and epsilon caprolactone (P(epsilonCL-TMC), 50: 50) have been synthesized by ring opening polymerization using rare earth alkoxides as initiator. Their potential use as nerve guide repairs has been assessed through indirect and direct in vitro biocompatibility tests and in vivo soft tissue response to EDI subclass macrophages. In vitro, we exposed monolayers of human skin fibroblasts and an established continuous cell line (Hela) to liquid extracts (either pure or diluted in the culture medium) of epsilonCL-TMC copolymer including positive (phenol) and negative controls. Then, colorimetric assays (Neutral red and MTT) were performed. The extracts of epsilonCL-TMC induced no significant cytotoxic effect. We also exposed in vitro Schwann cells to pieces of P(epsilonCL-TMC) and P(LA-GA) copolymers. We evaluated cell attachment at 1 and 3 h by measuring the activity of the lysosomal enzyme (N-acetyl-beta-hexosaminidase) and cell proliferation at 1, 3, 6 and 9 days by measuring the cell metabolic activity (MTT assay). Values for attachment slightly decreased between 1 and 3 h but were significantly higher than on agars (negative control). Cells plated on epsilonCL-TMC showed a rate of proliferation comparable with that of normalized controls and higher than on PGA-PLA at day 9. Finally, we evaluated in vivo the soft tissue response after implantation of cylindrical tubes of P(epsilonCL-TMC) and P(LA-GA) copolymers with an immunohistochemistry staining procedure for the newly recruited ED1 macrophages. An image analysis system automatically measured the optical density of labelled positive ED1 cells at 9, 21 and 60 days after implantation. epsilonCL-TMC copolymer showed a mild soft tissue reaction with no adverse chronic inflammatory reaction. These data allowed us to consider this conduit as a potential effective substitute in nerve repair. El sevier Science Ltd. All rights reserved.

Evolution of the local calcium content around irradiated beta-tricalcium phosphate ceramic implants: in vivo study in the rabbit.

To evaluate whether dissolved calcium from tricalcium phosphate implants contributes to osseous wound healing in bone defects, the authors used nuclear radioactivated materials. Six months after irradiation, the calcium was still radioactive. Samples of the material were prepared and placed in rabbit condyles for 1, 3 and 9 months. Over time the condyles were retrieved and treated for histology or radiocounting. Measurements of the radioactivity of the slices and histomorphometry of the implants and surrounding tissues were performed. The authors observed that the radioactivity decreased regularly. Connective tissue had penetrated the pores and totally invaded the implants, first at the periphery of the implants, then inside the pores. Comparison of the results of radioactivity and histomorphometry suggest that part of the calcium from the implants was re-used specifically in the new osseous tissue.

Embedded adrenal cells graft reduced local and early nonspecific inflammatory phenomena which follow agarose beads implantation.

Microencapsulation of adrenal cells is proposed for reducing the nonspecific inflammatory reaction observed around polymer implants. This hypothesis was tested by comparing both host cellular reaction and the surrounding graft cell populations which appeared either when agarose embedded cells or when empty agarose beads were implanted. Our results showed that the fibrotic material that surrounded the implanted empty agarose microbeads was not as severe and important when adrenal cells were present. Similarly, T lymphocyte population surrounding the graft was considerably reduced together with the percentage of CD4 and CD8 positive cell subpopulations. The activation macrophage marker IaD disappeared. Our results support the hypothesis that embedded adrenal cells may be a suitable solution for reducing early inflammatory events due to microcapsules implantation.

Relationships between striatal dopamine denervation and frontal executive tests in Parkinson's disease.

Indirect evidence from human and monkey investigations supports the idea that impaired frontal tasks in Parkinson's disease (PD) may result from striato-frontal disruption caused by dopamine (DA) denervation of the caudate nucleus. To directly investigate this hypothesis, we used PET with 11C-S-Nomifensine (11C-S-NMF), a sensitive marker of striatal DA denervation, in 10 non-demented PD patients in whom two frontal executive tests, the object alternation (OA) and the conditional associative learning (CAL) tasks, thought to reflect mainly set-shifting/inhibition and planning, respectively, were given. In addition, the central executive function of verbal working memory was assessed with the Brown Peterson paradigm (BPP). We found a highly significant correlation between right caudate 11C-S-NMF specific binding and OA performance, less significant and reverse-direction correlations between CAL performance and putamen 11C-S-NMF binding, and no significant correlation with BPP performance. Thus, caudate DA denervation may subtend poor set-shifting/inhibition process in PD. Our results also point to distinct and complex relationships between striatal DA and specific frontal tasks.

The effect of collagen shields on epithelial wound healing in rabbits.

We evaluated the effect of a collagen shield on epithelial healing in keratectomy wounds in rabbit eyes. Superficial keratectomies 6 mm in diameter were created in 12 eyes of six rabbits. Six eyes received collagen shields every eight hours; six eyes received no treatment (control group). Epithelial healing was significantly faster (P less than .01) in corneas treated with collagen shields (47.8 +/- 1.1 microns/hr) compared to untreated control corneas (40.8 +/- 1.6 microns/hr). Regression analysis gave a projected time for closure of treated corneas of 73.96 hours, compared to 83.41 hours for untreated corneas. Scanning electron microscopy of collagen shields after eight hours of wear showed a large number of polymorphonuclear leukocytes entrapped in the collagen matrix. Light microscopy of healed corneas showed that the appearance of the regenerated epithelium in treated and untreated corneas was similar. These results demonstrate that collagen shields speed reepithelialization of keratectomy wounds in the rabbit cornea.

"In vitro" dissolution of coral in peritoneal or fibroblast cell cultures.

Previous studies have shown that in vivo coral resorption involves a biphasic process: First, the edges of the coral block become powdery, then extracellular fluid and phagocytosis contribute to the dissolution of the crystals. The authors examined some types of cells that could be involved in phagocytosis, particularly the ability of both dermal fibroblasts and mouse-resident peritoneal cells to phagocytose and dissolve coral powder "in vitro". Radioactive coral was incubated for 24, 48, or 72 hrs with cells in the presence or absence of cytochalasin B (a phagocytic inhibitor) or chloroquine (a lysosomotropic agent). Furthermore, to specify the role of crystal cell contacts in the solubilization process, they incubated radioactive coral in conditioned media (obtained from two-day human fibroblastic or macrophagic cell culture in the presence or absence of non-radioactive coral) or at a distance from the cells using culture inserts. Measurements of the radioactivity in the different supernatants were performed. Transmission electron microscopy was carried out on the cells cultivated in the presence or absence of radioactive coral. The data suggest that both fibroblasts and macrophages dissolve the coral, and that the intracellular degradation in phagolysosomes is one of the mechanisms explaining coral powder dissolution.

Study of in vitro and in vivo stability of liposomes loaded with calcitonin or indium in the gastrointestinal tract.

Factors affecting liposome transport to the blood compartment after oral administration to rats were evaluated. A high entrapment of calcitonin (CT) was obtained when the vesicles were prepared by sonication and were composed of egg phosphatidylcholine, cholesterol and stearylamine. In vitro tests showed that the liposomes were stable in light acidic or basic buffers, but that they were partly lysed in pH 2.5, 10 mM bile salts and pancreatin. Oral administration of liposomes entrapping calcitonin in fasting rats showed that the vesicles facilitate transport of the hormone to the general circulation and that they increase the lifetime of 125I-CT in blood. Oral administration of liposomes entrapping radioactive indium in fasting rats did not induce radioactivity in blood. This could be explained by disruption of most of the vesicles in the enterocytes.