Reset of a critically disturbed microbial ecosystem: faecal transplant in recurrent Clostridium difficile infection

Recurrent Clostridium difficile infection (CDI) can be effectively treated by infusion of a healthy donor faeces suspension. However, it is unclear what factors determine treatment efficacy. By using a phylogenetic microarray platform, we assessed composition, diversity and dynamics of faecal microbiota before, after and during follow-up of the transplantation from a healthy donor to different patients, to elucidate the mechanism of action of faecal infusion. Global composition and network analysis of the microbiota was performed in faecal samples from nine patients with recurrent CDI. Analyses were performed before and after duodenal donor faeces infusion, and during a follow-up of 10 weeks. The microbiota data were compared with that of the healthy donors. All patients successfully recovered. Their intestinal microbiota changed from a low-diversity diseased state, dominated by Proteobacteria and Bacilli, to a more diverse ecosystem resembling that of healthy donors, dominated by Bacteroidetes and Clostridium groups, including butyrate-producing bacteria. We identified specific multi-species networks and signature microbial groups that were either depleted or restored as a result of the treatment. The changes persisted over time. Comprehensive and deep analyses of the microbiota of patients before and after treatment exposed a therapeutic reset from a diseased state towards a healthy profile. The identification of microbial groups that constitute a niche for C. difficile overgrowth, as well as those driving the reinstallation of a healthy intestinal microbiota, could contribute to the development of biomarkers predicting recurrence and treatment outcome, identifying an optimal microbiota composition that could lead to targeted treatment strategies.     

Phylogeny of some Fusarium species, as determined by large-subunit rRNA sequence comparison

Fifty-two strains from eight species of Fusarium were analyzed by rapid rRNA sequencing. Two highly variable stretches (138 and 214 nucleotides) of the 5' end of the 28S-like rRNA molecule were sequenced. Such stretches permit evaluation of the divergence between closely related species and even between varieties within a species. The phylogenetic tree computed from the number of nucleotide differences shows seven Fusarium species to be more closely related to one another than the eighth species, F. nivale, is to them. On the basis of these data, we discuss both the phylogenetic value of taxonomical criteria and the impact of our findings on the demarcation of the genus Fusarium. We conclude that this method is suitable for establishing a precise phylogeny between closely related species within a genus.      

Molecular models of NS3 protease variants of the Hepatitis C virus

Background  

Hepatitis C virus (HCV) currently infects approximately three percent of the world population. In view of the lack of vaccines against HCV, there is an urgent need for an efficient treatment of the disease by an effective antiviral drug. Rational drug design has not been the primary way for discovering major therapeutics. Nevertheless, there are reports of success in the development of inhibitor using a structure-based approach. One of the possible targets for drug development against HCV is the NS3 protease variants. Based on the three-dimensional structure of these variants we expect to identify new NS3 protease inhibitors. In order to speed up the modeling process all NS3 protease variant models were generated in a Beowulf cluster. The potential of the structural bioinformatics for development of new antiviral drugs is discussed.     

Staining of intracellular deposits of uranium in cultured murine macrophages

In our studies of the health effects of internalized depleted uranium, we developed a simple and rapid light microscopic method to stain specifically intracellular uranium deposits. Using J774 cells, a mouse macrophage line, treated with uranyl nitrate and the pyridylazo dye 2-(5-bromo-2- pyridylazo)-5-diethylaminophenol, uranium uptake by the cells was followed. Specificity of the stain for uranium was accomplished by using masking agents to prevent the interaction of the stain with other metals. Prestaining wash consisting of a mixture of sodium citrate and ethylenediaminetetraacetic acid eliminated staining of metals other than uranium. The staining solution consisted of the pyridylazo dye in borate buffer along with a quaternary ammonium salt, ethylhexadecyldimethylammonium bromide, and the aforementioned sodium citrate/ethylene-diaminetetraacetic acid mixture. The buffer was essential for maintaining the pH within the optimum range of 8 to 12, and the quaternary ammonium salt prevented precipitation of the dye. Staining was conducted at room temperature and was complete in 30 min. Staining intensity correlated with both uranyl nitrate concentration and incubation time. Our method provides a simple procedure for detecting intracellular uranium deposits in macrophages.     

Symmetrical breast reconstruction: is there a role for three-dimensional digital photography?

Clinical applications of three-dimensional digital photography include assessments of breast volume and contour. It was hypothesized that knowledge of preoperative and postoperative breast volumes might facilitate obtaining symmetry after reconstructions with autologous tissue or implants. Breast reconstruction was performed for 382 women during a 4-year period. Of those women, 334 completed all phases of the reconstruction and underwent symmetry analysis. Reconstructive procedures included the use of pedicle transverse rectus abdominis musculocutaneous flaps, free transverse rectus abdominis musculocutaneous flaps, deep inferior epigastric perforator flaps, superior gluteal artery perforator flaps, or latissimus dorsi flaps or expanders/implants. Three-dimensional digital photographic images were obtained for 33 women, whereas the remaining 301 women were not digitally photographed. The differences in symmetry after the initial reconstruction and after the secondary procedures were compared for all women and for the groups with and without three-dimensional photographic images. For the group with three-dimensional imaging, initial volume symmetry was obtained for 73 percent, initial contour symmetry was obtained for 27 percent, secondary procedures were necessary for 70 percent, final volume symmetry was obtained for 88 percent, and final contour symmetry was obtained for 79 percent. For the group without three-dimensional photographic images, initial volume symmetry was obtained for 57 percent, initial contour symmetry was obtained for 34 percent, secondary procedures were necessary for 50 percent, final volume symmetry was obtained for 80 percent, and final contour symmetry was obtained for 71 percent. The results demonstrated that there was no significant difference in final contour and volume symmetry between women who had or did not have three-dimensional digital photographic images taken. However, the results demonstrated that autologous tissue reconstructions resulted in improved contour and volume symmetry, compared with implant reconstructions.     

Quantitation of breast sensibility following reduction mammaplasty: a comparison of inferior and medial pedicle techniques

The preservation of sensitivity within the nipple-areola complex is of paramount importance to patients presenting for reconstructive and aesthetic breast procedures. Previous attempts to measure sensation in the breast before and after surgery have relied primarily on the Semmes-Weinstein monofilament test, which is an imprecise study that measures the logarithm of force necessary to bend a series of six to 20 filaments. Within the last 10 years, various authors have published normative pressure threshold data for the breast that have varied by a magnitude of greater than 10-fold. Recently, precise anatomic studies have been performed that have elucidated the innervation of the nipple-areola complex medially and laterally from cutaneous branches of the intercostal nerves. Despite this knowledge, no quantitative sensibility studies have yet been performed that compare postoperative sensation when medially versus laterally innervated pedicles have been used in reduction mammaplasty. The present study is the first to use computer-assisted neurosensory testing to generate normal breast sensation data and to compare sensory outcomes between the inferior and the medial pedicle techniques of reduction mammaplasty.A total of 34 patients were divided into four groups and underwent breast sensory testing (67 breasts total) using the Pressure-Specified Sensory Device, a computer-assisted force transducer that measures static and moving one and two-point discrimination. Sensation in the nipple and in the four quadrants of the areola was measured. Groups I and II were composed of 17 unoperated controls with breast sizes ranging from 34A to 36C (group I; 18 breasts) and 36DD to 46EE (group II; 16 breasts) who presented to a general plastic surgery clinic. Groups III and IV were composed of 17 patients who underwent either medial or inferior pedicle reduction mammaplasty between July of 1997 and March of 1999. Pressure thresholds in the most sensitive breasts were as low as 0.3 g/mm2, a marked contrast to data from previous studies using Semmes-Weinstein monofilaments documenting the lowest recordable pressure threshold as greater than 2 g/mm2. Several findings from previous studies using Semmes-Weinstein monofilament testing were confirmed in unoperated controls, including an inverse relationship between sensitivity and breast size, superior nipple sensitivity when compared with the areola, and significant interpatient variability with respect to static and moving two-point discrimination among women matched according to age and breast size. When comparing medial with inferior pedicle reduction mammaplasty patients, it was found that despite significantly greater reductions using the medial pedicle technique (mean of 1.7 kg versus 1.1 kg of breast tissue removed), there were no significant differences in postoperative sensory outcomes in the sample size of 17 patients. Furthermore, within each group of patients undergoing either the medial or inferior pedicle technique, the amount of breast tissue removed did not correlate with postoperative sensory outcomes.Computer-assisted quantitative neurosensory testing is a highly accurate technique for measuring sensibility. The use of this technology demonstrates a 10-fold difference in measurable sensory thresholds in normal patients from preexisting data using Semmes-Weinstein monofilaments. Advances in measurement methods have allowed the authors to compare postoperative sensory outcomes reliably using two popular techniques of reduction mammaplasty.     

Glycosylation sites and site-specific glycosylation in human Tamm- Horsfall glycoprotein

The N-glycosylation sites of human Tamm-Horsfall glycoprotein from one healthy male donor have been characterized, based on an approach using endoproteinase Glu-C (V-8 protease, Staphylococcus aureus ) digestion and a combination of chromatographic techniques, automated Edman sequencing, and fast atom bombardment mass spectrometry. Seven out of the eight potential N-glycosylation sites, namely, Asn52, Asn56, Asn208, Asn251, Asn298, Asn372, and Asn489, turned out to be glycosylated, and the potential glycosylation site at Asn14, being close to the N-terminus, is not used. The carbohydrate microheterogeneity on three of the glycosylation sites was studied in more detail by high-pH anion-exchange chromatographic profiling and 500 MHz1H-NMR spectroscopy. Glycosylation site Asn489 contains mainly di- and tri-charged oligosaccharides which comprise, among others, the GalNAc4 S (beta1-4)GlcNAc terminal sequence. Only glycosylation site Asn251 bears oligomannose-type carbohydrate chains ranging from Man5GlcNAc2to Man8GlcNAc2, in addition to a small amount of complex- type structures. Profiling of the carbohydrate moieties of Asn208 indicates a large heterogeneity, similar to that established for native human Tamm-Horsfall glycoprotein, namely, multiply charged complex-type carbohydrate structures, terminated by sulfate groups, sialic acid residues, and/or the Sda-determinant.      

Operative management and outcome of complex wounds following total knee arthroplasty.

This study describes the treatment protocol for and the outcome of the management of complex wounds around total knee replacements. An analysis of 28 patients (29 knees) with complex defects who had surgery between January 1, 1986, and July 30, 1996, was performed. A specific management protocol was applied to each knee on the basis of the size and depth of the wound, the presence of infection, and the quality of soft tissue. Primary treatment included local wound care, debridement, and skin grafting or coverage with a fasciocutaneous flap, pedicled muscle flap, or free muscle transfer. Postoperatively, knees were evaluated using the Knee Society objective score. Successful salvage of the lower extremity was obtained in 28 knees (97 percent) and of the knee prosthesis in 24 of 29 knees (83 percent). Secondary plastic surgery procedures were necessary in five knees (17 percent), and secondary orthopedic procedures were necessary in four knees (14 percent). Successful salvage of total knee arthroplasty in the presence of a complex wound requires early identification of infection, aggressive irrigation and debridement, and early appropriate soft-tissue coverage. The use of our proposed algorithm will facilitate management of these complex wounds.