Jaume Comas

Assessment of the Effects of Gramicidin, Formaldehyde, and Surfactants on Escherichia coli by Flow Cytometry Using Nucleic Acid and Membrane Potential Dyes

Two membrane potential sensitive dyes (Rhodamine 123 and bis-oxonol) and three nucleic acid dyes (propidium iodide, SYTO-13, and SYTO-17) were used to assess the effect of surfactants on Escherichia coli. The ability of E. coli to be stained by these probes was validated at different physiological states. Propidium iodide was used to assess the integrity of cell envelopes. Two double staining methods based on propidium iodide with SYTO-13 and bis-oxonol with SYTO-17 were used to improve the discrimination between bacteria and micelles or aggregated particles generated by the presence of surfactants. A rapid (1 h contact time between cells and surfactants, and less than 5 min for staining and obtaining data) Rhodamine 123 flow cytometric assay was developed to assess the bactericidal effect of surfactants.

Mitochondrial membrane potential measurement in rat cerebellar neurons by flow cytometry.

Mitochondrial membrane potential (MMP) in dissociated rat cerebellar neurons was measured using rhodamine 123 (Rh 123) as fluorescent dye, and flow cytometry. Dye distribution was studied by confocal scanning microscopy. Propidium iodide (PI)-marked cells (dead cells) were not stained by Rh 123, while the green fluorescence of living cells was restricted to mitochondria. Incubation of cells with different ionophores resulted in a maximal inhibition of Rh 123 fluorescence of 27.0 +/- 5.9% (valinomycin), 55.6 +/- 7.2% (ionomycin), and 37.3 +/- 5.1% (gramicidin). Ionophores decreased cell viability at high concentrations, measured as the number of propidium iodide-marked cells. Exposure of cell suspensions to the mitochondrial specific uncoupling agent CCCP caused a decrease in Rh 123 fluorescence (40 +/- 6.1%). Conversely, oxidative stress induced by H2O2 did not affect Rh 123 fluorescence. Impairment of glucose bioavailability reduced Rh 123 fluorescence. 2-Deoxy-D-glucose decreased the MMP with a maximal inhibition of 24.0 +/- 4.4%. Lack of glucose in the incubation medium also resulted in a decrease in MMP. Moreover, application of L-glutamate and N-methyl-D-aspartate (NMDA) (the excitatory amino acids) decreased Rh 123 uptake in a dose-dependent manner, which suggests that the measurement of MMP in dissociated cerebellar neurons by flow cytometry is a suitable method to detect the activity of drugs acting on glutamate receptors.

Comparison of four lymphocyte isolation methods applied to rodent T cell subpopulations and B cells

The aim of this study was to establish the validity of four lymphocyte isolation methods. The effects of three different erythrocyte lysing methods commonly used in the analysis of human cells, namely, lysis by ammonium chloride (AC), Becton Dickinson lysis (BDL) and the Coulter Q-Prep (CQP) preparation system were established by flow cytometry on rat lymphocyte subsets. The results were compared with those obtained with a Ficoll-Isopaque (FI) density gradient procedure adapted for use with rat cells. Lymphocyte isolation by AC or FI gradient was performed before labelling the lymphocyte subpopulations, whereas the BDL and CQP methods were performed after staining the cells in whole blood. The FI gradient yielded the lowest CD5+, CD4+ and CD25+ cell percentages. On the other hand AC lysis produced higher percentages of T cells and lower percentages of B cells than the other methods studied. The percentages obtained after BDL or CQP methods for T lymphocyte subsets and B cells were found to be reproducible. The commercial methods (BDL and CQP) are faster but rather expensive, whereas AC lysis and FI gradient separations are cheap and particularly useful when there is a requirement to culture the cells.

Infection during Natural Orifice Transluminal Endoscopic Surgery Peritoneoscopy: A Randomized Comparative Study in a Survival Porcine Model

BACKGROUND Infection in natural orifice transluminal endoscopic surgery (NOTES) remains controversial. OBJECTIVE To estimate the frequency of infection during NOTES peritoneoscopy with different routes of access and to compare with laparoscopy. DESIGN Prospective randomized controlled study (Canadian Classification type I). METHODS Forty female pigs were randomly assigned to 3 NOTES (transgastric, transrectal, and transvaginal) and laparoscopic groups. Antiseptic technique was used for NOTES, whereas laparoscopy was performed in a sterile environment. Preoperative and postoperative intravenous antibiotics were administered. Closure of the transluminal access site was performed in all animals. Peritoneal fluid was collected for culture at the end of surgery and at necropsy at day 14. RESULTS Thirty-nine peritoneoscopies were successfully completed. Necropsy confirmed complete healing of NOTES incisions, but 2 animals in the laparoscopy group had small abscesses in the abdominal incisions. There were no statistical differences in the presence of peritoneal adhesions. Positive culture results were seen in all groups at the end of the procedure and in all animals at necropsy, but this did not lead to clinical signs of gross infection. The most common organisms that colonized the peritoneum were gram-positive cocci and gram-negative bacilli from the normal swine gastrointestinal flora. LIMITATIONS Animal model and small sample size. CONCLUSIONS In these small series of animals and with the careful lavage and preparation used, NOTES appeared to be comparable to laparoscopy in terms of peritoneal contamination and clinical infection. Despite the adherence to a strict antiseptic protocol, peritoneal contamination occurs but does not lead to septic complications in the swine.

Flow cytometric narrow-angle light scatter and cell size during starvation of Escherichia coli in artificial sea water

Flow cytometry was used to study starvation of Escherichia coli in artificial sea water. Flow cytometric narrow‐angle light scatter was compared and assessed in relation to the cell sizes obtained by scanning electron microscopy at low temperature, and by image analysis. A correlation between narrow‐angle light scatter and cell size was not observed, although an acceptable correlation (γ= ‐0.845) between narrow‐angle light scatter and the starvation period was observed. On the other hand, the distribution of narrow‐angle light scatter at any given moment of culture is asymmetric and may be associated with the cell size distribution at the specific moment of starvation.

Resección conservadora de la región duodenopancreática en el tratamiento del tumor papilar mucinoso intraductal

Resumen Introduccion La tecnica quirurgica estandar para el tratamiento del tumor mucinoso papilar intraductal (TPMI) de las variedades del conducto dilatado (TPMI-CD) o ramificado (TPMI-R) es la duodenopancreatectomia cefalica (DPC). El TPMI-R tiene un crecimiento lento y con menor incidencia de malignidad. Objetivo Evaluar la utilidad de la cirugia conservadora, reseccion parcial de la cabeza pancreatica con preservacion duodenal (RPCP-PD), y la reseccion total de la cabeza pancreatica con duodenectomia segmentaria (RTCP-DS) en pacientes con TPMI-R. Pacientes y metodo Los resultados de la cirugia fueron evaluados en 8 pacientes, de los que a 4 se realizo la tecnica RTCP-PD y a 4, la tecnica RTCP-DS. Tambien se incluyo a 13 pacientes a los que se realizo la DPC segun tecnica de Whipple. Resultados La incidencia de complicaciones postoperatorias fue del 38% despues de Whipple, el 100% despues de RPCP-PD y el 25% despues de RTCP-DS. La estancia hospitalaria media fue 27 dias despues de RPCP-PD, 22 dias despues de Whipple y 16 dias despues de RTCP-DS. Un TPMI invasivo se encontro en el 38% despues de Whipple y un TPMI no invasivo, en el 100% de los pacientes sometidos a cirugia conservadora. Conclusiones La DPC es la tecnica de eleccion para pacientes con TPMI invasivo. La RTCP-DS parece ser un procedimiento seguro y de gran eficacia en el tratamiento de pacientes con TPMI-R localizado en la cabeza del pancreas.

Detection of Infectious Rotaviruses by Flow Cytometry

Human rotaviruses are considered the main cause of viral gastroenteritis in infants and young children throughout the world. Their transmission is through the fecal-oral route, mostly after ingestion of contaminated water and food. Since an extremely high number of virus particles are present in the feces during the acute gastroenteritis, methods based on electron microscopy, passive particle agglutination tests, or enzyme-linked immunosorbent assays are readily employed for clinical diagnosis. However, the sensitivity of these procedures is not high enough to detect the low number of viral particles sometimes present in the environment. In the case of environmental samples, amplification of viral nucleic acids by polymerase chain reaction assays coupled to reverse transcription (RT-PCR) has been increasingly applied to detect rotaviruses in water and shellfish samples. However, procedures based on molecular approaches have to face the drawback that they do not differentiate between infectious and noninfectious particles, which is of major relevance from the public health point of view. Virus propagation in cell culture prior to detection by immunological or molecular procedures accomplishes the dual purpose of increasing the amount of target material and incorporating an infectivity assay as well.Wild-type rotaviruses present difficulties in their in vitro replication, although some of them may be adapted to grow in several cell lines such as the monkey kidney cell line MA104 or the human intestinal cell line CaCo-2. More than a decade ago, an assay for the specific detection of infectious rotaviruses in environmental samples, involving an indirect immunofluorescence test (IIF) and optical microscopy (OM) counting of infected foci in infected MA-104 cell monolayers, was described. On the other hand, CaCo-2 cells have been successfully employed in our laboratory for infectivity assays of several fastidious enteric virus strains present in water samples.

Comparative Study of Microsporidian Spores by Flow Cytometric Analysis

ABSTRACT. Spore suspensions of microsporidian parasites of fish (Microsporidium ovoideum, Glugea stephani, Glugea atherinae and Spraguea lophii) have been analyzed by flow cytometry. Spore nuclei were dyed either by propidium iodide or bis‐benzimide (Hoechst 33342). By observation of forward light scatter and fluorescence the four species could be distinguished and the mono‐ and diplokaryotic populations of S. lophii identified. Staining of DNA by bis‐benzimide was better and easier than propidium iodide. Forward light scatter and fluorescence values were characteristic of each species and remained unchanged throughout the year, so flow cytometry can be used for distinction of spores of some microsporidian parasites once their flow cytometric parameters are known. However, special care has to be taken in tool calibration and material preparation for analysis because of the high precision of the technique.

On-demand endoscopic CO2 insufflation with feedback pressure regulation during natural orifice transluminal endoscopic surgery (NOTES) peritoneoscopy induces minimal hemodynamic and respiratory changes

BACKGROUND Endoscopic insufflation has been associated with marked increase in intra-abdominal pressure (IAP) and hemodynamic and respiratory changes during transgastric surgery. OBJECTIVE To investigate the hemodynamic and respiratory effects during intraperitoneal cavity exploration through 3 different natural orifice transluminal endoscopic surgery (NOTES) access locations compared with laparoscopy. DESIGN AND SETTING Survival experiments using 40 female pigs randomized to transgastric, transcolonic, transvaginal, and laparoscopic peritoneoscopy. INTERVENTIONS On-demand endoscopic insufflation of CO(2) with feedback pressure regulation was used in NOTES with a maximum pressure of 14 mm Hg. In the laparoscopy group, the IAP was maintained at 14 mm Hg. NOTES procedures were performed by an endoscopist (with the assistance of a gynecologist in the transvaginal group and a second endoscopist in the transgastric and transrectal groups) and laparoscopy by 2 surgeons. MAIN OUTCOME MEASUREMENTS Invasive hemodynamic and respiratory data. Blood samples were drawn for gas analyses. RESULTS All experiments except one in the transrectal group were completed. The IAP was significantly lower in all NOTES groups compared with the laparoscopy group. A significant increase in mean systemic arterial blood pressure was observed in the laparoscopy group at 15 and 30 minutes of intraperitoneal cavity exploration, but it remained unchanged during all NOTES procedures. An increase in airway pressures was observed at 15 and 30 minutes of peritoneoscopy in the animals undergoing laparoscopy, whereas those parameters remained unchanged in the NOTES groups. The laparoscopy group showed a significant impairment in pulmonary gas exchange (decrease in Pao(2), increase in Paco(2), and decrease in arterial pH) after 30 minutes of peritoneoscopy, whereas only a slight increase in Paco(2) was observed in the transrectal and transvaginal groups. LIMITATIONS Healthy animal model. CONCLUSION On-demand endoscopic insufflation of CO(2) with feedback pressure regulation can minimize the risk of hemodynamic and respiratory compromise caused by acute changes in IAP.

Gut Bacterial Community of the Xylophagous Cockroaches Cryptocercus punctulatus and Parasphaeria boleiriana

Cryptocercus punctulatus and Parasphaeria boleiriana are two distantly related xylophagous and subsocial cockroaches. Cryptocercus is related to termites. Xylophagous cockroaches and termites are excellent model organisms for studying the symbiotic relationship between the insect and their microbiota. In this study, high-throughput 454 pyrosequencing of 16S rRNA was used to investigate the diversity of metagenomic gut communities of C. punctulatus and P. boleiriana, and thereby to identify possible shifts in symbiont allegiances during cockroaches evolution. Our results revealed that the hindgut prokaryotic communities of both xylophagous cockroaches are dominated by members of four Bacteria phyla: Bacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria. Other identified phyla were Spirochaetes, Planctomycetes, candidatus Saccharibacteria (formerly TM7), and Acidobacteria, each of which represented 1–2% of the total population detected. Community similarity based on phylogenetic relatedness by unweighted UniFrac analyses indicated that the composition of the bacterial community in the two species was significantly different (P < 0.05). Phylogenetic analysis based on the characterized clusters of Bacteroidetes, Spirochaetes, and Deltaproteobacteria showed that many OTUs present in both cockroach species clustered with sequences previously described in termites and other cockroaches, but not with those from other animals or environments. These results suggest that, during their evolution, those cockroaches conserved several bacterial communities from the microbiota of a common ancestor. The ecological stability of those microbial communities may imply the important functional role for the survival of the host of providing nutrients in appropriate quantities and balance.