Optimization of fecal sample homogenization for untargeted metabolomics.

INTRODUCTION: Fecal samples are highly complex and heterogeneous, containing materials at various stages of digestion. The heterogeneity and complexity of feces make stool metabolomics inherently challenging. The level of homogenization influences the outcome of the study, affecting the metabolite profiles and reproducibility; however, there is no consensus on how fecal samples should be prepared to overcome the topographical discrepancy and obtain data representative of the stool as a whole. OBJECTIVES: Various combinations of homogenization conditions were compared to investigate the effects of bead size, addition of solvents and the differences between wet-frozen and lyophilized feces. METHODS: The homogenization parameters were systematically altered to evaluate the solvent usage, bead size, and whether lyophilization is required in homogenization. The metabolic coverage and reproducibility were compared among the different conditions. RESULTS: The current work revealed that a combination of mechanical and chemical lysis obtained by bead-beating with a mixture of big and small sizes of beads in an organic solvent is an effective way to homogenize fecal samples with adequate reproducibility and metabolic coverage. Lyophilization is required when bead-beating is not available. CONCLUSIONS: A comprehensive and systematical evaluation of various fecal matter homogenization conditions provides a profound understanding for the effects of different homogenization methods. Our findings would be beneficial to assist with standardization of fecal sample homogenization protocol.

Evaluation of fresh, frozen, and lyophilized fecal samples by SPME and derivatization methods using GC×GC-TOFMS.

INTRODUCTION: Feces is a highly complex matrix containing thousands of metabolites. It also contains live bacteria and enzymes, and does not have a static chemistry. Consequently, proper control of pre-analytical parameters is critical to minimize unwanted variations in the samples. However, no consensus currently exists on how fecal samples should be stored/processed prior to analysis. OBJECTIVE: The effects of sample handling conditions on fecal metabolite profiles and abundances were examined using comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC-TOFMS). METHODS: Solid-phase microextraction (SPME) and derivatization via trimethylsilylation (TMS) were employed as complementary techniques to evaluate fresh, frozen, and lyophilized fecal samples with expanded coverage of the fecal metabolome. The total number of detected peaks and the signal intensities were compared among the different handling conditions. RESULTS: Our analysis revealed that the metabolic profiles of fecal samples depend greatly on sample handling and processing conditions, which had a more pronounced effect on results obtained by SPME than by TMS derivatization. Overall, lyophilization resulted in a greater amount of total and class-specific metabolites, which may be attributed to cell lysis and/or membrane disintegration. CONCLUSIONS: A comprehensive comparison of the sample handling conditions provides a deeper understanding of the physicochemical changes that occur within the samples during freezing and lyophilization. Based on our results, snap-freezing at -80 °C would be preferred over lyophilization for handling samples in the field of fecal metabolomics as this imparts the least change from the fresh condition.

A novel chemical lysis method for maximum release of DNA from difficult-to-lyse bacteria.

Molecular detection of microorganisms requires releasing DNA from cells. However, since certain microbial organisms are refractory to lysis by chemical or enzymatic methods, mechanical lysis by bead-beating is typically employed to disrupt difficult-to-lyse microbes. A newly developed chemical lysis method called sporeLYSE enables release of DNA from difficult-to-lyse microbes without bead-beating. The sporeLYSE method was compared to bead-beating and an alkaline/detergent lysis solution for releasing DNA from microbes grown in vitro, including surrogates of Category A bioterrorism agents. sporeLYSE released 83% to 100% of DNA from Mycobacterium smegmatis, Francisella philomiragia, Yersinia enterocolitica, Bacillus thuringiensis, Pseudomonas aeruginosa, Moraxella catarrhalis and Klebsiella pneumoniae. qPCR results indicated that sporeLYSE extracted an equal or greater amount of DNA than either bead-beating or alkaline/detergent lysis from Gram-positive and Gram-negative bacteria. When sporeLYSE was used to extract DNA from saliva and sputum spiked with M. smegmatis and M. tuberculosis, respectively, the qPCR Ct values were 4-8 cycles lower than those for extractions via alkaline/detergent lysis and heat. Mean Ct values for sporesLYSE extractions from spores of Clostridium difficile and C. botulinum were approximately two cycles lower than those of MagNA Pure DNA extractions. Our results suggest that sporeLYSE is an easy-to-use liquid reagent that can efficiently release large amounts of DNA from a variety of bacteria, including spores.

Dairy product intake and bone properties in 70-year-old men and women.

In the present population-based study including 70-year-old men and women, total dairy product intake was associated with a weak positive association with tibia trabecular and cortical cross-sectional areas. PURPOSE: Milk consumption has recently been suggested to increase fracture risk. Therefore, we aimed to investigate associations between dairy product consumption and peripheral bone properties. Furthermore, we explored whether consumption of milk and fermented dairy products affected bone properties differently. METHODS: The Healthy Aging Initiative is a population-based, cross-sectional study investigating the health of 70-year-old men and women. Out of the 2904 individuals who met the inclusion criteria, data on self-reported daily dairy product consumption (dl/day), peripheral quantitative computed tomography (pQCT) examinations at the 4 and 66% scan sites of the tibia and radius, and dual-energy X-ray absorptiometry (DXA) scans were collected from 2040 participants. Associations between dairy product consumption and bone properties were examined using multiple linear regression models adjusted for sex, muscle area, meal size, dietary protein proportion, current smoking status, and objectively measured physical activity. RESULTS: Total dairy product intake was associated with larger trabecular (2.296 (95% CI, 0.552-4.039) mm2, per dl/day increase, p = 0.01) and cortical cross-sectional areas (CSAs) in the tibia (1.757 (95% CI, 0.683-2.830 mm2, p = 0.001) as measured by pQCT and higher areal bone mineral density (aBMD) of the radius (3.231 (95% CI, 0.764-5.698) mg/cm2, p = 0.01) as measured by DXA. No other measurement in the tibia, radius, femoral neck, or lower spine was associated significantly with dairy product intake. Bone properties did not differ according to the type of dairy product consumed. CONCLUSION: No evidence of a negative association between dairy product consumption and bone health was found. Furthermore, total dairy product consumption was associated with increased CSAs in the tibia, regardless of dairy product type. Collectively, our findings indicate the existence of a weak but significant positive association between dairy product consumption bone properties in older adults.

A method for assessing efficiency of bacterial cell disruption and DNA release.

BACKGROUND: DNA-based testing is becoming the preferred method both for identifying microorganisms and for characterizing microbial communities. However, no single DNA extraction method exists that is suitable for all types of microorganisms because bacteria are variable in their susceptibility to lysis by available extraction procedures. To develop and test new DNA extraction procedures, it would be helpful to determine their efficiencies. While the amount of extracted DNA can readily be measured by different methods, calculation of true efficiency requires knowledge of the initial amount of DNA in the starting bacterial sample, which cannot be done with precision by any existing method. In the process of developing a new extraction procedure, we developed a method that can be used to determine the total amount of both DNA and RNA in bacteria. The amount of DNA can be calculated from the amount of purines released after mild acid and alkali treatment. The amount of RNA in the same extract can also be calculated from the amount of ribonucleoside monophosphates. The released purines and ribonucleoside monophosphates can be quantified by absorbance using HPLC, with reference to appropriate standards. RESULTS: The acid/HPLC method was used to measure the efficiency of commonly used bead-beating and chemical protocols for releasing DNA from a particularly hardy organism, Mycobacterium smegmatis as well as several other species (Bacillus subtilis vegetative cells and spores; Francisella philomiragia; Pseudomonas aeruginosa; Moraxella catarrhalis; Bacillus thuringiensis; Staphylococcus aureus). Surprisingly large differences in efficiency between methods were found. CONCLUSIONS: The acid/HPLC method is a new tool to determine DNA extraction efficiencies and should aid in the development of improved protocols for releasing DNA from a broad range of microorganisms.

The European League Against Rheumatism (EULAR) - 16th Annual European Congress (June 10-13, 2015 - Rome, Italy).

The 16th Annual European Congress of Rheumatology, organized by the European League Against Rheumatism (EULAR), provided the latest advances in the field of rheumatologic diseases to around 14,000 participants from more than 120 countries. This congress has become the primary platform for exchange of scientific and clinical information and the biggest rheumatology event in Europe. The congress covered a broad spectrum of the rheumatic diseases through 400 lectures, workshops, 300 oral presentations, 2,000 posters, 350 invited speakers, and basic science and clinical symposia.

Spin-dependent electron scattering at graphene edges on Ni(111).

We investigate the scattering of surface electrons by the edges of graphene islands grown on Ni(111). By combining local tunneling spectroscopy and ab initio electronic structure calculations we find that the hybridization between graphene and Ni states results in strongly reflecting graphene edges. Quantum interference patterns formed around the islands reveal a spin-dependent scattering of the Shockley bands of Ni, which we attribute to their distinct coupling to bulk states. Moreover, we find a strong dependence of the scattering amplitude on the atomic structure of the edges, depending on the orbital character and energy of the surface states.

Yield and shape selection of graphene nanoislands grown on Ni(111).

The catalytic decomposition of hydrocarbons on transition-metal surfaces has attracted increasing interest as a method to prepare high quality graphene layers. Here, we study the optimal reaction path for the preparation of graphene nanoislands of selected shape using controlled decomposition of propene on Ni(111). Scanning tunneling microscopy performed at different stages of the reaction provides insight into the temperature and dose-dependent growth of graphene islands, which precedes the formation of monolayer graphene. The effect of postreaction annealing on the morphology of the islands is studied. By adjusting the initial propene dose, reaction temperature, and postannealing procedure, islands with a triangular or hexagonal shape can be selectively obtained.

The biochemical properties of the Francisella pathogenicity island (FPI)-encoded proteins IglA, IglB, IglC, PdpB and DotU suggest roles in type VI secretion.

The Francisella pathogenicity island (FPI) encodes proteins thought to compose a type VI secretion system (T6SS) that is required for the intracellular growth of Francisella novicida. In this work we used deletion mutagenesis and genetic complementation to determine that the intracellular growth of F. novicida was dependent on 14 of the 18 genes in the FPI. The products of the iglABCD operon were localized by the biochemical fractionation of F. novicida, and Francisella tularensis LVS. Sucrose gradient separation of water-insoluble material showed that the FPI-encoded proteins IglA, IglB and IglC were found in multiple fractions, especially in a fraction that did not correspond to a known membrane fraction. We interpreted these data to suggest that IglA, IglB and IglC are part of a macromolecular structure. Analysis of published structural data suggested that IglC is an analogue of Hcp, which is thought to form long nano-tubes. Thus the fractionation properties of IglA, IglB and IglC are consistent with the current model of the T6SS apparatus, which supposes that IglA and IglB homologues form an outer tube structure that surrounds an inner tube composed of Hcp (IglC) subunits. Fractionation of F. novicida expressing FLAG-tagged DotU (IcmH homologue) and PdpB (IcmF homologue) showed that these proteins localize to the inner membrane. Deletion of dotU led to the cleavage of PdpB, suggesting an interaction of these two proteins that is consistent with results obtained with other T6SSs. Our results may provide a mechanistic basis for many of the studies that have examined the virulence properties of Francisella mutants in FPI genes, namely that the observed phenotypes of the mutants are the result of the disruption of the FPI-encoded T6SS structure.

Optimization of human pancreatic islet isolation with a newly designed cooling system for COBE 2991.

OBJECTIVE: Islet purification is performed using the cell separator COBE 2991, which allows the purification of large amounts of islets through a continuous density gradient. However, the original cell separator COBE 2991 was not refrigerated, and islets were exposed to inappropriately high temperatures during the purification step of the isolation process. Our aim was to design a cooling system for the purification of human pancreatic islets using COBE 2991, to increase the viability and quality of the preparations. MATERIALS AND METHODS: We designed and adapted a cooling system for the COBE 2991 based on a cooling bath connected to a coil containing a recirculation solution with a temperature below 0 degrees C which was placed around the rotor of the COBE 2991. Cell viability was determined by acridine orange/propidium iodide staining, and islet cell function by measuring glucose-stimulated insulin secretion expressed as the insulin stimulation index. RESULTS: Pancreata from 10 consecutive organ donors (mean age, 42.8 +/- 4.3 years) were digested and purified using the newly generated cooling system for COBE 2991. At the end of the purification process, the temperature of the density gradient that contained the islet preparation was reduced by 8 degrees C to 10 degrees C compared with that of a system with no refrigeration. Islet viability increased to 83% +/- 4%, and the insulin stimulation index increased to 11.4 +/- 1.6 (average +/- SEM). CONCLUSION: This innovative cooling system for COBE 2991 achieved substantial reductions in temperature and improved the quality of human pancreatic islet preparations that were suitable for transplantation.

Gestión del transporte sanitario desde un servicio de urgencias / Emergency department management of ambulance services

Objetivo: Conocer si la gestión del transporte sanitario en ambulancia desde el servicio de urgencias hospitalario disminuye el tiempo de espera y las reclamaciones por la demora del mismo. Método: Análisis prospectivo y comparativo de dos periodos de tiempo, uno sin gestión del transporte sanitario (SGTS) en los meses de octubre y noviembre de 2007, y otro con gestión (CGTS) desde el servicio de urgencias en los meses de diciembre de2007 y enero de 2008. Se recogieron el número de transportes realizados, el tiempo de demora y las quejas recibidas en los dos periodos. Se excluyeron los transportes urgentes de pacientes en ambulancia a otros centros hospitalarios. Resultados: Durante el periodo SGTS se realizaron 544 evacuaciones y en el periodo CGTS 720. La demora media en el periodo SGTS fue de 2 horas y 4 minutos (espera máxima: 8 horas) y en el CGTS fue de 1 hora y 1 minuto (espera máxima: 4 horas). Durante el periodo SGTS se realizaron 5 quejas por escrito y 64 quejas orales en relación al transporte sanitario, mientras que en el CGTS no hubo quejas por escrito y se registraron 19 quejas orales. En este último periodo hubo el doble de transportes múltiples(2 pacientes en la misma ambulancia) que en el periodo SGTS. Conclusión: La gestión del transporte sanitario desde el servicio de urgencias en el periodo de estudio supuso una franca mejoría en las variables estudiadas (AU)

Objective: To determine whether hospital emergency service management of ambulance services reduces wait time and complaints about delayed transport. Methods: A prospective study comparing 2 periods of time, one in which ambulance service was not managed by the hospital emergency department (October and November 2007) and another in which the department did manage the ambulance service (December 2007 and January 2008). The number of transfers, transport time, and complaints received in the 2 periods were analyzed. Ambulance trips taking patients to other hospitals were excluded from the analysis. Results: During the first period, patients from 544 emergency calls were brought in; in the second period, the service responded to 720 calls for emergency transport. In the first study period, the mean wait time was 2 hours and 4 minutes(maximum wait time, 8 hours). In the second period, the mean wait time was 1 hour and 1 minute (maximum, 4hours). Five complaints about emergency transport (9 written, 64 verbal) were lodged in the first period. The second period saw no written complaints and 19 verbal complaints. Twice the number of double transports (2 patients in the same ambulance) took place in the second period with in the first period. Conclusion: Emergency department management of ambulance services brought considerable improvement in the variables studied (AU)

The Francisella pathogenicity island protein PdpD is required for full virulence and associates with homologues of the type VI secretion system.

Francisella tularensis is a highly infectious, facultative intracellular bacterial pathogen that is the causative agent of tularemia. Nearly a century ago, researchers observed that tularemia was often fatal in North America but almost never fatal in Europe and Asia. The chromosomes of F. tularensis strains carry two identical copies of the Francisella pathogenicity island (FPI), and the FPIs of North America-specific biotypes contain two genes, anmK and pdpD, that are not found in biotypes that are distributed over the entire Northern Hemisphere. In this work, we studied the contribution of anmK and pdpD to virulence by using F. novicida, which is very closely related to F. tularensis but which carries only one copy of the FPI. We showed that anmK and pdpD are necessary for full virulence but not for intracellular growth. This is in sharp contrast to most other FPI genes that have been studied to date, which are required for intracellular growth. We also showed that PdpD is localized to the outer membrane. Further, overexpression of PdpD affects the cellular distribution of FPI-encoded proteins IglA, IglB, and IglC. Finally, deletions of FPI genes encoding proteins that are homologues of known components of type VI secretion systems abolished the altered distribution of IglC and the outer membrane localization of PdpD.

Genetic elements for selection, deletion mutagenesis and complementation in Francisella spp.

Francisella novicida is a gram-negative pathogen that can induce disease in mice that mimics human tularemia, and is nearly identical to Francisella tularensis at the genomic level. In this work a number of antibiotic marker cassettes that incorporate a strong F. novicida promoter is constructed, which greatly enhances selection in F. novicida and F. tularensis. Two low-copy plasmid vectors based on a broad-host-range plasmid, and an integrating vector have also been made, and these can be used for genetic complementation. Two general approaches to deletion mutagenesis in F. novicida is also described.

The Francisella pathogenicity island protein IglA localizes to the bacterial cytoplasm and is needed for intracellular growth.

BACKGROUND: Francisella tularensis is a gram negative, facultative intracellular bacterium that is the etiological agent of tularemia. F. novicida is closely related to F. tularensis but has low virulence for humans while being highly virulent in mice. IglA is a 21 kDa protein encoded by a gene that is part of an iglABCD operon located on the Francisella pathogenicity island (FPI). RESULTS: Bioinformatics analysis of the FPI suggests that IglA and IglB are components of a newly described type VI secretion system. In this study, we showed that IglA regulation is controlled by the global regulators MglA and MglB. During intracellular growth IglA production reaches a maximum at about 10 hours post infection. Biochemical fractionation showed that IglA is a soluble cytoplasmic protein and immunoprecipitation experiments demonstrate that it interacts with the downstream-encoded IglB. When the iglB gene was disrupted IglA could not be detected in cell extracts of F. novicida, although IglC could be detected. We further demonstrated that IglA is needed for intracellular growth of F. novicida. A non-polar iglA deletion mutant was defective for growth in mouse macrophage-like cells, and in cis complementation largely restored the wild type macrophage growth phenotype. CONCLUSION: The results of this study demonstrate that IglA and IglB are interacting cytoplasmic proteins that are required for intramacrophage growth. The significance of the interaction may be to secrete effector molecules that affect host cell processes.

Original study of the biochemical and oil composition of the Cambodia nut Irvingia malayana.

Analysis of the biochemical composition of Irvingia malayana was carried out. This Cambodian nut contains 7.5% water and 70% oil. Most of the fatty acids are saturated and include 42% C12:0 and 41.8% C14:0; the sterol composition is similar to that of other vegetable oils. This oil is less rich in alpha-tocopherol than in gamma-tocopherol. Analysis of the solid content of the oil with respect to the temperature by NMR shows a fast fall of solid content around its fusion range at 38-39 degrees C. The main differences in the properties of the indigenous Cambodia nut from other known oleaginous seeds are in its selenium content, fatty acid composition, fusion temperature profile, and content of antioxidants. These important characteristics can soon make possible its application in pharmacology, cosmetics, the margarine industry, etc.

[Presentation of Burkitt's lymphoma in nasal sinus]. / Presentación nasosinusal del linfoma de Burkitt.

Burkitt' lymphoma is a malignant, non-Hodgkin's lymphoma of high grade that present two clinical from the african or endemic form affects near the middle of children of central africa and the american or sporadic form, that was first described in North American and the clinical setting was similar to endemic form. In our geographic area is an unusual entity. We present a clinical case of marrocan child that the first symptom was nasosinusal.

Ectopic thyroid tissue presenting as a submandibular mass.

BACKGROUND: To our knowledge, only three cases with ectopic submandibular thyroid have been reported. No patient with simultaneous presence of ectopic submandibular thyroid and normal thyroid gland has been reported. Literature on this topic is reviewed. METHODS: A 34-year-old woman with a longstanding asymptomatic right submandibular mass was evaluated by ecography, computer tomography scan, and fine-needle aspiration. Literature review was performed using EBSCO-Medline Results. The mass was hard, nontender, and not movable with swallowing. A fine-needle aspiration revealed hyperplastic thyroid tissue. A second ecographic-guided fine-needle aspiration confirmed cytologically ectopic thyroid tissue. A computer tomography scan showed a calcified mass with cystic areas beneath and medial to the submandibular gland. Rests of thyroid tissue lateral to the trajectory of thyroglossal duct were seen. Thyroid gland was atrophic. No metastatic lymph nodes were noted. Total serum thyroxine and triiodothyronine were normal. Basal and TRH-stimulated serum thyrotropin was suppressed. A 99Tc scintigraphy disclosed a nodular uptake of tracer in the submandibular area, and no uptake by normal thyroid. CONCLUSIONS: We describe the first case, to our knowledge, with lateral multiple aberrant thyroid tissue and hypoplastic thyroid gland.

Body composition of sedentary and physically active spinal cord injured individuals estimated from total body electrical conductivity.

Reliability and evidence of construct validity of Total Body Electrical Conductivity (TOBEC) for estimating body composition in spinal cord injured subjects was evaluated using 17 males with C6-L2 spinal cord transections. Subjects reporting regular exercise were categorized as active (n = 12); nonexercisers were considered sedentary (n = 5). Measures included body weight, length, circumferences, skinfolds, and three TOBEC readings. Reliability for percent fat of both single and multiple TOBEC trials (t = 3) ranged from .994 to .999. Average percent fat values were significantly (p < .009) higher in sedentary subjects. Sum of seven skinfolds was significantly correlated (r[15] = 0.73, p < .01) with percent fat measured by TOBEC. Results suggest TOBEC is reliable (rtt > 0.99) in estimating body composition in spinal cord injured individuals. High reliability estimates for single and multiple trials indicate use of a single trial will provide reliable body fat estimates. Construct validity evidence infers that TOBEC measured body composition discriminates between active and sedentary paraplegics.

Autoxidation of cholesterol in tallows heated under deep frying conditions: evaluation of oxysterols by GLC and TLC-FID.

The products of cholesterol autoxidation (oxysterols) in heated animal food fat were determined qualitatively and quantitatively to evaluate their toxicity and those of the foods in which they occur. Samples of beef tallow were taken from deep-fat fryers while they were in use. The oxysterols were identified and assayed by gas liquid chromatography and thin layer chromatography on Chromarods with flame ionization detection (TLC-FID). The two methods were compared and the TLC-FID method was found more convenient for a rapid estimation of autoxidation. Of the original cholesterol, 25% was destroyed during cooking and partly transformed into 3 beta-5-6 beta-trihydroxy-5 alpha-cholestane, 7 alpha-hydroxy-, 7 beta-hydroxy-, 7-oxo-cholesterol, 7-oxo-cholesta-3-5-diene and cholesterol epoxides. Certain other oxysterols were present in smaller quantities.