Influence of fecal collection conditions and 16S rRNA gene sequencing at two centers on human gut microbiota analysis

Published online: 12 March 2018To optimise fecal sampling for reproducible analysis of the gut microbiome, we compared different methods of sample collection and sequencing of 16S rRNA genes at two centers. Samples collected from six individuals on three consecutive days were placed in commercial collection tubes (OMNIgeneGut OMR-200) or in sterile screw-top tubes in a home fridge or home freezer for 6-24 h, before transfer and storage at -80 °C. Replicate samples were shipped to centers in Australia and the USA for DNA extraction and sequencing by their respective PCR protocols, and analysed with the same bioinformatic pipeline. Variation in gut microbiome was dominated by differences between individuals. Minor differences in the abundance of taxa were found between collection-processing methods and day of collection, and between the two centers. We conclude that collection with storage and transport at 4 °C within 24 h is adequate for 16S rRNA analysis of the gut microbiome. Other factors including differences in PCR and sequencing methods account for relatively minor variation compared to differences between individuals.Jocelyn Sietsma Penington, Megan A. S. Penno, Katrina M. Ngui, Nadim J. Ajami, Alexandra J. Roth-Schulze, Stephen A. Wilcox, Esther Bandala-Sanchez, John M. Wentworth, Simon C. Barry, Cheryl Y. Brown, Jennifer J. Couper, Joseph F. Petrosino, Anthony T. Papenfuss, Leonard C. Harrison and ENDIA Study Group (Lynne Giles and Rebecca L. Thomson

Women with type 1 diabetes exhibit a progressive increase in gut Saccharomyces cerevisiae in pregnancy associated with evidence of gut inflammation.

Aims: Studies of the gut microbiome have focused on its bacterial composition. We aimed to characterize the gut fungal microbiome (mycobiome) across pregnancy in women with and without type 1 diabetes. Methods: Faecal samples (n = 162) were collected from 70 pregnant women (45 with and 25 without type 1 diabetes) across all trimesters. Fungi were analysed by internal transcribed spacer 1 amplicon sequencing. Markers of intestinal inflammation (faecal calprotectin) and intestinal epithelial integrity (serum intestinal fatty acid binding protein; I-FABP), and serum antibodies to Saccharomyces cerevisiae (ASCA) were measured. Results: Women with type 1 diabetes had decreased fungal alpha diversity by the third trimester, associated with an increased abundance of Saccharomyces cerevisiae that was inversely related to the abundance of the anti-inflammatory butyrate-producing bacterium Faecalibacterium prausnitzii. Women with type 1 diabetes had higher concentrations of calprotectin, I-FABP and ASCA. Conclusions: Women with type 1 diabetes exhibit a shift in the gut mycobiome across pregnancy associated with evidence of gut inflammation and impaired intestinal barrier function. The relevance of these findings to the higher rate of pregnancy complications in type 1 diabetes warrants further study.Esther Bandala-Sanchez, Alexandra J. Roth-Schulze, Helena Oakey Megan A.S. Penno, Naiara G. Bediaga, Gaetano Naselli, Katrina M. Ngui, Alannah D. Smith, Dexing Huang, Enrique Zozaya-Valdes, Rebecca L. Thomson, James D. Brown, Peter J. Vuillermin, Simon C. Barry, Maria E. Craig, William D. Rawlinson, Elizabeth A. Davis, Mark Harris, Georgia Soldatos, Peter G. Colman, John M. Wentworth, Aveni Haynes, Grant Morahan, Richard O. Sinnott, Anthony T. Papenfuss, Jennifer J. Couper, Leonard C. Harrison, on behalf of the ENDIA Study Grou

Regulation of the Actin Cytoskeleton by an Interaction of IQGAP Related Protein GAPA with Filamin and Cortexillin I

Filamin and Cortexillin are F-actin crosslinking proteins in Dictyostelium discoideum allowing actin filaments to form three-dimensional networks. GAPA, an IQGAP related protein, is required for cytokinesis and localizes to the cleavage furrow during cytokinesis. Here we describe a novel interaction with Filamin which is required for cytokinesis and regulation of the F-actin content. The interaction occurs through the actin binding domain of Filamin and the GRD domain of GAPA. A similar interaction takes place with Cortexillin I. We further report that Filamin associates with Rac1a implying that filamin might act as a scaffold for small GTPases. Filamin and activated Rac associate with GAPA to regulate actin remodelling. Overexpression of filamin and GAPA in the various strains suggests that GAPA regulates the actin cytoskeleton through interaction with Filamin and that it controls cytokinesis through association with Filamin and Cortexillin

Influence of fecal collection conditions and 16S rRNA gene sequencing at two centers on human gut microbiota analysis

To optimise fecal sampling for reproducible analysis of the gut microbiome, we compared different methods of sample collection and sequencing of 16S rRNA genes at two centers. Samples collected from six individuals on three consecutive days were placed in commercial collection tubes (OMNIgeneGut OMR-200) or in sterile screw-top tubes in a home fridge or home freezer for 6-24 h, before transfer and storage at-80 °C. Replicate samples were shipped to centers in Australia and the USA for DNA extraction and sequencing by their respective PCR protocols, and analysed with the same bioinformatic pipeline. Variation in gut microbiome was dominated by differences between individuals. Minor differences in the abundance of taxa were found between collection-processing methods and day of collection, and between the two centers. We conclude that collection with storage and transport at 4 °C within 24 h is adequate for 16S rRNA analysis of the gut microbiome. Other factors including differences in PCR and sequencing methods account for relatively minor variation compared to differences between individuals

Environmental determinants of islet autoimmunity (ENDIA): a pregnancy to early life cohort study in children at-risk of type 1 diabetes

Members of ENDIA Study Group: Peter Baghurst, Simon Barry, Jodie Dodd, Maria Makrides for the University of Adelaide.BACKGROUND The incidence of type 1 diabetes has increased worldwide, particularly in younger children and those with lower genetic susceptibility. These observations suggest factors in the modern environment promote pancreatic islet autoimmunity and destruction of insulin-producing beta cells. The Environmental Determinants of Islet Autoimmunity (ENDIA) Study is investigating candidate environmental exposures and gene-environment interactions that may contribute to the development of islet autoimmunity and type 1 diabetes. METHODS/DESIGN ENDIA is the only prospective pregnancy/birth cohort study in the Southern Hemisphere investigating the determinants of type 1 diabetes in at-risk children. The study will recruit 1,400 unborn infants or infants less than six months of age with a first-degree relative (i.e. mother, father or sibling) with type 1 diabetes, across five Australian states. Pregnant mothers/infants will be followed prospectively from early pregnancy through childhood to investigate relationships between genotype, the development of islet autoimmunity (and subsequently type 1 diabetes), and prenatal and postnatal environmental factors. ENDIA will evaluate the microbiome, nutrition, bodyweight/composition, metabolome-lipidome, insulin resistance, innate and adaptive immune function and viral infections. A systems biology approach will be used to integrate these data. Investigation will be by 3-monthly assessments of the mother during pregnancy, then 3-monthly assessments of the child until 24 months of age and 6-monthly thereafter. The primary outcome measure is persistent islet autoimmunity, defined as the presence of autoantibodies to one or more islet autoantigens on consecutive tests. DISCUSSION Defining gene-environment interactions that initiate and/or promote destruction of the insulin-producing beta cells in early life will inform approaches to primary prevention of type 1 diabetes. The strength of ENDIA is the prospective, comprehensive and frequent systems-wide profiling from early pregnancy through to early childhood, to capture dynamic environmental exposures that may shape the development of islet autoimmunity. TRIAL REGISTRATION Australia New Zealand Clinical Trials Registry ACTRN12613000794707.Megan AS Penno, Jennifer J Couper, Maria E Craig, Peter G Colman, William D Rawlinson, Andrew M Cotterill, Timothy W Jones, Leonard C Harrison and ENDIA Study Grou

Photocatalytic Degradation of Estriol Using Iron-Doped TiO2 under High and Low UV Irradiation

Iron-doped TiO2 nanoparticles (Fe-TiO2) were synthesized and photocatalitically investigated under high and low fluence values of UV radiation. The Fe-TiO2 physical characterization was performed using X-ray Powder Diffraction (XRD), Brunauer-Emmett-Teller (BET) surface area analysis, Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), Diffuse Reflectance Spectroscopy (DRS), and X-ray Photoelectron Spectroscopy (XPS). The XPS evidenced that the ferric ion (Fe3+) was in the TiO2 lattice and unintentionally added co-dopants were also present because of the precursors of the synthetic method. The Fe3+ concentration played a key role in the photocatalytic generation of hydroxyl radicals ((OH)-O-center dot) and estriol (E3) degradation. Fe-TiO2 accomplished E3 degradation, and it was found that the catalyst with 0.3 at.% content of Fe (0.3 Fe-TiO2) enhanced the photocatalytic activity under low UV irradiation compared with TiO2 without intentionally added Fe (zero-iron TiO2) and Aeroxide (R) TiO2 P25. Furthermore, the enhanced photocatalytic activity of 0.3 Fe-TiO2 under low UV irradiation may have applications when radiation intensity must be controlled, as in medical applications, or when strong UV absorbing species are present in water

FPGA-based architecture for real-time data reduction of ultrasound signals

This paper describes a novel method for on-line real-time data reduction of radiofrequency (RF) ultrasound signals. The approach is based on a field programmable gate array (FPGA) system intended mainly for steel thickness measurements. Ultrasound data reduction is desirable when: (1) direct measurements performed by an operator are not accessible; (2) it is required to store a considerable amount of data; (3) the application requires measuring at very high speeds; and (4) the physical space for the embedded hardware is limited. All the aforementioned scenarios can be present in applications such as pipeline inspection where data reduction is traditionally performed on-line using pipeline inspection gauges (PIG). The method proposed in this work consists of identifying and storing in real-time only the time of occurrence (TOO) and the maximum amplitude of each echo present in a given RF ultrasound signal. The method is tested with a dedicated immersion system where a significant data reduction with an average of 96.5% is achieved. Crow

Role of organic matter, nitrogen and phosphorous on granulation and settling velocity in wastewater treatment

In this study, the ability of the aerobic biogranulation process to remove organic matter, total nitrogen, and total phosphorous from wastewater was investigated. The effect of organic matter load on the quality of the produced biogranules and settling velocity of the generated biomass was also investigated. Four different aerobic scenarios were studied in sequential batch bioreactors (SBR) to identify the best operating conditions. The experimental setup consisted of four bioreactors containing 300 mL samples (100 mL of sludge and 200 mL of sweet whey at four different concentrations) under constant stirring (150 rpm), room temperature, and controlled pH (e.g., 7.98 ± 0.25). The different organic matter loads were calculated as the chemical oxygen demand (COD) concentration at 1157 (A), 719 (B), 357 (C), and 202 mg/L (D), respectively. The process consisted of cycles of initial feast followed by 72 h of famine. During the experiments, COD, total nitrogen (TN), and total phosphorus (TP) were measured at the beginning (t0) and the end of the process. The results confirmed that the best conditions were found in the bioreactor with the highest COD load (1157 mg/L), which achieved 98.7 % reduction in organic matter, 45.6 % reduction in TP, and 99.4 % reduction in TN after 9 days with final average sludge settling velocity of 0.7 m/h. The biogranulation process achieved improved clarification of the supernatant, showing increased settling velocity in all of the bioreactors. Microscopic observation confirmed uniform morphology (e.g., spherical or cylindrical) with the biggest biogranules being observed in bioreactors A and B. Organic load was found to play a significant role in biogranules formation and the achievement of the highest organic matter removal, sludge volume reduction, and water clarification. The overall trend in final sludge settling velocity measured in the different bioreactors was C > B > D > A, and in all of them settling was faster than the untreated sample. The results of this study confirm the high potential of biogranulation process for the treatment of wastewater with high organic matter content.</p

Deep water ROV design for the Mexican oil industry

This paper describes both the design and technical integration of a deep water ROV (Remotely Operated Vehicle) that is able to employ visual inspection in deep water, as well as to take samples with an underwater arm. Visual inspection is the essential application for a ROV, which is useful for; underwater structure inspection, underwater oil production systems inspection and underwater pipeline inspection. This paper mainly describes the ROV design, and explains in general manner other elements; such as the SCU, LARS and TMS. It is important to mention that the design phase is complete and it was financed by PEMEX (Federal Mexican Oil Company). The project has stalled, since we are waiting for the financial support from PEMEX for the system construction. A future objective is the design and construction of Work-Class ROVs, which can be used to perform PEMEX specific tasks