Simultaneous Quantification of Multiple Bacteria by the BactoChip Microarray Designed to Target Species-Specific Marker Genes

Bacteria are ubiquitous throughout the environment, the most abundant inhabitants of the healthy human microbiome, and causal pathogens in a variety of diseases. Their identification in disease is often an essential step in rapid diagnosis and targeted intervention, particularly in clinical settings. At present, clinical bacterial detection and discrimination is primarily culture-based, requiring both time and microbiological expertise, especially for bacteria that are not easily cultivated. Higher-throughput molecular methods based on PCR amplification or, recently, microarrays are reaching the clinic as well. However, these methods are currently restricted to a small set of microbes or based on conserved phylogenetic markers such as the 16S rRNA gene, which are difficult to resolve at the species or strain levels. Here, we designed and experimentally validated the BactoChip, an oligonucleotide microarray for bacterial detection and quantification. The chip allows the culture-independent identification of bacterial species, also determining their relative abundances in complex communities as occur in the commensal microbiota or in clinical settings. The microarray successfully distinguished among bacterial species from 21 different genera using 60-mer probes targeting a novel set of in silico identified high-resolution marker genes. The BactoChip additionally proved accurate in determining species-level relative abundances over a 100-fold dynamic range in complex bacterial communities and with a low limit of detection (0.1%). In combination with the continually increasing number of sequenced bacterial genomes, future iterations of the technology could enable to highly accurate clinically-oriented tools for rapid assessment of bacterial community composition and relative abundances

Metagenomic microbial community profiling using unique clade-specific marker genes

Metagenomic shotgun sequencing data can identify microbes populating a microbial community and their proportions, but existing taxonomic profiling methods are inefficient for increasingly large datasets. We present an approach that uses clade-specific marker genes to unambiguously assign reads to microbial clades more accurately and >50× faster than current approaches. We validated MetaPhlAn on terabases of short reads and provide the largest metagenomic profiling to date of the human gu

The murine orthologue of the Golgi-localized TPTE protein provides clues to the evolutionary history of the human TPTE gene family

Abstract.: The human TPTE gene encodes a testis-specific protein that contains four potential transmembrane domains and a protein tyrosine phosphatase motif, and shows homology to the tumor suppressor PTEN/MMAC1. Chromosomal mapping revealed multiple copies of the TPTE gene present on the acrocentric chromosomes 13, 15, 21 and 22, and the Y chromosome. Zooblot analysis suggests that mice may possess only one copy of TPTE. In the present study, we report the isolation and initial characterization of the full-length cDNA of the mouse homologue Tpte. At least three different mRNA transcripts (Tpte.a, b, c) are produced via alternative splicing, encoding predicted proteins that would contain four potential transmembrane domains and a protein tyrosine phosphatase motif. Transfection of a 5′EGFP-TPTE fusion protein in Hela cells revealed an intracellular localization within the Golgi apparatus. Tpte was mapped by radiation hybrid to a region of mouse chromosome 8 that shows conserved synteny with human 13q14.2-q21 between NEK3 and SGT1. This region of the human genome was found to contain a partial, highly diverged copy of TPTE that is likely to represent the ancestral copy from which the other copies of TPTE arose through duplication events. The Y chromosome copy of TPTE is a pseudogene and is not therefore involved in the testis expression of this gene famil

Age and Gender Affect the Composition of Fungal Population of the Human Gastrointestinal Tract

The fungal component of the human gut microbiota has been neglected for long time due to the low relative abundance of fungi with respect to bacteria, and only recently few reports have explored its composition and dynamics in health or disease. The application of metagenomics methods to the full understanding of fungal communities is currently limited by the under representation of fungal DNA with respect to the bacterial one, as well as by the limited ability to discriminate passengers from colonizers. Here we investigated the gut mycobiota of a cohort of healthy subjects in order to reduce the gap of knowledge concerning fungal intestinal communities in the healthy status further screening for phenotypical traits that could reflect fungi adaptation to the host. We studied the fecal fungal populations of 111 healthy subjects by means of cultivation on fungal selective media and by amplicon-based ITS1 metagenomics analysis on a subset of 57 individuals. We then characterized the isolated fungi for their tolerance to gastrointestinal tract-like challenges and their susceptibility to antifungals. A total of 34 different fungal species were isolated showing several phenotypic characteristics associated with intestinal environment such as tolerance to body temperature (37°C), to acidic and oxidative stress and to bile salts exposure. We found a high frequency of azoles resistance in fungal isolates, with potential and significant clinical impact. Analyses of fungal communities revealed that the human gut mycobiota differs in function of individuals’ life stage in a gender-related fashion. The combination of metagenomics and fungal cultivation allowed an in-depth understanding of the fungal intestinal community structure associated to the healthy status and the commensalism-related traits of isolated fungi. We further discussed comparatively the results of sequencing and cultivation to critically evaluate the application of metagenomics-based approaches to fungal gut populations

Altered gut microbiota in Rett syndrome

Background The human gut microbiota directly affects human health, and its alteration can lead to gastrointestinal abnormalities and inflammation. Rett syndrome (RTT), a progressive neurological disorder mainly caused by mutations in MeCP2 gene, is commonly associated with gastrointestinal dysfunctions and constipation, suggesting a link between RTT’s gastrointestinal abnormalities and the gut microbiota. The aim of this study was to evaluate the bacterial and fungal gut microbiota in a cohort of RTT subjects integrating clinical, metabolomics and metagenomics data to understand if changes in the gut microbiota of RTT subjects could be associated with gastrointestinal abnormalities and inflammatory status. Results Our findings revealed the occurrence of an intestinal sub-inflammatory status in RTT subjects as measured by the elevated values of faecal calprotectin and erythrocyte sedimentation rate. We showed that, overall, RTT subjects harbour bacterial and fungal microbiota altered in terms of relative abundances from those of healthy controls, with a reduced microbial richness and dominated by microbial taxa belonging to Bifidobacterium, several Clostridia (among which Anaerostipes, Clostridium XIVa, Clostridium XIVb) as well as Erysipelotrichaceae, Actinomyces, Lactobacillus, Enterococcus, Eggerthella, Escherichia/Shigella and the fungal genus Candida. We further observed that alterations of the gut microbiota do not depend on the constipation status of RTT subjects and that this dysbiotic microbiota produced altered short chain fatty acids profiles. Conclusions We demonstrated for the first time that RTT is associated with a dysbiosis of both the bacterial and fungal component of the gut microbiota, suggesting that impairments of MeCP2 functioning favour the establishment of a microbial community adapted to the costive gastrointestinal niche of RTT subjects. The altered production of short chain fatty acids associated with this microbiota might reinforce the constipation status of RTT subjects and contribute to RTT gastrointestinal physiopathology

Effects of seagrasses and algae of the Caulerpa family on hydrodynamics and particle-trapping rates

The widespread decline of seagrass beds within the Mediterranean often results in the replacement of seagrasses by opportunistic green algae of the Caulerpa family. Because Caulerpa beds have a different height, stiffness and density compared to seagrasses, these changes in habitat type modify the interaction of the seafloor with hydrodynamics, influencing key processes such as sediment resuspension and particle trapping. Here, we compare the effects on hydrodynamics and particle trapping of Caulerpa taxifolia, C. racemosa, and C. prolifera with the Mediterranean seagrasses Cymodocea nodosa and Posidonia oceanica. All macrophyte canopies reduced near-bed volumetric flow rates compared to bare sediment, vertical profiles of turbulent kinetic energy revealed peak values around the top of the canopies, and maximum values of Reynolds stress increased by a factor of between 1.4 (C. nodosa) and 324.1 (P. oceanica) when vegetation was present. All canopies enhanced particle retention rates compared to bare sediment. The experimental C. prolifera canopy was the most effective at particle retention (m2 habitat); however, C. racemosa had the largest particle retention capacity per structure surface area. Hence, in terms of enhancing particle trapping and reducing hydrodynamic forces at the sediment surface, Caulerpa beds provided a similar or enhanced function compared to P.oceanica and C. nodosa. However, strong seasonality in the leaf area index of C. racemosa and C. taxifolia within the Mediterranean, combined with a weak rhizome structure, suggests that sediments maybe unprotected during winter storms, when most erosion occurs. Hence, replacement of seagrass beds with Caulerpa is likely to have a major influence on annual sediment dynamics at ecosystem scales.This research was funded by the European Network of Excellence ‘‘Marine Biodiversity and Ecosystem Function’’ (MarBEF); FP6, EC contract no. 505446 and a grant from the Fundacio ´n BBVA. EPM was supported by a European Union Marie Curie host fellowship for transfer of knowledge, MTKD-CT-2004-509254, the Spanish national project EVAMARIA (CTM2005-00395/MAR) and the regional government of Andalusia project FUNDIV(P07-RNM-2516)

Penggunaan Media Model dalam Pembelajaran IPA

This study aims to describe the increase in interest and student learning outcomes in science teaching in primary schools. Classroom action research was conducted in two cycles, each cycle consisted of two meetings with the various forms of energy and material use. Research subjects elementary school students grade IV No 20 Gunung Pangilun Padang Utara. The instrument of this study is the observation sheet student\u27s interests, learning activities observation sheets, and achievement test students\u27 interest in learning the instrument. The results showed that students\u27 interest in learning science in one cycle is 64.4 percent, and in the second cycle of 82.2 percent. Student learning outcomes in a single cycle on average 63.5 and 83.5 in the two cycle becomes. Besides that, it also revealed that an increasing mastery learning students from one cycle is 45.8 percent and in the second cycle of 91.6 percent. Analysis of teachers in implementing learning activities in a cycle that is 79.1 per cent and 91.6 per cent of the second cycle. The use of models in the media can increase interest in science learning, learning outcomes and teacher activities . Therefore, the model can be used medium primary school teachers as one of the media in learning science . Besides, teachers also need to make a good plan in accordance with the science curriculum in elementary schools

The new phylogenesis of the genus Mycobacterium

Abstract Phylogenetic knowledge of the genus Mycobacterium is based on comparative analysis of their genetic sequences. The 16S rRNA has remained for many years the only target of such analyses, but in the last few years, other housekeeping genes have been investigated and the phylogeny based on their concatenated sequences become a standard. It is now clear that the robustness of the phylogenetic analysis is strictly related to the size of the genomic target used. Whole genome sequencing (WGS) is nowadays becoming widely accessible and comparatively cheap. It was decided, therefore, to use this approach to reconstruct the ultimate phylogeny of the genus Mycobacterium . Over 50 types of strains of the same number of species of Mycobacterium were sequenced using the Illumina HiSeq platform. The majority of the strains of which the whole sequence was already available in GenBank were excluded from this panel with the aim of maximizing the number of the species with genome available. Following assembling and annotation with proper software, the phylogenetic analysis was conducted with PhyloPhlAn and the pan-genome analysis pipeline. The phylogenetic three which emerged was characterized by a clear-cut distinction of slowly and rapidly growing species with the latter being more ancestral. The species of the Mycobacterium terrae complex occupied an intermediate position between rapid and slow growers. Most of the species revealed clearly related and occupied specific phylogenetic branches. Thanks to the WGS technology, the genus Mycobacterium is finally approaching its definitive location

Genetic diversity and expanding nonindigenous range of the rhizocephalan Loxothylacus panopaei parasitizing mud crabs in the western North Atlantic

Nonindigenous parasite introductions and range expansions have become a major concern because of their potential to restructure communities and impact fisheries. Molecular markers provide an important tool for reconstructing the pattern of introduction. The parasitic castrator Loxothylacus panopaei, a rhizocephalan barnacle, infects estuarine mud crabs in the Gulf of Mexico and southeastern Florida. A similar parasite introduced into Chesapeake Bay before 1964, presumably via infected crabs associated with oysters from the Gulf of Mexico, was identified as L. panopaei. Our samples of this species during 2004 and 2005 show that the introduced range has expanded as far south as Edgewater, Florida, just north of the northern endemic range limit. The nonindigenous range expanded southward at a rate of up to 165 km/yr with relatively high prevalence, ranging from 30 to 93%. Mitochondrial DNA sequences from the cytochrome oxidase I gene showed that these nonindigenous L. panopaei are genetically distinct from the endemic parasites in southeastern Florida and the eastern Gulf of Mexico. The genetic difference was also associated with distinct host spectra. These results are incompatible with an eastern Gulf source population, but suggest that unrecognized genetic and phenotypic population structure may occur among Gulf of Mexico populations of Loxothvlacu