Clostridium leptum group bacteria abundance and diversity in the fecal microbiota of patients with inflammatory bowel disease: a case–control study in India

BACKGROUND: Alterations in the fecal bacterial flora occur in inflammatory bowel disease (IBD). We examined the abundance and diversity of Clostridium leptum group, an important group of carbohydrate-fermenting bacteria, in the feces of patients with IBD and compared them with healthy controls. METHODS: Seventeen healthy controls (HC), 20 patients with Crohn’s disease (CD) and 22 patients with ulcerative colitis (UC) participated in the study. DNA extracted from fecal samples was amplified by PCR targeting 16S rRNA gene sequences specific to C. leptum group. The PCR product was subjected to temporal temperature gradient electrophoresis (TTGE) and the number and position of individual bands were noted and diversity was estimated. The identity of bands at different positions was confirmed by cloning and sequencing. Real time quantitative PCR with Mesa Green, targeted at specific 16S rRNA gene sequences, was used to quantitate C. leptum group and its most prominent constituent, Faecalibacterium prausnitzii. RESULTS: Twenty five different operational taxonomic units (OTUs, equivalent to species) were identified constituting the C. leptum group in these participants. Their sequences were deposited in GenBank [accession numbers GQ465348 to GQ465370]. OTU number was significantly reduced in CD (7.7±3.7, mean±SD) and UC (9.0±3.0) compared to HC (11.9±2.2) (P=0.0005). The Simpson D index of alpha diversity was not significantly different between the three groups. Total numbers of C. leptum group bacteria and F. prausnitzii were reduced in both CD and UC compared to HC (P=0.0036 and P<0.0001 respectively). Disease activity did not influence numbers of C. leptum or F. prausnitzii in patients with CD or UC. CONCLUSION: C. leptum numbers and diversity were significantly reduced in both CD and UC suggesting that alterations noted were not specific to one disease. This could contribute to reduced short chain fatty acid production in IBD

ZifBASE: a database of zinc finger proteins and associated resources

<p>Abstract</p> <p>Background</p> <p>Information on the occurrence of zinc finger protein motifs in genomes is crucial to the developing field of molecular genome engineering. The knowledge of their target DNA-binding sequences is vital to develop chimeric proteins for targeted genome engineering and site-specific gene correction. There is a need to develop a computational resource of zinc finger proteins (ZFP) to identify the potential binding sites and its location, which reduce the time of <it>in vivo </it>task, and overcome the difficulties in selecting the specific type of zinc finger protein and the target site in the DNA sequence.</p> <p>Description</p> <p>ZifBASE provides an extensive collection of various natural and engineered ZFP. It uses standard names and a genetic and structural classification scheme to present data retrieved from UniProtKB, GenBank, Protein Data Bank, ModBase, Protein Model Portal and the literature. It also incorporates specialized features of ZFP including finger sequences and positions, number of fingers, physiochemical properties, classes, framework, PubMed citations with links to experimental structures (PDB, if available) and modeled structures of natural zinc finger proteins. ZifBASE provides information on zinc finger proteins (both natural and engineered ones), the number of finger units in each of the zinc finger proteins (with multiple fingers), the synergy between the adjacent fingers and their positions. Additionally, it gives the individual finger sequence and their target DNA site to which it binds for better and clear understanding on the interactions of adjacent fingers. The current version of ZifBASE contains 139 entries of which 89 are engineered ZFPs, containing 3-7F totaling to 296 fingers. There are 50 natural zinc finger protein entries ranging from 2-13F, totaling to 307 fingers. It has sequences and structures from literature, Protein Data Bank, ModBase and Protein Model Portal. The interface is cross linked to other public databases like UniprotKB, PDB, ModBase and Protein Model Portal and PubMed for making it more informative.</p> <p>Conclusion</p> <p>A database is established to maintain the information of the sequence features, including the class, framework, number of fingers, residues, position, recognition site and physio-chemical properties (molecular weight, isoelectric point) of both natural and engineered zinc finger proteins and dissociation constant of few. ZifBASE can provide more effective and efficient way of accessing the zinc finger protein sequences and their target binding sites with the links to their three-dimensional structures. All the data and functions are available at the advanced web-based search interface <url>http://web.iitd.ac.in/~sundar/zifbase</url>.</p

Correction: Single nucleotide polymorphisms in the human ATP7B gene modify the properties of the ATP7B protein

Correction for 'Single nucleotide polymorphisms in the human ATP7B gene modify the properties of the ATP7B protein' by Courtney J. McCann et al., Metallomics, 2019, 11, 1128-1139

Effect of Vibrio cholerae on Chemokine Gene Expression in HT29 cells and its Modulation by Lactobacillus GG

Epithelial cells participate in the innate immune response to pathogenic bacteria by elaborating chemokines. This study examined the effect of Vibrio cholerae and Lactobacillus rhamnosus GG on inflammatory chemokine gene expression in the HT29 human intestinal epithelial cell line. HT29 cells were exposed to V. cholerae 0139, Lactobacillus or both for 2 h and cultured further thereafter for 4 h. RNA was extracted from the cells and expression of genes for chemokines and related molecules was quantitated by real time PCR using a pathway-focused PCR array. TLR4 was silenced using shRNA and output of interleukin-8 (IL-8) into the media quantitated with and without V. cholerae exposure. NF&#954;B and p38 MAP kinase activation were determined by immunoblotting for I&#954;B&#945; and phosphorylated p38. Vibrio cholerae significantly upregulated gene expression for the neutrophil chemoattractant CXCL chemokines, IL-8, CXCL and CXCL in HT29 cells, while downregulating the expression of macrophage-attracting C-C chemokines. TLR4 silencing did not reduce IL-8 output from HT29 cells in response to V. cholerae. I&#954;B&#945; degradation was noted in the HT29 cells soon after exposure to V. cholerae and this recovered over time after removal of bacteria. p38 MAP kinase activation was not noted. Vibrio cholerae upregulated the expression of neutrophil attractant chemokines, most prominently IL-8, in HT29 cells, but downregulated macrophage-attracting chemokines. Probiotic lactobacilli modulated the IL-8, but not the other chemokine gene changes, in response to V. cholerae

Quantitative differences in intestinal Faecalibacterium prausnitzii in obese Indian children

Gut bacteria contribute to energy conservation in man through their ability to ferment unabsorbed carbohydrate. The present study examined the composition of predominant faecal microbiota in obese and non-obese children. The participants (n 28) aged 11-14 years provided fresh faecal samples and completed a dietary survey consisting of 24 h diet recall and a FFQ of commonly used foods taken over the previous 3 months. Faecal bacteria were quantitated by real-time PCR using primers targeted at 16S rDNA. Of the participants, fifteen (seven female) were obese, with median BMI-for-age at the 99th percentile (range 97 to&#60;99) while thirteen participants (seven female) were normal weight, with median BMI-for age being at the 50th percentile (range 1-85). Consumption of energy, carbohydrates, fat and protein was not significantly different between the obese and non-obese participants. There was no significant difference between the two groups in faecal levels of Bacteroides-Prevotella, Bifidobacterium species, Lactobacillus acidophilus group or Eubacterium rectale. Levels of Faecalibacterium prausnitzii were significantly higher in obese children than in non-obese participants (P = 0.0253). We concluded that the finding of increased numbers of F. prausnitzii in the faeces of obese children in south India adds to the growing information on alterations in faecal microbiota in obesity

The N-terminal domains of Bacillus subtilis CopA do not form a stable complex in the absence of their inter-domain linker

Copper-transporting P-type ATPases, which play important roles in trafficking Cu(I) across membranes for the biogenesis of copper proteins or for copper detoxification, contain a variable number of soluble metal-binding domains at their N-termini. It is increasingly apparent that these play an important role in regulating copper transport in a Cu(I)-responsive manner, but how they do this is unknown. CopA, a Cu(I)-transporter from Bacillus subtilis, contains two N-terminal soluble domains that are closely packed, with inter-domain interactions at two principal regions. Here, we sought to determine the extent to which the domains interact in the absence of their inter-domain covalent linker, and how their Cu(I)-binding properties are affected. Studies of a 1:1 mixture of separate CopAa and CopAb domains showed that the domains do not form a stable complex, with only indirect evidence of a weak interaction between them. Their Cu(I)-binding behaviour was distinct from that of the two domain protein and consistent with a lack of interaction between the domains. Cu(I)-mediated protein association was observed, but this occurred only between domains of the same type. Thus, the inter-domain covalent link between CopAa and CopAb is essential for inter-domain interactions and for Cu(I)-binding behaviour

Effect of yoghurt containing Bifidobacterium lactis Bb12® on faecal excretion of secretory immunoglobulin A and human beta-defensin 2 in healthy adult volunteers

<p>Abstract</p> <p>Background</p> <p>Probiotics are used to provide health benefits. The present study tested the effect of a probiotic yoghurt on faecal output of beta-defensin and immunoglobulin A in a group of young healthy women eating a defined diet.</p> <p>Findings</p> <p>26 women aged 18-21 (median 19) years residing in a hostel were given 200 ml normal yoghurt every day for a week, followed by probiotic yoghurt containing <it>Bifidobacterium lactis </it>Bb12^®(10⁹in 200 ml) for three weeks, followed again by normal yoghurt for four weeks. Stool samples were collected at 0, 4 and 8 weeks and assayed for immunoglobulin A and human beta-defensin-2 by ELISA. All participants tolerated both normal and probiotic yoghurt well. Human beta-defensin-2 levels in faeces were not altered during the course of the study. On the other hand, compared to the basal sample, faecal IgA increased during probiotic feeding (P = 0.0184) and returned to normal after cessation of probiotic yoghurt intake.</p> <p>Conclusions</p> <p><it>Bifidobacterium lactis </it>Bb12^®increased secretory IgA output in faeces. This property may explain the ability of probiotics to prevent gastrointestinal and lower respiratory tract infections.</p

ZFNGenome: A comprehensive resource for locating zinc finger nuclease target sites in model organisms

<p>Abstract</p> <p>Background</p> <p>Zinc Finger Nucleases (ZFNs) have tremendous potential as tools to facilitate genomic modifications, such as precise gene knockouts or gene replacements by homologous recombination. ZFNs can be used to advance both basic research and clinical applications, including gene therapy. Recently, the ability to engineer ZFNs that target any desired genomic DNA sequence with high fidelity has improved significantly with the introduction of rapid, robust, and publicly available techniques for ZFN design such as the Oligomerized Pool ENgineering (OPEN) method. The motivation for this study is to make resources for genome modifications using OPEN-generated ZFNs more accessible to researchers by creating a user-friendly interface that identifies and provides quality scores for all potential ZFN target sites in the complete genomes of several model organisms.</p> <p>Description</p> <p>ZFNGenome is a GBrowse-based tool for identifying and visualizing potential target sites for OPEN-generated ZFNs. ZFNGenome currently includes a total of more than 11.6 million potential ZFN target sites, mapped within the fully sequenced genomes of seven model organisms; <it>S. cerevisiae, C. reinhardtii, A. thaliana</it>, <it>D. melanogaster, D. rerio, C. elegans</it>, and <it>H. sapiens </it>and can be visualized within the flexible GBrowse environment. Additional model organisms will be included in future updates. ZFNGenome provides information about each potential ZFN target site, including its chromosomal location and position relative to transcription initiation site(s). Users can query ZFNGenome using several different criteria (e.g., gene ID, transcript ID, target site sequence). Tracks in ZFNGenome also provide "uniqueness" and ZiFOpT (Zinc Finger OPEN Targeter) "confidence" scores that estimate the likelihood that a chosen ZFN target site will function <it>in vivo</it>. ZFNGenome is dynamically linked to ZiFDB, allowing users access to all available information about zinc finger reagents, such as the effectiveness of a given ZFN in creating double-stranded breaks.</p> <p>Conclusions</p> <p>ZFNGenome provides a user-friendly interface that allows researchers to access resources and information regarding genomic target sites for engineered ZFNs in seven model organisms. This genome-wide database of potential ZFN target sites should greatly facilitate the utilization of ZFNs in both basic and clinical research.</p> <p>ZFNGenome is freely available at: <url>http://bindr.gdcb.iastate.edu/ZFNGenome</url> or at the Zinc Finger Consortium website: <url>http://www.zincfingers.org/</url>.</p

CPP-ZFN: A potential DNA-targeting anti-malarial drug

<p>Abstract</p> <p>Background</p> <p>Multidrug-resistant <it>Plasmodium </it>is of major concern today. Effective vaccines or successful applications of RNAi-based strategies for the treatment of malaria are currently unavailable. An unexplored area in the field of malaria research is the development of DNA-targeting drugs that can specifically interact with parasitic DNA and introduce deleterious changes, leading to loss of vital genome function and parasite death.</p> <p>Presentation of the hypothesis</p> <p>Advances in the development of zinc finger nuclease (ZFN) with engineered DNA recognition domains allow us to design and develop nuclease of high target sequence specificity with a mega recognition site that typically occurs only once in the genome. Moreover, cell-penetrating peptides (CPP) can cross the cell plasma membrane and deliver conjugated protein, nucleic acid, or any other cargo to the cytoplasm, nucleus, or mitochondria. This article proposes that a drug from the combination of the CPP and ZFN systems can effectively enter the intracellular parasite, introduce deleterious changes in its genome, and eliminate the parasite from the infected cells.</p> <p>Testing the hypothesis</p> <p>Availability of a DNA-binding motif for more than 45 triplets and its modular nature, with freedom to change number of fingers in a ZFN, makes development of customized ZFN against diverse target DNA sequence of any gene feasible. Since the <it>Plasmodium </it>genome is highly AT rich, there is considerable sequence site diversity even for the structurally and functionally conserved enzymes between <it>Plasmodium </it>and humans. CPP can be used to deliver ZFN to the intracellular nucleus of the parasite. Signal-peptide-based heterologous protein translocation to <it>Plasmodium</it>-infected RBCs (iRBCs) and different <it>Plasmodium </it>organelles have been achieved. With successful fusion of CPP with mitochondrial- and nuclear-targeting peptides, fusion of CPP with 1 more <it>Plasmodium </it>cell membrane translocation peptide seems achievable.</p> <p>Implications of the hypothesis</p> <p>Targeting of the <it>Plasmodium </it>genome using ZFN has great potential for the development of anti-malarial drugs. It allows the development of a single drug against all malarial infections, including multidrug-resistant strains. Availability of multiple ZFN target sites in a single gene will provide alternative drug target sites to combat the development of resistance in the future.</p