In vitro indices of tissue adherence in Staphylococcus intermedius

In vitro indices of adherence showed that strains of Staphylococcus intermedius from lesions of canine pyoderma differed from strains isolated from normal carrier sites in that a significantly greater proportion of pyoderma strains adhered to extracellular matrix proteins whilst fewer adhered to polystyrene. Slime production and a hydrophobicity index did not differ between the groups. This suggests that exposure of extracellular matrix proteins due to underlying disease may result in the selection of a narrower spectrum of strains from amongst those at carrier sites.     

Accuracy of Inferior Vena Cava Ultrasound for Predicting Dehydration in Children with Acute Diarrhea in Resource-Limited Settings

Introduction

Although dehydration from diarrhea is a leading cause of morbidity and mortality in children under five, existing methods of assessing dehydration status in children have limited accuracy.

Objective

To assess the accuracy of point-of-care ultrasound measurement of the aorta-to-IVC ratio as a predictor of dehydration in children.

Methods

A prospective cohort study of children under five years with acute diarrhea was conducted in the rehydration unit of the International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b). Ultrasound measurements of aorta-to-IVC ratio and dehydrated weight were obtained on patient arrival. Percent weight change was monitored during rehydration to classify children as having “some dehydration” with weight change 3–9% or “severe dehydration” with weight change > 9%. Logistic regression analysis and Receiver-Operator Characteristic (ROC) curves were used to evaluate the accuracy of aorta-to-IVC ratio as a predictor of dehydration severity.

Results

850 children were enrolled, of which 771 were included in the final analysis. Aorta to IVC ratio was a significant predictor of the percent dehydration in children with acute diarrhea, with each 1-point increase in the aorta to IVC ratio predicting a 1.1% increase in the percent dehydration of the child. However, the area under the ROC curve (0.60), sensitivity (67%), and specificity (49%), for predicting severe dehydration were all poor.

Conclusions

Point-of-care ultrasound of the aorta-to-IVC ratio was statistically associated with volume status, but was not accurate enough to be used as an independent screening tool for dehydration in children under five years presenting with acute diarrhea in a resource-limited setting.     

Identifying multi-locus chromatin contacts in human cells using tethered multiple 3C

Background

Several recently developed experimental methods, each an extension of the chromatin conformation capture (3C) assay, have enabled the genome-wide profiling of chromatin contacts between pairs of genomic loci in 3D. Especially in complex eukaryotes, data generated by these methods, coupled with other genome-wide datasets, demonstrated that non-random chromatin folding correlates strongly with cellular processes such as gene expression and DNA replication.

Results

We describe a genome architecture assay, tethered multiple 3C (TM3C), that maps genome-wide chromatin contacts via a simple protocol of restriction enzyme digestion and religation of fragments upon agarose gel beads followed by paired-end sequencing. In addition to identifying contacts between pairs of loci, TM3C enables identification of contacts among more than two loci simultaneously. We use TM3C to assay the genome architectures of two human cell lines: KBM7, a near-haploid chronic leukemia cell line, and NHEK, a normal diploid human epidermal keratinocyte cell line. We confirm that the contact frequency maps produced by TM3C exhibit features characteristic of existing genome architecture datasets, including the expected scaling of contact probabilities with genomic distance, megabase scale chromosomal compartments and sub-megabase scale topological domains. We also confirm that TM3C captures several known cell type-specific contacts, ploidy shifts and translocations, such as Philadelphia chromosome formation (Ph+) in KBM7. We confirm a subset of the triple contacts involving the IGF2-H19 imprinting control region (ICR) using PCR analysis for KBM7 cells. Our genome-wide analysis of pairwise and triple contacts demonstrates their preference for linking open chromatin regions to each other and for linking regions with higher numbers of DNase hypersensitive sites (DHSs) to each other. For near-haploid KBM7 cells, we infer whole genome 3D models that exhibit clustering of small chromosomes with each other and large chromosomes with each other, consistent with previous studies of the genome architectures of other human cell lines.

Conclusion

TM3C is a simple protocol for ascertaining genome architecture and can be used to identify simultaneous contacts among three or four loci. Application of TM3C to a near-haploid human cell line revealed large-scale features of chromosomal organization and multi-way chromatin contacts that preferentially link regions of open chromatin.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1236-7) contains supplementary material, which is available to authorized users.     

Unsupervised segmentation of continuous genomic data

The advent of high-density, high-volume genomic data has created the need for tools to summarize large datasets at multiple scales. HMMSeg is a command-line utility for the scale-specific segmentation of continuous genomic data using hidden Markov models (HMMs). Scale specificity is achieved by an optional wavelet-based smoothing operation. HMMSeg is capable of handling multiple datasets simultaneously, rendering it ideal for integrative analysis of expression, phylogenetic and functional genomic data. AVAILABILITY: http://noble.gs.washington.edu/proj/hmmseg     

Characterization of a Neochlamydia-like bacterium associated with epitheliocystis in cultured Arctic charr Salvelinus alpinus

Infections of branchial epithelium by intracellular gram-negative bacteria, termed epitheliocystis, have limited culture of Arctic charr Salvelinus alpinus. To characterize a bacterium associated with epitheliocystis in cultured charr, gills were sampled for histopathologic examination, conventional and immunoelectron microscopy, in situ hybridization, 16S ribosomal DNA (rDNA) amplification, sequence analysis and phylogenetic inference. Sampling was conducted at the Freshwater Institute (Shepherdstown, West Virginia, USA) during outbreaks of epitheliocystis in April and May 2002. Granular, basophilic, cytoplasmic inclusions in charr gill were shown to stain with Macchiavello, Lendrum's phloxine-tartrazine and Gimenez histochemical techniques. Ultrastructurally, inclusions were membrane-bound and contained round to elongate reticulate bodies that were immunoreactive to an antibody against chlamydial lipopolysaccharide, suggesting the presence of similar epitopes. DNA extracted from gills supported amplification of the most polymorphic and phylogenetically relevant region of the 16S rRNA gene, which had 97 to 100% identity with several uncultured clinical Neochlamydia spp. (order Chlamydiales) Clones WB13 (AY225593.1) and WB258 (AY225594.1). Sequence-specific riboprobes localized to inclusions during in situ hybridization experiments. Taxonomic affiliation was inferred by distance- and parsimony-based phylogenetic analyses of the 16S sequence, which branched with Neochlamydia hartmannellae in the order Chlamydiales with high confidence. This is the first molecular characterization of a chlamydia associated with epitheliocystis in Arctic charr and the fourth Neochlamydia spp. sequence to be associated with epitheliocystis. Presence of a clinical neochlamydial sequence, first identified from a cat, in Arctic charr suggests a possible mammalian and piscine host range for some environmental chlamydiae.     

Development of a statistically robust quantification method for microorganisms in mixtures using oligonucleotide microarrays

High-density oligonucleotide arrays can be extremely useful for identifying and quantifying specific targets (i.e., ribosomal RNA of microorganisms) in mixtures. However, current array identification schemes are severely compromised by nonspecific hybridization, resulting in numerous false-positive and false-negative calls, they lack an adequate internal control for assessing the quality of identification, and are dependent on amplification of specific target sequences which introduce biases. We have developed a novel approach for the routine quantification and identification of metabolically active microorganisms in mixed samples. The advantage of our approach over conventional ones is that it avoids designing, optimizing, validating, and selecting oligonucleotide probes for arrays; also, nonspecific hybridization is no longer a problem. The basic principle of the approach is that a fluorescence pattern of a mixed sample is a superposition of the fluorescent patterns for each target. The superposition can be quantitatively deconvoluted in terms of concentrations of each microbe. We demonstrated the utility of our approach by extracting rRNA from three microorganisms, making test mixtures, labeling the rRNA, and hybridizing each test mixture to DNA oligonucleotide (20-mers, n=346,608) arrays. Comparison of known concentrations of individual targets in mixtures to those estimated by the solution revealed highly consistent results. The goodness-of-fit of the solution revealed that about 90% of the variability in the data could be explained. A new analytical approach for microbial identification and quantification has been presented in this report. Our findings demonstrate that including signal intensity values from all duplexes on the array, which are essentially nonspecific to the target organisms, significantly improved predictions of known microbial targets. To our knowledge, this is the first study to report this phenomenon. In addition, we demonstrate that the method is a self-sufficient analytical procedure since it provides statistical confidence of the quantification.     

Structures of the Cd44-hyaluronan complex provide insight into a fundamental carbohydrate-protein interaction

Regulation of transient interactions between cells and the ubiquitous matrix glycosaminoglycan hyaluronan is crucial to such fundamental processes as embryonic development and leukocyte homing. Cd44, the primary cell surface receptor for hyaluronan, binds ligand via a lectin-like fold termed the Link module, but only after appropriate functional activation. The molecular details of the Cd44-hyaluronan interaction and hence the structural basis for this activation are unknown. Here we present the first crystal structure of Cd44 complexed with hyaluronan. This reveals that the interaction with hyaluronan is dominated by shape and hydrogen-bonding complementarity and identifies two conformational forms of the receptor that differ in orientation of a crucial hyaluronan-binding residue (Arg45, equivalent to Arg41 in human CD44). Measurements by NMR indicate that the conformational transition can be induced by hyaluronan binding, providing further insight into possible mechanisms for regulation of Cd44.