Advances in the use of terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes to characterize microbial communities

Terminal restriction fragment length polymorphism (T-RFLP) analysis is a popular high-throughput fingerprinting technique used to monitor changes in the structure and composition of microbial communities. This approach is widely used because it offers a compromise between the information gained and labor intensity. In this review, we discuss the progress made in T-RFLP analysis of 16S rRNA genes and functional genes over the last 10 years and evaluate the performance of this technique when used in conjunction with different statistical methods. Web-based tools designed to perform virtual polymerase chain reaction and restriction enzyme digests greatly facilitate the choice of primers and restriction enzymes for T-RFLP analysis. Significant improvements have also been made in the statistical analysis of T-RFLP profiles such as the introduction of objective procedures to distinguish between signal and noise, the alignment of T-RFLP peaks between profiles, and the use of multivariate statistical methods to detect changes in the structure and composition of microbial communities due to spatial and temporal variation or treatment effects. The progress made in T-RFLP analysis of 16S rRNA and genes allows researchers to make methodological and statistical choices appropriate for the hypotheses of their studies.     

Label-Free Density Measurements of Radial Peripapillary Capillaries in the Human Retina

Radial peripapillary capillaries (RPCs) comprise a unique network of capillary beds within the retinal nerve fibre layer (RNFL) and play a critical role in satisfying the nutritional requirements of retinal ganglion cell (RGC) axons. Understanding the topographical and morphological characteristics of these networks through in vivo techniques may improve our understanding about the role of RPCs in RGC axonal health and disease. This study utilizes a novel, non-invasive and label-free optical imaging technique, speckle variance optical coherence tomography (svOCT), for quantitatively studying RPC networks in the human retina. Six different retinal eccentricities from 16 healthy eyes were imaged using svOCT. The same eccentricities were histologically imaged in 9 healthy donor eyes with a confocal scanning laser microscope. Donor eyes were subject to perfusion-based labeling techniques prior to retinal dissection, flat mounting and visualization with the microscope. Capillary density and diameter measurements from each eccentricity in svOCT and histological images were compared. Data from svOCT images were also analysed to determine if there was a correlation between RNFL thickness and RPC density. The results are as follows: (1) The morphological characteristics of RPC networks on svOCT images are comparable to histological images; (2) With the exception of the nasal peripapillary region, there were no significant differences in RPC density measurements between svOCT and histological images; (3) Capillary diameter measurements were significantly greater in svOCT images compared to histology; (4) There is a positive correlation between RPC density and RNFL thickness. The findings in this study suggest that svOCT is a reliable modality for analyzing RPC networks in the human retina. It may therefore be a valuable tool for aiding our understanding about vasculogenic mechanisms that are involved in RGC axonopathies. Further work is required to explore the reason for some of the quantitative differences between svOCT and histology.     

Localization of the voltage-dependent anion channel-1 Ca2+-binding sites

Photoreactive azido ruthenium (AzRu) has been recently shown to specifically interact with Ca(2+)-binding proteins and to strongly inhibit their Ca(2+)-dependent activities. Upon UV irradiation, AzRu can bind covalently to such proteins. In this study, AzRu was used to localize and characterize Ca(2+)-binding sites in the voltage-dependent anion channel (VDAC). AzRu decreased the conductance of VDAC reconstituted into a bilayer while Ca(2+), in the presence of 1M NaCl, but not Mg(2+), prevented this effect. AzRu had no effect on mutated E72Q- or E202Q-VDAC1 conductance, and [(103)Ru]AzRu labeled native but not E72Q-VDAC1, suggesting that these residues are required for AzRu interaction with the VDAC Ca(2+)-binding site(s). AzRu protected against apoptosis induced by over-expression of native but not E72Q- or E202Q- murine VDAC1 in T-REx-293 cells depleted of endogenous hVDAC1. Chymotrypsin and trypsin digestion of AzRu-labeled VDAC followed by MALDI-TOF analysis revealed two AzRu-bound peptides corresponding to E72- and E202-containing sequences. These results suggest that the VDAC Ca(2+)-binding site includes E72 and E202, located, according to a proposed VDAC1 topology model, on two distinct cytosolic loops. Furthermore, AzRu protection against apoptosis involves interaction with these residues. Photoreactive AzRu represents an important tool for identifying novel Ca(2+)-binding proteins and localizing their Ca(2+)-binding sites.     

Statistical methods for characterizing diversity of microbial communities by analysis of terminal restriction fragment length polymorphisms of 16S rRNA genes

The analysis of terminal restriction fragment length polymorphisms (T-RFLP) of 16S rRNA genes has proven to be a facile means to compare microbial communities and presumptively identify abundant members. The method provides data that can be used to compare different communities based on similarity or distance measures. Once communities have been clustered into groups, clone libraries can be prepared from sample(s) that are representative of each group in order to determine the phylogeny of the numerically abundant populations in a community. In this paper methods are introduced for the statistical analysis of T-RFLP data that include objective methods for (i) determining a baseline so that 'true' peaks in electropherograms can be identified; (ii) a means to compare electropherograms and bin fragments of similar size; (iii) clustering algorithms that can be used to identify communities that are similar to one another; and (iv) a means to select samples that are representative of a cluster that can be used to construct 16S rRNA gene clone libraries. The methods for data analysis were tested using simulated data with assumptions and parameters that corresponded to actual data. The simulation results demonstrated the usefulness of these methods in their ability to recover the true microbial community structure generated under the assumptions made. Software for implementing these methods is available at http://www.ibest.uidaho.edu/tools/trflp_stats/index.php.     

Hyperhomocysteinemia increases arterial permeability and stiffness in mice

We have reported that hyperhomocysteinemia (HHcy) evoked by folate depletion increases arterial permeability and stiffness in rats and that low folate without HHcy increases arterial permeability in mice. In this study, we hypothesized that HHcy independently increases arterial permeability and stiffness in mice. C57BL/6J mice that received rodent chow and water [control (Con), n=12] or water supplemented with 0.5% L-methionine (HHcy, n=12) for 18+/-3 wk had plasma homocysteine concentrations of 8+/-1 and 41+/-1 microM, respectively (P<0.05), and similar liver folate (approximately 12+/-2 microg folate/g liver). Carotid arterial permeability, assessed as dextran accumulation using quantitative fluorescence microscopy, was greater in HHcy (3.95+/-0.4 ng.min-1.cm-2) versus Con (2.87+/-0.41 ng.min-1.cm-2) mice (P<0.05). Stress versus strain curves generated using an elastigraph indicated that 1) maximal stress (N/mm2), 2) physiological stiffness (low-strain Young's modulus, mN/mm), and 3) maximal stiffness (high-strain Young's modulus, N/mm) were higher (P<0.05) in aortas from HHcy versus Con mice. Thus, chronic HHcy increases arterial permeability and stiffness. Carotid arterial permeability also was assessed in age-matched C57BL/6J mice before and after incubation with 1) xanthine (0.4 mg/ml)/xanthine oxidase (0.2 mg/ml; X/XO) to generate superoxide anion (O2-) or 50 microM DL-homocysteine in the presence of 2) vehicle, 3) 300 microM diethylamine-NONOate (DEANO; a nitric oxide donor), or 4) 10(-3) M 4,5-dihydroxy-1,3-benzene disulfonic acid (tiron; a nonenzymatic intracellular O2- scavenger). Compared with preincubation values, X/XO and dl-homocysteine increased (P<0.05) permeability by 66+/-11% and 123+/-8%, respectively. DL-Homocysteine-induced increases in dextran accumulation were blunted (P<0.05) by simultaneous incubation with DEANO or tiron. Thus, acute HHcy increases arterial permeability by generating O2- to an extent whereby nitric oxide bioavailability is reduced.     

Mapping the ruthenium red-binding site of the voltage-dependent anion channel-1

We have previously shown that ruthenium red (RuR) binds to the voltage-dependent anion channel (VDAC) in the outer mitochondrial membrane, decreasing channel conductance and protecting against apoptotic cell death. In this report, we define the murine and yeast VDAC1 amino acid residues involved in the interaction with RuR. Binding of RuR to bilayer-reconstituted mVDAC1 and the resulting channel closure was inhibited upon mutation of specific VDAC1 residues. RuR protection against cell death, as induced by overexpression of native or mutated mVDAC1, was also diminished upon mutation of these amino acids. Moreover, RuR-mediated inhibition of cytochrome c release normally induced by staurosporine was not observed in cells expressing mutants VDAC1. We found that four glutamate residues, two each located in the first and third mVDAC1 cytosolic loops, are required for the interaction of VDAC1 with RuR and subsequent protection against cell death. Similar results were obtained with Q72E-yeast VDAC1, except that only three glutamate residues, located in two cytosolic loops were required. As a hexavalent reagent, RuR is expected to bind to more than one negatively charged group. Our results thus clearly indicate that RuR protects against cell death via a direct interaction with VDAC1 to inhibit cytochrome c release and subsequent cell death.     

DNA A-tracts bending: polarization effects on electrostatic interactions across their minor groove

Bending by the DNA A-tracts constitutes a contentious issue, suggesting deficiencies in the physics employed so far. Here, we inquire as to the importance in this bending of many-body polarization effects on the electrostatic interactions across their narrow minor groove. We have done this on the basis of the findings of Jarque and Buckingham who developed a procedure based on a Monte Carlo simulation for two charges of the same sign embedded in a polarizable medium. Remarkably, the present analysis reveals that for compact DNA conformations, which result from dynamic effects, an overall attractive interaction operates between the phosphate charges; this interaction is especially strong for the narrow minor groove of the A-tracts, suggesting a tendency for DNA to bend toward this groove. This tendency is in agreement with the conclusions of electrophoretic and NMR solution studies. The present analysis is also consistent with the experimental observations that the minor groove is much more easily compressible than the major groove and the bending propensity of the A-tracts is greatly reduced at “premelting” temperatures. By contrast, the dielectric screening model predicts a repulsion between the phosphate charges and is not consistent with the aforementioned bending tendency or experimental observations.     

Accounting for uncertainty in the tree topology has little effect on the decision-theoretic approach to model selection in phylogeny estimation

Currently available methods for model selection used in phylogenetic analysis are based on an initial fixed-tree topology. Once a model is picked based on this topology, a rigorous search of the tree space is run under that model to find the maximum-likelihood estimate of the tree (topology and branch lengths) and the maximum-likelihood estimates of the model parameters. In this paper, we propose two extensions to the decision-theoretic (DT) approach that relax the fixed-topology restriction. We also relax the fixed-topology restriction for the Bayesian information criterion (BIC) and the Akaike information criterion (AIC) methods. We compare the performance of the different methods (the relaxed, restricted, and the likelihood-ratio test [LRT]) using simulated data. This comparison is done by evaluating the relative complexity of the models resulting from each method and by comparing the performance of the chosen models in estimating the true tree. We also compare the methods relative to one another by measuring the closeness of the estimated trees corresponding to the different chosen models under these methods. We show that varying the topology does not have a major impact on model choice. We also show that the outcome of the two proposed extensions is identical and is comparable to that of the BIC, Extended-BIC, and DT. Hence, using the simpler methods in choosing a model for analyzing the data is more computationally feasible, with results comparable to the more computationally intensive methods. Another outcome of this study is that earlier conclusions about the DT approach are reinforced. That is, LRT, Extended-AIC, and AIC result in more complicated models that do not contribute to the performance of the phylogenetic inference, yet cause a significant increase in the time required for data analysis.     

Determinants of infant and young child feeding practices by mothers in two rural districts of Sindh,Pakistan: a cross-sectional survey

Background

Infant and young child feeding (IYCF) practices during the first two years of life are important for the growth and development of a child. The aim of this study was to assess IYCF practices and its associated factors in two rural districts of Pakistan.

Methods

A cross-sectional study was conducted in two rural districts of Sindh province, Pakistan as part of a stunting prevention project between May and August 2014. A standard questionnaire on IYCF practices recommended by World Health Organization was used to collect information from 2013 mothers who had a child aged between 0 and 23 months.

Results

Only 49% of mothers initiated breastfeeding within one hour of birth. Thirty-seven percent of mothers exclusively breastfed their infants for six months. Seventy-percent mothers introduced complementary feeding at 6–8 months of age. Eighty-two percent of mothers continued breastfeeding for at least one year and 75% for at least two years of age. IYCF practices were not significantly different for boys and girls in the study area. Being an employed mother (AOR 2.14; 95% CI 1.02, 4.51) was positively associated with the early initiation of breastfeeding. Children who were born at a health facility (AOR 0.65; 95% CI 0.50, 0.84) and were aged six to eleven months (AOR 0.70; 95% CI 0.54, 0.90) were less likely to be have an early initiation of breastfeeding. Mothers aged 25 to 29 years (AOR 1.83; 95% CI 1.05, 3.18), being literate (AOR 1.79; 95% CI 1.15, 2.78), and higher income (AOR 10.6; 95% CI 4.40, 25.30) were more likely to have an improved dietary diversity. Being an employed mother (AOR 2.18; 95% CI 1.77, 4.03) and higher income were more likely to have minimum acceptable diet (AOR 9.7; 95% CI 4.33, 21.71).

Conclusion

IYCF practices were below the acceptable level and associated with maternal age, maternal illiteracy, unemployment, and poor household wealth status. Emphasis should be given to improve maternal literacy and reduction in poverty to improve IYCF practices.