Identification and Characterization of the Major Porin of Desulfovibrio vulgaris Hildenborough.

Due in large part to their ability to facilitate the diffusion of a diverse range of solutes across the outer membrane (OM) of Gram-negative bacteria, the porins represent one of the most prominent and important bacterial membrane protein superfamilies. Notably, for the Gram-negative bacterium Desulfovibrio vulgaris Hildenborough, a model organism for studies of sulfate-reducing bacteria, no genes for porins have been identified or proposed in its annotated genome. Results from initial biochemical studies suggested that the product of the DVU0799 gene, which is one of the most abundant proteins of the D. vulgaris Hildenborough OM and purified as a homotrimeric complex, was a strong porin candidate. To investigate this possibility, this protein was further characterized biochemically and biophysically. Structural analyses via electron microscopy of negatively stained protein identified trimeric particles with stain-filled depressions and structural modeling suggested a ß-barrel structure for the monomer, motifs common among the known porins. Functional studies were performed in which crude OM preparations or purified DVU0799 was reconstituted into proteoliposomes and the proteoliposomes were examined for permeability against a series of test solutes. The results obtained establish DVU0799 to be a pore-forming protein with permeability properties similar to those observed for classical bacterial porins, such as those of Escherichia coli Taken together, these findings identify this highly abundant OM protein to be the major porin of D. vulgaris Hildenborough. Classification of DVU0799 in this model organism expands the database of functionally characterized porins and may also extend the range over which sequence analysis strategies can be used to identify porins in other bacterial genomes.IMPORTANCE Porins are membrane proteins that form transmembrane pores for the passive transport of small molecules across the outer membranes of Gram-negative bacteria. The present study identified and characterized the major porin of the model sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough, observing its preference for anionic sugars over neutral ones. Its predicted architecture appears to be novel for a classical porin, as its core ß-barrel structure is of a type typically found in solute-specific channels. Broader use of the methods employed here, such as assays for channel permeability and electron microscopy of purified samples, is expected to help expand the database of confirmed porin sequences and improve the range over which sequence analysis-based strategies can be used to identify porins in other Gram-negative bacteria. Functional characterization of these critical gatekeeping proteins from divergent Desulfovibrio species should offer an improved understanding of the physiological features that determine their habitat range and supporting activities.

High resolution mapping of Twist to DNA in Drosophila embryos: Efficient functional analysis and evolutionary conservation.

Cis-regulatory modules (CRMs) function by binding sequence specific transcription factors, but the relationship between in vivo physical binding and the regulatory capacity of factor-bound DNA elements remains uncertain. We investigate this relationship for the well-studied Twist factor in Drosophila melanogaster embryos by analyzing genome-wide factor occupancy and testing the functional significance of Twist occupied regions and motifs within regions. Twist ChIP-seq data efficiently identified previously studied Twist-dependent CRMs and robustly predicted new CRM activity in transgenesis, with newly identified Twist-occupied regions supporting diverse spatiotemporal patterns (>74% positive, n = 31). Some, but not all, candidate CRMs require Twist for proper expression in the embryo. The Twist motifs most favored in genome ChIP data (in vivo) differed from those most favored by Systematic Evolution of Ligands by EXponential enrichment (SELEX) (in vitro). Furthermore, the majority of ChIP-seq signals could be parsimoniously explained by a CABVTG motif located within 50 bp of the ChIP summit and, of these, CACATG was most prevalent. Mutagenesis experiments demonstrated that different Twist E-box motif types are not fully interchangeable, suggesting that the ChIP-derived consensus (CABVTG) includes sites having distinct regulatory outputs. Further analysis of position, frequency of occurrence, and sequence conservation revealed significant enrichment and conservation of CABVTG E-box motifs near Twist ChIP-seq signal summits, preferential conservation of ±150 bp surrounding Twist occupied summits, and enrichment of GA- and CA-repeat sequences near Twist occupied summits. Our results show that high resolution in vivo occupancy data can be used to drive efficient discovery and dissection of global and local cis-regulatory logic.

High-throughput isolation and characterization of untagged membrane protein complexes: outer membrane complexes of Desulfovibrio vulgaris.

Cell membranes represent the "front line" of cellular defense and the interface between a cell and its environment. To determine the range of proteins and protein complexes that are present in the cell membranes of a target organism, we have utilized a "tagless" process for the system-wide isolation and identification of native membrane protein complexes. As an initial subject for study, we have chosen the Gram-negative sulfate-reducing bacterium Desulfovibrio vulgaris. With this tagless methodology, we have identified about two-thirds of the outer membrane- associated proteins anticipated. Approximately three-fourths of these appear to form homomeric complexes. Statistical and machine-learning methods used to analyze data compiled over multiple experiments revealed networks of additional protein-protein interactions providing insight into heteromeric contacts made between proteins across this region of the cell. Taken together, these results establish a D. vulgaris outer membrane protein data set that will be essential for the detection and characterization of environment-driven changes in the outer membrane proteome and in the modeling of stress response pathways. The workflow utilized here should be effective for the global characterization of membrane protein complexes in a wide range of organisms.

Exploring Teacher Job Satisfaction in Rural China: Prevalence and Correlates.

Extant research continues to establish the importance of teacher job satisfaction to student performance, yet teacher job satisfaction remains under-investigated in rural China. In this paper, we examine the prevalence and correlates of teacher job satisfaction. Using data from 634 teachers across 120 schools in rural China, we find an alarmingly high prevalence of teacher job dissatisfaction: roughly 21% of rural teachers were less than satisfied with their jobs. In addition, we find that several individual- and school-level characteristics, including being a male teacher, being a homeroom teacher, not having a management role in school, being a middle-aged teacher, and a school's boarding status, are correlated with teacher job dissatisfaction. In sum, the results demonstrate a need for further research and policy interventions to improve teacher job satisfaction in rural schools.

Validation of two clinical color measuring instruments for use in dental research.

OBJECTIVE: There are several shade matching instruments developed for clinical use, but the validity of their use in dental research has not been thoroughly investigated. The objective of this study is to evaluate the accuracy of using two clinical color measuring instruments, VitaEasyshade and Spectroshade, against a referent laboratory color measuring instrument (Spectroradiometer PR670). METHODS AND MATERIALS: The validity and repeatability of the referent laboratory color measuring instrument was assessed using standard color patches with certified CIE L*a*b* values. 10% of the 240 color patches were randomly selected and measured ten times in a random order to test for repeatability. 16 metal ceramic specimens, fabricated from base metal alloy veneered with porcelain of different Vita Classic shades, were measured for L*a*b* values using the PR670, Vita EasyShade and the Spectroshade. The CIE L*a*b* values obtained from the three color measuring instruments were compared using repeated measures ANOVA and post hoc using the Bonferroni test. The color difference, CIEDE2000 (∆E00), between the gold standard (PR670) and the two color measuring instruments were also determined and compared against the known perceptible color difference. RESULTS: L*a*b* values varied significantly between the three instruments. When comparing the mean ΔE00, the differences in values between PR670 and Spectroshade were not clinically significantly. However, the mean ΔE00 values between PR670 and Vita Easyshade were both clinically and statistically significant. CONCLUSION: The lack of clinical significance in values obtained from the SpectroShade when compared to those obtained by the PR670 suggests that the SpectroShade may be recommended for use in dental color research. CLINICAL SIGNIFICANCE: The SpectroShade instrument and PR670 spectroradiometer provided values that lacked clinical significance, suggesting that its use may be highly beneficial for clinical shade matching as well as color research.

Developmental roles of 21 Drosophila transcription factors are determined by quantitative differences in binding to an overlapping set of thousands of genomic regions.

BACKGROUND: We previously established that six sequence-specific transcription factors that initiate anterior/posterior patterning in Drosophila bind to overlapping sets of thousands of genomic regions in blastoderm embryos. While regions bound at high levels include known and probable functional targets, more poorly bound regions are preferentially associated with housekeeping genes and/or genes not transcribed in the blastoderm, and are frequently found in protein coding sequences or in less conserved non-coding DNA, suggesting that many are likely non-functional. RESULTS: Here we show that an additional 15 transcription factors that regulate other aspects of embryo patterning show a similar quantitative continuum of function and binding to thousands of genomic regions in vivo. Collectively, the 21 regulators show a surprisingly high overlap in the regions they bind given that they belong to 11 DNA binding domain families, specify distinct developmental fates, and can act via different cis-regulatory modules. We demonstrate, however, that quantitative differences in relative levels of binding to shared targets correlate with the known biological and transcriptional regulatory specificities of these factors. CONCLUSIONS: It is likely that the overlap in binding of biochemically and functionally unrelated transcription factors arises from the high concentrations of these proteins in nuclei, which, coupled with their broad DNA binding specificities, directs them to regions of open chromatin. We suggest that most animal transcription factors will be found to show a similar broad overlapping pattern of binding in vivo, with specificity achieved by modulating the amount, rather than the identity, of bound factor.