How Thermophilic Gram-Positive Organisms Perform Extracellular Electron Transfer: Characterization of the Cell Surface Terminal Reductase OcwA.

Extracellular electron transfer is the key process underpinning the development of bioelectrochemical systems for the production of energy or added-value compounds. Thermincola potens JR is a promising Gram-positive bacterium to be used in these systems because it is thermophilic. In this paper, we describe the structural and functional properties of the nonaheme cytochrome OcwA, which is the terminal reductase of this organism. The structure of OcwA, determined at 2.2-Å resolution, shows that the overall fold and organization of the hemes are not related to other metal reductases and instead are similar to those of multiheme cytochromes involved in the biogeochemical cycles of nitrogen and sulfur. We show that, in addition to solid electron acceptors, OcwA can also reduce soluble electron shuttles and oxyanions. These data reveal that OcwA can work as a multipurpose respiratory enzyme allowing this organism to grow in environments with rapidly changing availability of terminal electron acceptors without the need for transcriptional regulation and protein synthesis.IMPORTANCE Thermophilic Gram-positive organisms were recently shown to be a promising class of organisms to be used in bioelectrochemical systems for the production of electrical energy. These organisms present a thick peptidoglycan layer that was thought to preclude them to perform extracellular electron transfer (i.e., exchange catabolic electrons with solid electron acceptors outside the cell). In this paper, we describe the structure and functional mechanisms of the multiheme cytochrome OcwA, the terminal reductase of the Gram-positive bacterium Thermincola potens JR found at the cell surface of this organism. The results presented here show that this protein can take the role of a respiratory "Swiss Army knife," allowing this organism to grow in environments with soluble and insoluble substrates. Moreover, it is shown that it is unrelated to terminal reductases found at the cell surface of other electroactive organisms. Instead, OcwA is similar to terminal reductases of soluble electron acceptors. Our data reveal that terminal oxidoreductases of soluble and insoluble substrates are evolutionarily related, providing novel insights into the evolutionary pathway of multiheme cytochromes.

Heterologous expression and purification of a multiheme cytochrome from a Gram-positive bacterium capable of performing extracellular respiration.

Microbial electrochemical technologies are emerging as environmentally friendly biotechnological processes. Recently, a thermophilic Gram-positive bacterium capable of electricity production in a microbial fuel cell was isolated. Thermincola potens JR contains several multiheme c-type cytochromes that were implicated in the process of electricity production. In order to understand the molecular basis by which Gram-positive bacteria perform extracellular electron transfer, the relevant proteins need to be characterized in detail. Towards this end, a chimeric gene containing the signal peptide from Shewanella oneidensis MR-1 small tetraheme cytochrome c (STC) and the gene sequence of the target protein TherJR_0333 was constructed. This manuscript reports the successful expression of this chimeric gene in the Gram-negative bacterium Escherichia coli and its subsequent purification and characterization. This methodology opens the possibility to study other multiheme cytochromes from Gram-positive bacteria, allowing the extracellular electron transfer mechanisms of this class of organisms to be unraveled.

Differential infiltration of CD8+ and NK cells in lip and oral cavity squamous cell carcinoma.

BACKGROUND: CD8+ and natural killer (NK) cells have been considered the most effective cells in the combat of cancer, contributing to better prognosis and longer survival. METHODS: The aim of this study was to evaluate the population of CD8+ and NK cells, by immunohistochemistry, in samples of oral cavity squamous cell carcinoma (OCSCC) and lip squamous cell carcinoma (LSCC), leukoplakia, actinic cheilitis, and healthy oral mucosa (control). The relationship of CD8+ and NK cells with survival data, lymph node metastasis, tumor size, and proliferative index was also evaluated. RESULTS: The number of peritumoral and intratumoral CD8+ and NK cells was significantly higher in LSCC, when compared with control, pre-malignant lesions, and OCSCC. A higher proportion of peritumoral CD8+ cells demonstrated correlation with a lower neoplastic proliferative index. Moreover, patients with OCSCC with a high density of peritumoral CD8+ cells showed a tendency towards a longer survival time. CONCLUSIONS: The differential CD8+ and NK cells infiltration in oral SCC might reflect a distinctive tumor microenvironment with a favorable local cytotoxic immune response against neoplastic cells.

Family-based association tests for qualitative and quantitative traits using single-nucleotide polymorphism and microsatellite data.

Using the Genetic Analysis Workshop 12 simulated data, we contrasted results for association tests in nuclear families and extended pedigrees using single-nucleotide polymorphism (SNP) data, and we compared results for different trait definitions, for outbred and isolate populations, and for SNP and microsatellite data. SNPs in major genes 1 and 6 were analyzed using transmission disequilibrium testing (TDT) [Spielman et al., Am J Hum Genet 52:506-16, 1993], sibship disequilibrium testing (SDT) [Horvath and Laird, Am J Hum Genet 63:1886-97, 1998], family-based association testing (FBAT) [Horvath et al., Eur J Hum Genet 9:301-6, 2001], and a chi-square analysis of founders. TDT and SDT were applied in a sample of independent nuclear families, while FBAT was applied in extended pedigrees. SNPs and microsatellites were analyzed with dichotomous and quantitative trait definitions using FBAT in the isolate and outbred populations. The results of the TDT, SDT, and FBAT analyses are comparable using SNP data to identify the disease gene. However, these tests of association were not helpful in discriminating between functional and non-functional SNPs in disequilibrium. SNP data were able to identify association with affection status in a gene that influences the liability directly (MG6), but did not perform as well when assessing association with affection status in a gene that influences the outcome only through a quantitative trait (MG1). Association with MG1 was observed using the SNP data when the outcome was defined quantitatively. Microsatellite data were relatively unsuccessful in identifying association with the markers in the region of a major gene. The magnitude of the associations between SNPs and the dichotomous or quantitative trait definitions were similar in the outbred and isolated populations.

Influence of marker heterozygosity and genetic heterogeneity on fine mapping.

The purpose of the current study was to utilize the Genetic Analysis Workshop 12 simulated data to evaluate fine-mapping strategies for quantitative traits. We approached the analysis as if it was a follow-up to a genome scan that had identified two regions of interest and used the provided 1-cM density microsatellite typing data to mimic fine mapping of these regions. As these investigators knew the true locations of the putative genes under study, we explored the effects of the informativeness of microsatellite markers (marker heterozygosity) and the effects of genetic heterogeneity across families for ten replicates of the data. These results shed a cautionary light on the reliability of fine-mapping efforts on refining mapping locations as the position and the strength of the lod score can be markedly affected by the sampling of the population, the amount of variation accounted for by the gene, and the informativeness of the marker. Our studies did not reveal a large effect of unlinked families on the shape of the lod score peak.

A scanning electron microscopy study of changes in rat colonic mucosa during carcinogenesis.

Scanning electron microscopy (SEM) has been used to examine morphological changes on the colonic mucosal surface during experimental carcinogenesis. Seventy-five rats were given weekly injections of dimethylhydrazine (DMH) and 25 rats acted as controls. Groups of DMH-treated and control rats were sacrificed at regular intervals and specimens of their colons were examined by SEM. The normal colonic mucosa had an orderly architectural pattern which remained constant throughout the experiment. Abnormalities in the mucosa of treated rats were detected as early as 6 weeks after the first injection of DMH and thereafter there was progressive distortion of the crypts, development of pericryptal fissures and mucosal surface irregularities, and eventually, formation of focal protuberances, polyps, and tumors. The findings indicate that there is a definite pattern of progressive mucosal abnormalities commencing long before the development of focal colon tumors and that SEM is useful in the detection of these abnormalities.

A scanning electron microscopy study of changes in the colonic mucus layer during chemical carcinogenesis.

Scanning electron microscopy was used to examine the appearances of the colonic mucus layer of rats during chemical carcinogenesis with dimethylhydrazine. The normal colonic mucus layer had a dense homogeneous appearance and provided a complete cover for the mucosal epithelium. At high magnifications tiny fenestrations could be seen in this mucus layer. During carcinogenesis these fenestrations enlarged, increased in number, and coalesced, causing focal defects in the mucus layer, which eventually broke into strands and clumps of mucus. The findings indicate that the colonic mucus layer develops progressive abnormalities during carcinogenesis which result in breakdown of its integrity and exposure of the mucosal epithelium to colonic contents.

A scanning electron microscopy study of ultrastructural changes in the colonic mucosa of patients with large bowel tumours.

Scanning electron microscopy (SEM) has been used to compare the appearances of the colonic mucosal surface and overlying mucus layer of patients with normal colons, adenomatous polyps and colorectal carcinoma. The normal colonic mucosa had a characteristic orderly arrangement of crypts and was completely covered by an intact mucus layer. Epithelial and cryptal irregularities were seen on the surface of both polyps and carcinomas, and, in addition, the mucus layer was fragmented, leaving areas of the underlying epithelium exposed. These changes were more marked in colorectal cancers than in polyps and were present to a lesser degree in the apparently normal mucosa adjacent to tumours. The findings indicate that SEM can detect minor subtle irregularities on the surface of the colon and so may be useful in detecting pre-neoplastic and early neoplastic changes.