Glut1 and Glut3 as Potential Prognostic Markers for Oral Squamous Cell Carcinoma

We associated clinical-pathological features of 142 OSCC with the expression pattern of GLUT1 and GLUT3 in order to estimate their prognostic value. Methods: Clinical-pathological features and overall survival data of 142 patients with Oral Squamous Cell Carcinoma (OSCC) were retrospectively reviewed from A. C. Camargo hospital records. A tissue microarray (TMA) was built for the immunohistochemical (IHC) analysis of GLUT 1 and GLUT 3. IHC results were evaluated according to the staining pattern and number of positive cells. Results: GLUT 1 was over expressed in 50.3% of OSSC cases showing membrane staining pattern. However, nuclear expression was observed in 49.7% of the analyzed cases. GLUT 3 over expression was detected in 21.1% of OSCC cases. The pattern of GLUT 1 expression showed significant association with alcohol consumption (p = 0.004). Positive cell membrane GLUT 3 protein expression was associated with advanced clinic-staging of tumours (p = 0.005) as well as with vascular embolization (p = 0.005). Positive expression of GLUT 3 was associated with unfavorable free-disease survival (p = 0.021). Conclusion: GLUT1 and GLUT3 protein expression evaluated by immunohistochemistry are, significantly, indicators of poor prognosis outcome in oral squamous cell carcinoma, probably due to the enhanced glycolytic metabolism of more aggressive neoplastic cells.

New Source of Muscle-Derived Stem Cells with Potential for Alveolar Bone Reconstruction in Cleft Lip and/or Palate Patients

Cleft lip and palate (CLP), one of the most frequent congenital malformations, affects the alveolar bone in the great majority of the cases, and the reconstruction of this defect still represents a challenge in the rehabilitation of these patients. One of the current most promising strategy to achieve this goal is the use of bone marrow stem cells (BMSC); however, isolation of BMSC or iliac bone, which is still the mostly used graft in the surgical repair of these patients, confers site morbidity to the donor. Therefore, in order to identify a new alternative source of stem cells with osteogenic potential without conferring morbidity to the donor, we have used orbicular oris muscle (OOM) fragments, which are regularly discarded during surgery repair (cheiloplasty) of CLP patients. We obtained cells from OOM fragments of four unrelated CLP patients (CLPMDSC) using previously described preplating technique. These cells, through flow cytometry analysis, were mainly positively marked for five mesenchymal stem cell antigens (CD29, CD90, CD105, SH3, and SH4), while negative for hematopoietic cell markers, CD14, CD34, CD45, and CD117, and for endothelial cell marker, CD31. After induction under appropriate cell culture conditions, these cells were capable to undergo chondrogenic, adipogenic, osteogenic, and skeletal muscle cell differentiation, as evidenced by immunohistochemistry. We also demonstrated that these cells together with a collagen membrane lead to bone tissue reconstruction in a critical-size cranial defects previously induced in non-immunocompromised rats. The presence of human DNA in the new bone was confirmed by PCR with human-specific primers and immunohistochemistry with human nuclei antibodies. In conclusion, we showed that cells from OOM have phenotypic and behavior characteristics similar to other adult stem cells, both in vitro and in vivo. Our findings suggest that these cells represent a promising source of stem cells for alveolar bone grafting treatment, particularly in young CLP patients.

FAM5C Contributes to Aggressive Periodontitis

Aggressive periodontitis is characterized by a rapid and severe periodontal destruction in young systemically healthy subjects. A greater prevalence is reported in Africans and African descendent groups than in Caucasians and Hispanics. We first fine mapped the interval 1q24.2 to 1q31.3 suggested as containing an aggressive periodontitis locus. Three hundred and eighty-nine subjects from 55 pedigrees were studied. Saliva samples were collected from all subjects, and DNA was extracted. Twenty-one single nucleotide polymorphisms were selected and analyzed by standard polymerase chain reaction using TaqMan chemistry. Non-parametric linkage and transmission distortion analyses were performed. Although linkage results were negative, statistically significant association between two markers, rs1935881 and rs1342913, in the FAM5C gene and aggressive periodontitis (p = 0.03) was found. Haplotype analysis showed an association between aggressive periodontitis and the haplotype A-G (rs1935881-rs1342913; p = 0.009). Sequence analysis of FAM5C coding regions did not disclose any mutations, but two variants in conserved intronic regions of FAM5C, rs57694932 and rs10494634, were found. However, these two variants are not associated with aggressive periodontitis. Secondly, we investigated the pattern of FAM5C expression in aggressive periodontitis lesions and its possible correlations with inflammatory/immunological factors and pathogens commonly associated with periodontal diseases. FAM5C mRNA expression was significantly higher in diseased versus healthy sites, and was found to be correlated to the IL-1 beta, IL-17A, IL-4 and RANKL mRNA levels. No correlations were found between FAM5C levels and the presence and load of red complex periodontopathogens or Aggregatibacter actinomycetemcomitans. This study provides evidence that FAM5C contributes to aggressive periodontitis.

Prevalence of Metabolic Syndrome in Japanese-Brazilians According to Specific Definitions for Ethnicity

Background: The American Heart Association/National Heart, Lung, and Blood Institute (AHA/NHLBI), revising the National Cholesterol Evaluation Program for Adult Treatment Panel III (NCEP ATP III), and the International Diabetes Federation (IDF) have proposed definitions of metabolic syndrome that take into account waist circumference thresholds according to ethnicity. In this study we estimated the prevalence of metabolic syndrome in a Japanese-Brazilian population using NCEP definitions for Westerners (NCEPwe) and Asians (NCEPas), and IDF for Japanese (IDF). Methods: A total of 650 Japanese-Brazilians living in a developed Brazilian city and aged 30-88 years were included. Results: Metabolic syndrome prevalence according to NCEPwe, NCEPas, and IDF was, respectively, 46.5%, 56.5%, and 48.3%. Only 43.5% of subjects did not have metabolic syndrome by any of the 3 definitions, and 38.3% fulfilled metabolic syndrome criteria for all 3 definitions. Ten percent of subjects were positive for metabolic syndrome based on NCEPas and IDF, but not for NCEPwe. Because IDF requires abdominal obesity as a criterion, the frequency of subjects without metabolic syndrome according to IDF, but with metabolic syndrome by NCEPwe and NCEPas was 8.2%. Conclusions: Independent of the metabolic syndrome definition, Japanese-Brazilians present an elevated metabolic syndrome prevalence, which was higher when using NCEP criteria for Asians, followed by the IDF definition for Japanese.

Sequential analysis of global gene expression profiles in immature and in vitro matured bovine oocytes: potential molecular markers of oocyte maturation

Background: Without intensive selection, the majority of bovine oocytes submitted to in vitro embryo production (IVP) fail to develop to the blastocyst stage. This is attributed partly to their maturation status and competences. Using the Affymetrix GeneChip Bovine Genome Array, global mRNA expression analysis of immature (GV) and in vitro matured (IVM) bovine oocytes was carried out to characterize the transcriptome of bovine oocytes and then use a variety of approaches to determine whether the observed transcriptional changes during IVM was real or an artifact of the techniques used during analysis. Results: 8489 transcripts were detected across the two oocyte groups, of which similar to 25.0% (2117 transcripts) were differentially expressed (p < 0.001); corresponding to 589 over-expressed and 1528 under-expressed transcripts in the IVM oocytes compared to their immature counterparts. Over expression of transcripts by IVM oocytes is particularly interesting, therefore, a variety of approaches were employed to determine whether the observed transcriptional changes during IVM were real or an artifact of the techniques used during analysis, including the analysis of transcript abundance in oocytes in vitro matured in the presence of a-amanitin. Subsets of the differentially expressed genes were also validated by quantitative real-time PCR (qPCR) and the gene expression data was classified according to gene ontology and pathway enrichment. Numerous cell cycle linked (CDC2, CDK5, CDK8, HSPA2, MAPK14, TXNL4B), molecular transport (STX5, STX17, SEC22A, SEC22B), and differentiation (NACA) related genes were found to be among the several over-expressed transcripts in GV oocytes compared to the matured counterparts, while ANXA1, PLAU, STC1and LUM were among the over-expressed genes after oocyte maturation. Conclusion: Using sequential experiments, we have shown and confirmed transcriptional changes during oocyte maturation. This dataset provides a unique reference resource for studies concerned with the molecular mechanisms controlling oocyte meiotic maturation in cattle, addresses the existing conflicting issue of transcription during meiotic maturation and contributes to the global goal of improving assisted reproductive technology.

PRACTICAL USE OF MOLECULAR MARKERS IN BEEF AND DAIRY ZEBU CATTLE IN BRAZIL

Selection of zebu (Bos indicus) beef and dairy cattle in Brazil and the validation process of genetic markers for growth, carcass and meat quality traits and also for milk production, fat and protein milk content are discussed as concerned to the concepts and details of their use as auxiliary tools in selection processes. It is highlighted, also, the importance of right selection of ova donor cows for production of embryos to be transferred.

Random X Inactivation and Extensive Mosaicism in Human Placenta Revealed by Analysis of Allele-Specific Gene Expression along the X Chromosome

Imprinted inactivation of the paternal X chromosome in marsupials is the primordial mechanism of dosage compensation for X-linked genes between females and males in Therians. In Eutherian mammals, X chromosome inactivation (XCI) evolved into a random process in cells from the embryo proper, where either the maternal or paternal X can be inactivated. However, species like mouse and bovine maintained imprinted XCI exclusively in extraembryonic tissues. The existence of imprinted XCI in humans remains controversial, with studies based on the analyses of only one or two X-linked genes in different extraembryonic tissues. Here we readdress this issue in human term placenta by performing a robust analysis of allele-specific expression of 22 X-linked genes, including XIST, using 27 SNPs in transcribed regions. We show that XCI is random in human placenta, and that this organ is arranged in relatively large patches of cells with either maternal or paternal inactive X. In addition, this analysis indicated heterogeneous maintenance of gene silencing along the inactive X, which combined with the extensive mosaicism found in placenta, can explain the lack of agreement among previous studies. Our results illustrate the differences of XCI mechanism between humans and mice, and highlight the importance of addressing the issue of imprinted XCI in other species in order to understand the evolution of dosage compensation in placental mammals.

Phylogeography of the common vampire bat (Desmodus rotundus): Marked population structure, Neotropical Pleistocene vicariance and incongruence between nuclear and mtDNA markers

Background: The common vampire bat Desmodus rotundus is an excellent model organism for studying ecological vicariance in the Neotropics due to its broad geographic range and its preference for forested areas as roosting sites. With the objective of testing for Pleistocene ecological vicariance, we sequenced a mitocondrial DNA (mtDNA) marker and two nuclear markers (RAG2 and DRB) to try to understand how Pleistocene glaciations affected the distribution of intraspecific lineages in this bat. Results: Five reciprocally monophyletic clades were evident in the mitochondrial gene tree, and in most cases with high bootstrap support: Central America (CA), Amazon and Cerrado (AMC), Pantanal (PAN), Northern Atlantic Forest (NAF) and Southern Atlantic Forest (SAF). The Atlantic forest clades formed a monophyletic clade with high bootstrap support, creating an east/west division for this species in South America. On the one hand, all coalescent and non-coalescent estimates point to a Pleistocene time of divergence between the clades. On the other hand, the nuclear markers showed extensive sharing of haplotypes between distant localities, a result compatible with male-biased gene flow. In order to test if the disparity between the mitochondrial and nuclear markers was due to the difference in mutation rate and effective size, we performed a coalescent simulation to examine the feasibility that, given the time of separation between the observed lineages, even with a gene flow rate close to zero, there would not be reciprocal monophyly for a neutral nuclear marker. We used the observed values of theta and an estimated mutation rate for the nuclear marker gene to perform 1000 iterations of the simulation. The results of this simulation were inconclusive: the number of iterations with and without reciprocal monophyly of one or more clades are similar. Conclusions: We therefore conclude that the pattern exhibited by the common vampire bat, with marked geographical structure for a mitochondrial marker and no phylogeographic structure for nuclear markers is compatible with a historical scenario of complete isolation of refuge-like populations during the Pleistocene. The results on demographic history on this species is compatible with the Carnaval-Moritz model of Pleistocene vicariance, with demographic expansions in the southern Atlantic forest.

The constancy of global regulation across a species: the concentrations of ppGpp and RpoS are strain-specific in Escherichia coli

Background: Sigma factors and the alarmone ppGpp control the allocation of RNA polymerase to promoters under stressful conditions. Both ppGpp and the sigma factor sigma(S) (RpoS) are potentially subject to variability across the species Escherichia coli. To find out the extent of strain variation we measured the level of RpoS and ppGpp using 31 E. coli strains from the ECOR collection and one reference K-12 strain. Results: Nine ECORs had highly deleterious mutations in rpoS, 12 had RpoS protein up to 7-fold above that of the reference strain MG1655 and the remainder had comparable or lower levels. Strain variation was also evident in ppGpp accumulation under carbon starvation and spoT mutations were present in several low-ppGpp strains. Three relationships between RpoS and ppGpp levels were found: isolates with zero RpoS but various ppGpp levels, strains where RpoS levels were proportional to ppGpp and a third unexpected class in which RpoS was present but not proportional to ppGpp concentration. High-RpoS and high-ppGpp strains accumulated rpoS mutations under nutrient limitation, providing a source of polymorphisms. Conclusions: The ppGpp and sigma(S) variance means that the expression of genes involved in translation, stress and other traits affected by ppGpp and/or RpoS are likely to be strain-specific and suggest that influential components of regulatory networks are frequently reset by microevolution. Different strains of E. coli have different relationships between ppGpp and RpoS levels and only some exhibit a proportionality between increasing ppGpp and RpoS levels as demonstrated for E. coli K-12.

Leishmania-Specific Surface Antigens Show Sub-Genus Sequence Variation and Immune Recognition

Background: A family of hydrophilic acylated surface (HASP) proteins, containing extensive and variant amino acid repeats, is expressed at the plasma membrane in infective extracellular (metacyclic) and intracellular (amastigote) stages of Old World Leishmania species. While HASPs are antigenic in the host and can induce protective immune responses, the biological functions of these Leishmania-specific proteins remain unresolved. Previous genome analysis has suggested that parasites of the sub-genus Leishmania (Viannia) have lost HASP genes from their genomes. Methods/Principal Findings: We have used molecular and cellular methods to analyse HASP expression in New World Leishmania mexicana complex species and show that, unlike in L. major, these proteins are expressed predominantly following differentiation into amastigotes within macrophages. Further genome analysis has revealed that the L. (Viannia) species, L. (V.) braziliensis, does express HASP-like proteins of low amino acid similarity but with similar biochemical characteristics, from genes present on a region of chromosome 23 that is syntenic with the HASP/SHERP locus in Old World Leishmania species and the L. (L.) mexicana complex. A related gene is also present in Leptomonas seymouri and this may represent the ancestral copy of these Leishmania-genus specific sequences. The L. braziliensis HASP-like proteins (named the orthologous (o) HASPs) are predominantly expressed on the plasma membrane in amastigotes and are recognised by immune sera taken from 4 out of 6 leishmaniasis patients tested in an endemic region of Brazil. Analysis of the repetitive domains of the oHASPs has shown considerable genetic variation in parasite isolates taken from the same patients, suggesting that antigenic change may play a role in immune recognition of this protein family. Conclusions/Significance: These findings confirm that antigenic hydrophilic acylated proteins are expressed from genes in the same chromosomal region in species across the genus Leishmania. These proteins are surface-exposed on amastigotes (although L. (L.) major parasites also express HASPB on the metacyclic plasma membrane). The central repetitive domains of the HASPs are highly variant in their amino acid sequences, both within and between species, consistent with a role in immune recognition in the host.

Metal-insulator transition in Nd(1-x) Eu(x) NiO(3) probed by specific heat and anelastic measurements

Oxides RNiO(3) (R - rare-earth, R not equal La) exhibit a metal-insulator (MI) transition at a temperature T(MI) and an antiferromagnetic (AF) transition at T(N). Specific heat (C(P)) and anelastic spectroscopy measurements were performed in samples of Nd(1-x)Eu(x)NiO(3), 0 <= x <= 0.35. For x - 0, a peak in C(P) is observed upon cooling and warming at essentially the same temperature T(MI) - T(N) similar to 195 K, although the cooling peak is much smaller. For x >= 0.25, differences between the cooling and warming curves are negligible, and two well defined peaks are clearly observed: one at lower temperatures that define T(N), and the other one at T(MI). An external magnetic field of 9 T had no significant effect on these results. The elastic compliance (s) and the reciprocal of the mechanical quality factor (Q(-1)) of NdNiO(3), measured upon warming, showed a very sharp peak at essentially the same temperature obtained from C(P), and no peak is observed upon cooling. The elastic modulus hardens below T(MI) much more sharply upon warming, while the cooling and warming curves are reproducible above T(MI). Conversely, for the sample with x - 0.35, s and Q(-1) curves are very similar upon warming and cooling. The results presented here give credence to the proposition that the MI phase transition changes from first to second order with increasing Eu doping. (C) 2011 American Institute of Physics. [doi: 10.1063/1.3549615]

Identification of Novel Immunoregulatory Molecules in Human Thymic Regulatory CD4(+)CD25(+) T Cells by Phage Display

Thymic CD4(+)CD25(+) cells play an important role in immune regulation and are continuously developed in the thymus as an independent lineage. How these cells are generated, what are their multiple pathways of suppressive activity and which are their specific markers are questions that remain unanswered. To identify molecules involved in the function and development of human CD4(+)CD25(+) T regulatory cells we targeted thymic CD4(+)CD25(+) cells by peptide phage display. A phage library containing random peptides was screened ex vivo for binding to human thymic CD4(+)CD25(+) T cells. After four rounds of selection on CD4(+)CD25(+) enriched populations of thymocytes, we sequenced several phage displayed peptides and selected one with identity to the Vitamin D Receptor (VDR). We confirmed the binding of the VDR phage to active Vitamin D in vitro, as well as the higher expression of VDR in CD4(+)CD25(+) cells. We suggest that differential expression of VDR on natural Tregs may be related to the relevance of Vitamin D in function and ontogeny of these cells.

Sequence-specific electrochemical detection of Alicyclobacillus acidoterrestris DNA using electroconductive polymer-modified fluorine tin oxide electrodes

This study outlines the quantification of low levels of Alicyclobacillus acidoterrestris in pure cultures, since this bacterium is not inactivated by pasteurization and may remain in industrialized foods and beverages. Electroconductive polymer-modified fluorine tin oxide (FTO) electrodes and multiple nanoparticle labels were used for biosensing. The detection of A. acidoterrestris in pure cultures was performed by reverse transcription polymerase chain reaction (RT-PCR) and the sensitivity was further increased by asymmetric nested RT-PCR using electrochemical detection for quantification of the amplicon. The quantification of nested RT-PCR products by Ag/Au-based electrochemical detection was able to detect 2 colony forming units per mL (CFU mL(-1)) of spores in pure culture and low detection and quantification limits (7.07 and 23.6 nM, respectively) were obtained for the target A. acidoterrestris on the electrochemical detection bioassay.

Evaluating Theobroma grandiflorum for comparative genomic studies with Theobroma cacao

The seeds of Theobroma cacao (cacao) are the source of cocoa, the raw material for the multi-billion dollar chocolate industry. Cacao`s two most important traits are its unique seed storage triglyceride (cocoa butter) and the flavor of its fermented beans (chocolate). The genome of T. cacao is being sequenced, and to expand the utility of the genome sequence to the improvement of cacao, we are evaluating Theobroma grandiflorum, the closest economically important species of Theobroma for its potential use in a comparative genomic study. T. grandiflorum differs from cacao in important agronomic traits such as flavor of the fermented beans, disease resistance to witches` broom and abscission of mature fruits. By comparing genomic sequences and analyzing viable inter-specific hybrids, we hope to identify the key genes that regulate cacao`s most important traits. We have investigated the utility in T. grandiflorum of three types of markers (microsatellite markers, single-strand conformational polymorphism markers and single nucleotide polymorphism (SNP) markers) developed in cacao. Through sequencing of amplicons of 12 diverse individuals of both cacao and T. grandiflorum, we have identified new intra- and inter-specific SNPs. Two markers which had no overlap of alleles between the species were used to genotype putative inter-specific hybrid seedlings. Sequence conservation was significant and species-specific differences numerous enough to suggest that comparative genomics of T. grandiflorum and T. cacao will be useful in elucidating the genetic differences that lead to a variety of important agronomic trait differences.

Disparity between Multilocus Enzyme Electrophoresis, Microsatellite Markers and Pulsed-Field Gel Electrophoresis in epidemiological tracking of Candida albicans

Various molecular systems are available for epidemiological, genetic, evolutionary, taxonomic and systematic studies of innumerable fungal infections, especially those caused by the opportunistic pathogen C. albicans. A total of 75 independent oral isolates were selected in order to compare Multilocus Enzyme Electrophoresis (MLEE), Electrophoretic Karyotyping (EK) and Microsatellite Markers (Simple Sequence Repeats - SSRs), in their abilities to differentiate and group C. albicans isolates (discriminatory power), and also, to evaluate the concordance and similarity of the groups of strains determined by cluster analysis for each fingerprinting method. Isoenzyme typing was performed using eleven enzyme systems: Adh, Sdh, M1p, Mdh, Idh, Gdh, G6pdh, Asd, Cat, Po, and Lap (data previously published). The EK method consisted of chromosomal DNA separation by pulsed-field gel electrophoresis using a CHEF system. The microsatellite markers were investigated by PCR using three polymorphic loci: EF3, CDC3, and HIS3. Dendrograms were generated by the SAHN method and UPGMA algorithm based on similarity matrices (S(SM)). The discriminatory power of the three methods was over 95%, however a paired analysis among them showed a parity of 19.7-22.4% in the identification of strains. Weak correlation was also observed among the genetic similarity matrices (S(SM)(MLEE) x S(SM)(EK) x S(SM)(SSRs)). Clustering analyses showed a mean of 9 +/- 12.4 isolates per cluster (3.8 +/- 8 isolates/taxon) for MLEE, 6.2 +/- 4.9 isolates per cluster (4 +/- 4.5 isolates/taxon) for SSRs, and 4.1 +/- 2.3 isolates per cluster (2.6 +/- 2.3 isolates/taxon) for EK. A total of 45 (13%), 39(11.2%), 5 (1.4%) and 3 (0.9%) clusters pairs from 347 showed similarity (Si) of 0.1-10%, 10.1-20%, 20.1-30% and 30.1-40%, respectively. Clinical and molecular epidemiological correlation involving the opportunistic pathogen C. albicans may be attributed dependently of each method of genotyping (i.e., MLEE, EK, and SSRs) supplemented with similarity and grouping analysis. Therefore, the use of genotyping systems that give results which offer minimum disparity, or the combination of the results of these systems, can provide greater security and consistency in the determination of strains and their genetic relationships. (C) 2010 Elsevier B.V. All rights reserved.