Bacteriophage SPP1 portal protein is a large cyclical homo-oligomer composed of 13 subunits. The solution structure and assembly behavior of this protein with high-point rotational symmetry was characterized. The purified protein was present as a monodisperse population of 13-mers, named gp6H, at univalent salt concentrations in the hundred millimolar range >>/= 250 mM NaCl) or in the presence of bivalent cations in the millimolar range >>/= 5 mM MgCl2). Gp6H had a slightly higher sedimentation coefficient, a smaller shape-dependent frictional ratio, and a higher rate of intersubunit cross-linking in the presence of magnesium than in its absence. In the absence of bivalent cations and at univalent salt concentrations below 250 mM, the 13-mer molecules dissociated partially into stable monomers, named gp6L. The monomer had a somewhat different shape from the subunit present in the 13-mer, but maintained a defined tertiary structure. The association-dissociation equilibrium was mainly between the monomer and the 13-mer with a minor population of intermediate oligomers. Their interconversion was strongly influenced by the ionic environment. Under physiological conditions, the concentration of Mg2+ found in the Bacillus subtilis cytoplasm (10-50 mM) probably promotes complete association of gp6 into 13-mer rings with a compact conformation.

Previous studies have shown that bone marrow beta 2m(-)/Thy-1+ hepatic stem cells (BMHSCs) were able to engraft in vivo and differentiate into functioning hepatocytes in vitro. Our transcriptomic profiling on BMHSCs derived from rats subjected to common bile duct ligation (CBDL) demonstrated CBDL-derived beta 2m(-)/Thy-1+ BMHSCs expressed hepatocyte-like genes and shared more commonly expressed genes with hepatocytes, suggesting that an "on-site" priming of BMHSCs into hepatocyte lineage was initiated under the condition of CBDL. In this paper, transcriptomic profiling was carried out on livers from rats with CBDL to identify candidate factors released from cholestatic livers possibly involved in the priming of BMHSCs using Affymetrix Rat Genome U34A arrays. In CBDL rat livers, 1,091 probe sets were differentially expressed, of which 188 up-regulated probe sets were annotated as "extracellular" components. Gene ontology analysis showed many up-regulated genes belonged to cytokines, chemokines and growth factors, including Il1b, Il18, Ptn, Spp1, Grn, Ccl2, Cxcl1, Pf4, Tgfb, and Tgfb3. Cell differentiation and proliferation regulation factors such as Dmbt1, Efna1, Lgals1, Lep, Pmp2, and Gas6 were also induced in CBDL livers. Furthermore, many proteolysis and peptidolysis genes such as Mmp2, Mmp12, Mmp14, and Mmp23 were up-regulated in CBDL livers. Gene expression profiling showed that many cytokine-, chemokine-, growth factor- as well as certain extracellular protein-related genes were induced in CBDL livers, suggesting that these genes may be involved in hepatic BMHSCs priming.

Vascular disruption that occurs as a consequence of bone fracture, leads to hypoxia at the site of damage. Hypoxia regulates the expression of a number of genes that can modulate energy conservation, cell survival, tissue regeneration and angiogenesis. In this study we investigated the expression of Angiopoietin-like 4, an adipocytokine that has additional roles in angiogenesis, at the fracture site. We demonstrate that Angptl4 mRNA expression increased early during fracture healing (day 3) returning close to baseline at day14. In the callus, Angptl4 mRNA was visualized in areas of condensing mesenchymal cells, callus cartilage and was especially high in mineralizing osteoblasts located in areas of new bone formation. In vitro, Angptl4 mRNA expression in osteoblasts increased under hypoxic conditions and in cells treated with the hypoxia mimetic desferrioxamine. Angptl4 levels were strongly induced at day 14 in differentiating MC3T3-E1 osteoblastic cells. Exogenous ANGPTL4 increased expression of Runx2, Spp1, vegfa, and Alp mRNA in differentiating osteoblasts. We suggest that the distribution of Angptl4 in the callus may be driven by hypoxia and that Angptl4 may play a role in osteoblastic differentiation, and possibly angiogenesis via regulation of VEGF. Further studies could reveal a dual role for Angptl4 in angiogenesis and osteogenesis.

One of the major cardiovascular risk factor which predisposes to and accelerates atherosclerosis is arterial hypertension (AH). To determine the molecular basis of the crosslink between AH and atherosclerosis for the development of new treatment strategies large-scale transcriptome analysis of the cells implicated in atherogenesis is needed. We used cDNA microarray technique for simultaneous analysis of gene expression in human abdominal aorta normal sites and atherosclerotic lesions of different histological types, as well as in peripheral blood leukocytes from patients with essential hypertension (EH) and donors. The microarray data were verified by quantitative RT-PCR (reverse transcription coupled with polymerase chain reaction) and immunohistochemical analysis. Differential expression of 40 genes has been found, among which twenty two genes demonstrated up-regulation and 18 genes demonstrated down-regulation in atherosclerotic aorta compared with normal vessel. New gene-candidates, implicated in atherogenesis, have been identified - FPRL2, CD37, CD53, RGS1, LCP1, SPI1, CTSA, EPAS1, FHL1, GEM, RHOB, SPARCL1, ITGA8, PLN, and COL14A1. These genes participate in cell migration and adhesion, phenotypic changes of smooth muscle cells, immune and inflammatory reactions, oxidative processes and extracellular matrix remodeling. We have found increased expression levels of CD53, SPI1, FPRL2, SPP1, CTSD, ACP5, LCP1, CTSA and LIPA genes in peripheral blood leukocytes from EH patients and in atherosclerotic lesions of human aorta. The majority of these genes significantly (p<0.005) positively (r>0.5) correlated with AH stage as well as with histological grading of atherosclerotic lesions.

Surface modifications of titanium alloys are useful methods to enhance the biological stability of intraosseous implants and to promote a well succeeded osseointegration in the early stages of implantation. This work aims to investigate the influence of chemically modified surfaces of Ti-6Al-4V-ELI (extra-low interstitial) on the gene expression of human osteoblastic (HOb) cells. The surface treatments by acid etching or acid etching plus alkaline treatment were carried out to modify the topography, effective area, contact angle and chemical composition of the samples. The surface morphology was investigated using: scanning electron microscopy (SEM) and confocal laser-scanning microscope (CLSM). Roughness measurements and effective surface area were obtained using the CLSM. Surface composition was analysed by energy dispersive X-ray spectroscopy (EDX) and by X-Ray Diffraction (XRD). The expression levels of some bone related genes (ALPL, COL1A1, COL3A1, SPP1, RUNX2, and SPARC) were analysed using real-time Reverse Transcription Polymerase Chain Reaction (real-time RT-PCR). The results showed that all the chemical modifications studied in this work influenced the surface morphology, wettability, roughness, effective area and gene expression of human osteoblasts. Acid phosphoric combined to alkaline treatment presented a more accelerated gene expression after 7days while the only phosphoric etching or chloride etching combined to alkaline treatment presented more effective responses after 15days.

Osteoporosis is a common complex disorder with reduced bone mineral density (BMD) and increased susceptibility to fracture. Peak BMD is one of the primary determinants of osteoporotic fracture risk, and is under substantial genetic control. Extracellular matrix, a major component of the bone, influences BMD by regulating mineral deposition and maintaining cellular activity. It contains several SIBLING family proteins, null mutations of which cause mineralization defects in humans. In this study, we tested 59 single-nucleotide polymorphisms (SNPs) located in the 5 SIBLING family genes (DSPP, DMP1, IBSP, MEPE and SPP1) for association with normal variation in peak BMD in healthy men and women. We measured femoral neck (FN) and lumbar spine (LS) areal BMD by dual energy x-ray absorptiometry (DXA) in 1692 premenopausal European-American women, 512 premenopausal African-American women and 715 European-American men. SNPs were tested for association with FN and LS-BMD in the 3 subsamples. In the European-American women, we observed association (p≤0.005) with LS-BMD for SNPs in DSPP, IBSP and MEPE, and for FN-BMD with SNPs in DMP1 and IBSP. Allele-specific regulation of gene expression (ASE) is an important mechanism in which an allele giving rise to modest influence in transcript abundance might result in a predisposition to disease. To identify whether there was ASE of SIBLING family genes at these SNPs, we examined 52 human bone samples obtained from the femoral neck during surgical hip replacement (27 female, 25 male; 44 European-American and 8 African-American). We observed unidirectional ASE for the IBSP gene, with lower expression of the G allele compared to the A allele for SNP rs17013181. Our data suggest that SNPs within the SIBLING genes may contribute to normal variation of peak BMD. Further studies are necessary to identify the functional variants and to determine the mechanisms underlying the differences in ASE and how these differences relate to the pathophysiology of osteoporosis.

The B. subtilis bacteriophage SPP1 terminase, encoded by genes 1 and 2, is required for the initiation of headful packaging. The DNA segment to which gene 1 product (G/P) binds includes the pacL and pacR sites and the late PL1 and PL2 promoters from which genes 1 to 7 are transcribed. When SPP1wt or SPP1sus115 (gene 6-) phages were used to infect a B. subtilis sup0 strain, the gene 1 to 7 mRNA synthesis was reduced at late times of infection. This was not observed, however, when either chloramphenicol was added 7 min after infection with SPP1wt or when SPP1sus114 (gene 1-) or SPP1sus19 (gene 2-) were used to infect B. subtilis sup0 cells. These results suggest that the terminase enzyme functions as a repressor of its own transcription. G/P and B. subtilis RNA polymerase (RP) bind to the pacL segment, which contains the PL1 and PL2 promoter region. The binding of G/P to the pacL site does not seem to exclude RP from the promoters, despite of the overlapping of their binding sites. It is likely that the terminase protein does not repress transcription by a mere steric hindrance of RP binding.

Egg formation and embryonic development occur as the yolk passes through the magnum, isthmus, and shell gland of the oviduct before oviposition in hens. The present study identified candidate genes associated with secretory function of the chicken oviduct after ovulation and contributing to egg formation and oviposition. Hens (n = 5 per time point) were euthanized to recover the reproductive tract when the egg was in the magnum (3 h after ovulation) and the shell gland (20 h after ovulation). Total RNA was extracted from each segment of the oviducts and subjected to Affymetrix chicken GeneChip analysis. Quantitative PCR and in situ hybridization analyses of selected genes confirmed the validity of the gene expression patterns detected using microarray analysis. In particular, ACP1, CALB1, CYP26A1, PENK, RCAN1 and SPP1 expression increased significantly in the shell gland between 3 h and 20 h postovulation, whereas only RCNA1 expression increased significantly in the magnum between 3 h and 20 h postovulation. Results of the high-throughput analysis revealed cell-specific and temporal changes in gene expression in the oviduct at 3 h and 20 h postovulation in laying hens provide novel insight into changes at the molecular and cellular levels of candidate genes related to formation of the egg and oviposition.

Aberrant modifications of proteins occur during disease development and elicit disease-specific antibody responses. We have developed a protein array platform that enables the modification of many proteins in parallel and assesses their immunogenicity without the need to express, purify, and modify proteins individually. We used anticitrullinated protein antibodies (ACPAs) in rheumatoid arthritis (RA) as a model modification and profiled antibody responses to ∼190 citrullinated proteins in 20 RA patients. We observed unique antibody reactivity patterns in both clinical anticyclic citrullinated peptide assay positive (CCP+) and CCP- RA patients. At individual antigen levels, we detected antibodies against known citrullinated autoantigens and discovered and validated five novel antibodies against specific citrullinated antigens (osteopontin (SPP1), flap endonuclease (FEN1), insulin like growth factor binding protein 6 (IGFBP6), insulin like growth factor I (IGF1) and stanniocalcin-2 (STC2)) in RA patients. We also demonstrated the utility of our innovative array platform in the identification of immune-dominant epitope(s) for citrullinated antigens. We believe our platform will promote the study of post-translationally modified antigens at a breadth that has not been achieved before, by both identifying novel autoantigens and investigating their roles in disease development. The developed platforms can potentially be used to study many autoimmune disease-relevant modifications and their immunogenicity.

OBJECTIVE

Transcriptome patterns associated with acute myocardial infarction at the site of coronary occlusion are largely unknown. The aim of this study was to decipher the angiogenic, atherosclerotic, and inflammatory mRNA profiles in whole blood samples collected at the site of coronary occlusion in patients with ST-elevation myocardial infarction (STEMI).

RESULTS

In five consecutive patients with STEMI, blood was sampled at the site of occlusion (local) and in the systemic circulation (peripheral) during primary percutaneous coronary intervention. RNA was extracted from whole blood samples. Among 221 genes involved in angiogenesis, inflammation and atherosclerosis, 24 were shown to be differentially modulated locally, by analysis with custom-designed DNA array technology. Validation in 28 distinct STEMI patients using real-time quantitative PCR identified seven out of these 24 genes to be consistently and significantly upregulated in local versus peripheral blood (p<0.05). Three genes were chemokine family members (CCL2, CCL18 and CXCL12), three genes belonged to the cell-cell and cell-extracellular matrix family (FN1, CDH5 and SPP1), and one gene was representative of the lipoprotein family (APOE).

CONCLUSIONS

We identified a set of whole blood transcripts induced at the site of coronary occlusion in the acute phase of myocardial infarction. Resolved genes indicate a predominant role for chemokines, cell-extracellular matrix, and lipoprotein alterations in the pathophysiology of acute myocardial infarction and the initial response to myocardial injury.

Cytological and genetic evidence suggests that the Bacillus subtilis DNA uptake machinery localizes at a single cell pole and takes up single-stranded (ss) DNA. The integration of homologous donor DNA into the recipient chromosome requires RecA, while plasmid establishment, which is independent of RecA, requires at least RecO and RecU. RecA and RecN colocalize at the polar DNA uptake machinery, from which RecA forms filamentous structures, termed threads, in the presence of chromosomal DNA. We show that the transformation of chromosomal and of plasmid DNA follows distinct pathways. In the absence of DNA, RecU accumulated at a single cell pole in competent cells, dependent on RecA. Upon addition of any kind of DNA, RecA formed highly dynamic thread structures, which rapidly grew and shrank, and RecU dissipated from the pole. RecO visibly accumulated at the cell pole only upon addition of plasmid DNA, and, to a lesser degree, of phage DNA, but not of chromosomal DNA. RecO accumulation was weakly influenced by RecN, but not by RecA. RecO annealed ssDNA complexed with SsbA in vitro, independent of any nucleotide cofactor. The DNA end-joining Ku protein was also found to play a role in viral and plasmid transformation. On the other hand, transfection with SPP1 phage DNA required functions from both chromosomal and plasmid transformation pathways. The findings show that competent bacterial cells possess a dynamic DNA recombination machinery that responds in a differential manner depending if entering DNA shows homology with recipient DNA or has self-annealing potential. Transformation with chromosomal DNA only requires RecA, which forms dynamic filamentous structures that may mediate homology search and DNA strand invasion. Establishment of circular plasmid DNA requires accumulation of RecO at the competence pole, most likely mediating single-strand annealing, and RecU, which possibly down-regulates RecA. Transfection with SPP1 viral DNA follows an intermediate route that contains functions from both chromosomal and plasmid transformation pathways.

The present study was aimed to explore possible key genes and bioprocess affected by age during fracture healing. GSE589, GSE592 and GSE1371 were downloaded from gene expression omnibus database. The time-series genes of three age levels rats were firstly identified with hclust function in R. Then functional and pathway enrichment analysis for selected time-series genes were performed. Finally, the VennDiagram package of R language was used to screen overlapping n time-series genes. The expression changes of time-series genes in the rats of three age levels were classified into two types: one was higher expressed at 0 day, decreased at 3 day to 2 week, and increased from 4 to 6 week; the other was the opposite. Functional and pathways enrichment analysis showed that 12 time-series genes of adult and old rats were significantly involved in ECM-receptor interaction pathway. The expression changes of 11 genes were consistent with time axis, 10 genes were up-regulated at 3 days after fracture, and increased slowly in 6 week, while Itga2b was down-regulated. The functions of 106 overlapping genes were all associated with growth and development of bone after fracture. The key genes in ECM-receptor interaction pathway including Spp1, Ibsp, Tnn and Col3a1 have been reported to be related to fracture in literatures. The difference during fracture healing in three age levels rats is mainly related to age. The Spp1, Ibsp, Tnn and Col3a1 are possible potential age-related genes and ECM-receptor interaction pathway is the potential age-related process during fracture healing.

The seeding of tumor cells is a critical step in the process of metastasis, but whether and how the microenvironment of target organs affects metastatic seeding remain largely unknown. Based on cell and mouse models, we found that the metastatic seeding and outgrowth of tumor cells were significantly enhanced in fibrotic lungs. The conditioned medium from both fibrotic lungs and the fibrotic lung-derived fibroblasts (CM-FLF) had a strong activity to chemoattract tumor cells and to inhibit the apoptosis of tumor cells. Subsequent investigations revealed that the levels of fibronectin 1 (FN1) and secreted phosphoprotein 1 (SPP1) were significantly increased in fibrotic lungs. Silencing of FN1 in the fibrotic lung-derived fibroblasts dramatically decreased the chemoattracting activity of CM-FLF, while silencing of FN1 or SPP1 in fibroblasts attenuated the anti-apoptosis activity of CM-FLF. Moreover, the CM-FLF-induced apoptosis resistance or chemotaxis of tumor cells was attenuated when ITGAV, the common receptor of FN1 and SPP1, was silenced by RNA interference or blocked by GRGDS treatment in tumor cells. Consistently, ITGAV silencing or GRGDS treatment significantly inhibited the seeding and outgrowth of tumor cells in fibrotic lungs in vivo. Collectively, we suggest that fibrotic microenvironment may enhance the metastatic seeding of tumor cells in the lung by chemoattracting tumor cells and inhibiting their apoptosis via activating the FN1/SPP1-ITGAV signaling. These findings give a novel insight into the regulatory mechanisms of cancer metastasis and provide a potential target for anti-metastasis therapy.

UNASSIGNED

Chronic wounds remain a major clinical challenge. Human cryopreserved viable amniotic membrane (hCVAM) is among the most successful therapies, but the mechanisms of action remain loosely defined. Because proper regulation of macrophage behavior is critical for wound healing with biomaterial therapies, we hypothesized that hCVAM would positively regulate macrophage behavior in vitro, and that soluble factors released from the hCVAM would be important for this effect.

UNASSIGNED

Primary human pro-inflammatory (M1) macrophages were seeded directly onto intact hCVAM or cultured in separation via transwell inserts (Soluble Factors) in the presence of pro-inflammatory stimuli (interferon-γ and lipopolysaccharide) to simulate the chronic wound environment. Macrophages were characterized after 1 and 6 days using multiplex gene expression analysis of 37 macrophage phenotype- and angiogenesis-related genes via NanoString™, and protein content from conditioned media collected at days 1, 3 and 6 was analyzed via enzyme linked immunosorbent assays.

UNASSIGNED

Gene expression analysis showed that Soluble Factors promoted significant upregulation of pro-inflammatory marker IL1B on day 1 yet downregulation of TNF on day 6 compared to the M1 macrophage control. In contrast, intact hCVAM, which includes both extracellular matrix, viable cells, and soluble factors, promoted downregulation of pro-inflammatory markers TNF, CCL5 and CCR7 on day 1 and endothelial receptor TIE1 on day 6, and upregulation of the anti-inflammatory marker IL10 on day 6 compared to the M1 Control. Other genes related to inflammation and angiogenesis (MMP9, VEGF, SPP1, TGFB1, etc.) were differentially regulated between the Soluble Factors and intact hCVAM groups at both time points, though they were not expressed at significantly different levels compared to the M1 Control. Interestingly, Soluble Factors promoted increased secretion of the proinflammatory cytokine tumor necrosis factor-α (TNF-α), while direct contact with hCVAM inhibited secretion of TNF, relative to the M1 Control. Both Soluble Factors and intact hCVAM inhibited secretion of MMP9 and VEGF, pro-inflammatory proteins that are critical for angiogenesis and remodeling, compared to the M1 Control, with intact hCVAM having a stronger effect.

UNASSIGNED

In a simulated pro-inflammatory environment, intact hCVAM has distinct anti-inflammatory effects on primary human macrophages, and direct macrophage contact with intact hCVAM is required for these effects. These findings are important for the design of next generation immunomodulatory biomaterials for wound repair and regenerative medicine that may include living cells, soluble factors, or a controlled drug delivery system.

Tumor progression usually proceeds through several sequential stages, any of which could be targets for interrupting the progression process if one understood these steps at the molecular level. We extracted nascent plasma cell tumor (PCT) cells from within inflammatory oil granulomas (OG) isolated from IP pristane-injected BALB/c.iMyc(Eμ) mice at 5 different time points during tumor progression. We used laser capture microdissection to collect incipient PCT cells and analyzed their global gene expression on Affymetrix Mouse Genome 430A microarrays. Two independent studies were performed with different sets of mice. Analysis of the expression data used ANOVA and Bayesian estimation of temporal regulation. Genetic pathway analysis was performed using MetaCore (GeneGo) and IPA (Ingenuity). The gene expression profiles of PCT samples and those of undissected OG samples from adjacent sections showed that different genes and pathways were mobilized in the tumor cells during tumor progression, compared with their stroma. Our analysis implicated several genetic pathways in PCT progression, including biphasic (up- and then down-regulation) of the Spp1/osteopontin-dependent network and up-regulation of mRNA translation/protein synthesis. The latter led to a biologic validation study that showed that the AMPK-activating diabetes drug, metformin, was a potent specific PCT inhibitor in vitro.

Retinoic acid (RA), the main active metabolite of vitamin A, regulates vertebrate morphogenesis through signaling pathways not yet fully understood. Such process involves the specific activation of retinoic acid and retinoid X receptors (RARs and RXRs), which are nuclear receptors of the steroid/thyroid hormone receptor superfamily. Teleost fish are suitable models to study vertebrate development, such as skeletogenesis. Cell systems capable of in vitro mineralization have been developed for several fish species and may provide new insights into the specific cellular and molecular events related to vitamin A activity in bone, complementary to in vivo studies. This work aims at investigating the in vitro effects of RA (0.5 and 12.5 μM) on proliferation, differentiation and extracellular matrix (ECM) mineralization of two gilthead seabream bone-derived cell lines (VSa13 and VSa16), and at identifying molecular targets of its action through gene expression analysis. RA induced phenotypic changes and cellular proliferation was inhibited in both cell lines in a cell type-dependent manner (36-59% in VSa13 and 17-46% in VSa16 cells). While RA stimulated mineral deposition in VSa13 cell cultures (50-62% stimulation), it inhibited the mineralization of extracellular matrix in VSa16 cells (11-57% inhibition). Expression of hormone receptor genes (rars and rxrs), and extracellular matrix-related genes such as matrix and bone Gla proteins (mgp and bglap), osteopontin (spp1) and type I collagen (col1a1) were differentially regulated upon exposure to RA in proliferating, differentiating and mineralizing cultures of VSa13 and VSa16 cells. Altogether, our results show: (i) RA affects proliferative and mineralogenic activities in two fish skeletal cell types and (ii) that during phenotype transitions, specific RA nuclear receptors and bone-related genes are differentially expressed in a cell type-dependent manner.

We have constructed lambda/SPP1 hybrid phages by in vitro ligation of EcoRI fragments of the Bacillus subtilis phage SPP1 DNA to a lambdoid bacteriophage vector. EcoRI digestion of SPP1 generated 15 DNA fragments of which 13 could be cloned. The SPP1 DNA of such hybrids was stably maintained and replicated in Escherichia coli, as indicated by marker rescue experiments in B. subtilis. EcoRI fragment 1 of SPP1 could not be cloned although subfragments of fragment 1 resulting from spontaneous deletions which occurred during the cloning regime were consistently obtained. A region within EcoRI fragment 1 responsible for its incompatibility with replication in E. coli was defined by these experiments.

Surface modifications of commercially pure titanium (Cp-Ti), a material widely used to produce dental implants, can induce specific responses on osteoblastic cells after implantation. This work aims to investigate the influence of chemically modified surfaces of Cp-Ti by acid etching or acid etching plus alkaline treatment on the gene expression of human osteoblastic (Hob) cells. Roughness and contact angle measurements were carried out to evaluate the surface properties of the samples. The surface morphology was investigated with scanning electron microscopy. Chemical composition was analyzed by energy dispersive X-ray spectroscopy (EDS). The expression levels of some bone-related genes (ALPL, COL1A1, COL3A1, SPP1, RUNX2, and SPARC) were analyzed using real time Reverse Transcription-Polymerase Chain Reaction (real time RT-PCR). The results showed that all the chemical modifications studied in this work influenced the surface morphology, wettability, roughness and induced an osteoconductive behavior. The samples that were acid etched and alkaline treated showed a more pronounced effect.

OBJECTIVE

To evaluate genetic variations associated with kidney stone disease in Northeastern Thai patients.

METHODS

Altogether, 67 single nucleotide polymorphisms (SNP) distributed within 8 candidate genes, namely TFF1, S100A8, S100A9, S100A12, AMBP, SPP1, UMOD, and F2, which encode stone inhibitor proteins, including trefoil factor 1, calgranulin (A, B, and C), bikunin, osteopontin, tamm-Horsfall protein, and prothrombin, respectively, were initially genotyped in 112 individuals each and in additional subjects to consist of 164 patients and 216 control subjects in total.

RESULTS

We found that minor allele and homozygous genotype frequencies of 8 of 10 SNPs distributed within the F2 gene were significantly higher in the control group than in the patient group. Two F2 haplotypes were found to be dually associated with kidney stone risk, one (TGCCGCCGCG) with increased disease risk and the other (CGTTCCGCTA) with decreased disease risk. However, these 2 haplotypes were associated with the disease risks in only the female, not the male, group.

CONCLUSIONS

The results of our study indicate that genetic variation of F2 is associated with kidney stone risk in Northeastern Thai female patients.

Serum tumor markers for the diagnosis of esophageal squamous cell carcinoma (ESCC) have low sensitivity. This study aims to identify new serum markers for ESCC diagnosis from RNA sequencing (RNA-seq) data. RNA-seq was performed using six pairs of ESCC and matched normal tissues. The candidates for ESCC were screened from the differentially expressed genes. The candidates were analyzed by ELISA from the serum of a test group and a validation group. Real-time PCR, Western blotting and immunohistochemistry were used to detect the expression of the candidates in tumor cell lines and tumor tissues. Ten genes were selected from the RNA-seq data. Serum levels of ADAM12, CHI3L1, MMP13 and SPP1 were significantly higher in the ESCC patients than in the healthy controls. A diagnostic model combining CHI3L1, MMP13, and SPP1 was established. The area under the curve (AUC) values for serum CHI3L1, MMP13, and SPP1 and the diagnostic model for discriminating ESCC patients from controls were 0.732, 0.881, 0.661 and 0.928, respectively. In the validation cohort, the AUC values were 0.753, 0.789, 0.696 and 0.843, respectively. Moreover, the AUC of the model for classifying patients with early ESCC was 0.918 in the test group and 0.857 in the validation group. Overexpression of CHI3L1, MMP13 and SPP1 was observed in the tumor cell lines and tissues. The diagnostic model composed of CHI3L1, MMP13 and SPP1 discriminates ESCC patients with high sensitivity. Our data highlight the potential of this diagnostic model for the noninvasive diagnosis of ESCC.

Partially purified extracts from sporulating Bacillussubtilis cultures transcribed different natural DNAs with different efficiencies. This template specificity results in an increased or a decreased synthetic activity with respect to extracts from vegetative cells, depending on the template used. With SPP1 DNA a decrease in activity occurs, whereas with T7 DNA an increased activity was observed, which is due to a higher efficiency of initiation. This is not an intrinsic property of RNA polymerase, but is due to some fraction(s) which can be separated from the enzyme. Together with invivo experiments on transcription and SPP1 phage production during sporulation, these results suggest a possible role of promoter recognition in sporulation.

In vitro transcription of SPP1 DNA occurred on only one of the two strands, the same which is predominantly transcribed in SPP1-infected cells. Transcripts were distributed in several size classes. Analysis of elongation kinetics and of size distribution, coupled with hybridization to DNA restriction fragments, showed that some regions of the template have more initiation sites than others; some have none. Some regions were transcribed directly, some were transcribed from initiation sites located in other regions, and one was never transcribed. Several transcription initiation sites on SPP1 DNA are located on EcoRI fragment 1; four to five others are distributed among other fragments. Cutting the DNA with EcoRI did not introduce artifactual initiation sites. In vitro transcription units can be localized and oriented with respect to the EcoRI restriction map of SPP1 DNA.

Secreted phosphoprotein 1 (Spp1) is located within quantitative trait loci associated with lung function that was previously identified by contrasting C3H/HeJ and JF1/Msf mouse strains that have extremely divergent lung function. JF1/Msf mice with diminished lung function had reduced lung SPP1 transcript and protein during the peak stage of alveologenesis (postnatal day [P]14-P28) as compared with C3H/HeJ mice. In addition to a previously identified genetic variant that altered runt-related transcription factor 2 (RUNX2) binding in the Spp1 promoter, we identified another promoter variant in a putative RUNX2 binding site that increased the DNA protein binding. SPP1 induced dose-dependent mouse lung epithelial-15 cell proliferation. Spp1((-/-)) mice have decreased specific total lung capacity/body weight, higher specific compliance, and increased mean airspace chord length (Lm) compared with Spp1((+/+)) mice. Microarray analysis revealed enriched gene ontogeny categories, with numerous genes associated with lung development and/or respiratory disease. Insulin-like growth factor 1, Hedgehog-interacting protein, wingless-related mouse mammary tumor virus integration site 5A, and NOTCH1 transcripts decreased in the lung of P14 Spp1((-/-)) mice as determined by quantitative RT-PCR analysis. SPP1 promotes pneumocyte growth, and mice lacking SPP1 have smaller, more compliant lungs with enlarged airspace (i.e., increased Lm). Microarray analysis suggests a dysregulation of key lung developmental transcripts in gene-targeted Spp1((-/-)) mice, particularly during the peak phase of alveologenesis. In addition to its known roles in lung disease, this study supports SPP1 as a determinant of lung development in mice.

Prostate cancer (PCa) is the most commonly diagnosed malignancy in men. The current prevalent diagnosis method, prostate-specific antigen (PSA) screening test, has low sensitivity, specificity and is poor at predicting the grade of disease. Thus, new biomarkers are urgently needed to improve the PCa diagnosis and staging for the management of patients. The aim of this study is to investigate the first voided urinary sample after massage for biomarker discovery for PCa. In this work, untargeted metabolomic profiling of the first voided urinary sample after massage from 28 confirmed prostate cancer patients, 20 benign enlarged prostate patients and 6 healthy volunteers was performed using liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-MS/MS). Single and multiple peptide protein and cross-linking molecules were identified using PEAKS software. Analytical and diagnostic performance was tested using the Student's t test, Benjamini Hochberg correction and the receiver operating characteristic (ROC) curves. Using differential display analysis to compare peptides and cross-linking molecules of urinary samples between patients with benign, enlarged prostate and malignant cancer, we identified multiple peptides derived from osteopontin (SPP1) and prothrombin (F2) that are lower in PCa patients than in benign and enlarged prostate. The diagnosis accuracies of SPP1 and F2 peptides are 0.65-0.77 and 0.68-0.72, respectively. In addition to this, there are significant differences between PCa and benign/enlarged prostate patients in pyridinoline (PYD) and deoxypyridinoline (DPD) (p value = 0.001). Differences also, as shown in the excretion of these molecules for different stages of PCa (p value = 0.04) as the level of DPD and DPD/PYD ratio, were high in patients with locally advanced tumours. The study underscores the importance of proteomics analysis, and our results demonstrate that a urinary-based in depth proteomic approach allows the potential identification of dysregulated pathways and diagnostic biomarkers.