Germ-line stem cells (GSCs) constitute a stem cell population with remarkable stability and proliferative potential in vitro and are a useful model for studying the mechanism of self-renewal and "stemness" function of committed tissue stem cells. To identify GSC-specific genes, we performed subtractive hybridization using cDNA from GSCs, testis, and embryonic stem (ES) cells, and successfully identified 11 genes highly expressed in GSCs. Histological analysis confirmed expression of Cry alpha b, Mcpt8, Cxcl5, Fth1, Ctla2 alpha, and Spp1 in undifferentiated spermatogonia on the basement membrane area of the seminiferous epithelium of the testis, where the GSC niche is thought to be located. Among GSC-specific genes, quantitative PCR analysis showed seven genes-Fth1, Cry alpha b, Spp1, Bcap31, Arhgap1, Ctla2 alpha, and Serpina3g-to be common transcripts highly expressed in hematopoietic stem cells (HSCs). Histological analysis confirmed that Ctla2 alpha-, Serpina3g-, and Spp1-expressing cells were observed in the trabecular bone region of the bone marrow, where the HSC niche is located. Furthermore, histological analysis revealed that only Spp1 was expressed in the hair follicle bulge in the area of the hair follicle stem cell niche. Thus, identifying stemness genes by comparative analysis to GSCs is a powerful tool with which to explore the fundamental commonalities of HSCs and other stem cell types.

The proper development of atrioventricular (AV) valves is critical for heart morphogenesis and for the formation of the cardiac conduction system. Defects in AV valve development are the most common type of congenital heart defect. Cardiac troponin I ( ctnni), a structural and regulatory protein involved in cardiac muscle contraction, is a subunit of the troponin complex, but the functions and molecular mechanisms of ctnni during early heart development remain unclear. We created a knockout zebrafish model in which troponin I type 1b ( tnni1b) ( Tnni-HC, heart and craniofacial) was deleted using the clustered regularly interspaced short palindromic repeat/clustered regularly interspaced short palindromic repeat-associated protein system. In the homozygous mutant, the embryos showed severe pericardial edema, malformation of the heart tube, reduction of heart rate without contraction and with almost no blood flow, heart cavity congestion, and lack of an endocardial ring or valve leaflet, resulting in 88.8 ± 6.0% lethality at 7 d postfertilization. Deletion of tnni1b caused the abnormal expression of several markers involved in AV valve development, including bmp4, cspg2, has2, notch1b, spp1, and Alcam. Myocardial re-expression of tnni1b in mutants partially rescued the pericardial edema phenotype and AV canal (AVC) developmental defects. We further showed that tnni1b knockout in zebrafish and ctnni knockdown in rat h9c2 myocardial cells inhibited cardiac wnt signaling and that myocardial reactivation of wnt signaling partially rescued the abnormal expression of AVC markers caused by the tnni1b deletion. Taken together, our data suggest that tnni1b plays a vital role in zebrafish AV valve development by regulating the myocardial wnt signaling pathway.-Cai, C., Sang, C., Du, J., Jia, H., Tu, J., Wan, Q., Bao, B., Xie, S., Huang, Y., Li, A., Li, J., Yang, K., Wang, S., Lu, Q. Knockout of tnni1b in zebrafish causes defects in atrioventricular valve development via the inhibition of myocardial wnt signaling pathway.

Goat farming in Pakistan depends on indigenous breeds that have adapted to specific agro-ecological conditions. Pakistan has a rich resource of goat breeds, and the genetic diversity of these goat breeds is largely unknown. In this study, genetic diversity and population structure were characterized from seven indigenous goat breeds using the goat 50K SNP chip. The genetic diversity analysis showed that Bugi toori goats have the highest inbreeding level, consistent with the highest linkage disequilibrium, lowest diversity and long run of heterozygosity segments. This indicates that this breed should be prioritized in future conservation activities. The population structure analysis revealed four fairly distinct clusters (including Bugi toori, Bari, Black Tapri and some Kamori) and three other breeds that are seemingly the results of admixture between these or related groups (some Kamori, Pateri, Tapri and White Tapri). The selection signatures were evaluated in each breed. A total of 2508 putative selection signals were reported. The 26 significant windows were identified in more than four breeds, and selection signatures spanned several genes that directly or indirectly influence traits included coat colour variation (KIT), reproduction (BMPR1B, GNRHR, INSL6, JAK2 and EGR4), body size (SOCS2), ear size (MSRB3) and milk composition (ABCG2, SPP1, CSN1S2, CSN2, CSN3 and PROLACTIN).

The Set1 family of histone H3 lysine 4 (H3K4) methyltransferases is highly conserved from yeast to human. Here we show that the Set1 complex (Set1C) directly binds RNA in vitro through the regions that comprise the double RNA recognition motifs (dRRM) and N-SET domain within Set1 and its subunit Spp1. To investigate the functional relevance of RNA binding, we performed UV RNA crosslinking (CRAC) for Set1 and RNA polymerase II in parallel with ChIP-seq experiments. Set1 binds nascent transcripts through its dRRM. RNA binding is important to define the appropriate topology of Set1C distribution along transcription units and correlates with the efficient deposition of the H3K4me3 mark. In addition, we uncovered that Set1 binds to different classes of RNAs to levels that largely exceed the levels of binding to the general population of transcripts, suggesting the Set1 persists on these RNAs after transcription. This class includes RNAs derived from SET1, Ty1 retrotransposons, specific transcription factors genes and snRNAs (small nuclear RNAs). We propose that Set1 modulates adaptive responses, as exemplified by the post-transcriptional inhibition of Ty1 retrotransposition.

Biomarkers that assess treatment response for patients with the autoimmune disorder, myasthenia gravis (MG), have not been evaluated to a significant extent. We hypothesized the pro-inflammatory cytokine, osteopontin (OPN), may be associated with variability of response to glucocorticoids (GCs) in patients with MG. A cohort of 250 MG patients treated with standardized protocol of GCs was recruited, and plasma OPN and polymorphisms of its gene, secreted phosphoprotein 1 (SPP1), were evaluated. Mean OPN levels were higher in patients compared to healthy controls. Carriers of rs11728697*T allele (allele definition: one of two or more alternative forms of a gene) were more frequent in the poorly GC responsive group compared to the GC responsive group indicating an association of rs11728697*T allele with GC non-responsiveness. One risk haplotype (AGTACT) was identified associated with GC non-responsiveness compared with GC responsive MG group. Genetic variations of SPP1 were found associated with the response to GC among MG patients.

Portal proteins are cyclical oligomers which play essential roles in bacteriophage pro-capsid formation, DNA packaging, and in connector formation. Bacteriophage SPP1 portal protein (gp6) is a turbine-like molecule with 13-fold symmetry [Dube et al. (1993) EMBO J. 12, 1303-1309]. The purified protein was crystallized with polyethylene glycol 400 as the precipitating agent using the vapor-diffusion method. Salt conditions were selected based on the properties of gp6 in different ionic environments. X-ray diffraction data up to a resolution of 7.85 A were measured from frozen crystals with orthorhombic space group C2221 and cell dimensions a = 180.5 (5), b = 223.5 (5), c = 417 (1) A. The asymmetric unit contains one tridecameric portal protein with 57.3 kDa subunits. The self-rotation searches confirm the 13-fold symmetry of the crystallized protein.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by hyperplastic megakaryopoiesis and myelofibrosis. We recently described the upregulation of MAF (v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog) in PMF CD34+ hematopoietic progenitor cells (HPCs) compared to healthy donor. Here we demonstrated that MAF is also upregulated in PMF compared with the essential thrombocytemia (ET) and polycytemia vera (PV) HPCs. MAF overexpression and knockdown experiments shed some light into the role of MAF in PMF pathogenesis, by demonstrating that MAF favors the megakaryocyte and monocyte/macrophage commitment of HPCs and leads to the increased expression of proinflammatory and profibrotic mediators. Among them, we focused our further studies on SPP1 and LGALS3. We assessed SPP1 and LGALS3 protein levels in 115 PMF, 47 ET and 24 PV patients plasma samples and we found that SPP1 plasma levels are significantly higher in PMF compared with ET and PV patients. Furthermore, in vitro assays demonstrated that SPP1 promotes fibroblasts and mesenchymal stromal cells proliferation and collagen production. Strikingly, clinical correlation analyses uncovered that higher SPP1 plasma levels in PMF patients correlate with a more severe fibrosis degree and a shorter overall survival. Collectively our data unveil that MAF overexpression contributes to PMF pathogenesis by driving the deranged production of the profibrotic mediator SPP1.

OBJECTIVE

Ageing is a major risk factor for development of metabolic diseases such as type 2 diabetes. Identification of the mechanisms underlying this association could help to elucidate the relationship between age-associated progressive loss of metabolic health and development of type 2 diabetes. We aimed to determine molecular signatures during ageing in the endocrine pancreas.

METHODS

Global gene transcription was measured in pancreatic islets isolated from young and old rats by Ilumina BeadChip arrays. Promoter DNA methylation was measured by Sequenom MassArray in 46 genes that showed differential expression with age, and correlations with expression were established. Alterations in morphological and cellular processes with age were determined by immunohistochemical methods.

RESULTS

Age-related changes in gene expression were found at 623 loci (>1.5-fold, false discovery rate [FDR] <5%), with a significant (FDR < 0.05) enrichment in genes previously implicated in islet-cell function (Enpp1, Abcc8), type 2 diabetes (Tspan8, Kcnq1), inflammatory processes (Cxcl9, Il33) and extracellular matrix organisation (Col3a1, Dpt). Age-associated transcriptional differences negatively correlated with promoter DNA methylation at several loci related to inflammation, glucose homeostasis, cell proliferation and cell-matrix interactions (Il33, Cxcl9, Gpr119, Fbp2, Col3a1, Dpt, Spp1).

CONCLUSIONS

Our findings suggest that a significant proportion of pancreatic islets develop a low-grade 'chronic' inflammatory status with ageing and this may trigger altered functional plasticity. Furthermore, we identified changes in expression of genes previously linked to type 2 diabetes and associated changes in DNA methylation that could explain their age-associated dysregulation. These findings provide new insights into key (epi)genetic signatures of the ageing process in islets.

Estrogen plays a key role in maintaining the morphology and function of the efferent ductules. We previously demonstrated that the antiestrogen fulvestrant markedly affected gene expression in the rat efferent ductules. The mechanism of fulvestrant action to modulate gene expression may involve not only the blockade of ESR1 and ESR2 estrogen receptors, but also the activation of ESR1 and ESR2 when the receptors are tethered to AP-1 or SP1 transcription factors, or the activation of the G protein-coupled estrogen receptor 1. We therefore compared the effects of two strategies to interfere with estrogen action in the rat efferent ductules: treatment with fulvestrant or with the aromatase inhibitor anastrozole. Whereas fulvestrant markedly increased Mmp7 and Spp1, and reduced Nptx1 mRNA levels, no changes were observed with anastrozole. Fulvestrant caused changes in epithelial morphology that were not seen with anastrozole. Fulvestrant shifted MMP7 immunolocalization in the epithelial cells from the supranuclear to the apical region; this effect was less pronounced with anastrozole. In vitro studies of (35)S-methionine incorporation showed that protein release was increased, whereas tissue protein content in the efferent ductules of fulvestrant-treated rats was decreased. Although fulvestrant markedly affected gene expression, no changes were observed on AP-1 and SP1 DNA-binding activity. The blockade of ESRs seems to be the major reason explaining the differences between both treatments. At least some of the effects of fulvestrant appear to result from compensatory mechanisms activated by the dramatic changes caused by ESR1 blockade.

This study examined polymorphisms of the secreted phosphoprotein 1 (SPP1) gene and its association with growth and carcass traits in the F(2) population of the crossbred Landrace × Jeju (Korea) Black pig. The authors detected the presence/absence polymorphisms of short interspersed nuclear element in the SPP1 intron 6 of the population; they then designated the longer fragment as allele A and the shorter one as allele B. The SPP1 A/B heterozygous pigs evidenced significantly heavier body weight at birth and on days 21 and 70, and a higher level of average daily gain during the early developmental period than was seen in the A/A and B/B homozygous pigs (P < 0.05). Further, the SPP1 A/B heterozygous pigs evidenced significantly greater body length, less backfat thickness measured at three different sites, and larger loin muscle area than the homozygotes (P < 0.05). On the other hand, the levels of late average daily gain, 140th-day body weight, and marbling score were not significantly associated (P>> 0.05). The results of this study reveal faster growth rate and differences in pig productivity according to genotypes of the SPP1 gene. These findings demonstrate that SPP1 genotypes may effectively function as molecular genetic markers for the improvement of Jeju Black pig-related crossbreeding systems.

Recent studies from mammalian, fish, and in vitro models have identified bone and cartilage development as sensitive targets for dioxins and other aryl hydrocarbon receptor ligands. In this study, we assess how embryonic 2,3,7,8-tetrachlorochlorodibenzo-p-dioxin (TCDD) exposure impacts axial osteogenesis in Japanese medaka (Oryzias latipes), a vertebrate model of human bone development. Embryos from inbred wild-type Orange-red Hd-dR and 3 transgenic medaka lines (twist:EGFP, osx/sp7:mCherry, col10a1:nlGFP) were exposed to 0.15 nM and 0.3 nM TCDD and reared until 20 dpf. Individuals were stained for mineralized bone and imaged using confocal microscopy to assess skeletal alterations in medial vertebrae in combination with a qualitative spatial analysis of osteoblast and osteoblast progenitor cell populations. Exposure to TCDD resulted in an overall attenuation of vertebral ossification characterized by truncated centra, and reduced neural and hemal arch lengths. Effects on mineralization were consistent with modifications in cell number and cell localization of transgene-labeled osteoblast and osteoblast progenitor cells. Endogenous expression of osteogenic regulators runt-related transcription factor 2 (runx2) and osterix (osx/sp7), and extracellular matrix genes osteopontin (spp1), collagen type I alpha I (col1), collagen type X alpha I (col10a1), and osteocalcin (bglap/osc) was significantly diminished at 20 dpf following TCDD exposure as compared with controls. Through global transcriptomic analysis more than 590 differentially expressed genes were identified and mapped to select pathological states including inflammatory disease, connective tissue disorders, and skeletal and muscular disorders. Taken together, results from this study suggest that TCDD exposure inhibits axial bone formation through dysregulation of osteoblast differentiation. This approach highlights the advantages and sensitivity of using small fish models to investigate how xenobiotic exposure may impact skeletal development.

Understanding the process of ex vivo embryonic stem (ES) cell differentiation is important for generating higher yields of desirable cell types or lineages and for understanding fundamental aspects of ES differentiation. We used DNA microarray analysis to investigate the differentiation of mouse ES cells cultured under three differentiation conditions. Embryoid body (EB) formation was compared to differentiation on surfaces coated with either gelatin (GEL) or matrigel (MAT). Based on the transcriptional patterns of a list of literature-based "stemness" genes, ES cell differentiation on the two coated surfaces appeared similar but not identical to EB differentiation. A notable difference was the GEL and MAT upregulation but EB downregulation of nine such stemness genes, which are related to cell adhesion and epithelial differentiation. Further, GEL and MAT differentiation showed higher expression of bone formation-related genes (Spp1, Csf1, Gsn, Bmp8b, Crlf1). Gene ontology analysis shows an increase in the expression of genes related to migration and cell structure in all three conditions. Overall, GEL and MAT conditions resulted in a more similar to each other transcriptional profile than to the EB condition, and such differences are apparently related to higher nutrient and metabolite gradients and limitations in the EB versus the GEL or MAT cultures.

Bacillus subtilis competent cells were transfected with SPP1 heteroduplices having pyrimidine dimers in one of the strands. The data obtained reveal that excision repair of the pyrimidine dimers influences the ratio of wild type versus mutant progeny observed in "normal" heteroduplex transfection. With increased exposure of one strand to UV dose the percentage of infective centers having the unirradiated strand genotype shows an increase. A comparison of the transfection data in her+ and her- host excludes asymmetric replication as the cause of the observed changes in the conversion pattern. The data can be explained on the basis of a dimer induced co-excision of the mismatched region. In addition transfection data from wild type/deletion mutant heteroduplices where the strand of mutant origin was irradiated exclude the possibility of the wild type loop being excised during uptake.

Severe cutaneous injuries continue to result from exposure to sulfur mustard [bis(2-chloroethyl)sulfide; HD] and thermal burns. Microarray analysis was utilized in this study to evaluate transcriptional changes in porcine skin assessing the underlying repair mechanisms of HD and thermal injury involved in wound healing. Four ventral abdominal sites on each of 4 weanling swine were exposed to 400 microL undiluted HD or a heated brass rod (70 degrees C) for 8 minutes and 45-60 seconds, respectively. At 7 days postexposure, skin samples were excised and total RNA was isolated, labeled, and hybridized to Affymetrix GeneChip (Santa Clara, CA, USA) Porcine Genome Arrays (containing 20,201 genes). Based on the gene expression patterns in HD- and thermal-exposed skin at 7 days, the transcriptional profiles do not differ greatly. HD and thermal exposures promoted similar changes in transcription, where 270 and 283 transcripts were increased with HD and thermal exposures, respectively. Both exposures promoted decreases in 317 and 414 transcripts, respectively. Of the significantly increased transcripts, at least 77% were commonly expressed in both HD- and thermal-exposed skin, whereas at least 67% of decreased transcripts were common between both exposure types. Six of the top 10 biological functions were common to HD and thermal injury in which 9 canonical pathways were shared. The present study illustrates the similarities found between HD and thermal injury with respect to transcriptional response and wound healing and identifies specific genes (CXCL2, CXCR4, FGFR2, HMOX1, IGF1, PF4, PLAU, PLAUR, S100A8, SPP1, and TNC) that may be useful as potential therapeutic targets to promote improved wound healing.

Secreted phosphoprotein 1 (SPP1) is a phosphorylated acidic glycoprotein. It is broadly expressed in a variety of tissues, and it is involved in a number of physiological and pathological events, including cancer metastasis, tissues remodeling, pro-inflammation regulation, and cell survival. SPP1 has shown its function of protecting tissues and organs against injury and wound, giving itself potentials to become a therapy target or giving its antibodies of other counter-acting reagents potentials to become drug candidates. Non-tagged (native) recombinant SPP1 would be valuable in therapeutic and pharmaceutical researches. In our study, mouse Spp1 DNA fragment without signal peptide was built in pET28a(+) vector and transformed into Escherichia coli BL21 (DE3). The recombinant mouse SPP1 (rmSPP1) was then expressed in bacteria upon induction by isopropyl β-D-thiogalactopyranoside (IPTG). The abundance of rmSPP1 was increased using isoelectric precipitation and ammonium sulfate fractionation methods, and anion and cation exchange chromatography was employed to further purify rmSPP1. Finally, we got rmSPP1 product with 12.8 % productivity, 97 % purity, satisfactory bioactivity, and low endotoxin content.

The use of C(60) fullerenes is expected to increase in various industrial fields. Little is known about the potential toxicological mechanism of action of water-soluble C(60) fullerenes. In our previous research, gene expression profiling of the rat lung was performed after whole-body inhalation exposure to C(60) fullerenes to gain insights into the molecular events. These DNA microarray-based data closely matched the pathological findings that C(60) fullerenes caused no serious adverse pulmonary effects under the inhalation exposure condition. Taking advantage of this, we attempted to characterize time-dependent changes in the gene expression profiles after intratracheal instillation with C(60) fullerenes at different dosages and to identify the candidate expressed genes as potential biomarkers. The hierarchical cluster analysis revealed that the up- or downregulation of genes after intratracheal instillation with 1.0 mg C(60) fullerene particles in rat lung tissue was significantly over-represented in the "response to stimulus" and "response to chemical stimulus" categories of biological processes and in the "extracellular space" category of the cellular component. These results were remarkable for 1 week after the instillation with C(60) fullerenes. In the lung tissues instilled with 1.0 mg C(60) fullerene particles, many representative genes involved in "inflammatory response," such as the Cxcl2, Cxcl6, Orm1, and Spp1 genes, and in "matrix metalloproteinase activity," such as the Mmp7 and Mmp12 genes, were upregulated for over 6 months. The expression levels of 89 and 21 genes were positively correlated with the C(60) fullerene dose at 1 week and 6 months after the instillation, respectively. Most of them were involved in "inflammatory response", and the Ccl17, Ctsk, Cxcl2, Cxcl6, Lcn6, Orm1, Rnase9, Slc26a4, Spp1, Mmp7, and Mmp12 genes were overlapped. Meanwhile, the expression levels of 16 and 4 genes were negatively correlated with the C(60) fullerene dose at 1 week and 6 months after the instillation, respectively. Microarray-based gene expression profiling suggested that the expression of some genes is correlated with the dose of intratracheally instilled C(60) fullerenes. We propose that these genes are useful for identifying potential biomarkers in acute-phase or persistent responses to C(60) fullerenes in the lung tissue.

Secreted phosphoprotein one (SPP1, osteopontin) may regulate conceptus implantation and placentation. We investigated effects of progesterone (P(4)) and the conceptus on expression and localization of SPP1 in the ovine uterus. Steady-state levels of SPP1 mRNA in the endometrium of unilaterally pregnant ewes did not differ significantly between nongravid and gravid horns within their respective days of pregnancy; however, levels did increase as pregnancy progressed. SPP1 mRNA was detectable in the glandular epithelium (GE) of both nongravid and gravid horns via in situ hybridization. SPP1 protein was localized to the apical surface of the luminal epithelium of both nongravid and gravid uterine horns. Gravid horns exhibited extensive stromal SPP1 on Days 40 through 120, whereas SPP1 was markedly lower in the stroma of nongravid uterine horns through Day 80 of pregnancy. By Day 120, stromal expression of SPP1 between nongravid and gravid horns was similar. Long-term P(4) treatment of ovariectomized ewes induced SPP1 in the uterine stroma and GE. A bioactive 45-kDa SPP1 fragment was purified from uterine secretions and promoted ovine trophectoderm cell attachment in vitro. Interestingly, increased stromal cell expression of SPP1 was positively associated with vascularization as assessed by von Willebrand factor staining. Finally, ovine uterine artery endothelial cells produced SPP1 during outgrowth into three-dimensional collagen matrices in an in vitro model system that recapitulates angiogenesis. Collectively, P(4) induces and the conceptus further stimulates SPP1 in uterine GE and stroma, where SPP1 likely influences histotrophic and hematotrophic support of conceptus development.

In all mammalian species, critical events, including uterine receptivity and development of the conceptus (embryo/fetus and its associated extraembryonic membranes), must be intricately orchestrated and carefully timed during the window of implantation. Otherwise, failure of conceptuses to implant is inevitable, which accounts for 50%-75% of failures to establish pregnancy. Unlike human and rodent blastocysts, the blastocysts of pigs and ruminants undergo rapid transitions from spherical to tubular and filamentous conceptuses in response to histotroph during the peri-implantation period of pregnancy. Both arginine and secreted phosphoprotein 1 (SPP1; also known as osteopontin) are multifunctional molecules that increase significantly in ovine uterine histotroph during early pregnancy; however, little is known about their relationship and synergistic effects on conceptus development. Therefore, we conducted in vitro experiments using our established ovine trophectoderm cell line (oTr1) isolated from Day 15 ovine conceptuses to determine their migratory and adhesive responses to individual and combined effects of arginine and recombinant SPP1 (rSPP1) that contains an Arg-Gly-Asp (RGD) binding sequence. Migration and adhesion of oTr1 cells were significantly stimulated by rSPP1, whereas arginine alone only induced a significant increase in cell migration. However, the combination of arginine and rSPP1 had an additive effect on migration, and a synergistic effect on adhesion of oTr1 cells. Those cooperative effects of arginine and SPP1 were mediated by focal adhesion assembly-MTORC2-cytoskeletal reorganization and MAPK pathways. Collectively, results suggest that arginine and SPP1 in histotroph affect cellular events required for rapid elongation of ovine conceptuses during the peri-implantation period of pregnancy.

Matrix Metalloproteinase-2 (Mmp2) is a collagenase known to be important in the development of renal fibrosis. In unilateral ureteral obstruction (UUO) the obstructed kidney (OK) develops fibrosis, while the contralateral (CL) does not. In this study we investigated the effect of UUO on gene expression, fibrosis and pelvic remodeling in the kidneys of Mmp2 deficient mice (Mmp2-/-), heterozygous animals (Mmp2+/-) and wild-type mice (Mmp2+/+). Sham operated animals served as controls (Cntrl). UUO was prepared under isoflurane anaesthesia, and the animals were sacrificed after one week. UUO caused hydronephrosis, dilation of renal tubules, loss of parenchymal thickness, and fibrosis. Damage was most severe in Mmp2+/+ mice, while both Mmp2-/- and Mmp2+/- groups showed considerably milder hydronephrosis, no tubular necrosis, and less tubular dilation. Picrosirius red quantification of fibrous collagen showed 1.63±0.25% positivity in OK and 0.29±0.11% in CL (p<0.05) of Mmp2+/+, Mmp2-/- OK and Mmp2-/- CL exhibited only 0.49±0.09% and 0.23±0.04% (p<0.05) positivity, respectively. Mmp2+/- OK and Mmp2+/- CL showed 0.43±0.09% and 0.22±0.06% (p<0.05) positivity, respectively. Transcriptomic analysis showed that 26 genes (out of 48 examined) were differentially expressed by ANOVA (p<0.05). 25 genes were upregulated in Mmp2+/+ OK compared to Mmp2+/+ CL: Adamts1, -2, Col1a1, -2, -3a1, -4a1, -5a1, -5a2, Dcn, Fbln1, -5, Fmod, Fn1, Itga2, Loxl1, Mgp, Mmp2, -3, Nid1, Pdgfb, Spp1, Tgfb1, Timp2, Trf, Vim. In Mmp2-/- and Mmp2+/- 18 and 12 genes were expressed differentially between OK and CL, respectively. Only Mmp2 was differentially regulated when comparing Mmp2-/- OK and Mmp2+/- OK. Under stress, it appears that Mmp2+/- OK responds with less Mmp2 upregulation than Mmp2+/+ OK, suggesting that there is a threshold level of Mmp2 necessary for damage and fibrosis to occur. In conclusion, reduced Mmp2 expression during UUO protects mice against hydronephrosis and renal fibrosis.

OBJECTIVE

Gliomas are the most common primary tumours of the central nervous system. Snake venom, such as candoxin (CDX) isolated from Bungarus candidus, inhibits glioma cell proliferation. This study explored the gene regulation profile of CDX-treated human glioma Hs683 cells.

METHODS

Using microarray technology and bioinformatics analyses the underlying molecular mechanism of action of CDX was evaluated by constructing gene regulation and protein-protein interaction co expression networks.

RESULTS

CDX treatment induced a large number of related genes at the transcriptional level. The MYC gene (v-myc myelocytomatosis viral oncogene homologue [avian]) had a key role in the response of Hs683 cells to CDX treatment. Activation of MYC upregulated NDRG1 (N-myc downstream regulated 1), WNT10B (wingless-type mouse mammary tumour virus integration site family, member 10B), CASP9 (caspase 9, apoptosis-related cysteine peptidase) and CDKN2A (cyclin-dependent kinase inhibitor 2A), and downregulated ID3 (inhibitor of DNA binding 3, dominant negative helix-loop-helix protein) and SLC1A4 (solute carrier family 1 [glutamate/neutral amino acid transporter], member 4). In addition, a subnetwork was constructed among SPP1 (secreted phosphoprotein 1), SDC1 (syndecan 1) and CD44 based on protein-protein interactions, and these genes were predicted to be involved in glioma cell invasion.

CONCLUSIONS

These findings might provide novel therapeutic targets for glioma chemotherapy.

The DNA packaging motor of dsDNA bacterial viruses contains a head-tail connector with a channel for the genome to enter during assembly and to exit during host infection. The DNA packaging motor of bacterial virus phi29 was recently reported to use the "One-way revolving" mechanism for DNA packaging. This raises a question of how dsDNA is ejected during infection if the channel acts as a one-way inward valve. Here we report a three step conformational change of the portal channel that is common among DNA translocation motors of bacterial viruses T3, T4, SPP1, and phi29. The channels of these motors exercise three discrete steps of gating, as revealed by electrophysiological assays. The data suggest that the three step channel conformational changes occur during DNA entry process, resulting in a structural transition in preparation for DNA movement in the reverse direction during ejection.

OBJECTIVE

Osteopontin (OPN) is aglyco-phosphoprotein, involved in tissue remodeling, inflammation and boneresorption. In various adult neoplasms OPN was shown to correlate with cancer progression, invasiveness and metastasis.

OBJECTIVE

to define the role of OPN in malignancies of children and young adults.

METHODS

a structured PubMed and Google Scholar literature analysis based on reports published in English between I'1995 and XII'2015.

RESULTS

14 studies (four on hematological malignancies, four on bone tumors, three on CNS tumors, two on dendritic proliferative diseases and one on renal tumors) were identified. Higher levels of serum and cerebro-spinal fluid OPN protein, and high expressions of OPN mRNA and SPP1 gene were present in more aggressive and advanced childhood malignancies. In children with acute lymphoblastic leukemia with CNS involvement and with atypical teratoid/rhabdoid tumor (AT/RT) and medulloblastoma, the serum and CSF OPN levels reflected tumor bulk and response to therapy, while in children with AT/RT and multisystem Langerhans cell histiocytosis with high-risk organs involvement, high OPN serum levels correlated with poorer survival. To the contrary, in osteosarcoma, high OPN mRNA and SPP1 gene expressions correlated with better survival and good response to chemotherapy.

CONCLUSIONS

The literature review suggests that OPN may play important roles in the development and progression of selected cancers of children and young adults, including acute lymphoblastic leukemia, malignant gliomas, AT/RT and Langerhans cell histiocytosis. However, limited number of published studies prevents from definite concluding on the clinical utility of OPN as a marker of diagnosis, prognosis and treatment monitoring in these pediatric cancers. Further studies performed in more numerous groups of patients with particular types of cancers of children and young adults are warranted.

OBJECTIVE

Abcb4 (-/-) mice secrete phosphatidylcholine-deficient bile and develop sclerosing cholangitis (SC), a condition that involves differential hepatic transcription of genes governing inflammation, tissue remodelling and fibrosis. The objective of this study was to test the hypothesis that genes involved in the regulation of tissue inflammation and fibrosis display transcription rates that parallel differences in abcb4 (-/-) SC activity. The activity of abcb4 (-/-) SC can be altered through dietary intervention: abcb4 (-/-) mice fed cholic acid (CA) display high SC activity, whereas ursodeoxycholic acid (UDCA)-fed mice display low SC activity.

METHODS

Differential hepatic transcription of genes was measured in abcb4 (-/-) mice maintained on CA- and UDCA-supplemented diets using cDNA microarrays. Abcb4 (+/+) mice served as controls. Differential transcription of selected genes was verified by real-time polymerase chain reaction. Liver tissue pathology was quantified by histopathology scoring.

RESULTS

Histopathology score, reflecting increased inflammation and fibrosis, was increased in CA-fed mice compared with UDCA-fed mice. cDNA microarray analysis showed up-regulation of 1582 genes in livers of CA-fed mice in contrast to 573 genes in UDCA-fed mice. Differential transcription of Ccl2, Ccl20, Cxcl10, Nfkappab1, Nfkappab2, Tgfbeta1, Tgfbeta2, Sparc, Ctgf, Lgals3, Elf3, Spp1, Pdgfa, Pdgfrb, Col1a1, Col1a2 and Col4a1 genes paralleled the unequal SC activities of CA- and UDCA-fed abcb4 (-/-) mice.

CONCLUSIONS

The numbers of differentially transcribed genes and the transcriptional activity of genes relating to inflammation, tissue remodelling and fibrosis parallel disease activity in CA- and UDCA-fed abcb4 (-/-) mice harbouring SC. Data on their hepatic transcription can gauge SC disease activity.

While hepatitis C exemplifies the role of host genetics in infectious diseases outcomes, there is no comprehensive overview of polymorphisms influencing spontaneous and/or treatment-induced hepatitis C virus clearance. We performed a systematic review and meta-analysis of host polymorphisms associated with these phenotypes. Literature search was conducted using combinations of keywords in three databases. Studies were reviewed and relevant data systematically extracted for subsequent meta-analyses. Polymorphisms from candidate gene studies were tested in two cohorts of HCV-infected patients with available genomic data. The literature search yielded 8'294 citations, among which 262 studies were selected. In the meta-analysis of 27 HLA studies, the most significant associations with spontaneous hepatitis C virus clearance included DQB1*02, DQB1*03, DRB1*04 and DRB1*11. In the meta-analysis of 16 studies of KIR genes and their HLA-ligands, KIR2DS3 was associated with both spontaneous and treatment-induced clearance, and the HLA-C2 ligand with failure to spontaneously clear the virus. In a pooled analysis of 105 candidate genes and two genome-wide association studies, we observed associations of single nucleotide polymorphisms from nine genes (EIF2AK2, IFNAR2, ITPA, MBL2, MX1, OASL, SPP1, TGFB1, TNK2) with response to interferon-based therapy. Meta-analysis of 141 studies confirmed the association of IFNL3/4 polymorphisms with spontaneous and treatment-induced hepatitis C virus clearance, even in previously underpowered groups, such as hepatitis C virus genotypes 2/3-infected patients. This study may contribute to a better understanding of hepatitis C virus immunopathogenesis and highlights the complex role of host genetics in hepatitis C virus clearance.