OBJECTIVE

To investigate the gene expression profile of human gingiva following surgical wounding.

METHODS

Ten volunteers had one side of the palate wounded. Five days later, biopsies were harvested from both wounded (healing gingiva) and contra-lateral site (normal gingiva). Tissue samples were processed for gene expression (RNA-Seq, real-time PCR) and immunohistochemistry. Gene set enrichment/pathway analysis was also performed.

RESULTS

Seven hundred genes were significantly differentially expressed in healing gingiva. Among genes with>>twofold change (FC) in expression, 399 genes were up-regulated and 88 down-regulated, several not previously reported expressed in gingiva. Most increased in expression (≥30-FC) were MMP1, CCL18, SPP1, MUC21, CTHRC1, MMP10, and SERPINE1; most decreased (≥7-FC) were COCH, SIAH3, MT4, IGFL3, KY, and SYT16. Real-time PCR confirmed significantly changed mRNA levels for selective genes tested. Gene set enrichment analysis revealed several significantly enriched biological pathways. Immunohistochemistry confirmed protein expression of MUC21, CTHRC1, CTGF, and SYT16 in normal and healing gingiva.

CONCLUSIONS

This first comprehensive analysis of the human gingival transcriptome during surgical wound healing offers novel insights into the participating molecular and biological mechanisms. The present results could serve as basis for future investigations into gingival wound healing following surgical, traumatic, or other type of injury.

The mdx mouse is a good genetic and molecular murine model for Duchenne Muscular Dystrophy (DMD), a progressive and devastating muscle disease. However, this model is inappropriate for testing new therapies due to its mild phenotype. Here, we transferred the mdx mutation to the 129/Sv strain with the aim to create a more severe model for DMD. Unexpectedly, functional analysis of the first three generations of mdx129 showed a progressive amelioration of the phenotype, associated to less connective tissue replacement, and more regeneration than the original mdxC57BL. Transcriptome comparative analysis was performed to identify what is protecting this new model from the dystrophic characteristics. The mdxC57BL presents three times more differentially expressed genes (DEGs) than the mdx129 (371 and 137 DEGs respectively). However, both models present more overexpressed genes than underexpressed, indicating that the dystrophic and regenerative alterations are associated with the activation rather than repression of genes. As to functional categories, the DEGs of both mdx models showed a predominance of immune system genes. Excluding this category, the mdx129 model showed a decreased participation of the endo/exocytic pathway and homeostasis categories, and an increased participation of the extracellular matrix and enzymatic activity categories. Spp1 gene overexpression was the most significant DEG exclusively expressed in the mdx129 strain. This was confirmed through relative mRNA analysis and osteopontin protein quantification. The amount of the 66 kDa band of the protein, representing the post-translational product of the gene, was about 4,8 times higher on western blotting. Spp1 is a known DMD prognostic biomarker, and our data indicate that its upregulation can benefit phenotype. Modeling the expression of the DEGs involved in the mdx mutation with a benign course should be tested as a possible therapeutic target for the dystrophic process.

Kidney cancer (Renal Cell Carcinoma (RCC)) is one of the most deadly malignancies due to frequent late diagnosis and poor treatment options. Histologically, RCC embraces a wide variety of different subtypes with the clear cell variant (ccRCC) being the most common, accounting for 75-90% of all RCCs. At present, the surveillance protocols for follow-up of RCC patients after radical nephrectomy are based on the American Joint Committee on Cancers (AJCC) pathological tumor-node-metastasis (TNM) classification system. Other comprehensive staging modalities have emerged and have been implemented in an attempt to improve prognostication by combining other pathological and clinical variables, including Fuhrman nuclear grade and Leibovich score. However, even early stage tumors remain at risk of metastatic progression after surgical resection and 20-40% of patients undergoing nephrectomy for clinically localized RCC will develop a recurrence. Identifying this high-risk group of RCC patients remains a challenge. Hence, novel molecular prognostic biomarkers are needed to better predict clinical outcomes. An intensive search within this field has been ongoing in the past few years, and the three main predictive and prognostic markers validated in RCC are Von Hippel Lindau (VHL), vascular endothelial growth factor (VEGF) and carbonic anhydrase IX (CAIX). Nonetheless, the use of these is still debated and none of them have yet been implemented in clinical routine. RCC is resistant to conventional oncological therapies, such as chemotherapy and radiation. The availability of novel targeted therapies directed against tumorigenic and angiogenic pathways have increased over the last years, and the outcome of patients with advanced RCC has significantly improved as a consequence. Unfortunately, all patients eventually become resistant. Thus, the development of novel targeted therapies is of great importance. The aim of this thesis was therefore to contribute in the search for novel prognostic molecular markers in RCC and to identify novel targeted therapies by in-vitro studies. This was specifically conducted by investigating; 1) The impact of symptom presentation of RCC on prognosis, 2) The expression of Calcium-activated potassium channels in RCC, the correlation of KCa3.1 to prognosis in ccRCC and the ability of TRAM-34, RA-2 and Paxilline to inhibit the proliferation of ccRCC cell lines in-vitro, 3) The gene expression and prognostic value of 19 selected genes in ccRCC and 4) The expression of the protein kinase CK subunits in subtypes of RCC, the prognostic impact of high protein expression of the CK2α subunit in ccRCC and the ability of CX-4945 and E9 to inhibit ccRCC growth in-vitro. Our molecular study cohort consisted of 155 patients with different subtypes of RCC and the benign renal neoplasm, oncocytoma. They were diagnosed in Region of Southern Denmark in 2001-2013. Frozen tissue from tumor and normal renal cortex parenchyma, together with paraffin-embedded tissue was available for every patient. We performed gene expression analysis by qRT-PCR, immunohistochemical staining of Tissue Micro Arrays, protein kinase activity analysis and functional studies. Study I was performed as a descriptive observational study focusing on the prognostic impact of symptom presentation in RCC. We included 204 patients with renal neoplasms diagnosed in 2011-2012. Incidentally discovered RCC without symptomatic presentation had overall a better prognosis, and presented with smaller tumors, a lower T-stage, lower Fuhrman grade and lower Leibovich score. In addition, the non-symptomatic patient group experienced metastatic disease less frequently. In study II we focused on the expression of two calcium-activated potassium channels in ccRCC and oncocytoma. Both KCa3.1 and KCa1.1 were higher expressed in ccRCC compared to oncocytoma. High expression of KCa3.1 was moreover correlated with poor progression free survival of ccRCC. Functional studies provided new insights since we could detect currents compatible with KCa3.1 and KCa1.1 in the cell membrane of primary and commercial ccRCC cell lines. Nonetheless, we were not able to show any significant inhibition of cell growth by the selective inhibitors of KCa3.1 and KCa1.1, TRAM-34, RA-2 and Paxilline. In study III our aim was to investigate the prognostic role of 19 genes selected on the basis of an earlier study done by the group. We used Taqman® Low Density Array to perform a quantitative real-time PCR analysis. By selecting an optimal cut-point and correct for overestimation of the p-value, we could identify three genes with impact on prognosis of ccRCC in both univariate and multivariate analysis. High expression of the genes SPP1 and CSNK2A1 (encoding Osteopontin and CK2α respectively) correlated with poor prognosis while high expression of DEFB1 (encoding β-Defensin) correlated with better prognosis. Study IV focused on validating the results obtained in Paper III by investigating the protein expression of CK2α (Protein kinase 2, alpha subunit) in the different subtypes of RCC and oncocytoma. Furthermore, we investigated whether protein expression of CK2α in ccRCC correlated with prognosis. Here we could show, that a positive nuclear staining was a marker of poor prognosis in high-stage ccRCC. Moreover, enzyme activity analysis revealed a higher activity of the protein kinase in tumor tissue of ccRCC than in normal renal cortex. Novel insights were provided in a proliferation study where we investigated the selective inhibitors of CK2α, CX-4945 and E9. CX-4945 was able to inhibit ccRCC cell growth by nearly 50%. All together the studies presented in this thesis add additional information to the ongoing research within identification of novel prognostic markers in ccRCC. We have discovered four new molecular markers, which reliably can predict prognosis at the time of diagnosis. Additionally, we identified CK2α as a novel therapeutic target of ccRCC. The studies suggest further research to validate the findings on larger cohorts and thereby obtain more insight into the involved pathways. Future research initiatives based on the results presented in this thesis could clarify the potential role of CX-4945 as a novel targeted treatment of ccRCC patients.

Bateriophage (phi29, SPP1, or SPO1)-infected, toluene-treated minicells of Bacillus subtilis are capable of limited amounts of non-replicative DNA synthesis as measured by incorporation of [3H]dTTP into a trichloroacetic acid-precipitable form. The [3H]dTTP is covalently incorporated into small DNA fragments which result from the degradation of a small percentage of the infecting phage genomes (molecular weights in the range of 2 . 10(5)). Short exposure of the DNA molecules containing the incorporated [3H]dTMP to Escherichia coli exonuclease III results in over 90% of the E13H]dTMP being converted to a trichloroacetic acid-soluble form. The synthesis is totally dependent on host-cell enzymes and is not inhibited by the addition of chloramphenicol, rifampicin, nalidixic acid and mitomycin C and only slightly (approx. 20%) inhibited by the addition of 6-(p-hydroxyphenylazo)-uracil.

Tax1-binding protein 1 (Tax1bp1) negatively regulates NF-κB by editing the ubiquitylation of target molecules by its catalytic partner A20. Genetically engineered TAX1BP1-deficient (KO) mice develop age-dependent inflammatory constitutions in multiple organs manifested as valvulitis or dermatitis and succumb to premature death. Laser capture dissection and gene expression microarray analysis on the mitral valves of TAX1BP1-KO mice (8 and 16 week old) revealed 588 gene transcription alterations from the wild type. SAA3 (serum amyloid A3), CHI3L1, HP, IL1B and SPP1/OPN were induced 1,180-, 361-, 187-, 122- and 101-fold respectively. WIF1 (Wnt inhibitory factor 1) exhibited 11-fold reduction. Intense Saa3 staining and significant I-κBα reduction were reconfirmed and massive infiltration of inflammatory lymphocytes and edema formation were seen in the area. Antibiotics-induced 'germ free' status or the additional MyD88 deficiency significantly ameliorated TAX1BP1-KO mice's inflammatory lesions. These pathological conditions, as we named 'pseudo-infective endocarditis' were boosted by the commensal microbiota who are usually harmless by their nature. This experimental outcome raises a novel mechanistic linkage between endothelial inflammation caused by the ubiquitin remodeling immune regulators and fatal cardiac dysfunction.

Bone defect is an important topic in public health. Novel therapies are based on osteogenic induction by natural antiosteoporotic compounds including plant-derived estrogens. In the current study, the osteogenic potential of Sophora pachycarpa root extract (SPRE) was explored on human adipose-derived mesenchymal stem cells. Herein, adipose-derived mesenchymal stem cells were osteoinducted in the presence of increased concentrations of the extract for 21 days. Then, cell viability was evaluated by MTT assay, and the differentiated cells were stained by Alizarin Red S for calcium deposition and subjected to alkaline phosphatase (ALP) assay for enzymatic activity. To assess the expression of bone-related genes, treated cells were evaluated by real-time polymerase chain reaction. The MTT test demonstrated that SPRE had no toxic effects on the cell viability. Treating the cells with SPRE noticeably promoted ALP activity, mineralization, and mRNA expression of runt-related transcription factor 2 (RUNX2), bone gamma-carboxyglutamate protein (BGLAP), secreted phosphoprotein 1 (SPP1), and collagen type I alpha 1 (COL1A1). Additionally, cells subjected to 0.1 μg/mL SPRE showed the highest osteogenic effects. According to high-performance liquid chromatography fingerprinting of SPRE, the osteoprotective effects of SPRE is probably due to presence of phytochemicals with estrogen-like activity in the extract. Thus, SPRE might be a suitable therapeutic agent for bone defects therapy in the future research. Copyright © 2017 John Wiley & Sons, Ltd.

OBJECTIVE

Diabetes mellitus type 2 (2DM) is associated with altered bone quality. In order to analyze associated changes on a molecular level, we investigated the gene expression of key factors of osteoblast metabolism in type 2 diabetics.

METHODS

Total mRNA and protein of bone samples from 2DM patients and non-diabetic patients were isolated, and subsequently, reverse transcription polymerase chain reaction (RT-PCR) or Western blot was performed. Furthermore, pro- and anti-inflammatory serum cytokine levels were determined using a cytokine array.

RESULTS

Expression of runt-related transcription factor 2 (RUNX2) was increased by 53 %. Expression of the bone sialoproteins, secreted phosphoprotein 1 (SPP1; osteopontin), and integrin-binding sialoprotein (IBSP), was elevated by more than 50 %, and activating transcription factor 4 (ATF4) expression was 13 % lower in the investigated diabetes group compared to the control group. Similarly, the expression of versican (VCAN) and decorin (DCN) was upregulated twofold in the diabetic group. At the same time, 2DM patients and controls show alterations in pro- and anti-inflammatory cytokine levels in the serum.

CONCLUSIONS

This study identifies considerable changes in the expression of transcription factors and extracellular matrix (ECM) components of bone in 2DM patients. Furthermore, the analysis of key differentiation factors of osteoblasts revealed significant alterations in gene expression of these factors, which may contribute to the dysregulation of energy metabolism in 2DM.

Analysis of the nucleotide sequence of the genome of the lactococcal bacteriophage r1t showed that it may encode at least two proteins involved in DNA replication. On the basis of its similarity with the G38P protein encoded by the Bacillus subtilis phage SPP1, the product of orf11 (Pro11) is thought to be involved in the initiation of phage DNA replication. This protein was overexpressed in Lactococcus lactis and partially purified. Gel retardation analysis using various r1t DNA fragments indicates that Pro11 specifically binds to a sequence located within its cognate gene. DNase I footprinting showed that Pro11 protects a stretch of DNA of 47 bp. This region spans four 6-bp short direct repeats, which suggests that the region contains four binding sites for Pro11. 1,10-Phenanthroline-copper footprinting confirmed the protection of the hexamers. An asymmetric protection pattern of each strand was observed, suggesting that Pro11 contacts each DNA strand separately at contiguous hexamers. We propose a model for the binding of Pro11 to its target sites that may account for the torsion strain required for strand opening at the origin of replication.

In the preceding paper (Amann et al. 1981) we described the in vitro construction of hybrids between Escherichia coli phage lambda NM607 imm434 and B. subtilis phage SPP1. These lambda/SPP1 hybrids have been used to infect minicells produced by E. coli strain DS410. Analysis on polyacrylamide gels of 35S-methionine labeled proteins synthesized in infected minicells revealed the expression of both lambda and SPP1 genes. Infection of E. coli minicells carrying plasmid pGY101, which encodes and expresses the repressor gene of phage 434, results in the selective expression of the cloned SPP1 DNA. This has resulted in the assignment of 26 out of a total of 46 known SPP1 polypeptides (Mertens et al. 1979) to individual SPP1 DNA fragments. In addition, several lambda/SPP1 fusion peptides whose transcription either originates from lambda promoters or from promoters located on the inserted SPP1 fragment, were identified.

GBK2 is a bacteriophage, isolated from a backyard compost pile, that infects the thermophile Geobacillus kaustophilus. GBK2 has a circularly permuted genome of 39,078 bp with a G+C content of 43 %. Annotation of the genome reveals 62 putative open reading frames (ORFs), 25 of which (40.3 %) show homology to known proteins and 37 of which (59.7 %) are proteins with unknown functions. Twelve of the identified ORFs had the greatest homology to genes from the phage SPP1, a phage that infects the mesophile Bacillus subtilis. The overall genomic arrangement of GBK2 is similar to that of SPP1, with the majority of GBK2 SPP1-like genes coding for proteins involved in DNA replication and metabolism.

Macrophages are the main drivers of obesity-induced adipose tissue (AT) inflammation that causes insulin resistance. Macrophages polarize toward different inflammatory (M1) or protective (M2) phenotypes. Osteopontin (OPN) is an inflammatory cytokine highly expressed in AT in obesity and known to be involved in chronic inflammatory processes. It was hypothesized that OPN polarizes macrophages into a proinflammatory phenotype.

AT macrophages (ATMs) of OPN-deficient (Spp1(-/-) ) and wild-type C57BL/6 (WT) mice with obesity and bone marrow-derived macrophages (BMDMs) of Spp1(-/-) and WT mice as well as human monocyte-derived macrophages (MDMs) polarized in the presence of OPN were investigated.

While ATM infiltration was lower in Spp1(-/-) upon high-fat diet, Spp1(-/-) ATMs expressed more M1 and less M2 markers but less tumor necrosis factor-α compared with WT. There was no effect of OPN deficiency on BMDM polarization. In human MDMs, the presence of OPN during polarization ambiguously altered M1/M2-related marker expression and diminished LPS-induced inflammatory cytokine production. Strikingly, phagocytic activity was elevated by the presence of OPN during polarization in both human MDMs and murine BMDMs.

In contradiction to our hypothesis, data indicated that OPN does not induce inflammatory macrophages but was a signal to induce phagocytosis, which may be required due to increased adipocyte death in obesity.

SPP1 is a siphophage infecting the gram-positive bacterium Bacillus subtilis. The SPP1 tail electron microscopy (EM) reconstruction revealed that it is mainly constituted by conserved structural proteins such as the major tail proteins (gp17.1), the tape measure protein (gp18), the Distal tail protein (Dit, gp19.1), and the Tail associated lysin (gp21). A group of five small genes (22-24.1) follows in the genome but it remains to be elucidated whether their protein products belong or not to the tail. Noteworthy, an unassigned EM density accounting for ~245 kDa is present at the distal end of the SPP1 tail-tip. We report here the gp23.1 crystal structure at 1.6 A resolution, a protein that lacks sequence identity to any known protein. We found that gp23.1 forms a hexamer both in the crystal lattice and in solution as revealed by light scattering measurements. The gp23.1 hexamer does not fit well in the unassigned SPP1 tail-tip EM density and we hypothesize that this protein might act as a chaperone.

OBJECTIVE

The aim of this study was to analyze the capacity of a new modified laser surface to stimulate calvarial osteoblasts isolated from neonatal mouse bones to differentiate and form mineralized nodules.

METHODS

Titanium discs were subjectezd or not to laser irradiation according to specific parameters and characterized. Osteoblasts isolated from neonatal mouse calvaria were cultured over the discs, and the capacity of these cells to proliferate (MTT assay), form mineralized nodules (Alizarin red assay), and enhance alkaline phosphatase activity (ALPase activity) was analyzed. Real-time PCR was used for quantification of gene expression.

RESULTS

Laser-irradiated titanium discs (L) presented a rough nano-to-micrometric oxidized surface contrasting with the smooth pattern on polished discs (P). The Ra on the micrometric level increased from 0.32 ± 0.01 μm on P surfaces to 10.57 ± 0.39 μm on L surfaces. When compared with P, L promoted changes in osteoblast morphology, increased mineralized nodule formation in osteoblasts cultured on the surfaces for 14 days, and enhanced ALPase activity at days 7 and 14. Transcription factors triggering osteoblast differentiation (Runx2 and Sp7) and genes encoding the bone extracellular matrix proteins collagen type-1 (Col1a1), osteopontin (Spp1), and osteocalcin (Bglap) were upregulated in cells on L surfaces compared with those on P surfaces at days 1-14.

CONCLUSIONS

Laser treatment of titanium surfaces created a rough surface that stimulated osteoblast differentiation.

CONCLUSIONS

Laser treatment of titanium generates a reproducible and efficient surface triggering osteoblast differentiation that can be of importance for osteointegration.

The present study investigated the effects of CHs on osteogenic activities and MAPK-regulation on bone matrix gene expressions. The effects of CHs on cell proliferation, alkaline phosphatase (ALP) activity, collagen synthesis, and mineralization were measured in human osteoblastic MG-63 cells. Activation of MAPKs and downstream transcription factors such as extracellular-signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase 1/2 (JNK1/2), p38, ELK1, and cJUN was examined using Western blot analysis. The expressions of osteogenic genes were measured by quantitative real-time PCR. CHs dose-dependently increased MG-63 cell proliferation, ALP activity, collagen synthesis, and calcium deposition. CHs activated ERK1/2, JNK1/2, p38, and ELK1 phosphorylation except cJUN. The COL1A1 (collagen, type I, alpha 1), ALPL (alkaline phosphatase), BGLAP (osteocalcin), and SPP1 (secreted phosphoprotein 1, osteopontin) gene expressions were increased by CH treatment. The ERK1/2 inhibitor (PD98509) blocked the CH-induced COL1A1 and ALPL gene expression, as well as ELK1 phosphorylation. The JNK1/2 inhibitor (SP600125) abolished CH-induced COL1A1 expression. The p38 inhibitor (SB203580) blocked CH-induced COL1A1 and SPP1 gene expression. In conclusion, CH treatment stimulates the osteogenic activities and increases bone matrix gene expressions via the MAPK/ELK1 signaling pathway. These results could provide a mechanistic explanation for the bone-strengthening effects of CHs.

In previous studies, fluorapatite (FA) crystal-coated surfaces have been shown to stimulate the differentiation and mineralization of human dental pulp stem cells (DPSCs) in two-dimensional cell culture. However, whether the FA surface can recapitulate these properties in three-dimensional culture is still unknown. This study examined the differences in behavior of human DPSCs cultured on electrospun polycaprolactone (PCL) NanoECM nanofibers with or without the FA crystals. Under near-physiologic conditions, the FA crystals were synthesized on the PCL nanofiber scaffolds. The FA crystals were evenly distributed on the scaffolds. DPSCs were cultured on the PCL+FA or the PCL scaffolds for up to 28 days. Scanning electron microscope images showed that DPSCs attached well to both scaffolds after the initial seeding. However, it appeared that more multicellular aggregates formed on the PCL+FA scaffolds. After 14 days, the cell proliferation on the PCL+FA was slower than that on the PCL-only scaffolds. Interestingly, even without any induction of mineralization, from day 7, the upregulation of several pro-osteogenic molecules (dmp1, dspp, runx2, ocn, spp1, col1a1) was detected in cells seeded on the PCL+FA scaffolds. A significant increase in alkaline phosphatase activity was also seen on FA-coated scaffolds compared with the PCL-only scaffolds at days 14 and 21. At the protein level, osteocalcin expression was induced only in the DPSCs on the PCL+FA surfaces at day 21 and then significantly enhanced at day 28. A similar pattern was observed in those specimens stained with Alizarin red and Von Kossa after 21 and 28 days. These data suggest that the incorporation of FA crystals within the three-dimensional PCL nanofiber scaffolds provided a favorable extracellular matrix microenvironment for the growth, differentiation, and mineralization of human DPSCs. This FA-modified PCL nanofiber scaffold shows promising potential for future bone, dental, and orthopedic regenerative applications.

Human cells, when exposed to both real and simulated microgravity (s-µg), form 3D tissue constructs mirroring in vivo architectures (e.g., cartilage, intima constructs, cancer spheroids and others). In this study, we exposed human foetal osteoblast (hFOB 1.19) cells to a Random Positioning Machine (RPM) for 7 days and 14 days, with the purpose of investigating the effects of s-µg on biological processes and to engineer 3D bone constructs. RPM exposure of the hFOB 1.19 cells induces alterations in the cytoskeleton, cell adhesion, extra cellular matrix (ECM) and the 3D multicellular spheroid (MCS) formation. In addition, after 7 days, it influences the morphological appearance of these cells, as it forces adherent cells to detach from the surface and assemble into 3D structures. The RPM-exposed hFOB 1.19 cells exhibited a differential gene expression of the following genes: transforming growth factor beta 1 (TGFB1, bone morphogenic protein 2 (BMP2), SRY-Box 9 (SOX9), actin beta (ACTB), beta tubulin (TUBB), vimentin (VIM), laminin subunit alpha 1 (LAMA1), collagen type 1 alpha 1 (COL1A1), phosphoprotein 1 (SPP1) and fibronectin 1 (FN1). RPM exposure also induced a significantly altered release of the cytokines and bone biomarkers sclerostin (SOST), osteocalcin (OC), osteoprotegerin (OPG), osteopontin (OPN), interleukin 1 beta (IL-1β) and tumour necrosis factor 1 alpha (TNF-1α). After the two-week RPM exposure, the spheroids presented a bone-specific morphology. In conclusion, culturing cells in s-µg under gravitational unloading represents a novel technology for tissue-engineering of bone constructs and it can be used for investigating the mechanisms behind spaceflight-related bone loss as well as bone diseases such as osteonecrosis or bone injuries.

Gene expression and associations were examined in a model of heart failure with preserved ejection fraction (HFpEF), a condition with minimal effective treatment. Genes with at least two studies showing significant changes in Dahl rat with heart failure were examined by meta-analysis. Significantly increased in expression were iNOS, p47phox, ADM, ANP, OPN, ACE, MCP-1, GP91PHOX, ICAM-1, TGF-β1, CTGF, ET-1, p22phox, ETB, BNP, ETA, MMP13, Col1a1, MMP2, TIMP1, Col3a1, Il-1β, β-MHC, ECE1, MMP14, AGT, and MMP9. In contrast, GLUT4, VEGF, eNOS, HIF-1α, and PGC1-α were significantly decreased in expression. The top biological process clusters identified in Database for Annotation, Visualization and Integrated Discovery, ToppGene, and PANTHER were collagen metabolic process, cellular ion homeostasis, regulation of cell migration, and response to decreased oxygen levels. These data suggest refocusing understanding of the pathophysiology of HFpEF to pathways involved in collagen metabolism, cell migration likely for inflammatory cells, and responses to decreased oxygen levels. Abbreviations Inducible nitric oxide synthase (INOS), neutrophil cytosolic factor 1 (p47phox), adrenomedullin (ADM), atrial natriuretic peptide (ANP), osteopontin (OPN/SPP1), angiotensin converting enzyme (ACE), monocyte chemotactic protein 1 (MCP-1), cytochrome b-245 beta polypeptide (gp91phox), intercellular adhesion molecule 1 (ICAM-1), transforming growth factor beta 1 (TGF-β1), connective tissue growth factor (CTGF), endothelin-1 (ET-1), cytochrome B-245, alpha polypeptide (p22phox), endothelin receptor type B (ETB/EDNRB), brain natriuretic peptide (BNP), endothelin receptor type A (ETA/EDNRA), matrix metallopeptidase 13 (MMP13), type I collagen (Col1a1), matrix metallopeptidase 2 (MMP2), TIMP metallopeptidase inhibitor 1 (TIMP1), Type III collagen (Col3a1), interleukin 1 beta (IL-1β), beta myocin heavy chain (β-MHC), endothelin converting enzyme 1 (ECE1) matrix metallopeptidase 14 (MMP14), angiotensinogen (AGT), angiotensin II receptor Type 1 (AT1R), cytochrome C oxidase I (COX1), fms-like tyrosine kinase 1 (FLT1), TIMP metallopeptidase inhibitor 2 (TIMP2), phospholamban (PLN), vascular cell adhesion molecule 1 (VCAM1), extracellular matrix (ECM).

Reverse transcription (RT) quantitative real-time PCR (qPCR) is the most accurate and easy-to-perform technique to measure the expression level of a selected gene of interest by quantifying mRNA transcripts. The use of reference genes is commonly accepted as the most reliable approach to normalize RT-qPCR data and reduce possible errors generated in the quantification of gene expression. The optimal number and choice of reference genes are experimentally validated for specific tissues or cell types and experimental designs. To date, data on qPCR normalization in goats are scarce and the most suitable reference genes in this species have been identified for only a limited number of tissues. The aim of this study was to determine an optimal combination of stably expressed reference genes in caprine milk somatic cells (MSC) from healthy and infected mammary glands. For the purpose, we performed RT-qPCR for 10 commonly used reference genes from various functional classes and then determined their expression level in MSC from goats intramammary challenged with Staphylococcus aureus and in MSC from healthy controls, with a view to select genes whose stability would be unaffected under infection conditions. The geNorm and NormFinder algorithms were used for validating the reference genes. Furthermore, to demonstrate the importance of normalization of gene expression with appropriate reference genes, we tested the effect of using a combination of the least stable genes for expression analysis evaluation. On the basis of our evaluation, we recommend the use of a panel of reference genes that should include G6PD, YWHAZ, and ACTB for caprine MSC gene expression profiling. The expression of the 2 genes of interest, pentraxin-related protein (PTX3) and secreted phosphoprotein 1 (SPP1), was evaluated by RT-qPCR in all samples collected pre- and postinfection, and the recommended reference genes were used to normalize the data. Our study provides a validated panel of optimal reference genes for the identification of genes differentially expressed by qRT-PCR in caprine MSC. Moreover, we provided a set of intron-spanning primer sequences that could be suitable for gene expression experiments using SYBR Green chemistry on other caprine tissues and cells.

Cholangiocarcinoma (CCA) continues to harbor a difficult prognosis and it is difficult to diagnose in its early stages. The molecular mechanisms of CCA oncogenesis and progression are poorly understood. This study aimed to identify candidate biomarkers for CCA. Integrated analysis of microarray data sets was performed to identify differentially expressed genes (DEGs) between CCA and normal tissues. Gene Ontology enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were then performed to identify the functions of DEGs. Furthermore, the protein-protein interaction (PPI) network of DEGs was constructed. The expressions of DEGs were validated in human CCA tissues by qRT-PCR. A set of 712 DEGs were identified in CCA compared with normal tissues, including 306 upregulated and 406 downregulated DEGs. It can be shown from the KEGG pathway analysis that some pathways may have important roles in pathology of CCA, including peroxisome proliferator-activated receptor signaling pathway, bile secretion, cell cycle, fat digestion and absorption. PPI network indicated that the significant hub proteins were PKM, SPP1 and TPM1. The abnormally overexpression PKM, SPP1 and TPM1 were closely related to oncogenesis and progression of CCA. PKM, SPP1, TPM1, COL1A1 and COL1A2 may serve as candidate biomarkers for diagnosis and prognosis of CCA.

In our previous study, calcitriol and its analogs PRI-2191 and PRI-2205 stimulated 4T1 mouse mammary gland cancer metastasis. Therefore, we aimed to analyze the inflammatory response in 4T1-bearing mice treated with these compounds. Gene expression analysis of the splenocytes and regional lymph nodes demonstrated prevalence of the T helper lymphocytes (Th2) response with an increased activity of regulatory T (Treg) lymphocytes in mice treated with these compounds. We also observed an increased number of mature granulocytes and B lymphocytes and a decreased number of TCD4⁺, TCD4⁺CD25⁺, and TCD8⁺, as well as natural killer (NK) CD335⁺, cells in the blood of mice treated with calcitriol and its analogs. Among the splenocytes, we observed a significant decrease in NK CD335⁺ cells and an increase in TCD8⁺ cells. Calcitriol and its analogs decreased the levels of interleukin (IL)-1β and IL-10 and increased the level of interferon gamma (IFN-γ) in the plasma. In the tumor tissue, they caused an increase in the level of IL-10. Gene expression analysis of lung tissue demonstrated an increased level of osteopontin (Spp1) and transforming growth factor β (TGF-β) mRNA. The expression of Spp1 was also elevated in lymph nodes. Calcitriol and its analogs caused prevalence of tumor-conducive changes in the immune system of 4T1 tumor-bearing mice, despite the induction of some tumor-disadvantageous effects.

Overexpression of osteopontin (OPN) could contribute to tumorigenesis and metastasis in hepatocellular carcinoma (HCC). Previous studies have shown that OPN-positive cancer cells are often localized in the periphery of cancer nodules adjacent to stromal cells. To identify the difference in intratumor genomic aberration pattern between OPN-positive and OPN-negative HCC cells, we adopted microarray-based comparative genomic hybridization (array-CGH) to achieve high-resolution analysis of genome-wide aberrations. Our present study indicates that, compared to OPN-negative cancer cells, OPN-positive cancer cells show much more amplification of chromosomal regions, including 4q13.1-q13.3, 4q21.23-q22.1 and 13q32.1-q32.3. Some candidate tumor related genes, such as SMR3B, MUC7, EPHA5, SPP1 and CLDN10, were detected with over 1.5-fold amplification. Together, the intratumor genomic heterogeneity may suggest that OPN-positive cancer cells play a more important role in the development of HCC malignancy than their OPN-negative counterparts.

The uterine endometrium plays a critical role in regulating the estrous cycle and the establishment and maintenance of pregnancy in mammalian species. Many studies have investigated the expression and function of genes in the uterine endometrium, but the global expression pattern of genes and relationships among genes differentially expressed in the uterine endometrium during gestation in pigs remain unclear. Thus, this study investigated global gene expression profiles using microarray in pigs. Diverse transcriptome analyses including clustering, network, and differentially expressed gene (DEG) analyses were performed to detect endometrial gene expression changes during the different gestation stages. In total, 6,991 genes were found to be differentially expressed by comparing genes expressed on day (D) 12 of pregnancy with those on D15, D30, D60, D90 and D114 of pregnancy, and clustering analysis of detected DEGs distinguished 8 clusters. Furthermore, several pregnancy-related hub genes such as ALPPL2, RANBP17, NF1B, SPP1, and CST6 were discovered through network analysis. Finally, detected hub genes were technically validated by quantitative RT-PCR. These results suggest the complex network characteristics involved in uterine endometrial gene expression during pregnancy and indicate that diverse patterns of stage-specific gene expression and network connections may play a critical role in endometrial remodeling and in placental and fetal development to establish and maintenance of pregnancy in pigs.

BACKGROUND

Additional accurate non-invasive biomarkers are needed in the clinical setting to improve prostate cancer (PCa) diagnosis. Here we have developed a new and improved multiplex mRNA urine test to detect prostate cancer (PCa). Furthermore, we have validated the PCA3 urinary transcript and some panels of urinary transcripts previously reported as useful diagnostic biomarkers for PCa in our cohort.

METHODS

Post-prostatic massage urine samples were prospectively collected from PCa patients and controls. Expression levels of 42 target genes selected from our previous studies and from the literature were studied in 224 post-prostatic massage urine sediments by quantitative PCR. Univariate logistic regression was used to identify individual PCa predictors. A variable selection method was used to develop a multiplex biomarker model. Discrimination was measured by ROC curve AUC for both, our model and the previously published biomarkers.

RESULTS

Seven of the 42 genes evaluated (PCA3, ELF3, HIST1H2BG, MYO6, GALNT3, PHF12 and GDF15) were found to be independent predictors for discriminating patients with PCa from controls. We developed a four-gene expression signature (HIST1H2BG, SPP1, ELF3 and PCA3) with a sensitivity of 77% and a specificity of 67% (AUC = 0.763) for discriminating between tumor and control urines. The accuracy of PCA3 and previously reported panels of biomarkers is roughly maintained in our cohort.

CONCLUSIONS

Our four-gene expression signature outperforms PCA3 as well as previously reported panels of biomarkers to predict PCa risk. This study suggests that a urinary biomarker panel could improve PCa detection. However, the accuracy of the panels of urinary transcripts developed to date, including our signature, is not high enough to warrant using them routinely in a clinical setting.