Recent studies have shown that osteopontin, a cytokine with suggested immunoregulatory functions, may contribute to pathogenesis of asthma. To determine whether single-nucleotide polymorphisms (SNPs) in SPP1, the gene encoding osteopontin, are associated with risk of asthma, we genotyped 6 known SNPs in SPP1 in the well-characterized Genetics of Asthma in Latino Americans population of 294 Mexican and 365 Puerto Rican parent-child asthma trios. The associations between SNPs and asthma or asthma-related phenotypes were examined by transmission disequilibrium tests as implemented in the family-based association test program. Three polymorphisms, 1 in exon 7 (rs1126616C) and 2 in the 3'-untranslated region (rs1126772A and rs9138A) of SPP1, were associated with diagnosis of asthma, severity of asthma, asthma in subjects with elevated immunoglobulin E (IgE) (IgE >100 IU/mL), and postbronchodilator FEV(1) in Puerto Ricans (P values=0.00007-0.04). The CC genotype of rs1126616 conferred an odds ratio of 1.7 (95% CI=[1.3, 2.3], P value adjusted for multiple comparisons=0.001) for asthma compared with the CT and TT genotypes. Furthermore, haplotype analysis identified rs1126616C-rs1126772A-rs9138A to be associated with an increased risk for asthma, severity of asthma, and asthma in subjects with elevated IgE (P=0.03). There was no association between the SPP1 SNPs and asthma outcomes in Mexicans. Our findings suggest that the SPP1 gene is a risk factor for asthma and asthma-related phenotypes in Puerto Ricans, and are consistent with previous animal and human studies on the role of osteopontin in pathogenesis of asthma.

Altered epigenetic control of gene expression plays a substantial role in tumor development and progression. Accumulating studies suggest that somatic mutations of CREB binding proteins (CBP)/p300 occur in some cancer cells. CBP/p300 possess histone acetyltransferase (HAT) activity, and are involved in many cellular processes. In this study, we investigated the expression and functional role of CBP/p300 in hepatocellular carcinoma (HCC) using the specific inhibitor C646 of CBP/p300 HAT activity. We examined its effect on several apoptosis-related proteins and invasion-related genes. The results showed that CBP/p300 were highly expressed in HCC tissues and that expression of p300, but not of CBP, was strongly correlated with the malignant character of HCC. C646 inhibited proliferation of HCC cell lines in a dose dependent manner. C646 significantly augmented TRAIL-induced apoptotic sensitivity, which was accompanied by reduced levels of survivin, in HepG2, HLE and SK-HEP1 cells. C646 significantly inhibited invasion of Huh7, HLE and SK-HEP1 cells. The level of matrix metallopeptidase 15 (MMP15) mRNA expression was significantly reduced, whereas the level of laminin alpha 3 (LAMA3) and secreted phosphoprotein 1 (SPP1) mRNA expression was significantly increased in Huh7 cells following exposure to C646. In conclusion, our results suggest that CBP/p300 HAT activity has an important role in malignant transformation, proliferation, apoptotic sensitivity and invasion in HCC. CBP/p300 could be a promising therapeutic target in HCC.

The aim of this study was to compare cytokine expression on both gene and protein levels in acute and chronic phase of HIV type 1 (HIV-1) infection. Thirty four patients were enrolled for cytokine expression analysis on protein level in acute and chronic stage of HIV-1 infection. Using PCR array technology, expression of 84 cytokine genes was measured in 3 patients in acute and 3 patients in chronic stage of HIV-1 infection. Bead-based cytometry was used to quantify levels of Th1/Th2/Th9/Th17/Th22 cytokines. The results showed statistically significant increase of 13 cytokine gene expression (cd40lg, csf2, ifna5, il12b, il1b, il20, lta, osm, spp1, tgfa, tnfsf 11, 14 and 8) and downregulation of the il12a expression in chronic HIV type 1 infection. Concentrations of IL-10, IL-4 and TNF-α were increased in the acute HIV type 1 infection when compared to control group. During chronic HIV type 1 infection there was an increase of IL-10, TNF-α, IL-2, IL-6, IL-13 and IL-22 levels when compared to control group. Comparison of cytokine expression between two stages of infection showed a significant decrease in IL-9 concentration. This study showed changes in cytokine profiles on both gene and protein levels in different stages of HIV-infection.

Lysosome membrane permeabilization (LMP) mediates cell death in a variety of cancer cells. However, little is known about lysosomes and LMP in chronic lymphocytic leukemia (CLL). Owing to drug resistance and toxicity in CLL patients, better treatment strategies are required. Our results show that CLL cells were sensitive to the lysosomotropic agent siramesine. Furthermore, this drug was more effective in CLL cells, regardless of prognostic factors, compared with normal B cells. Siramesine caused LMP, lipid peroxidation and transcription factor EB nuclear translocation followed by mitochondrial membrane potential loss and reactive oxygen species release. Siramesine-induced cell death was blocked by lipid antioxidants, but not by soluble antioxidants or protease inhibitors. To determine whether CLL cells had altered lysosomes, we investigated sphingolipid metabolism as the lysosome is a hub for lipid metabolism. We found that CLL cells had more lysosomes, increased sphingosine-1-phosphate phosphatase 1 (SPP1) expression, and increased levels of sphingosine compared with normal B cells. Raising sphingosine levels increased LMP and cell death in CLL cells, but not in normal B cells. Together, these results show that excess sphingosine in CLL cells could contribute to their sensitivity toward LMP. Thus, targeting the lysosome could be a novel therapeutic strategy in CLL.

Gastric cancer (GC) is a common heterogeneous disease. The critical roles of microRNA-340 (miR-340) in the development and progression of GC were emphasized in accumulating studies. This study aims to examine the regulatory mechanism of miR-340 in GC cellular processes. Initially, microarray technology was used to identify differentially expressed genes and regulatory miRs in GC. After that, the potential role of miR-340 in GC was determined via ectopic expression, depletion, and reporter assay experiments. Expression of secreted phosphoprotein 1 (SPP1), miR-340, phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway, and epithelial-mesenchymal transition (EMT)-related genes was measured. Moreover, to further explore the function of miR-340 in vivo and in vitro, proliferation, apoptosis, migration, invasion, and tumorigenic capacity were evaluated. SPP1 was a target gene of miR-340 which could then mediate the PI3K/AKT signaling pathway by targeting SPP1 in GC. Furthermore, miR-340 levels were reduced and SPP1 was enriched in GC tissues and cells, with the PI3K/AKT signaling pathway being activated. Inhibitory effects of upregulated miR-340 on SPP1 and the PI3K/AKT signaling pathway were confirmed in vivo and in vitro. Overexpression of miR-340 or the silencing of SPP1 inhibited GC cell proliferation, invasion, migration, and EMT process, but promoted apoptosis of GC cells. Typically, targeting of SPP1 by miR-340 may contribute to the inhibition of proliferation, migration, invasion, and EMT of GC cells via suppression of PI3K/AKT signaling pathway.

BACKGROUND

In Duchenne muscular dystrophy (DMD), abnormal cardiac function is typically preceded by a decade of skeletal muscle disease. Molecular reasons for differences in onset and progression of these muscle groups are unknown. Human biomarkers are lacking.

METHODS

We analyzed cardiac and skeletal muscle microarrays from normal and golden retriever muscular dystrophy (GRMD) dogs (ages 6, 12, or 47+ mo) to gain insight into muscle dysfunction and to identify putative DMD biomarkers. These biomarkers were then measured using human DMD blood samples.

RESULTS

We identified GRMD candidate genes that might contribute to the disparity between cardiac and skeletal muscle disease, focusing on brain-derived neurotropic factor (BDNF) and osteopontin (OPN/SPP1, hereafter indicated as SPP1). BDNF was elevated in cardiac muscle of younger GRMD but was unaltered in skeletal muscle, while SPP1 was increased only in GRMD skeletal muscle. In human DMD, circulating levels of BDNF were inversely correlated with ventricular function and fibrosis, while SPP1 levels correlated with skeletal muscle function.

CONCLUSIONS

These results highlight gene expression patterns that could account for differences in cardiac and skeletal disease in GRMD. Most notably, animal model-derived data were translated to DMD and support use of BDNF and SPP1 as biomarkers for cardiac and skeletal muscle involvement, respectively.

BACKGROUND

We are using ACI and BN rats, which differ markedly in their susceptibility to 17β-estradiol (E2)-induced mammary cancer, to identify genetic variants and environmental factors that determine mammary cancer susceptibility. The objective of this study was to characterize the cellular and molecular responses to E2 in the mammary glands of ACI and BN rats to identify qualitative and quantitative phenotypes that associate with and/or may confer differences in susceptibility to mammary cancer.

METHODS

Female ACI and BN rats were treated with E2 for 1, 3 or 12 weeks. Mammary gland morphology and histology were examined by whole mount and hematoxylin and eosin (H&E) staining. Cell proliferation and epithelial density were evaluated by quantitative immunohistochemistry. Apoptosis was evaluated by quantitative western blotting and flow cytometry. Mammary gland differentiation was examined by immunohistochemistry. Gene expression was evaluated by microarray, qRT-PCR and quantitative western blotting assays. Extracellular matrix (ECM) associated collagen was evaluated by Picrosirius Red staining and Second Harmonic Generation (SHG) microscopy.

RESULTS

The luminal epithelium of ACI rats exhibited a rapid and sustained proliferative response to E2. By contrast, the proliferative response exhibited by the mammary epithelium of BN rats was restrained and transitory. Moreover, the epithelium of BN rats appeared to undergo differentiation in response to E2, as evidenced by production of milk proteins as well as luminal ectasia and associated changes in the ECM. Marked differences in expression of genes that encode proteins with well-defined roles in mammary gland development (Pgr, Wnt4, Tnfsf11, Prlr, Stat5a, Areg, Gata3), differentiation and milk production (Lcn2, Spp1), regulation of extracellular environment (Mmp7, Mmp9), and cell-cell or cell-ECM interactions (Cd44, Cd24, Cd52) were observed.

CONCLUSIONS

We propose that these cellular and molecular phenotypes are heritable and may underlie, at least in part, the differences in mammary cancer susceptibility exhibited by ACI and BN rats.

The costimulatory receptor Slamf6 partially controls lupus-related autoimmunity in congenic Sle1b mice; for instance, the presence of the protein isoform Slamf6-H1 in Sle1b.Slamf6-H1 mice mitigates disease. Here, we report that young Sle1b mice, but not Sle1b.Slamf6-H1 or B6 mice, contain a memory T-helper cell subset identified by ]mt]2-fold increase in expression of 17 genes, chief among which is Spp1, encoding the cytokine osteopontin (OPN). These T follicular helper (TFH) cells, including OPN(+) TFH cells, expand concomitantly with severity of the disease. By contrast, Sle1b.Slamf6-H1 or Sle1b.SAP(-)/(-) mice do not develop autoantibodies and the number of T(FH) cells is 5 times lower than in age-matched Sle1b mice. By comparing Sle1b and Sle1b.OPN(-)/(-) mice, we find that the lack of OPN expression impedes early autoantibody production. Furthermore, on the adoptive transfer of Sle1b.OPN(-)/(-) CD4(+) T cells into bm12 recipients autoantibody production and germinal center formation is reduced compared to recipients of Sle1b.OPN(+/+) CD4(+) T cells. We propose a model in which OPN provides a survival signal for a precursor T(FH) cell subset, which is a key factor in autoimmunity.

BACKGROUND

The aim of this study was to identify key genes and novel potential therapeutic targets related to gastric cancer (GC) by comparing cancer tissue samples and healthy control samples using DNA microarray analysis.

METHODS

Microarray data set GSE19804 was downloaded from Gene Expression Omnibus. Preprocessing and differential analysis were conducted with of R statistical software packages, and a number of differentially expressed genes (DEGs) were obtained. Cluster analysis was also done with gene expression values. Functional enrichment analysis was performed for all the DEGs with DAVID tools. The significantly up- and downregulated genes were selected out and their interactors were retrieved with STRING and HitPredict, followed by construction of networks. For all the genes in the two networks, GeneCodis was chosen for gene function annotation.

RESULTS

A total of 638 DEGs were identified, and we found that SPP1 and FABP4 were the markedly up- and downregulated genes, respectively. Cell cycle and regulation of proliferation were the most significantly overrepresented functional terms in up- and downregulated genes. In addition, extracellular matrix-receptor interaction was found to be significant in the SPP1-included interaction network.

CONCLUSIONS

A range of DEGs were obtained for GC. These genes not only provided insights into the pathogenesis of GC but also could develop into biomarkers for diagnosis or treatment.

The aim of this study is to identify candidate biomarkers for the detection of hepatocellular carcinoma (HCC) through pre-cancerous gene expression analysis in an HBx transgenic mouse model. The gene expression profiles of liver and tumor tissues from 6-, 12- and 18-month-old HBx transgenic and littermate control mice were monitored using DNA oligo microarrays. Data analysis revealed changes in 684 genes in the tumor tissues from HBx transgenic mice. Based on their pre-cancerous expression profiles, two separate gene groups, corresponding to HCC specific and non-specific groups respectively, were identified. Gene ontology analysis identified 47 genes encoding secretory or transmembrane proteins among 155 upregulated genes in the HCC-specific group. Among these, four genes encoding TFF3, IGF2, LPL and SPP1 were found to be promising biomarker candidates for the detection of HCC as compared with AFP. This work describes a new way to identify novel biomarker candidates for HCC diagnosis.

Transcriptional complexes formed in vitro using DNA of B. subtilis phage SPP1 as template and E. coli and B. subtilis RNA polymerases were analyzed by electron microscopy. Both enzymes recognize the same five strong promoters in the early region of the genome. Strand selection at these sites was identical with both enzymes. These results correlate well with data obtained from in vivo transcription studies. Transcriptional activity in the late region of the genome was very low, not permitting the identification of promoter sites.

OBJECTIVE

To identify genes and molecular mechanisms associated with photoreceptor degeneration in a canine model of XLRP caused by an RPGR exon ORF15 microdeletion. Methods. Expression profiles of mutant and normal retinas were compared by using canine retinal custom cDNA microarrays. qRT-PCR, Western blot analysis, and immunohistochemistry (IHC) were applied to selected genes, to confirm and expand the microarray results.

RESULTS

At 7 and 16 weeks, respectively, 56 and 18 transcripts were downregulated in the mutant retinas, but none were differentially expressed (DE) at both ages, suggesting the involvement of temporally distinct pathways. Downregulated genes included the known retina-relevant genes PAX6, CHML, and RDH11 at 7 weeks and CRX and SAG at 16 weeks. Genes directly or indirectly active in apoptotic processes were altered at 7 weeks (CAMK2G, NTRK2, PRKCB, RALA, RBBP6, RNF41, SMYD3, SPP1, and TUBB2C) and 16 weeks (SLC25A5 and NKAP). Furthermore, the DE genes at 7 weeks (ELOVL6, GLOD4, NDUFS4, and REEP1) and 16 weeks (SLC25A5 and TARS2) are related to mitochondrial functions. qRT-PCR of 18 genes confirmed the microarray results and showed DE of additional genes not on the array. Only GFAP was DE at 3 weeks of age. Western blot and IHC analyses also confirmed the high reliability of the transcriptomic data.

CONCLUSIONS

Several DE genes were identified in mutant retinas. At 7 weeks, a combination of nonclassic anti- and proapoptosis genes appear to be involved in photoreceptor degeneration, whereas at both 7 and 16 weeks, the expression of mitochondria-related genes indicates that they may play a relevant role in the disease process.

The left end of the genome of Bacillus subtilis bacteriophage SPP1 is represented by EcoRI DNA fragments 12 and 1 (EcoRI-12 and EcoRI-1). A number of different deletions were identified in EcoRI-1. A detailed physical and genetic map of EcoRI-1 from wild-type (wt) phage and SPP1 deletion mutants was constructed. Genes encoding essential products involved in late and early stages of phage DNA metabolism were mapped at the left and right ends of the 8.5-kb EcoRI-1, respectively. Deletions fell within the internal 5157-bp DNA segment of EcoRI-1. The nucleotide (nt) sequence of this region and of the endpoints of two deletions, delta X and delta L, were determined. The nt sequence of the junctions in SPP1 delta X and SPP1 delta L showed that, in these deletions, a segment of DNA between short directly repeated sequences of 10 and 13 bp, located 3427 and 4562 bp apart in the wt sequence, had been eliminated. In both cases, the copy of the repeated sequence was retained in the deletion mutant, consistent with the hypothesis that the deletions originated by homologous intramolecular recombination. The corresponding region in wt phage had fifteen presumptive open reading frames (orfs) and the previously identified SPP1 early promoters (PE1). The poor growth phenotype associated with the SPP1 deletion mutants was attributed to premature transcriptional read through from promoter(s) of the early region into late operon brought into close vicinity of the late genes due to the deletion event.

Bacillus subtilis bacteriophage SPP1 G40P hexameric replicative DNA helicase unidirectionally translocates with a 5'-->3' polarity while separating the DNA strands. A G40P mutant derivative lacking the N-terminal domain (containing amino acid residues 110-442 from G40P, G40PDeltaN109) was purified and characterized. G40PDeltaN109 showed an ATPase activity that was dependent on the presence of single-stranded (ss) DNA. Unlike G40P, G40PDeltaN109 was shown to bind with similar affinity both ssDNA arms of forked structures by nuclease protection assays. In a pH-dependent manner, G40PDeltaN109 unwound a branched double-arm substrate preferentially with a 3'-->5' polarity. Our results show that the linker region and the C-terminal domain of G40P are sufficient to render an enzyme capable of encircling the ssDNA tails of the forked DNA and to unwind DNA with both 5'-->3' and 3'-->5' polarity. The presence of the N-terminal domain, which does not play an essential role in helicase action, might be required indirectly for strand discrimination and polarity of translocation.

Fibroblast growth factor 8 (FGF-8) is expressed at an increased level in a high proportion of prostate cancers and it is associated with a poor prognosis of the disease. Our aim was to study the effects of FGF-8b on proliferation of PC-3 prostate cancer cells and growth of PC-3 tumors, and to identify FGF-8b-associated molecular targets. Expression of ectopic FGF-8b in PC-3 cells caused a 1.5-fold increase in cell proliferation in vitro and a four- to fivefold increase in the size of subcutaneous and orthotopic prostate tumors in nude mice. Tumors expressing FGF-8b showed a characteristic morphology with a very rich network of capillaries. This was associated with increased spread of the cancer cells to the lungs as measured by RT-qPCR of FGF-8b mRNA. Microarray analyses revealed significantly altered, up- and downregulated, genes in PC-3 cell cultures (169 genes) and in orthotopic PC-3 tumors (61 genes). IPA network analysis of the upregulated genes showed the strongest association with development, cell proliferation (CRIP1, SHC1), angiogenesis (CCL2, DDAH2), bone metastasis (SPP1), cell-to-cell signaling and energy production, and the downregulated genes associated with differentiation (DKK-1, VDR) and cell death (CYCS). The changes in gene expression were confirmed by RT-qPCR. In conclusion, our results demonstrate that FGF-8b increases the growth and angiogenesis of orthotopic prostate tumors. The associated gene expression signature suggests potential mediators for FGF-8b actions on prostate cancer progression and metastasis.

Two inhibitors of replicative deoxyribonucleic acid (DNA) synthesis, nalidixic acid (NAL) and 6-(p-hydroxyphenylazo)-uracil (HPUra), showed different effects on genetic recombination and DNA repair in Bacillus subtilis. Previous work (Pedrini et al., 1972) showed that NAL does not interfere with the transformation process of B. subtilis. The results reported in this work demonstrated that the drug was also without effect on the transfection by SPP1 or SPO-1 phage DNA (a process that requires a recombination event). The drug was also ineffective on the host cell reactivation of ultraviolet-irradiated SPP1 phage, as well as on transfection with ultraviolet-irradiated DNA of the same phage. HPUra instead markedly reduced the transformation process, as well as transfection, by SPO-1 DNA, but it did not affect the host cell reactivation of SPO-1 phage. In conclusion, whereas the NAL target seems to be specific for replicative DNA synthesis, the HPUra target (i.e., the DNA polymerase III of B. subtilis) seems to be involved also in recombination, but not in the excision repair process. The mutations conferring NAL and HPUra resistance used in this work were mapped by PBS-1 transduction.

The Bacillus subtilis bacteriophage SPP1 gene 40 product (G40P), which belongs to the DnaB-like family of helicases, is essential for SPP1 genome replication. The active form of the enzyme is the hexamer, capable of DNA unwinding with a 5' to 3' polarity fueled by the hydrolysis of a nucleoside 5'-triphosphate. We have used electron microscopy of negatively stained G40P samples and image processing techniques to study the structural characteristics of the hexameric assemblies of this protein. Our results provide the first low resolution data on a hexameric helicase of a Gram-positive bacterial origin. A novel approach has been adopted to analyze possible symmetry heterogeneities, an unsupervised method based on a neural network self-organizing algorithm, which has led to the detection of different subclasses of G40P views. Two different quaternary states of G40P homohexamers sharing a C3 symmetry organization have been found, as well as a minor class that seems to reflect an alternative C6 symmetry architecture. These forms show general features known for other hexameric helicases, such as the ring-like arrangement of monomers around a central hole. A clear structural handedness has also been detected in some of these forms. An analysis of these quaternary states and a model for the structural organization of G40P are presented.

A new procedure for the purification of B. subtilis RNA polymerase, based on mild lysis of cells, low speed centrifugation, gel filtration, DEAE-Sephadex chromatography and affinity chromatography on DNA-cellulose, yields three forms of enzyme referred here as enzyme A, B and C. As revealed by SDS gel electrophoresis, enzyme A has the subunit structure of core polymerase plus some small polypeptides. Its catalytic properties are similar to those of core polymerase. Enzyme B has the composition of core polymerase. Both enzymes A and B can be stimulated by the addition of beta factor. Enzyme C has the holo-enzyme composition. The pattern of sensitivity of the three forms of enzyme towards KCl are very different: enzymes A and B, even at low concentration of salt, are inhibited with all the DNA templates tested, whereas enzyme C shows a pattern of stimulation specific for each DNA tested. The transcripts of the three enzymes on phage SPP1 DNA template have been analyzed by hybridization to the separated strands. Only enzyme C selectively transcribed the H strands.

In Saccharomyces cerevisiae, all H3K4 methylation is performed by a single Set1 Complex (Set1C) that is composed of the catalytic (Set1) and seven other subunits (Swd1, Swd2, Swd3, Bre2, Sdc1, Spp1 and Shg1). It has been known for quite some time that trimethylated H3K4 (H3K4me3) is enriched in the vicinity of meiotic double-strand breaks (DSBs), but the link between H3K4me3 and the meiotic nuclease Spo11 was uncovered only recently. The PHD-containing subunit Spp1, by interacting with H3K4me3 and Mer2, was shown to promote the recruitment of potential meiotic DSB sites to the chromosomal axis allowing their subsequent cleavage by Spo11. Therefore, Spp1 emerged as a key regulator of the H3K4 trimethylation catalyzed by Set1C and of the formation of meiotic DSBs. These findings illustrate the remarkable multifunctionality of Spp1, which not only regulates the catalytic activity of the enzyme (Set1), but also interacts with the deposited mark, and mediates its biological effect (meiotic DSB formation) independently of the complex. As it was previously described for Swd2, and now for Spp1, we anticipate that other Set1C subunits, in addition to regulating H3K4 methylation, may participate in diverse biological functions inside or outside of the complex.

KMT2/Set1 is the catalytic subunit of the complex of proteins associated with Set1 (COMPASS) that is responsible for the methylation of lysine 4 of histone H3 (H3K4) in Saccharomyces cerevisiae. Whereas monomethylated H3K4 (H3K4me1) is found throughout the genome, di- (H3K4me2) and tri- (H3K4me3) methylated H3K4 are enriched at specific loci, which correlates with the promoter and 5'-ends of actively transcribed genes in the case of H3K4me3. The COMPASS subunits contain a number of domains that are conserved in homologous complexes in higher eukaryotes and are reported to interact with modified histones. However, the exact organization of these subunits and their role within the complex have not been elucidated. In this study we showed that: (1) subunits Swd1 and Swd3 form a stable heterodimer that dissociates upon binding to a modified H3K4me2 tail peptide, suggesting a regulatory role in COMPASS; (2) the affinity of the subunit Spp1 for modified histone H3 substrates is much higher than that of Swd1 and Swd3; (3) Spp1 has a preference for H3K4me2/3 methylation state; and (4) Spp1 contains a high-affinity DNA-binding domain in the previously uncharacterised C-terminal region. These data allow us to suggest a mechanism for the regulation of COMPASS activity at an actively transcribed gene.

BACKGROUND

Tumor cell invasion into the surrounding matrix has been well documented as an early event of metastasis occurrence. However, the dynamic expression patterns of proteins during early invasion of hepatocellular carcinoma (HCC) are largely unknown. Using a three-dimensional HCC invasion culture model established previously, we investigated the dynamic expression patterns of identified proteins during early invasion of HCC.

METHODS

Highly metastatic MHCC97H cells and a liver tissue fragment were long-term co-cultured in a rotating wall vessel (RWV) bioreactor to simulate different pathological states of HCC invasion. The established spherical co-cultures were collected on days 0, 5, 10, and 15 for dynamic expression pattern analysis. Significantly different proteins among spheroids at different time points were screened and identified using quantitative proteomics of iTRAQ labeling coupled with LC-MS/MS. Dynamic expression patterns of differential proteins were further categorized by K-means clustering. The expression modes of several differentially expressed proteins were confirmed by Western blot and qRT-PCR.

RESULTS

Time course analysis of invasion/metastasis gene expressions (MMP2, MMP7, MMP9, CD44, SPP1, CXCR4, CXCL12, and CDH1) showed remarkable, dynamic alterations during the invasion process of HCC. A total of 1,028 proteins were identified in spherical co-cultures collected at different time points by quantitative proteomics. Among these proteins, 529 common differential proteins related to HCC invasion were clustered into 25 types of expression patterns. Some proteins displayed significant dynamic alterations during the early invasion process of HCC, such as upregulation at the early invasion stage and downregulation at the late invasion stage (e.g., MAPRE1, PHB2, cathepsin D, etc.) or continuous upregulation during the entire invasion process (e.g., vitronectin, Met, clusterin, ICAM1, GSN, etc.).

CONCLUSIONS

Dynamic expression patterns of candidate proteins during the early invasion process of HCC facilitate the discovery of new molecular targets for early intervention to prevent HCC invasion and metastasis.

Bone strength traits in chickens are gaining importance due to economic losses and welfare concerns associated with bone fractures and other abnormalities. A chicken F2 resource population was generated from layer and broiler genetic lines, and traits relating to bone strength, egg production, egg quality and growth rate were measured in approximately 500 F2 hens. Four biological candidate genes (vitamin D receptor, VDR; insulin, INS; insulin-like growth factor 1, IGF1; and osteopontin, SPP1) were selected for investigation. Single nucleotide polymorphisms (SNPs) were identified for each candidate gene by comparing sequences between grandparent lines. Polymerase chain reaction restriction-fragment length polymorphism or SNaPshot assays were developed to genotype the F2 population and to evaluate associations between each SNP genotype and multiple phenotypes. Significant associations (P < 0.0125) were found between VDR and bone mineral content of the humerus at 35 weeks of age; between IGF1 and SPP1 and 5-week body weight; and between INS and 55-week body weight.

Complex viruses that encode their own initiation proteins and subvert the host's elongation apparatus have provided valuable insights into DNA replication. Using purified bacteriophage SPP1 and Bacillus subtilis proteins, we have reconstituted a rolling circle replication system that recapitulates genetically defined protein requirements. Eleven proteins are required: phage-encoded helicase (G40P), helicase loader (G39P), origin binding protein (G38P) and G36P single-stranded DNA-binding protein (SSB); and host-encoded PolC and DnaE polymerases, processivity factor (β(2)), clamp loader (τ-δ-δ') and primase (DnaG). This study revealed a new role for the SPP1 origin binding protein. In the presence of SSB, it is required for initiation on replication forks that lack origin sequences, mimicking the activity of the PriA replication restart protein in bacteria. The SPP1 replisome is supported by both host and viral SSBs, but phage SSB is unable to support B. subtilis replication, likely owing to its inability to stimulate the PolC holoenzyme in the B. subtilis context. Moreover, phage SSB inhibits host replication, defining a new mechanism by which bacterial replication could be regulated by a viral factor.

The circular, reversible conversion of the mammary gland during pregnancy and involution is a paradigm of physiological tissue plasticity. The two most prominent cell types in mammary gland, adipocytes and epithelial cells, interact in an orchestrated way to coordinate this process. Previously, we showed that this conversion is at least partly achieved by reciprocal transdifferentiation between mammary adipocytes and lobulo-alveolar epithelial cells. Here, we aim to shed more light on the regulators of mammary transdifferentiation. Using immunohistochemistry with cell type-specific lipid droplet-coating markers (Perilipin1 and 2), we show that cells with an intermediate adipoepithelial phenotype exist during and after pregnancy. Nuclei of cells with similar transitional structural characteristics are highly positive for Elf5, a master regulator of alveologenesis. In cultured adipocytes, we could show that transient and stable ectopic expression of Elf5 induces expression of the milk component whey acidic protein, although the general adipocyte phenotype is not affected suggesting that additional pioneering factors are necessary. Furthermore, the lack of transdifferentiation of adipocytes during pregnancy after clearing of the epithelial compartment indicates that transdifferentiation signals must emanate from the epithelial part. To explore candidate genes potentially involved in the transdifferentiation process, we devised a high-throughput gene expression study to compare cleared mammary fat pads with developing, contralateral controls at several time points during pregnancy. Incorporation of bioinformatic predictions of secretory proteins provides new insights into possible paracrine signaling pathways and downstream transdifferentiation factors. We discuss a potential role for osteopontin (secreted phosphoprotein 1 [Spp1]) signaling through integrins to induce adipoepithelial transdifferentiation.