Cadmium is a toxic metal able to enter the cells through channels and transport pathways dedicated to essential ions, leading, among others, to the dysregulation of divalent ions homeostasis. Despite its recognized human carcinogenicity, the mechanisms are still under investigation. A powerful tool for mechanistic studies of carcinogenesis is the Cell Transformation Assay (CTA). We have isolated and characterized by whole genome microarray and bioinformatics analysis of differentially expressed genes (DEGs) cadmium-transformed cells from different foci (F1, F2, and F3) at the end of CTA (6 weeks). The systematic analysis of up- and down-regulated transcripts and the comparison of DEGs in transformed cells evidence different functional targets and the complex picture of cadmium-induced transformation. Only 34 in common DEGs are found in cells from all foci, and among these, only 4 genes are jointly up-regulated (Ccl2, Ccl5, IL6 and Spp1), all responsible for cytokines/chemokines coding. Most in common, DEGs are down-regulated, suggesting that the switching-off of specific functions plays a major role in this process. In addition, the comparison of dysregulated pathways immediately after cadmium treatment with those in transformed cells provides a valuable means to the comprehension of the overall process.

Biological systems contain highly-ordered structures performing diverse functions. The elegant structures of biomachines have inspired the development of nanopores as single molecule sensors. Over the years, the utility of nanopores for detecting a wide variety of analytes have rapidly emerged for sensing, sequencing and diagnostic applications. Several protein channels with diverse shapes and sizes, such as motor channels from bacteriophage Phi29, SPP1, T3, and T4, as well as α-hemolysin, MspA, aerolysin, FluA, OmpF/G, CsgG, ClyA, have been continually investigated and developed as nanopores. Herein, we focus on advances in biological nanopores for single molecule sensing and DNA sequencing from a protein engineering standpoint for changing pore sizes, altering charge distributions, enhancing sensitivity, improving stability, and imparting new detection capabilities.

Background: Endometrial carcinoma (EC) is the most common gynecological malignant tumors which poses a serious threat to women health. This study aimed to screen the candidate genes differentially expressed in EC by bioinformatics analysis.

Methods: GEO database and GEO2R online tool were applied to screen the differentially expressed genes (DEGs) of EC from the microarray datasets. Protein-protein interaction (PPI) network for the DEGs was constructed to further explore the relationships among these genes and identify hub DEGs. Gene ontology and KEGG enrichment analyses were performed to investigate the biological role of DEGs. Besides, correlation analysis, genetic alteration, expression profile, and survival analysis of these hub DEGs were also investigated to further explore the roles of these hub gene in mechanism of EC tumorigenesis. qRT-PCR analysis was also performed to verify the expression of identified hub DEGs.

Results: A total of 40 DEGs were screened out as the DEGs with 3 upregulated and 37 downregulated in EC. The gene ontology analysis showed that these genes were significantly enriched in cell adhesion, response to estradiol, and growth factor activity, etc. The KEGG pathway analysis showed that DEGs were enriched in focal adhesion, leukocyte transendothelial migration, PI3K-Akt signaling pathway, and ECM-receptor interaction pathway. More importantly, COL1A1, IGF1, COL5A1, CXCL12, PTEN, and SPP1 were identified as the hub genes of EC. The genetic alteration analysis showed that hub genes were mainly altered in mutation and deep deletion. Expression validation by bioinformatic analysis and qRT-PCR also proved the expression of these six hub genes were differentially expressed in EC. Additionally, significantly better overall survival and disease-free survival were observed with six hub genes altered, and survival outcome in high expression of COL1A1, IGF1, and PTEN patients was also significantly better than low expression patients.

Conclusions: COL1A1, IGF1, COL5A1, CXCL12, PTEN, and SPP1 involved in the pathogenesis of EC and might be candidate genes for diagnosis of EC.

Keywords: Bioinformatics analysis; Biomarker; Differentially expressed gene; Endometrial carcinoma; Pathway.

To study hepatocyte injure through establishing the rat model of acute hepatic failure (ALF). ALF rat model was established by administration with D-galactosamine and LPS, and then giving lowering blood ammonia (LBA) treatment. Besides, the intervention groups were injected with ornithine and aspartate. The control groups were injected saline. Blood ammonia, ALT, AST, TNF-α and IL-6 in blood samples were test at 12 hrs and 24 hrs after treatment with LBA. Hepatocyte apoptosis were tested by TUNEL and DNA Ladder. Expression of P53 and SPP1 were detected by RT-PCR.

RESULTS

showed that blood ammonia in hepatic failure group and intervention group compared with blank control group was significantly increased at 12 h, 24 h; intervention group compared with hepatic failure group was significantly reduced (P<0.05). Serum ALT, AST in 24 h group were higher than 12 h. 12 h intervention group was decreased compared with hepatic failure group, but there was no significant statistically difference (P>0.05). 24 h intervention group compared with hepatic failure group was significantly reduced (P<0.05). Except the control group, DNA ladder and the TUNEL results showed hepatocyte apoptosis rate increased in 24 h compared with 12 h. Intervention group compared with hepatic failure group was significantly reducing (P<0.05). IL-6, TNF-α, p53 expression levels were increased with time (24 h>12 h). The hepatic failure and intervention group compared with blank control group was significantly increased; Intervention group compared with hepatic failure group was significantly reducing (P<0.05). SPP1 gene was high expression in ALF rat model. SPP1 level in hepatic failure and intervention group compared with control group was significantly increased, and intervention group compared with hepatic failure group was significantly reducing (P<0.05). In conclusion, hepatocyte apoptosis is an important pathological change in ALF rat mode, and lowing ammonia can reduce liver injury and apoptosis. Blood TNF-α, IL-6 and SPP1 may be more sensitive injure indicators.

The International Society of Nephrology/Renal Pathology Society (ISN/RPS) lupus nephritis (LN) classification is under reconsideration, given challenges with inter-rater reliability and resultant inconsistent relationship with treatment response. Integration of molecular classifiers into histologic evaluation can improve diagnostic precision and identify therapeutic targets. This study described the relationship between histological and molecular phenotypes and clinical responses in LN. Renal compartmental mRNA abundance was measured in 54 biopsy specimens from LN patients and correlated to ISN/RPS classification and individual histologic lesions. A subset of transcripts was also evaluated in sequential biopsies of a separate longitudinal cohort of 36 patients with paired samples obtained at the time of flare and at follow up. Unsupervised clustering based on mRNA abundance did not demonstrate a relationship with the (ISN/RPS) classification, nor did univariate statistical analysis. Exploratory analyses suggested a correlation with individual histologic lesions. Glomerular FN1 (fibronectin), SPP1 (secreted phosphoprotein 1), and LGALS3 (galectin 3) abundance correlated with disease activity and changed following treatment. Exploratory analyses suggested relationships between specific transcripts and individual histologic lesions, with the important representation of interferon-regulated genes. Our findings suggested that the current LN classification could be refined by the inclusion of molecular descriptors. Combining molecular and pathologic kidney biopsy phenotypes may hold promise to better classify disease and identify actionable treatment targets and merits further exploration in larger cohorts.

Osteoarthritis (OA) is a common cause of morbidity and disability worldwide. However, the pathogenesis of OA is unclear. Therefore, this study was conducted to characterize the pathogenesis and implicated genes of OA. The gene expression profiles of GSE82107 and GSE55235 were downloaded from the Gene Expression Omnibus database. Altogether, 173 differentially expressed genes including 68 upregulated genes and 105 downregulated genes in patients with OA were selected based on the criteria of ∣log fold-change | >1 and an adjusted p value < 0.05. Protein-protein interaction network analysis showed that FN1, COL1A1, IGF1, SPP1, TIMP1, BGN, COL5A1, MMP13, CLU, and SDC1 are the top ten genes most closely related to OA. Quantitative reverse transcription-polymerase chain reaction showed that the expression levels of COL1A1, COL5A1, TIMP1, MMP13, and SDC1 were significantly increased in OA. This study provides clues for the molecular mechanism and specific biomarkers of OA.

In order to identify potential diagnostic and prognostic biomarkers, and treatment targets for head and neck squamous cell carcinoma (HNSCC), the present study obtained the gene expression profiles in HNSCC through public data mining, and core genes were identified using a series of bioinformatics analysis methods and databases. A total of nine hub genes (SPP1, ITGA6, TMPRSS11D, MMP1, LAMC2, FAT1, ACTA1, SERPINE1 and CEACAM1) were identified to be significantly correlated with HNSCC. Furthermore, overall survival analysis demonstrated that the expression values of hub genes were associated with overall survival in HNSCC. Furthermore, certain of the identified genes, including, TMPRSS11D, ACTA1 and CEACAM1, have not been thoroughly investigated in HNSCC previously. Taken together, the nine hub genes obtained by screening in the present study may serve as potential tumor markers and important prognostic indicators for HNSCC.

Lung adenocarcinoma (LUAD) is one of the most malignant tumors with high morbidity and mortality worldwide due to the lack of reliable methods for early diagnosis and effective treatment. It's imperative to study the mechanism of its development and explore new biomarkers for early detection of LUAD. In this study, the Gene Expression Omnibus (GEO) dataset GSE43458 and The Cancer Genome Atlas (TCGA) were used to explore the differential co-expressed genes between LUAD and normal samples. Three hundred sixity-six co-expressed genes were identified by differential gene expression analysis and Weighted Gene Co-expression Network Analysis (WGCNA) method. Those genes were mainly enriched in ameboidal-type cell migration (biological process), collagen-containing extracellular matrix (cell component), and extracellular matrix structure constituent (molecular function). The protein-protein network (PPI) was constructed and 10 hub genes were identified, including IL6, VWF, CDH5, PECAM1, EDN1, BDNF, CAV1, SPP1, TEK, and SELE. The expression level of hub genes was validated in the GEPIA database, compared with normal tissues, VWF is lowly expressed and SPP1 is upregulated in LUAD tissues. The survival analysis showed increased expression of SPP1 indicated unfavorable prognosis whereas high expression of VWF suggested favorable prognosis in LUAD (p < 0.05). Based on the immune infiltration analysis, the relationship between SPP1 and VWF expression and macrophage, neutrophil, and dendritic cell infiltration was weak in LUAD. Quantitative real-time PCR (qRT-PCR) and western blotting were used to validate the expression of VWF and SPP1 in normal human bronchial epithelial (HBE) cell and three LUAD cell lines, H1299, H1975, and A549. Immunohistochemistry (IHC) was further performed to detect the expression of VWF in 10 cases LUAD samples and matched normal tissues. In summary, the data suggest that VWF is a potential novel biomarker for prognosis of LUAD.

Keywords: VWF; bioinformatics; co-expression analysis; differential expression genes analysis; lung adenocarcinoma.

Breast cancer (BC) mortality is mainly due to metastatic disease, which is primarily driven by cancer stem cells (CSC). The chemokine C-X-C motif ligand-1 (CXCL1) is involved in BC metastasis, but the question of whether it regulates breast cancer stem cell (BCSC) behavior is yet to be explored. Here, we demonstrate that BCSCs express CXCR2 and produce CXCL1, which stimulates their proliferation and self-renewal, and that CXCL1 blockade inhibits both BCSC proliferation and mammosphere formation efficiency. CXCL1 amplifies its own production and remarkably induces both tumor-promoting and immunosuppressive factors, including SPP1/OPN, ACKR3/CXCR7, TLR4, TNFSF10/TRAIL and CCL18 and, to a lesser extent, immunostimulatory cytokines, including IL15, while it downregulates CCL2, CCL28, and CXCR4. CXCL1 downregulates TWIST2 and SNAI2, while it boosts TWIST1 expression in association with the loss of E-Cadherin, ultimately promoting BCSC epithelial-mesenchymal transition. Bioinformatic analyses of transcriptional data obtained from BC samples of 1,084 patients, reveals that CXCL1 expressing BCs mostly belong to the Triple-Negative (TN) subtype, and that BC expression of CXCL1 strongly correlates with that of pro-angiogenic and cancer promoting genes, such as CXCL2-3-5-6, FGFBP1, BCL11A, PI3, B3GNT5, BBOX1, and PTX3, suggesting that the CXCL1 signaling cascade is part of a broader tumor-promoting signaling network. Our findings reveal that CXCL1 functions as an autocrine growth factor for BCSCs and elicits primarily tumor progression and immune escape programs. Targeting the CXCL1/CXCR2 axis could restrain the BCSC compartment and improve the treatment of aggressive BC.

Keywords: CXCL1; breast cancer stem cells; chemokines; immunity genes; triple-negative breast cancer; tumor microenvironment.

Background We previously reported that osteopontin plays an essential role in accelerating both reparative fibrosis and clearance of dead cells (efferocytosis) during tissue repair after myocardial infarction (MI) and galectin-3^hiCD206⁺ macrophages is the main source of osteopontin in post-MI heart. Interleukin-10- STAT3 (signal transducer and activator of transcription 3)-galectin-3 axis is essential for Spp1 (encoding osteopontin) transcriptional activation in cardiac macrophages after MI. Here, we investigated the more detailed mechanism responsible for functional maturation of osteopontin-producing macrophages. Methods and Results In post-MI hearts, Spp1 transcriptional activation occurred almost exclusively in MerTK (Mer tyrosine kinase)⁺ galectin-3^hi macrophages. The induction of MerTK on galectin-3^hi macrophages is essential for their functional maturation including efferocytosis and Spp1 transcriptional activity. MerTK⁺galectin-3^hi macrophages showed a strong activation of both STAT3 and ERK (extracellular signal-regulated kinase). STAT3 inhibition suppressed the differentiation of osteopontin-producing MerTK⁺galectin-3^hi macrophages, however, STAT3 activation was insufficient at inducing Spp1 transcriptional activity. ERK inhibition suppressed Spp1 transcriptional activation without affecting MerTK or galectin-3 expression. Concomitant activation of ERK is required for transcriptional activation of Spp1. In Il-10 knockout enhanced green fluorescent protein-Spp1 knock-in mice subjected to MI, osteopontin-producing macrophages decreased but did not disappear entirely. Interleukin-10 and macrophage colony-stimulating factor synergistically activated STAT3 and ERK and promoted the differentiation of osteopontin-producing MerTK⁺galectin-3^hi macrophages in bone marrow-derived macrophages. Coadministration of anti-interleukin-10 plus anti-macrophage colony-stimulating factor antibodies substantially reduced the number of osteopontin-producing macrophages by more than anti-interleukin-10 antibody alone in post-MI hearts. Conclusions Interleukin-10 and macrophage colony-stimulating factor act synergistically to activate STAT3 and ERK in cardiac macrophages, which in turn upregulate the expression of galectin-3 and MerTK, leading to the functional maturation of osteopontin-producing macrophages.

Keywords: MerTK; galectin‐3; macrophage; macrophage colony‐stimulating factor; myocardial infarction; osteopontin.

Lack of physical exercise is considered an important risk factor for chronic diseases. On the contrary, physical exercise reduces the morbidity rates of obesity, diabetes, bone disease, and hypertension. In order to gain novel molecular and cellular clues, we analyzed the effects of physical exercise on differentiation of mesenchymal circulating progenitor cells (M-CPCs) obtained from runners. We also investigated autophagy and telomerase-related gene expression to evaluate the involvement of specific cellular functions in the differentiation process. We performed cellular and molecular analyses in M-CPCs, obtained by a depletion method, of 22 subjects before (PRE RUN) and after (POST RUN) a half marathon performance. In order to prove our findings, we performed also in vitro analyses by testing the effects of runners' sera on a human bone marrow-derived mesenchymal stem (hBM-MSC) cell line. PCR array analyses of PRE RUN versus POST RUN M-CPC total RNAs put in evidence several genes which appeared to be modulated by physical activity. Our results showed that physical exercise promotes differentiation. Osteogenesis-related genes as RUNX2, MSX1, and SPP1 appeared to be upregulated after the run; data showed also increased levels of BMP2 and BMP6 expressions. SOX9, COL2A1, and COMP gene enhanced expression suggested the induction of chondrocytic differentiation as well. The expression of telomerase-associated genes and of two autophagy-related genes, ATG3 and ULK1, was also affected and correlated positively with MSC differentiation. These data highlight an attractive cellular scenario, outlining the role of autophagic response to physical exercise and suggesting new insights into the benefits of physical exercise in counteracting chronic degenerative conditions.

Over the past 15 years, several biological and pathological characteristics proved their significance in pediatric anaplastic lymphoma kinase (ALK)-positive anaplastic large-cell lymphoma (ALCL) prognostic stratification. However, the identification of new non-invasive disease biomarkers, relying on the most important disease mechanisms, is still necessary. In recent years, plasmatic circulating small extracellular vesicles (S-EVs) gathered great importance both as stable biomarker carriers and active players in tumorigenesis. In the present work, we performed a comprehensive study on the proteomic composition of plasmatic S-EVs of pediatric ALCL patients compared to healthy donors (HDs). By using a mass spectrometry-based proteomics approach, we identified 50 proteins significantly overrepresented in S-EVs of ALCL patients. Gene Ontology enrichment analysis disclosed cellular components and molecular functions connected with S-EV origin and vesicular trafficking, whereas cell adhesion, glycosaminoglycan metabolic process, extracellular matrix organization, collagen fibril organization and acute phase response were the most enriched biological processes. Of importance, consistently with the presence of nucleophosmin (NPM)-ALK fusion protein in ALCL cells, a topological enrichment analysis based on Reactome- and Kyoto Encyclopedia of Genes and Genomes (KEGG)-derived networks highlighted a dramatic increase in proteins of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway in ALCL S-EVs, which included heat shock protein 90-kDa isoform alpha 1 (HSP90AA1), osteopontin (SPP1/OPN) and tenascin C (TNC). These results were validated by Western blotting analysis on a panel of ALCL and HD cases. Further research is warranted to better define the role of these S-EV proteins as diagnostic and, possibly, prognostic parameters at diagnosis and for ALCL disease monitoring.

Keywords: ALCL; HSP90; osteopontin; proteomics; small EVs; tenascin C.

Assembly of tailed bacteriophages and herpesviruses starts with formation of procapsids (virion precursors without DNA). Scaffolding proteins (SP) drive assembly by chaperoning the major capsid protein (MCP) to build an icosahedral lattice. Here we report near-atomic resolution cryo-EM structures of the bacteriophage SPP1 procapsid, the intermediate expanded procapsid with partially released SPs, and the mature capsid with DNA. In the intermediate state, SPs are bound only to MCP pentons and to adjacent subunits from hexons. SP departure results in the expanded state associated with unfolding of the MCP N-terminus and straightening of E-loops. The newly formed extensive inter-capsomere bonding appears to compensate for release of SPs that clasp MCP capsomeres together. Subsequent DNA packaging instigates bending of MCP A domain loops outwards, closing the hexons central opening and creating the capsid auxiliary protein binding interface. These findings provide a molecular basis for the sequential structural rearrangements during viral capsid maturation.

Arsenic is a known human carcinogen. Early-life exposure to inorganic arsenic induces tumors in humans and in C3H mice. We hypothesized that arsenic exposure in utero may induce epigenetic changes at the level of DNA methylation and miRNA alterations that could lead to greater postnatal susceptibility to cancer. To test this hypothesis, pregnant C3H mice were given sodium arsenite at doses known to cause liver cancer (42.5 and 85 ppm in the drinking water) from gestation day 8 to 19, and the livers from male fetal mice were collected for analysis. The antibody against 5-methylcytosine was used to perform chromatin-immunoprecipitation coupled with sequencing (ChIP-Seq) to determine genome-wide methylation alterations. In utero arsenic exposure produced global DNA hypomethylation and an array of gene-specific DNA methylation changes, including hypomethylation of Cyclin D1 and hypermethylation of Tp53. Illumina Correlation Engine analysis revealed 260 methylation alterations that would affect 143 microRNAs. MicroRNA array further revealed 140 aberrantly expressed miRNAs out of the 718 miRNAs. The increased expression of miR-205, miR-203, miR-215, miR-34a, and decreased expression of miR-217 were confirmed by qPCR. Comparison of the methylation changes to those of microarray analyses indicates little if any correspondence between gene methylation and gene expression. The increased expression of Xist, Prrc2, Krit1, Nish, and decreased expression of Prss2, Spp1, Col1a2, and Lox were confirmed by qPCR. In summary, in utero arsenic exposure induced global alterations in DNA methylation and aberrant miRNA expression that might contribute to adult adverse outcomes including liver cancer.

BACKGROUND

Sarcoidosis is a systemic inflammatory disease of unknown etiology. Osteopontin (SPP1, OPN) is an extra cellular matrix glycoprotein and cytokine with a known role in granuloma formation and in autoimmune and inflammatory diseases.

OBJECTIVE

To determine whether plasma OPN levels are elevated in patients with sarcoidosis and compare the frequency of four single nucleotide polymorphism (SNPs) variants in the OPN gene in sarcoidosis patients compared to healthy controls.

METHODS

Demographic and clinical information, radiological studies and pulmonary function tests were evaluated in 113 patients with sarcoidosis and in 79 healthy controls. Blood samples were analyzed for SNPs of the OPN gene and for plasma OPN and CRP levels. Association between clinical features of disease and OPN levels as well as SNP frequencies was determined.

RESULTS

Plasma OPN levels were higher in sarcoidosis patients than in healthy subjects, (median: 217 vs 122ng/ml, p<0.001). Area under the curve for receiver operator curves (ROC) was 0.798 (0.686-0.909 95% CI.) No differences were observed between sarcoidosis patients and controls in the frequency of any of the SNPs evaluated. Presence of lung parenchymal involvement was associated with SNP distribution at rs1126772 (p = 0.02). We found no correlation between SNPs distribution and plasma OPN levels.

CONCLUSIONS

Osteopontin protein levels are elevated in sarcoidosis. We found no evidence for an association between SNPs on the osteopontin gene and plasma OPN levels or the presence of sarcoidosis, however, an association between genotype and several phenotypic clinical parameters of disease was observed.

Although deficiencies in porcine blastocyst elongation play a significant role in early embryonic mortality and establishment of within-litter developmental variation, the exact mechanisms of elongation are poorly understood. Secreted phosphoprotein 1 (SPP1) is increased within the uterine milieu during early porcine pregnancy and contains an Arg-Gly-Asp (RGD) peptide sequence that binds to cell surface integrins on the uterine endometrium and trophectoderm, promoting cell adhesion and migration. The aim of the present study was to evaluate the development of preimplantation porcine blastocysts encapsulated and cultured within alginate hydrogels either supplemented with SPP1 or conjugated with RGD. Blastocysts encapsulated within alginate hydrogels supplemented with SPP1 or conjugated with RGD had increased survival compared with non-encapsulated control blastocysts. In addition, the percentage of blastocysts encapsulated within RGD hydrogels that underwent morphological changes was greater than that of blastocysts encapsulated within standard alginate hydrogels or SPP1-supplemented hydrogels. Finally, only blastocysts encapsulated within RGD hydrogels had both increased expression of steroidogenic and immune responsiveness transcripts and increased 17β-oestradiol production, consistent with blastocysts undergoing elongation in vivo. These results illustrate the importance of the integrin-binding RGD peptide sequence for stimulating the initiation of blastocyst elongation.

MiR-140-5p is high expressed in normal fracture healing, but its specific role and mechanism in tissue-to-bone healing are rarely reported. Therefore, this study investigated the effects of miR-140-5p on tissue-to-bone healing. Clone formation experiment, flow cytometry, Alizarin Red S Staining and Oil Red O Staining were performed to investigate the biological characteristics of mouse embryonic bone marrow mesenchymal stem cells C3H10T1/2. MiR-140-5p mimic was transfected into osteogenic medium (OS)-treated C3H10T1/2 cells to investigate the effects of miR-140-5p on osteogenic differentiation. MiR-140-5p transgenic mouse model and the transgenic fracture model were established, and the effects of miR-140-5p on osteogenic differentiation, bone mineral density (BMD) and bone mass of bone tissues were detected by haematoxylin and eosin staining and computed tomography scan. The expressions of osteocalcin, differentiation-related genes (Runx2, ALP, Spp1 and Bglap3) and miR-140-5p were determined by quantitative real-time polymerase chain reaction. C3H10T1/2 cells showed the abilities of forming cloned differentiation of osteogenesis, fat cells, and its phenotypes including CD44, CD90.1 and Sca-1 but excluding CD45 haematopoietic stem cell marker. Overexpression of miR-140-5p promoted the expressions of differentiation-related genes and calcium deposition of OS-treated C3H10T1/2 cells. MiR-140-5p increased the expression of osteocalcin, BMD and bone mass and promoted bone healing of miR-140-5p-transgenic mice with fracture. MiR-140-5p promoted osteogenic differentiation of mouse embryonic bone marrow mesenchymal stem cells and post-fracture healing in mice. SIGNIFICANCE OF THE STUDY: C3H10T1/2 cells showed the abilities of forming cloned differentiation of osteogenesis, fat cells and its phenotypes including CD44, CD90.1 and Sca-1 but excluding CD45 haematopoietic stem cell marker. Overexpression of miR-140-5p promoted the expressions of differentiation-related genes and calcium deposition of osteogenic medium-treated C3H10T1/2 cells. MiR-140-5p increased the expression of osteocalcin and bone mineral density and bone mass and promoted bone healing of miR-140-5p-transgenic mice with fracture. Our results showed that miR-140-5p promoted osteogenic differentiation of mouse embryonic bone marrow mesenchymal stem cells and post-fracture healing in mice, which may be a therapeutic target for treating fractures and promoting bone healing.

Keywords: C3H10T1/2 cells; bone healing; fracture; miR-140-5p; osteogenic differentiation.

Poly(L-lactide-co-ε-caprolactone) scaffolds were functionalised by 10 or 20 µg/mL of human demineralised dentine matrix. Release kinetics up to 21 days and their osteogenic potential on human bone marrow stromal cells after 7 and 21 days were studied. A total of 390 proteins were identified by mass spectrometry. Bone regeneration proteins showed initial burst of release. Human bone marrow stromal cells were cultured on scaffolds physisorbed with 20 µg/mL and cultured in basal medium (DDM group) or physisorbed and cultured in osteogenic medium or cultured on non-functionalised scaffolds in osteogenic medium. The human bone marrow stromal cells proliferated less in demineralised dentine matrix group and activated ERK/1/2 after both time points. Cells on DDM group showed highest expression of IL-6 and IL-8 at 7 days and expressed higher collagen type 1 alpha 2, SPP1 and bone morphogenetic protein-2 until 21 days. Extracellular protein revealed higher collagen type 1 and bone morphogenetic protein-2 at 21 days in demineralised dentine matrix group. Cells on DDM group showed signs of mineralisation. The functionalised scaffolds were able to stimulate osteogenic differentiation of human bone marrow stromal cells.

Enzyme modified non-oxidative LDL (ELDL) is effectively taken up by vascular smooth muscle cells (SMC) and mediates transition into foam cells and produces phenotypic changes in SMC function. Our data show that incubation of human coronary artery SMC (HCASMC) with low concentration of ELDL (10 μg/ml) results in significantly enhanced foam cell formation compared to oxidized LDL (200 μg/ml; p < 0.01) or native LDL (200 μg/ml; p < 0.01). Bioinformatic network analysis identified activation of p38 MAPK, NFkB, ERK as top canonical pathways relevant for biological processes linked to cell migration and osteoblastic differentiation in ELDL-treated cells. Functional studies confirmed increased migration of HCASMC upon stimulation with ELDL (10 μg/ml) or Angiopoietin like protein 4, (ANGPTL4, 5 μg/ml), and gain in osteoblastic gene profile with significant increase in mRNA levels for DMP-1, ALPL, RUNX2, OPN/SPP1, osterix/SP7, BMP and reduction in mRNA for MGP and ENPP1. Enhanced calcification of HCASMC by ELDL was demonstrated by Alizarin Red staining. In summary, ELDL is highly potent in inducing foam cells in HCASMC and mediates a phenotypic switch with enhanced migration and osteoblastic gene profile. These results point to the potential of ELDL to induce migratory and osteoblastic effects in human smooth muscle cells with potential implications for migration and calcification of SMCs in human atherosclerosis.

The SPP1, LAP3, and LCORL are located on chromosome 6 of sheep and a domain of 36.15-38.56 Mb, which plays an essential role in tissue and embryonic growth. In this study, we cloned the complete coding sequences of SPP1 and partial coding regions of LAP3 and LCORL from Hu sheep (Gansu Province, China) and analyzed their genomic structures. The RT-qPCR showed that the three genes were expressed widely in the different tissues of Hu sheep. The SPP1 expression was significantly higher in the kidney (p < 0.01) and LAP3 expression was significantly higher in the spleen, lung, kidney, and duodenum than in the other tissues (heart, liver, rumen, muscle, fat, and ovary; p < 0.05). The LCORL was preferentially expressed in the spleen, duodenum, and lung (p < 0.05). In addition, the nucleotide substitution NM_001009224.1:c.132A>C was found in SPP1; an association analysis showed that it was associated with birth weight and yearling weight (p < 0.05), and NM_001009224.1:c.132C was the dominant allele. Two mutations XM_012179698.3:c.232C>G and XM_012179698.3:c.1154C>T were identified in LAP3. The nucleotide substitution XM_012179698.3:c.232C>G was confirmed to be associated with birth weight, 1-month weight, 3-month weight (p < 0.05), and 2-month weight (p < 0.01). The nucleotide substitution XM_012179698.3:c.1154C>T was associated with birth weight (p < 0.01), 1-month weight, and 2-month weight (p < 0.05). The LAP3 gene XM_012179698.3:c.232C>G mutation with the C allele has higher body weight than other sheep, and CC genotype individuals show higher birth weight, 1-month weight, and weaning weight than the GG genotype individuals (p < 0.05). Our results support the conclusion that the mutations on ovine SPP1 and LAP3 successfully track functional alleles that affect growth in sheep, and these genes could be used as candidate genes for improving the growth traits of sheep during breeding.

It is well known that dermal papilla cells (DPCs) are crucial for hair follicle growth and regeneration. However, dermal papilla cells in 2D culture could lose their ability of regeneration after several passage intervals. As opposed to DPCs in 2D culture, the DPCs in 3D culture could passage extensively. However, the molecular mechanisms of DPCs' regeneration in 3D culture remain unclear. Accordingly, gene sequencing is recommended for the investigation of hair regeneration between 2D and 3D culture, the three groups were established including DPCs in passage 2 in 2D culture, DPCs in passage 8 in 2D culture and DPCs in passage 8 in 3D culture. The differentially expressed genes (DEGs) were identified using the Venn diagram of these three groups, which included 1642 known and 359 novel genes, respectively. A total of 1642 known genes were used for Gene Ontology (GO), Kyoto Gene, Genomic Encyclopedia (KEGG) pathway enrichment and protein-protein interaction (PPI) analyses, respectively. The functions and pathways of DEGs were enriched in biological regulation, signal transduction and immune system, etc. The key module and the top 10 hub genes (IL1B, CXCL12, HGF, EGFR, APP, CCL2, PTGS2, MMP9, NGF and SPP1) were also identified using the Cytoscape application. Furthermore, the qRT-PCR results of the three groups validated that the hub genes were crucial for hair growth. In conclusion, the ten identified hub genes and related pathways in the current study can be used to understand the molecular mechanism of hair growth, and those provided a possibility for hair regeneration.

Keywords: 3D culture; RNA-seq; dermal papilla cells; hair follicle; regeneration.

Nephrotoxicity limits the use of cisplatin (CP) in cancer therapy; however, current clinical measures of renal health do not reflect low degrees of kidney injury. Therefore, discovering new biomarkers for CP-induced acute kidney injury (AKI) is essential for monitoring kidney health during therapy. To identify novel candidate biomarkers in urine for reduced renal function due to CP therapy, we conducted a pilot study on cancer patients eligible for CP treatment. Urine from 30 patients was collected before (baseline) and after 3d of intravenous CP infusion. Urine samples were subjected to Isobaric Tag for Relative Absolute Quantitation (iTRAQ) analysis. Biological roles and pathways for the proteins with altered urine concentrations were identified using bioinformatic tools ITRAQ analysis detected 1411 proteins, 12 of which showed significantly altered levels. Growth differentiation factor-15 (GDF15), leucine-rich alpha-2-glycoprotein 1 (LRG1), and secreted phosphoprotein 1 (SPP1) were identified as potential candidate markers by proteomic analysis and were validated by ELISA in another 30 patients and in a CP-induced AKI mouse model. Therefore, GDF15, LRG1, and SPP1 may be applied as novel candidate urinary markers of kidney injury after cisplatin treatment. These findings may facilitate the development of new methods to monitor kidney function, particularly in CP-based chemotherapy. SIGNIFICANCE: Cisplatin is pivotal for cancer treatment; however, nephrotoxicity limits its use. Clinical measures for renal health are not reflective of early signs of acute kidney injury. Thus, new indicators of the state of renal health following cisplatin treatment will have to be discovered. This study reports the use of proteomics to mine for candidate markers of kidney injury in the urine of patients undergoing treatment with cisplatin. Results were experimentally validated using patient urine and a mouse model of cisplatin-induced acute kidney injury. The novel candidate biomarkers reported in this study may be used for the non-invasive monitoring of renal health and in mitigating the side-effects of cisplatin during the course of cancer treatment.

The SIBLINGs are a subfamily of the secreted calcium-binding phosphoproteins and comprise five small integrin-binding ligand N-linked glycoproteins [dentin matrix protein-1 (DMP1), secreted phosphoprotein-1 (SPP1) also called osteopontin (OPN), integrin-binding sialoprotein (IBSP) also called bone sialoprotein (BPS), matrix extracellular phosphoglycoprotein (MEPE), and dentin sialophosphoprotein (DSPP)]. Each SIBLING has at least one "acidic, serine- and aspartic acid-rich motif" (ASARM) and multiple Ser-x-Glu/pSer sequences that when phosphorylated promote binding of the protein to hydroxyapatite for regulation of biomineralization. Mendelian disorders from loss-of-function mutation(s) of the genes that encode the SIBLINGs thus far involve DSPP causing various autosomal dominant dysplasias of dentin but without skeletal disease, and DMP1 causing autosomal recessive hypophosphatemic rickets, type 1 (ARHR1). No diseases have been reported from gain-of-function mutation(s) of DSPP or DMP1 or from alterations of SPP1, IBSP, or MEPE. Herein, we describe severe hypophosphatemic osteosclerosis and hyperostosis associated with skeletal deformity, short stature, enthesopathy, tooth loss, and high circulating FGF23 levels in a middle-aged man and young woman from an endogamous family living in southern India. Both shared novel homozygous mutations within two genes that encode a SIBLING protein: stop-gain ("non-sense") DMP1 (c.556G > T, p.Glu186Ter) and missense SPP1 (c.769C > T, p.Leu266Phe). The man alone also carried novel heterozygous missense variants within two additional genes that condition mineral homeostasis and are the basis for autosomal recessive disorders: CYP27B1 underlying vitamin D dependent rickets, type 1, and ABCC6 underlying both generalized arterial calcification of infancy, type 2 and pseudoxanthoma elasticum (PXE). By immunochemistry, his bone contained high amounts of OPN, particularly striking surrounding osteocytes. We review how our patients' disorder may represent the first digenic SIBLING protein osteopathy.