BACKGROUND

Osteopontin (OPN) or secreted phosphoprotein 1 (SPP1) is a matricellular glycoprotein whose expression is elevated in various types of cancer and has been shown to be involved in tumourigenesis and metastasis in many malignancies, including follicular cell-derived thyroid carcinomas. Its role in C-cell-derived thyroid lesions and tumours remains to be established.

OBJECTIVE

The objective of this study is to clarify the role of OPN expression in the development of medullary thyroid carcinoma (MTC).

METHODS

OPN expression was analysed in a series of 116 MTCs by immunohistochemistry and by qPCR mRNA quantification of the 3 OPN isoforms (OPNa, OPNb and OPNc) in six cases from which fresh frozen tissue was available. Statistical tests were used to evaluate the relationship of OPN expression and the clinicopathological and molecular characteristics of patients and tumours.

RESULTS

OPN expression was detected in 91 of 116 (78.4%) of the MTC. We also observed high OPN expression in C-cell hyperplasia as well as in C-cells scattered in the thyroid parenchyma adjacent to the tumours. OPN expression was significantly associated with smaller tumour size, PTEN nuclear expression and RAS status, and suggestively associated with non-invasive tumours. OPNa isoform was expressed significantly at higher levels in tumours than in non-tumour samples. OPNb and OPNc presented similar levels of expression in all samples. Furthermore, OPNa isoform overexpression was significantly associated with reduced growth and viability in the MTC-derived cell line (TT).

CONCLUSIONS

The expression of OPN in normal C-cells and C-cell hyperplasia suggests that OPN is a differentiation marker of C-cells, rather than a marker of biological aggressiveness in this setting. At variance with other cancers, OPN expression is associated with good prognostic features in MTC.

Recently, more and more studies show that long non-coding RNAs (lncRNAs) play a very important role in various biological processes. However, research on lncRNA in the tumor cell drug resistance of it is seldom reported. In this study, gemcitabine-resistant pancreatic cancer cell line SWl990/GZ was obtained by treating parental cell line SWl990 in vitro with increasing dosage of gemcitabine in culture medium intermittently for ten months. We identified 4983 of 13310 detected lncRNAs demonstrated>> 2-fold abnormally expressed in response to the gemcitabine-resistant, among of them, 1993 and 2990 lncRNAs were upregulated and downregulated. Meanwhile, 4759 mRNAs exhibited at least a 2-fold, of these, 2671 and 2088 mRNAs were upregulated and downregulated. Gene Ontology analysis and Pathway analysis revealed that differential expression mRNA involved in significant biological regulatory function and some genes may be particular to pancreatic cancer chemotherapy resistance. Quantitative real time PCR confirmed the changes of six lncRNAs (RP11-58D2.1, lincRNA-ZNF532, AP000221.1, CTC-338M12.5, CR619813, DDX6P) and nine mRNAs (SYT1, FAM171B, ZNF331, FAM187B, CYP1A1, SRXN1, HIST1H2BL, TOMM40L and SPP1) in SW1990 and SW1990/GZ. We also found that the upregulating of gemcitabine on the expression of lincRNA-ZNF532 was time-dependent. Gemcitabine at a range from 1.0 μM to 16.0 μM induced a increase of lincRNA-ZNF532 in SW1990 cells. The relative level of DDX6P is opposite to that of lincRNA-ZNF53 in the same circumstance. In conclusion, the dysregulated lncRNAs and mRNAs identified in this work may represent good candidates for future diagnostic or prognostic biomarkers and therapeutic targets.

Macrophages are key innate immune cells in the tumor microenvironment that regulate primary tumor growth, vascularization, metastatic spread and response to therapies. Macrophages can polarize into two different states (M1 and M2) with distinct phenotypes and functions. To investigate the known tumoricidal effects of M1 macrophages, we obtained RNA expression profiles and clinical data from The Cancer Genome Atlas Thyroid Cancer (TCGA-THCA). The proportions of immune cells in tumor samples were assessed using CIBERSORT, and weighted gene co-expression network analysis (WGCNA) was used to identify M1 macrophage-related modules. Univariate Cox analysis and LASSO-Cox regression analysis were performed, and four genes (SPP1, DHRS3, SLC11A1, and CFB) with significant differential expression were selected through GEPIA. These four genes can be considered hub genes. The four-gene risk-scoring model may be an independent prognostic factor for THCA patients. The validation cohort and the entire cohort confirmed the results. Univariate and multivariate Cox analysis was performed to identify independent prognostic factors for THCA. Finally, a prognostic nomogram was built based on the entire cohort, and the nomogram combining the risk score and clinical prognostic factors was superior to the nomogram with individual clinical prognostic factors in predicting overall survival. Time-dependent ROC curves and DCA confirmed that the combined nomogram is useful. Gene set enrichment analysis (GSEA) was used to elucidate the potential molecular functions of the high-risk group. Our study identified four genes associated with M1 macrophages and established a prognostic nomogram that predicts overall survival for patients with THCA, which may help determine clinical treatment options for different patients.

Keywords: CIBERSORT; M1 macrophages; nomogram; thyroid cancer; weighted gene co-expression network analysis.

Osteopontin (OPN) has been shown to promote colorectal cancer (CRC) progression; however, the mechanism of OPN-induced CRC progression is largely unknown. In this study, we found that OPN overexpression led to enhanced anchorage-independent growth, cell migration and invasion in KRAS gene mutant cells but to a lesser extent in KRAS wild-type cells. OPN overexpression also induced PI3K signalling, expression of Snail and Matrix metallopeptidase 9 (MMP9), and suppressed the expression of E-cadherin in KRAS mutant cells. In human CRC specimens, a high-level expression of OPN significantly predicted poorer survival in CRC patients and OPN expression was positively correlated with MMP9 expression, and negatively correlated with E-cadherin expression. Furthermore, we have found that 15 genes were co-upregulated in OPN highly expression CRC and a list of candidate drugs that may have potential to reverse the secreted phosphoprotein 1 (SPP1) gene signature by connectivity mapping. In summary, OPN is a potential prognostic indicator and therapeutic target for colon cancer.

OBJECTIVE

To determine whether orthodontic treatment with removable aligners vs fixed orthodontic appliances is associated with a different frequency of orthodontically induced external apical root resorption (OIEARR) when genetic, radiographic, and clinical factors are accounted for.

METHODS

Three hundred seventy-two orthodontic patients treated with removable aligners (Invisalign) or fixed appliances were genetically screened for interleukin 1B gene (IL1B) (rs1143634), interleukin 1 receptor antagonist gene (IL1RN) (rs419598), and osteopontin gene (SPP1) (rs9138/rs11730582). Twelve clinical variables, potentially associated with OIEARR, were also considered. Subjects were divided according to the presence of radiographically determined OIEARR (>2 mm). The association between OIEARR and appliance type, and radiographic, clinical and genetic factors, was assessed using backward stepwise conditional logistic regression. Odds ratios (ORs) and 95% confidence intervals (CIs) were reported.

RESULTS

Reliability of the methods was adequate. Clinical case complexity (American Board of Orthodontics [ABO] Discrepancy Index) (OR: 1.032; 95% CI: 1.005-1.061; P = .021) and extent of incisor apical displacement in the sagittal plane (OR: 1.478; 95% CI: 1.285-1.699; P = .001) were associated with an increased OIEARR risk. After adjusting for associations between clinical/radiographic/genetic factors, there were no statistically significant differences with respect to OIEARR or type of orthodontic appliance used, whether removable aligners or fixed appliances (OR: 1.662; 95% CI: 0.945-2.924; P = .078). Only subjects homozygous for the T allele of IL1RN (rs419598) were more prone to OIEARR during orthodontic treatment (OR: 3.121; CI: 1.93-5.03; P < .001).

CONCLUSIONS

A similar OIEARR predisposition was identified using either removable aligners (Invisalign) or fixed appliances.

Osteosarcoma is the most common type of primary bone cancer in children and young adults. The prognosis of osteosarcoma is very poor when it is diagnosed with metastasis. Lysosomal‑associated membrane protein 3 (LAMP3) is a tumor‑specific protein induced by hypoxia, which stimulates invasion and metastasis of various cancer cells via hypoxia‑inducible factor (HIF). A previous study from our group has reported that expression of LAMP3 is significantly increased in lung metastatic osteosarcoma compared with primary osteosarcoma using microarray analysis, suggesting that LAMP3 may be involved in metastatic osteosarcoma. The present study therefore aimed to investigate the role of LAMP3 in osteosarcoma metastasis. Knockdown of LAMP3 decreased the invasion of two osteosarcoma cell lines in vitro. Furthermore, knockdown of LAMP3 increased the expression of secreted phosphoprotein 1 (SPP1), cadherin 1, and keratin 19, while it decreased the expression of matrix metallopeptidase 2, collagen type III α 1, twist family bHLH transcription factor 1 and cadherin 2. Concurrent knockdown of SPP1 and LAMP3 attenuated the changes in gene expression profile induced by LAMP3 knockdown alone. Gene ontology and KEGG analysis demonstrated that SPP1 was involved in cell adhesion, focal adhesion, and extracellular matrix‑receptor interaction. In conclusion, the present results suggest that LAMP3 may be involved in the invasion and metastasis of osteosarcoma via regulating signaling downstream of SPP1. Thus, LAMP3/SPP1 signaling may serve as a potential target in the future to prevent osteosarcoma metastasis.

Atrophic nonunion, a complicated failure of fracture healing, is still obscure regarding its molecular pathological mechanisms. Carboxyl-terminal binding proteins (CtBPs), an NADH-sensitive transcriptional corepressor family, are involved in many diseases, such as cancer and inflammation. Here, we found that CtBP2, but not CtBP1, was significantly overexpressed in atrophic nonunion tissues compared to healthy controls. Using a mass spectrometry assay, we found that CtBP2 can form a complex with histone acetyltransferase p300 and transcription factor Runx2. The lower NADH level in atrophic nonunion tissues disrupted CtBP2 dimerization and enhanced the blockage of the accessibility of the p300-Runx2 complex to the promoters of a series of bone-related target genes, such as OSC, ALPL, COL1A1, IBSP, SPP1 and MMP13. The expression of these genes can be reversed by a forced increase in NADH with CoCl₂ treatment. In conclusion, our study revealed that NADH levels determine the expression of bone formation and development of related genes through affecting the dissociation or binding of CtBP2 to the p300-Runx2 complex. These results represent a conserved mechanism, by which CtBP2 serves as a NADH-dependent repressor of the p300-Runx2 transcriptional complex and thus affects bone formation.

OBJECTIVE

Periodontal ligament (PDL) cells play an important role in maintaining periodontal homeostasis upon force loading caused by mastication or orthodontic force. Previous studies revealed stretch-induced realignment of human PDL cells, but the mechanism for this phenomenon still remains unclear. As extracellular matrix (ECM) and adhesion molecules play critical roles in cell migration and alignment, this study aimed to identify mechanoresponsive genes related to ECM and adhesion in human PDL cells.

METHODS

Human PDL cells were exposed to 10% stretch strain for 6 or 24 h, and the expression of 84 genes related to ECM and adhesion were analyzed with real-time PCR array. The protein expression of integrin α5 was examined by Western blot and flow cytometric analysis.

RESULTS

Among the genes screened, 6 were up-regulated and 3 were down-regulated after 6 h stretch. There were 12 up-regulated and 2 down-regulated genes after 24 h stretch. These differentially expressed genes included genes encoding cell-cell adhesion molecules (CD44, ICAM1), cell-matrix adhesion molecules (ITGA5, ITGA6, ITGAL, ITGB2, SPP1), basement membrane constituents (SPARC, TNC), collagens and ECM constituents (COL5A1, COL11A1, FN1), ECM proteases (ADAMTS1, ADAMTS8, MMP8) and inhibitors (TIMP1), as well as other adhesion-related molecules (CTGF, CTNND2, TGFBI, CLEC3B). Both the cytosolic and membrane integrin α5 protein levels were up-regulated in response to stretch.

CONCLUSIONS

This study identified several force-sensitive genes related to ECM and adhesion in stretched human PDL cells and should facilitate future studies on the stretch-induced cell realignment and mechanic force related periodontal remodelling by providing potential target genes.

An effective circulating tumour marker is needed for melanoma especially with the advent of targeted therapies. Gene expression studies examining primary melanomas have shown that increased expression of osteopontin (SPP1) is associated with poor prognosis. Studies subsequently reported higher blood levels in melanoma patients with metastatic disease than those without. This study was designed to determine whether osteopontin plasma concentrations in disease-free patients after initial treatment predict survival. An enzyme-linked immunosorbent assay (ELISA) was used to measure osteopontin levels in stored plasma samples (N=215) from participants in the Leeds Melanoma Cohort. AJCC stage at sampling was statistically significant associated with osteopontin levels (p=0.03). Participants with untreated stage IV disease at sampling (n=10) had higher median osteopontin levels compared to those with treated stage I-III disease (n=158) (p<0.001) confirming previous findings. There was a trend for increased risk of death with increasing osteopontin levels but this was not statistically significant. If a level of 103.14 ng/ml (95th centile of healthy controls) was taken as the upper end of the normal range then 2.5% of patients with treated stage I-III (4/110), 17.6% of patients with untreated stage III (3/17) and 30% of patients with untreated stage IV disease (3/10) had higher levels. These findings suggest that plasma osteopontin levels warrant investigation as a tumour marker in a larger study in which the significance of change in levels over time should be studied in relation to detectable disease recurrence.

An optimal environment for fertilization and early embryonic development is provided by the mammalian oviduct and uterus. The secretory cells lining the lumen of the oviduct and uterus synthesize and secrete proteins that have been shown to interact with and influence the activities of gametes and embryos. Western blotting in this study demonstrated that a 50-kDa secreted phosphoprotein 1 (SPP1) form was present in the uterus on Days 0, 3, and 5 in pregnant and nonbred gilts, and the concentration of SPP1 on Day 0 was higher than on Days 3 and 5 in pregnant gilts, but in nonbred gilts the concentration of SPP1 on Day 0 was higher than Day 3, but not Day 5. In addition, we show that addition of 0.1 microg/ml SPP1 to the culture medium after fertilization increased the percent cleaved (24 hr: 23.6 +/- 1.29(a) vs. 18.7 +/- 0.65(b) (2-cell %)), and the percent blastocyst (37.2 +/- 1.12(a) vs. 30.9 +/- 0.56(b)) derived from IVF (P < 0.05). In parthenogenetic-derived embryos the percent cleaved was increased due to SPP1 at 24 hr (24.0 +/- 1.59(a) vs. 19.7 +/- 1.59(b) (>2-cell %)), and at 48 hr (72.9+/- 2.99(a) vs. 63.3 +/- 2.99(b)), but not the percent blastocyst. By TUNEL assay, SPP1 decreased both apoptosis (7.9 +/- 0.04(a) vs. 13.1 +/- 0.02(b)) and the percent fragmentation (45.2 +/- 0.07(a) vs. 58.8 +/- 0.03(b)). We conclude that SPP1 can improve development in vitro possibly by reducing the rate of apoptosis.

Cellular and molecular immunoinflammatory changes in gingival tissues drive alveolar bone loss in periodontitis. Since ageing is a risk factor for periodontitis, we sought to identify age-related gingival transcriptome changes associated with bone metabolism in both healthy and in naturally occurring periodontitis.

Adult (12-16 years) and aged (18-23 years) non-human primates (M. mulatta) (n = 24) were grouped into healthy and periodontitis. Gingival tissue samples were obtained and subjected to microarray analysis using the Gene Chip Macaque Genome Array. Gene expression profiles involved in osteoclast/osteoblast proliferation, adhesion and function were evaluated and compared across and between the age groups. QPCR was also performed on selected genes to validate microarray data.

Healthy aged tissues showed a gene profile expression that suggest enhancement of osteoclastic adhesion, proliferation/survival and function (SPP1, TLR4, MMP8 and TFEC) and impaired osteoblastic activity (SMEK3P and SMAD5). The gingival transcriptome in both adult and aged animals with naturally occurring periodontitis (FOS, IL6, TLR4, MMP9, MMP10 and SPP1 genes) was consistent with a local inflammatory response driving towards bone/connective tissue destruction.

A pro-osteoclastogenic gingival transcriptome is associated with periodontitis irrespective of age; however; a greater bone-destructive molecular environment is associated with ageing in healthy tissues.

Colorectal cancer (CRC) is one of the leading causes of death by cancer worldwide. Bowel cancer screening programs enable us to detect early lesions and improve the prognosis of patients with CRC. However, they also generate a significant number of problematic polyps, e.g., adenomas with epithelial misplacement (pseudoinvasion) which can mimic early adenocarcinoma. Therefore, biomarkers that would enable us to distinguish between adenoma with epithelial misplacement (pseudoinvasion) and adenoma with early adenocarcinomas (true invasion) are needed. We hypothesized that the former are genetically similar to adenoma and the latter to adenocarcinoma and we used bioinformatics approach to search for candidate genes that might be potentially used to distinguish between the two lesions. We used publicly available data from Gene Expression Omnibus database and we analyzed gene expression profiles of 252 samples of normal mucosa, colorectal adenoma, and carcinoma. In total, we analyzed 122 colorectal adenomas, 59 colorectal carcinomas, and 62 normal mucosa samples. We have identified 16 genes with differential expression in carcinoma compared to adenoma: COL12A1, COL1A2, COL3A1, DCN, PLAU, SPARC, SPON2, SPP1, SULF1, FADS1, G0S2, EPHA4, KIAA1324, L1TD1, PCKS1, and C11orf96. In conclusion, our in silico analysis revealed 16 candidate genes with different expression patterns in adenoma compared to carcinoma, which might be used to discriminate between these two lesions.

BACKGROUND

Mammalian target of rapamycin (mTOR) inhibitors have anti-tumor effects against renal cell carcinoma, pancreatic neuroendocrine cancer and breast cancer. In this study, we analyzed the antitumor effects of mTOR inhibitors in small cell lung cancer (SCLC) cells and sought to clarify the mechanism of resistance to mTOR inhibitors.

METHODS

We analyzed the antitumor effects of three mTOR inhibitors including everolimus in 7 SCLC cell lines by MTS assay. Gene-chip analysis, receptor tyrosine kinases (RTK) array and Western blotting analysis were performed to identify molecules associated with resistance to everolimus.

RESULTS

Only SBC5 cells showed sensitivity to everolimus by MTS assay. We established two everolimus resistant-SBC5 cell lines (SBC5 R1 and SBC5 R10) by continuous exposure to increasing concentrations of everolimus stepwise. SPP1 and MYC were overexpressed in both SBC5 R1 and SBC5 R10 by gene-chip analysis. High expression levels of eukaryotic translation initiation factor 4E (eIF4E) were observed in 5 everolimus-resistant SCLC cells and SBC5 R10 cells by Western blotting. MYC siRNA reduced eIF4E phosphorylation in SBC5 cells, suggesting that MYC directly activates eIF4E by an mTOR-independent bypass pathway. Importantly, after reduction of MYC or eIF4E by siRNAs, the SBC5 parent and two SBC5-resistant cells displayed increased sensitivity to everolimus relative to the siRNA controls.

CONCLUSIONS

These findings suggest that eIF4E has been shown to be an important factor in the resistance to everolimus in SCLC cells. Furthermore, a link between MYC and mTOR-independent eIF4E contribute to the resistance to everolimus in SCLC cells. Control of the MYC-eIF4E axis may be a novel therapeutic strategy for everolimus action in SCLC.

Equilibrative nucleoside transporter 1 (ENT1) mediates passage of adenosine across the plasma membrane. We reported previously that mice lacking ENT1 (ENT1(-/-)) exhibit progressive ectopic mineralization of spinal tissues resembling diffuse idiopathic skeletal hyperostosis (DISH) in humans. Here, we investigated mechanisms underlying aberrant mineralization in ENT1(-/-) mice. Micro-CT revealed ectopic mineralization of spinal tissues in both male and female ENT1(-/-) mice, involving the annulus fibrosus of the intervertebral discs (IVDs) of older mice. IVDs were isolated from wild-type and ENT1(-/-) mice at 2months of age (prior to disc mineralization), 4, and 6months of age (disc mineralization present) and processed for real-time PCR, cell isolation, or histology. Relative to the expression of ENTs in other tissues, ENT1 was the primary nucleoside transporter expressed in wild-type IVDs and mediated the functional uptake of [(3)H]2-chloroadenosine by annulus fibrosus cells. No differences in candidate gene expression were detected in IVDs from ENT1(-/-) and wild-type mice at 2 or 4months of age. However, at 6months of age, expression of genes that inhibit biomineralization Mgp, Enpp1, Ank, and Spp1 were reduced in IVDs from ENT1(-/-) mice. To assess whether changes detected in ENT1(-/-) mice were cell autonomous, annulus fibrosus cell cultures were established. Compared to wild-type cells, cells isolated from ENT1(-/-) IVDs at 2 or 6months of age demonstrated greater activity of alkaline phosphatase, a promoter of biomineralization. Cells from 2-month-old ENT1(-/-) mice also showed greater mineralization than wild-type. Interestingly, altered localization of alkaline phosphatase activity was detected in the inner annulus fibrosus of ENT1(-/-) mice in vivo. Alkaline phosphatase activity, together with the marked reduction in mineralization inhibitors, is consistent with the mineralization of IVDs seen in ENT1(-/-) mice at older ages. These findings establish that both cell-autonomous and systemic mechanisms contribute to ectopic mineralization in ENT1(-/-) mice.

Colorectal cancer (CRC) is one of the most common malignancies, giving rise to serious financial burden globally. This study was designed to explore the potential mechanisms implicated with CRC and identify some key biomarkers. CRC-associated gene expression dataset (GSE32323) was downloaded from GEO database. The differentially expressed genes (DEGs) were selected out based on the GEO2R tool. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were employed to search the enriched pathways of these DEGs. Additionally, a protein-protein interaction (PPI) network was also constructed to visualize interactions between these DEGs. Quantitative Real-time PCR (qPCR) was further performed to valid the top5 up-regulated and top5 down-regulated genes in patients with CRC. Finally, the survival analysis of the top5 up-regulated and top5 down-regulated genes was conducted using GEPIA, aiming to clarify their potential effects on CRC. In this study, a total of 451 DEGs were captured (306 down-regulated genes and 145 up-regulated genes). Among these DEGs, the top5 up-regulated genes were DPEP1, KRT23, CLDN1, LGR5 and FOXQ1 while the top5 down-regulated genes were CLCA4, ZG16, SLC4A4, ADH1B and GCG. GO analysis revealed that these DEGs were mainly enriched in cell adhesion, cell proliferation, RNA polymerase II promoter and chemokine activity. KEGG analysis disclosed that the enriched pathway included mineral absorption, chemokine signaling pathway, transcriptional misregulation in cancer, pathways in cancer and PPAR signaling pathway. Survival analysis showed that the expression level of ZG16 may correlate with the prognosis of CRC patients. Furthermore, according to the connectivity degree of these DEGs, we selected out the top15 hub genes, namely MYC, CXCR1, TOP2A, CXCL12, SST, TIMP1, SPP1, PPBP, CDK1, THBS1, CXCL1, PYY, LPAR1, BMP2 and MMP3, which were expected to be promising therapeutic target in CRC. Collectively, our analysis unveiled potential biomarkers and candidate targets in CRC, which could be helpful to the diagnosis and treatment of CRC.

The expression of genes and their regulation during lactation in buffaloes remains less understood. To understand the interplay of various genes and pathways, the milk transcriptome from three lactation stages of Murrah buffalo was analyzed by RNA sequencing. The filtered reads were mapped to the Bubalus bubalis as well as Bos taurus reference assemblies. The average mapping rate to water buffalo and Btau 4.6 reference sequence, was 75.5% and 75.7% respectively. Highly expressed genes (RPKM > 3000), throughout lactation included CSN2, CSN1S1, CSN3, LALBA, SPP1 and TPT1. A total of 12833 transcripts were common across all the stages, while 271, 205 and 418 were unique to early, mid and late lactation respectively. Majority of the genes throughout lactation were linked to biological functions like protein metabolism, transport and immune response. A discernible shift from metabolism in early stage to metabolism and immune response in mid stage, and an increase in immune response functions in late lactation was observed. The results provide information of candidate genes and pathways involved in the different stages of lactation in buffalo. The study also identified 14 differentially expressed and highly connected genes across the three lactation stages, which can be used as candidates for future research.

Alzheimer's disease (AD) is a chronic neurodegenerative disease characterized by intracellular formation of neurofibrillary tangles and extracellular deposition of β-amyloid protein (Aβ) in the extracellular matrix. The pathogenesis of AD has not yet been fully elucidated and little is known about global alterations in the brain proteome that are related to AD. To identify and quantify such AD-related changes in the brain, we employed a tandem mass tags (TMT) approach coupled to high-resolution mass spectrometry. We compared the proteomes of frontal cortex from AD patients with corresponding age-matched brain samples. LC-MS/MS analysis carried out on an Orbitrap Fusion Lumos Tribrid mass spectrometer led to identification of 8,066 proteins. Of these, 432 proteins were observed to be significantly altered (>1.5 fold) in their expression in AD brains. Proteins whose abundance was previously known to be altered in AD were identified including secreted phosphoprotein 1 (SPP1), somatostatin (SST), SPARC related modular calcium binding 1 (SMOC1), dual specificity phosphatase 26 (DUSP26) and neuronal pentraxin 2 (NPTX2). In addition, we identified several novel candidates whose association with AD has not been previously described. Of the novel molecules, we validated chromogranin A (CHGA), inner membrane mitochondrial protein (IMMT) and RAS like proto-oncogene A (RALA) in an additional set of 20 independent brain samples using targeted parallel reaction monitoring (PRM) mass spectrometry assays. The differentially expressed proteins discovered in our study, once validated in larger cohorts, should help discern the pathogenesis of AD.

Keywords: AD; Neurodegeneration; Proteome; TMT.

BACKGROUND

Osteoporosis is a disease characterized by low bone mineral density (BMD) and increased risk of fractures. Studies have demonstrated the use of phytoestrogens, or plant-derived estrogens, such as genistein and daidzein, to effectively increase osteogenic activity of bone marrow-derived mesenchymal stem cells (BMSCs). Herein, the effects of daidzein analogs on the osteogenic differentiation efficiency of human BMSC and adipose-derived stromal/stem cells (ASC) were explored.

METHODS

BMSCs and ASCs underwent osteogenic differentiation in the presence of vehicle, 17β-estradiol (E2), phytoestrogens, or daidzein analogs. Cells were stained for alkaline phosphatase (ALP) enzymatic activity, calcium deposition by alizarin red s, and phosphate mineralization by silver nitrate. Gene expression analysis was conducted on cells treated with daidzein analogs.

RESULTS

Cells treated with E2, daidzein, or genistein increased calcium deposition by 1.6-, 1.5-, and 1.4-fold, respectively, relative to vehicle-treated BMSCs and 1.6-, 1.7-, and 1.4-fold relative to vehicle-treated ASCs, respectively. BMSCs treated with daidzein analog 2c, 2g, and 2l demonstrated a 1.6-, 1.6-, and 1.9-fold increase in calcium deposition relative to vehicle-treated BMSCs, respectively, while ASCs treated with daidzein analog 2c, 2g, or 2l demonstrated a 1.7-, 2.0-, and 2.2-fold increase in calcium deposition relative to vehicle-treated ASCs, respectively. Additional analysis with BMSCs and ASCs was conducted in the more efficient compounds: 2g and 2l. ALP activity and phosphate mineralization was increased in 2g- and 2l-treated cells. The analysis of lineage specific gene expression demonstrated increased expression of key osteogenic genes (RUNX2, c-FOS, SPARC, DLX5, SPP1, COL1A1, IGF1, SOST, and DMP1) and earlier induction of these lineage specific genes, following treatment with 2g or 2l, relative to vehicle-treated cells. Estrogen receptor (ER) inhibitor studies demonstrated that ER antagonist fulvestrant inhibited the osteogenic differentiation of 2g in BMSCs and ASCs, while fulvestrant only attenuated the effects of 2l, suggesting that 2l acts by both ER dependent and independent pathways.

CONCLUSIONS

These studies provide support for exploring the therapeutic efficacy of daidzein derivatives for the treatment of osteoporosis. Furthermore, the patterns of gene induction differed following treatment with each daidzein analog, suggesting that these daidzein analogs activate distinct ER and non-ER pathways to induce differentiation in BMSCs and ASCs.

Progressive lung fibrosis is a major cause of mortality in systemic sclerosis (SSc) patients, but the underlying mechanisms remain unclear. We demonstrate that immune complexes (ICs) activate human monocytes to promote lung fibroblast migration partly via osteopontin (OPN) secretion, which is amplified by autocrine monocyte colony stimulating factor (MCSF) and interleukin-6 (IL-6) activity. Bulk and single-cell RNA sequencing demonstrate that elevated OPN expression in SSc lung tissue is enriched in macrophages, partially overlapping with CCL18 expression. Serum OPN is elevated in SSc patients with interstitial lung disease (ILD) and prognosticates future lung function deterioration in SSc cohorts. Serum OPN levels decrease following tocilizumab (monoclonal anti-IL-6 receptor) treatment, confirming the connection between IL-6 and OPN in SSc patients. Collectively, these data suggest a plausible link between autoantibodies and lung fibrosis progression, where circulating OPN serves as a systemic proxy for IC-driven profibrotic macrophage activity, highlighting its potential as a promising biomarker in SSc ILD.

Keywords: IL-6; ILD; SPP1; SSc; biomarker; fibrosis; immune complex; macrophages; osteopontin; systemic sclerosis.

BACKGROUND

Sleeve gastrectomy (SG) constitutes an effective procedure for the treatment of morbid obesity. The aim of the present study was to establish in rats the effects of surgically induced weight loss on circulating concentrations and mRNA expression in adipose tissue and liver of osteopontin (OPN), a proinflammatory protein involved in the development of obesity.

METHODS

Eighty male diet-induced obese Wistar rats were subjected to surgical interventions [sham operation (SH), SG, or pair-fed to the amount of food eaten by SG animals] and dietary interventions [fed ad libitum with a normal chow diet (ND) or a high-fat diet (HFD)]. Body, epididymal adipose tissue (EWAT), and liver weights were determined. Circulating OPN concentrations and the transcript levels of Spp1 (OPN) in EWAT and liver were analyzed.

RESULTS

Rats undergoing SG showed decreased body weight (P < 0.001) and fat mass (P < 0.001) and greater excess weight loss (P < 0.001). The HFD significantly decreased serum OPN levels (P < 0.001). However, SG did not change serum OPN concentrations. OPN expression was dramatically increased in animals fed HFD (P < 0.001) in EWAT, but was unaffected by SG. The expression of OPN in the liver was not affected by HFD or SG.

CONCLUSIONS

Circulating OPN levels decreased with HFD feeding remaining unaltered after SG. The expression of Spp1 in EWAT and liver was not modified by SG. The global improvement of metabolism after SG appears not to involve changes in serum OPN concentrations as well as in EWAT and liver expression in rats.

Dental pulp cells play an important role in maintaining dental mineralized tissue throughout life. Supplementary mineralization such as reparative dentin and pulp stone frequently occurs after primary dentin formation. Dental pulp cells are thought to be closely associated with such mineralization. We found that clonal rat dental pulp cells, RDP4-1 and RPC-C2A, produce and secrete osteopontin, but do not synthesize phosphophoryn which is a major noncollagenous protein found in dentin. The dental pulp osteopontin was highly phosphorylated and identified by thrombin susceptibility and immunoprecipitation with osteopontin/2ar antibody. Osteopontin synthesis markedly increased by 12-O-tetradecanoylphorbol-13-acetate (TPA) as observed in many osteoblastic cells. This study indicates that these cells can produce osteopontin as a major phosphoprotein and suggests that the synthesis of osteopontin could be used as a characteristic marker of dental pulp cells.