Pirfenidone and nintedanib exert additive antifibrotic effects by the SPP1-AKT pathway in macrophages and fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive disease with high mortality rates. It has been shown that pirfenidone (PFD) and nintedanib (Ofev) can slow down the decline in lung function of IPF patients, but their efficacy remains suboptimal. Some studies have suggested that the combination of PFD and Ofev may yield promising results. However, there is a lack of research on the combined application of these two medications in the treatment of IPF. A mouse model of bleomycin-induced (BLM) pulmonary fibrosis was established to investigate the impact of combination therapy on pulmonary fibrosis of mice. The findings demonstrated a significant reduction in lung tissue damage in mice treated with the combination therapy. Subsequent transcriptome analysis identified the differential gene secreted phosphoprotein 1 (SPP1), which was found to be associated with macrophages and fibroblasts based on multiple immunofluorescence staining results. Analysis of a phosphorylated protein microarray indicated that SPP1 plays a regulatory role in macrophages and fibroblasts via the AKT pathway. Consequently, the regulation of macrophages and fibroblasts in pulmonary fibrosis by the combination of PFD and Ofev is mediated by SPP1 through the AKT pathway, potentially offering a novel therapeutic option for IPF patients. Further investigation into the targeting of SPP1 for the treatment of pulmonary fibrosis is warranted.

SPP1 expression indicates outcome of immunotherapy plus tyrosine kinase inhibition in advanced renal cell carcinoma.

Clinical guidelines have recently advised combination therapy involving immunotherapy (IO) and tyrosine kinase inhibitors (TKI) as the first-line therapy approach for advanced renal cell carcinoma (RCC). Nevertheless, there is currently no available biomarker that can effectively distinguish the progression-free survival (PFS). RNA-sequencing and immunohistochemistry were conducted on our cohort of metastatic RCC patients, namely ZS-MRCC, who received combination therapy consisting of IO and TKI. We further applied RNA-sequencing, immunohistochemistry, and flow cytometry to examine the immune cell infiltration and functionality inside the tumor microenvironment of high-risk localized RCC samples. SPP1 expression was significantly higher in non-responders to IO-TKI therapy. Elevated levels of SPP1 were associated with poor PFS in both the ZS-MRCC cohort (HR = 2.73, p = .018) and validated in the JAVELIN Renal 101 cohort (HR = 1.61, p = .004). By multivariate Cox analysis, SPP1 was identified as a significant independent prognosticator. Furthermore, there existed a negative correlation between elevated levels of SPP1 and the presence of GZMB+CD8+ T cells (Spearman's ρ= -0.48, p < .001). Conversely, SPP1 expression is associated with T cell exhaustion markers. A significant increase in the abundance of Tregs was observed in tumors with high levels of SPP1. Additionally, a machine-learning-based model was constructed to predict the benefit of IO-TKI treatment. High SPP1 is associated with therapeutic resistance and unfavorable PFS in IO-TKI therapy. SPP1 expression have also been observed to be indicative of malfunction and exhaustion in T cells. Increased SPP1 expression has the potential to serve as a potential biomarker for treatment selection of metastatic RCC.

Functionalizable bacterial cellulose composite membrane for guided tissue regeneration.

This research aims to develop guided tissue regeneration (GTR) membranes from bacterial cellulose (BC), a natural polysaccharide-based biopolymer. A double-layered BC composite membrane was prepared by coating the BC membrane with mixed carboxymethyl cellulose/poly(ethylene oxide) (CMC/PEO) fibers via electrospinning. The CMC/PEO-BC membranes were then characterized for their chemical and physical characteristics. The 8 % (wt/v) CMC/PEO (1:1) aqueous solution yielded well-defined electrospun CMC/PEO nanofibers (125 ± 10 nm) without beads. The CMC/PEO-BC membranes exhibited good mechanical and swelling properties as well as good cytocompatibility against human periodontal ligament cells (hPDLs). Its functionalizability via carboxyl entities in CMC was tested using the calcium-binding domain of plant-derived recombinant human osteopontin (p-rhOPN-C122). As evaluated by enzyme-linked immunosorbent assay, a 98-99 % immobilization efficiency was achieved in a concentration-dependent manner over an applied p-rhOPN-C122 concentration range of 7.5-30 ng/mL. The biological function of the membrane was assessed by determining the expression levels of osteogenic-related gene transcripts using quantitative real-time reverse-transcriptase polymerase chain reaction. Mineralization assay indicated that the p-rhOPN-C122 immobilized CMC/PEO-BC membrane promoted hPDLs osteogenic differentiation. These results suggested that the developed membrane could serve as a promising GTR membrane for application in bone tissue regeneration.

HOXB9 promotes osteosarcoma cell survival and malignancy under glucose starvation via upregulating SPP1 expression.

Homeobox B9 (HOXB9) has been shown to play a critical role in several tumors. However, the precise biological mechanisms and functions of HOXB9 in osteosarcoma remain largely unknown. In this study, we found that HOXB9 was increased upon glucose starvation. Elevated HOXB9 suppressed osteosarcoma cell death and supported cell growth and migration under glucose starvation. Further mechanistic studies demonstrated that HOXB9 directly bound to the promoter of secreted phosphoprotein 1 (SPP1) and transcriptionally upregulated SPP1 expression which then led cell death decrease and cell growth increase under glucose deprivation environment. Clinically, HOXB9 was significantly upregulated in osteosarcoma compared with normal tissues and increase of HOXB9 expression was positively associated with the elevation of SPP1 in osteosarcoma. Overall, our study illustrates that HOXB9 contributes to malignancy in osteosarcoma and inhibits cell death through transcriptional upregulating SPP1 under glucose starvation.

Osteopontin drives neuroinflammation and cell loss in MAPT-N279K frontotemporal dementia patient neurons.

Frontotemporal dementia (FTD) is an incurable group of early-onset dementias that can be caused by the deposition of hyperphosphorylated tau in patient brains. However, the mechanisms leading to neurodegeneration remain largely unknown. Here, we combined single-cell analyses of FTD patient brains with a stem cell culture and transplantation model of FTD. We identified disease phenotypes in FTD neurons carrying the MAPT-N279K mutation, which were related to oxidative stress, oxidative phosphorylation, and neuroinflammation with an upregulation of the inflammation-associated protein osteopontin (OPN). Human FTD neurons survived less and elicited an increased microglial response after transplantation into the mouse forebrain, which we further characterized by single nucleus RNA sequencing of microdissected grafts. Notably, downregulation of OPN in engrafted FTD neurons resulted in improved engraftment and reduced microglial infiltration, indicating an immune-modulatory role of OPN in patient neurons, which may represent a potential therapeutic target in FTD.

Expression and Impact of Fibronectin, Tenascin-C, Osteopontin, and Type XIV Collagen in Fuchs Endothelial Corneal Dystrophy.

Purpose: Fuchs endothelial corneal dystrophy (FECD) is characterized by Descemet's membrane (DM) abnormalities, namely an increased thickness and a progressive appearance of guttae and fibrillar membranes. The goal of this study was to identify abnormal extracellular matrix (ECM) proteins expressed in FECD DMs and to evaluate their impact on cell adhesion and migration. Methods: Gene expression profiles from in vitro (GSE112039) and ex vivo (GSE74123) healthy and FECD corneal endothelial cells were analyzed to identify deregulated matrisome genes. Healthy and end-stage FECD DMs were fixed and analyzed for guttae size and height. Immunostaining of fibronectin, tenascin-C, osteopontin, and type XIV collagen was performed on ex vivo specimens, as well as on tissue-engineered corneal endothelium reconstructed using healthy and FECD cells. An analysis of ECM protein expression according to guttae and fibrillar membrane was performed using immunofluorescent staining and phase contrast microscopy. Finally, cell adhesion was evaluated on fibronectin, tenascin-C, and osteopontin, and cell migration was studied on fibronectin and tenascin-C. Results: SPP1 (osteopontin), FN1 (fibronectin), and TNC (tenascin-C) genes were upregulated in FECD ex vivo cells, and SSP1 was upregulated in both in vitro and ex vivo FECD conditions. Osteopontin, fibronectin, tenascin-C, and type XIV collagen were expressed in FECD specimens, with differences in their location. Corneal endothelial cell adhesion was not significantly affected by fibronectin or tenascin-C but was decreased by osteopontin. The combination of fibronectin and tenascin-C significantly increased cell migration. Conclusions: This study highlights new abnormal ECM components in FECD, suggests a certain chronology in their deposition, and demonstrates their impact on cell behavior.

Tumor-associated macrophage subtypes on cancer immunity along with prognostic analysis and SPP1-mediated interactions between tumor cells and macrophages.

Tumor-associated macrophages (TAM) subtypes have been shown to impact cancer prognosis and resistance to immunotherapy. However, there is still a lack of systematic investigation into their molecular characteristics and clinical relevance in different cancer types. Single-cell RNA sequencing data from three different tumor types were used to cluster and type macrophages. Functional analysis and communication of TAM subpopulations were performed by Gene Ontology-Biological Process and CellChat respectively. Differential expression of characteristic genes in subpopulations was calculated using zscore as well as edgeR and Wilcoxon rank sum tests, and subsequently gene enrichment analysis of characteristic genes and anti-PD-1 resistance was performed by the REACTOME database. We revealed the heterogeneity of TAM, and identified eleven subtypes and their impact on prognosis. These subtypes expressed different molecular functions respectively, such as being involved in T cell activation, apoptosis and differentiation, or regulating viral bioprocesses or responses to viruses. The SPP1 pathway was identified as a critical mediator of communication between TAM subpopulations, as well as between TAM and epithelial cells. Macrophages with high expression of SPP1 resulted in poorer survival. By in vitro study, we showed SPP1 mediated the interactions between TAM clusters and between TAM and tumor cells. SPP1 promoted the tumor-promoting ability of TAM, and increased PDL1 expression and stemness of tumor cells. Inhibition of SPP1 attenuated N-cadherin and ß-catenin expression and the activation of AKT and STAT3 pathway in tumor cells. Additionally, we found that several subpopulations could decrease the sensitivity of anti-PD-1 therapy in melanoma. SPP1 signal was a critical pathway of communication between macrophage subtypes. Some specific macrophage subtypes were associated with immunotherapy resistance and prognosis in some cancer types.

Pharmacological effects of methysticin and L-sulforaphane through the Nrf2/ARE signaling pathway in MLO-Y4 osteocytes: in vitro study.

BACKGROUND: Oxidative stress plays a crucial role in the pathogenesis of many skeletal diseases by inducing osteocyte death. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is a master regulator of various antioxidant gene expressions through antioxidant response element (ARE) against cellular oxidative stress and can be induced by various stimulants, including the phytochemicals methysticin (MET) and L-sulforaphane (SFN). This study aimed to establish an osteocyte in vitro model to investigate the pharmacological effects of MET and SFN on the Nrf2/ARE pathway. METHODS: MLO-Y4 murine osteocytes and the stably transduced MLO-Y4-SIN-lenti-ARE reporter gene cell line were used. MET and SFN were used as Nrf2 inducers. The cytotoxicity of MET, SFN, and hydrogen peroxide (H2O2) was evaluated using the CytoTox-Glo™ Assay. Time- and dose-dependent ARE induction was examined by Monoluciferase Assay. The mRNA and protein expressions of Nrf2 target markers, such as heme-oxygenase 1 (Ho-1), NADPH quinone dehydrogenase 1 (Nqo1), and thioredoxin reductase 1 (Txnrd1), were detected by RT-qPCR, Western Blot, and immunofluorescence staining, respectively. Osteogenesis markers, osteopontin, and osteocalcin were compared with and without treatment by immunofluorescence staining. RESULTS: The experimental data showed that MET and SFN induced ARE activity in a time- and dose-dependent manner and increased the mRNA and protein expression of antioxidant markers compared to vehicle-treated controls. The protein expression of osteopontin and osteocalcin in the samples treated with SFN were significantly higher than without treatment, and the number of cell death treated with SFN was significantly lower than without treatment under H2O2-induced stress conditions. CONCLUSIONS: Nrf2 inducers MET and SFN increased the mRNA expression of antioxidant genes through the Nrf2/ARE pathway in osteocytes. Notably, SFN increased the protein expression of osteocyte-associated osteogenic markers and suppressed cell death under H2O2-induced stress condition. Thus, Nrf2 stimulators can exert stress-relieving and osteogenic effects on osteocytes.

Serum secreted phosphoprotein 1 level is associated with plaque vulnerability in patients with coronary artery disease.

Background: Vulnerable plaque was associated with recurrent cardiovascular events. This study was designed to explore predictive biomarkers of vulnerable plaque in patients with coronary artery disease. Methods: To reveal the phenotype-associated cell type in the development of vulnerable plaque and to identify hub gene for pathological process, we combined single-cell RNA and bulk RNA sequencing datasets of human atherosclerotic plaques using Single-Cell Identification of Subpopulations with Bulk Sample Phenotype Correlation (Scissor) and Weighted gene co-expression network analysis (WGCNA). We also validated our results in an independent cohort of patients by using intravascular ultrasound during coronary angiography. Results: Macrophages were found to be strongly correlated with plaque vulnerability while vascular smooth muscle cell (VSMC), fibrochondrocyte (FC) and intermediate cell state (ICS) clusters were negatively associated with unstable plaque. Weighted gene co-expression network analysis showed that Secreted Phosphoprotein 1 (SPP1) in the turquoise module was highly correlated with both the gene module and the clinical traits. In a total of 593 patients, serum levels of SPP1 were significantly higher in patients with vulnerable plaques than those with stable plaque (113.21 [73.65 - 147.70] ng/ml versus 71.08 [20.64 - 135.68] ng/ml; P < 0.001). Adjusted multivariate regression analysis revealed that serum SPP1 was an independent determinant of the presence of vulnerable plaque. Receiver operating characteristic curve analysis indicated that the area under the curve was 0.737 (95% CI 0.697 - 0.773; P < 0.001) for adding serum SPP1 in predicting of vulnerable plaques. Conclusion: Elevated serum SPP1 levels confer an increased risk for plaque vulnerability in patients with coronary artery disease.

Enhancing immune regulation in vitro: the synergistic impact of 3'-sialyllactose and osteopontin in a nutrient blend following influenza virus infection.

Natural components of breast milk, human milk oligosaccharides (HMOs) and osteopontin (OPN) have been shown to have a variety of functional activities and are widely used in infant formulas. However, the preventive and therapeutic effects of both on influenza viruses are not known. In this study, antiviral assays using a human laryngeal carcinoma cell line (HEP-2) showed that 3'-sialyllactose (3'-SL) and OPN had the best antiviral ability with IC50 values of 33.46 µM and 1.65 µM, respectively. 3'-SL (10 µM) and OPN (4 µM) were used in combination to achieve 75% inhibition. Further studies found that the combination of 200 µg/mL of 3'-SL with 500 µg/mL of OPN exerted the best antiviral ability. The reason for this was related to reduced levels of the cytokines TNF-α, IL-6, and iNOS in relation to mRNA expression. Plaque assay and TCID50 assay found the same results and verified synergistic effects. Our research indicates that a combination of 3'-SL and OPN can effectively reduce inflammatory storms and exhibit anti-influenza virus effects through synergistic action.

SOCS3 regulates pathological retinal angiogenesis through modulating SPP1 expression in microglia and macrophages.

Pathological ocular angiogenesis has long been associated with myeloid cell activation. However, the precise cellular and molecular mechanisms governing the intricate crosstalk between the immune system and vascular changes during ocular neovascularization formation remain elusive. In this study, we demonstrated that the absence of the suppressor of cytokine signaling 3 (SOCS3) in myeloid cells led to a substantial accumulation of microglia and macrophage subsets during the neovascularization process. Our single-cell RNA sequencing data analysis revealed a remarkable increase in the expression of the secreted phosphoprotein 1 (Spp1) gene within these microglia and macrophages, identifying subsets of Spp1-expressing microglia and macrophages during neovascularization formation in angiogenesis mouse models. Notably, the number of Spp1-expressing microglia and macrophages exhibited further elevation during neovascularization in mice lacking myeloid SOCS3. Moreover, our investigation unveiled the Spp1 gene as a direct transcriptional target gene of signal transducer and activator of transcription 3. Importantly, pharmaceutical activation of SOCS3 or blocking of SPP1 resulted in a significant reduction in pathological neovascularization. In conclusion, our study highlights the pivotal role of the SOCS3/STAT3/SPP1 axis in the regulation of pathological retinal angiogenesis.

METTL3/16-mediated m6A modification of ZNNT1 promotes hepatocellular carcinoma progression by activating ZNNT1/osteopontin/S100A9 positive feedback loop-mediated crosstalk between macrophages and tumour cells.

Macrophages are the major components of tumour microenvironment, which play critical roles in tumour development. N6-methyladenosine (m6A) also contributes to tumour progression. However, the potential roles of m6A in modulating macrophages in hepatocellular carcinoma (HCC) are poorly understood. Here, we identified ZNNT1 as an HCC-related m6A modification target, which was upregulated and associated with poor prognosis of HCC. METTL3 and METTL16-mediated m6A modification contributed to ZNNT1 upregulation through stabilizing ZNNT1 transcript. ZNNT1 exerted oncogenic roles in HCC. Furthermore, ZNNT1 recruited and induced M2 polarization of macrophages via up-regulating osteopontin (OPN) expression and secretion. M2 Macrophages-recruited by ZNNT1-overexpressed HCC cells secreted S100A9, which further upregulated ZNNT1 expression in HCC cells via AGER/NF-κB signaling. Thus, this study demonstrates that m6A modification activated the ZNNT1/OPN/S100A9 positive feedback loop, which promoted macrophages recruitment and M2 polarization, and enhanced malignant features of HCC cells. m6A modification-triggered ZNNT1/OPN/S100A9 feedback loop represents potential therapeutic target for HCC.

Targeted Macrophage CRISPR-Cas13 mRNA Editing in Immunotherapy for Tendon Injury.

CRISPR-Cas13 holds substantial promise for tissue repair through its RNA editing capabilities and swift catabolism. However, conventional delivery methods fall short in addressing the heightened inflammatory response orchestrated by macrophages during the acute stages of tendon injury. In this investigation, macrophage-targeting cationic polymers are systematically screened to facilitate the entry of Cas13 ribonucleic-protein complex (Cas13 RNP) into macrophages. Notably, SPP1 (OPN encoding)-producing macrophages are recognized as a profibrotic subtype that emerges during the inflammatory stage. By employing ROS-responsive release mechanisms tailored for macrophage-targeted Cas13 RNP editing systems, the overactivation of SPP1 is curbed in the face of an acute immune microenvironment. Upon encapsulating this composite membrane around the tendon injury site, the macrophage-targeted Cas13 RNP effectively curtails the emergence of injury-induced SPP1-producing macrophages in the acute phase, leading to diminished fibroblast activation and mitigated peritendinous adhesion. Consequently, this study furnishes a swift RNA editing strategy for macrophages in the inflammatory phase triggered by ROS in tendon injury, along with a pioneering macrophage-targeted carrier proficient in delivering Cas13 into macrophages efficiently.

Anoikis and SPP1 in idiopathic pulmonary fibrosis: integrating bioinformatics, cell, and animal studies to explore prognostic biomarkers and PI3K/AKT signaling regulation.

OBJECTIVE: This study aims to explore the relevance of anoikis in idiopathic pulmonary fibrosis (IPF) and identify associated biomarkers and signaling pathways. METHOD: Unsupervised consensus cluster analysis was employed to categorize IPF patients into subtypes. We utilized Weighted Gene Co-Expression Network Analysis (WGCNA) and Protein-Protein Interaction network construction to identify anoikis-related modules and key genes. A prognostic signature was developed using Lasso and multivariate Cox regression analysis. Single-cell sequencing assessed hub gene expression in various cell types, and both cell and animal experiments confirmed IPF-related pathways. RESULTS: We identified two distinct anoikis-associated subtypes with differing prognoses. WGCNA revealed essential hub genes, with SPP1 being prominent in the anoikis-related signature. The anoikis-related signature is effective in determining the prognosis of patients with IPF. Single-cell sequencing highlighted significant differences in SPP1 expression, notably elevated in fibroblasts derived from IPF patients. In vivo and in vitro experiments demonstrated that SPP1 enhances fibrosis in mouse lung fibroblasts by regulating p27 through the PI3K/Akt pathway. CONCLUSION: Our research demonstrates a robust prognostic signature associated with anoikis and highlights SPP1 as a pivotal regulator of the PI3K/AKT signaling pathway in pulmonary fibrosis.

Overexpression of SPP1 is a prognostic indicator of immune infiltration in lung adenocarcinoma.

OBJECTIVE: The extracellular phosphoprotein, secreted phosphoprotein 1 (SPP1), plays a crucial role in various tumors and regulating the immune system. This study aimed to evaluate its prognostic value and relationship to immune infiltration in lung adenocarcinoma (LUAD). METHODS: In the TCGA and GEO datasets, the information on clinic and transcriptome analysis of SPP1 in non-small-cell lung cancer (NSCLC) was examined accordingly. The association of SPP1 expression with overall survival and clinicopathologic characteristics was investigated by univariate and multivariate analysis. CancerSEA database was utilized to investigate the role of SPP1 at the cellular level by single-cell analysis. Additionally, the CIBERSORT algorithm was utilized to assess the correlation among the immune cells that infiltrated. RESULTS: NSCLC tissues exhibited a notable rise in SPP1 expression compared with that of normal tissues. Furthermore, the overexpression of SPP1 was substantially associated with clinicopathological features and unfavorable survival outcomes in individuals with LUAD, whereas no such correlation was observed in lung squamous cell carcinoma. Immune cells that infiltrate tumors and their corresponding genes were associated with SPP1 expression levels in LUAD. CONCLUSIONS: SPP1 is a reliable indicator for assessing LUAD immune infiltration status and prognosis. With this approach, SPP1 can help earlier LUAD diagnosis and act as a possible immunotherapy target.

Gastro-Intestinal Digested Bovine Milk Osteopontin Modulates Gut Barrier Biomarkers In Vitro.

SCOPE: Osteopontin (OPN) is a multifunctional protein naturally present in mammals' milk, associated with immune homeostasis and intestinal maturation. This study aims to investigate the protein digestion pattern and the cellular bioactivity of bovine milk OPN digesta in vitro. METHODS AND RESULTS: A modified INFOGEST static in vitro infant digestion protocol and a Caco-2/HT-29 co-culture cell model are employed to evaluate the digestion properties and the anti-inflammatory effects of OPN. OPN is resistant to gastric hydrolysis but degraded into large peptides during intestinal digestion. Its 10 kDa digesta permeate with predicted extensive bioactivities protects the co-culture cell model from the inflammation-induced dysfunction by dose-dependently recovering the expression of occludin, claudin-3, and ZO-1. Low dosage of OPN significantly decreases the production of IL-8 and IL-6, and downregulates the mRNA and protein expression of MyD88, NF-κB p65, and IκB-α, whereas a high dose evokes a mild pro-inflammatory response. Interestingly, anti-inflammatory effect of OPN digesta is stronger than lactoferrin and whey protein concentrate counterparts. CONCLUSION: The findings demonstrate that the bioactive peptides released from in vitro infant gastrointestinal digestion of bovine milk OPN alleviates intestinal epithelial cell inflammation by inhibiting NF-κB pathway activation and potentiates the barrier function of the intestinal epithelium.

Immunoregulation of bovine lactoferrin together with osteopontin promotes immune system development and maturation.

The immune system of infants is partly weak and immature, and supplementation of infant formula can be of vital importance to boost the development of the immune system. Lactoferrin (LF) and osteopontin (OPN) are essential proteins in human milk with immunoregulation function. An increasing number of studies indicate that proteins have interactions with each other in milk, and our previous study found that a ratio of LF : OPN at 1 : 5 (w/w, denoted as LOP) had a synergistic effect on intestinal barrier protection. It remains unknown whether LOP can also exert a stronger effect on immunoregulation. Hence, we used an in vitro model of LPS-induced macrophage inflammation and in vivo models of LPS-induced intestinal inflammation and early life development. We showed that LOP increased the secretion of the granulocyte-macrophage colony-stimulating factor (132%), stem cell factor (167%) and interleukin-3 (176%) in bone marrow cells, as well as thymosin (155%) and interleukin-10 (161%) in the thymus, more than LF or OPN alone during development, and inhibited changes in immune cells and cytokines during the LPS challenge. In addition, analysis of the components of digested proteins in vitro revealed that differentially expressed peptides may provide immunoregulation. Lastly, LOP increased the abundance of Rikenellaceae, Muribaculum, Faecalibaculum, and Elisenbergiella in the cecum content. These results imply that LOP is a potential immunomodifier for infants and offers a new theoretical basis for infant formula innovation.

Effects of recombinant osteopontin expressed in Escherichia coli on the recovery of the endometrial epidermal growth factor profile and fertility in repeat breeder dairy cows.

Endometrial epidermal growth factor (EGF) shows a cyclic change with two peaks on days 2-4 and days 13-14 of the estrous cycle. In repeat breeder cows, loss of the peaks has been associated with reduced fertility. By infusing seminal plasma (SP) and osteopontin (OPN) derived from SP and milk into the vagina, their EGF profile and fertility are restored. However, SP is difficult to obtain, and both SP and OPN can transmit infectious diseases. While OPN can be sourced from recombinant protein without this risk, recombinant bovine OPN (rOPN) expressed in Escherichia coli should be examined for its effects on the EGF profile, since it does not undergo posttranslational modification, which is important for its biological activity. In study 1, PBS, SP (0.5 mL), and rOPN (0.3 mg) were infused into the vagina at estrus (day 0) in 74, 37, and 105 repeat breeder Holstein cows, respectively, with an altered EGF profile. The endometrial EGF concentrations were measured on day 3. Some cows (n = 58, 20, and 83, respectively) were inseminated immediately before the infusion and then diagnosed for pregnancy between days 30 and 35. The normalization rate of the EGF profile and conception rate in the rOPN group (58.1 % and 47.0 %, respectively) were not significantly different from those in the SP group (62.2 % and 45.0 %, respectively) but higher than those in PBS group (29.7 % and 28.1 %, respectively) (P < 0.05). In study 2, repeat breeder cows with an altered EGF profile were infused with PBS (n = 18) and rOPN (n = 17), while fertile controls with a normal EGF profile (n = 18) were infused with PBS. Two or three embryos were transferred into cows on day 7 and then recovered on day 14. Embryo recovery rates of the rOPN and fertile groups were comparable (58.7 % vs. 58.3 %) but higher than that of the PBS group (58.7 % vs. 32.0 %) (P < 0.05). The embryo recovery rate of cows with normalized EGF profile was higher than that of cows with unnormalized EGF profile (64.4 % vs. 16.7 %) (P < 0.05). The embryo sizes of cows in the rOPN and fertile groups were comparable but larger than those in the PBS group (P < 0.05). However, the embryo size was not correlated to the corresponding endometrial EGF concentrations. In conclusion, rOPN without posttranslational modifications normalized the EGF profile in repeat breeder cows. Improved fertility by normalization of the EGF profile could be attributed partly to the increased embryo viability up to day 14.

SPP1, a potential therapeutic target and biomarker for lung cancer: functional insights through computational studies.

NIH reported 128 different types of cancer of which lung cancer is the leading cause of mortality. Globally, it is estimated that on average one in every seventeen hospitalized patients was deceased. There are plenty of studies that have been reported on lung cancer draggability and therapeutics, but yet a protein that plays a central specific to cure the disease remains unclear. So, this study is designed to identify the possible therapeutic targets and biomarkers that can be used for the potential treatment of lung cancers. In order to identify differentially expressed genes, 39 microarray datasets of lung cancer patients were obtained from various demographic regions of the GEO database available at NCBI. After annotating statistically, 6229 up-regulated genes and 10324 down-regulated genes were found. Out of 17 up-regulated genes and significant genes, we selected SPP1 (osteopontin) through virtual screening studies. We found functional interactions with the other cancer-associated genes such as VEGF, FGA, JUN, EGFR, and TGFB1. For the virtual screening studies,198 biological compounds were retrieved from the ACNPD database and docked with SPP1 protein (PDBID: 3DSF). In the results, two highly potential compounds secoisolariciresinol diglucoside (-12.9 kcal/mol), and Hesperidin (-12.0 kcal/mol) showed the highest binding affinity. The stability of the complex was accessed by 100 ns simulation in an SPC water model. From the functional insights obtained through these computational studies, we report that SPP1 could be a potential biomarker and successive therapeutic protein target for lung cancer treatment.

Osteopontin: A Novel Therapeutic Target for Respiratory Diseases.

Osteopontin (OPN) is a multifunctional phosphorylated protein that is involved in physiological and pathological events. Emerging evidence suggests that OPN also plays a critical role in the pathogenesis of respiratory diseases. OPN can be produced and secreted by various cell types in lungs and overexpression of OPN has been found in acute lung injury/acute respiratory distress syndrome (ALI/ARDS), pulmonary hypertension (PH), pulmonary fibrosis diseases, lung cancer, lung infection, chronic obstructive pulmonary disease (COPD), and asthma. OPN exerts diverse effects on the inflammatory response, immune cell activation, fibrosis and tissue remodeling, and tumorigenesis of these respiratory diseases, and genetic and pharmacological moudulation of OPN exerts therapeutic effects in the treatment of respiratory diseases. In this review, we summarize the recent evidence of multifaceted roles and underlying mechanisms of OPN in these respiratory diseases, and targeting OPN appears to be a potential therapeutic intervention for these diseases.