Enzyme-free and rapid colorimetric analysis of osteopontin via triple-helix aptamer probe coupled with catalytic hairpin assembly reaction.

BACKGROUND: Osteopontin (OPN) is closely associated with tumorigenesis, growth, invasion, and immune escape and it serves as a plasma biomarker for hepatocellular carcinoma (HCC). Nevertheless, the accurate and rapid detection of low-abundance OPN still poses significant challenges. Currently, the majority of protein detection methods rely heavily on large precision instruments or involve complex procedures. Therefore, developing a simple, enzyme-free, rapid colorimetric analysis method with high sensitivity is imperative. RESULTS: In this study, we have developed a portable colorimetric biosensor by integrating the triple-helix aptamer probe (THAP) and catalytic hairpin assembly (CHA) strategy, named as T-CHA. After binding to the OPN, the trigger probe can be released from THAP, then initiates the CHA reaction and outputs the signal through the formation of a G-quadruplex/Hemin DNAzyme with horseradish peroxidase-like activity. Consequently, this colorimetric sensor achieves visual free-labeled detection without additional fluorophore modification and allows for accurate quantification by measuring the optical density of the solution at 650 nm. Under optimal conditions, the logarithmic values of various OPN concentrations exhibit satisfactory linearity in the range of 5 pg mL-1 to 5 ng mL-1, with a detection limit of 2.04 pg mL-1. Compared with the widely used ELISA strategy, the proposed T-CHA strategy is rapid (∼105 min), highly sensitive, and cost-effective. SIGNIFICANCE: The T-CHA strategy, leveraging the low background leakage of THAP and the high catalytic efficiency of CHA, has been successfully applied to the detection of OPN in plasma, demonstrating significant promise for the early diagnosis of HCC in point-of-care testing. Given the programmability of DNA and the universality of T-CHA, it can be readily modified for analyzing other useful tumor biomarkers.

Effects of Ziziphus Honey on Bone Healing of the Extracted Tooth Socket by Evaluating Osteopontin Levels.

OBJECTIVE: To evaluate the effect of Ziziphus honey on the healing of post-extraction alveolar sockets by estimating the levels of osteopontin (OPN) in humans. STUDY DESIGN: Randomised controlled trial. Place and Duration of the Study: Dental section of the Lahore General Hospital, Lahore, Pakistan, from March 2020 to February 2021. METHODOLOGY: A total of 30 patients were included in the study. The mean age was 35 ± 0.28 years. The participants were adults undergoing permanent molar extraction, randomly divided into two groups, a control group and an experimental group. After tooth extractions in both groups, 1ml of Ziziphus honey was administered into the extracted tooth socket of the experimental group while no intervention was done to the control group. Saliva samples were collected on day 0 before tooth extraction and on days 3 and 7 after tooth extractions. Enzyme-linked immunosorbent assay (ELISA) technique was used to measure the levels of OPN in the saliva sample. Radiographic evaluation was also done with the help of periapical radiographs using Image J® software. To find out the significance of the outcome in experimental and control groups, an unpaired t-test was applied. A p-value <0.05 was considered statistically significant. RESULTS: A total of 30 participants were selected for the study, of which 16 were females and 14 were males. The OPN levels between the control vs. experimental groups were (22.55 ± 2.45 vs. 23.31 ± 2.38; p = 0.4) on day 0, (30.95 ± 2.96 vs. 53.29 ± 4.69; p = 0.001) on day 3, and (55.33 ± 4.52 vs. 81.90 ± 4.49; p = 0.001) on day 7. CONCLUSION: Increased salivary levels of the OPN in the experimental group with the use of Ziziphus honey suggests better bone healing as compared to the control group. KEY WORDS: Extraction tooth, Honey, Osteopontin, Ziziphus, Bone healing.

An Immunohistochemical Analysis of Osteopontin and S100 Calcium-binding Protein P is Useful for Subclassifying Large- and Small-duct Type Intrahepatic Cholangiocarcinomas.

Intrahepatic cholangiocarcinoma (iCCA) has been newly subclassified into two different subtypes: large-duct (LD) type and small-duct (SD) type. However, many cases are difficult to subclassify, and there is no consensus regarding subclassification criteria. LD type expresses the highly sensitive diagnostic marker S100 calcium-binding protein P (S100P), while SD type lacks sensitive markers. We identified osteopontin (OPN) as a highly sensitive marker for SD type. This study aimed to develop new subclassification criteria for LD-type and SD-type iCCA. We retrospectively investigated 74 patients with iCCA and subclassified them based on whole-section immunostaining of S100P and OPN. Of the 74 cases, 41 were subclassified as LD type, 32 as SD type, and one was indeterminate. Notably, all S100P-negative cases had OPN positivity. Seventy-three of the 74 cases (98.6%) were clearly and easily subclassified as LD or SD type using only these 2 markers. We also determined the value of immunohistochemistry in cases that were difficult to diagnose based on hematoxylin-eosin and Alcian blue-periodic acid-Schiff staining. Furthermore, we analyzed the clinicopathological characteristics and prognoses of these 2 subtypes. LD type was a poor prognostic factor on univariate analysis; it had significantly worse overall survival ( P = 0.007) and recurrence-free survival ( P < 0.001) than the SD type. In conclusion, we propose new subclassification criteria for iCCA based on immunostaining of S100P and OPN. These criteria may help pathologists to diagnose subtypes of iCCA, supporting future clinical trials and the development of medications for these 2 subtypes as distinct cancers.

Noninvasive and Continuous Monitoring of On-Chip Stem Cell Osteogenesis Using a Reusable Electrochemical Immunobiosensor.

Noninvasive monitoring of biofabricated tissues during the biomanufacturing process is needed to obtain reproducible, healthy, and functional tissues. Measuring the levels of biomarkers secreted from tissues is a promising strategy to understand the status of tissues during biofabrication. Continuous and real-time information from cultivated tissues enables users to achieve scalable manufacturing. Label-free biosensors are promising candidates for detecting cell secretomes since they can be noninvasive and do not require labor-intensive processes such as cell lysing. Moreover, most conventional monitoring techniques are single-use, conducted at the end of the fabrication process, and, challengingly, are not permissive to in-line and continual detection. To address these challenges, we developed a noninvasive and continual monitoring platform to evaluate the status of cells during the biofabrication process, with a particular focus on monitoring the transient processes that stem cells go through during in vitro differentiation over extended periods. We designed and evaluated a reusable electrochemical immunosensor with the capacity for detecting trace amounts of secreted osteogenic markers, such as osteopontin (OPN). The sensor has a low limit of detection (LOD), high sensitivity, and outstanding selectivity in complex biological media. We used this OPN immunosensor to continuously monitor on-chip osteogenesis of human mesenchymal stem cells (hMSCs) cultured 2D and 3D hydrogel constructs inside a microfluidic bioreactor for more than a month and were able to observe changing levels of OPN secretion during culture. The proposed platform can potentially be adopted for monitoring a variety of biological applications and further developed into a fully automated system for applications in advanced cellular biomanufacturing.

Changes in biomarkers levels from gingival crevicular fluid in pre- and postmenopausal women undergoing orthodontic treatment : A systematic review.

PURPOSE: This study aimed to verify whether there is a difference in biomarker levels in the gingival crevicular fluid between premenopausal and postmenopausal women undergoing orthodontic treatment. METHODS: As eligibility criteria, prospective or retrospective observational studies evaluating women undergoing orthodontic treatment (P), comparing postmenopausal (E) and premenopausal (C) women, and analyzing differences in gingival crevicular fluid biomarkers (O) were included. An electronic search was conducted in seven databases (PubMed, Scopus, Web of Science, LILACS, The Cochrane Library, Embase, and EBSCO: Dentistry & Oral Science) and one grey literature source (Google Scholar). All databases were searched from September 2022 to March 2023. After duplicate exclusion and data extraction, the Newcastle-Ottawa scale was applied to assess the quality and risk of bias, and the Grading of Recommendations Assessment, Development and Evaluation (GRADE) tool was used to verify the certainty of evidence. RESULTS: Three case-control studies that analyzed receptor activator of nuclear factor kappa­B ligand (RANKL), osteopontin (OPN), and interleukin (IL)-17A levels were included. One study reported a significant difference for RANKL and another for OPN levels. A third study reported that there was a higher expression of IL17­A in the postmenopausal group. However, the small number of articles limits our systematic review. The heterogeneity and imprecision in the study results cast doubt on the findings' internal validity. CONCLUSION: The studies reported alterations in biomarker levels but differed in their conclusions. Therefore, further studies must include other types of bone and inflammatory biomarkers in female patients who are pre- or postmenopausal and undergoing orthodontic treatment. REGISTRATION: The review was registered at the Open Science Framework ( https://doi.org/10.17605/OSF.IO/Q9YZ8 ).

Prognostic and clinicopathological value of osteopontin expression in non-small cell lung cancer: a meta-analysis and systematic review.

BACKGROUND: Although Osteopontin (OPN) has been reported to be associated with many different human cancers, the data on non-small cell lung cancer (NSCLC) are not definitive. This study aimed to explore the prognostic effect of OPN expression and clinicopathological characteristics in patients with NSCLC. METHODS: This study followed all aspects of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) report. PubMed, Embase and the Cochrane Library were searched to identify the relative studies. The pooled hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated to estimate the prognostic value of the OPN in patients with NSCLC. The odds ratio (OR) was calculated to represent the relationship between OPN expression and clinicopathological parameters. RESULTS: A total of fifteen studies with 2173 participants were finally included. The results revealed that high expression of OPN was significantly associated with poorer overall survival (OS) (HR = 1.89; 95%CI = 1.68-2.11; p < 0.001). Moreover, a significant correlation was observed between increased OPN expression and poorly differentiated (well and moderately differentiated vs. poorly differentiated; pooled OR = 0.38; 95% CI = 0.23-0.64; p < 0.001), lymph node metastasis (absence vs. presence; pooled OR = 0.49; 95%CI = 0.32-0.74; p < 0.001), and distant metastasis (absence vs. presence; pooled OR = 0.18; 95%CI = 0.11-0.29; p < 0.001). CONCLUSION: This meta-analysis implies that OPN might be a valuable biomarker for a poor prognosis and poor clinicopathological outcomes for patients with NSCLC.

Our findings suggest that osteopontin is an important biomarker for poor prognosis and poor clinicopathological outcome in Non-small cell lung cancer (NSCLC) patients.Increased expression of osteopontin in NSCLC patients is associated not only with poorer survival but also with tumor differentiation, lymph node metastasis, and distant metastasis.This may be due to that osteopontin promotes multiple pathological processes including cancer cell proliferation, invasion, tumor progression, and metastasis in NSCLC.

CXCL9:SPP1 macrophage polarity identifies a network of cellular programs that control human cancers.

Tumor microenvironments (TMEs) influence cancer progression but are complex and often differ between patients. Considering that microenvironment variations may reveal rules governing intratumoral cellular programs and disease outcome, we focused on tumor-to-tumor variation to examine 52 head and neck squamous cell carcinomas. We found that macrophage polarity-defined by CXCL9 and SPP1 (CS) expression but not by conventional M1 and M2 markers-had a noticeably strong prognostic association. CS macrophage polarity also identified a highly coordinated network of either pro- or antitumor variables, which involved each tumor-associated cell type and was spatially organized. We extended these findings to other cancer indications. Overall, these results suggest that, despite their complexity, TMEs coordinate coherent responses that control human cancers and for which CS macrophage polarity is a relevant yet simple variable.

Effects of bovine milk osteopontin on in vitro enamel remineralization as a topical application prior to immersion in remineralizing solutions with/without fluoride.

The aim of the present study was to investigate the effects of bovine milk osteopontin (OPN) on enamel remineralization as a topical application prior to immersion in remineralizing solutions with/without fluoride. Bovine enamel blocks were demineralized then were divided into the following 3 groups: OPN (2.7 and 5.4 µM) solutions and deionized water (control). Each group was divided into 2 groups (remineralizing solution with or without 1 ppm of fluoride (F)). The specimens were analyzed by micro-CT and scanning electron microscope (SEM). The percentage of remineralization was higher in remineralization solution with than without F (p<0.05). The present results suggest that bovine milk OPN inhibits remineralization in solution without F, but 5.4 µM bovine milk OPN does not inhibit remineralization of the demineralized body using solution containing F by interrupting mineral deposition on the enamel surface.

Curcumin alleviated oxidation stress injury by mediating osteopontin in nephrolithiasis rats

Purpose: To explore the role and mechanism of curcumin (Cur) in reducing oxidative stress damage in rats with nephrolithiasis induced by ethylene glycol (EG). Methods: Thirty male rats were divided into normal control, model, positive (10% potassium citrate), Cur-10 (10 mg/kg curcumin) and Cur-20 (20 mg/kg curcumin) groups. Results: The results of kidney tissue section stained by hematoxylin-eosin and von Kossa showed that curcumin treatment can inhibit the formation of kidney stones. The biochemical test results showed that the urea (Ur), creatinine (Cr), uric acid (UA), inorganic phosphorus and Ca2+ concentrations in urine decreased after being treated with curcumin. There were significant differences between different doses of curcumin (P < 0.05). Compared with the Cur-10 group, Cur-20 had a more significant inhibitory effect on malondialdehyde (MDA) (P < 0.05). In addition, reverse transcription polymerase chain reaction (PCR) detection and immunohistochemical results indicated that the osteopontin (OPN) in the kidney was significantly reduced after curcumin treatment. Conclusion: Curcumin could reduce the oxidative stress damage caused by EG-induced kidney stones.

[Concentration of osteopontin in human milk and associated factors in Chinese populations from 2011 to 2013].

OBJECTIVE: To analyze content of human milk osteopontin(OPN) and to explore associated factors in Chinese populations. METHODS: The samples and data were extracted from the database for human milk composition in China between 2011 and 2013. A sub-sample of 459 mothers was randomly selected after stratification according to lactation stage, and human milk OPN concentrations were determined by ultra performance liquid chromatography-tandem mass spectrometer(UPLC/MS). RESULTS: The average OPN concentration(M(P25, P75)) in breast milk was 44.0(30.1-72.0) mg/L within 0-330 days postpartum. OPN concentrations were independent of lactation stage, which were 45.6(31.8, 80.7) mg/L in colostrum, 41.3(29.2, 70.0) mg/L in transitional milk and 46.9(30.2, 71.9) mg/L in mature milk, corresponding to 0.40%、0.42% and 0.65% of the total milk protein content(OPN/protein%). The percentage of OPN to total protein in milk showed an increasing trend with lactation progression(r=0.21, P<0.001). Multivariate analysis showed that sleep quality of mothers within one week prior to milk collection was correlated with the breast milk OPN level(P=0.04). The OPN level in breast milk from mothers with good sleep quality was significantly higher than that from mothers with poor sleep quality(46.5 mg/L vs.34.7 mg/L). The median level of milk OPN concentration in mothers from Yunnan was higher than mothers from Beijing(50.5 mg/L vs.36.1 mg/L, P=0.03). Maternal age, mode of delivery, prepregnancy body mass index, weight gain during pregnancy, passive smoking and outdoor activities 24 hours prior to milk collection were not correlated with milk OPN concentration. OPN concentration in breast milk was not related to preterm birth. Also, milk OPN concentration did not correlate with diarrhoea, respiratory disease, or allergic disease in infants during two weeks before milk collection. CONCLUSION: The concentration of OPN in breast milk of Chinese woman may be similar among different lactation stages. Geographic region and sleep quality of mothers may be related to the milk OPN concentration.

Immunohistochemical expression of osteopontin and collagens in choroid plexus of human brains.

Evidence showing the functional significance of the choroid plexus is accumulating. Although it is clinically well-known that calcification is frequently seen in the choroid plexus of aged human brains, it is unclear why calcification occurs in the aged choroid plexus and what exert effects on the calcification has. In this study, immunohistochemical localizations of collagens and other molecules related to fibrosis or calcification were investigated on the choroid plexus of autopsied human brains. Densely fibrous or calcified materials were located in the stroma just below the epithelial cells of the choroid plexus of all human brains examined. Immunoreactivity for collagen type I was identified in the stroma just below the epithelial cells, consistent with the densely fibrous or calcified area, whereas that for collagen type III was observed in almost all stroma other than the densely fibrous or calcified areas. Linear or membranous immunoreactivity for collagen type IV was intermittently localized on the epithelium-facing side of the materials, suggesting an injured basement membrane. In addition, clear immunoreactivity for osteopontin was localized on the epithelium-facing side of the fibrous or calcified materials as well as in the cytoplasm of epithelial cells. These findings indicate that collagen type I exists in contact with osteopontin in and around the densely fibrous or calcified materials in the choroid plexus. They suggest that the densely fibrous or calcified materials are deposited in the subepithelial stroma just below an injured basement membrane of epithelial cells via the collagen type I and osteopontin.

Structural changes and expression of hepatic fibrosis-related proteins in coculture of Echinococcus multilocularis protoscoleces and human hepatic stellate cells.

BACKGROUND: Echinococcus multilocularis is the causative agent of human hepatic alveolar echinococcosis (AE). AE can cause damage to several organs, primarily the liver, and have severe outcomes, such as hepatic failure and encephalopathy. The main purpose of this study was to explore the interactions between hepatic stellate cells (HSCs) and E. multilocularis protoscoleces (PSCs). The results of this study provide an experimental basis for further examination of the pathogenesis of hepatic fibrosis due to AE infection. METHODS: We investigated the role of Echinococcus multilocularis (Echinococcus genus) PSCs in hepatic fibrosis by examining structural changes and measuring hepatic fibrosis-related protein levels in cocultures of PSCs and human HSCs. Structural changes were detected by transmission electron microscopy (TEM), and levels of the hepatic fibrosis-related proteins collagen I (Col-I), alpha-smooth muscle actin (α-SMA) and osteopontin (OPN) were measured by western blotting and enzyme-linked immunosorbent assay (ELISA). RESULTS: Under coculture (1) both PSCs and HSCs exhibited morphological changes, as observed by TEM; (2) Col-I, α-SMA, and OPN expression levels, which were determined by western blotting and ELISA, significantly increased after 3 days of incubation. CONCLUSIONS: The results of this study provide insights into the molecular mechanisms of AE-induced hepatic fibrosis.

Osteopontin in Cardiovascular Diseases.

Unprecedented advances in secondary prevention have greatly improved the prognosis of cardiovascular diseases (CVDs); however, CVDs remain a leading cause of death globally. These findings suggest the need to reconsider cardiovascular risk and optimal medical therapy. Numerous studies have shown that inflammation, pro-thrombotic factors, and gene mutations are focused not only on cardiovascular residual risk but also as the next therapeutic target for CVDs. Furthermore, recent clinical trials, such as the Canakinumab Anti-inflammatory Thrombosis Outcomes Study trial, showed the possibility of anti-inflammatory therapy for patients with CVDs. Osteopontin (OPN) is a matricellular protein that mediates diverse biological functions and is involved in a number of pathological states in CVDs. OPN has a two-faced phenotype that is dependent on the pathological state. Acute increases in OPN have protective roles, including wound healing, neovascularization, and amelioration of vascular calcification. By contrast, chronic increases in OPN predict poor prognosis of a major adverse cardiovascular event independent of conventional cardiovascular risk factors. Thus, OPN can be a therapeutic target for CVDs but is not clinically available. In this review, we discuss the role of OPN in the development of CVDs and its potential as a therapeutic target.

Transcriptional Suppression of Diabetic Nephropathy with Novel Gene Silencer Pyrrole-Imidazole Polyamides Preventing USF1 Binding to the TGF-ß1 Promoter.

Upstream stimulatory factor 1 (USF1) is a transcription factor that is increased in high-glucose conditions and activates the transforming growth factor (TGF)-ß1 promoter. We examined the effects of synthetic pyrrole-imidazole (PI) polyamides in preventing USF1 binding on the TGF-ß1 promoter in Wistar rats in which diabetic nephropathy was established by intravenous administration of streptozotocin (STZ). High glucose induced nuclear localization of USF1 in cultured mesangial cells (MCs). In MCs with high glucose, USF1 PI polyamide significantly inhibited increases in promoter activity of TGF-ß1 and expression of TGF-ß1 mRNA and protein, whereas it significantly decreased the expression of osteopontin and increased that of h-caldesmon mRNA. We also examined the effects of USF1 PI polyamide on diabetic nephropathy. Intraperitoneal injection of USF1 PI polyamide significantly suppressed urinary albumin excretion and decreased serum urea nitrogen in the STZ-diabetic rats. USF1 PI polyamide significantly decreased the glomerular injury score and tubular injury score in the STZ-diabetic rats. It also suppressed the immunostaining of TGF-ß1 in the glomerulus and proximal tubules and significantly decreased the expression of TGF-ß1 protein from kidney in these rats. These findings indicate that synthetic USF1 PI polyamide could potentially be a practical medicine for diabetic nephropathy.

Factors influencing milk osteopontin concentration based on measurements from Danish Holstein cows.

Our objective was to determine the content of the bioactive protein osteopontin (OPN) in bovine milk and identify factors influencing its concentration. OPN is expressed in many tissues and body fluids, with by far the highest concentrations in milk. OPN plays a role in immunological and developmental processes and it has been associated with several milk production traits and lactation persistency in cows. In the present study, we report the development of an enzyme linked immunosorbent assay (ELISA) for measurement of OPN in bovine milk. The method was used to determine the concentration of OPN in milk from 661 individual Danish Holstein cows. The median OPN level was determined to 21.9 mg/l with a pronounced level of individual variation ranging from 0.4 mg/l to 67.8 mg/l. Breeding for increased OPN in cow's milk is of significant interest, however, the heritability of OPN in milk was found to be relatively low, with an estimated value of 0.19 in the current dataset. The variation explained by the herd was also found to be low suggesting that OPN levels are not affected by farm management or feeding. Interestingly, the concentration of OPN was found to increase with days in milk and to decrease with parity.

Quantitative determination of osteopontin in bovine, buffalo, yak, sheep and goat milk by Ultra-high performance liquid chromatography-tandem mass spectrometry and stable isotope dimethyl labeling.

Osteopontin (OPN) is a multifunctional protein present in different tissues, body fluids and milk. Different milk has different level of OPN content. To determine the amount of osteopontin in bovine, buffalo, yak, sheep and goat milk, we developed an ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method to detect an osteopontin signature peptide. The signature peptides selected by searching Uniprot database for trypsin digested osteopontin. The sample preparation procedure includes trypsin digestion, dimethyl labeling of tryptic peptides, purification and concentration of labeled tryptic peptide with solid phase extraction. The limit of detection and limit of quantification are 0.5 mg L-1 and 2.0 mg L-1, respectively. The method has satisfactory analytical performance with a linearity of R2 ≥ 0.998, recoveries of 103.7-111.0%, and precision of 1.8-6.2%. It is also validated and successfully applied to quantifying osteopontin content in bovine, buffalo, yak, sheep and goat milk.

An Analytical Method to Quantify Osteopontin in Dairy Powders and Infant Formulas by Signature Peptide Quantification with UHPLC-MS/MS.

BACKGROUND: Osteopontin (OPN) is an important protein in human milk, and is of growing interest to infant formula (IF) manufacturers. OPN is present at low quantities in bovine milk and its derived ingredients, and there is a need for an accurate quantitative method in complex matrixes such as IF and growing-up milks (GUMs). OBJECTIVE: The objective of this work was to validate a method to quantify OPN in several dairy powders produced from bovine milk, including skimmed milk powder (SMP), whey protein concentrate (WPC), demineralized WPC and α-lactalbumin-enriched WPC (α-lac WPC). The method was further validated in intact-protein IF and GUM powders produced using combinations of these ingredients. METHODS: Test samples were digested using trypsin, and the most appropriate peptide fragmentation transitions were identified by UHPLC-MS/MS. Quantification was made against a standard curve constructed from OPN reference material, and isotopically-labelled peptide standards were used as internal standards. Curve linearity was assessed, and samples were spiked at two OPN levels. RESULTS: The validation parameters were met in almost all cases, with precision RSDr and RSDiR values ranging from 0.26-7.43% and 1.22-12.70%, respectively, and spike recoveries ranging from 88-102%. The method was used to accurately measure OPN in bovine milk-based IF and GUM powders with intact protein systems, based on comparisons with mass balance calculations. CONCLUSIONS: The results from this study show that the method is fit-for-purpose to support IF and GUM manufacturers in evaluating OPN contents of raw materials and products containing whole, intact protein systems from bovine milk. HIGHLIGHTS: An LC-MS/MS method was developed to measure OPN in dairy powders, IF and GUMs containing whole, intact protein systems from bovine milk.

[Inhibitory Effects of Dabigatran on Airway Inflammation Induced by Lipopolysaccharide in Mice].

Asthma and chronic obstructive pulmonary disease (COPD) are characterised by chronic inflammation in the lung that is associated with airway obstruction. Inhaled therapy with a combination of corticosteroid and a long-acting ß2-agonist is an effective anti-inflammatory medicine for asthma, but in patients with severe asthma and COPD fails to completely control these symptoms with current therapies. The inflammatory process in these diseases, which involves activation of the coagulation and fibrinolytic system in the lung, offers the opportunity for alternative anti-inflammatory therapies. In this study, we investigated the effects of anti-coagulants on lipopolysaccharide (LPS)-induced airway inflammation in mice. A/J mice were exposed to LPS, a bacterial endotoxin, intranasally and accumulation of inflammatory cells, TNF-α, C-X-C motif chemokine (CXCL) 1, and osteopontin in bronchoalveolar lavage fluid (BALF) was monitored by flow cytometry and an enzyme-linked immunosorbent assay. LPS exposure induced airway neutrophilia and accumulation of TNF-α, CXCL1, and osteopontin in BALF. This LPS-induced airway inflammation was not relieved using a corticosteroid, fluticasone propionate (FP), or a direct inhibitor of Factor Xa, rivaroxaban. In contrast, a direct thrombin inhibitor, dabigatran, inhibited LPS-induced airway neutrophilia and decreased inflammatory cytokine production in a dose dependent manner. Furthermore, combination of dabigatran and FP elicited stronger inhibition of LPS-induced airway inflammation. Therefore, these results suggest that dabigatran could be an effective new therapy for severe respiratory diseases.

Identification and verification of three key genes associated with survival and prognosis of COAD patients via integrated bioinformatics analysis.

BACKGROUND: Colorectal cancer (CRC) is the third most lethal malignancy in the world, wherein colon adenocarcinoma (COAD) is the most prevalent type of CRC. Exploring biomarkers is important for the diagnosis, treatment, and prevention of COAD. METHODS: We used GEO2R and Venn online software for differential gene screening analysis. Hub genes were screened via Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and Cytoscape, following Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Finally, survival analysis and RNA expression validation were performed via UALCAN online software and real-time PCR. Immunohistochemistry (IHC) was performed to verify the protein expression level of hub genes from tissues of COAD patients. RESULTS: In the present study, we screened 323 common differentially expressed genes (DEGs) from four GSE datasets. Furthermore, four hub genes were selected for survival correlation analysis and expression level verification, three of which were shown to be statistically significant. CONCLUSION: Our study suggests that Serpin Family E Member 1 (SERPINE1), secreted phosphoprotein 1 (SPP1) and tissue inhibitor of metalloproteinase 1 (TIMP1) may be biomarkers closely related to the prognosis of CRC patients.