Technical development and validation of a clinically applicable microenvironment classifier as a biomarker of tumour hypoxia for soft tissue sarcoma.

BACKGROUND: Soft tissue sarcomas (STS) are rare, heterogeneous tumours and biomarkers are needed to inform management. We previously derived a prognostic tumour microenvironment classifier (24-gene hypoxia signature). Here, we developed/validated an assay for clinical application. METHODS: Technical performance of targeted assays (Taqman low-density array, nanoString) was compared in 28 prospectively collected formalin-fixed, paraffin-embedded (FFPE) biopsies. The nanoString assay was biologically validated by comparing to HIF-1α/CAIX immunohistochemistry (IHC) in clinical samples. The Manchester (n = 165) and VORTEX Phase III trial (n = 203) cohorts were used for clinical validation. The primary outcome was overall survival (OS). RESULTS: Both assays demonstrated excellent reproducibility. The nanoString assay detected upregulation of the 24-gene signature under hypoxia in vitro, and 16/24 hypoxia genes were upregulated in tumours with high CAIX expression in vivo. Patients with hypoxia-high tumours had worse OS in the Manchester (HR 3.05, 95% CI 1.54-5.19, P = 0.0005) and VORTEX (HR 2.13, 95% CI 1.19-3.77, P = 0.009) cohorts. In the combined cohort, it was independently prognostic for OS (HR 2.24, 95% CI 1.42-3.53, P = 0.00096) and associated with worse local recurrence-free survival (HR 2.17, 95% CI 1.01-4.68, P = 0.04). CONCLUSIONS: This study comprehensively validates a microenvironment classifier befitting FFPE STS biopsies. Future uses include: (1) selecting high-risk patients for perioperative chemotherapy; and (2) biomarker-driven trials of hypoxia-targeted therapies.

A specific visual-volumetric sensor for mercury ions based on smart hydrogel.

On the basis of the "seeing is believing" concept and the existing theory of Hg2+ coordination chemistry, for the first time, we innovatively designed and synthesized a visual-volumetric sensor platform with fluorescein and uracil functionalized polyacrylamide hydrogel. Without the aid of any complicated instruments and power sources, the sensor-enabled quantitative µM-level Hg2+ detection Hg2+ by reading graduation on a pipette with the naked eye. The sensor undergoes volumetric response and shows a wide linear response range to Hg2+ (1.0 × 10-6-5.0 × 10-5 mol L-1) with 2.8 × 10-7 mol L-1 as the detection limit. The highly selective (easily distinguished Hg2+ from other common metal ions), rapid response (∼30 min), and acceptable repeatability (RSD < 5% in all cases) demonstrated that the developed sensor is suitable for onsite practical use for the determination of Hg2+ while being low-cost, simple, and portable. The design principles of the obtained materials and the construction techniques and methods of the sensors described in our study provide a new idea for the research and development of smart materials and a series of visual-volumetric sensors for other analytes.

Discovery and therapeutic implications of bioactive dihydroxylated phenolic acids in patients with severe heart disease and conditions associated with inflammation and hypoxia.

Our initial studies detected elevated levels of 3,4-dihydroxyphenyllactic acid (DHPLA) in urine samples of patients with severe heart disease when compared with healthy subjects. Given the reported anti-inflammatory properties of DHPLA and related dihydroxylated phenolic acids (DPAs), we embarked on an exploratory multi-centre investigation in patients with no urinary tract infections to establish the possible pathophysiological significance and therapeutic implications of these findings. Chinese and Caucasian patients being treated for severe heart disease or those conditions associated with inflammation (WBC ≥ 10 ×109/L or hsCRP ≥ 3.0 mg/L) and/or hypoxia (PaO2 ≤ 75 mmHg) were enrolled; their urine samples were analyzed by HPLC, HPLC-MS, GC-MS and biotransformation assays. DHPLA was detected in urine samples of patients, but undetectable in healthy volunteers. Dynamic monitoring of inpatients undergoing treatment showed their DHPLA levels declined in proportion to their clinical improvement. In DHPLA-positive patients' fecal samples, Proteus vulgaris and P. mirabilis were more abundant than healthy volunteers. In culture, these gut bacteria were capable of reversible interconversion between DOPA and DHPLA. Furthermore, porcine and rodent organs were able to metabolize DOPA to DHPLA and related phenolic acids. The elevated levels of DHPLA in these patients suggest bioactive DPAs are generated de novo as part of a human's defense mechanism against disease. Because DHPLA isolated from Radix Salvia miltiorrhizae has a multitude of pharmacological activities, these data underpin the scientific basis of this medicinal plant's ethnopharmacological applications as well as highlighting the therapeutic potential of endogenous, natural or synthetic DPAs and their derivatives in humans.

Repurposing FDA approved drugs as radiosensitizers for treating hypoxic prostate cancer.

BACKGROUND: The presence of hypoxia is a poor prognostic factor in prostate cancer and the hypoxic tumor microenvironment promotes radioresistance. There is potential for drug radiotherapy combinations to improve the therapeutic ratio. We aimed to investigate whether hypoxia-associated genes could be used to identify FDA approved drugs for repurposing for the treatment of hypoxic prostate cancer. METHODS: Hypoxia associated genes were identified and used in the connectivity mapping software QUADrATIC to identify FDA approved drugs as candidates for repurposing. Drugs identified were tested in vitro in prostate cancer cell lines (DU145, PC3, LNCAP). Cytotoxicity was investigated using the sulforhodamine B assay and radiosensitization using a clonogenic assay in normoxia and hypoxia. RESULTS: Menadione and gemcitabine had similar cytotoxicity in normoxia and hypoxia in all three cell lines. In DU145 cells, the radiation sensitizer enhancement ratio (SER) of menadione was 1.02 in normoxia and 1.15 in hypoxia. The SER of gemcitabine was 1.27 in normoxia and 1.09 in hypoxia. No radiosensitization was seen in PC3 cells. CONCLUSION: Connectivity mapping can identify FDA approved drugs for potential repurposing that are linked to a radiobiologically relevant phenotype. Gemcitabine and menadione could be further investigated as potential radiosensitizers in prostate cancer.

Independence of HIF1a and androgen signaling pathways in prostate cancer.

BACKGROUND: Therapeutic targeting of the androgen signaling pathway is a mainstay treatment for prostate cancer. Although initially effective, resistance to androgen targeted therapies develops followed by disease progression to castrate-resistant prostate cancer (CRPC). Hypoxia and HIF1a have been implicated in the development of resistance to androgen targeted therapies and progression to CRCP. The interplay between the androgen and hypoxia/HIF1a signaling axes was investigated. METHODS: In vitro stable expression of HIF1a was established in the LNCaP cell line by physiological induction or retroviral transduction. Tumor xenografts with stable expression of HIF1a were established in castrated and non-castrated mouse models. Gene expression analysis identified transcriptional changes in response to androgen treatment, hypoxia and HIF1a. The binding sites of the AR and HIF transcription factors were identified using ChIP-seq. RESULTS: Androgen and HIF1a signaling promoted proliferation in vitro and enhanced tumor growth in vivo. The stable expression of HIF1a in vivo restored tumor growth in the absence of endogenous androgens. Hypoxia reduced AR binding sites whereas HIF binding sites were increased with androgen treatment under hypoxia. Gene expression analysis identified seven genes that were upregulated both by AR and HIF1a, of which six were prognostic. CONCLUSIONS: The oncogenic AR, hypoxia and HIF1a pathways support prostate cancer development through independent signaling pathways and transcriptomic profiles. AR and hypoxia/HIF1a signaling pathways independently promote prostate cancer progression and therapeutic targeting of both pathways simultaneously is warranted.

Metabolite identification of tanshinol borneol ester in rats by high-performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight mass spectrometry.

Tanshinol borneol ester (DBZ) is a potential drug candidate composed of danshensu and borneol. It shows anti-ischemic and anti-atherosclerosis activity. However, little is known about its metabolism in vivo. This research aimed to elucidate the metabolic profile of DBZ through analyzing its metabolites using high-performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight mass spectrometry. Chromatographic separation was performed on an Agilent TC-C18 column (150 × 4.6 mm, 5.0 µm) with gradient elution using methanol and water containing 0.2% (v/v) formic acid as the mobile phase. Metabolite identification involved analyzing the retention behaviors, changes in molecular weights and MS/MS fragment patterns of DBZ and its metabolites. As a result, 20 potential metabolites were detected and tentatively identified in rat plasma, urine and feces after administration of DBZ. DBZ could be metabolized to O-methylated DBZ, DBZ-O-glucuronide, O-methylated DBZ-O-glucuronide, hydroxylated DBZ and danshensu. Danshensu, a hydrolysis product of DBZ, could further be transformed into 12 metabolites. The proposed method was confirmed to be a reliable and sensitive alternative for characterizing metabolic pathways of DBZ and providing valuable information on its druggability.

Investigation on temperature-induce conformational change of immobilized ß2 adrenergic receptor.

The ß2 adrenergic receptor (ß2-AR) is a prototypical family A G protein-coupled receptor (GPCR) and an excellent model system for studying the mechanism of GPCR activation. Purified ß2-AR was immobilized on macroporous silica gel to obtain liquid chromatographic stationary phase. The resulting phase was packed into a stainless steel column (4.6 × 50 mm, 7 µm) and used for on-line chromatographic system. When column oven temperature increased from 20.0 °C to 40.0 °C, uncomplete separate chromatographic peaks of ephedrine and pseudoephedrine as receptor conformational probe were gradually merged into one peak, meanwhile retention time and resolution of the probes were reduced correspondingly, which suggested that temperature could regulate protein conformation. Temperature-induced conformational change of immobilized ß2-AR, especially changes at higher temperatures, indicated that constructed receptor chromatography could simulate fever disease state of human body and clarify receptor conformation change at pathological condition. At the same time this study could also provide new ideas for screening active components in pathological conditions.

Identification of metabolites of isopropyl 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate in rats by high performance liquid chromatography combined with electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

Isopropyl 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate (IDHP) is an investigational new drug having the capacity for treating ailments in cardiovascular and cerebrovascular system. In this work, a rapid and sensitive method using high performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (HPLC-ESI-Q-TOF-MS) was developed to reveal the metabolic profile of IDHP in rats after oral administration. The method involved pretreatment of the samples by formic acid-methanol solution (v:v, 5:95), chromatographic separation by an Agilent Eclipse XDB-C18 column (150 × 4.6 mm i.d., 5 µm) and online identification of the metabolites by Q-TOF-MS equipped with electrospray ionizer. A total of 16 metabolites from IDHP, including 4 phase I metabolites and 12 phase II metabolites, were detected and tentatively identified from rat plasma, urine and feces. Among these metabolites, Danshensu (DSS), a hydrolysis product of IDHP, could be further transformed to 11 metabolites. These results indicated that DSS was the main metabolite of IDHP in rats and the major metabolic pathways of IDHP in vivo were hydrolysis, O-methylation, sulfation, glucuronidation and reduction. The results also demonstrated that renal route was the main pathway of IDHP clearance in rat. The present study provided valuable information for better understanding the efficacy and safety of IDHP. This article is protected by copyright. All rights reserved.

Estimation of interaction between oriented immobilized green fluorescent protein and its antibody by high performance affinity chromatography and molecular docking.

Although green fluorescence protein (GFP) and its antibody are widely used to track a protein or a cell in life sciences, the binding behavior between them remains unclear. In this work, diazo coupling method that synthesized a new stationary GFP was oriented immobilized on the surface of macro-porous silica gel by a phase. The stationary phase was utilized to confirm the validation of injection amount-dependent analysis in exploring protein-protein interaction that use GFP antibody as a probe. GFP antibody was proved to have one type of binding site on immobilized GFP. The number of binding site and association constant were calculated to be (6.41 ± 0.76) × 10(-10) M and (1.39 ± 0.12) × 10(9) M(-1). Further analysis by molecular docking showed that the binding of GFP to its antibody is mainly driven by hydrogen bonds and salt bridges. These results indicated that injection amount-dependent analysis is capable of exploring the protein-protein interactions with the advantages of ligand and time saving. It is a valuable methodology for the ligands, which are expensive or difficult to obtain.

Pathway activity inference for multiclass disease classification through a mathematical programming optimisation framework.

BACKGROUND: Applying machine learning methods on microarray gene expression profiles for disease classification problems is a popular method to derive biomarkers, i.e. sets of genes that can predict disease state or outcome. Traditional approaches where expression of genes were treated independently suffer from low prediction accuracy and difficulty of biological interpretation. Current research efforts focus on integrating information on protein interactions through biochemical pathway datasets with expression profiles to propose pathway-based classifiers that can enhance disease diagnosis and prognosis. As most of the pathway activity inference methods in literature are either unsupervised or applied on two-class datasets, there is good scope to address such limitations by proposing novel methodologies. RESULTS: A supervised multiclass pathway activity inference method using optimisation techniques is reported. For each pathway expression dataset, patterns of its constituent genes are summarised into one composite feature, termed pathway activity, and a novel mathematical programming model is proposed to infer this feature as a weighted linear summation of expression of its constituent genes. Gene weights are determined by the optimisation model, in a way that the resulting pathway activity has the optimal discriminative power with regards to disease phenotypes. Classification is then performed on the resulting low-dimensional pathway activity profile. CONCLUSIONS: The model was evaluated through a variety of published gene expression profiles that cover different types of disease. We show that not only does it improve classification accuracy, but it can also perform well in multiclass disease datasets, a limitation of other approaches from the literature. Desirable features of the model include the ability to control the maximum number of genes that may participate in determining pathway activity, which may be pre-specified by the user. Overall, this work highlights the potential of building pathway-based multi-phenotype classifiers for accurate disease diagnosis and prognosis problems.

Danshensu inhibits ß-adrenergic receptors-mediated cardiac fibrosis by ROS/p38 MAPK axis.

Danshensu, the effective ingredient of the plant Salvia miltiorrhiza (Danshen), has been widely used for treatment of cardiovascular diseases. Cardiac fibrosis is an important process in pathological cardiac remodeling and leads to heart failure. We investigated the effect of Danshensu on ß-adrenergic receptor (ß-AR)-mediated cardiac fibrosis and the involved signaling transduction. Danshensu inhibited cardiofibroblast proliferation and collagen I synthesis induced by isoproterenol (ISO), a selective ß-AR agonist. Phosphorylation of p38 mitogen-activated protein kinase (MAPK), which mediates ISO-induced cardiac fibrosis, was negatively regulated in this process. The negative regulation depended on the ISO inhibition of reactive oxygen species (ROS) production. Taken together, Danshensu may inhibit ß-AR-mediated cardiac fibrosis by negative regulation of ROS-p38 MAPK signaling.

Srs11-92, a ferrostatin-1 analog, improves oxidative stress and neuroinflammation via Nrf2 signal following cerebral ischemia/reperfusion injury.

AIM: Ferroptosis is increasingly becoming to be considered as an important mechanism of pathological cell death during stroke, and specific exogenous ferroptosis inhibitors have the ability to reverse cerebral ischemia/reperfusion injury. However, research on Srs11-92 (AA9), a ferrostatin-1 (Fer-1) analog, in preclinical studies is limited. METHODS: In the middle cerebral artery occlusion-reperfusion (MCAO/R) mice model or oxygen-glucose deprivation/reperfusion (OGD/R) cell model, Fer-1, AA9, and/or ML385 were administered, and brain infarct size, neurological deficits, neuronal damage, oxidative stress, and neuroinflammation were determined after the damage, in vitro and in vivo. RESULTS: Fer-1 and AA9 improved brain infarct size, neuronal damage, and neurological deficits in mice model of MCAO/R, and inhibited the overloaded iron deposition, ROS accumulation, and neuroinflammation response: it also increased the expression of GPx4, Nrf2, and HO-1 and suppressed the expression of HMGB1 and NF-κB p65 in the epicenter of injured hippocampal formation. However, Nrf2 inhibitor ML385 reversed the neuroprotective effect of AA9, including the oxidative stress and neuroinflammation. In vitro studies showed that AA9 relieved OGD/R-induced neuronal oxidative stress and neuroinflammation via the Nrf2 pathway, which was impaired by ML385 in primary neurons. CONCLUSION: The findings imply that Fer-1 analog AA9 may be suitable for further translational studies for the protection of neuronal damage via Nrf2 signal pathway-mediated oxidative stress and neuroinflammation in stroke and others neurological diseases.

Akkermansia muciniphila-Nlrp3 is involved in the neuroprotection of phosphoglycerate mutase 5 deficiency in traumatic brain injury mice.

Introduction: Gut-microbiota-brain axis is a potential treatment to decrease the risk of chronic traumatic encephalopathy following traumatic brain injury (TBI). Phosphoglycerate mutase 5 (PGAM5), a mitochondrial serine/threonine protein phosphatase, resides in mitochondrial membrane and regulates mitochondrial homeostasis and metabolism. Mitochondria mediates intestinal barrier and gut microbiome. Objectives: This study investigated the association between PGAM5 and gut microbiota in mice with TBI. Methods: The controlled cortical impact injury was established in mice with genetically-ablated Pgam5 (Pgam5-/-) or wild type, and WT male mice were treated with fecal microbiota transplantation (FMT) from male Pgam5-/- mice or Akkermansia muciniphila (A. muciniphila). Then the gut microbiota abundance, blood metabolites, neurological function, and nerve injury were detected. Results: Treated with antibiotics for suppressing gut microbiota in Pgam5-/- mice partially relieved the role of Pgam5 deficiency in the improvement of initial inflammatory factors and motor dysfunction post-TBI. Pgam5 knockout exhibited an increased abundance of A. muciniphila in mice. FMT from male Pgam5-/- mice enabled better maintenance of amino acid metabolism and peripherial environment than that in TBI-vehicle mice, which suppressed neuroinflammation and improved neurological deficits, and A. muciniphila was negatively associated with intestinal mucosal injury and neuroinflammation post-TBI. Moreover, A. muciniphila treatment ameliorated neuroinflammation and nerve injury by regulating Nlrp3 inflammasome activation in cerebral cortex with TBI. Conclusion: Thus, the present study provides evidence that Pgam5 is involved in gut microbiota-mediated neuroinflammation and nerve injury, with A. muciniphila-Nlrp3 contributing to peripheral effects.

Development and clinical applications of an enclosed automated targeted NGS library preparation system.

The rapid development of next-generation sequencing (NGS) technology has promoted its wide clinical application in precision medicine for oncology. However, laborious and time-consuming manual operations, highly skilled personnel requirements, and cross-contamination are major challenges for the clinical implementation of NGS technology-based tests. The Automated NGS Diagnostic Solutions (ANDiS) 500 system is a fully enclosed cassette-dependent automated NGS library preparation system. This platform could produce qualified targeted amplicon library in three steps with only 15 min of hands-on time. Rigorous cross-contamination test using simulated contaminant plasmids confirmed that the design of disposable cassette guarantees zero sample cross-contamination. The BRCA1 and BRCA2 mutation detection panel and gastrointestinal cancer-related gene analysis panel for the ANDiS 500 platform showed 100% accuracy and precision in detecting germ-line mutations and somatic mutations respectively. Furthermore, those panels showed 100% concordance with verified methods in a prospective cohort study enrolling 363 patients and a cohort of 45 pan-cancer samples. In conclusion, the ANDiS 500 automated platform could overcome major challenges for implementing NGS assays clinically and is eligible for routine clinical tests.

Simultaneous and rapid analysis of chiral Danshensu and its ester derivatives by supercritical fluid chromatography.

The analysis and separation of chiral compounds with wide polar range by supercritical fluid chromatography is of major importance in the process of drug development and quality control. In this work, a fast and reliable enantioselective method for the simultaneous quantitative determination of 8 DBZ-related enantiomers has been successfully developed by supercritical fluid chromatography using an amylose-based reversed-chiral stationary phase. Within less than seven minutes all target compounds could be baseline resolved, using a mobile phase comprising supercritical carbon dioxide and methanol with 0.05 % H3PO4. The optimum chiral stationary phase showed to be a CHIRALPAK® AD-RH column, operated at flow rate of 3.0 mL/min, back pressure of 150 bar and temperature of 40 °C. Method validation confirmed that the developed procedure was selective, linear (r2 > 0.998), accurate (recovery rates: 98.02-100.02 %), and precise (intra-day: 0.05-1.98 %, inter-day: 0.08-1.98 %); the limit of detection and limit of quantification were 0.13-0.55 µg/mL and 0.37-1.68 µg/mL on column, respectively. After initial evaluation of stability according to the ICH Q1A (R2) guideline, R-DBZ showed good stability. Thus, this developed method can be used for assessing the stability of bulk DBZ samples, dosage forms of DBZ and also for monitoring the synthetic procedures of DBZ.

Long-Term Outcomes of Radical Radiation Therapy with Hypoxia Modification with Biomarker Discovery for Stratification: 10-Year Update of the BCON (Bladder Carbogen Nicotinamide) Phase 3 Randomized Trial (ISRCTN45938399).

PURPOSE: Many muscle-invasive bladder cancers are hypoxic, which limits the efficacy of radiation therapy. Hypoxia modification using carbogen and nicotinamide has been tested in a phase 3 trial, Bladder Carbogen Nicotinamide. We present mature follow-up data with biomarker predictions of outcomes. METHODS AND MATERIALS: Bladder Carbogen Nicotinamide is a prospective, phase 3, multicenter, randomized, 2-arm, nonblinded clinical trial. Participants were randomized to receive radical radiation therapy (RT; control arm) alone or with the addition of carbogen (98% O2; 2% CO2) and nicotinamide (CON). Patients with muscle-invasive or high-grade non-muscle invasive bladder cancer were included. Tumor tissue was collected at entry and was analyzed for tumor necrosis, hypoxia (24-gene signature), and basal and luminal tumor molecular subtypes. Overall survival (OS) and disease-free survival and relationships with biomarker status outcomes are analyzed using multivariable Cox regression and log-rank analysis. RESULTS: We analyzed 333 patients with a median follow-up of 10.3 years. The 10-year OS rates were 30% (95% confidence interval [CI], 0.23-0.39) in RT + CON patients and 24% (95% CI, 0.18-0.33) in the RT-alone patients (hazard ratio [HR], 0.80; 95% CI, 0.61-1.04; P = .08). The greatest benefit from CON was seen in patients with tumor necrosis (n = 79; 5-year OS, 53% vs. 33% in patients without tumor necrosis; HR, 0.59; 95% CI, 0.36-0.99; P = .04). Cases with a high hypoxia gene score (n = 75) had a 5-year OS rate of 51%, compared to 34% for a low score (HR, 0.64; 95% CI, 0.38-1.08; P = .09); those with the basal molecular subtype (n = 70) had a 5-year OS rate of 58%, compared to 38% for those with the luminal subtype (HR, 0.58; 95% CI, 0.32-1.06; P = .08). CONCLUSIONS: Although the improvement in long-term OS in the whole population is not statistically significant, patients selected by necrosis and high hypoxia gene score benefitted from hypoxia modification.

Developing Tumor Radiosensitivity Signatures Using LncRNAs.

Long non-coding RNAs (lncRNAs) are involved in diverse biological processes, including DNA damage repair, and are of interest as potential biomarkers of radiosensitivity. We investigated whether lncRNA radiosensitivity signatures could be derived for use in cancer patients treated with radiotherapy. Signature development involved radiosensitivity measurements for cell lines and primary tumor samples, and patient outcome after radiotherapy. A 10-lncRNA signature trained on radiosensitivity measurements in bladder cell lines showed a trend towards independent validation. In multivariable analyses, patients with tumors classified as radioresistant by the lncRNA signature had poorer local relapse-free survival (P = 0.065) in 151 patients with muscle-invasive bladder cancer who underwent radiotherapy. An mRNA-based radiosensitivity index signature performed similarly to the lncRNA bladder signature for local relapse-free survival (P = 0.055). Pathway analysis showed the lncRNA signature associated with molecular processes involved in radiation responses. Knockdown of one of the lncRNAs in the signature showed a modest increase in radiosensitivity in one cell line. An alternative approach involved training on primary cervical tumor radiosensitivity or local control after radiotherapy. Both approaches failed to generate a cervix lncRNA radiosensitivity signature, which was attributed to the age of samples in our cohorts. Our work highlights challenges in validating lncRNA signatures as biomarkers in archival tissue from radiotherapy cohorts, but supports continued investigation of lncRNAs for a role in radiosensitivity.

miR-142-5p as a CXCR4-Targeted MicroRNA Attenuates SDF-1-Induced Chondrocyte Apoptosis and Cartilage Degradation via Inactivating MAPK Signaling Pathway.

Osteoarthritis (OA) is a chronic joint function disorder with characteristics of chondrocytes reduction and extracellular matrix (ECM) components destruction. MicroRNAs (miRNAs) and the SDF-1/CXCR4 axis are essential factors of chondrocyte apoptosis and ECM degeneration. However, very few studies have investigated the correlation between miRNAs and the SDF-1/CXCR4 axis in osteoarthritis so far. Here, through miRNAs microarray and bioinformatics analyses, we identified miR-142-5p as a CXCR4-targeted and dramatically downregulated miRNA in cartilage from OA patients, as well as in SDF-1-induced OA chondrocytes in vitro. In SDF-1-treated primary human OA chondrocytes that were transfected with a miR-142-5p mimic or inhibitor, the expression of CXCR4 was found to be inversely correlated with the expression of miR-142-5p. The dual luciferase reporter assay further verified the target relationship between miR-142-5p and CXCR4. Overexpression of miR-142-5p alleviated OA pathology by suppressing chondrocyte apoptosis, even in CXCR4 overexpressed OA chondrocytes. This was associated with decreased cartilage matrix degradation, reduced cartilage inflammation, and inactivated MAPK signaling pathway. Our study suggests that upregulated expression of CXCR4-targeted miR-142-5p can inhibit apoptosis, inflammation, and matrix catabolism and inactivate the MAPK signaling pathway in OA chondrocytes. Our work provides important insight into targeting miR-142-5p and the SDF-1/CXCR4 axis in OA therapy.

Hypoxia gene expression signatures as predictive biomarkers for personalising radiotherapy.

Hypoxia is a generic micro-environmental factor of solid tumours. High levels of hypoxia lead to resistance to radiotherapy, which can be targeted by adding hypoxia-modifying therapy to improve clinical outcomes. Not all patients benefit from hypoxia-modifying therapy, and there is a need for biomarkers to enable progression to biologically personalised radiotherapy. Gene expression signatures are a relatively new category of biomarkers that can reflect tumour hypoxia. This article reviews the published hypoxia gene signatures, summarising their development and validation. The challenges of gene signature derivation and development, and advantages and disadvantages in comparison with other hypoxia biomarkers are also discussed. Current evidence supports investment in gene signatures as a promising hypoxia biomarker approach for clinical utility.

Screening of bioactive components from traditional Chinese medicine by immobilized ß2 adrenergic receptor coupled with high performance liquid chromatography/mass spectrometry.

Traditional Chinese medicine (TCM) represents a valuable resource for lead compounds discovery. Given the complexity of TCM components, analytical methods play a key role in novel drug development. In our study, we established a high specific and reliable bio-active components screen system, where ß2 adrenergic receptor (ß2-AR) was immobilized on silica by non-covalent bonds and packed into a stainless steel column (4.6 × 50 mm, 7 µm) to form ß2-AR chromatography column. The column was further coupled with high performance liquid chromatography-time of flight tandem mass spectrometry (TOF-MS/MS). By utilizing this strategy, we successfully identified four ß2-AR-targeting compounds: tetrahydroberberine, tetrahydrocolumbamine, fumarine and corydaline from Corydalis Rhizome. The association constants between ß2-AR and tetrahydroberberine (9.04 × 104/M) as well as fumarine (4.30 × 104/M) were determined by frontal chromatography. We also found that these two compounds shared the identical binding site on immobilized ß2-AR with corresponding concentrations of 6.67 × 10-4 M and 5.88 × 10-4 M, respectively. The newly established method represents an efficient tool to identify the target specific natural compounds.