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author:("Xing, dangme")
1.  Detection of Circulating Tumor DNA in Early- and Late-Stage Human Malignancies 
Science translational medicine  2014;6(224):224ra24.
The development of noninvasive methods to detect and monitor tumors continues to be a major challenge in oncology. We used digital polymerase chain reaction–based technologies to evaluate the ability of circulating tumor DNA (ctDNA) to detect tumors in 640 patients with various cancer types. We found that ctDNA was detectable in >75% of patients with advanced pancreatic, ovarian, colorectal, bladder, gastroesophageal, breast, melanoma, hepatocellular, and head and neck cancers, but in less than 50% of primary brain, renal, prostate, or thyroid cancers. In patients with localized tumors, ctDNA was detected in 73, 57, 48, and 50% of patients with colorectal cancer, gastroesophageal cancer, pancreatic cancer, and breast adenocarcinoma, respectively. ctDNA was often present in patients without detectable circulating tumor cells, suggesting that these two biomarkers are distinct entities. In a separate panel of 206 patients with metastatic colorectal cancers, we showed that the sensitivity of ctDNA for detection of clinically relevant KRAS gene mutations was 87.2% and its specificity was 99.2%. Finally, we assessed whether ctDNA could provide clues into the mechanisms underlying resistance to epidermal growth factor receptor blockade in 24 patients who objectively responded to therapy but subsequently relapsed. Twenty-three (96%) of these patients developed one or more mutations in genes involved in the mitogen-activated protein kinase pathway. Together, these data suggest that ctDNA is a broadly applicable, sensitive, and specific biomarker that can be used for a variety of clinical and research purposes in patients with multiple different types of cancer.
PMCID: PMC4017867  PMID: 24553385
2.  Clinical Research of Traditional Chinese Medicine Needs to Develop Its Own System of Core Outcome Sets 
Currently, quality issues concerning clinical research of traditional Chinese medicine (TCM) have come into the spotlight. It has been recognized that poorly-devised research methodology largely restricted the development of clinical research in TCM. The choice of appropriate outcome measurements is key to the success of clinical research; however, the current procedure for outcomes selection in clinical research of TCM is problematic due to the underdevelopment of clinical methodology. Under this circumstance, we propose the introduction to the concept of Core Outcome Set (COS) and discuss the feasibility of developing a COS system that caters for clinical studies in TCM, in the hope that the outcome evaluation system could be up to international standards.
PMCID: PMC3838829  PMID: 24312133
3.  The key role of Shenyan Kangfu tablets, a Chinese patent medicine for diabetic nephropathy: study protocol for a randomized, double-blind and placebo-controlled clinical trial 
Trials  2013;14:165.
Diabetic nephropathy (DN) is a major microvascular complication with diabetes. In China, an estimated 34.7 percent of people diagnosed with diabetes have renal complications and a further 50 percent die of renal failure. Hence, identification of alternative treatments for these patients should be given priority. The Shenyan Kangfu tablet (SYKFT) is a new formulation of an existing and widely acclaimed Chinese herbal tea for treating qi-yin deficiency syndrome. Because a considerable portion of DN patients presenting with symptoms of swelling, fatigue and weak limbs would be diagnosed with qi-yin deficiency syndrome according to the traditional Chinese medicine (TCM) diagnostic criteria, we hypothesize that SYKFT may represent a complementary drug for DN patients with the corresponding syndrome. In view of this, we have designed a trial to assess the efficacy and safety of SYKFT for patients with diabetic nephropathy exhibiting signs of qi and yin deficiency.
This is a multicenter, double-blind, randomized controlled trial (RCT). The total target sample size is planned at 80 participants, with a balanced (1:1) treatment allocation. The experimental intervention will be SYKFY plus irbesartan (SI regimen) and the control intervention will be a placebo plus irbesartan (PI regimen). Participants will receive two courses of medication treatment each lasting 8 weeks. The primary outcome will be the composite of the quantitative 24-hour urinary protein level and urinary albumin excretion rate (UAER). Changes in urine albumin-to-creatinine ratio (UACR) and DN staging, and TCM symptom improvement will be the secondary outcome measures. Adverse events (AEs) will be monitored throughout the trial.
This study will be the first placebo-controlled RCT to assess whether SYKFT plus irbesartan will have beneficial effects on enhancing overall response rate (ORR), changing DN staging, improving clinical symptoms, and reducing the frequency of AEs for DN patients with qi-yin deficiency syndrome. The results of this trial will help to provide evidence-based recommendations for clinicians.
Trial registration
Chinese Clinical Trials Register, Identifier: ChiCTR-TRC-12002182
PMCID: PMC3679981  PMID: 23738508
Shenyan Kangfu tablets; Efficacy; Safety; Diabetic nephropathy; Randomized controlled trial
4.  Hypoxia and Hypoxia-Inducible Factor in the Burn Wound 
The importance of hypoxia-inducible factor (HIF) in promoting angiogenesis and vasculogenesis during wound healing has been demonstrated. It is widely accepted that HIF activity can be promoted by many factors, including hypoxia in the wound or cytokines from inflammatory cells infiltrating the wound. However, there has not been a systematic exploration of the relationship between HIF activity and hypoxia in the burn wound. The location of the hypoxic tissue has not been clearly delineated. The time course of the appearance of hypoxia and the increased activity of HIF and appearance of HIF’s downstream transcription products has not been described. The aim of this study was to utilize pimonidazole, a specific tissue hypoxia marker, to characterize the spatial and temporal course of hypoxia in a murine burn model and correlate this with the appearance of HIF-1α and its important angiogenic and vasculogenic transcription products VEGF and SDF-1. Hypoxia was found in the healing margin of burn wounds beginning at 48 hours after burn and peaking at day 3 after burn. On sequential sections of the same tissue block, positive staining of HIF-1α, SDF-1, and VEGF all occurred at the leading margin of the healing area and peaked at day 3, as did hypoxia. Immunohistochemical analysis was used to explore the characteristics of the hypoxic region of the wound. The localization of hypoxia was found to be related to cell growth and migration, but not to proliferation or inflammatory infiltration.
PMCID: PMC3075089  PMID: 21362088
ypoxia; Hypoxia-inducible factor -10α; Burn; Wound Healing; Ki67 Cell Proliferation; Keratin17
5.  Hypoxia preconditioning protects corneal stromal cells against induced apoptosis 
Experimental eye research  2005;82(5):780-787.
The purpose of this study, was to determine whether hypoxia preconditioning can protect corneal stromal cells from UV stress and cytokine mediated apoptosis. Two models were implemented. First, primary cultured bovine corneal fibroblasts were preconditioned with 0.5–1.5% O2 for 4 hr and stressed with UV-irradiation or stimulation of Fas receptor. Second, bovine eyes were preconditioned with 0.5% O2 for 4 hr and stressed by epithelial scraping to induce anterior keratocyte apoptosis. Cell fate was analyzed at 4 hr after stress using quantitative TUNEL or condensed nuclei assays. Cell apoptotic rates in hypoxia preconditioned groups were significantly lower (50–80%) than that of normoxia control groups. Hypoxia prevented the degradation of the transcription factor HIF-1α. CoCl2 (100–200 μM), a chemical inducer of HIF-1α, also produced strong protection against UV and Fas induced apoptosis. Moreover, hypoxia preconditioned media protected cells against UV-induced apoptosis. These findings demonstrate that hypoxia preconditioning has a generalized protective effect against stromal fibroblast and keratocyte apoptosis and suggest that HIF-1α mediated expression and secretion of protective factors is involved.
PMCID: PMC3085538  PMID: 16364292
cornea; keratocytes; apoptosis; hypoxia preconditioning
6.  Effect of cAMP on TGFβ1-Induced Corneal Keratocyte–Myofibroblast Transformation 
TGFβ is the major mediator to induce myofibroblast differentiation in the corneal wound-healing process. Elevated cAMP can reduce TGFβ-induced fibrosis in other tissues. This study was conducted to determine whether elevated cAMP can inhibit TGFβ1-induced rabbit corneal keratocyte–myofibroblast transformation.
Primary isolated rabbit corneal keratocytes were cultured in serum-free medium. The effects of the adenylate cyclase agonist forskolin (FSK; 2 μM) on TGFβ1 (5 ng/mL)-induced α-smooth muscle actin (α-SMA) expression was examined by immunofluorescence, flow cytometry, and immunochemistry 72 hours after treatment. The effects of TGFβ+FSK on activated pSmad3, CREB binding protein (CBP), MAPKs, and RhoA were determined by coimmunoprecipitation and Western blot.
FSK significantly reduced the myofibroblast phenotype and α-SMA expression induced by TGFβ1 in rabbit corneal keratocytes. TGFβ1 increased the phosphorylation of ERK and Smad3. TGFβ1-induced α-SMA expression was reduced by MEK inhibition (U0126); however, the levels of pERK, pSmad3, or the extent of the interaction between pSmad3 and CBP induced by TGFβ1 were not affected by FSK. TGFβ1 also activated RhoA and ROCK (Y27632) inhibition reduced α-SMA expression. Activation of RhoA was significantly reduced by FSK.
Raising cAMP by FSK treatment inhibits the TGFβ1-induced corneal myofibroblast transformation and α-SMA expression and thereby provides a promising method to control corneal fibrosis. The data suggest that cAMP-dependent inhibition does not occur by altering Smads or MAPK signaling, but possibly by reducing the activation of RhoA.
PMCID: PMC2810623  PMID: 18936144
7.  Hypoxia preconditioning protection of corneal stromal cells requires HIF1α but not VEGF 
Molecular Vision  2009;15:1020-1027.
Hypoxia preconditioning protects corneal stromal cells from stress-induced death. This study determined whether the transcription factor HIF-1α (Hypoxia Inducible Factor) is responsible and whether this is promulgated by VEGF (Vascular Endothelial Growth Factor).
Cultured bovine stromal cells were preconditioned with hypoxia in the presence of cadmium chloride, a chemical inhibitor of HIF-1α, and HIF-1α siRNA to test if HIF-1α activity is needed for hypoxia preconditioning protection from UV-irradiation induced cell death. TUNEL assay was used to detect cell apoptosis after UV-irradiation. RT-PCR and western blot were used to detect the presence of HIF-1α and VEGF in transcriptional and translational levels.
During hypoxia (0.5% O2), 5 μM cadmium chloride completely inhibited HIF-1α expression and reversed the protection by hypoxia preconditioning.  HIF-1α siRNA (15 nM) reduced HIF-1α expression by 90% and produced a complete loss of protection provided by hypoxia preconditioning.  Since VEGF is induced by hypoxia, can be HIF-1α dependent, and is often protective, we examined the changes in transcription of VEGF and its receptors after 4 h of hypoxia preconditioning.  VEGF and its receptors Flt-1 and Flk-1 are up-regulated after hypoxia preconditioning.  However, the transcription and translation of VEGF were paradoxically increased by siHIF-1α, suggesting that VEGF expression in stromal cells is not down-stream of HIF-1α.
These findings demonstrate that hypoxia preconditioning protection in corneal stromal cells requires HIF-1α, but that VEGF is not a component of the protection.
PMCID: PMC2684561  PMID: 19461932
8.  Hypoxia reduces TGFβ1-induced corneal keratocyte myofibroblast transformation 
Molecular Vision  2009;15:1827-1834.
The purpose of this study was to determine whether transient hypoxia had an effect on transforming growth factor β1 (TGFβ1)-induced rabbit corneal keratocyte myofibroblast transformation.
Primary isolated rabbit corneal keratocytes were cultured in a serum-free medium. The effect of transient hypoxia treatment (1% oxygen, 4 h/day) on TGFβ1 (5 ng/ml)-induced α-smooth muscle actin (α-SM actin) expression was examined by immunofluorescence, flow cytometry, and immunocytochemistry 72 h after treatment. We found that hypoxia treatment significantly reduced the myofibroblast phenotype and α-SM actin expression that was induced by TGFβ1. To explore the possible mechanism for this effect, we screened for the effects of hypoxia on several early TGFβ-dependent signaling events including activated pSmad3, CREB (cAMP response element binding) binding protein (CBP), MAPKs (Mitogen-activated protein kinase), and RhoA by co-immunoprecipitation and western blotting.
Hypoxia alone increased α-SM actin expression and the association of pSmad3 to CBP, but it did not induce the myofibroblast phenotype. The levels of pERK (the extracellular signal-regulated protein kinase) and pSmad3 or the extent of the interaction between pSmad3 and CBP induced by TGFβ1 were not affected by hypoxia whereas the activation of RhoA induced by TGFβ1 was significantly reduced.
We conclude that hypoxia can inhibit TGFβ1-induced corneal myofibroblast transformation and α-SM actin expression. Our data show that this inhibition does not occur by altering Smads or MAPK signaling but possibly by reducing the early activation of RhoA.
PMCID: PMC2742637  PMID: 19753310

Results 1-8 (8)