[Formulation of expert consensus on clinical application of external ethnic medicine: taking Baimai Ointment as an example].

The concept of ethnic medicine is divided into a broad sense and a narrow sense. The broad concept refers to the traditional medicine of the Chinese nation, and the narrow concept refers to the traditional medicine of Chinese ethnic minorities. The external medicine is one of the main forms of ethnic medicine, and it is also the important content of ethnic medicine for external use, which is widely used in clinical practice. As the theory of ethnic medicine is unique, the application methods have certain characteristics, which are the key technical parts of clinical practice. However, the existing traditional Chinese medicine consensus formulation me-thods cannot meet the needs of the consensus formulation of the external ethnic medicine. Therefore, the methods suitable for expert consensus on external ethnic medicine are required. This article took Expert opinion on clinical application of Baimai Ointment as an exa-mple, and explorde a reasonable, effective, multi-dimensional, and multi-stage method to formulate expert consensus on the external ethnic medicine. In this research, three-dimensional sources of information, including ancient classics, clinical research evidence, and expert application experiences, were systematically and scientifically collected. After organization and analysis, the information was formed into comprehensive evidence. In a formal consensus meeting, part of the recommendations reached consensus. As to the issues that did not reach agreement, in-depth interviews were used to explore the reasons for the differences and resolve the disagreements. Finally, unanimous recommendations were reached. There are common problems during the formulation process of Expert opinion on clinical application of Baimai Ointment. This study is expected to provide references for the formulation of expert consensus on other external ethnic medicine.

[Systematic review of efficacy and safety of Ginkgo biloba extract 50 in treatment of ischemic stroke].

The present study aimed to systematically evaluate the efficacy and safety of Ginkgo biloba extract 50(GBE50) in the treatment of ischemic stroke. The databases including CNKI, Wanfang, VIP, SinoMed, PubMed, EMbase, and Cochrane Library were searched for randomized controlled trial(RCT) of GBE50 for the treatment of ischemic stroke reported between database inception and May 2020. The methodological quality of the included RCTs was evaluated via the Cochrane risk of bias tool. The RevMan 5.4 was used for Meta-analysis. Sixteen RCTs were included, involving 1 615 patients with acute ischemic stroke. Most of the included RCTs reported the methods of random sequence generation, but only two performed the concealment of random sequence. All RCTs failed in blinding. Two RCTs reported the information of cases lost to follow-up and drop-outs. Since the number was small, the baselines of groups remained balanced. All RCTs reported key outcomes of ischemic stroke, which made selective reporting bias in a low risk. Meta-analysis results revealed that GBE50 combined with routine therapies could effectively lower the score of the National Institutes of Health stroke scale(NIHSS) and restore cognitive function and daily activity in ischemic stroke patients. Compared with routine therapies, the combination is advantageous in treating patients with ischemic stroke. However, high-quality multicenter RCTs with large sample sizes are still required for verification.

Traditional Chinese Medicine in Cancer Care: An Overview of 5834 Randomized Controlled Trials Published in Chinese.

BACKGROUND: Traditional Chinese medicine (TCM) is widely integrated into cancer care in China. An overview in 2011 identified 2384 randomized and non-randomized controlled trials (RCTs, non-RCTs) on TCM for cancer published in the Chinese literature. This article summarizes updated evidence of RCTs on TCM for cancer care. METHODS: We searched 4 main Chinese databases: China National Knowledge Infrastructure, Chinese Scientific Journal Database, SinoMed, and Wanfang. RCTs on TCM used in cancer care were analyzed in this bibliometric study. RESULTS: Of 5834 RCTs (477 157 cancer patients), only 62 RCTs were indexed in MEDLINE. The top 3 cancers treated were lung, stomach, and breast cancer. About 4752 RCTs (81.45%) tested TCM combined with conventional treatment, and 1082 RCTs (18.55%) used TCM alone for treating symptoms and side-effects. Herbal medicine was the most frequently used TCM modality (5087 RCTs; 87.20%). The most frequently reported outcome was symptom improvement (3712 RCTs; 63.63%) followed by quality of life (2725 RCTs; 46.71%), and biomarkers (2384 RCTs; 40.86%). The majority of RCTs (4051; 69.44%) concluded there were beneficial effects using either TCM alone or TCM plus conventional treatment compared with conventional treatment. CONCLUSION: Substantial randomized trials demonstrated different types/stages of cancer were treated by various TCM modalities, alone or in combination with conventional medicine. Further evaluation on the effects and safety of TCM modalities focusing on outcomes such as quality of life is required.

Methylenetetrahydrofolate reductase genetic polymorphism and the risk of diabetic nephropathy in type 2 diabetic patients.

BACKGROUND: As indicated by numerous studies, there exists a relationship between the polymorphism of methylenetetrahydrofolate reductase (MTHFR) and susceptibility to diabetic nephropathy (DN) in various populations; nonetheless, the findings remain inconsistent. Therefore, we carried out a meta-analysis to determine the relationship between the MTHFR gene polymorphism and DN susceptibility. MATERIALS AND METHOD: Related studies were identified from PubMed, Cochrane Library, EMBASE, and the China National Knowledge Infrastructure database (time period: from building the library to October 2019). The strength of the association was examined using odds ratios (ORs) with 95% confidence intervals (95% CIs). RESULTS: The findings illustrated that the C677T gene polymorphism was significantly associated with an enhanced susceptibility to DN compared to that with diabetes mellitus in allelic (OR = 1.64, 95% CI = 1.34-2.00, P < .001), dominant (OR = 1.85, 95% CI = 1.40-2.46, P < .001), codominant (heterozygote: OR = 1.67, 95% CI = 1.27-2.21, P < .001; homozygote: OR = 2.55, 95% CI = 1.82-3.57, P < .001), and recessive (OR = 1.89, 95% CI = 1.50-2.38, P < .001) models of the overall population. Moreover, as compared with the healthy controls, a significantly augmented susceptibility to DN was found in all 5 genetic comparison models (allelic: OR = 2.06, 95% CI = 1.58-2.67, P < .001; dominant: OR = 2.52, 95% CI = 1.73-3.69, P < .001; codominant: OR = 3.78, 95% CI = 2.50-5.70, P < .001; recessive: OR = 2.41, 95% CI = 1.96-2.97, P < .001). Furthermore, stratifying data by ethnicity revealed substantially augmented vulnerability to DN in not only Caucasian but also Asian populations. CONCLUSION: The present study suggests that the C677T polymorphism was associated with an augmented susceptibility to DN.

[Consideration on establishing evaluation system of clinical practice guidelines in traditional Chinese medicine].

Standardization is the technical support for the development of traditional Chinese medicine(TCM), and the guidelines have become the main component of the core standards of TCM technology. With the rise and development of evidence-based medicine in China, more than 500 guidelines have been issued in China, and the number is still increasing, but the quality of guidelines still lags far behind the international level. Similarly, the formulation of evidence-based clinical practice guidelines for TCM has gradually attracted the attention of the industry, but the quality is not so good, and most guidelines are not really evidence-based guidelines. Only reliable guidelines can fully and effectively play the role of clinical guidance. In order to comprehensively improve the scientificity and credibility of the guidelines, guideline evaluation can be used as a means to improve the quality of the guidelines. For the development of traditional Chinese medicine, it has become an urgent task to establish a complete evaluation standard system of guidelines, especially the evaluation standard system that conforms to the technical characteristics of traditional Chinese medicine. In this paper, the advantages and limitations of a series of domestic and foreign guideline evaluation tools were systematically analyzed, and the thinking and difficulties to establish the evaluation system of TCM guidelines were put forward, with a purpose to further improve the quality of TCM clinical practice guidelines, so that they can be better applied in clinical practice to enhance the clinical efficacy of TCM and ensure the quality of medical services.

[Quality evaluation recommendation list and interpretation of traditional Chinese medicine clinical practice guidelines].

Based on the idea of establishing a complete guideline evaluation system applicable to the field of traditional Chinese medicine(TCM), the author believes that a complete guideline evaluation system of traditional Chinese medicine clinical practice should be divided into three parts: quality evaluation, clinical applicability evaluation and clinical application investigation. According to the different purposes, different evaluators, different evaluation methods and different evaluation time points in the guideline evaluation, the quality evaluation recommendation list, the clinical applicability evaluation recommendation list and the clinical application questionnaire were formed. Among them, the purpose of quality evaluation is to evaluate the methodological quality in the guideline development process, in order to measure whether the entire guideline development process is scientifically rigorous. The evaluators must be the methodologists with an evidence-based medicine background. Therefore, a logical, detailed and comprehensive guideline quality evaluation list will provide good evaluation tools for the TCM guideline formulation team and play an important role in promoting the quality and application of the guidelines. By referring to the internationally recognized development process and methods of evaluation tools, as well as the proof by authoritative TCM clinical experts and methodologists, the author worked out the quality evaluation list of clinical practice guidelines applicable to the field of TCM by considering the characteristics of TCM field. In this paper, the author introduces the whole list of quality evaluation recommendations, and interprets each item in details, hoping to provide reference for the standardization of TCM clinical practice guidelines in the future.

[Applicability evaluation recommendation list and interpretation of traditional Chinese medicine clinical practice guidelines].

The development of the guidelines should not only meet the rigorous methodological requirements, but also ensure the credibility or enforceability of the guideline recommendations when they are applied in clinical practice. Based on the idea of establi-shing a perfect guideline evaluation system applicable to the field of traditional Chinese medicine(TCM), the author believed that a complete evaluation system of clinical practice guidelines in the field of TCM shall be divided into three parts: quality evaluation, applicability evaluation and clinical application investigation. Among them, applicability evaluation refers to the evaluation of the degree of fit between the guideline and clinical practice, that is, whether the guidelines have good readability and clinical applicability to promote clinical application. The evaluators are clinical experts in the related fields of TCM. Therefore, a logical, detailed and comprehensive guideline quality evaluation list will provide good evaluation tools for the TCM guideline formulation team and play an important role in promoting the quality and application of the guidelines. Based on the internationally recognized development process and methods of evaluation tools, as well as the proof by authoritative TCM clinical experts and methodologists, the author worked out the quality evaluation list of clinical practice guidelines applicable to the field of TCM. In this paper, the author introduces the whole list of quality evaluation, and interprets each item in details, hoping to provide reference for the standardization of TCM clinical practice guidelines in the future.

[Systematic review on clinical efficacy and safety of Cheezheng Pain Relieving Plaster for soft tissue injury].

To systematically evaluate the clinical efficacy and safety of Cheezheng Pain Relieving Plaster in the treatment of soft tissue injury. Four Chinese databases(namely CNKI, WanFang, VIP, CBM) and 2 English databases(namely PubMed, Cochrane Library) were retrieved from the establishment of each database to March 2019. The randomized controlled trials of Cheezheng Pain Relieving Plaster compared with routine therapy in treatment of soft tissue injury were included. The quality of the included studies was assessed using the Cochrane Risk Assessment Tool. Five studies were included, and 367 patients were enrolled. None of the included studies reported randomized concealment, blinding, follow-up and dropping off. The results showed that Cheezheng Pain Relieving Plaster may have advantages in alleviating joint pain, swelling, tenderness and dysfunction and other symptoms, with no serious adverse reaction. Compared with routine therapy, Cheezheng Pain Relieving Plaster may have advantages in the treatment of soft tissue injury. However, due to the quality of the included RCTs, the conclusions of this study were limited. In addition, to produce high-quality evidences for the clinical application of Cheezheng Pain Relieving Plaster, the conclusions of this study shall be further verified with large-sample, scientifically designed and strictly implemented clinical trials.

[Bibliometric analysis and quality assessment of clinical evidence on Xiaotong Tiegao].

Many clinical studies on Cheezheng Xiaotong Tiegao have been accumulated since it was launched in 1993,but they have not been comprehensively analyzed and evaluated. This study systematically retrieved relevant studies in six databases at home and abroad as of December 2017. This study analyzed the statistics of the included studies in several aspects,including publication time,region,fund,disease category and type of study. In this study,various tools were used to evaluate the methodological quality of included studies,such as the Cochrane collaboration's tool for assessing the risk of bias in randomized trials,MINORS,IHE,AMSTAR2.The results showed that the literatures were mainly published from 2010 to 2011,and a total of 28 projects were financially supported.The most involved disease was arthropathy. The randomized controlled trials were the majority in the included studies,but the quality was low,and most of the literatures didn't report the allocation concealment and blinding. This study comprehensively reflected the current situations and shortcomings of the clinical studies of Cheezheng Xiaotong Tiegao,and put forward several suggestions,in the expectation of providing a reference for the future clinical research direction of Cheezheng Xiaotong Tiegao.

Evidence that miR-146a attenuates aging- and trauma-induced osteoarthritis by inhibiting Notch1, IL-6, and IL-1 mediated catabolism.

Primary osteoarthritis (OA) is associated with aging, while post-traumatic OA (PTOA) is associated with mechanical injury and inflammation. It is not clear whether the two types of osteoarthritis share common mechanisms. We found that miR-146a, a microRNA-associated with inflammation, is activated by cyclic load in the physiological range but suppressed by mechanical overload in human articular chondrocytes. Furthermore, miR-146a expression is decreased in the OA lesions of human articular cartilage. To understand the role of miR-146a in osteoarthritis, we systemically characterized mice in which miR-146a is either deficient in whole body or overexpressed in chondrogenic cells specifically. miR-146a-deficient mice develop early onset of OA characterized by cartilage degeneration, synovitis, and osteophytes. Conversely, miR-146a chondrogenic overexpressing mice are resistant to aging-associated OA. Loss of miR-146a exacerbates articular cartilage degeneration during PTOA, while chondrogenic overexpression of miR-146a inhibits PTOA. Thus, miR-146a inhibits both OA and PTOA in mice, suggesting a common protective mechanism initiated by miR-146a. miR-146a suppresses IL-1ß of catabolic factors, and we provide evidence that miR-146a directly inhibits Notch1 expression. Therefore, such inhibition of Notch1 may explain suppression of inflammatory mediators by miR-146a. Chondrogenic overexpression of miR-146a or intra-articular administration of a Notch1 inhibitor alleviates IL-1ß-induced catabolism and rescues joint degeneration in miR-146a-deficient mice, suggesting that miR-146a is sufficient to protect OA pathogenesis by inhibiting Notch signaling in the joint. Thus, miR-146a may be used to counter both aging-associated OA and mechanical injury-/inflammation-induced PTOA.

PRBC-derived plasma induces non-muscle myosin type IIA-mediated neutrophil migration and morphologic change.

CONTEXT: Neutrophils are the primary effector cells in the pathogenesis of transfusion-related acute lung injury or multiple organ failure after blood transfusion. OBJECTIVE: We aimed to investigate the effect of fresh (1 day preparation) and aged (42 day preparation) PRBC-derived plasma on neutrophil morphology, migration and phagocytosis. MATERIALS AND METHODS: We evaluated the production of reactive oxygen species (ROS) and the expression of non-muscle myosin heavy chain IIA (MYH9) in neutrophils treated with PRBC-derived plasma. We used western blots and antibody arrays to evaluate changes in signal transduction pathways in plasma-treated neutrophils. RESULTS: Aged PRBC-derived plasma elicited a stronger oxidative burst in neutrophils when compared with fresh PRBC-derived plasma (p < 0.05). Antibody arrays showed increased phosphorylation of NF-ĸB proteins (p105, p50 and Ikk) in aged PRBC-derived plasma-treated neutrophils. The expression of non-muscle myosin IIA (MYH9), a cytoskeleton protein involved in immune cell migration and morphological change, was also significantly upregulated in neutrophils treated with aged PRBC-derived plasma compared to fresh plasma (p < 0.05). Pretreatment of neutrophils with blebbistatin (a specific type II myosin inhibitor), ascorbic acid (an antioxidant), or staurosporine (a protein tyrosine kinase inhibitor), effectively abrogated the morphological changes, neutrophil migration, and phagocytosis induced by aged PRBC-derived plasma. CONCLUSION: Upregulation of MYH9 in neutrophils treated with aged PRBC-derived plasma and abrogation of neutrophil migration in blebbistatin-treated neutrophils suggested a functional role of MYH9 in the directional migration of immune cells. Our data help elucidate the cellular and molecular mechanisms of transfusion-related injury.

MiR-365: a mechanosensitive microRNA stimulates chondrocyte differentiation through targeting histone deacetylase 4.

Mechanical stress plays an essential role in tissue development and remodeling. In this study, we determined the role of microRNA in chondrocyte mechanotransduction. Using microarray, we identified miR-365 as a mechanoresponsive microRNA in parallel to mechanical induction of Indian hedgehog (Ihh) in primary chicken chondrocytes cultured in 3-dimensional collagen scaffoldings under cyclic loading (1 Hz, 5% elongation). Interestingly, expression of miR-365 is elevated in the prehypertrophic zone of the growth plate, coinciding with the Ihh expression region in vivo. MiR-365 significantly stimulates chondrocyte proliferation and differentiation. MiR-365 increases expression of Ihh and the hypertrophic marker type X collagen, whereas anti-miR-365 inhibits the expression of these genes. We identified histone deacetylase 4 (HDAC4), an inhibitor of chondrocyte hypertrophy, as a target of miR-365. MiR-365 inhibits both endogenous HDAC4 protein levels as well as the activity of a reporter gene bearing the 3'-untranslated region of HDAC4 mRNA. Conversely, inhibition of endogenous miR-365 relieves the repression of HDAC4. Mutation of the miR-365 binding site in HDAC4 mRNA abolishes miR-365-mediated repression of the reporter gene activity. Overexpression of HDAC4 reverses miR-365 stimulation of chondrocyte differentiation markers including Ihh, Col X, and Runx2. Moreover, inhibition of miR-365 abolishes mechanical stimulation of chondrocyte differentiation. Taken together, miR-365 is the first identified mechanically responsive microRNA that regulates chondrocyte differentiation via directly targeting HDAC4.

Phospho-SXXE/D motif mediated TNF receptor 1-TRADD death domain complex formation for T cell activation and migration.

In TNF-treated cells, TNFR1, TNFR-associated death domain protein (TRADD), Fas-associated death domain protein, and receptor-interacting protein kinase proteins form the signaling complex via modular interaction within their C-terminal death domains. In this paper, we report that the death domain SXXE/D motifs (i.e., S381DHE motif of TNFR1-death domain as well as S215LKD and S296LAE motifs of TRADD-death domain) are phosphorylated, and this is required for stable TNFR1-TRADD complex formation and subsequent activation of NF-κB. Phospho-S215LKD and phospho-S296LAE motifs are also critical to TRADD for recruiting Fas-associated death domain protein and receptor-interacting protein kinase. IκB kinase ß plays a critical role in TNFR1 phosphorylation of S381, which leads to subsequent T cell migration and accumulation. Consistently, we observed in inflammatory bowel disease specimens that TNFR1 was constitutively phosphorylated on S381 in those inflammatory T cells, which had accumulated in high numbers in the inflamed mucosa. Therefore, SXXE/D motifs found in the cytoplasmic domains of many TNFR family members and their adaptor proteins may serve to function as a specific interaction module for the α-helical death domain signal transduction.

Acetylation-dependent signal transduction for type I interferon receptor.

Cytokine-activated receptors initiate intracellular signaling by recruiting protein kinases that phosphorylate the receptors on tyrosine residues, thus enabling docking of SH2 domain-bearing activating factors. Here we report that in response to type 1 interferon (IFNalpha), IFNalpha receptors recruit cytoplasmic CREB-binding protein (CBP). By binding to IFNalphaR2 within the region where two adjacent proline boxes bear phospho-Ser364 and phospho-Ser384, CBP acetylates IFNalphaR2 on Lys399, which in turn serves as the docking site for interferon regulatory factor 9 (IRF9). IRF9 interacts with the acetyl-Lys399 motif by means of its IRF homology2 (IH2) domain, leading to formation of the ISGF3 complex that includes IRF9, STAT1, and STAT2. All three components are acetylated by CBP. Remarkably, acetylation within the DNA-binding domain (DBD) of both IRF9 and STAT2 is critical for the ISGF3 complex activation and its associated antiviral gene regulation. These results have significant implications concerning the central role of acetylation in cytokine receptor signal transduction.

Stat3 cleavage by caspases: impact on full-length Stat3 expression, fragment formation, and transcriptional activity.

Stat3 and its isoforms belong to a family of cytoplasmic transcription factors that affect the synthesis of various proteins. Caspases are cysteinyl-aspartate proteases that function under apoptotic and non-apoptotic conditions. We now report that, in addition to transcriptional splicing, Stat3 fragmentation can be mediated by caspases. Caspase activation in DU145 cells was achieved by staurosporine (STS) exposure, and Western analysis revealed a reduction in full-length Stat3 (fl-Stat3) expression that was caspase-mediated. This proteolytic relationship was further studied by exposing purified Stat3 protein to a mixture of active caspases under cell-free conditions. This demonstrated that caspases directly cleaved Stat3 and Stat3 cleavage was accompanied by the apparent formation of cleavage fragment(s). Stat3 cleavage fragments, reflecting multiple caspase cleavage sites, also were observed in vitro following STS exposure in DU145 cells and in HEK293T cells transfected to express Stat3 truncation mutants. The impact of cleavage on Stat3 transcriptional activity next was assessed and revealed that cleavage of fl-Stat3 was accompanied by reductions in Stat3-DNA binding, Stat3-driven reporter protein (luciferase) activity, and the expression of selected Stat3-dependent genes. Further, reduced Stat3 expression correlated with increased sensitivity to apoptotic stimuli. In concomitant experiments, reporter activity was assessed in Stat3 truncation mutant-expressing HEK293T cells and revealed that, under non-apoptotic conditions, expression of different Stat3 fragments induced differential effects on Stat3-driven luciferase activity. These findings demonstrate that fl-Stat3 undergoes proteolytic processing by caspases that reduces its expression and leads to the formation of cleavage fragments that may modulate Stat3 transcriptional activity.

Ankyrin repeat and SOCS box 3 (ASB3) mediates ubiquitination and degradation of tumor necrosis factor receptor II.

Ankyrin repeat and SOCS box (ASB) family members have a C-terminal SOCS box and an N-terminal ankyrin-related sequence of variable repeats belonging to the SOCS superfamily. While SH2-domain-bearing SOCS proteins are mainly involved in the negative feedback regulation of the protein tyrosine kinase-STAT pathway in response to a variety of cytokines, the roles of ASB family members remain largely unknown. To investigate ASB functions, we screened for ASB3-interacting factors by using antibody array technology and identified tumor necrosis factor receptor II (TNF-R2) as an ASB3 binding target. ASB3 expression and activities are required for (i) TNF-R2 ubiquitination both in vivo and in vitro, (ii) TNF-R2 proteolysis via the proteasome pathway, and (iii) the inhibition of TNF-R2-mediated Jun N-terminal protein kinase (JNK) activation. While the ankyrin repeats of ASB3 interact with the C-terminal 37 amino acids of TNF-R2, the SOCS box of ASB3 is responsible for recruiting the E3 ubiquitin ligase adaptors Elongins-B/C, leading to TNF-R2 ubiquitination on multiple lysine residues within its C-terminal region. Downregulation of ASB3 expression by a small interfering RNA inhibited TNF-R2 degradation and potentiated TNF-R2-mediated cytotoxicity. The data presented here implicate ASB3 as a negative regulator of TNF-R2-mediated cellular responses to TNF-alpha by direct targeting of TNF-R2 for ubiquitination and proteasome-mediated degradation.

Stat3 dimerization regulated by reversible acetylation of a single lysine residue.

Upon cytokine treatment, members of the signal transducers and activators of transcription (STAT) family of proteins are phosphorylated on tyrosine and serine sites within the carboxyl-terminal region in cells. We show that in response to cytokine treatment, Stat3 is also acetylated on a single lysine residue, Lys685. Histone acetyltransferase p300-mediated Stat3 acetylation on Lys685 was reversible by type I histone deacetylase (HDAC). Use of a prostate cancer cell line (PC3) that lacks Stat3 and PC3 cells expressing wild-type Stat3 or a Stat3 mutant containing a Lys685-to-Arg substitution revealed that Lys685 acetylation was critical for Stat3 to form stable dimers required for cytokine-stimulated DNA binding and transcriptional regulation, to enhance transcription of cell growth-related genes, and to promote cell cycle progression in response to treatment with oncostatin M.

Central role of the threonine residue within the p+1 loop of receptor tyrosine kinase in STAT3 constitutive phosphorylation in metastatic cancer cells.

The receptor tyrosine kinases (RTKs) RET, MET, and RON all carry the Met(p+1loop)-->Thr point mutation (i.e., 2B mutation), leading to the formation of tumors with high metastatic potential. Utilizing a novel antibody array, we identified constitutive phosphorylation of STAT3 in cells expressing the 2B mutation but not wild-type RET. MET or RON with the 2B mutation also constitutively phosphorylated STAT3. Members of the EPH, the only group of wild-type RTK that carry Thr(p+1loop) residue, are often expressed unexpectedly in different types of cancers. Ectopic expression of wild-type but not Thr(p+1loop)-->Met substituted EPH family members constitutively phosphorylated STAT3. In both RTK(Metp+1loop) with 2B mutation and wild-type EPH members the Thr(p+1loop) residue is required for constitutive kinase autophosphorylation and STAT3 recruitment. In multiple endocrine neoplasia 2B (MEN-2B) patients expressing RET(M918T), nuclear enrichment of STAT3 and elevated expression of CXCR4 was detected in metastatic thyroid C-cell carcinoma in the liver. In breast adenocarcinoma cell lines expressing multiple EPH members, STAT3 constitutively bound to the promoters of MUC1, MUC4, and MUC5B genes. Inhibiting STAT3 expression resulted in reduced expression of these metastasis-related genes and inhibited mobility. These findings provide insight into Thr(p+1loop) residue in RTK autophosphorylation and constitutive activation of STAT3 in metastatic cancer cells.

Antibodies immobilized as arrays to profile protein post-translational modifications in mammalian cells.

Previously, we demonstrated that antibodies printed on a solid support were able to detect protein-protein interaction in mammalian cells. Here we further developed the antibody array system for detecting proteins with various post-translational modifications in mammalian cells. In this novel approach, immunoprecipitated proteins were labeled with fluorescent dye followed by incubation over antibody arrays. Targeted proteins, captured by the antibodies immobilized on PVDF membrane or glass slide, were detected by means of near infrared fluorescent scanner or fluorescent microscopy. To demonstrate the application of the antibody arrays in protein post-translational modifications, we profiled protein tyrosine phosphorylation, ubiquitination, and acetylation in mammalian cells under different conditions. Our results indicate that antibody array technology can provide a powerful means of profiling a large number of proteins with different post-translational modifications in cells.