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Esophageal squamous cell carcinoma (ESCC) is one of the most fatal malignancies worldwide. Recently, our group identified purine-rich element binding protein alpha (PURα), a single-stranded DNA/RNA-binding protein, to be significantly associated with the progression of ESCC. Additional immunofluorescence staining demonstrated that PURα forms cytoplasmic stress granules to suppress mRNA translation initiation. The expression level of cytoplasmic PURα in ESCC tumor tissues was significantly higher than that in adjacent epithelia and correlated with a worse patient survival rate by immunohistochemistry. Functionally, PURα strongly preferred to bind to UG-/U-rich motifs and mRNA 3´UTR by CLIP-seq analysis. Moreover, PURα knockout significantly increased the protein level of insulin-like growth factor binding protein 3 (IGFBP3). In addition, it was further demonstrated that PURα-interacting proteins are remarkably associated with translation initiation factors and ribosome-related proteins and that PURα regulates protein expression by interacting with translation initiation factors, such as PABPC1, eIF3B and eIF3F, in an RNA-independent manner, while the interaction with ribosome-related proteins is significantly dependent on RNA. Specifically, PURα was shown to interact with the mRNA 3´UTR of IGFBP3 and inhibit its expression by suppressing mRNA translation initiation. Together, this study identifies cytoplasmic PURα as a modulator of IGFBP3, which could be a promising therapeutic target for ESCC treatment.

The mechanisms of mast cell (MC) degranulation and MC-driven skin symptoms are well-described. In contrast, data about the role of mitochondrial respiration for immune functions of human skin MCs are lacking. Oxygen consumption rate (OCR) in primary human skin MCs during IgE-mediated activation in the absence of glucose was examined using a metabolic flux analyzer. Effects of the inhibition of mitochondrial complex I (by rotenone A) and III (by myxothiazol) on degranulation and cytokine secretion (IL-4, IL-5, IL-6, IL-13, TNF-α, and GM-CSF) were explored by the β-hexosaminidase release assay and multiplex ELISA. IgE-mediated activation rapidly increased the mitochondrial OCR and extracellular acidification; the contribution of non-mitochondrial oxygen consumption remained unchanged at lower levels. Both myxothiazol and rotenone A reduced OCR, the mitochondrial parameters, and extracellular acidification; however, myxothiazol did not affect degranulation and cytokine secretion. In contrast, degranulation and the secretion of IL-6, IL-13, TNF-α, and GM-CSF were reduced by rotenone A, whereas the secretion of IL-4 and IL-5 was not significantly affected. The inhibitors did not affect cell viability. Our results highlight the important role played by mitochondrial respiration in primary human skin MCs and allow for a conclusion on a hierarchy of their effector functions. Drugs targeting specific pathways in mitochondria may provide future options to control MC-driven skin symptoms.

Hypertrophic scar is a serious skin disorder, which reduces the patient's quality of life. 5-aminolevulinic acid (5-ALA)-mediated photodynamic therapy has been used to treat patients with hypertrophic scar. However, the poor skin retention of 5-ALA limited the therapeutic effect. In this study, we constructed the 5-ALA-hyaluronic acid (HA) complex to potentially prolong the skin retention of 5-ALA for improving the therapeutic efficacy. HA is a polysaccharide with viscoelasticity and the carboxyl groups could conjugate with amino groups of 5-ALA via electrostatic interaction. The protoporphyrin IX (PpIX) assay revealed that 5-ALA-HA complexes markedly enhanced the skin retention, resulting in increased generation and accumulation of endogenous photosensitizer PpIX. Furthermore, 5-ALA-HA complexes allowed PpIX to be maintained at a high level for 12 h, much longer than the 3 h of 5-ALA alone. And then, the accumulative PpIX induced by 5-ALA-HA in human hypertrophic scar fibroblasts (HSF) was triggered by laser irradiation to produce sufficient reactive oxygen species, leading to efficient necrosis and apoptosis of HSF. In vivo therapeutic efficacy study indicated that 5-ALA-HA effectively reduced the appearance and scar thickness, and the scar elevation index with 5-ALA-HA treatment was significantly lower than other groups, suggesting that the 5-ALA-HA-treated scar became flattened and was closely matched to the unwounded tissues. Moreover, 5-ALA-HA treatment markedly downregulated the gene expression levels of α-SMA and TGF-β1, demonstrating attenuated the scar formation and growth. Therefore, the 5-ALA-HA complex enhancing skin retention and PpIX accumulation at the lesion site provide a promising therapeutic strategy for hypertrophic scar.

Cellular metabolism modulates effector functions in human CD4+ T (Th) cells by providing energy and building blocks. Conversely, cellular metabolic responses are modulated by various influences, e.g., age. Thus, we hypothesized that metabolic reprogramming in human Th cells during aging modulates effector functions and contributes to "inflammaging", an aging-related, chronic, sterile, low-grade inflammatory state characterized by specific proinflammatory cytokines. Analyzing the metabolic response of human naive and memory Th cells from young and aged individuals, we observed that memory Th cells exhibit higher glycolytic and mitochondrial fluxes than naive Th cells. In contrast, the metabolism of the latter was not affected by donor age. Memory Th cells from aged donors showed a higher respiratory capacity, mitochondrial content, and intracellular ROS production than those from young donors without altering glucose uptake and cellular ATP levels, which finally resulted in higher secreted amounts of proinflammatory cytokines, e.g., IFN-γ, IP-10 from memory Th cells taken from aged donors after TCR-stimulation which were sensitive to ROS inhibition. These findings suggest that metabolic reprogramming in human memory Th cells during aging results in an increased expression of proinflammatory cytokines through enhanced ROS production, which may contribute to the pathogenesis of inflammaging. ispartof: FRONTIERS IN IMMUNOLOGY vol:13 ispartof: location:Switzerland status: published

After trauma, the formed fracture hematoma within the fracture gap contains all the important components (immune/stem cells, mediators) to initiate bone regeneration immediately. Thus, it is of great importance but also the most susceptible to negative influences. To study the interaction between bone and immune cells within the fracture gap, up-to-date in vitro systems should be capable of recapitulating cellular and humoral interactions and the physicochemical microenvironment (eg, hypoxia). Here, we first developed and characterized scaffold-free bone-like constructs (SFBCs), which were produced from bone marrow-derived mesenchymal stromal cells (MSCs) using a macroscale mesenchymal condensation approach. SFBCs revealed permeating mineralization characterized by increased bone volume (mu CT, histology) and expression of osteogenic markers (RUNX2, SPP1, RANKL). Fracture hematoma (FH) models, consisting of human peripheral blood (immune cells) mixed with MSCs, were co-cultivated with SFBCs under hypoxic conditions. As a result, FH models revealed an increased expression of osteogenic (RUNX2, SPP1), angiogenic (MMP2, VEGF), HIF-related (LDHA, PGK1), and inflammatory (IL6, IL8) markers after 12 and 48 hours co-cultivation. Osteogenic and angiogenic gene expression of the FH indicate the osteoinductive potential and, thus, the biological functionality of the SFBCs. IL-6, IL-8, GM-CSF, and MIP-1 beta were detectable within the supernatant after 24 and 48 hours of co-cultivation. To confirm the responsiveness of our model to modifying substances (eg, therapeutics), we used deferoxamine (DFO), which is well known to induce a cellular hypoxic adaptation response. Indeed, DFO particularly increased hypoxia-adaptive, osteogenic, and angiogenic processes within the FH models but had little effect on the SFBCs, indicating different response dynamics within the co-cultivation system. Therefore, based on our data, we have successfully modeled processes within the initial fracture healing phase in vitro and concluded that the cross-talk between bone and immune cells in the initial fracture healing phase is of particular importance for preclinical studies. (c) 2021 American Society for Bone and Mineral Research (ASBMR).

OBJECTIVES—: To quantify the bias of shear wave speed (SWS) measurements between different commercial ultrasonic shear elasticity systems and a magnetic resonance elastography (MRE) system in elastic and viscoelastic phantoms. METHODS—: Two elastic phantoms, representing healthy through fibrotic liver, were measured with 5 different ultrasound platforms, and 3 viscoelastic phantoms, representing healthy through fibrotic liver tissue, were measured with 12 different ultrasound platforms. Measurements were performed with different systems at different sites, at 3 focal depths, and with different appraisers. The SWS bias across the systems was quantified as a function of the system, site, focal depth, and appraiser. A single MRE research system was also used to characterize these phantoms using discrete frequencies from 60 to 500 Hz. RESULTS—: The SWS from different systems had mean difference 95% confidence intervals of ±0.145 m/s (±9.6%) across both elastic phantoms and ± 0.340 m/s (±15.3%) across the viscoelastic phantoms. The focal depth and appraiser were less significant sources of SWS variability than the system and site. Magnetic resonance elastography best matched the ultrasonic SWS in the viscoelastic phantoms using a 140 Hz source but had a − 0.27 ± 0.027-m/s (−12.2% ± 1.2%) bias when using the clinically implemented 60-Hz vibration source. CONCLUSIONS—: Shear wave speed reconstruction across different manufacturer systems is more consistent in elastic than viscoelastic phantoms, with a mean difference bias of < ±10% in all cases. Magnetic resonance elastographic measurements in the elastic and viscoelastic phantoms best match the ultrasound systems with a 140-Hz excitation but have a significant negative bias operating at 60 Hz. This study establishes a foundation for meaningful comparison of SWS measurements made with different platforms.

Antiretroviral therapy (ART) prevents human immunodeficiency virus (HIV)-1 onward transmission and disease progression, leading to excellent prognosis in people living with HIV-1 (PWH). However, side effects, complications, and impaired immune reconstitution persist in some patients treated with ART. We aimed to profile proteome changes in plasma before and after ART to identify the molecular pathways altered by ART.Quantitative proteomics analysis based on tandem mass tag (TMT) labeling was used to profile proteome changes of paired plasma samples from HIV-1 patients before receiving ART and after ART treatment.A total of 1398 protein groups (PGs) were identified, in which 18 proteins were downregulated and 50 were upregulated in plasma from ART treated patients. Based on Ingenuity Pathway analysis (IPA), gap junction signaling and actin cytoskeleton signaling were enriched among upregulated proteins, while downregulated proteins were mainly participated in IL-15 signaling pathway. Patients with the low level of CSF1R and the high levels of MINPP1 and TGM3 showed better CD4The present study provided plasma proteome changes after ART to elucidate the underlying mechanistic pathways in response to ART, and also identified potential targets to prompt immune reconstitution.

Although there are a lot of chemical drugs to treat breast cancer, increasing drug resistance of cancer cells has strongly hindered the effectiveness of chemotherapy. ATP-binding cassette transporters represented by P-glycoprotein (P-gp), multidrug resistance associated protein 1 (MRP1) and breast cancer resistance protein (BCRP) play an important role in drug resistance. This study aims to investigate the effect of 7-iO/i-geranylquercetin (GQ) combining microRNA-451(miR-451) on reversing drug resistance of breast cancer and reveal the mechanism related to P-gp. Real-time RT-PCR and western blot assays showed that miR-326, miR-328, miR-451 and miR-155 inhibitor down-regulated the expression of genes MRP1, BCRP, MDR1 and the corresponding proteins MRP1, BCRP, P-gp, respectively. Cell counting kit-8 (CCK-8) assay indicated that these miRNAs reversed the resistance of MCF-7/ADR cells to Adriamycin (ADR), and miR-451 showed the greatest reversal effect. Combination of GQ and miR-451 enhanced the inhibitory effects of ADR on the proliferation and migration of MCF-7/ADR cells, and attenuated the expression of MDR1 and P-gp in MCF-7/ADR cells. A xenograft tumor model was used to show that GQ and miR-451 amplified the antitumor effect of ADR in nude mice, while western blot and immunohistochemical assays revealed the decreased expression of P-gp in tumor tissues. These results suggest that GQ and miR-451 have synergistic effect on reversing drug resistance through reducing the expression of MDR1 and P-gp in breast cancer MCF-7/ADR cells.

Human cytomegalovirus (HCMV) infection in the respiratory tract leads to pneumonitis in immunocompromised hosts without available vaccine. Considering cytomegalovirus (CMV) mainly invades through the respiratory tract, CMV‐specific pulmonary mucosal vaccine development that provides a long‐lasting protection against CMV challenge gains our attention. In this study, N‐terminal domain of GP96 (GP96‐NT) was used as a mucosal adjuvant to enhance the induction of pulmonary‐resident CD8 T cells elicited by MCMV glycoprotein B (gB) vaccine. Mice were intranasally co‐immunized with 50 μg pgB and equal amount of pGP96‐NT vaccine 4 times at 2‐week intervals, and then i.n. challenged with MCMV at 16 weeks after the last immunization. Compared with pgB immunization alone, co‐immunization with pgB/pGP96‐NT enhanced a long‐lasting protection against MCMV pneumonitis by significantly improved pneumonitis pathology, enhanced bodyweight, reduced viral burdens and increased survival rate. Moreover, the increased CD8 T cells were observed in lung but not spleen from pgB/pGP96‐NT co‐immunized mice. The increments of pulmonary CD8 T cells might be mainly due to non‐circulating pulmonary‐resident CD8 T‐cell subset expansion but not circulating CD8 T‐cell populations that home to inflammation site upon MCMV challenge. Finally, the deterioration of MCMV pneumonitis by depletion of pulmonary site‐specific CD8 T cells in mice that were pgB/pGP96‐NT co‐immunization might be a clue to interpret the non‐circulating pulmonary‐resident CD8 T subset expansion. These data might uncover a promising long‐lasting prophylactic vaccine strategy against MCMV‐induced pneumonitis.

The transition zone (TZ) of the cilium/flagellum serves as a diffusion barrier that controls the entry/exit of ciliary proteins. Mutations of the TZ proteins disrupt barrier function and lead to multiple human diseases. However, the systematic regulation of ciliary composition and signaling-related processes by different TZ proteins is not completely understood. Here, we reveal that loss of TCTN1 in Chlamydomonas reinhardtii disrupts the assembly of wedge-shaped structures in the TZ. Proteomic analysis of cilia from WT and three TZ mutants, tctn1, cep290, and nphp4, shows a unique role of each TZ subunit in the regulation of ciliary composition, explaining the phenotypic diversity of different TZ mutants. Interestingly, we find that defects in the TZ impair the formation and biological activity of ciliary ectosomes. Collectively, our findings provide systematic insights into the regulation of ciliary composition by TZ proteins and reveal a link between the TZ and ciliary ectosomes.

Background Death and disability from coronary heart disease (CHD) can be largely reduced by improving risk factor management. However, adhering to evidence-based recommendations is challenging and requires interventions at the level of the patient, provider, and health system. Objective The aim of this study was to develop an Intelligent Individualized Cardiovascular App for Risk Elimination (iCARE) to facilitate adherence to health behaviors and preventive medications, and to test the usability of iCARE. Methods We developed iCARE based on a user-centered design approach, which included 4 phases: (1) function design, (2) iterative design, (3) expert inspections and walkthroughs of the prototypes, and (4) usability testing with end users. The usability testing of iCARE included 2 stages: stage I, which included a task analysis and a usability evaluation (January to March 2019) of the iCARE patient app using the modified Health Information Technology Usability Survey (Health-ITUES); and stage II (June 2020), which used the Health-ITUES among end users who used the app for 6 months. The end users were individuals with a confirmed diagnosis of CHD from 2 university-affiliated hospitals in Beijing, China. Results iCARE consists of a patient app, a care provider app, and a cloud platform. It has a set of algorithms that trigger tailored feedback and can send individualized interventions based on data from initial assessment and health monitoring via manual entry or wearable devices. For stage I usability testing, 88 hospitalized patients (72% [63/88] male; mean age 60 [SD 9.9] years) with CHD were included in the study. The mean score of the usability testing was 90.1 (interquartile range 83.3-99.0). Among enrolled participants, 90% (79/88) were satisfied with iCARE; 94% (83/88) and 82% (72/88) reported that iCARE was useful and easy to use, respectively. For stage II usability testing, 61 individuals with CHD (85% [52/61] male; mean age 53 [SD 8.2] years) who were from an intervention arm and used iCARE for at least six months were included. The mean total score on usability testing based on the questionnaire was 89.0 (interquartile distance: 77.0-99.5). Among enrolled participants, 89% (54/61) were satisfied with the use of iCARE, 93% (57/61) perceived it as useful, and 70% (43/61) as easy to use. Conclusions This study developed an intelligent, individualized, evidence-based, and theory-driven app (iCARE) to improve patients’ adherence to health behaviors and medication management. iCARE was identified to be highly acceptable, useful, and easy to use among individuals with a diagnosis of CHD. Trial Registration Chinese Clinical Trial Registry ChiCTR-INR-16010242; https://tinyurl.com/2p8bkrew

Fadraciclib (CYC065) is a second-generation aminopurine CDK2/9 inhibitor with increased potency and selectivity toward CDK2 and CDK9 compared to seliciclib (R-roscovitine). In chronic lymphocytic leukemia (CLL), a disease that depends on the over-expression of anti-apoptotic proteins for its survival, inhibition of CDK9 by fadraciclib reduced phosphorylation of the C-terminal domain of RNA polymerase II and blocked transcription in vitro; these actions depleted the intrinsically short-lived anti-apoptotic protein Mcl-1 and induced apoptosis. While the simulated bone marrow and lymph node microenvironments induced Mcl-1 expression and protected CLL cells from apoptosis, these conditions did not prolong the turnover rate of Mcl-1, and fadraciclib efficiently abrogated the protective effect. Further, fadraciclib was synergistic with the Bcl-2 antagonist venetoclax, inducing more profound CLL cell death, especially in samples with 17p deletion. While fadraciclib, venetoclax, and the combination each had distinct kinetics of cell death induction, their activities were reversible, as no additional cell death was induced upon removal of the drugs. The best combination effects were achieved when both drugs were maintained together. Altogether, this study provides a rationale for the clinical development of fadraciclib in CLL, either alone or in combination with a Bcl-2 antagonist.

Background Mobile health-based individualized interventions have shown potential effects in managing cardiovascular risk factors. This study aims to assess whether or not mHealth based individualized interventions delivered by an Individualized Cardiovascular Application system for Risk Elimination (iCARE) could reduce the incidence of major cardiovascular events in individuals with coronary heart disease. Methods This study is a large-scale, multi-center, parallel-group, open-label, randomized controlled clinical trial. This study will be conducted from September 2019 to December 2025. A total of 2820 patients with coronary heart disease will be recruited from two clinical sites and equally randomized into three groups: the intervention group and two control groups. All participants will be informed of six-time points (at 1, 3, 6, 12, 24, and 36 months after discharge) for follow-up visits. Over a course of 36 months, patients who are randomized to the intervention arm will receive individualized interventions delivered by a fully functional iCARE that using various visualization methods such as comics, videos, pictures, text to provide individualized interventions in addition to standard care. Patients randomized to control group 1 will receive interventions delivered by a modified iCARE that only presented in text in addition to routine care. Control group 2 will only receive routine care. The primary outcome is the incidence of major cardiovascular events within 3 years of discharge. Main secondary outcomes include changes in health behaviors, medication adherence, and cardiovascular health score. Discussion If the iCARE trial indeed demonstrates positive effects on patients with coronary heart disease, it will provide empirical evidence for supporting secondary preventive care in this population. Results will inform the design of future research focused on mHealth-based, theory-driven, intelligent, and individualized interventions for cardiovascular risk management. Trial registration Trial registered 24th December 2016 with the Chinese Clinical Trial Registry (ChiCTR-INR-16010242). URL: http://www.chictr.org.cn/showproj.aspx?proj=17398.

Chemotherapy occupies a prominent position in combination treatments of melanoma. However, the severe systemic side effects and the pro-tumorigenic microenvironment limited its therapeutic efficacy. In the present study, polysaccharide hydrogels (SCOD) were constructed by N-succinyl chitosan and oxidized dextran through Schiff-base formation to deliver doxorubicin (Dox) locally. The gelation time and mechanical properties of SCOD hydrogels could be fine-tuned by varying concentration of precursor solutions. Rheological data revealed that SCOD hydrogels possessed injectable shear-shinning property and remarkable self-healing capability. It also could be degraded by lysozyme widely present in body fluids. Moreover, SCOD hydrogels were readily loaded with Dox in precursor solutions and released drug over 1 week in a pH-dependent manner. The ability of Dox-loaded SCOD hydrogels to inhibit the growth of murine B16 and human A375 melanoma was verified by in vitro assays. Strikingly, Dox-loaded SCOD hydrogels were found to efficiently induce polarization of tumor-associated macrophages towards M1 phenotype that favors an anti-tumorigenic tumor microenvironment. Notably, in vivo experiments demonstrated that locally injected Dox-loaded SCOD hydrogels exhibited excellent anti-tumor activity against B16 melanoma, outperforming Dox at equivalent doses administrated intravenously. Therefore, the injectable and self-healing polysaccharide hydrogels are a promising strategy to improve locoregional control in melanoma.

Background Out-of-hospital cardiac arrest (OHCA), a global health problem with a survival rate ranging from 2 to 22% across different countries, has been a leading cause of premature death for decades. The aim of this study was to evaluate the trends of survival after OHCA over time and its relationship with bystander cardiopulmonary resuscitation (CPR), initial shockable rhythm, return of spontaneous circulation (ROSC), and survived event. Methods In this prospective observational study, data of OHCA patients were collected following the “Utstein style” by the Beijing, China, Emergency Medical Service (EMS) from January 2011 (data from February to June in 2011 was not collected) to October 2016. Patients who had a cardiac arrest and for whom an ambulance was dispatched were included in this study. All cases were followed up to determine hospital discharge or death. The trend of OHCA survival was analyzed using the Chi-square test. The relationship among bystander CPR, initial shockable rhythm, ROSC, survived event, and OHCA survival rate was analyzed using multivariate path analyses with maximum standard likelihood estimation. Results A total of 25,421 cases were transferred by the Beijing EMS; among them, 5042 (19.8%) were OHCA (median age: 78 years, interquartile range: 63–85, 60.1% male), and 484 (9.6%) received bystander CPR. The survival rate was 0.6%, which did not improve from 2012 to 2015 (P = 0.569). Overall, bystander CPR was indirectly associated with an 8.0% (β = 0.080, 95% confidence interval [CI] = 0.064–0.095, P = 0.002) increase in survival rate. The indirect effect of bystander CPR on survival rate through survived event was 6.6% (β = 0.066, 95% CI = 0.051–0.081, P = 0.002), which accounted for 82.5% (0.066 of 0.080) of the total indirect effect. With every 1 increase in survived event, the possibility of survival rate will directly increase by 53.5% (β = 0.535, 95% CI = 0.512–0.554, P = 0.003). Conclusions The survival rate after OHCA was low in Beijing which has not improved between 2012 and 2015. The effect of bystander CPR on survival rate was mainly mediated by survived event. Trial registration Chinese Clinical Trial Registry: ChiCTR-TRC-12002149 (2 May, 2012, retrospectively registered). http://www.chictr.org.cn/showproj.aspx?proj=7400

Objective To test the hypothesis that a prediction rule including levels of interleukin-6 in pericardial drainage (pdIL-6) would improve the discrimination in classifying patients undergoing coronary artery bypass grafting (CABG) into different postoperative atrial fibrillation (POAF) risk levels. Design Prospective cohort study. Setting A university-affiliated tertiary hospital. Participants Patients undergoing CABG. Interventions None. Measurements and Main Results We prospectively recruited patients who underwent CABG into derivation and validation cohorts. The independent predictors were identified in the derivation cohort using multiple logistic regression and tested in the validation cohort. The performance of the predictive model was tested using area under the receiver operating characteristic curve (AUC) in both cohorts. A prediction rule was created by assigning points to each predictor. Patients were classified in various risk levels according to their total risk scores. We enrolled 302 and 207 patients in the derivation and validation cohorts, respectively. Multiple logistic regression analysis identified six predictors: age ≥61 y, left atrial diameter ≥49 mm, right atrial diameter ≥45 mm, number of grafts ≥3, and serum uric acid ≥226 µmol/L and pdIL-6 levels ≥166 ng/mL at postoperative 12 h. The AUC of the model was 0.78 and 0.77 for the derivation and validation cohort, respectively, which was greatly increased by adding pdIL-6. Patients were stratified into low-risk, moderate-risk and high-risk groups. Conclusions A POAF prediction rule including pdIL-6 had good performance for stratifying CABG patients into various risk groups for POAF. The inclusion of pdIL-6 resulted in clinically meaningful improvement in risk prediction.

Background The mortality of coronary heart disease can be largely reduced by modifying unhealthy lifestyles. However, the long-term effectiveness of interventions for modifying unhealthy diet and physical inactivity of patients with coronary heart disease remain unsatisfactory worldwide. This study aims to systematically design a set of theory-based and evidence-based, individualized, and intelligent interventions for promoting the adoption and maintenance of a healthy diet and physical activity level in patients with coronary heart disease. Methods The interventions will be delivered by a mobile health care system called Individualized, Intelligent and Integrated Cardiovascular Application for Risk Elimination. Three steps of the intervention mapping framework were used to systematically develop the interventions. Step 1: needs assessment, which was carried out by a literature review, in-depth interviews and focus group discussions. Step 2: development of objective matrix for diet and physical activity changes, based on the intersection of objectives and determinants from the Contemplation-Action-Maintenance behavior change model. Step 3: formulation of evidence-based methods and strategies, and practical applications, through a systematic review of existing literature, research team discussions, and consultation with multidisciplinary expert panels. Results Three needs relevant to content of the intervention, one need relevant to presentation modes of the intervention, and four needs relevant to functional features of the application were identified. The objective matrix includes three performance objectives, and 24 proximal performance objectives. The evidence-based and theory-based interventions include 31 strategies, 61 evidence-based methods, and 393 practical applications. Conclusions This article describes the development of theory-based and evidence-based interventions of the mobile health care system for promoting the adoption and maintenance of a healthy diet and physical activity level in a structured format. The results will provide a theoretical and methodological basis to explore the application of intervention mapping in developing effective behavioral mobile health interventions for patients with coronary heart disease. Trial registration Chinese Clinical Trial Registry: ChiCTR-INR-16010242. Registered 24 December 2016. http://www.chictr.org.cn/index.aspx

Ovarian cancer (OC) is the most lethal gynecological carcinoma due to the lack of diagnostic markers and effective drug targets. Discovery of new therapeutic targets in OC to improve the treatment outcome is urgently needed. We performed proteomic analysis of OC specimens and the paired normal tissues and revealed that proteins associated with mitochondrial proteostasis and protein translation were highly expressed in ovarian tumor tissues, indicating that mitochondria are required for tumor progression of OC. Heat shock protein 60 (HSP60), an important mitochondrial chaperone, was upregulated in ovarian tumors. HSP60 silencing significantly attenuated growth of OC cells in both cells and mice xenografts. Proteomic analysis revealed that HSP60 silencing downregulated proteins involved in mitochondrial functions and protein synthesis. Metabolomic analysis revealed that HSP60 silencing resulted in a more than 100-fold increase in cellular adenine levels, leading to increased adenosine monophosphate and an activated AMPK pathway, and consequently reduced mTORC1-mediated S6K and 4EBP1 phosphorylation to inhibit protein synthesis that suppressed the proliferation of OC cells. These results suggest that HSP60 knockdown breaks mitochondrial proteostasis, and inactivates the mTOR pathway to inhibit OC progression, suggesting that HSP60 is a potential therapeutic target for OC treatment.

BRCA1-associated protein 1 (BAP1) is a member of the ubiquitin C-terminal hydrolase family of deubiquitinating enzymes and is implicated in transcriptional regulation. The BAP1 gene is mutated in about 10% of patients with ccRCC, the most common form of renal cancer, suggesting that BAP1 is a tumor suppressor. However, whether BAP1 influences the progression of ccRCC tumors expressing wild-type (WT) BAP1 is unclear. Here, we assessed the expression and function of BAP1 using human ccRCC specimens and cell lines. Analysis of datasets in The Cancer Genome Atlas revealed that lower BAP1 expression is correlated with longer overall survival of ccRCC patients. We established human ccRCC cell lines with stable BAP1 knockout and performed multiomic analysis of BAP1-mediated cellular processes. BAP1 knockout downregulated proteins associated with protein synthesis, resulting in decreased cell growth. Importantly, loss of BAP1 decreased the formation of stress fibers and membrane protrusions and induced migration and invasion defects. BAP1 knockout in ccRCC cells also downregulated the expression of transcriptional repressor protein Snail and decreased the activity of Rho family GTPases, promoting the cells to undergo mesenchymal-epithelial transition. Unexpectedly, quantitative proteomics also showed that BAP1 knockout increased expression of several amino acid transporters and multiple tyrosine kinases, including the epidermal growth factor receptor. Overall, our results suggest that BAP1 regulates multiple cellular processes, and we also uncover a new role for BAP1 in controlling mesenchymal-epithelial transition in ccRCC cells.

Objective To assess the diagnostic delay (DD) and physician-related DD (pDD) in patients with axial spondyloarthritis (SpA) and the potential benefits of a multidisciplinary clinic (MDC) approach. Methods A retrospective study was undertaken among patients with axial SpA, which aimed to analyse DD, pDD and their risk factors. The influence of pDD on disease outcomes was examined. The pDDs among consecutive SpA patients in an MDC cohort were compared with propensity score matched historical controls (1:1). Results A total of 208 patients with axial SpA formed the historical control group and 49 patients with axial SpA formed the MDC cohort after introduction of the MDC. The median DD and pDD in the historical controls were 25.5 and 10.0 months, respectively. A cut-off of pDD > 4 months was associated with more active disease and functional impairment. An initial visit to a non-rheumatologist was the most significant risk factor for pDD. Following MDC introduction, the median pDD decreased from 13 months to 1 month after adjustments were made for confounders such as sex, education level, history of smoking, human leukocyte antigen-B27 status and SpA/ankylosing spondylitis classification criteria. Conclusion The MDC was a promising approach that resulted in a reduced pDD among patients with axial SpA.

The viability of chronic lymphocytic leukemia (CLL) is critically dependent upon staving off death by apoptosis, a hallmark of CLL pathophysiology. The recognition that Mcl-1, a major component of the anti-apoptotic response, is intrinsically short-lived and must be continually resynthesized suggested a novel therapeutic approach. Pateamine A (PatA), a macrolide marine natural product, inhibits cap-dependent translation by binding to the initiation factor eIF4A. In this study, we demonstrated that a synthetic derivative of PatA, des-methyl des-amino PatA (DMDAPatA), blocked mRNA translation, reduced Mcl-1 protein and initiated apoptosis in CLL cells. This action was synergistic with the Bcl-2 antagonist ABT-199. However, avid binding to human plasma proteins limited DMDAPatA potency, precluding further development. To address this, we synthesized a new series of PatA analogs and identified three new leads with potent inhibition of translation. They exhibited less plasma protein binding and increased cytotoxic potency toward CLL cells than DMDAPatA, with greater selectivity towards CLL cells over normal lymphocytes. Computer modeling analysis correlated their structure-activity relationships and suggested that these compounds may act by stabilizing the closed conformation of eIF4A. Thus, these novel PatA analogs hold promise for application to cancers within the appropriate biological context, such as CLL.

BACKGROUND: As a novel class of endogenous ncRNAs, Circular RNAs (circRNAs) have been verified to be involved in the carcinogenesis and tumor progression. OBJECTIVE: This study aimed to investigate the potential function of a candidate circRNA hsa_circ_0036988 in oral squamous cell carcinoma (OSCC). METHODS: The altered expression of hsa_circ_0036988 was validated by quantitative real-time polymerase chain reaction (qRT-PCR) in OSCC samples and OSCC cell lines. The associations between the levels of hsa_circ_0036988 and the clinicopathological features were statistically analysed. The function of hsa_circ_0036988 in OSCC were evaluated via a series of in vitro experiments by using constructed plasmids or siRNA. Western blotting assays were conducted to evaluate changes in protein expression levels. RESULTS: Hsa_circ_0036988 was significantly downregulated in OSCC tissues compared with adjacent normal tissues. While low expression of hsa_circ_0036988 was highly correlated with lymph nodes metastasis. Overexpression or knockdown of hsa_circ_0036988 significantly affected the proliferation, migration and invasion of OSCC cells. Furthermore, the altered expression of hsa_circ_0036988 have an impact on the epithelial-to-mesenchymal transition (EMT)-related protein expression levels. CONCLUSIONS: Our findings indicated that hsa_circ_0036988 may affect cell proliferation, migration and invasion by regulating EMT progress, which might provide a therapeutic strategy for the treatment of OSCC.

The constitutive activation of B-cell receptor (BCR) signaling, together with the overexpression of the Bcl-2 family anti-apoptotic proteins, represents two hallmarks of chronic lymphocytic leukemia (CLL) that drive leukemia cell proliferation and sustain their survival. TG02 is a small molecule multi-kinase inhibitor that simultaneously targets both of these facets of CLL pathogenesis. First, its inhibition of cyclin-dependent kinase 9 blocked the activation of RNA polymerase II and transcription. This led to the depletion of Mcl-1 and rapid induction of apoptosis in the primary CLL cells. This mechanism of apoptosis was independent of CLL prognostic factors or prior treatment history, but dependent on the expression of BAX and BAK. Second, TG02, which inhibits the members of the BCR signaling pathway such as Lck and Fyn, blocked BCR-crosslinking-induced activation of NF-κB and Akt, indicating abrogation of BCR signaling. Finally, the combination of TG02 and ibrutinib demonstrated moderate synergy, suggesting a future combination of TG02 with ibrutinib, or use in patients that are refractory to the BCR antagonists. Thus, the dual inhibitory activity on both the CLL survival pathway and BCR signaling identifies TG02 as a unique compound for clinical development in CLL and possibly other B cell malignancies.

In Location-Based Social Networks (LBSNs), registered users submit their reviews for visited point-of-interests (POIs) to the system providers (SPs). The SPs anonymously publish submitted reviews to build reputations for POIs. Unfortunately, the user profile and trajectory contained in reviews can be easily obtained by adversaries who SPs has compromised with. Even worse, existing techniques, such as cryptography and generalization, etc., are infeasible due to the necessity of public publication of reviews and the facticity of reviews. Inspired by pseudonym techniques, we propose an approach to exchanging reviews before users submit reviews to SPs. In our approach, we introduce two attacks, namely review-based location correlation attack (RLCA) and semantic-based long-term statistical attack (SLSA). RLCA can be exploited to link the real user by reconstructing the trajectory, and SLSA can be launched to establish a connection between locations and users through the difference of semantic frequency. To resist RLCA, we design a method named User Selection to Resist RLCA (USR-RLCA) to exchange reviews. We propose a metric to measure the correlation between a user and a trajectory. Based on the metric, USR-RLCA can select reviews resisting RLCA to exchange by suppressing the number of locations on each reconstructed trajectory below the correlation. However, USR-RLCA fails to resist SLSA because of ignoring the essential semantics. Hence, we design an enhanced USR-RLCA named User Selection to Resist SLSA (USR-SLSA). We first propose a metric to measure the indistinguishability of locations concerning the difference of semantic frequency in a long term. Then, USR-SLSA can select reviews resisting SLSA to exchange by allowing two reviews whose indistinguishability is below the probability difference after the exchange to be exchanged. Evaluation results verify the effectiveness of our approach in terms of privacy and utility.

A post-synthetic integration of polypyrrole onto NU-1000 MOF (PPy@NU-1000) was done by pyrrole adsorption, followed by oxidative polymerization. The synthesized materials were characterized by XRD, SEM, BET, and FTIR. The ultra-high specific surface area with high-density catalytic sites of NU-1000 (2223 m2 g−1) was combined with the electrical conductivity of PPy (2–100 S cm−1). PPy@NU-1000 provides superior electrocatalytic activity and charge transfer properties compared to an individual component. The PPy@NU-1000-modified GCE was applied to detect the biomolecule Levodopa (LD). The DPV oxidation peak of LD was strongest at 272 ± 10 mV vs. Ag/AgCl reference electrode. Under the optimized experimental condition, the fabricated electrochemical sensor exhibited a wide quantification range of 0.005–70 μM with a sub-nanomolar detection limit of 0.0001 μM (S/N 3). The described sensor exhibits high sensitivity (2.08 μA μM−1 cm−2) with reasonable stability, reproducibility, and selectivity for the detection LD in the presence of potentially interfering compounds. Furthermore, human serum analysis showed excellent recovery values within the range 99.3–101.6%. Validation of the method was performed against HPLC. Graphical abstract

Glutathionylation is an important posttranslational modification that protects proteins from further oxidative damage as well as influencing protein structure and activity. In the present study, we demonstrate that the cysteine-42 residue in protein arginine N-methyltransferase 5 (PRMT5) is glutathionylated in aged mice or in cells that have been exposed to oxidative stress. Deglutathionylation of this protein is catalyzed by glutaredoxin-1 (Grx1). Using mutagenesis and subsequent biochemical analyses, we show that glutathionylation decreased the binding affinity of PRMT5 with methylosome protein-50 (MEP50) and reduced the methyltransferase activity of PRMT5. Furthermore, overexpression of PRMT5-C42A mutant caused a significant increase in histone methylation in HEK293T and A549 cells and promoted cell growth, whereas overexpression of the PRMT5-C42D mutant, a mimic of glutathionylated PRMT5, inhibited cell proliferation. Taken together, our results demonstrate a new mechanism of regulation of PRMT5 methyltransferases activity and suggest that PRMT5 glutathionylation is partly responsible for reactive oxygen species-mediated cell growth inhibition.

The aim of the current study is to explore the mechanism by which deviant peer affiliation affects substance abusers' substance abstention motivation and the mediating role of perceived social support in the relationship between these concepts. Moreover, we also investigated whether collective identity moderates the relations among deviant peer affiliation, perceived social support, and substance rehabilitation. The participants were 430 male substance abstainers who completed a battery of questionnaires. The Chinese versions of the Deviant Peer Affiliation Questionnaire, Motivation for Abstention Scale, Perceived Social Support Scale and Identity Orientation Scale were used. The results showed that all the dimensions of deviant peer affiliation were negatively associated with the dimensions of substance abstention motivation. Moreover, perceived social support partially mediated the relations between deviant peer affiliation and substance abstention motivation. Additionally, collective identity was a significant moderator of the relations between perceived social support and substance abstention motivation. These findings provide a clearer understanding regarding the impact of deviant peer affiliation and perceived social support on substance abstention motivation in individuals with substance use disorder.

B cells undergo two types of genomic alterations to increase antibody diversity: introduction of point mutations into immunoglobulin heavy- and light-chain (IgH and IgL) variable regions by somatic hypermutation (SHM) and alteration of antibody effector functions by changing the expressed IgH constant region exons through IgH class switch recombination (CSR). SHM and CSR require the B cell–specific activation-induced cytidine deaminase (AID) protein, the transcription of germline noncoding RNAs, and the activity of the 3′ regulatory region (3′RR) super-enhancer. Although many transcription regulatory elements (e.g., promoters and enhancers) reside inside the IgH and IgL sequences, the question remains whether clusters of regulatory elements outside IgH control CSR. Using RNA exosome-deficient mouse B cells where long noncoding RNAs (lncRNAs) are easily detected, we identified a cluster of three RNA-expressing elements that includes lncCSR (IgA) (that expresses lncRNA-CSR(IgA)). B cells isolated from a mouse model lacking lncRNA-CSR(IgA) transcription fail to undergo normal levels of CSR to IgA both in B cells of the Peyer’s patches and grown in ex vivo culture conditions. lncRNA-CSR(IgA) is expressed from an enhancer site (lncCSR(IgA)) to facilitate the recruitment of regulatory proteins to a nearby CTCF site (CTCF(lncCSR)) that alters the chromosomal interactions inside the TAD(lncCSRIgA) and long-range interactions with the 3′RR super-enhancer. Humans with IgA deficiency show polymorphisms in the lncCSR(IgA) locus compared with the normal population. Thus, we provide evidence for an evolutionarily conserved topologically associated domain (TAD(lncCSRIgA)) that coordinates IgA CSR in Peyer’s patch B cells through an lncRNA (lncRNA-CSR(IgA)) transcription-dependent mechanism.

Summary: Mitogen-activated protein kinases (MAPKs) are inactivated by dual-specificity phosphatases (DUSPs), the activities of which are tightly regulated during cell differentiation. Using knockdown screening and single-cell transcriptional analysis, we demonstrate that DUSP4 is the phosphatase that specifically inactivates p38 kinase to promote megakaryocyte (Mk) differentiation. Mechanistically, PRMT1-mediated methylation of DUSP4 triggers its ubiquitinylation by an E3 ligase HUWE1. Interestingly, the mechanistic axis of the DUSP4 degradation and p38 activation is also associated with a transcriptional signature of immune activation in Mk cells. In the context of thrombocytopenia observed in myelodysplastic syndrome (MDS), we demonstrate that high levels of p38 MAPK and PRMT1 are associated with low platelet counts and adverse prognosis, while pharmacological inhibition of p38 MAPK or PRMT1 stimulates megakaryopoiesis. These findings provide mechanistic insights into the role of the PRMT1-DUSP4-p38 axis on Mk differentiation and present a strategy for treatment of thrombocytopenia associated with MDS.

The mechanism of action of CNDAC (2′-C-cyano-2′-deoxy-1-β-d-arabino-pentofuranosyl-cytosine) is unique among deoxycytidine analogs because upon incorporation into DNA it causes a single strand break which is converted to a double strand break after DNA replication. This lesion requires homologous recombination (HR) for repair. CNDAC, as the parent nucleoside, DFP10917, and as an oral prodrug, sapacitabine, are undergoing clinical trials for hematological malignancies and solid tumors. The purpose of this study is to investigate the potential of CNDAC for the therapy of ovarian cancer (OC). Drug sensitivity was evaluated using a clonogenic survival assay. Drug combination effects were quantified by median effect analysis. OC cells lacking function of the key HR genes, BRCA1 or BRCA2, were more sensitive to CNDAC than corresponding HR proficient cells. The sensitization was associated with greater levels of DNA damage in response to CNDAC at clinically achievable concentrations, manifested as chromosomal aberrations. Three classes of CNDAC-based drug combinations were investigated. First, the PARP1 inhibitors, rucaparib and talazoparib, were selectively synergistic with CNDAC in BRCA1/2 deficient OC cells (combination index

To investigate the cellular mechanisms of multiple myeloma (MM), we used liquid chromatography-tandem mass spectrometry for proteomics analysis of CD138+ plasma cells from patients with MM and healthy controls. We found that the 60-kDa heat shock protein (HSP60, also known as HSPD1) was significantly upregulated in myeloma cells. HSP60 is an important chaperone protein that regulates the homeostasis of mitochondrial proteins and maintains mitochondrial function. Knockdown (KD) of HSP60 in myeloma cells resulted in inhibition of proliferation and reduced the quality of the mitochondria. Mitochondrial stress tests showed that HSP60 KD inhibited glycolysis and mitochondrial activity. Metabolomics showed a decrease in glycolysis and tricarboxylic acid cycle metabolites, and inhibited the formation of creatine and phosphocreatine by the reaction of S-adenosylmethionine (SAM) with amino acids mediated by demethyladenosine transferase 1, mitochondrial (TFB1M) and reduced energy storage substances. Moreover, HSP60 silencing influenced the synthesis of ribonucleotides and nicotinamide adenine dinucleotide phosphate (NADPH) by the pentose phosphate pathway to inhibit cell proliferation. HSP60 KD inhibited 5' adenosine monophosphate-activated protein kinase (AMPK), which inhibited the key enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), effecting the metabolism of fatty acids by inhibiting malonyl-coenzyme A. Our data suggest that reduced HSP60 expression alters metabolic reprogramming in MM, inhibits tumour progression and reduces mitochondrial-dependent biosynthesis, suggesting that HSP60 is a potential therapeutic target for MM treatment.

BACKGROUND Serum level of interleukin 6 (IL-6) is known to be associated with postoperative delirium. However, no consensus has emerged on the postoperative time point at which IL-6 level may predict postoperative delirium after coronary artery bypass graft surgery. OBJECTIVES To compare trends in IL-6 levels in patients with and without postoperative delirium and to examine the relationship between IL-6 levels at different times and postoperative delirium after coronary artery bypass graft. METHODS A prospective cohort study of patients who underwent their first elective isolated coronary artery bypass graft between November 2013 and August 2015 at a cardiac intensive care unit in Beijing, China. Concentrations of IL-6 were measured before the operation and at the 6th, 12th, and 18th postoperative hours. Participants were assessed for postoperative delirium twice daily for 5 days. Univariate and multivariate logistic regression analyses were done to determine associations between IL-6 levels at different time points, postoperative changes in IL-6 levels, and the occurrence of postoperative delirium. RESULTS Postoperative delirium was diagnosed in 85 of 266 patients (32%). Levels of IL-6 were significantly higher in patients with postoperative delirium than in patients without it at the 6th, 12th, and 18th postoperative hours (P = .03, .004, and .001, respectively). Change in IL-6 level (odds ratio, 2.97; 95% CI, 1.20-7.31; P = .02) and IL-6 level of 583 pg/mL or higher at the 18th postoperative hour (odds ratio, 5.20; 95% CI, 1.84-14.70; P = .002) were associated with higher incidence of postoperative delirium. CONCLUSION Interleukin 6 level (≥ 583 pg/mL) at the 18th postoperative hour may serve as a potent predictor of postoperative delirium in coronary artery bypass graft patients.

Objectives The aim of this study was to assess the occurrence of new-onset diabetes mellitus (DM) after chronic pancreatitis (CP) diagnosis via systematic review and meta-analysis. Methods A systematic review of literature and meta-analysis of relevant reports were performed. The primary outcome measures studied were newly diagnosed DM and DM treated with insulin. For the binary outcomes, pooled prevalence and 95% confidence interval (CI) were calculated. Methods Fifteen studies involving 8970 patients were eligible. The incidence of new-onset DM after CP diagnosis was 30% (95% CI, 27%-33%). Among all patients, 17% (95% CI, 13%-22%) developed insulin-dependent new-onset DM. The prevalence of newly diagnosed DM after CP diagnosis increased from 15% within 36 months to 33% after 60 months. The proportion of alcoholic CP, sex, age, and body mass index had minimal effect on the studied outcomes. Conclusions This systematic review identified a clinically relevant risk of new-onset DM after CP diagnosis. Therefore, patients should be informed of the risk of DM and monitored.

Porphyrinic Metal-Organic Frameworks (porph-MOFs) are attracting attention due to the redox activity in the porphyrin subunit. Herein, we report the design of a novel core-shell structure hybrid material with a sea-cucumber morphology, namely PMeTh, containing the poly(3-methythiophene) conducting polymer coated on the surface of iron-based porph-MOFs PCN-222(Fe) via in-situ oxidative chemical polymerization. The porous PCN-222(Fe) serves as the electrocatalytic sites, while the poly(3-methythiophene) conducting polymer functions as the charge collector to facilitate the charge transport to the redox active sites. The resulting PMeTh composite demonstrates an excellent electrochemical response towards the levodopa detection. The sensitivity towards the L-dopa detection is estimated to be 1.868 μA ⋅ μM −1 ⋅ cm −2 in the linear concentration of 0.05 - 7.0 μmol ⋅ L −1 and 0.778 μA ⋅ μM −1 ⋅ cm −2 in the linear concentration of 7.00 - 100 μmol ⋅ L −1 , respectively. Additionally, the levodopa sensor exhibits a low detection limit of 2 nmol L −1 as well as excellent stability after 120 cycles in 10 μmol L −1 levodopa. The feasibility of this novel L-dopa sensor was evaluated in human urine samples by standard addition. The satisfactory recoveries were in the range of 97.0 - 104.5% with the R.S.D. value lower than 4.4%. The method of intergrating porph-MOFs and conducting polymers can efficiently expand the porph-MOFs based composites in bioanalysis.

Context Diagnosis of non-chromosomal type disorders of sex development (DSD) has long been challenging. There is still no research on overview of a large Chinese DSD cohort. Objective To determine the etiologic diagnosis through unbiased large-scale panel sequencing and whole-exome sequencing (WES) within a large Chinese DSD cohort. Design Patients were recruited according to the inclusion criteria of DSD. The applied panel contains 2742 known disease-causing genes, including all known diagnostic genes for DSD. Methods Targeted panel sequencing (TPS) was performed, and identified candidate variants were verified. Variant pathogenicities were evaluated according to established guidelines. WES was performed for randomly selected negative samples. Results This study included 125 patients. Seventy-five variants were identified by TPS and 31 variants were reported for the first time. Pathogenic and likely pathogenic variants accounted for 38.7 and 30.7%, respectively. On the basis of clinical certainty, etiologic diagnostic rates of 46.9 and 10.3% were obtained for 46,XY and 46,XX DSD patients, respectively. We reported novel candidate genes (BMPR1B, GNAS, GHR) and regions of copy number variants outside the expected DSD genotype–phenotype correlation and determined a founder mutation (SRD5A2 p.R227Q) in patients with 5α-reductase deficiency. Further WES in randomly selected negative samples identified only one among 14 negative samples as a variant of uncertain significance, indicating that WES did not improve the diagnostic rate. Conclusions This is the first report of the applying unbiased TPS in a large Chinese cohort of patients with 46,XY and 46,XX DSD. Our findings expand the gene, mutation and phenotype spectra of the rare types of DSD in the Chinese population and provide new insight into the current understanding of the etiologies of DSD.

Adaptive immune responses that occur in infection, cancer, and autoimmune as well as allergic diseases involve the participation of T cells. T cells travel throughout the body searching for antigens, which are recognized via the major histocompatibility complexes. In the healthy organism, these T cells maintain metabolic quiescence until they encounter a potentially cognate antigen. Once activated, e.g., during an infection or tissue damage, T cells switch their metabolic program to gain energy and building blocks to maintain cellular homeostasis and to fulfill their specific immune functions involving clonal expansion and/or differentiation into effector and memory T cells to ultimately ensure host survival. Thus, differences in metabolism in healthy and pathogenic T cells provide an explanation for dysfunctionality of T-cell responses in metabolic disorders, autoimmunity, and cancer. Here, we summarize current knowledge on T-cell metabolism during the maintenance of homeostasis, activation, and differentiation as well as over the course of time that memory is generated in health and in diseased states such as autoimmunity and cancer.

Aims To investigate the antitumor effect of 7-O-geranylquercetin (GQ) combining with survivin siRNA (siSuvi) or IL-10 siRNA (siIL-10) to breast cancer. Main methods Xenograft tumor model was established by subcutaneously inoculating human breast cancer MCF-7 cells in BALB/c nude mice. Transfection efficiency of siRNA mediated by cationic liposome CDO14 in MCF-7 cells and tumor bearing mice was measured by flow cytometer and living imaging sysytem, respectively. Cell viability was detected using CCK-8 assay. Cell apoptosis was determined by Hoechst33342 staining and AV-PI staining. Tumors bearing mice were administered with GQ by gavage, and/or with liposome CDO14 mediated siRNAs via tail intravenous injection. Expression levels of proteins and cytokines were detected by western blot and ELISA, respectively. Key findings Liposome CDO14 could deliver siRNA to tumor effectively. Combination of GQ and siSuvi promoted the antiproliferation and pro-apoptosis effects of GQ or siSuvi to MCF-7 cells, and reduced the level of survivin and raised the level of caspase-7 in cells. GQ combining with siSuvi inhibited the growth of tumor, down-regulated the expression of survivin and up-regulated the expression of caspase-7 in tumor tissue. Similarly, GQ combining with siIL-10 inhibited the growth of tumor, decreased the level of IL-10 and increased the level of TNF-α. These results revealed that GQ enhanced the pro-apoptosis effect of siSuvi on tumor cells and the modulating effect of siIL-10 on tumor microenvironment. Significances Synergistic anti-tumor effect of GQ and siRNAs against breast cancer proved that chemical drugs combining with siRNAs is a promising antitumor strategy.

Plague is a zoonotic disease that primarily infects rodents via fleabite. Transmission from flea to host niches requires rapid adaption of Yersinia pestis to the outer environments to establish infection. Here, quantitative proteome and secretome analyses of Y. pestis grown under conditions mimicking the two typical niches, i.e., the mammalian host (Mh) and the flea vector (Fv), were performed to understand the adaption strategies of this deadly pathogen. A secretome of Y. pestis containing 308 proteins has been identified using TMT-labeling mass spectrometry analysis. Although some proteins are known to be secreted, such as the type III secretion substrates, PsaA and F1 antigen, most of them were found to be secretory proteins for the first time. Comparative proteomic analysis showed that membrane proteins, chaperonins and stress response proteins are significantly upregulated under the Mh condition, among which the previously uncharacterized proteins YP_3416∼YP_3418 are remarkable because they cannot only be secreted but also translocated into HeLa cells by Y. pestis. We further demonstrated that the purified YP_3416 and YP_3418 exhibited E3 ubiquitin ligase activity in in vitro ubiquitination assay and yp_3416∼3418 deletion mutant of Y. pestis showed significant virulence attenuation in mice. Taken together, our results represent the first Y. pestis secretome, which will promote the better understanding of Y. pestis pathogenesis, as well as the development of new strategies for treatment and prevention of plague., Graphical Abstract, Highlights • Quantitative proteome analysis of Y. pestis was performed to understand its adaption strategies. • A secretome of Y. pestis containing 308 proteins has been identified. • YP_3416 and YP_3418 exhibited strong E3 ubiquitin ligase activity. • yp_3416∼3418 deletion mutant of Y. pestis showed significant virulence attenuation in mice., In Brief Rapid adaption to the environments is critical for microbes to establish infection. Quantitative proteome and secretome analyses of Y. pestis grown under conditions mimicking its two typical natural niches were performed to understand the adaption strategies of this deadly pathogen. We identified three secreted proteins that can be translocated into host cells and further demonstrated that two of them show strong E3 ubiquitin ligase activity and contribute significantly to the virulence of Y. pestis.

// Hong Qu 1, * , Yuling Chen 2, 3, * , Guangming Cao 1 , Chongdong Liu 1 , Jiatong Xu 3 , Haiteng Deng 3 , Zhenyu Zhang 1 1 Department of Obstetrics & Gynecology, Beijing Chao-yang Hospital Affiliated to Capital Medical University, Beijing, China 2 Tsinghua University-Peking University Joint Center for Life Sciences, Beijing, China 3 MOE Key Laboratory of Bioinformatics, School of Life Sciences, Tsinghua University, Beijing, China * These authors have contributed equally to this work Correspondence to: Zhenyu Zhang, email: zhenyuzhang2000@163.com Haiteng Deng, email: dht@mail.tsinghua.edu.cn Keywords: proteomics, epithelial ovarian cancer, CLIC1, LGALS3BP, therapeutic target Received: May 02, 2016 Accepted: October 19, 2016 Published: November 4, 2016 ABSTRACT Ovarian cancer is the most lethal gynecological malignant tumor because of its high recurrence rate. In the present work, in order to find new therapeutic targets, we identified 8480 proteins in thirteen pairs of ovarian cancer tissues and normal ovary tissues through quantitative proteomics. 498 proteins were found to be differentially expressed in ovarian cancer, which involved in various cellular processes, including metabolism, response to stimulus and biosynthetic process. The expression levels of chloride intracellular channel protein 1 (CLIC1) and lectin galactoside-binding soluble 3 binding protein (LGALS3BP) in epithelial ovarian cancer tissues were significantly higher than those in normal ovary tissues as confirmed by western blotting and immunohistochemistry. The knockdown of CLIC1 in A2780 cell line downregulated expression of CTPS1, leading to the decrease of CTP and an arrest of cell cycle G1 phase, which results into a slower proliferation. CLIC1-knockdown can also slow down the tumor growth in vivo. Besides, CLIC1-knockdown cells showed an increased sensitivity to hydrogen peroxide and cisplatin, suggesting that CLIC1 was involved in regulation of redox and drug resistance in ovarian cancer cells. These results indicate CLIC1 promotes tumorgenesis, and is a potential therapeutic target in epithelial ovarian cancer treatment.

Cisplatin and other platinum-based drugs have been widely used in the treatment of ovarian cancer, but most patients acquire the drug resistance that greatly compromises the efficacy of drugs. Understanding the mechanism of drug resistance is important for finding new therapeutic approaches. In the present study, we found that the expression of vimentin was downregulated in drug-resistant ovarian cancer cell lines A2780-DR and HO-8910 as compared to their respective control cells. Overexpression of vimentin in A2780-DR cells markedly increased their sensitivity to cisplatin, whereas knockdown of vimentin in A2780, HO-8910-PM and HO-8910 cells increased the resistance to cisplatin, demonstrating that vimentin silencing enhanced cisplatin resistance in ovarian cancer cells. Quantitative proteomic analysis identified 95 differentially expressed proteins between the vimentin silenced A2780 cells (A2780-VIM-KN) and the control cells, in which downregulation of endocytic proteins and the upregulation of exocytotic proteins CHMP2B and PDZK1 were proposed to contribute the decreased cisplatin accumulation in vimentin knockdown cells. Silencing of vimentin induced upregulation of cancer stem cell markers and both A2780-DR and A2780-VIM-KN cells were more facile to form spheroids than control cells under serum-free culture condition. Our results also revealed that vimentin knockdown increased the 14-3-3 mediated retention of Cdc25C in the cytoplasm, leading to inactivation of Cdk1 and the prolonged G2 phase arrest that allowed the longer period of time for cells to repair cisplatin-damaged DNA. Taken together, we demonstrated that vimentin silencing enhanced cells' resistance to cisplatin via prolonged G2 arrest and increased exocytosis, suggesting that vimentin is a potential target for treatment of drug resistant ovarian cancer.

(-)-Agelastatin A (AglA, 1), a member of the pyrrole-aminoimidazole marine alkaloid (PAI) family, possesses a unique tetracyclic structure and is one of the most potent anticancer PAIs isolated to date. In efforts to expand the SAR of these agents and delineate sites that tolerate modification while retaining activity, we synthesized several derivatives and tested their anticancer activity. The cytotoxic effects of these derivatives were measured against several cancer cell lines including cervical cancer (HeLa), epidermoid carcinoma (A431), ovarian (Igrov and Ovcar3), osteosarcoma (SJSA1), acute T cell leukemia (A3), epidermoid carcinoma (A431) in addition to primary human chronic lymphocytic leukemia (CLL) cells. New positions for modification of AglA and new substitutions were explored leading to novel derivatives, 14-chloro AglA (3) and 14-methyl AglA (12), that retained activity toward various cancer cell lines with decreased toxicity toward B- and T-cells. The SAR data informed the synthesis of a trifunctional probe bearing an alkyne and a diazirine potentially useful for cellular target identification.

Aberrant mitophagy has been implicated in a broad spectrum of disorders. PINK1, Parkin, and ubiquitin have pivotal roles in priming mitophagy. However, the entire regulatory landscape and the precise control mechanisms of mitophagy remain to be elucidated. Here, we uncover fundamental mitophagy regulation involving PINK1 and a non-canonical role of the mitochondrial Tu translation elongation factor (TUFm). The mitochondrion-cytosol dual-localized TUFm interacts with PINK1 biochemically and genetically, which is an evolutionarily conserved Parkin-independent route toward mitophagy. A PINK1-dependent TUFm phosphoswitch at Ser222 determines conversion from activating to suppressing mitophagy. PINK1 modulates differential translocation of TUFm because p-S222-TUFm is restricted predominantly to the cytosol, where it inhibits mitophagy by impeding Atg5-Atg12 formation. The self-antagonizing feature of PINK1/TUFm is critical for the robustness of mitophagy regulation, achieved by the unique kinetic parameters of p-S222-TUFm, p-S65-ubiquitin, and their common kinase PINK1. Our findings provide new mechanistic insights into mitophagy and mitophagy-associated disorders.

Craniofacial autologous bone grafts offer superior outcomes to long bone grafts in the reconstruction of maxillofacial bone defects, but the mechanism responsible for this superiority has not yet been illustrated clearly. Osteoblasts play vital roles in bone development and regeneration. However, presently, only a few studies have compared the osteogenic ability of osteoblasts from craniofacial and long bones, and the results are contradictory. Additionally, the angiogenic characteristics of osteoblasts from these different bones remain unknown. We obtained osteoblasts from the rat mandible (MOBs) and femur (FOBs) to investigate their proliferative capacity and osteogenic potential, and using a co-culture system with human umbilical vein endothelial cells (HUVECs), we explored their angiogenic capabilities in vitro. FOBs exhibited higher alkaline phosphatase activity and increased matrix mineralization and expressed more osteogenic related marker genes, while MOBs proliferated at the highest rate and showed elevated expression of angiogenesis-related factors. Conditioned media from MOBs enhanced the expression of angiogenesis-related factors in HUVECs. Furthermore, the conditioned media generated from MOBs showed stronger promotion of proliferation, migration, and tube-like structure formation in HUVECs, suggesting that MOBs had a stronger pro-angiogenic effect on HUVECs than FOBs. Taken together, these results indicate that osteoblasts possess skeletal site-specific differences in osteogenic and angiogenic capabilities, and this might lead to a better understanding of the molecular impact of bone cells from different bone entities on maxillofacial bone reconstructions.

SIRT3 is a NAD+-dependent mitochondrial protein deacetylase implicated in the regulation of central metabolism and mitochondrial proteostasis. SIRT3 is downregulated in clear cell renal cell carcinoma (ccRCC), which is the most common form of renal cancer. Although ccRCC is characterized by a typical Warburg-like phenotype, mitochondrial dysfunction and elevated fat deposition, it is unknown whether SIRT3 plays a role in tumorigenesis and the development of this disease. In the present study, we found that SIRT3 overexpression and knockdown had opposing effects on the growth of ccRCC cells, decreasing and increasing the rate of cell proliferation, respectively. SIRT3 overexpression also increased mitochondrial mass in ccRCC cells. Unexpectedly, SIRT3 overexpression increased ROS levels, and sensitized cells to oxidative stress. Metabolomics and quantitative proteomics showed that SIRT3 overexpression alterd cellular metabolism and reversed the Warburg effect in ccRCC cells. Further studies demonstrated that SIRT3 promoted mitochondrial biogenesis by increasing both the expression and deacetylation of TFAM (transcription factor A, mitochondrial). Mutagenesis experiments revealed that acetylation of TFAM at K154 impaired TFAM interaction with mitochondrial DNA, thereby decreasing the activity of the protein and, consequently, mitochondrial biogenesis. Overall, our results suggest that SIRT3 regulates mitochondrial biogenesis and that its downregulation promotes a Warburg phenotype in ccRCC.

SummaryProtein glutathionylation is an important post-translational modification that regulates many cellular processes, including energy metabolism, signal transduction, and protein homeostasis. Global profiling of glutathionylated proteins (denoted as glutathionylome) is crucial for understanding redox-regulated signal transduction. Here, we developed a novel method based on click reaction and proteomics to enrich and identify the glutathionylated peptides in Escherichia coli and Drosophila lysates, in which 937 and 1,930 potential glutathionylated peptides were identified, respectively. Bioinformatics analysis showed that the cysteine residue next to negatively charged amino acid residues has a higher frequency of glutathionylation. Importantly, we found that most proteins associated with metabolic pathways were glutathionylated and that the glutathionylation sites of metabolic enzymes were highly conserved among different species. Our results indicate that the glutathione analog is a useful tool to characterize protein glutathionylation, and glutathionylation of metabolic enzymes, which play important roles in regulating cellular metabolism, is conserved.

Background: The molecular mechanisms underlying the endometriosis are still not completely understood. In order to test the hypothesis that the approaches in phosphoproteomics might contribute to the identification of key biomarkers to assess disease pathogenesis and drug targets, we carried out a phosphoproteomics analysis of human endometrium. Methods: A large-scale differential phosphoproteome analysis, using peptide enrichment of titanium dioxide purify and sequential elution from immobilized metal affinity chromatography with linear trap quadrupole-tandem mass spectrometry, was performed in endometrium tissues from 8 women with or without endometriosis. Results: The phosphorylation profiling of endometrium from endometriosis patients had been obtained, and found that identified 516 proteins were modified at phosphorylation level during endometriosis. Gene ontology annotation analysis showed that these proteins were enriched in cellular processes of binding and catalytic activity. Further pathway analysis showed that ribosome pathway and focal adhesion pathway were the top two pathways, which might be deregulated during the development of endometriosis. Conclusions: That large-scale phosphoproteome quantification has been successfully identified in endometrium tissues of women with or without endometriosis will provide new insights to understand the molecular mechanisms of the development of endometriosis.

Motor dysfunction is a major consequence after stroke and it is generally believed that the loss of motor ability is caused by the impairments in neural network that controls movement. To explore the abnormal mechanisms how the brain controls shoulder abduction and elbow flexion in “flexion synergy” following stroke, we used the functional corticomuscular coupling (FCMC) between the brain and the muscles as a tool to identify the temporal evolution of corticomuscular interaction between the synkinetic and separate phases. 59-channel electroencephalogram (EEG) over brain scalp and 2-channel electromyogram (EMG) from biceps brachii (BB)/deltoid (DT) were recorded in sixteen stroke patients with motor dysfunction and eight healthy controls during a task of uplifting the arm (stage 1) and maintaining up to the chest (stage 2). As a result, compared to healthy controls, stroke patients had abnormally reduced coherence in EEG-BB combination and increased coherence in EEG-DT combination. Compared to synkinetic stroke patients, separate ones exhibited higher coupling at gamma-band during stage 1 and higher at beta-band during stage 2 in EEG-BB combination, but lower at beta-band during stage 2 in EEG-DT combination. Therefore, we infer that the disorders of efferent control and afferent proprioception in sensorimotor system for stroke patients effect on the oscillation at beta and gamma bands. Patients need integrate more information for shoulder abduction to compensate for the functional loss of elbow flexion in the recovery process, so that partial cortical cortex controlling on the elbow flexion may work on the shoulder abduction during “flexion synergy”. Such researches could provide new perspective on the temporal evolution of corticomuscular interaction after stroke and add to our understanding of possible pathomechanisms how the brain abnormally controls shoulder abduction and elbow flexion in “flexion synergy”., Highlights • We explored the mechanisms how the brain controlled the shoulder abduction when the elbow flexed for stroke by FCMC analysis. • Abnormal increased coupling occurred in EEG-DT combination and reduced coupling was in EEG-BB combination for stroke. • Separate stroke patients exhibited higher coupling compared to synkinetic ones.

By capturing and sequencing the RNA fragments protected by translating ribosomes, ribosome profiling provides snapshots of translation at subcodon resolution. The growing needs for comprehensive annotation and characterization of the context-dependent translatomes are calling for an efficient and unbiased method to accurately recover the signal of active translation from the ribosome profiling data. Here we present our new method, RiboCode, for such purpose. Being tested with simulated and real ribosome profiling data, and validated with cell type-specific QTI-seq and mass spectrometry data, RiboCode exhibits superior efficiency, sensitivity, and accuracy for de novo annotation of the translatome, which covers various types of ORFs in the previously annotated coding and non-coding regions. As an example, RiboCode was applied to assemble the context-specific translatomes of yeast under normal and stress conditions. Comparisons among these translatomes revealed stress-activated novel upstream and downstream ORFs, some of which are associated with translational dysregulations of the annotated main ORFs under the stress conditions.

Summary 8-chloro-adenosine (8-Cl-Ado) is currently in phase-I clinical trials for acute myeloid leukaemia and chronic lymphocytic leukaemia (CLL). Previously, we demonstrated that treatment with 8-Cl-Ado leads to diminished ATP levels. We hypothesized that AMP-activated protein kinase (AMPK) signalling would be initiated in these cells, leading to induction of autophagy. AMPK activation and induction of autophagy were demonstrated during preclinical incubations in CLL cells with the analogues. Importantly, we extended similar observations in CLL lymphocytes during an 8-Cl-Ado phase-I trial. In conclusion, 8-Cl-Ado treatment induces autophagy in CLL lymphocytes in vitro as well as in vivo during clinical trial.