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Shexiang Baoxin Pill (SBP) and Suxiao Jiuxin Pill (SJP) are traditional Chinese medicines used to treat cardiovascular disease (CVD) in China. However, the mechanism of their therapeutic effect on CVD has not been clearly elucidated yet.The aim of this study is to investigate the cardioprotective effect of SBP and SJP in the treatment of acute myocardial infarction (AMI) model rats by applying serum proteomic approach.The rat model of AMI was generated by ligating the left anterior descending coronary artery. 42 rats were randomly divided into four groups: sham-operating (Sham, n = 10) group, model (Mod, n = 8) group, Shexiang Baoxin pills pretreatment (SBP, n = 12) group and Suxiao Jiuxin pills pretreatment (SJP, n = 12) group. Data Independent Acquisition (DIA) proteomic approach was utilized to investigate the serum proteome from the rat individuals. The differentially expressed proteins were subsequently obtained with bioinformatic analysis.DIA-MS identified 415 proteins within 42 samples, and 84 differentially expressed proteins may contribute to the therapeutic effects of SBP and SJP. GOBP and KEGG pathway analysis of 84 differentially expressed proteins revealed that the proteins were mainly involved in platelet activation and adhesion processes. All 84 differentially expressed proteins presented the same changing tendency in the SBP and SJP groups when compared with the Mod group. Among these 84 proteins, 25 proteins were found to be related to CVD. Among these 25 proteins, ACTB, ACTG1, FGA, FGB, FGG, PF4 and VWF were found to be involved in platelet aggregation and activation. FN1, HSPA5 and YWHAZ were associated with adhesion.The results of our study suggest that the cardioprotective effects of SBP and SJP are achieved through the modulation of focal adhesion, platelet activation pathways.

We performed proteogenomic characterization of intrahepatic cholangiocarcinoma (iCCA) using paired tumor and adjacent liver tissues from 262 patients. Integrated proteogenomic analyses prioritized genetic aberrations and revealed hallmarks of iCCA pathogenesis. Aflatoxin signature was associated with tumor initiation, proliferation, and immune suppression. Mutation-associated signaling profiles revealed that TP53 and KRAS co-mutations may contribute to iCCA metastasis via the integrin-FAK-SRC pathway. FGFR2 fusions activated the Rho GTPase pathway and could be a potential source of neoantigens. Proteomic profiling identified four patient subgroups (S1-S4) with subgroup-specific biomarkers. These proteomic subgroups had distinct features in prognosis, genetic alterations, microenvironment dysregulation, tumor microbiota composition, and potential therapeutics. SLC16A3 and HKDC1 were further identified as potential prognostic biomarkers associated with metabolic reprogramming of iCCA cells. This study provides a valuable resource for researchers and clinicians to further identify molecular pathogenesis and therapeutic opportunities in iCCA.

Staphylococcus epidermidis is a common causative of nosocomial infections associated with indwelling medical devices. To date, the mechanisms of the pathogenicity and drug resistance of S. epidermidis have not been clearly elucidated. AbfR has been previously identified as an oxidation-sensing regulator that regulates bacterial aggregation and biofilm formation by responding to oxidative stress in S. epidermidis; however, the regulatory pathways of AbfR are underexplored. In this study, we investigated the oxidation-sensing regulatory mechanism of AbfR using TMT10-plex labelling quantitative proteomic and untargeted metabolomic approaches. Integrated analysis of two omics datasets indicated that abfR depletion influenced nucleic acid metabolism and activated the DNA mismatch repair pathway. In addition, several energy-related metabolic pathways, including tricarboxylic acid (TCA) cycle, glycolysis, and arginine metabolism, were remarkably impacted by the deletion of abfR. This study revealed the regulatory networks of the transcription factor AbfR from a multi-omics view and demonstrated that AbfR played a broad role in not only mismatch repair but also energy metabolism, enabling S. epidermidis to constantly sense and adapt to environmental stress. SIGNIFICANCE: Staphylococcus epidermidis has emerged as a major nosocomial infection causing pathogen. AbfR, a transcription factor of S. epidermidis, plays an important role in oxidative stress, cell aggregation, and biofilm formation; however, the regulatory mechanism of AbfR is unknown. Using proteomic and metabolomic approaches, this study unveils the global regulatory networks of AbfR, and demonstrates that AbfR not only regulates the DNA mismatch repair pathway by an oxidation sensing mechanism but also affects energy metabolism. This study expands the body of knowledge related to regulatory transcription factors in staphylococci and lays a foundation for future research on clinical infections caused by S. epidermidis.

Cancer has no borders: Generation and analysis of molecular data across multiple centers worldwide is necessary to gain statistically significant clinical insights for the benefit of patients. Here we conceived and standardized a proteotype data generation and analysis workflow enabling distributed data generation and evaluated the quantitative data generated across laboratories of the international Cancer Moonshot consortium. Using harmonized mass spectrometry (MS) instrument platforms and standardized data acquisition procedures, we demonstrated robust, sensitive, and reproducible data generation across eleven sites in nine countries on seven consecutive days in a 24/7 operation mode. The data presented from the high-resolution MS1-based quantitative data-independent acquisition (HRMS1-DIA) workflow shows that coordinated proteotype data acquisition is feasible from clinical specimens using such standardized strategies. This work paves the way for the distributed multi-omic digitization of large clinical specimen cohorts across multiple sites as a prerequisite for turning molecular precision medicine into reality.

Profiling of biological relationships between different molecular layers dissects regulatory mechanisms that ultimately determine cellular function. To thoroughly assess the role of protein post‐translational turnover, we devised a strategy combining pulse stable isotope‐labeled amino acids in cells (pSILAC), data‐independent acquisition mass spectrometry (DIA‐MS), and a novel data analysis framework that resolves protein degradation rate on the level of mRNA alternative splicing isoforms and isoform groups. We demonstrated our approach by the genome‐wide correlation analysis between mRNA amounts and protein degradation across different strains of HeLa cells that harbor a high grade of gene dosage variation. The dataset revealed that specific biological processes, cellular organelles, spatial compartments of organelles, and individual protein isoforms of the same genes could have distinctive degradation rate. The protein degradation diversity thus dissects the corresponding buffering or concerting protein turnover control across cancer cell lines. The data further indicate that specific mRNA splicing events such as intron retention significantly impact the protein abundance levels. Our findings support the tight association between transcriptome variability and proteostasis and provide a methodological foundation for studying functional protein degradation., Molecular Systems Biology, 16 (3), ISSN:1744-4292

Thermogenesis is a promising approach to limit weight gain in response to excess nutrition. In contrast to cold-induced thermogenesis, the molecular and cellular mechanisms of diet-induced thermogenesis (DIT) have not been fully characterized. Here, we explored the response of brown adipose tissue (BAT) and subcutaneous white adipose tissue (sWAT) to high fat diet (HFD) using proteome and phosphoproteome analysis. We observed that after HFD, Uncoupling protein 1 (UCP1) and its phosphorylation were only increased in BAT. Furthermore, proteins involved in fatty acid oxidation, tricarboxylic acid cycle, and oxidative phosphorylation were also upregulated in BAT. Nevertheless, most metabolic related proteins were downregulated in sWAT. We found that these metabolic changes accompanied with different variation of mitochondrial proteins between BAT and sWAT. After HFD, most mitochondrial proteins were decreased in sWAT, but not in BAT. This effect was correlated with decreased mitochondrial ribosomal proteins in sWAT. Finally, through phosphoproteomic analysis, we predicted the activities of kinases in HFD mice and observed that there were more kinases inactivated in sWAT. Finally, this dataset provides a valuable resource for molecular researchers in the fields of obesity and obesity-related disease. SIGNIFICANCE: Thermogenesis is a promising approach to combat obesity in response to excess energy. Nevertheless, the molecular and cellular mechanisms of DIT have not been fully characterized. Herein, we employed mass spectrometry (MS)-based proteomics and phosphoproteomics to identify differentially regulated proteins and phosphosites in BAT and sWAT of mice fed with HFD. This study unveils the differential regulatory networks of HFD in BAT and sWAT, which provides reference omics data to future researchers.

The juridification of Chinese society has gone through four distinct historical phases: an early stage based on Confucian moral law, the introduction of German civil law, followed by socialist (Sov...

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Human adipose-derived stem cells (hADSCs) are increasingly presumed to be a prospective stem cell source for cell replacement therapy in various degenerative and/or traumatic diseases. The potential of trans-differentiating hADSCs into motor neuron cells indisputably provides an alternative way for spinal cord injury (SCI) treatment. In the present study, a stepwise and efficient hADSC trans-differentiation protocol with retinoic acid (RA), sonic hedgehog (SHH), and neurotrophic factors were developed. With this protocol hADSCs could be converted into electrophysiologically active motoneuron-like cells (hADSC-MNs), which expressed both a cohort of pan neuronal markers and motor neuron specific markers. Moreover, after being primed for neuronal differentiation with RA/SHH, hADSCs were transplanted into SCI mouse model and they survived, migrated, and integrated into injured site and led to partial functional recovery of SCI mice. When ablating the transplanted hADSC-MNs harboring HSV-TK-mCherry overexpression system with antivirial Ganciclovir (GCV), functional relapse was detected by motor-evoked potential (MEP) and BMS assays, implying that transplanted hADSC-MNs participated in rebuilding the neural circuits, which was further confirmed by retrograde neuronal tracing system (WGA). GFP-labeled hADSC-MNs were subjected to whole-cell patch-clamp recording in acute spinal cord slice preparation and both action potentials and synaptic activities were recorded, which further confirmed that those pre-conditioned hADSCs indeed became functionally active neurons in vivo. As well, transplanted hADSC-MNs largely prevented the formation of injury-induced cavities and exerted obvious immune-suppression effect as revealed by preventing astrocyte reactivation and favoring the secretion of a spectrum of anti-inflammatory cytokines and chemokines. Our work suggests that hADSCs can be readily transformed into MNs in vitro, and stay viable in spinal cord of the SCI mouse and exert multi-therapeutic effects by rebuilding the broken circuitry and optimizing the microenvironment through immunosuppression.

Background Mass spectrometry (MS) has become a promising analytical technique to acquire proteomics information for the characterization of biological samples. Nevertheless, most studies focus on the final proteins identified through a suite of algorithms by using partial MS spectra to compare with the sequence database, while the pattern recognition and classification of raw mass-spectrometric data remain unresolved. Results We developed an open-source and comprehensive platform, named MSpectraAI, for analyzing large-scale MS data through deep neural networks (DNNs); this system involves spectral-feature swath extraction, classification, and visualization. Moreover, this platform allows users to create their own DNN model by using Keras. To evaluate this tool, we collected the publicly available proteomics datasets of six tumor types (a total of 7,997,805 mass spectra) from the ProteomeXchange consortium and classified the samples based on the spectra profiling. The results suggest that MSpectraAI can distinguish different types of samples based on the fingerprint spectrum and achieve better prediction accuracy in MS1 level (average 0.967). Conclusion This study deciphers proteome profiling of raw mass spectrometry data and broadens the promising application of the classification and prediction of proteomics data from multi-tumor samples using deep learning methods. MSpectraAI also shows a better performance compared to the other classical machine learning approaches.

Parkinson’s disease (PD) is an aging-related degenerative disorder arisen from the loss of dopaminergic neurons in substantia nigra. Although many genetic mutations have been implicated to be genetically linked to PD, the low incidence of familial PD carried with mutations suggests that there must be other factors such as oxidative stress, mitochondrial dysfunction, accumulation of misfolded proteins, and enhanced inflammation, which are contributable to the pathophysiology of PD. The major efforts of current research have been devoted to unravel the toxic effect of multiple factors, which directly cause the degeneration of dopaminergic neurons in adulthood. Until recently, several studies have demonstrated that NSCs had compromised proliferation and differentiation capacity in PD animal models or PD patient-derived iPS models, suggesting that the pathology of PD may be rooted in some cellular aberrations at early developmental stage but the mechanism remains to be elusive. Based on the early-onset PD patient-specific iPSCs, we found that PD-patient iPSC-derived NSCs were more susceptible to stress and became functionally compromised by radiation or oxidative insults. We further unraveled that stress-induced SIRT1 downregulation leading to autophagic dysfunction, which were responsible for these deficits in PD-NSCs. Mechanistically, we demonstrated that stress-induced activation of p38 MAPK suppressed SIRT1 expression, which in turn augmented the acetylation of multiple ATG proteins of autophagic complex and eventually led to autophagic deficits. Our studies suggest that early developmental deficits may, at least partially, contribute to the pathology of PD and provide a new avenue for developing better therapeutic interventions to PD.

Rationale Therapeutic agents for memory enhancement in psychiatric disorders, such as schizophrenia, are urgently needed. Objective The aim of this study is to characterize the preclinical profile of ITI-214, a potent inhibitor of phosphodiesterase 1 (PDE1). Methods ITI-214 was assayed for inhibition of PDE1 versus other PDE enzyme families using recombinant human PDE enzymes and for off-target binding to 70 substrates (General SEP II diversity panel; Caliper Life Sciences). Effects of ITI-214 (0.1–10 mg/kg, po) on memory performance were assayed in rats using the novel object recognition (NOR) paradigm, with drug given at specified time points prior to or following exposure to objects in an open field. ITI-214 was evaluated for potential drug-drug interaction with risperidone in rats using conditioned avoidance response (CAR) and pharmacokinetic assessments. Results ITI-214 inhibited PDE1A (Ki = 33 pmol) with >1000-fold selectivity for the nearest other PDE family (PDE4D) and displayed minimal off-target binding interactions in a 70-substrate selectivity profile. By using specific timing of oral ITI-214 administration, it was demonstrated in the NOR that ITI-214 is able to enhance acquisition, consolidation, and retrieval memory processes. All memory effects were in the absence of effects on exploratory behavior. ITI-214 did not disrupt the risperidone pharmacokinetic profile or effects in CAR. Conclusions ITI-214 improved the memory processes of acquisition, consolidation, and retrieval across a broad dose range (0.1–10 mg/kg, po) without disrupting the antipsychotic-like activity of a clinical antipsychotic medication, specifically risperidone. Clinical development of ITI-214 is currently in progress.

The expression of let-7 family members was differentiated in ischemic stroke (IS), functioning as an important regulating molecular in the pathophysiology of stroke. We hypothesized that genetic polymorphism in the promoters of let-7 family may be associated with the risk of IS. To test this hypothesis, we investigated the association of the rs10877887 and rs13293512 in the promoters of let-7 family with the susceptibility to IS. A hospital-based case-control study was performed. The rs10877887 genotype was determined by using a polymerase chain reaction-restriction fragment length polymorphism assay, and the rs13293512 genotype was determined by using a TaqMan assay. We found that the rs13293512CC genotype was associated with a reduced risk of IS (CC vs. TT: adjusted OR = 0.43, 95 % CI 0.26-0.71; dominant model: adjusted OR = 0.70, 95 % CI 0.49-0.98; recessive model: adjusted OR = 0.45, 95 % CI, 0.28-0.73). Stratification analysis showed that the rs10877887TT carriers had a higher level of total cholesterol compared to rs10877887TC/CC carriers (P = 0.03). Combined analysis showed that the rs10877887TC/CC and rs13293512TC/CC genotypes had a reduced risk of IS risk (adjusted OR = 0.58, 95 % CI 0.36-0.95). Our findings suggest that the rs13293512 polymorphism may be a protective factor for the development of IS.

A diverse set of 3-aminopyrazolo[3,4-d]pyrimidinones was designed and synthesized. The structure-activity relationships of these polycyclic compounds as phosphodiesterase 1 (PDE1) inhibitors were studied along with their physicochemical and pharmacokinetic properties. Systematic optimizations of this novel scaffold culminated in the identification of a clinical candidate, (6aR,9aS)-2-(4-(6-fluoropyridin-2-yl)benzyl)-5-methyl-3-(phenylamino)-5,6a,7,8,9,9a-hexahydrocyclopenta[4,5]imidazo[1,2-a]pyrazolo[4,3-e]pyrimidin-4-(2H)-one phosphate (ITI-214), which exhibited picomolar inhibitory potency for PDE1, demonstrated excellent selectivity against all other PDE families and showed good efficacy in vivo. Currently, this investigational new drug is in Phase I clinical development and being considered for the treatment of several indications including cognitive deficits associated with schizophrenia and Alzheimer's disease, movement disorders, attention deficit and hyperactivity disorders, and other central nervous system (CNS) and non-CNS disorders.

Background: Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related mortality. Artemisinin (ART) and SOMCL-14-221 (221), a spirobicyclic analogue of ART, have been reported to inhibit the proliferation of A549 cells; yet, the underlying mechanism remains unclear. Methods: Both in vitro and in vivo experiments were performed to examine the anti-proliferative effects of ART and 221. The TMT-based quantitative proteomics and NMR-based metabolomics were conducted to analyze the global proteomic and metabolomic changes in ART and 221 groups, respectively. The potential mechanism of ART and 221 in suppressing tumor growth was studied by knockdown of asparagine synthetase (ASNS) and inhibition of ER stress. Findings: Both ART and 221 inhibited the proliferation of NSCLC cells in vitro and in vivo. The proteomic data revealed 114 upregulated proteins in ART and 221 groups, in which biological process of “response to endoplasmic reticulum stress” were highly represented. ASNS was identified as a key node protein in this process. Interestingly, knockdown of ASNS improved the cytotoxicity of ART and 221, and administration with ART and 221 disordered amino acid metabolism. Moreover, ART and 221 activated ER stress, and inhibition of ER stress abolished the anti-proliferative effects of ART and 221. Interpretation: This study demonstrates that ART and 221 suppress tumor growth by triggering ER stress, and the inhibition of ASNS enhances the antitumor activity of ART and 221, which provides new strategy of combined drug therapy. Funding Statement: This work was supported by the National Key Research and Development Program from the Ministry of Science and Technology of China (No. 2017YFC1700200), the National Science & Technology Major Project "Key New Drug Creation and Manufacturing Program" of China (Grant No. 2018ZX09711002), National Natural Science Foundation of China (No. 31800693, No. 21877120, 81430080) and the ''Personalized Medicines-Molecular Signature-based Drug Discovery and Development" of Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDA12020320). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: All animal studies (including the mice euthanasia procedure) were done in compliance with the regulations and guidelines of Shanghai Institute of Materia Medica (Chinese Academy of Sciences) institutional animal care and conducted according to the AAALAC and the IACUC guidelines.

Background: Hepatic stellate cell (HSC) activation induced by transforming growth factor β1 (TGF-β1) plays a pivotal role in fibrogenesis, while the complex downstream mediators of TGF-β1 in such process are largely unknown. Methods: We performed proteomic profiling of mice liver tissues from control, carbon tetrachloride (CCl4)-induced fibrosis and NPLC0393 administrated groups. Our target Mat2a was overexpressed and knocked down in vivo by tail vein injection of AAV vectors. We examined NF-κB transcriptional and MAT2A promoter activation via luciferase assay. Intracellular SAM contents were analyzed by LC/MS method. Findings: We found that methionine adenosyltransferase 2A (MAT2A) is significantly upregulated in the CCl4-induced fibrosis mice model and application of NPLC0393, a known small molecule inhibitor of TGF-β1 signaling pathway, inhibits the upregulation of MAT2A. Mechanistically, TGF-β1 induces phosphorylation of p65 and activation of NF-κB, thereby promoting mRNA transcription and protein expression of MAT2A and reduces S-adenosylmethionine (SAM) concentration in HSCs. Consistently, in vivo and in vitro knockdown of MAT2A alleviates CCl4- and TGF-β1-induced HSC activation, whereas in vivo overexpression of MAT2A facilitates hepatic fibrosis and abolishes therapeutic effect of NPLC0393. Interpretation: This study identifies TGF-β1/p65/MAT2A pathway that is involved in the regulation of intracellular SAM concentration and liver fibrogenesis, suggesting that this pathway is a potential therapeutic target for hepatic fibrosis. Funding: This work was supported by National Natural Science Foundation of China (No. 81500469, 81573873 and 81774196), Zhejiang Provincial Natural Science Foundation of China (No. Y15H030004), the National Key Research and Development Program from the Ministry of Science and Technology of China (No. 2017YFC1700200) and the Key Program of National Natural Science Foundation of China (No. 8153000502). Declaration of Interest: The authors declare no conflict of interest. Ethical Approval: The animal experiment was approved by Science and Technology Commission of Shanghai Municipality, and all experimental procedures were performed according to the ethical guidelines of Animal care and use committee, Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

Gastric cancer is the third leading cause of cancer-related death worldwide. The regulatory mechanisms underlying gastric cancer cell proliferation are largely unclear. Here, we show that the transcription factor GFI1 is associated with advanced clinical gastric cancer progression and promoted gastric cancer cell proliferation partially through inhibition of gastrokine-2 (GKN2) transcription. GFI1 was a degrading substrate of FBXW7, whose loss was observed in gastric cancer. Mechanistically, GSK3β-mediated GFI1 S94/S98 phosphorylation triggered its interaction with FBXW7, resulting in SCFFBXW7-mediated ubiquitination and degradation. A nondegradable GFI1 S94A/S98A mutant was more potent in driving gastric cancer cell proliferation and tumorigenesis than wild-type GFI1. Overall, this study reveals the oncogenic role of GFI1 in gastric cancer and provides mechanistic insights into the tumor suppressor function of FBXW7. SIGNIFICANCE: These findings demonstrate the oncogenic role of the transcription factor GFI1 and the tumor suppressive function of FBXW7 in gastric cancer.

In conventional data-dependent acquisition (DDA) mode used for phosphoproteome analysis by mass spectrometry, a considerable number of phosphosites are localized ambiguously. We found that data-independent acquisition (DIA) method could localize phosphosite accurately and confidently due to its acquisition nature of repeated and unbiased mode. Here, we present a robust and promising DIA workflow for modification sites determination. Through dedicated collection and analyses of site-determining ions following their chromatographic profilings, phosphosites could be assigned or rectified unambiguously.

Dermal fibroblast is one of the major constitutive cells of skin and plays a central role in skin senescence. The replicative senescence of fibroblasts may cause skin aging, bad wound healing, skin diseases and even cancer. In this study, a label-free quantitative proteomic approach was employed to analyzing the serial passaged human skin fibroblast (CCD-1079Sk) cells, resulting in 3371 proteins identified. Of which, 280 proteins were significantly changed in early passage (6 passages, P6), middle passage (12 passages, P12) and late passage (21 passages, P21), with a time-dependent decrease or increase tendency. Bioinformatic analysis demonstrated that the chromosome condensin complex, including structural maintenance of chromosomes protein 2 (SMC2) and structural maintenance of chromosomes protein 4 (SMC4), were down-regulated in the serially passaged fibroblast cells. The qRT-PCR and Western Blot experiments confirmed that the expression of these two proteins were significantly down-regulated in a time-dependent manner in the subculture of human skin fibroblasts (HSFb cells). In summary, we used serially passaged human skin fibroblast cells coupled with quantitative proteomic approach to profile the protein expression pattern in the temporal progress of replicative senescence in HSFb cells and revealed that the down-regulation of the chromosome condensin complex subunits, such as SMC2 and SMC4, may play an important role in the fibroblast senescence.

Cancer metastasis is the primary cause of morbidity and mortality, and accounts for up to 95% of cancer-related deaths1. Cancer cells often reprogram their metabolism to efficiently support cell proliferation and survival2,3. However, whether and how those metabolic alterations contribute to the migration of tumour cells remain largely unknown. UDP-glucose 6-dehydrogenase (UGDH) is a key enzyme in the uronic acid pathway, and converts UDP-glucose to UDP-glucuronic acid4. Here we show that, after activation of EGFR, UGDH is phosphorylated at tyrosine 473 in human lung cancer cells. Phosphorylated UGDH interacts with Hu antigen R (HuR) and converts UDP-glucose to UDP-glucuronic acid, which attenuates the UDP-glucose-mediated inhibition of the association of HuR with SNAI1 mRNA and therefore enhances the stability of SNAI1 mRNA. Increased production of SNAIL initiates the epithelial–mesenchymal transition, thus promoting the migration of tumour cells and lung cancer metastasis. In addition, phosphorylation of UGDH at tyrosine 473 correlates with metastatic recurrence and poor prognosis of patients with lung cancer. Our findings reveal a tumour-suppressive role of UDP-glucose in lung cancer metastasis and uncover a mechanism by which UGDH promotes tumour metastasis by increasing the stability of SNAI1 mRNA. UDP-glucose has a tumour-suppressive role by inhibiting the association between HuR and SNAI1 mRNA, whereas UGDH-mediated metabolism of UDP-glucose leads to increased SNAI1 mRNA stability and expression, thereby promoting tumour cell migration and lung cancer metastasis.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin fragility disorder caused by mutations in the Col7a1 gene. Patients with RDEB suffer from recurrent erosions in skin and mucous membranes and have a high risk for developing cutaneous squamous cell carcinoma (cSCCs). TGFβ signaling has been associated with fibrosis and malignancy in RDEB. In this study, the activation of TGFβ signaling was demonstrated in col7a1−/− mice as early as a week after birth starting in the interdigital folds of the paws, accompanied by increased deposition of collagen fibrils and elevated dermal expression of matrix metalloproteinase (MMP)-9 and MMP-13. Furthermore, human cord blood-derived unrestricted somatic stem cells (USSCs) that we previously demonstrated to significantly improve wound healing and prolong the survival of col7a1−/− mice showed the ability to suppress TGFβ signaling and MMP-9 and MMP-13 expression meanwhile upregulating anti-fibrotic TGFβ3 and decorin. In parallel, we cocultured USSCs in a transwell with RDEB patient-derived fibroblasts, keratinocytes, and cSCC, respectively. The patient-derived cells were constitutively active for STAT, but not TGFβ signaling. Moreover, the levels of MMP-9 and MMP-13 were significantly elevated in the patient derived-keratinocytes and cSCCs. Although USSC coculture did not inhibit STAT signaling, it significantly suppressed the secretion of MMP-9 and MMP-13, and interferon (IFN)-γ from RDEB patient-derived cells. Since epithelial expression of these MMPs is a biomarker of malignant transformation and correlates with the degree of tumor invasion, these results suggest a potential role for USSCs in mitigating epithelial malignancy, in addition to their anti-inflammatory and anti-fibrotic functions.

Aging of neural stem cell, which can affect brain homeostasis, may be caused by many cellular mechanisms. Autophagy dysfunction was found in aged and neurodegenerative brains. However, little is known about the relationship between autophagy and human neural stem cell (hNSC) aging. The present study used 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) to treat neural precursor cells (NPCs) derived from human embryonic stem cell (hESC) line H9 and investigate related molecular mechanisms involved in this process. MPTP-treated NPCs were found to undergo premature senescence [determined by increased senescence-associated-β-galactosidase (SA-β-gal) activity, elevated intracellular reactive oxygen species level, and decreased proliferation] and were associated with impaired autophagy. Additionally, the cellular senescence phenotypes were manifested at the molecular level by a significant increase in p21 and p53 expression, a decrease in SOD2 expression, and a decrease in expression of some key autophagy-related genes such as Atg5, Atg7, Atg12, and Beclin 1. Furthermore, we found that the senescence-like phenotype of MPTP-treated hNPCs was rejuvenated through treatment with a well-known autophagy enhancer rapamycin, which was blocked by suppression of essential autophagy gene Beclin 1. Taken together, these findings reveal the critical role of autophagy in the process of hNSC aging, and this process can be reversed by activating autophagy.

Autophagy is the main catabolic pathway in cells for the degradation of impaired proteins and organelles. Accumulating evidence supports the hypothesis that dysfunction of autophagy, leading to an imbalance of proteostasis and the accumulation of toxic proteins in neurons, is a central player in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS). The clinical pathology of ALS is complex and many genes associated with autophagy and RNA processing are mutated in patients with the familial form. But a causal relationship between autophagic dysfunction and ALS has not been fully established. More importantly, studies on the pathological mechanism of ALS are mainly based on animal models that may not precisely recapitulate the disease itself in human beings. The development of human iPSC techniques allows us to address these issues directly in human cell models that may profoundly influence drug discovery for ALS.

Once urinary bladder cancer (UBC) develops into muscle-invasive bladder cancer, its mortality rate increases dramatically. However, the molecular mechanisms of UBC invasion and metastasis remain largely unknown. Herein, using 5637 UBC cells, we generated two sublines with low (5637 NMI) and high (5637 HMI) invasive capabilities. Mass spectrum analyses revealed that the Wnt family protein Wnt7a is more highly expressed in 5637 HMI cells than in 5637 NMI cells. We also found that increased Wnt7a expression is associated with UBC metastasis and predicted worse clinical outcome in UBC patients. Wnt7a depletion in 5637 HMI and T24 cells reduced UBC cell invasion and decreased levels of active β-catenin and its downstream target genes involved in the epithelial-to-mesenchymal transition (EMT) and extracellular matrix (ECM) degradation. Consistently, treating 5637 NMI and J82 cells with recombinant Wnt7a induced cell invasion, EMT, and expression of ECM degradation–associated genes. Moreover, TOP/FOPflash luciferase assays indicated that Wnt7a activated canonical β-catenin signaling in UBC cells, and increased Wnt7a expression was associated with nuclear β-catenin in UBC samples. Wnt7a ablation suppressed matrix metalloproteinase 10 (MMP10) expression, and Wnt7a overexpression increased MMP10 promoter activity through two TCF/LEF promoter sites, confirming that Wnt7a-mediated MMP10 activation is mediated by the canonical Wnt/β-catenin pathway. Of note, the microRNA miR-370-3p directly repressed Wnt7a expression and thereby suppressed UBC cell invasion, which was partially restored by Wnt7a overexpression. Our results have identified an miR-370-3p/Wnt7a axis that controls UBC invasion through canonical Wnt/β-catenin signaling, which may offer prognostic and therapeutic opportunities.

The X-linked deubiquitinase, USP9X, is implicated in multiple cancers by targeting various substrates. Increased expression of USP9X is observed in non-small-cell lung cancer (NSCLC) and is correlated with poor prognosis. However, the molecular mechanism for USP9X regulation of tumor cell survival and tumorigenesis in NSCLC is less defined. Methods: In this study, chemical labeling, quantitative proteomic screening was applied to analyze A549 cells with or without USP9X RNA interference. Functional in vitro and in vivo experiments were performed to confirm the oncogenic effects of USP9X in NSCLC and to investigate the underlying mechanisms. Results: The resulting data suggested that dual specificity protein kinase TTK is a potential substrate of USP9X. Further experimental evidences confirmed that USP9X stabilized TTK via direct interaction and efficient deubiquitination of TTK on K48 ubiquitin chain. Moreover, knockdown of USP9X or TTK inhibited cell proliferation, migration and tumorigenesis, and the immunohistochemical analysis of clinical NSCLC samples showed that the protein expression levels of USP9X and TTK were significantly elevated and positively correlated in tumor tissues. Conclusions: In summary, our data demonstrated that the USP9X-TTK axis may play a critical role in NSCLC, and could be considered as a potential therapeutic target.

Recessive dystrophic epidermolysis bullosa (RDEB) is a devastating inherited skin blistering disease caused by mutations in the COL7A1 gene that encodes type VII collagen (C7), a major structural component of anchoring fibrils at the dermal-epidermal junction (DEJ). We recently demonstrated that human cord blood-derived unrestricted somatic stem cells promote wound healing and ameliorate the blistering phenotype in a RDEB (col7a1−/−) mouse model. Here, we demonstrate significant therapeutic effect of a further novel stem cell product in RDEB, that is, human placental-derived stem cells (HPDSCs), currently being used as human leukocyte antigen-independent donor cells with allogeneic umbilical cord blood stem cell transplantation in patients with malignant and nonmalignant diseases. HPDSCs are isolated from full-term placentas following saline perfusion, red blood cell depletion, and volume reduction. HPDSCs contain significantly higher level of both hematopoietic and nonhematopoietic stem and progenitor cells than cord blood and are low in T cell content. A single intrahepatic administration of HPDSCs significantly elongated the median life span of the col7a1−/− mice from 2 to 7 days and an additional intrahepatic administration significantly extended the median life span to 18 days. We further demonstrated that after intrahepatic administration, HPDSCs engrafted short-term in the organs affected by RDEB, that is, skin and gastrointestinal tract of col7a1−/− mice, increased adhesion at the DEJ and deposited C7 even at 4 months after administration of HPDSCs, without inducing anti-C7 antibodies. This study warrants future clinical investigation to determine the safety and efficacy of HPDSCs in patients with severe RDEB.

Pluripotent stem cells (PSCs) harbor constitutive DNA replication stress during their rapid proliferation and the consequent genome instability hampers their applications in regenerative medicine. It is therefore important to understand the regulatory mechanisms of replication stress response in PSCs. Here, we report that mouse embryonic stem cells (ESCs) are superior to differentiated cells in resolving replication stress. Specifically, ESCs utilize a unique Filia-Floped protein complex-dependent mechanism to efficiently promote the restart of stalled replication forks, therefore maintaining genomic stability. The ESC-specific Filia-Floped complex resides on replication forks under normal conditions. Replication stress stimulates their recruitment to stalling forks and the serine 151 residue of Filia is phosphorylated in an ATR-dependent manner. This modification enables the Filia-Floped complex to act as a functional scaffold, which then promotes the stalling fork restart through a dual mechanism: both enhancing recruitment of the replication fork restart protein, Blm, and stimulating ATR kinase activation. In the Blm pathway, the scaffolds recruit the E3 ubiquitin ligase, Trim25, to the stalled replication forks, and in turn Trim25 tethers and concentrates Blm at stalled replication forks through ubiquitination. In differentiated cells, the recruitment of the Trim25-Blm complex to replication forks and the activation of ATR signaling are much less robust due to lack of the ESC-specific Filia-Floped scaffold. Thus, our study reveals that ESCs utilize an additional and unique regulatory layer to efficiently promote the stalled fork restart and maintain genomic stability.

We report the synthesis and structure-activity relationships of a class of tetracyclic butyrophenones that exhibit potent binding affinities to serotonin 5-HT(2A) and dopamine D2 receptors. This work has led to the discovery of 4-((6bR,10aS)-3-methyl-2,3,6b,9,10,10a-hexahydro-1H,7H-pyrido[3',4':4,5]pyrrolo[1,2,3-de]quinoxalin-8-yl)-1-(4-fluorophenyl)-butan-1-one 4-methylbenzenesulfonate (ITI-007), which is a potent 5-HT(2A) antagonist, postsynaptic D2 antagonist, and inhibitor of serotonin transporter. This multifunctional drug candidate is orally bioavailable and exhibits good antipsychotic efficacy in vivo. Currently, this investigational new drug is under clinical development for the treatment of neuropsychiatric and neurological disorders.

We performed the first proteogenomic characterization of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) using paired tumor and adjacent liver tissues from 159 patients. Integrated proteogenomic analyses revealed consistency and discordance among multi-omics, activation status of key signaling pathways, and liver-specific metabolic reprogramming in HBV-related HCC. Proteomic profiling identified three subgroups associated with clinical and molecular attributes including patient survival, tumor thrombus, genetic profile, and the liver-specific proteome. These proteomic subgroups have distinct features in metabolic reprogramming, microenvironment dysregulation, cell proliferation, and potential therapeutics. Two prognostic biomarkers, PYCR2 and ADH1A, related to proteomic subgrouping and involved in HCC metabolic reprogramming, were identified. CTNNB1 and TP53 mutation-associated signaling and metabolic profiles were revealed, among which mutated CTNNB1-associated ALDOA phosphorylation was validated to promote glycolysis and cell proliferation. Our study provides a valuable resource that significantly expands the knowledge of HBV-related HCC and may eventually benefit clinical practice.

The structural and magnetic properties of Nd0.5-xPrxSr0.5MnO3 (x = 0, 0.1,0.2,0.3,0.4 and 0.5) system have been investigated. With the substitution of Pr in Nd0.5Sr0.5MnO3, it shows a gradual structure transformation from the Imma orthorhombic symmetry to the tetragonal 14/mcm phase, and the crystallographic transition remains incomplete, even in Pr0.5Sr0.5MnO3. A large bifurcation between zero-field-cooled (ZFC) and field-cooled (FC) susceptibility has been observed below Curie temperature (T-C), which is characteristic of coexistence of ferromagnetism (FM) and antiferromagnetism (AFM) at low temperature region. The magnetization of Pr0.5Sr0.5MnO3 is larger than that of Nd0.5Sr0.5MnO3, while Nd0.5Sr0.5MnO3 with more CE-type AFM shows larger magnetization than Nd0.3Pr0.2Sr0.5MnO3, which mixed with CE-type (majority) and A-type (minority) AFM at low temperature, indicating that the magnetization of Nd0.5-xPrxSr0.5MnO3 system is affected by A-site disorder combined with orbital ordering of A-type AFM and CE-type AFM. (C) 2011 Elsevier B.V. All rights reserved.

Two distinct families of small molecules were discovered as novel alpha7 nicotinic acetylcholine receptor (nAChR) antagonists by pharmacophore-based virtual screening. These novel antagonists exhibited selectivity for the neuronal alpha7 subtype over other nAChRs and good brain penetration. Neuroprotection was demonstrated by representative compounds 7i and 8 in a mouse seizure-like behavior model induced by the nerve agent diisopropylfluorophosphate (DFP). These novel nAChR antagonists have potential use as antidote for organophosphorus nerve agent intoxication.

The mortality rate of urinary bladder cancer (UBC) increases dramatically, once low grade non-muscle invasive bladder cancer develops into muscle invasive bladder cancer. Hence, understanding the mechanisms underlying UBC invasion/metastasis and identification of biomarkers for invasion/metastasis will help clinicians to determine how to treat patients. Herein we generated two sublines from parental 5637 UBC cells with low invasive and high invasive capacities, which were designated as 5637 NMI and 5637 HMI, respectively. Mass spectrometry analysis was performed to identify the invasion-associated proteins. 5,987 proteins were quantified across all six samples with the coefficients among different channels all nearly 1.00, suggesting good biological repeatability and minor differences between 5637 HMI and NMI samples. Fold change > 1.2 and p value Depletion of Wnt7a in 5637 HMI and T24 cells reduced UBC cell invasion, with the reduction of active β-Catenin and its downstream target genes involved in epithelial-to-mesenchymal transition (EMT) and extracellular matrix (ECM) degradation. Consistently, introduction recombinant Wnt7a protein to 5637 NMI and J82 cells induced cell invasion, EMT, and the expression of ECM degradation-associated genes. Using luciferase reporter driven by the wildtype or mutants of matrix metalloproteinase 10 (MMP10) promoter, a canonical Wnt/β-Catenin regulation pattern was demonstrated. Four miRNAs downregulated in UBCs were predicted directly targeting Wnt7a using bioinformatics analysis. miRNA mimic could decrease Wnt7a expression, reduce the activation of MMP 10 reporter and suppress the invasion of 5637 HMI cells. Treatment of UBC cells with Wnt7a partially abrogated such miRNAs-induced invasion suppression. Taken together, our data identify a new miRNA/Wnt7a axis to mediate cell invasion and metastasis of UBC through canonical Wnt/β-Catenin pathway, which may offer prognostic and therapeutic opportunity. Citation Format: Ruimin Huang, Xiaojing Huang, Hongwen Zhu, Junlong Zhuang, Yu Dong, Hu Zhou, Hongqian Guo, Jun Yan. Wnt7a promotes bladder cancer cell invasion via canonical Wnt/β-Catenin signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3163.

Organophosphorus (OP) compounds cause toxic symptoms, including convulsions, coma, and death, as the result of irreversible inhibition of acetylcholinesterase (AChE). The development of effective treatments to block these effects and attenuate long-term cognitive and motor disabilities that result from OP intoxication is hampered by a limited understanding of the CNS pathways responsible for these actions. We employed a candidate method (called CNSProfile™) to identify changes in the phosphorylation state of key neuronal phosphoproteins evoked by the OP compound, diisopropyl fluorophosphate (DFP). Focused microwave fixation was used to preserve the phosphorylation state of phosphoproteins in brains of DFP-treated mice; hippocampus and striatum were analyzed by immunoblotting with a panel of phospho-specific antibodies. DFP exposure elicited comparable effects on phosphorylation of brain phosphoproteins in both C57BL/6 and FVB mice. DFP treatment significantly altered phosphorylation at regulatory residues on glutamate receptors, including Serine897 (S897) of the NR1 NMDA receptor. NR1 phosphorylation was bi-directionally regulated after DFP in striatum versus hippocampus. NR1 phosphorylation was reduced in striatum, but elevated in hippocampus, compared with controls. DARPP-32 phosphorylation in striatum was selectively increased at the Cdk5 kinase substrate, Threonine75 (T75). Phencynonate hydrochloride, a muscarinic cholinergic antagonist, prevented seizure-like behaviors and the observed changes in phosphorylation induced by DFP. The data reveal region-specific effects of nerve agent exposure on intracellular signaling pathways that correlate with seizure-like behavior and which are reversed by the muscarinic receptor blockade. This approach identifies specific targets for nerve agents, including substrates for Cdk5 kinase, which may be the basis for new anti-convulsant therapies.

The convergence of the three nets of telecommunication, television and Internet is a general trend of development. Though the “convergence of three nets” promotes the competition in the relevant markets, it causes relatively big impacts on the existing legal order. To meet the challenges brought forth by the “convergence of three nets”, many countries and regions have successively adopted effective measures for legal transformation, not only integrating laws and regulations on the telecommunication industry, but also attaching high importance to the transformation of the legal adjustment and control mode where increasingly more emphasis is laid on the influence and functions of competition law in the “convergence of three nets”. China’s antimonopoly law shall also play an important role during the process of the “convergence of three nets”. With respect to the definition of the relevant market and identification of monopoly practices, we shall closely combine the features of the “convergence of three nets” and pointedly apply the antimonopoly law.

Multiple genes are thought to influence both susceptibility to nicotine dependence and its comorbid behavioral traits in humans. However, which specific genes contribute to this pleiotropic effect is poorly understood. Previous rodent studies have shown that many addictive substances and stressful stimuli increase the expression of the transcription factor FosB in limbic and associated regions and that the protein products of fosB contribute to certain behavioral effects of cocaine and morphine. However, the role of this gene in nicotine-regulated behaviors and dependence-related behavioral traits is unknown. We tested the hypothesis that a constitutive level of FosB affects nicotine-regulated behaviors and comorbid behavioral traits using constitutive fosB knockout (KO) mice. Following repeated or prolonged nicotine administration, but not a single acute administration, KO mice were impaired in conditioned place preference, oral nicotine intake and motor suppression. In wild-type mice, repeated nicotine injections, but not a single acute injection, increased the expression of FosB and its truncated variant DeltaFosB in the targets but not at the origins of the mesolimbic and nigrostriatal dopamine pathways; no detectable level of FosB/DeltaFosB was found in KO mice. In tasks designed to assess behavioral traits, KO mice exhibited more pronounced behavioral abnormalities when stress levels were high than when they were minimized. Our results suggest that the constitutive absence of fosB has a pleiotropic influence on the behavioral effects of repeated or prolonged nicotine administration and on stress-related behavioral traits in mice.

HOXD13, the homeobox-containing gene located at the most 5' end of the HOXD cluster, plays a critical role in limb development. It has been shown that mutations in human HOXD13 can give rise to limb malformations, with variable expressivity and a wide spectrum of clinical manifestations. Polyalanine expansions in HOXD13 cause synpolydactyly, whereas amino acid substitutions in the homeodomain are associated with brachydactyly types D and E. We describe two large Han Chinese families with different limb malformations, one with syndactyly type V and the other with limb features overlapping brachydactyly types A4, D, and E and mild syndactyly of toes 2 and 3. Two-point linkage analysis showed LOD scores3 (theta =0) for markers within and/or flanking the HOXD13 locus in both families. In the family with syndactyly type V, we identified a missense mutation in the HOXD13 homeodomain, c.950A--G (p.Q317R), which leads to substitution of the highly conserved glutamine that is important for DNA-binding specificity and affinity. In the family with complex brachydactyly and syndactyly, we detected a deletion of 21 bp in the imperfect GCN (where N denotes A, C, G, or T) triplet-containing exon 1 of HOXD13, which results in a polyalanine contraction of seven residues. Moreover, we found that the mutant HOXD13 with the p.Q317R substitution was unable to transactivate the human EPHA7 promoter. Molecular modeling data supported these experimental results. The calculated interactions energies were in agreement with the measured changes of the activity. Our data established the link between HOXD13 and two additional limb phenotypes--syndactyly type V and brachydactyly type A4--and demonstrated that a polyalanine contraction in HOXD13, most likely, led to other digital anomalies but not to synpolydactyly. We suggest the term "HOXD13 limb morphopathies" for the spectrum of limb disorders caused by HOXD13 mutations.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin blistering disease caused by mutations in COL7A1-encoding type VII collagen (C7). Currently, there is no curative therapy for patients with RDEB. Our previous studies demonstrated that human umbilical cord blood (HUCB) derived unrestricted somatic stem cells (USSCs) express C7 and facilitate wound healing in a murine wounding model. The primary objective of this study is to investigate the therapeutic functions of USSCs in the C7 null (Col7a1−/−) C57BL6/J mice, a murine model of RDEB. We demonstrated that intrahepatic administration of USSCs significantly improved the blistering phenotype and enhanced the life span in the recipients. The injected USSCs trafficked to the sites of blistering and were incorporated in short-term in the recipients' skin and gastrointestinal tract. Consistent with an overall histological improvement in the epidermal-dermal adherence following USSC treatment, the expression of C7 at the basement membrane zone was detected and the previously disorganized integrin α6 distribution was normalized. We also demonstrated that USSCs treatment induced an infiltration of macrophages with a regenerative “M2” phenotype. Our data suggest that HUCB-derived USSCs improved the RDEB phenotype through multiple mechanisms. This study has warranted future clinical investigation of USSCs as a novel and universal allogeneic stem cell donor source in selected patients with RDEB. Stem Cells 2015;33:1807–1817

Human umbilical cord blood (CB)-derived unrestricted somatic stem cells (USSCs) have previously been demonstrated to have a broad differentiation potential and regenerative beneficial effects when administered in animal models of multiple degenerative diseases. Here we demonstrated that USSCs could be induced to express genes that hallmark keratinocyte differentiation. We also demonstrated that USSCs express type VII collagen (C7), a protein that is absent or defective in patients with an inherited skin disease, recessive dystrophic epidermolysis bullosa (RDEB). In mice with full-thickness excisional wounds, a single intradermal injection of USSCs at a 1-cm distance to the wound edge resulted in significantly accelerated wound healing. USSC-treated wounds displayed a higher density of CD31+ cells, and the wounds healed with a significant increase in skin appendages. These beneficial effects were demonstrated without apparent differentiation of the injected USSCs into keratinocytes or endothelial cells. In vivo bioluminescent imaging (BLI) revealed specific migration of USSCs modified with a luciferase reporter gene, from a distant intradermal injection site to the wound, as well as following systemic injection of USSCs. These data suggest that CB-derived USSCs could significantly contribute to wound repair and be potentially used in cell therapy for patients with RDEB.

To solve the problem of how to make better the matching of enterprise scale and its IT level, we have chosen 94 enterprises with different sizes in Heilongjiang province as sample, analyzed the correlation and the synergy between enterprise scale and IT level. Our research has found out that in the case of large sample, it shows the significant positive correlation between enterprise scale and IT level, but with the decreasing of sample size, the correlation is no longer significant. At last, we have determined that the optimal order parameter between enterprise scale and IT level is 1.49.

This letter presents a new one-dimensional chaotic map with infinite collapses. Theoretical analyses show that the map has complicated dynamical behavior and ideal distribution. The map can be applied in chaotic spreading spectrum communication and chaotic cipher.