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  • Exploring the causal pathway from ischemic stroke to atrial fibrillation: a network Mendelian randomization study
    Mol. Med. (IF 2.991) Pub Date : 2020-01-15
    Lei Hou; Mingqing Xu; Yuanyuan Yu; Xiaoru Sun; Xinhui Liu; Lu Liu; Yunxia Li; Tonghui Yuan; Wenchao Li; Hongkai Li; Fuzhong Xue

    Previous studies have found ischemic stroke is associated with atrial fibrillation. However, the causal association between ischemic stroke and atrial fibrillation is not clear. Furthermore, the network relationship among ischemic stroke, atrial fibrillation and its risk factors need further attention. This study aims to examine the potential causal association between ischemic stroke and atrial fibrillation and further to explore potential mediators in the causal pathway from ischemic stroke to atrial fibrillation. Summary statistics from the ISGC (case = 10,307 and control = 19,326) were used as ischemic stroke genetic instruments, AFGen Consortium data (case = 65,446 and control = 522,744) were used for atrial fibrillation, and other consortia data were used for potential mediators (fasting insulin, white blood cell count, procalcitonin, systolic and diastolic blood pressure, body mass index, waist circumference, and height). Under the framework of network Mendelian randomization, two-sample Mendelian randomization study was performed using summary statistics from several genome-wide association studies. Inverse-variance weighted method was performed to estimate causal effect. Blood pressure mediates the causal pathways from ischemic stroke to atrial fibrillation. The total odds ratio of ischemic stroke on atrial fibrillation was 1.05 (95% confidence interval [CI], 1.02 to 1.07; P = 1.3 × 10−5). One-unit increase of genetically determined ischemic stroke was associated with 0.02 (DBP: 95% CI, 0.001 to 0.034, P = 0.029; SBP: 95% CI, 0.006 to 0.034, P = 0.003) upper systolic and diastolic blood pressure levels. Higher genetically determined systolic and diastolic blood pressure levels were associated with higher atrial fibrillation risk (DBP: RR, 1.18; 95% CI, 1.03 to 1.35; P = 0.012. SBP: RR, 1.18; 95% CI, 1.01 to 1.38; P = 0.04). Specially, we also found the bidirectional causality between blood pressure and ischemic stroke. Our study provided a strong evidence that raised blood pressure in stroke patients increases the risk of atrial fibrillation and active acute blood pressure lowering can improve the outcome in ischemic stroke patients.

    更新日期:2020-01-15
  • Retinal pigment epithelium degeneration caused by aggregation of PRPF31 and the role of HSP70 family of proteins
    Mol. Med. (IF 2.991) Pub Date : 2019-12-31
    Lourdes Valdés-Sánchez; Sofia M. Calado; Berta de la Cerda; Ana Aramburu; Ana Belén García-Delgado; Simone Massalini; Adoración Montero-Sánchez; Vaibhav Bhatia; Eduardo Rodríguez-Bocanegra; Andrea Diez-Lloret; Daniel Rodríguez-Martínez; Christina Chakarova; Shom S. Bhattacharya; Francisco J. Díaz-Corrales

    Mutations in pre-mRNA splicing factor PRPF31 can lead to retinitis pigmentosa (RP). Although the exact disease mechanism remains unknown, it has been hypothesized that haploinsufficiency might be involved in the pathophysiology of the disease. In this study, we have analyzed a mouse model containing the p.A216P mutation in Prpf31 gene. We found that mutant Prpf31 protein produces cytoplasmic aggregates in the retinal pigment epithelium and decreasing the protein levels of this splicing factor in the nucleus. Additionally, normal protein was recruited in insoluble aggregates when the mutant protein was overexpressed in vitro. In response to protein aggregation, Hspa4l is overexpressed. This member of the HSP70 family of chaperones might contribute to the correct folding and solubilization of the mutant protein, allowing its translocation to the nucleus. Our data suggests that a mechanism haploinsufficiency and dominant-negative is involved in retinal degeneration due to mutations in PRPF31. HSP70 over-expression might be a new therapeutic target for the treatment of retinal degeneration due to PRPF31 mutations.

    更新日期:2019-12-31
  • Role of 5-HT7 receptors in the immune system in health and disease
    Mol. Med. (IF 2.991) Pub Date : 2019-12-31
    Alejandro Quintero-Villegas; Sergio Iván Valdés-Ferrer

    In mammalians, serotonin (5-HT) has critical roles in the central nervous system (CNS), including mood stability, pain tolerance, or sleep patterns. However, the vast majority of serotonin is produced by intestinal enterochromaffin cells of the gastrointestinal tract and circulating blood platelets, also acting outside of the CNS. Serotonin effects are mediated through its interaction with 5-HT receptors (5-HTRs), a superfamily with a repertoire of at least fourteen well-characterized members. 5-HT7 receptors are the last 5-HTR member to be identified, with well-defined functions in the nervous, gastrointestinal, and vascular systems. The effects of serotonin on the immune response are less well understood. Mast cells are known to produce serotonin, while T cells, dendritic cells, monocytes, macrophages and microglia express 5-HT7 receptor. Here, we review the known roles of 5-HT7 receptors in the immune system, as well as their potential therapeutic implication in inflammatory and immune-mediated disorders.

    更新日期:2019-12-31
  • Inhibition of microRNA-29a alleviates hyperoxia-induced bronchopulmonary dysplasia in neonatal mice via upregulation of GAB1
    Mol. Med. (IF 2.991) Pub Date : 2019-12-31
    Yu Hu; Liang Xie; Jing Yu; Hongling Fu; Dan Zhou; Hanmin Liu

    The main features of bronchopulmonary dysplasia (BPD) are alveolar simplification, pulmonary growth arrest, and abnormal lung function. Multiple studies have highlighted microRNA-29 (miR-29) as a potential biomarker for lung diseases and cancers. Upregulation of miR-29a has been known to downregulate GRB2-associated-binding protein 1 (GAB1), which is often highly expressed in the lung. The current study was designed to investigate the potential role of miR-29a in hyperoxia-induced BPD by targeting GAB1 in a neonatal mouse model. The expression of miR-29a and GAB1 in lung tissues of neonatal mice with hyperoxia-induced BPD and mouse alveolar epithelial cells (MLE-12) was determined using RT-qPCR and western blot analysis. Subsequently, the relationship between miR-29a and GAB1 was verified using in silico analysis. In order to assess the effects of miR-29a or GAB1 on BPD, the pathological characteristics of alveoli, as well as proliferation and apoptosis of cells were measured through gain- and loss-of-function studies. Upregulation of miR-29a and downregulation of GAB1 were evident in both lung tissues and MLE-12 cells following BPD modeling. GAB1 was a direct target gene of miR-29a. Inhibition of miR-29a and overexpression of GAB1 were shown to alleviate lung injury, promote cell proliferation and inhibit apoptosis but reduce chord length in lung tissues of neonatal mice following hyperoxia-induced BPD modeling. Altogether, down-regulation of miR-29a can potentially elevate GAB1 expression, reducing cell apoptosis and stimulating proliferation, ultimately retarding the development of BPD in mice. This study highlights the potential of a promising new target for preventing BPD.

    更新日期:2019-12-31
  • Disorders of FZ-CRD; insights towards FZ-CRD folding and therapeutic landscape
    Mol. Med. (IF 2.991) Pub Date : 2019-12-31
    Reham M. Milhem; Bassam R. Ali

    The ER is hub for protein folding. Proteins that harbor a Frizzled cysteine-rich domain (FZ-CRD) possess 10 conserved cysteine motifs held by a unique disulfide bridge pattern which attains a correct fold in the ER. Little is known about implications of disease-causing missense mutations within FZ-CRD families. Mutations in FZ-CRD of Frizzled class receptor 4 (FZD4) and Muscle, skeletal, receptor tyrosine kinase (MuSK) and Receptor tyrosine kinase-like orphan receptor 2 (ROR2) cause Familial Exudative Vitreoretinopathy (FEVR), Congenital Myasthenic Syndrome (CMS), and Robinow Syndrome (RS) respectively. We highlight reported pathogenic inherited missense mutations in FZ-CRD of FZD4, MuSK and ROR2 which misfold, and traffic abnormally in the ER, with ER-associated degradation (ERAD) as a common pathogenic mechanism for disease. Our review shows that all studied FZ-CRD mutants of RS, FEVR and CMS result in misfolded proteins and/or partially misfolded proteins with an ERAD fate, thus we coin them as “disorders of FZ-CRD”. Abnormal trafficking was demonstrated in 17 of 29 mutants studied; 16 mutants were within and/or surrounding the FZ-CRD with two mutants distant from FZ-CRD. These ER-retained mutants were improperly N-glycosylated confirming ER-localization. FZD4 and MuSK mutants were tagged with polyubiquitin chains confirming targeting for proteasomal degradation. Investigating the cellular and molecular mechanisms of these mutations is important since misfolded protein and ER-targeted therapies are in development. The P344R-MuSK kinase mutant showed around 50% of its in-vitro autophosphorylation activity and P344R-MuSK increased two-fold on proteasome inhibition. M105T-FZD4, C204Y-FZD4, and P344R-MuSK mutants are thermosensitive and therefore, might benefit from extending the investigation to a larger number of chemical chaperones and/or proteasome inhibitors. Nonetheless, FZ-CRD ER-lipidation it less characterized in the literature and recent structural data sheds light on the importance of lipidation in protein glycosylation, proper folding, and ER trafficking. Current treatment strategies in-place for the conformational disease landscape is highlighted. From this review, we envision that disorders of FZ-CRD might be receptive to therapies that target FZ-CRD misfolding, regulation of fatty acids, and/or ER therapies; thus paving the way for a newly explored paradigm to treat different diseases with common defects.

    更新日期:2019-12-31
  • HMGB1 concentration measurements in trauma patients: assessment of pre-analytical conditions and sample material
    Mol. Med. (IF 2.991) Pub Date : 2019-12-31
    William Ottestad; Ingrid N. Rognes; Erlend Skaga; Cassandra Frisvoll; Guttorm Haraldsen; Torsten Eken; Peter Lundbäck

    HMGB1 is a mediator of systemic inflammation in sepsis and trauma, and a promising biomarker in many diseases. There is currently no standard operating procedure for pre-analytical handling of HMGB1 samples, despite that pre-analytical conditions account for a substantial part of the overall error rate in laboratory testing. We hypothesized that the considerable variations in reported HMGB1 concentrations and kinetics in trauma patients could be partly explained by differences in pre-analytical conditions and choice of sample material. Trauma patients (n = 21) admitted to a Norwegian Level I trauma center were prospectively included. Blood was drawn in K2EDTA coated tubes and serum tubes. The effects of delayed centrifugation were evaluated in samples stored at room temperature for 15 min, 3, 6, 12, and 24 h respectively. Plasma samples subjected to long-term storage in − 80 °C and to repeated freeze/thaw cycles were compared with previously analyzed samples. HMGB1 concentrations in simultaneously acquired arterial and venous samples were also compared. HMGB1 was assessed by standard ELISA technique, additionally we investigated the suitability of western blot in both serum and plasma samples. Arterial HMGB1 concentrations were consistently lower than venous concentrations in simultaneously obtained samples (arterial = 0.60 x venous; 95% CI 0.30–0.90). Concentrations in plasma and serum showed a strong linear correlation, however wide limits of agreement. Storage of blood samples at room temperature prior to centrifugation resulted in an exponential increase in plasma concentrations after ≈6 h. HMGB1 concentrations were fairly stable in centrifuged plasma samples subjected to long-term storage and freeze/thaw cycles. We were not able to detect HMGB1 in either serum or plasma from our trauma patients using western blotting. Arterial and venous HMGB1 concentrations cannot be directly compared, and concentration values in plasma and serum must be compared with caution due to wide limits of agreement. Although HMGB1 levels in clinical samples from trauma patients are fairly stable, strict adherence to a pre-analytical protocol is advisable in order to protect sample integrity. Surprisingly, we were unable to detect HMGB1 utilizing standard western blot analysis.

    更新日期:2019-12-31
  • Post-sepsis syndrome – an evolving entity that afflicts survivors of sepsis
    Mol. Med. (IF 2.991) Pub Date : 2019-12-31
    Zachary Mostel; Abraham Perl; Matthew Marck; Syed F. Mehdi; Barbara Lowell; Sagar Bathija; Ramchandani Santosh; Valentin A. Pavlov; Sangeeta S. Chavan; Jesse Roth

    The sequelae of sepsis were once thought to be independent of sepsis itself and assumed to be either comorbid to sick patients or complications of critical illness. Recent studies have reported consistent patterns of functional disabilities in sepsis survivors that can last from months to years after symptoms of active sepsis had resolved. Post-sepsis syndrome is an emerging pathological entity that has garnered significant interest amongst clinicians and researchers over the last two decades. It is marked by a significantly increased risk of death and a poor health-related quality of life associated with a constellation of long-term effects that persist following the patient’s bout with sepsis. These include neurocognitive impairment, functional disability, psychological deficits, and worsening medical conditions. This “post-sepsis syndrome” has been the subject of active preclinical and clinical research providing new mechanistic insights and approaches linked to survivor well-being. Here we review important aspects of these research efforts and goals of care for patients who survive sepsis.

    更新日期:2019-12-31
  • The neuroprotective mechanisms of ginkgolides and bilobalide in cerebral ischemic injury: a literature review
    Mol. Med. (IF 2.991) Pub Date : 2019-12-21
    Zili Feng; Qian Sun; Wang Chen; Yu Bai; Daihua Hu; Xin Xie

    The incidence and mortality of strokes have increased over the past three decades in China. Ischemic strokes can cause a sequence of detrimental events in patients, including increased permeability and dysfunction of the blood-brain barrier, brain edema, metabolic disturbance, endoplasmic reticulum stress, autophagy, oxidative stress, inflammation, neuron death and apoptosis, and cognitive impairment. Thrombolysis using recombinant tissue plasminogen activator (rtPA) and mechanical embolectomy with a retrievable stent are two recognized strategies to achieve reperfusion after a stroke. Nevertheless, rtPA has a narrow therapeutic timeframe, and mechanical embolectomy has limited rates of good neurological outcomes. EGb761 is a standardized and extensively studied extract of Ginkgo biloba leaves. The ginkgolides and bilobalide that constitute a critical part of EGb761 have demonstrated protective properties towards cerebral injury. Ginkgolides include Ginkgolide A (GA), Ginkgolide B (GB), Ginkgolide C (GC), Ginkgolide J (GJ), Ginkgolide K (GK), Ginkgolide L (GL), and Ginkgolide M (GM). This review seeks to elucidate the neuroprotective effects and mechanisms of ginkgolides, especially GA and GB, and bilobalide in cerebral injury following ischemic strokes.

    更新日期:2019-12-21
  • Deficiency of sphingomyelin synthase 1 but not sphingomyelin synthase 2 reduces bone formation due to impaired osteoblast differentiation
    Mol. Med. (IF 2.991) Pub Date : 2019-12-17
    Goichi Matsumoto; Chieko Hashizume; Ken Watanabe; Makoto Taniguchi; Toshiro Okazaki

    There are two isoforms of sphingomyelin synthase (SMS): SMS1 and SMS2. SMS1 is located in the Golgi apparatus only while SMS2 is located in both the plasma membrane and the Golgi apparatus. SMS1 and SMS2 act similarly to generate sphingomyelin (SM). We have undertaken the experiments reported here on SMS and osteoblast differentiation in order to better understand the role SMS plays in skeletal development. We analyzed the phenotype of a conditional knockout mouse, which was generated by mating a Sp7 promoter-driven Cre-expressing mouse with an SMS1-floxed SMS2-deficient mouse (Sp7-Cre;SMS1f/f;SMS2−/− mouse). When we compared Sp7-Cre;SMS1f/f;SMS2−/− mice with C57BL/6, SMS2-deficient mice (SMS1f/f;SMS2−/−) and SP7-Cre positive control mice (Sp7-Cre, Sp7-Cre;SMS1+/+;SMS2+/− and Sp7-Cre;SMS1+/+;SMS2−/−), we found that although cartilage formation is normal, Sp7-Cre;SMS1f/f;SMS2−/− mice showed reduced trabecular and cortical bone mass, had lower bone mineral density, and had a slower mineral apposition rate than control mice. Next, we have used a tamoxifen-inducible knockout system in vitro to show that SMS1 plays an important role in osteoblast differentiation. We cultured osteoblasts derived from ERT2-Cre;SMS1f/f SMS2−/− mice. We observed impaired differentiation of these cells in response to Smad1/5/8 and p38 that were induced by bone morphogenic protein 2 (BMP2). However, Erk1/2 phosphorylation was unaffected by inactivation of SMS1. These findings provide the first genetic evidence that SMS1 plays a role in bone development by regulating osteoblast development in cooperation with BMP2 signaling. Thus, SMS1 acts as an endogenous signaling component necessary for bone formation.

    更新日期:2019-12-18
  • Long non-coding RNA MCM3AP-AS1 promotes growth and migration through modulating FOXK1 by sponging miR-138-5p in pancreatic cancer
    Mol. Med. (IF 2.991) Pub Date : 2019-12-12
    Ming Yang; Shijuan Sun; Yao Guo; Junjie Qin; Guangming Liu

    Pancreatic cancer (PC) is a type of malignant gastrointestinal tumor. Long non-coding RNA MCM3AP antisense RNA 1 (MCM3AP-AS1) has been reported to stimulate proliferation, migration and invasion in several types of tumors. However, the role of MCM3AP-AS1 in PC remains unclear. MCM3AP-AS1, microRNA miR-138-5p (miR-138-5p) and FOXK1 levels were detected using quantitative real time PCR. Cell proliferation, migration and invasion were analyzed. Dual luciferase reporter assay was used to confirm the relationship between MCM3AP-AS1 and miR-138-5p, between miR-138-5p and FOXK1. Protein levels were identified using western blot analysis. MCM3AP-AS1 overexpression promoted proliferation, migration and invasion in PC cells. MCM3AP-AS1 silencing showed a suppressive effect on cell growth in PC cells. Moreover, MCM3AP-AS1 knockdown suppressed tumor growth in mice. Dual luciferase reporter assay demonstrated MCM3AP-AS1 could sponge microRNA-138-5p (miR-138-5p), and FOXK1 could bind with miR-138-5p. Positive correlation between MCM3AP-AS1 and FOXK1 was testified, as well as negative correlation between miR-138-5p and FOXK1. MCM3AP-AS1 promoted FOXK1 expression by targeting miR-138-5p, and MCM3AP-AS1 facilitated growth and invasion in PC cells by FOXK1. MCM3AP-AS1 promoted growth and migration through modulating miR-138-5p/FOXK1 axis in PC, providing insights into MCM3AP-AS1/miR-138-5p/FOXK1 axis as novel candidates for PC therapy from bench to clinic.

    更新日期:2019-12-13
  • Edaravone reduces oxidative stress and intestinal cell apoptosis after burn through up-regulating miR-320 expression
    Mol. Med. (IF 2.991) Pub Date : 2019-12-11
    Jiaxiang Ke; Xi Bian; Hu Liu; Bei Li; Ran Huo

    Intestinal mucosa barrier dysfunction after burn injury is an important factor for causing mortality of burn patients. The current study established a burn model in rats and used a free radical scavenger edaravone (ED) to treat the rats, so as to investigate the effect of edaravone on intestinal mucosa barrier after burn injury. Anesthetized rats were subjected to 40% total body surface area water burn immediately, followed by treatment with ED, scrambled antagomir, or antagomiR-320. Intestinal mucosa damage was observed by hematoxylin-eosin staining and graded by colon mucosal damage index (CMDI) score. The contents of total sulfhydryl (TSH), superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) were determined by spectrophotometry. Cell apoptosis, protein relative expression,and the in situ expressions of p-Akt and p-Bad were detected by flow cytometry, Western blotting and immunohistochemistry, respectively. The miR-320 expression was determined by quantitative real-time polymerase chain reaction. ED alleviated intestinal mucosal damage caused by burn injury, down-regulated the levels of MDA, cytochrome C, cleaved caspase-9 and cleaved caspase-3, but up-regulated the levels of TSH, SOD, CAT and Bcl-2. We also found that ED could reduce oxidative stress, inhibit cell apoptosis, increase the expressions of p-Akt, p-Bad and miR-320, and decrease PTEN expression. PTEN was predicted to be the target gene for miR-320, and cell apoptosis could be promoted by inhibiting miR-320 expression. ED regulates Akt/Bad/Caspase signaling cascade to reduce apoptosis and oxidative stress through up-regulating miR-320 expression and down-regulating PTEN expression, thus protecting the intestinal mucosal barrier of rats from burn injury.

    更新日期:2019-12-11
  • Adverse transverse-tubule remodeling in a rat model of heart failure is attenuated with low-dose triiodothyronine treatment
    Mol. Med. (IF 2.991) Pub Date : 2019-12-06
    Shimin An; Nimra Gilani; Yuan Huang; Adam Muncan; Youhua Zhang; Yi-Da Tang; A. Martin Gerdes; Kaie Ojamaa

    Pre-clinical animal studies have shown that triiodothyronine (T3) replacement therapy improves cardiac contractile function after myocardial infarction (MI). We hypothesized that T3 treatment could prevent adverse post-infarction cardiomyocyte remodeling by maintaining transverse-tubule (TT) structures, thus improving calcium dynamics and contractility. Myocardial infarction (MI) or sham surgeries were performed on female Sprague-Dawley rats (aged 12 wks), followed by treatment with T3 (5μg/kg/d) or vehicle in drinking water for 16 wks (n = 10–11/group). After in vivo echocardiographic and hemodynamic analyses, left ventricular myocytes were isolated by collagenase digestion and simultaneous calcium and contractile transients in single cardiomyocytes were recorded using IonOptix imaging. Live cardiomyocytes were stained with AlexaFluor-488 conjugated wheat germ agglutinin (WGA-488) or di-8-ANEPPS, and multiple z-stack images per cell were captured by confocal microscopy for analysis of TT organization. RTqPCR and immunoblot approaches determined expression of TT proteins. Echocardiography and in vivo hemodynamic measurements showed significant improvements in systolic and diastolic function in T3- vs vehicle-treated MI rats. Isolated cardiomyocyte analysis showed significant dysfunction in measurements of myocyte relengthening in MI hearts, and improvements with T3 treatment: max relengthening velocity (Vmax, um/s), 2.984 ± 1.410 vs 1.593 ± 0.325, p < 0.05 and time to Vmax (sec), 0.233 ± 0.037 vs 0.314 ± 0.019, p < 0.001; MI + T3 vs MI + Veh, respectively. Time to peak contraction was shortened by T3 treatment (0.161 ± 0.021 vs 0.197 ± 0.011 s., p < 0.01; MI + T3 vs MI + Veh, respectively). Analysis of TT periodicity of WGA- or ANEPPS-stained cardiomyocytes indicated significant TT disorganization in MI myocytes and improvement with T3 treatment (transverse-oriented tubules (TE%): 9.07 ± 0.39 sham, 6.94 ± 0.67 MI + Veh and 8.99 ± 0.38 MI + T3; sham vs MI + Veh, p < 0.001; MI + Veh vs MI + T3, p < 0.01). Quantitative RT-PCR showed that reduced expression of BIN1 (Bridging integrator-1), Jph2 (junctophilin-2), RyR2 (ryanodine receptor) and Cav1.2 (L-type calcium channel) in the failing myocardium were increased by T3 and immunoblot analysis further supporting a potential T3 effect on the TT-associated proteins, BIN1 and Jph2. In conclusion, low dose T3 treatment initiated immediately after myocardial infarction attenuated adverse TT remodeling, improved calcium dynamics and contractility, thus supporting the potential therapeutic utility of T3 treatment in heart failure.

    更新日期:2019-12-07
  • Btk inhibitor ibrutinib reduces inflammatory myeloid cell responses in the lung during murine pneumococcal pneumonia
    Mol. Med. (IF 2.991) Pub Date : 2019-01-15
    Alexander P. de Porto; Zhe Liu; Regina de Beer; Sandrine Florquin; Onno J. de Boer; Rudi W. Hendriks; Tom van der Poll; Alex F. de Vos

    Streptococcus pneumoniae is a major causative agent in community-acquired pneumonia and sepsis. Overwhelming lung inflammation during pneumococcal pneumonia may hamper lung function. Ibrutinib is an irreversible inhibitor of Bruton’s tyrosine kinase (Btk), a key signaling protein controlling the activation of various immune cells, including macrophages and neutrophils. The aim of this study was to determine whether ibrutinib treatment ameliorates acute lung inflammation during pneumococcal pneumonia. Mice were treated orally with ibrutinib and the effect on acute pulmonary inflammation elicited by the gram-positive bacterial cell wall component lipoteichoic acid (LTA) and during ceftriaxone-treated pneumococcal pneumonia was assessed. Treatment with ibrutinib prior to and after intranasal LTA instillation reduced alveolar macrophage activation, neutrophil influx, cytokine release and plasma leakage into the lung. Postponed treatment with ibrutinib supplementing antibiotic therapy during ongoing pneumococcal pneumonia did not impair bacterial killing in lung, blood and spleen. In this setting, ibrutinib reduced alveolar macrophage and systemic neutrophil activation and substantially diminished further monocyte and neutrophil influx in the lung. In vitro, ibrutinib inhibited macrophage TNF secretion and neutrophil activation upon LTA and pneumococcal stimulation. Taken together, these data indicate that the Btk inhibitor ibrutinib reduces inflammatory myeloid cell responses during acute pulmonary inflammation evoked by LTA and antibiotic-treated pneumococcal pneumonia and suggest that ibrutinib has the potential to inhibit ongoing lung inflammation in an acute infectious setting.

    更新日期:2019-11-28
  • Molecular expression, characterization and mechanism of ALAS2 gain-of-function mutants
    Mol. Med. (IF 2.991) Pub Date : 2019-01-24
    Vassili Tchaikovskii; Robert J. Desnick; David F. Bishop

    X-linked protoporphyria (XLP) (MIM 300752) is an erythropoietic porphyria due to gain-of-function mutations in the last exon (Ducamp et al., Hum Mol Genet 22:1280-88, 2013) of the erythroid-specific aminolevulinate synthase gene (ALAS2). Five ALAS2 exon 11 variants identified by the NHBLI Exome sequencing project (p.R559H, p.E565D, p.R572C, p.S573F and p.Y586F) were expressed, purified and characterized in order to assess their possible contribution to XLP. To further characterize the XLP gain-of-function region, five novel ALAS2 truncation mutations (p.P561X, p.V562X, p.H563X, p.E569X and p.F575X) were also expressed and studied. Site-directed mutagenesis was used to generate ALAS2 mutant clones and all were prokaryotically expressed, purified to near homogeneity and characterized by protein and enzyme kinetic assays. Standard deviations were calculated for 3 or more assay replicates. The five ALAS2 single nucleotide variants had from 1.3- to 1.9-fold increases in succinyl-CoA Vmax and 2- to 3-fold increases in thermostability suggesting that most could be gain-of-function modifiers of porphyria instead of causes. One SNP (p.R559H) had markedly low purification yield indicating enzyme instability as the likely cause for XLSA in an elderly patient with x-linked sideroblastic anemia. The five novel ALAS2 truncation mutations had increased Vmax values for both succinyl-CoA and glycine substrates (1.4 to 5.6-fold over wild-type), while the Kms for both substrates were only modestly changed. Of interest, the thermostabilities of the truncated ALAS2 mutants were significantly lower than wild-type, with an inverse relationship to Vmax fold-increase. Patients with porphyrias should always be assessed for the presence of the ALAS2 gain-of-function modifier variants identified here. A key region of the ALAS2 carboxyterminal region is identified by the truncation mutations studied here and the correlation of increased thermolability with activity suggests that increased molecular flexibility/active site openness is the mechanism of enhanced function of mutations in this region providing further insights into the role of the carboxyl-terminal region of ALAS2 in the regulation of erythroid heme synthesis.

    更新日期:2019-11-28
  • Angiopoietin 1 influences ischemic reperfusion renal injury via modulating endothelium survival and regeneration
    Mol. Med. (IF 2.991) Pub Date : 2019-02-13
    Wen-Chih Chiang; Yu-Chin Huang; Ten-I Fu; Ping-Min Chen; Fan-Chi Chang; Chun-Fu Lai; Vin-Cent Wu; Shuei-Liong Lin; Yung-Ming Chen

    Damage to the endothelium due to ischemia reperfusion injury (IRI) leads to a disruption of the microvasculature, which could be influenced by angiopoietin 1 via its effects on endothelium. We investigated the physiological and therapeutic roles of angiopoietin 1 in renal IRI using angiopoietin 1 knockout and over-expression mice. Renal IRI was induced by clamping the right renal artery seven days after left uninephrectomy for 25 min followed by reperfusion. A whole body angiopoietin 1 knockout was achieved by induction with tamoxifen. The renal tubule over-expression of angiopoietin 1 was induced by doxycycline. In the normal mice, the renal expression of angiopoietin 1 increased 7 days to 14 days after IRI. The angiopoietin 1 knockout caused a delay in the recovery of renal function, less tubular regeneration and more residual tubular necrosis. The endothelial density was lower and the VE-cadherin protein loss was greater in the knockout mice. The over-expression of angiopoietin 1 attenuated the tubular necrosis and renal function impairment 1 and 3 days after IRI. The loss of the endothelium was ameliorated in the over-expression mice. This protective effect was associated with the up-regulation of the gene expression of epidermal growth factor, hepatocyte growth factor, and insulin like growth factor-1 and less tubular apoptosis. The over-expression of angiopoietin 1 stimulated tumor necrosis factor-α, C-C chemokine receptor type 2 and CX3C chemokine receptor 1 inflammatory gene expression, but did not influence macrophage infiltration. Altogether, the augmentation and downregulation of angiopoietin 1 attenuated renal damage and impaired renal recovery, respectively, by influencing the survival/regeneration of the endothelium. The manipulation of angiopoietin 1 represents a novel therapeutic approach for the treatment of ischemic kidney injury.

    更新日期:2019-11-28
  • Further corroboration of distinct functional features in SCN2A variants causing intellectual disability or epileptic phenotypes
    Mol. Med. (IF 2.991) Pub Date : 2019-02-27
    Anaïs Begemann; Mario A. Acuña; Markus Zweier; Marie Vincent; Katharina Steindl; Ruxandra Bachmann-Gagescu; Annette Hackenberg; Lucia Abela; Barbara Plecko; Judith Kroell-Seger; Alessandra Baumer; Kazuhiro Yamakawa; Yushi Inoue; Reza Asadollahi; Heinrich Sticht; Hanns Ulrich Zeilhofer; Anita Rauch

    Deleterious variants in the voltage-gated sodium channel type 2 (Nav1.2) lead to a broad spectrum of phenotypes ranging from benign familial neonatal-infantile epilepsy (BFNIE), severe developmental and epileptic encephalopathy (DEE) and intellectual disability (ID) to autism spectrum disorders (ASD). Yet, the underlying mechanisms are still incompletely understood. To further elucidate the genotype-phenotype correlation of SCN2A variants we investigated the functional effects of six variants representing the phenotypic spectrum by whole-cell patch-clamp studies in transfected HEK293T cells and in-silico structural modeling. The two variants p.L1342P and p.E1803G detected in patients with early onset epileptic encephalopathy (EE) showed profound and complex changes in channel gating, whereas the BFNIE variant p.L1563V exhibited only a small gain of channel function. The three variants identified in ID patients without seizures, p.R937C, p.L611Vfs*35 and p.W1716*, did not produce measurable currents. Homology modeling of the missense variants predicted structural impairments consistent with the electrophysiological findings. Our findings support the hypothesis that complete loss-of-function variants lead to ID without seizures, small gain-of-function variants cause BFNIE and EE variants exhibit variable but profound Nav1.2 gating changes. Moreover, structural modeling was able to predict the severity of the variant impact, supporting a potential role of structural modeling as a prognostic tool. Our study on the functional consequences of SCN2A variants causing the distinct phenotypes of EE, BFNIE and ID contributes to the elucidation of mechanisms underlying the broad phenotypic variability reported for SCN2A variants.

    更新日期:2019-11-28
  • LncRNA PVT1 regulates atrial fibrosis via miR-128-3p-SP1-TGF-β1-Smad axis in atrial fibrillation
    Mol. Med. (IF 2.991) Pub Date : 2019-03-20
    Feng Cao; Zhe Li; Wen-mao Ding; Ling Yan; Qing-yan Zhao

    Long non-coding RNAs (lncRNA) plasmacytoma variant translocation 1 (PVT1) has been shown to be associated with liver fibrosis. Nevertheless, the role of PVT1 in atrial fibrosis remains undefined. This study aims to elucidate the pathophysiological role of lncRNA PVT1 in the regulation of atrial fibrosis and to explore the underlying mechanism. Expression of PVT1, miR-128-sp, and Sp1 were examined in human atrial muscle tissues and angiotensin-II (Ang-II)-induced human atrial fibroblasts. Furthermore, the role of PVT1 in regulating atrial fibrosis in Ang-II-treated human atrial fibroblasts and Ang-II-induced atrial fibrosis in mice was investigated. Moreover, the interaction among PVT1, miR-128-3p, and Sp1 were examined using bioinformatics, expression correlation analysis, gain- or loss-of-function assays, RIP assays, and luciferase reporter assays. The involvement of transforming growth factor beta 1 (TGF-β1)/Smad pathway in this process was also explored. PVT1 was increased in atrial muscle tissues from AF patients and positively with collagen I and collagen III. In vitro assay revealed that PVT1 overexpression facilitated the Ang-II-induced atrial fibroblasts proliferation, collagen production, and TGF-β1/Smad signaling activation, whereas PVT1 knockdown caused the opposite effect. In vivo assay further confirmed that PVT1 knockdown attenuated the Ang-II-induced mouse atrial fibrosis. Mechanically, PVT1 acted as a sponge for miR-128-3p to facilitate Sp1 expression, thereby activating the TGF-β1/Smad signaling pathway. LncRNA PVT1 promotes atrial fibrosis via miR-128-3p-SP1-TGF-β1-Smad axis in atrial fibrillation.

    更新日期:2019-11-28
  • Association between admission plasma 2-oxoglutarate levels and short-term outcomes in patients with acute heart failure: a prospective cohort study
    Mol. Med. (IF 2.991) Pub Date : 2019-03-28
    Zhengliang Peng; Qiong Zhan; Xiangkun Xie; Hanlin Li; Yan Tu; Yujia Bai; Xingfu Huang; Wenyan Lai; Boxin Zhao; Qingchun Zeng; Dingli Xu

    2-oxoglutarate (2OG), an intermediate metabolite in the tricarboxylic acid cycle, has been found to associate with chronic heart failure (HF), but its effect on short-term adverse outcomes in patients with acute HF (AHF) is uncertain. This prospective cohort study included 411 consecutive hospitalized patients with AHF. During hospitalization, fasting plasma samples were collected within the first 24 h of admission. Plasma 2OG levels were measured by hydrophilic interaction liquid chromatography-liquid chromatography tandem mass spectrometry (HILIC-LC/MS/MS). All participants were followed up for six months. Multiple logistic regression was used to determine the odds ratio (OR) and 95% confidence interval (CI) for primary outcomes. The AHF cohort consisted of HF with preserved ejection fraction (EF) (64.7%), mid-range EF (16.1%), and reduced EF (19.2%), the mean age was 65 (±13) years, and 65.2% were male. Participants were divided into two groups based on median 2OG levels (μg/ml): low group (< 6.0, n = 205) and high group (≥6.0, n = 206). There was a relatively modest correlation between 2OG and N-terminal pro B-type natriuretic peptide (NT-proBNP) levels (r = 0.25; p < 0.001). After adjusting for age, sex, and body mass index, we found that the progression of the NYHA classification was associated with a gradual increase in plasma 2OG levels (p for trend< 0.001). After six months of follow-up, 76 (18.5%) events were identified. A high baseline 2OG level was positively associated with a short-term rehospitalization and all-cause mortality (OR: 2.2, 95% CI 1.3–3.7, p = 0.003), even after adjusting for NT-proBNP and estimated glomerular filtration rate (eGFR) (OR: 1.9, 95% CI 1.1–3.4, p = 0.032). After a similar multivariable adjustment, the OR was 1.4 (95% CI 1.1–1.7, p = 0.018) for a per-SD increase in 2OG level. High baseline 2OG levels are associated with adverse short-term outcomes in patients with AHF independent of NT-proBNP and eGFR. Hence plasma 2OG measurements may be helpful for risk stratification and treatment monitoring in AHF. ChiCTR-ROC-17011240 . Registered 25 April 2017.

    更新日期:2019-11-28
  • Differentiation of adult human retinal pigment epithelial cells into dopaminergic-like cells in vitro and in the recipient monkey brain
    Mol. Med. (IF 2.991) Pub Date : 2019-03-29
    Sha Li; Han Zhang; Aifang Wang; Yan Liu; Houqi Liu; Feng Yue; Xianmixinuer Abulaiti; Caiqiao Zhang; Lingsong Li

    Cell therapy is proposed to be a potential treatment for Parkinson’s disease (PD). Although fetal retinal pigment epithelial (RPE) cells have been tested in trials for treating PD patients, controversy has been raised over the issue of whether such cells can be reprogrammed into dopamine-producing cells for therapeutic efficacy. Here, we aim to investigate whether adult human RPE cells can be reprogrammed into dopamine-producing cells both in vitro and in the recipient monkey brain. The RPE layer was isolated from frozen posterior eyeball tissue after penetrating keratoplasty surgery. The tumorigenicity of RPE cells was examined by G-banding and a tumor formation assay in nude mice. Immunogenicity was measured using a one-way mixed lymphocyte reaction (MLR) assay. Dopamine-production in chemically reprogrammed RPE cells was measured by HPLC. Finally, RPE cells were grafted into the brains of monkeys with MPTP-induced PD in order to investigate the potential of such cells treating PD patients in the future. RPE cell lines have been successively established from adult human eye tissues. Such cells can be chemically reprogrammed into dopamine-producing cells in vitro. Moreover, after being grafted into the brain caudate putamen of monkeys with MPTP-induced PD, RPE cells became tyrosine hydroxylase-positive cells, and recipient PD monkeys showed significant improvement of clinical conditions. This preclinical study using a primate model indicates that human adult RPE cells could be a potential cell source for the treatment of PD in the future.

    更新日期:2019-11-28
  • HDAC6 is associated with the formation of aortic dissection in human
    Mol. Med. (IF 2.991) Pub Date : 2019-03-29
    Xian Guo; Ze-Min Fang; Xiang Wei; Bo Huo; Xin Yi; Cai Cheng; Jun Chen; Xue-Hai Zhu; Anas Omar Khalil Abu Bokha; Ding-Sheng Jiang

    The pathological features of aortic dissection (AD) include vascular smooth muscle cell (VSMC) loss, elastic fiber fraction, and inflammatory responses in the aorta. However, little is known about the post-translational modification mechanisms responsible for these biological processes. A total of 72 aorta samples, used for protein detection, were collected from 36 coronary artery disease (CAD, served as the control) patients and 36 type A AD (TAAD) patients. Chromatin immunoprecipitation (ChIP)-PCR was used to identify the genes regulated by H3K23ac, and tubastatin A, an inhibitor of HDAC6, was utilized to clarify the downstream mechanisms regulated by HDAC6. We found that the protein level of histone deacetylase HDAC6 was reduced in the aortas of patients suffering from TAAD and that the protein levels of H4K12ac, and H3K23ac significantly increased, while H3K18ac, H4K8ac, and H4K5ac dramatically decreased when compared with CAD patients. Although H3K23ac, H3K18ac, and H4K8ac increased in the human VSMCs after treatment with the HDAC6 inhibitor tubastatin A, only H3K23ac showed the same results in human tissues. Notably, the results of ChIP-PCR demonstrated that H3K23ac was enriched in extracellular matrix (ECM)-related genes, including Col1A2, Col3A1, CTGF, POSTN, MMP2, TIMP2, and ACTA2, in the aortic samples of TAAD patients. In addition, our results showed that HDAC6 regulates H4K20me2 and p-MEK1/2 in the pathological process of TAAD. These results indicate that HDAC6 is involved in human TAAD formation by regulating H3K23ac, H4K20me2 and p-MEK1/2, thus, providing a strategy for the treatment of TAAD by targeting protein post-translational modifications (PTMs), chiefly histone PTMs.

    更新日期:2019-11-28
  • PGK1 facilities cisplatin chemoresistance by triggering HSP90/ERK pathway mediated DNA repair and methylation in endometrial endometrioid adenocarcinoma
    Mol. Med. (IF 2.991) Pub Date : 2019-03-29
    Jing-Wei Zhou; Juan-Juan Tang; Wei Sun; Hui Wang

    Endometrial carcinoma represents one of the most common cancer types of the female reproductive tract. If diagnosed at an early stage, the 5-year survival rate is promising. However, recurrence and chemoresistance remain problematic for at least 15% of the patients. In the present study, we aim to reveal the mechanism by which PGK1 regulates chemoresistance in endometrial carcinoma. qPCR was performed to detect expression of PGK1 in clinical tissue samples of endometrial carcinoma. Specific shRNAs were employed to knockdown PGK1 expression in endometrial cancer cell lines. MTT assay was used to evaluate cell viability and cisplatin sensitivity of endometrial carcinoma cell lines. Western blot was performed to assess the effects of PGK1 knockdown on the expression levels of HSP90, DNA repair-associated proteins (c-JUN, FOSL1, and POLD1), and DNA methylation-related enzymes (DNMT1, DNMT3A and DNMT3B). Immunoprecipitation was performed to verify direct binding between PGK1 and HSP90. We first showed that PGK1 expression is elevated in tumor tissues of endometrial cancer, and high PGK1 levels are associated with clinical stages and metastasis. Knockdown of PGK1 inhibits proliferation of endometrial cancer cells, and enhances the inhibitory effect of cisplatin on cell viability. In addition, knockdown of PGK1 down-regulates the expression of DNA repair-related proteins, methylation-related enzymes, and total cellular methylation level. PGK1 was next shown to interact directly with HSP90 and exhibit pro-tumor effects by modulating the ATPase activity of HSP90. We propose that PGK1 mediates DNA repair and methylation through the HSP90/ERK pathway, and eventually enhances the chemoresistance to cisplatin. The results provide new insights on functions of PGK1 and HSP90, which might make them as promising targets for endometrial cancer chemotherapy.

    更新日期:2019-11-28
  • Mus musculus deficient for secretory antibodies show delayed growth with an altered urinary metabolome
    Mol. Med. (IF 2.991) Pub Date : 2019-04-03
    Kim R. Simpfendorfer; Nancy Wang; Dedreia L. Tull; David P. De Souza; Amsha Nahid; Andre Mu; Dianna M. Hocking; John S. Pedersen; Odilia L. C. Wijburg; Malcolm J. McConville; Richard A. Strugnell

    The polymeric immunoglobulin receptor (pIgR) maintains the integrity of epithelial barriers by transporting polymeric antibodies and antigens through the epithelial mucosa into the lumen. In this study, we examined the role of pIgR in maintaining gut barrier integrity, which is important for the normal development in mice. Cohorts of pIgR−/− mice and their wildtype controls were housed under Specific Pathogen Free (SPF) conditions and monitored for weight gain as an indicator of development over time. The general physiology of the gastrointestinal tract was analysed using immunohistochemistry in young (8–12 weeks of age) and aged mice (up to 18 months of age), and the observed immunopathology in pIgR−/− mice was further characterised using flow cytometry. Urinary metabolites were analysed using gas chromatography-mass spectrometry (GC-MS), which revealed changes in metabolites that correlated with age-related increase in gut permeability in pIgR−/− mice. We observed that pIgR−/− mice exhibited delayed growth, and this phenomenon is associated with low-grade gut inflammation that increased with ageing. The gross intraepithelial lymphocytic (IEL) infiltration characteristic of pIgR−/− mice was redefined as CD8α+αβ+ T cells, the majority of which expressed high levels of CD103 and CD69 consistent with tissue resident memory T cells (TRM). Comparison of the urinary metabolome between pIgR−/− and wild-type mice revealed key changes in urinary biomarkers fucose, glycine and Vitamin B5, suggestive of altered mucosal permeability. A significant increase in gut permeability was confirmed by analysing the site-specific uptake of sugar probes in different parts of the intestine. Our data show that loss of the secretory antibody system in mice results in enhanced accumulation of inflammatory IELs in the gut, which likely reflects ongoing inflammation in reaction to gut microbiota or food antigens, leading to delayed growth in pIgR−/− mice. We demonstrate that this leads to the presence of a unique urinary metabolome profile, which may provide a biomarker for altered gut permeability.

    更新日期:2019-11-28
  • Inhibition of HMGB1/RAGE-mediated endocytosis by HMGB1 antagonist box A, anti-HMGB1 antibodies, and cholinergic agonists suppresses inflammation
    Mol. Med. (IF 2.991) Pub Date : 2019-04-11
    Huan Yang; Hui Liu; Qiong Zeng; Gavin H. Imperato; Meghan E. Addorisio; Jianhua Li; Mingzhu He; Kai Fan Cheng; Yousef Al-Abed; Helena E. Harris; Sangeeta S. Chavan; Ulf Andersson; Kevin J. Tracey

    Extracellular high mobility group box 1 protein (HMGB1) serves a central role in inflammation as a transporter protein, which binds other immune-activating molecules that are endocytosed via the receptor for advanced glycation end-products (RAGE). These pro-inflammatory complexes are targeted to the endolysosomal compartment, where HMGB1 permeabilizes the lysosomes. This enables HMGB1-partner molecules to avoid degradation, to leak into the cytosol, and to reach cognate immune-activating sensors. Lipopolysaccharide (LPS) requires this pathway to generate pyroptosis by accessing its key cytosolic receptors, murine caspase 11, or the human caspases 4 and 5. This lytic, pro-inflammatory cell death plays a fundamental pathogenic role in gram-negative sepsis. The aim of the study was to identify molecules inhibiting HMGB1 or HMGB1/LPS cellular internalization. Endocytosis was studied in cultured macrophages using Alexa Fluor-labeled HMGB1 or complexes of HMGB1 and Alexa Fluor-labeled LPS in the presence of an anti-HMGB1 monoclonal antibody (mAb), recombinant HMGB1 box A protein, acetylcholine, the nicotinic acetylcholine receptor subtype alpha 7 (α7 nAChR) agonist GTS-21, or a dynamin-specific inhibitor of endocytosis. Images were obtained by fluorescence microscopy and quantified by the ImageJ processing program (NIH). Data were analyzed using student’s t test or one-way ANOVA followed by the least significant difference or Tukey’s tests. Anti-HMGB1 mAb, recombinant HMGB1 antagonist box A protein, acetylcholine, GTS-21, and the dynamin-specific inhibitor of endocytosis inhibited internalization of HMGB1 or HMGB1-LPS complexes in cultured macrophages. These agents prevented macrophage activation in response to HMGB1 and/or HMGB1-LPS complexes. These results demonstrate that therapies based on HMGB1 antagonists and the cholinergic anti-inflammatory pathway share a previously unrecognized molecular mechanism of substantial clinical relevance.

    更新日期:2019-11-28
  • Validation of impaired Transient Receptor Potential Melastatin 3 ion channel activity in natural killer cells from Chronic Fatigue Syndrome/ Myalgic Encephalomyelitis patients
    Mol. Med. (IF 2.991) Pub Date : 2019-04-23
    H. Cabanas; K. Muraki; C. Balinas; N. Eaton-Fitch; D. Staines; S. Marshall-Gradisnik

    Chronic Fatigue Syndrome/ Myalgic Encephalomyelitis (CFS/ME) is a complex multifactorial disorder of unknown cause having multi-system manifestations. Although the aetiology of CFS/ME remains elusive, immunological dysfunction and more particularly reduced cytotoxic activity in natural killer (NK) cells is the most consistent laboratory finding. The Transient Receptor Potential (TRP) superfamily of cation channels play a pivotal role in the pathophysiology of immune diseases and are therefore potential therapeutic targets. We have previously identified single nucleotide polymorphisms in TRP genes in peripheral NK cells from CFS/ME patients. We have also described biochemical pathway changes and calcium signaling perturbations in NK cells from CFS/ME patients. Notably, we have previously reported a decrease of TRP cation channel subfamily melastatin member 3 (TRPM3) function in NK cells isolated from CFS/ME patients compared with healthy controls after modulation with pregnenolone sulfate and ononetin using a patch-clamp technique. In the present study, we aim to confirm the previous results describing an impaired TRPM3 activity in a new cohort of CFS/ME patients using a whole cell patch-clamp technique after modulation with reversible TRPM3 agonists, pregnenolone sulfate and nifedipine, and an effective TRPM3 antagonist, ononetin. Indeed, no formal research has commented on using pregnenolone sulfate or nifedipine to treat CFS/ME patients while there is evidence that clinicians prescribe calcium channel blockers to improve different symptoms. Whole-cell patch-clamp technique was used to measure TRPM3 activity in isolated NK cells from twelve age- and sex-matched healthy controls and CFS/ME patients, after activation with pregnenolone sulfate and nifedipine and inhibition with ononetin. We confirmed a significant reduction in amplitude of TRPM3 currents after pregnenolone sulfate stimulation in isolated NK cells from another cohort of CFS/ME patients compared with healthy controls. The pregnenolone sulfate-evoked ionic currents through TRPM3 channels were again significantly modulated by ononetin in isolated NK cells from healthy controls compared with CFS/ME patients. In addition, we used nifedipine, another reversible TRPM3 agonist to support the previous findings and found similar results confirming a significant loss of the TRPM3 channel activity in CFS/ME patients. Impaired TRPM3 activity was validated in NK cells isolated from CFS/ME patients using different pharmacological tools and whole-cell patch-clamp technique as the gold standard for ion channel research. This investigation further helps to establish TRPM3 channels as a prognostic marker and/ or a potential therapeutic target for CFS/ME.

    更新日期:2019-11-28
  • Tag-based next generation sequencing: a feasible and reliable assay for EGFR T790M mutation detection in circulating tumor DNA of non small cell lung cancer patients
    Mol. Med. (IF 2.991) Pub Date : 2019-04-27
    Mariella Dono; Giuseppa De Luca; Sonia Lastraioli; Giorgia Anselmi; Maria Giovanna Dal Bello; Simona Coco; Irene Vanni; Francesco Grossi; Antonella Vigani; Carlo Genova; Manlio Ferrarini; Jean Louis Ravetti; Simona Zupo

    The demonstration of EGFR T790M gene mutation in plasma is crucial to assess the eligibility of Non Small Cell Lung Cancer (NSCLC) patients, who have acquired resistance to first or second generation Tyrosine Kinase Inhibitors (TKIs), to receive a subsequent treatment with osimertinib. Since circulating tumor DNA (ctDNA) is present in very low amounts in plasma, high sensitive and specific methods are required for molecular analysis. Improving sensitivity of T790M mutation detection in plasma ctDNA enables a larger number of NSCLC patients to receive the appropriate therapy without any further invasive procedure. A tag-based next generation sequencing (NGS) platform capable of tagging rare circulating tumor DNA alleles was employed in this study for the identification of T790M mutation in 42 post-TKI NSCLC patients. Compared to Real Time PCR, tag-based NGS improved the T790M detection rate (42.85% versus 21.4%, respectively), especially in those cases with a low median mutation abundance (i.e. 0.24, range 0.07–0.78). Moreover, the tag-based NGS identified EGFR activating mutations more efficiently than Real Time PCR (85.7% versus 61.9% detection rate, respectively), particularly of the L858R variant type (0.06–0.75 mutation abundance range). Patients in whom the T790M mutation was detected in plasma, achieved an objective response to osimertinib (9/14, 64.28%). Tag-based NGS represents an accurate and sensitive tool in a clinical setting for non-invasive assessment and monitoring of T790M variant in NSCLC patients.

    更新日期:2019-11-28
  • Increased proteinase 3 and neutrophil elastase plasma concentrations are associated with non-alcoholic fatty liver disease (NAFLD) and type 2 diabetes
    Mol. Med. (IF 2.991) Pub Date : 2019-05-02
    Andreea-Manuela Mirea; Erik J. M. Toonen; Inge van den Munckhof; Isabelle D. Munsterman; Eric T. T. L. Tjwa; Martin Jaeger; Marije Oosting; Kiki Schraa; Joost H. W. Rutten; Marinette van der Graaf; Niels P. Riksen; Jacqueline de Graaf; Mihai G. Netea; Cees J. Tack; Triantafyllos Chavakis; Leo A. B. Joosten

    Non-alcoholic fatty liver disease (NAFLD) is becoming a major health problem worldwide. Inflammation plays an important role in disease pathogenesis and recent studies have shown a potential role for the neutrophil serine proteases (NSPs) proteinase-3 (PR3) and neutrophil elastase (NE) in NAFLD as well as an imbalance between NSPs and their natural inhibitor alpha-1 antitrypsin (AAT). The aim of this study was to investigate whether PR3 and NE plasma concentrations are associated with NAFLD and/or type 2 diabetes. To explore this hypothesis we used several cohorts: a cohort of 271 obese individuals with liver steatosis, a cohort of 41 patients with biopsy-proven NAFLD, a cohort of 401 obese type 2 diabetes patients and a cohort of 205 lean healthy controls; and measured PR3 and NE plasma concentrations. In addition, we measured AAT plasma concentrations in order to investigate if the ratios between NSPs and their natural inhibitor were altered in NAFLD and type 2 diabetes when compared to healthy controls. Our data shows an increase in PR3 and NE concentrations and a decrease in AAT concentrations in obese patients when compared to controls. Moreover, PR3 plasma concentrations are increased in patients with liver steatosis. Furthermore, PR3 and NE concentrations in the liver are associated with the advanced stages of NAFLD characterized by NASH and/ or liver fibrosis. Additionally, PR3 and NE concentrations were up-regulated in patients with type 2 diabetes when compared to lean and obese controls. We conclude that circulating levels of NSPs associate with obesity-related metabolic disorders. Further research is needed to clearly establish the role of these proteases and investigate whether they could be used as non-invasive markers for NAFLD and/or type 2 diabetes.

    更新日期:2019-11-28
  • Silencing of SAA1 inhibits palmitate- or high-fat diet induced insulin resistance through suppression of the NF-κB pathway
    Mol. Med. (IF 2.991) Pub Date : 2019-05-06
    Yong Wang; Feng Cao; Yang Wang; Gang Yu; Ben-Li Jia

    Obesity is one of the leading causes of insulin resistance. Accumulating reports have highlighted that serum amyloid A-1 (SAA1) is a potential candidate that is capable of attenuating insulin resistance. Hence, we conducted the current study with aims of investigating our proposed hypothesis that silencing SAA1 could inhibit the progression of obesity-induced insulin resistance through the NF-κB pathway. Gene expression microarray analysis was initially performed to screen differentially expressed genes (DEGs) associated with obesity. Palmitate (PA)-induced insulin resistance Huh7 cell models and high-fat diet (HFD)-induced mouse models were established to elucidate the effect of SAA1/Saa1 on insulin resistance. The NF-κB pathway-related expression was subsequently determined through the application of reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis. Saa1 was identified as an obesity-related gene based on the microarray data of GSE39549. Saa1 was determined to be highly expressed in HFD-induced insulin resistance mouse models. PA-induced Huh7 cells, treated with silenced SAA1 or NF-κB pathway inhibition using BAY 11–7082, displayed a marked decrease in both Saa1 and SOCS3 as well as an elevation in 2DG, IRS1 and the extent of IRS1 phosphorylation. HFD mice treated with silenced Saa1 or inhibited NF-κB pathway exhibited improved fasting blood glucose (FBG) levels as well as fasting plasma insulin (FPI) levels, glucose tolerance and systemic insulin sensitivity. Saa1/SAA1 was determined to show a stimulatory effect on the transport of the NF-κBp65 protein from the cytoplasm to the nucleus both in vivo and in vitro, suggesting that Saa1/SAA1 could activate the NF-κB pathway. Taken together, our key findings highlight a novel mechanism by which silencing of SAA1 hinders PA or HFD-induced insulin resistance through inhibition of the NF-κB pathway.

    更新日期:2019-11-28
  • Circulating miR-26a-1, miR-146a and miR-199a-1 are potential candidate biomarkers for acute myocardial infarction
    Mol. Med. (IF 2.991) Pub Date : 2019-05-15
    Sheng Xue; Wenjie Zhu; Dacheng Liu; Zhe Su; Liwei Zhang; Qing Chang; Peifeng Li

    Acute myocardial infarction (AMI) was considered to be one of the major causes of morbidity and mortality worldwide. In order to manage the acute myocardial infarction outbreaks, accurate biomarkers for risk prediction are needed. Circulating microRNAs (miRNAs) may act as diagnostic and prognostic biomarkers for cardiovascular events. This study aimed to determine the possibility of circulating miRNAs used as biomarkers for AMI and their dynamic expression levels before and after percutaneous coronary intervention (PCI) in patients. Circulating miR-26a-1, miR-27a, miR-30d, miR-146a, miR-199a-1 and miR-423 were selected and validated in 31 AMI patients and 27 matched controls by quantitative real-time PCR (qPCR). The expression levels of plasma miR-26a-1, miR-146a and miR-199a-1 were significantly increased in AMI patients. Receiver operating characteristic (ROC) analysis indicated that miR-26a-1, miR-146a and miR-199a-1 showed considerable diagnostic efficiency for predicting AMI. Furthermore, we demonstrated that the combination of miR-26a-1, miR-146a and miR-199a-1 facilitated AMI diagnosis. Our findings suggest that circulating miR-26a-1, miR-146a and miR-199a-1 have the potential to be used as biomarkers for AMI diagnosis.

    更新日期:2019-11-28
  • Temporal progression of gene regulation of peripheral white blood cells explains gender dimorphism of critically ill patients after trauma
    Mol. Med. (IF 2.991) Pub Date : 2019-05-16
    Amol Kolte; Rainer König

    The immune response of the critically ill after severe trauma is sex-specific and may explain the different progression of the disease. This may be explained by a different gene regulatory program of their peripheral immune cells. We investigated the progression of the transcription profiles of peripheral immune cells of the patients to elucidate their distinct physiological response and clinical course. We compared transcription profiles of whole blood of male and female patients from a larger longitudinal study of critically ill patients after trauma. We developed a statistical analysis pipeline that synchronized the time lapse of the profiles based on the temporal severity score of each patient. This enabled to categorize the temporal progression of the disease into two pre-acute, an acute and two post-acute phases. Comparing gene regulation of male and female patients at each phase, we identified distinctively regulated molecular processes mainly in the immune response, but also in the regulation of metabolism allowing to cluster these discriminative gene sets into sets of highly related cellular processes. Compared to male patients and healthy controls, female patients showed upregulation of gene sets of innate immunity in the early phase, upregulation of wound healing processes during the acute phase and upregulation of adaptive immunity in the late phase indicating early recovery. In turn, during the pre-acute and acute phase, male patients showed less suppression of gene sets coding for enzymes of energy metabolism and anabolism, most prominently the tricarboxylic acid cycle and β-oxidation, and cellular maintenance, such as cell cycle, DNA replication and damage response, and RNA metabolism. A stronger innate immune response at the very early phase of the disease may support early clearance of the pathogen and its associated molecular patterns. Upregulation of wound healing processes may explain reduced multiple organ failure during the acute phase. Down regulated energy metabolism during the acute phase may make female patients less susceptible to oxidative stress, the upregulated adaptive immune system reflects an earlier recovery and rebuilding of the adaptive immune system that may protect them from secondary infections. Follow up studies need to be performed confirming these observations experimentally.

    更新日期:2019-11-28
  • Astragalus polysaccharides suppresses high glucose-induced metabolic memory in retinal pigment epithelial cells through inhibiting mitochondrial dysfunction-induced apoptosis by regulating miR-195
    Mol. Med. (IF 2.991) Pub Date : 2019-05-22
    Ping Liu; Qing-Hua Peng; Ping Tong; Wen-Jie Li

    Metabolic memory contributes to the development of diabetic retinopathy (DR), which is the complication of diabetes. But it’s still unknown how to prevent the metabolic memory to treat the DR. In our study, we want to examine the function of Astragalus polysaccharides (APS) in the metabolic memory of retinal pigment epithelium (RPE) pretreated with high glucose (HG). ARPE-19 and PRPE cells were exposed to HG followed by normal glucose (NG) treatment with or without APS. QPCR was used to examine the levels of miR-195 and Bcl-2. MDA and SOD detection assays were used to examine the oxidative stress level. Western blotting and immunostaining were applied to detect the protein level of mitochondrial damage and apoptotic signaling pathway. Flow cytometry and TUNEL staining were used to analyze cell apoptosis. Luciferase assay was used to examine the direct target of miR-195. APS treatment significantly decreased the expression of miR-195, while increased the expression of Bcl-2 with optimized dosages which were induced by HG treatment, even after replacing the HG with NG. And we found Bcl-2 was the direct target of miR-195. APS alleviated the oxidative stress, mitochondrial damage and cell apoptosis induced by HG and HG + NG treatments in RPE cells via regulating miR-195. Furthermore, we found overexpression of miR-195 abolished the alleviated effects of APS on the HG-treated RPE cells. APS suppressed high glucose-induced metabolic memory in retinal pigment epithelial cells through inhibiting mitochondrial dysfunction-induced apoptosis by regulating miR-195.

    更新日期:2019-11-28
  • Electroacupuncture attenuated cerebral ischemic injury and neuroinflammation through α7nAChR-mediated inhibition of NLRP3 inflammasome in stroke rats
    Mol. Med. (IF 2.991) Pub Date : 2019-05-22
    Tao Jiang; Meiyan Wu; Zhanqin Zhang; Chaoying Yan; Zhi Ma; Shan He; Wei Yuan; Kairui Pu; Qiang Wang

    Our previous research confirmed that electroacupuncture (EA) stimulus elicits neuroprotective effects against cerebral ischemic injury through α7 nicotinic acetylcholine receptor (α7nAChR)-mediated inhibition of high-mobility group box 1 release mechanism. This study investigated whether the signal transducer of α7nAChR and inhibition of NLRP3 inflammasome are involved in the neuroprotective effects of EA stimulus. In adult male Sprague-Dawley rats, the focal cerebral ischemic injury was induced by middle cerebral artery occlusion (MCAO) models for 1.5 h. The expression of NLRP3 inflammasome in the penumbral tissue following reperfusion was assessed by western blotting and immunoflourescent staining. The infarct size, neurological deficit score, TUNEL staining and the expression of proinflammatory factors or anti-inflammatory cytokines were evaluated at 72 h after reperfusion in the presence or absence of either α7nAChR antagonist (α-BGT) or agonist (PHA-543,613). The contents of inflammasome proteins were gradually increased after cerebral ischemia/reperfusion (I/R). EA stimulus attenuated NLRP3 inflammasome mediated inflammatory reaction and regulated the balance between proinflammatory factors and anti-inflammatory cytokines. The agonist of α7nAChR induced similar neuroprotective effects as EA stimulus. In contrast, α7nAChR antagonist reversed not only the neuroprotective effects, but also the inhibitory effects of NLRP3 inflammasome and the regulatory effects on the balance between proinflammatory factors and anti-inflammatory cytokines. These results provided compelling evidence that α7nAChR played a pivotal role in regulating the activation and expression of NLRP3 inflammasome in neurons after cerebral I/R. These findings highlighted a novel anti-inflammatory mechanism of EA stimulus by α7nAChR modulating the inhibition of NLRP3 inflammasome, suggesting that α7nAChR-dependent cholinergic anti-inflammatory system and NLRP3 inflammasome in neurons might act as potential therapeutic targets in EA induced neuroprotection against cerebral ischemic injury.

    更新日期:2019-11-28
  • CKD-602, a topoisomerase I inhibitor, induces apoptosis and cell-cycle arrest and inhibits invasion in cervical cancer
    Mol. Med. (IF 2.991) Pub Date : 2019-05-28
    Sungha Lee; Jung Yoon Ho; Jing Jing Liu; Hyewon Lee; Jae Young Park; Minwha Baik; Minji Ko; Seon Ui Lee; Youn Jin Choi; Soo Young Hur

    Cervical cancer is the third most common gynecological malignancy. Conventional treatment options are known to be ineffective for the majority of patients with advanced or recurrent cervical cancer. Therefore, novel therapeutic agents for cervical cancer are necessary. In this study, the effects of CKD-602 in cervical cancer were investigated. Three established human, immortalized, cervical cancer cell lines (CaSki, HeLa and SiHa) were used in this study. Following treatment with CKD-602, apoptosis was quantified using fluorescein isothiocyanate Annexin V-FITC and propidium iodide (PI) detection kit and cell cycle analysis was analyzed using fluorescence activated cell sorting (FACS). Transwell chambers were used for invasion assays. Western blot assay was performed to analyze proteomics. CaSki cells were subcutaneously injected into BALB/c-nude mice and cervical cancer xenograft model was established to elucidate the antitumor effect of CKD-602 in vivo. Treatment with CKD-602 induced apoptosis and increased expression of the enzyme PARP, cleaved PARP, and BAX. In addition, expression of phosphorylated p53 increased. Cell cycle arrest at G2/M phase and inhibition of invasion were detected after treatment with CKD-602. A significant decrease in cervical cancer tumor volume was observed in this in vivo model, following treatment with CKD-602. This is the first report of CKD-602 having an antitumor effect in cervical cancer in both an in vitro and in vivo models. The results of this study indicate that CKD-602 may be a novel potential drug, targeting cervical cancer, providing new opportunities in the development of new therapeutic strategies.

    更新日期:2019-11-28
  • Extracellular cold inducible RNA-binding protein mediates binge alcohol-induced brain hypoactivity and impaired cognition in mice
    Mol. Med. (IF 2.991) Pub Date : 2019-05-30
    Asha Jacob; Yilong Ma; Elham Nasiri; Mahendar Ochani; Joseph Carrion; Shichun Peng; Max Brenner; Patricio T. Huerta; Ping Wang

    Alcohol abuse affects the brain regions responsible for memory, coordination and emotional processing. Binge alcohol drinking has shown reductions in brain activity, but the molecular targets have not been completely elucidated. We hypothesized that brain cells respond to excessive alcohol by releasing a novel inflammatory mediator, called cold inducible RNA-binding protein (CIRP), which is critical for the decreased brain metabolic activity and impaired cognition. Male wild type (WT) mice and mice deficient in CIRP (CIRP−/−) were studied before and after exposure to binge alcohol level by assessment of relative brain glucose metabolism with fluorodeoxyglucose (18FDG) and positron emission tomography (PET). Mice were also examined for object-place memory (OPM) and open field (OF) tasks. Statistical Parametric Analysis (SPM) of 18FDG-PET uptake revealed marked decreases in relative glucose metabolism in distinct brain regions of WT mice after binge alcohol. Regional analysis (post hoc) revealed that while activity in the temporal (secondary visual) and limbic (entorhinal/perirhinal) cortices was decreased in WT mice, relative glucose metabolic activity was less suppressed in the CIRP−/− mice. Group and condition interaction analysis revealed differing responses in relative glucose metabolism (decrease in WT mice but increase in CIRP−/− mice) after alcohol in brain regions including the hippocampus and the cortical amygdala where the percent changes in metabolic activity correlated with changes in object discrimination performance. Behaviorally, alcohol-treated WT mice were impaired in exploring a repositioned object in the OPM task, and were more anxious in the OF task, whereas CIRP−/− mice were not impaired in these tasks. CIRP released from brain cells could be responsible for regional brain metabolic hypoactivity leading to cognitive impairment under binge alcohol conditions.

    更新日期:2019-11-28
  • IgG Galactosylation status combined with MYOM2-rs2294066 precisely predicts anti-TNF response in ankylosing spondylitis
    Mol. Med. (IF 2.991) Pub Date : 2019-06-13
    Jing Liu; Qi Zhu; Jing Han; Hui Zhang; Yuan Li; Yanyun Ma; Dongyi He; Jianxin Gu; Xiaodong Zhou; John D. Reveille; Li Jin; Hejian Zou; Shifang Ren; Jiucun Wang

    Tumor necrosis factor (TNF) blockers have a high efficacy in treating Ankylosing Spondylitis (AS), yet up to 40% of AS patients show poor or even no response to this treatment. In this paper, we aim to build an approach to predict the response prior to clinical treatment. AS patients during the active progression were included and treated with TNF blocker for 3 months. Patients who do not fulfill ASASAS40 were considered as poor responders. The Immunoglobulin G galactosylation (IgG-Gal) ratio representing the quantity of IgG galactosylation was calculated and candidate single nucleotide polymorphisms (SNPs) in patients treated with etanercept was obtained. Machine-learning models and cross-validation were conducted to predict responsiveness. Both IgG-Gal ratio at each time point and differential IgG-Gal ratios between week 0 and weeks 2, 4, 8, 12 showed significant difference between responders and poor-responders. Area under curve (AUC) of the IgG-Gal ratio prediction model was 0.8 after cross-validation, significantly higher than current clinical indexes (C-reactive protein (CRP) = 0.65, erythrocyte sedimentation rate (ESR) = 0.59). The SNP MYOM2-rs2294066 was found to be significantly associated with responsiveness of etanercept treatment. A three-stage approach consisting of baseline IgG-Gal ratio, differential IgG-Gal ratio in 2 weeks, and rs2294066 genotype demonstrated the ability to precisely predict the response of anti-TNF therapy (100% for poor-responders, 98% for responders). Combination of different omics can more precisely to predict the response of TNF blocker and it is potential to be applied clinically in the future.

    更新日期:2019-11-28
  • miR-122 promotes hepatic lipogenesis via inhibiting the LKB1/AMPK pathway by targeting Sirt1 in non-alcoholic fatty liver disease
    Mol. Med. (IF 2.991) Pub Date : 2019-06-13
    Jun-Ke Long; Wen Dai; Ya-Wen Zheng; Shui-Ping Zhao

    Non-alcoholic fatty liver disease (NAFLD) is a common hepatic disease with an increasing prevalence but an unclear aetiology. This study aimed to investigate the functional implications of microRNA-122 (miR-122) in the pathogenesis of NAFLD and the possible molecular mechanisms. Both in vitro and in vivo models of NAFLD were generated by treating HepG2 and Huh-7 cells with free fatty acids (FFA) and by feeding mice a high-fat diet (HFD), respectively. HE and Oil Red O staining were used to examine liver tissue morphology and lipid deposition, respectively. Immunohistochemical (IHC) staining was used to examine Sirt1 expression in liver tissues. qRT-PCR and Western blotting were employed to measure the expression of miR-122, Sirt1, and proteins involved in lipogenesis and the AMPK pathway. Enzyme-linked immunosorbent assay (ELISA) was used to quantify triglyceride (TG) levels in HepG2 and Huh-7 cells and in liver tissues. The interaction between miR-122 and the Sirt1 gene was further examined by a dual luciferase reporter assay and RNA-immunoprecipitation (RIP). NAFLD hepatic tissues and FFA-treated HepG2 and Huh-7 cells presented excess lipid production and TG secretion, accompanied by miR-122 upregulation, Sirt1 downregulation, and potentiated lipogenesis-related genes. miR-122 suppressed Sirt1 expression via binding to its 3′-untranslated region (UTR). Knockdown of miR-122 effectively mitigated excessive lipid production and suppressed the expression of lipogenic genes in FFA-treated HepG2 and Huh-7 cells via upregulating Sirt1. Furthermore, miR-122 knockdown activated the LKB1/AMPK signalling pathway. The inhibition of miR-122 protects hepatocytes from lipid metabolic disorders such as NAFLD and suppresses lipogenesis via elevating Sirt1 and activating the AMPK pathway. These data support miR-122 as a promising biomarker and drug target for NAFLD.

    更新日期:2019-11-28
  • Curcumin induced oxidative stress attenuation by N-acetylcysteine co-treatment: a fibroblast and epithelial cell in-vitro study in idiopathic pulmonary fibrosis
    Mol. Med. (IF 2.991) Pub Date : 2019-06-13
    L. R. Rodriguez; S. N. Bui; R. T. Beuschel; E. Ellis; E. M. Liberti; M. K. Chhina; B. Cannon; M. Lemma; S. D. Nathan; G. M. Grant

    Idiopathic Pulmonary Fibrosis (IPF) is a fatal lung disease of unknown etiology with only two federally approved drug options. Given the complex molecular pathogenesis of IPF involving multiple cell types and multiple pathways, we explore the effects of a potential antifibrotic and antioxidant drug combination. Curcumin is a polyphenolic compound derived from turmeric with significant biological activity including a potential antifibrotic capacity. N-acetylcysteine (NAC) is a precursor to the antioxidant glutathione. To advance our understanding of these molecules, and to identify a clinical application, we present a small number of focused experiments that interrogates the effect of curcumin and NAC on pathways relevant to IPF in both fibroblasts and epithelial cells. Primary epithelial cell and fibroblasts isolated from patients with IPF were challenged with a combination treatment of NAC and curcumin. Evaluation of the antifibrotic potential and effect on oxidative stress was performed through QPCR gene expression analysis and functional assays including scratch tests, viability assays, and measurement of induced reactive oxygen species. We demonstrate that curcumin alone does have antifibrotic potential, but that effect is accompanied by proapoptotic increases in oxidative stress. Coupled with this, we find that NAC alone can reduce oxidative stress, but that epithelial cell viability is decreased through this treatment. However, co-administration of these two molecules decreases oxidative stress and maintains high cell viability in both cell types. In addition, this co-treatment maintains an antifibrotic potential. These findings suggest a novel application for these molecules in IPF and encourage further exploration of this potential therapeutic approach.

    更新日期:2019-11-28
  • CircRNA-9119 suppresses poly I:C induced inflammation in Leydig and Sertoli cells via TLR3 and RIG-I signal pathways
    Mol. Med. (IF 2.991) Pub Date : 2019-06-13
    Le Qin; Jie Lin; Xiaoxiao Xie

    Circular RNAs (circRNAs) contribute to the epigenetic modulation of pathological and physiological conditions. The understanding of the impact of circRNAs on generation of testicular inflammatory reactions is insufficient. Our research adopted a poly I:C-triggered testicular inflammation murine model and cell assays. Microarray data and quantitative evaluation revealed the elevation in the concentrations of Toll-like receptor 3 (TLR3), circRNA-9119, and retinoic acid inducible gene-I (RIG-I) and repression in the levels of miR-136 and miR-26a. Inhibition of circRNA-9119 expression impaired the inflammatory reactions in the separated Leydig and Sertoli cells subjected to poly I:C treatment. CircRNA-9119 suppressed the expression of miR-136 and miR-26a by acting as a microRNA sponge. miR-136 and miR-26a repressed the expression of RIG-I and TLR3 through the expected target region in Leydig and Sertoli cells in vitro. Inhibition of miR-136 and miR-26a expression, at least in part, restored the expression of inflammatory cytokines, which were inhibited upon circRNA-9119 expression silencing. Furthermore, the expression of circRNA-9119 was positively associated with RIG-I and TLR3 mRNA and protein levels. The expression of inflammatory genes triggered by poly I:C treatment was noticeably suppressed after RIG-I and TLR3 knockout. Our results suggest that circRNA-9119 may serve as a competing endogenous RNA that insulated miR-136 and miR-26a and consequently defended RIG-I and TLR3 mRNAs against miR-26a/miR-136-mediated inhibition of testicular cells. Moreover, RIG-I and TLR3 contributed to the modulation of poly I:C-triggered inflammatory cytokine generation during orchitis in testicular cells.

    更新日期:2019-11-28
  • FBXW7 suppresses HMGB1-mediated innate immune signaling to attenuate hepatic inflammation and insulin resistance in a mouse model of nonalcoholic fatty liver disease
    Mol. Med. (IF 2.991) Pub Date : 2019-06-18
    Cheng Zhang; Feng Chen; Li Feng; Qun Shan; Gui-Hong Zheng; Yong-Jian Wang; Jun Lu; Shao-Hua Fan; Chun-Hui Sun; Dong-Mei Wu; Meng-Qiu Li; Bin Hu; Qing-Qing Wang; Zi-Feng Zhang; Yuan-Lin Zheng

    Innate immune dysfunction contributes to the development and progression of nonalcoholic fatty liver disease (NAFLD), however, its pathogenesis is still incompletely understood. Identifying the key innate immune component responsible for the pathogenesis of NAFLD and clarifying the underlying mechanisms may provide therapeutic targets for NAFLD. Recently, F-box- and WD repeat domain-containing 7 (FBXW7) exhibits a regulatory role in hepatic glucose and lipid metabolism. This study aims to investigate whether FBXW7 controls high-mobility group box 1 protein (HMGB1)-mediated innate immune signaling to improve NAFLD and the mechanism underlying this action. Mice were fed a high-fat diet (HFD) for 12 or 20 weeks to establish NAFLD model. Hepatic overexpression or knockdown of FBXW7 was induced by tail-vein injection of recombinant adenovirus. Some Ad-FBXW7-injected mice fed a HFD were injected intraperitoneally with recombinant mouse HMGB1 to confirm the protective role of FBXW7 in NAFLD via inhibition of HMGB1. FBXW7 improves NAFLD and related metabolic parameters without remarkable influence of body weight and food intake. Moreover, FBXW7 markedly ameliorated hepatic inflammation and insulin resistance in the HFD-fed mice. Furthermore, FBXW7 dramatically attenuated the expression and release of HMGB1 in the livers of HFD-fed mice, which is associated with inhibition of protein kinase R (PKR) signaling. Thereby, FBXW7 restrains Toll-like receptor 4 (TLR4) and receptor for advanced glycation end products (RAGE) signaling in HFD-fed mouse livers. In addition, exogenous HMGB1 treatment abolished FBXW7-mediated inhibition of hepatic inflammation and insulin resistance in HFD-fed mouse livers. Our results demonstrate a protective role of FBXW7 in NAFLD by abating HMGB1-mediated innate immune signaling to suppress inflammation and consequent insulin resistance, suggesting that FBXW7 is a potential target for therapeutic intervention in NAFLD development.

    更新日期:2019-11-28
  • A rare functional variant of SHARPIN attenuates the inflammatory response and associates with increased risk of late-onset Alzheimer’s disease
    Mol. Med. (IF 2.991) Pub Date : 2019-06-20
    Yuya Asanomi; Daichi Shigemizu; Akinori Miyashita; Risa Mitsumori; Taiki Mori; Norikazu Hara; Kaoru Ito; Shumpei Niida; Takeshi Ikeuchi; Kouichi Ozaki

    Late-onset Alzheimer’s disease (LOAD), the most common form of dementia, results from complicated interactions among multiple environmental and genetic factors. Despite recent advances in genetic analysis of LOAD, more than half of the heritability for the disease remains unclear. Although genetic studies in Caucasians found rare risk variants for LOAD with large effect sizes, these variants are hardly detectable in the Japanese population. To identify rare variants possibly explaining part of the genetic architecture for LOAD in Japanese people, we performed whole-exome sequencing analyses of 202 LOAD individuals without the APOE ε4 risk allele, a major genetic factor for LOAD susceptibility. We also implemented in vitro functional analyses of the variant(s) to reveal possible functions associated with LOAD risk. Via step-by-step selection of whole-exome variants, we found seven candidate risk variants. We then conducted a case-control association study in a large Japanese cohort consisting of 4563 cases and 16,459 controls. We finally identified a rare nonsynonymous variant, rs572750141 (NM_030974.3:p.Gly186Arg), in SHARPIN that was potentially associated with increased risk of LOAD (corrected P = 8.05 × 10− 5, odds ratio = 6.1). The amino acid change in SHARPIN resulted in aberrant cellular localization of the variant protein and attenuated the activation of NF-κB, a central mediator of inflammatory and immune responses. Our work identified a rare functional SHARPIN variant as a previously unknown genetic risk factor for LOAD. The functional alteration in SHARPIN induced by the rare coding variant is associated with an attenuated inflammatory/immune response that may promote LOAD development.

    更新日期:2019-11-28
  • Active repurposing of drug candidates for melanoma based on GWAS, PheWAS and a wide range of omics data
    Mol. Med. (IF 2.991) Pub Date : 2019-06-20
    Ali Khosravi; B. Jayaram; Bahram Goliaei; Ali Masoudi-Nejad

    Drug repurposing is a swift, safe, and cheap drug discovery method. Melanoma disorders present low survival and high mortality rates and are challenging to diagnose and treat. Moreover, there is a high volume of worldwide investigations that are attempting to find melanoma-related genes of influence, which can be identified as responsive targets for reliable treatment. In this study, we used a wide range of data analyses to analyze over 1100 genes and proteins of influence with respect to cutaneous malignant melanoma. Our analysis included various investigational results from genome- and phenome-wide association studies (GWAS and PheWAS, respectively), biomedical, transcriptomic, and metabolomic datasets. We then researched the DrugBank for potential melanoma targets from the selected list. We excluded known melanoma targets to obtain a list of druggable proteins. We performed a precise analysis of the drugs’ pathogenesis and checked the expression profiles of the selected drugs having high associations with known anti-melanoma drugs. We found 35 drugs that interacted with 20 unique targets. These drugs appear to have high melanoma treatment potentials. We confirmed our results with previous studies and found supporting references for 30 of these drugs. In conclusion, this investigation can be applied to various diseases for the efficient and economical repurposing of various drug compounds. For further validation, the results may be applicable for in vivo tests and clinical trials.

    更新日期:2019-11-28
  • Dystrophin R16/17 protein therapy restores sarcolemmal nNOS in trans and improves muscle perfusion and function
    Mol. Med. (IF 2.991) Pub Date : 2019-07-02
    Junling Zhao; Hsiao Tung Yang; Lakmini Wasala; Keqing Zhang; Yongping Yue; Dongsheng Duan; Yi Lai

    Delocalization of neuronal nitric oxide synthase (nNOS) from the sarcolemma leads to functional muscle ischemia. This contributes to the pathogenesis in cachexia, aging and muscular dystrophy. Mutations in the gene encoding dystrophin result in Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). In many BMD patients and DMD patients that have been converted to BMD by gene therapy, sarcolemmal nNOS is missing due to the lack of dystrophin nNOS-binding domain. Dystrophin spectrin-like repeats 16 and 17 (R16/17) is the sarcolemmal nNOS localization domain. Here we explored whether R16/17 protein therapy can restore nNOS to the sarcolemma and prevent functional ischemia in transgenic mice which expressed an R16/17-deleted human micro-dystrophin gene in the dystrophic muscle. The palmitoylated R16/17.GFP fusion protein was conjugated to various cell-penetrating peptides and produced in the baculovirus-insect cell system. The best fusion protein was delivered to the transgenic mice and functional muscle ischemia was quantified. Among five candidate cell-penetrating peptides, the mutant HIV trans-acting activator of transcription (TAT) protein transduction domain (mTAT) was the best in transferring the R16/17.GFP protein to the muscle. Systemic delivery of the mTAT.R16/17.GFP protein to micro-dystrophin transgenic mice successfully restored sarcolemmal nNOS without inducing T cell infiltration. More importantly, R16/17 protein therapy effectively prevented treadmill challenge-induced force loss and improved muscle perfusion during contraction. Our results suggest that R16/17 protein delivery is a highly promising therapy for muscle diseases involving sarcolemmal nNOS delocalizaton.

    更新日期:2019-11-28
  • YAP promotes the malignancy of endometrial cancer cells via regulation of IL-6 and IL-11
    Mol. Med. (IF 2.991) Pub Date : 2019-07-12
    Jing Wang; Tiefang Song; Suiyang Zhou; Xianchao Kong

    Emerging evidence shows that Hippo signal pathways can regulate the progression of various cancer. While the roles of Yes-associated protein (YAP), the key transducer of Hippo signals, in the development of endometrial cancer (EC) are rarely investigated. The expression of YAP in endometrial cancer cells and tissues was measured. Its roles in proliferation and expression of interleukins (ILs) were investigated by use of its specific siRNA or inhibitor (verteporfin, VP). YAP was upregulated in endometrial cancer cells and tissues. Knockdown of YAP or VP can suppress the proliferation while increase its chemo-sensitivity of EC cells. We found that targeted inhibition of YAP can decrease the expression of interleukin-6 (IL-6) and IL-11 in EC cells. Recombinant IL-6 or IL-11 can attenuate si-YAP suppressed proliferation of EC cells. Chromatin immunoprecipitation (ChIP) assay suggested that YAP can directly bind with the promoter of IL-6 and induce its transcription. As to IL-11, inhibitor of NF-κB (BAY 11–7082) can significantly down regulate the mRNA expression of IL-11. Over expression of p65 abolished si-YAP suppressed transcription of IL-11. It suggested that NF-κB was involved in the YAP regulated expression of IL-11. YAP can regulate the proliferation and progression of EC cells. It suggested that targeted inhibition of YAP might be a potent potential approach for EC therapy.

    更新日期:2019-11-28
  • Fimasartan reduces neointimal formation and inflammation after carotid arterial injury in apolipoprotein E knockout mice
    Mol. Med. (IF 2.991) Pub Date : 2019-07-15
    Jong-Ho Kim; I-Rang Lim; Hyung Joon Joo; Chi-Yeon Park; Seung-Cheol Choi; Han Saem Jeong; Soon Jun Hong

    The beneficial effects of angiotensin II type 1 receptor blockers (ARBs) on atherosclerosis have been demonstrated in numerous studies. We investigated the effects of fimasartan on reducing neointimal formation and systemic inflammation after carotid artery (CA) injury in Apolipoprotein E knockout (ApoE KO) mice. ApoE KO mice were randomly allocated to Group I (without CA injury), Group II (without CA injury + Fimasartan), Group III (CA injury), and Group IV (CA injury + Fimasartan). Fimasartan was orally administered everyday starting 3 days before iatrogenic left CA injury. At 28 days, neointimal hyperplasia and the inflammatory cytokines including TNFα, IL-6, ICAM, and MMP-9 in the peripheral blood were significantly reduced in Groups II and IV compared to Groups I and III, respectively. All fimasartan-administered groups revealed significant increases of CD4+CD25+Foxp3+ regulatory T (Treg) cells with increased plasma levels of IL-10 and TGFβ. In addition, increased CD8+ T cells by fimasartan were correlated with reduced smooth muscle cell (SMC) proliferation in the neointima in Groups II and IV. Furthermore, the populations of Treg and CD8+ T cells in total splenocytes were increased in Groups II and IV compared to Groups I and III, respectively. The enlargement of spleens due to CA injury in the Group III was attenuated by fimasartan, as shown in the Group IV. These data indicate that fimasartan significantly reduced SMC proliferation in neointima and increased Treg cells in ApoE KO CA injury mice. This study suggests fimasartan could be an efficient strategy for reduction of atherosclerotic progression, with a decrease in immune response and systemic inflammation.

    更新日期:2019-11-28
  • Correction to: Plasma Fibrinogen Is a Natural Deterrent to Amyloid β-Induced Platelet Activation and Neuronal Toxicity
    Mol. Med. (IF 2.991) Pub Date : 2019-07-30
    Vijay K. Sonkar; Paresh P. Kulkarni; Susheel N. Chaurasia; Ayusman Dash; Abhishek Jauhari; Devendra Parmar; Sanjay Yadav; Debabrata Dash

    Following publication of the original article [1], the author reported an error in Figure 1. The correct version of Figure 1 is as follows:

    更新日期:2019-11-28
  • Gut microbiota promote the inflammatory response in the pathogenesis of systemic lupus erythematosus
    Mol. Med. (IF 2.991) Pub Date : 2019-08-01
    Yiyangzi Ma; Xiaoxue Xu; Mengtao Li; Jun Cai; Qiang Wei; Haitao Niu

    Systemic lupus erythematosus (SLE) is a chronic autoimmune disease whose onset and progression are affected by genetic and environmental factors. The purpose of this study is to identify the influence of gut microbiota in the pathogenesis of SLE, and to investigate the mechanism involved. Fecal microbiota from C57/BL6 mice and SLE prone mice were examined using next-generation sequencing (NGS). Germ free mice were given fecal microbiota transplantation (FMT), and their gut microbiome and gene expression in recipients’ colons were examined by NGS. The anti-double stranded DNA (anti-dsDNA) antibodies in recipients were determined using an enzyme-linked immunosorbent assay (ELISA). The immune cell profiles of mice were analyzed by flow cytometry at the 3rd week after FMT, and the expression of genes associated with SLE after FMT was determined using quantitative real-time PCR (qRT-PCR). The fecal microbiota of SLE mice had lower community richness and diversity than healthy mice. Fecal microbiota of recipient mice were similar to their donors. Fecal microbiome from SLE mice could lead to a significant increase of anti-dsDNA antibodies and promote the immune response in recipient mice. Our results also indicated that fecal microbiome from SLE mice resulted in significant changes in the distribution of immune cells and upregulated expression of certain lupus susceptibility genes. SLE is associated with alterations of gut microbiota. Fecal microbiome from SLE mice can induce the production of anti-dsDNA antibodies in germ free mice and stimulate the inflammatory response, and alter the expression of SLE susceptibility genes in these mice.

    更新日期:2019-11-28
  • Differential network analysis and protein-protein interaction study reveals active protein modules in glucocorticoid resistance for infant acute lymphoblastic leukemia
    Mol. Med. (IF 2.991) Pub Date : 2019-08-01
    Zaynab Mousavian; Abbas Nowzari-Dalini; Yasir Rahmatallah; Ali Masoudi-Nejad

    Acute lymphoblastic leukemia (ALL) is the most common type of cancer diagnosed in children and Glucocorticoids (GCs) form an essential component of the standard chemotherapy in most treatment regimens. The category of infant ALL patients carrying a translocation involving the mixed lineage leukemia (MLL) gene (gene KMT2A) is characterized by resistance to GCs and poor clinical outcome. Although some studies examined GC-resistance in infant ALL patients, the understanding of this phenomenon remains limited and impede the efforts to improve prognosis. This study integrates differential co-expression (DC) and protein-protein interaction (PPI) networks to find active protein modules associated with GC-resistance in MLL-rearranged infant ALL patients. A network was constructed by linking differentially co-expressed gene pairs between GC-resistance and GC-sensitive samples and later integrated with PPI networks by keeping the links that are also present in the PPI network. The resulting network was decomposed into two sub-networks, specific to each phenotype. Finally, both sub-networks were clustered into modules using weighted gene co-expression network analysis (WGCNA) and further analyzed with functional enrichment analysis. Through the integration of DC analysis and PPI network, four protein modules were found active under the GC-resistance phenotype but not under the GC-sensitive. Functional enrichment analysis revealed that these modules are related to proteasome, electron transport chain, tRNA-aminoacyl biosynthesis, and peroxisome signaling pathways. These findings are in accordance with previous findings related to GC-resistance in other hematological malignancies such as pediatric ALL. Differential co-expression analysis is a promising approach to incorporate the dynamic context of gene expression profiles into the well-documented protein interaction networks. The approach allows the detection of relevant protein modules that are highly enriched with DC gene pairs. Functional enrichment analysis of detected protein modules generates new biological hypotheses and may help in explaining the GC-resistance in MLL-rearranged infant ALL patients.

    更新日期:2019-11-28
  • FOXD1 mutations are related to repeated implantation failure, intra-uterine growth restriction and preeclampsia
    Mol. Med. (IF 2.991) Pub Date : 2019-08-08
    Paula Quintero-Ronderos; Karen Marcela Jiménez; Clara Esteban-Pérez; Diego A. Ojeda; Sandra Bello; Dora Janeth Fonseca; María Alejandra Coronel; Harold Moreno-Ortiz; Diana Carolina Sierra-Díaz; Elkin Lucena; Sandrine Barbaux; Daniel Vaiman; Paul Laissue

    Human reproductive disorders consist of frequently occurring dysfunctions including a broad range of phenotypes affecting fertility and women’s health during pregnancy. Several female-related diseases have been associated with hypofertility/infertility phenotypes, such as recurrent pregnancy loss (RPL). Other occurring diseases may be life-threatening for the mother and foetus, such as preeclampsia (PE) and intra-uterine growth restriction (IUGR). FOXD1 was defined as a major molecule involved in embryo implantation in mice and humans by regulating endometrial/placental genes. FOXD1 mutations in human species have been functionally linked to RPL’s origin. FOXD1 gene mutation screening, in 158 patients affected by PE, IUGR, RPL and repeated implantation failure (RIF), by direct sequencing and bioinformatics analysis. Plasmid constructs including FOXD1 mutations were used to perform in vitro gene reporter assays. Nine non-synonymous sequence variants were identified. Functional experiments revealed that p.His267Tyr and p.Arg57del led to disturbances of promoter transcriptional activity (C3 and PlGF genes). The FOXD1 p.Ala356Gly and p.Ile364Met deleterious mutations (previously found in RPL patients) have been identified in the present work in women suffering PE and IUGR. Our results argue in favour of FOXD1 mutations’ central role in RPL, RIF, IUGR and PE pathogenesis via C3 and PlGF regulation and they describe, for the first time, a functional link between FOXD1 and implantation/placental diseases. FOXD1 could therefore be used in clinical environments as a molecular biomarker for these diseases in the near future. Recurrent pregnancy loss, Preeclampsia, Intra-uterine growth restriction, FOXD1

    更新日期:2019-11-28
  • High co-expression of TNF-α and CARDS toxin is a good predictor for refractory Mycoplasma pneumoniae pneumonia
    Mol. Med. (IF 2.991) Pub Date : 2019-08-09
    Gang Li; Liping Fan; Yuqing Wang; Li Huang; Meijuan Wang; Canhong Zhu; Chuangli Hao; Wei Ji; Hansi Liang; Yongdong Yan; Zhengrong Chen

    Early distinction between refractory M. pneumoniae pneumonia (RMPP) and non-RMPP (NRMPP) is still difficult. The community-acquired respiratory distress syndrome (CARDS) toxin can induce inflammatory and histopathological phenotypes associated with M. pneumoniae infection. This study aimed to investigate the clinical significance of CARDS toxin and pro-inflammatory cytokines in children with RMPP and to explore whether CARDS toxin can induce TNF-α expression. Levels of CARDS toxin and cytokines in BALF from control and children with MPP were determined by real-time PCR and ELISA, respectively. A receiver-operating characteristic (ROC) analysis was performed to assess the diagnostic values of CARDS toxin, TNF-α, and IL-6 in RMPP. The recombinant CARDS toxin was constructed and prepared at different concentrations for stimulation of RAW264.7 cells. After co-culture with CARDS toxin, cytokines were detected by ELISA and the mRNA levels were measured by real-time PCR. Effects of CARDS toxin and TNF-α on inflammatory cell infiltration and mucus secretion in mouse lungs were also evaluated. Levels of CARDS toxin, TNF-α and IL-6 in bronchoalveolar lavage fluid (BALF) were significantly higher in RMPP cases compared with NRMPP cases. Furthermore, TNF-α had better diagnostic ability for differentiation of RMPP with AUC of 0.824 and Youden index of 0.692 compared with CARDS toxin and IL-6. Moreover, CARDS toxin was positively correlated with TNF-α level in MPP cases. In vitro assay revealed that CARDS toxin induced RAW264.7 macrophages to secrete TNF-α. Further in vivo assay showed that TNF-α deletion partially abrogated the CARDS toxin-mediated induction of inflammatory cell infiltration and mucus secretion in mouse lungs. The high co-expression of TNF-α and CARDS toxin in BALF is a good diagnostic biomarker for differentiating children with RMPP and NRMPP.

    更新日期:2019-11-28
  • The cholinergic anti-inflammatory pathway in resistant hypertension treated with renal denervation
    Mol. Med. (IF 2.991) Pub Date : 2019-08-15
    Marie Hilderman; Abdul Rashid Qureshi; Farhad Abtahi; Nils Witt; Christina Jägren; Joakim Olbers; Martin Delle; Kaj Lindecrantz; Annette Bruchfeld

    Renal denervation (RDN) reduces sympathetic tone and may alter the sympathetic-parasympathetic balance. The autonomic nervous system is partly a regulator of innate immunity via the cholinergic anti-inflammatory pathway (CAP) which inhibits inflammation via the vagus nerve. Placental Growth Factor (PlGF) influences a neuro-immunological pathway in the spleen which may contribute to hypertension. The aim of this study was to investigate if modulation of renal sympathetic nerve activity affects CAP in terms of cytokine release as well as levels of PlGF. Ten patients treated with RDN (St Jude EnligHTN™), were analyzed for TNF, IL-1b and IL-10 and Lipopolysaccharide (LPS)-stimulated cytokine release before RDN, 1 day after and at 3- and 6-months follow-up. Four patients who underwent elective coronary angiography served as disease controls (DC). Baseline TNF was significantly lower 1 day after RDN (p = 0.03). LPS-stimulated (0, 10 and 100 ng/mL) TNF and IL-1b were significantly lower 1 day after RDN (TNF p = 0.0009, p = 0.0009 and p = 0.001, IL-1b; p = 0.0001, p = 0.002 and p = 0.005). IL-10 was significantly higher one day after RDN (p = ns, p = 0.02 and p = 0.01). These differences however declined during follow up. A more marked TNF reduction was achieved with a cholinergic analogue, GTS-21, in LPS-stimulated whole blood as compared with samples without GTS-21. Cytokine levels in controls did not differ before and 1 day after coronary angiography. PlGF was significantly higher in RDN patients and DC compared with healthy controls but did not change during follow-up. RDN has an immediate effect on TNF in vivo and cytokine release ex vivo but seems to wane over time suggesting that current RDN techniques may not have long-lasting immunomodulatory effect. Repeated and extended stimulation of CAP in resistant hypertension by targeting neural circuits may be a potential therapeutic strategy for treatment of both hypertension and inflammation.

    更新日期:2019-11-28
  • Metabolomics analysis elucidates unique influences on purine / pyrimidine metabolism by xanthine oxidoreductase inhibitors in a rat model of renal ischemia-reperfusion injury
    Mol. Med. (IF 2.991) Pub Date : 2019-08-22
    Takashi Tani; Ken Okamoto; Megumi Fujiwara; Akira Katayama; Shuichi Tsuruoka

    Clinically applied as anti-gout drugs, xanthine oxidoreductase (XOR) inhibitors, especially the potent, selective, non-purine-analog XOR inhibitors febuxostat and topiroxostat, exert organ-protective effects. We tested the hypothesis that preservation of tissue concentrations of high-energy phosphates, such as ATP and ADP, contributes to organ-protective effects through CE-TOFMS metabolomics. Rats were subjected to 30 min of renal ischemia-reperfusion (I/R) injury 60 min after oral administration of 10 mg/kg febuxostat, 10 mg/kg topiroxostat, 50 mg/kg allopurinol, or vehicle. In non-purine-analog XOR inhibitor-treated groups, renal concentrations of high-energy phosphates were greater before and after I/R injury, and renal adenine compounds were less depleted by I/R injury than in the vehicle and allopurinol groups. These findings were well in accordance with the proposed hypothesis that the recomposition of high-energy phosphates is promoted by non-purine-analog XOR inhibitors via the salvage pathway through blockade of hypoxanthine catabolism, whereas non-specific inhibitory effects of allopurinol on purine/pyrimidine enzymes impede this re-synthesis process. This metabolic approach shed light on the physiology of the organ-protective effects of XOR inhibitors.

    更新日期:2019-11-28
  • Tamoxifen and bone morphogenic protein-7 modulate fibrosis and inflammation in the peritoneal fibrosis model developed in uremic rats
    Mol. Med. (IF 2.991) Pub Date : 2019-08-28
    Filipe M. O. Silva; Elerson C. Costalonga; Cleonice Silva; Ana C. O. Carreira; Samirah A. Gomes; Mari C. Sogayar; Camilla Fanelli; Irene L. Noronha

    Peritoneal fibrosis (PF) represents a long-term complication of peritoneal dialysis (PD), affecting peritoneal membrane (PM) integrity and function. Understanding the mechanisms underlying PF development in an uremic environment aiming alternative therapeutic strategies for treating this process is of great interest. The aim of this study was to analyze the effects of tamoxifen (TAM) and recombinant BMP7 (rBMP7) in an experimental model of PF developed in uremic rats. To mimic the clinical situation of patients on long-term PD, a combo model, characterized by the combination of PF and CKD with severe uremia, was developed in Wistar rats. PF was induced by intraperitoneal (IP) injections of chlorhexidine gluconate (CG), and CKD was induced by an adenine-rich diet. Uremia was confirmed by severe hypertension, increased blood urea nitrogen (BUN> 120 mg/dL) and serum creatinine levels (> 2 mg/dL). Uremic rats with PF were treated with TAM (10 mg/Kg by gavage) or BMP7 (30 μg/Kg, IP). Animals were followed up for 30 days. CG administration in uremic rats induced a striking increase in PM thickness, neoangiogenesis, demonstrated by increased capillary density, and failure of ultrafiltration capacity. These morphological and functional changes were blocked by TAM or rBMP7 treatment. In parallel, TAM and rBMP7 significantly ameliorated the PM fibrotic response by reducing α-SMA, extracellular matrix proteins and TGF-ß expression. TAM or rBMP7 administration significantly inhibited peritoneal Smad3 expression in uremic rats with PF, prevented Smad3 phosphorylation, and induced a remarkable up-regulation of Smad7, an intracellular inhibitor of TGFβ/Smad signaling, contributing to a negative modulation of profibrotic genes. Both treatments were also effective in reducing local inflammation, possibly by upregulating IκB-α expression in the PM of uremic rats with PF. In vitro experiments using primary peritoneal fibroblasts activated by TGF-ß confirmed the capacity of TAM or rBMP7 in blocking inflammatory mediators, such as IL-1ß expression. In conclusion, these findings indicate important roles of TGF-ß/Smad signaling in PF aggravated by uremia, providing data regarding potential therapeutic approaches with TAM or rBMP7 to block this process.

    更新日期:2019-11-28
  • Visualisation of HER2 homodimers in single cells from HER2 overexpressing primary formalin fixed paraffin embedded tumour tissue
    Mol. Med. (IF 2.991) Pub Date : 2019-08-28
    Diana B. Peckys; Daniela Hirsch; Timo Gaiser; Niels de Jonge

    HER2 is considered as one of the most important, predictive biomarkers in oncology. The diagnosis of HER2 positive cancer types such as breast- and gastric cancer is usually based on immunohistochemical HER2 staining of tumour tissue. However, the current immunohistochemical methods do not provide localized information about HER2’s functional state. In order to generate signals leading to cell growth and proliferation, the receptor spontaneously forms homodimers, a process that can differ between individual cancer cells. HER2 overexpressing tumour cells were dissociated from formalin-fixed paraffin-embedded (FFPE) patient’s biopsy sections, subjected to a heat-induced antigen retrieval procedure, and immobilized on microchips. HER2 was specifically labelled via a two-step protocol involving the incubation with an Affibody-biotin compound followed by the binding of a streptavidin coated quantum dot (QD) nanoparticle. Cells with membrane bound HER2 were identified using fluorescence microscopy, coated with graphene to preserve their hydrated state, and subsequently examined by scanning transmission electron microscopy (STEM) to obtain the locations at the single molecule level. Label position data was statistically analysed via the pair correlation function, yielding information about the presence of HER2 homodimers. Tumour cells from two biopsies, scored HER2 3+, and a HER2 negative control sample were examined. The specific labelling protocol was first tested for a sectioned tissue sample of HER2-overexpressing tumour. Subsequently, a protocol was optimized to study HER2 homodimerization in single cells dissociated from the tissue section. Electron microscopy data showed membrane bound HER2 in average densities of 201–689 proteins/μm2. An automated, statistical analysis of well over 200,000 of measured protein positions revealed the presence of HER2 homodimers in 33 and 55% of the analysed images for patient 1 and 2, respectively. We introduced an electron microscopy method capable of measuring the positions of individually labelled HER2 proteins in patient tumour cells from which information about the functional status of the receptor was derived. This method could take HER2 testing a step further by examining HER2 homodimerization directly out of tumour tissue and may become important for adjusting a personalized antibody-based drug therapy.

    更新日期:2019-11-28
  • Melatonin prevents bone destruction in mice with retinoic acid–induced osteoporosis
    Mol. Med. (IF 2.991) Pub Date : 2019-08-28
    Xudong Wang; Tongzhou Liang; Yuanxin Zhu; Jincheng Qiu; Xianjian Qiu; Chengjie Lian; Bo Gao; Yan Peng; Anjing Liang; Hang Zhou; Xiaoming Yang; Zhiheng Liao; Yongyong Li; Caixia Xu; Peiqiang Su; Dongsheng Huang

    The protective effect of melatonin against bone metabolism imbalance in osteoporosis (OP) induced by drugs such as retinoic acid (RA) is unclear. The aim of this study was to explore the role of melatonin in bone destruction based on a mouse model. RA-induced OP model mice were established. To assess the effect of melatonin on these mice, micro-CT was used to characterize the trabecular structure of normal mice and those treated with RA (model), RA + low-dose melatonin (Mlt-L), RA + high-dose melatonin (Mlt-H), and RA + alendronate sodium (positive control). The shape of the trabecular bone, the length and diameter of the femoral head and the height and diameter of vertebra(L1) of each group were also measured and the number of osteoclasts was determined by Tartrate-resistant acid phosphatase (TRACP) staining. Meanwhile, the expression of alkaline phosphatase (ALP) was evaluated by immunohistochemistry assays. The differences between groups in terms of liver and kidney oxidation–related indexes and serum and urinary indicators related to bone metabolism were also analyzed. Furthermore, qRT-PCR and western blotting were used to evaluate the effect of melatonin on osteogenic and osteoclastic differentiation in MC3T3-E1 and RAW264.7 cells, respectively. RA induction led to a decrease in the amount and density of trabecular bone, a decrease in the length and diameter of the femur and height, diameter of the vertebra (L1), a decrease in bone mass and density and the expression of ALP, and an increase in the number of osteoclasts. Melatonin treatment alleviated these effects induced by RA, increasing the amount of trabecular bone in OP mice, improving the microstructure of the femur and vertebra(L1) and increasing bone mass bone density and the expression of ALP, as well as decreasing the number of osteoclasts. Additionally, blood and urinary bone metabolism-related indicators showed that melatonin promoted bone formation and inhibited bone resorption. Determination of oxidant and antioxidant biomarkers in the livers and kidneys of the mice revealed that melatonin promoted the antioxidant level and suppressed the level of oxidant molecules in these organs. In vitro, RA promoted osteoclasts and inhibit osteogenesis by increasing oxidative stress levels in the RAW264.7 and MC3T3-E1 cells, but melatonin reversed this effect. Melatonin may, therefore, play a role in the ERK/SMAD and NF-κB pathways. Melatonin can alleviate bone loss in RA-induced OP model mice, repair the trabecular microstructure, and promote bone formation. These effects may be related to reducing oxidation levels in vivo and vitro through the ERK/SMAD and NF-κB pathways.

    更新日期:2019-11-28
  • PRSS1 mutation: a possible pathomechanism of pancreatic carcinogenesis and pancreatic cancer
    Mol. Med. (IF 2.991) Pub Date : 2019-09-14
    Qicai Liu; Ling Guo; Sheng Zhang; Jingwen Wang; Xinhua Lin; Feng Gao

    Previous studies revealed somatic mutations of the cationic trypsinogen gene (PRSS1) in patients with chronic pancreatitis and pancreatic cancer. However, whether PRSS1 mutations trigger pancreatic cancer and/or promote malignant proliferation and metastasis in pancreatic cancer remains largely unclear, as well as the potential underlying mechanisms. In the present study, whole-exome sequencing was applied for screening, and the R116C mutation was validated by Sanger sequencing. Phosphorylation antibody array, RNA-Seq, and RT-qPCR were adopted to screen and validate that R116C mutation promoted pancreatic cancer progression via the JAK1-STAT5 pathway. It showed that migration and invasion were significantly increased in R116C-bearing PANC-1 cells compared with wild type counterparts. In a transgenic mouse model of iZEG-PRSS1_R116C, primary pancreatic intraepithelial neoplasia (PanINs) was observed in the pancreatic duct. These findings suggested a novel pathway mediating pancreatic cancer development, with PRSS1 mutation and overexpression playing an “inside job” role in pancreatic carcinogenesis and tumor development.

    更新日期:2019-11-28
  • Club cell protein 16 in sera from trauma patients modulates neutrophil migration and functionality via CXCR1 and CXCR2
    Mol. Med. (IF 2.991) Pub Date : 2019-10-30
    Baolin Xu; Andrea Janicova; Jan Tilmann Vollrath; Philipp Störmann; Lukas Martin; Ingo Marzi; Sebastian Wutzler; Frank Hildebrand; Sabrina Ehnert; Borna Relja

    Club Cell protein (CC)16 correlates with lung injury and respiratory complications, which are in part triggered by polymorphonuclear leukocytes (PMNL) in severely traumatized patients (TP). CC16 exerts anti-inflammatory and immunosuppressive effects, however, its influence on PMNL functions after trauma is unknown. Here, we evaluated whether CC16 present in sera from TP could modify the biological functions of PMNL. Sera from 16 severely injured TP without pneumonia (no P, n = 8) or with pneumonia (P, n = 8) were collected at admission to emergency department (ED) and 1 day prior pneumonia and pre-incubated with or without anti-CC16 antibody for CC16 neutralization. Samples from the equal post-injury days in the corresponding no P group were used. Neutrophils were isolated from healthy volunteers (HV, n = 5) and incubated with 20% of the serum medium from TP, respectively. In PMNL, CD62L, CD11b/CD18 and CD31 expression, migratory capacity, phagocytosis rate, oxidative burst and apoptosis were investigated. In isolated PMNL, CXCR1 and CXCR2 were neutralized before stimulation with CC16, and oxidative burst, phagocytosis and apoptosis were analyzed in neutrophils and their subsets. Serum from the P group enhanced significantly PMNL migration compared to no P group, while CC16-neutralization further increased the migratory rate of PMNL in both groups. CC16-neutralization increased significantly the expression of CD62L in the P group at ED. Oxidative burst was significantly increased in the P group vs. no P during the study period. CC16 seemed to have no influence on oxidative burst and phagocytosis in TP. However, in a more controlled study design, CC16 induced a significant increase of oxidative burst and a decrease of apoptosis of CD16+ granulocytes. These effects were markedly observed in mature CD16brightCD62Lbright and immune suppressive CD16brightCD62Ldim neutrophils. In mature subset, CXCR1 and CXCR2 neutralization diminished CC16-induced effects. CC16 in sera from multiply traumatized patients, notably of those with pneumonia, has significant effects on PMNL. The results suggest an association of CC16 with CXCR1 and CXCR2. Our data suggest that CC16 reduces the migratory capacity of PMNL and thus modulates their function in patients with respiratory complications after trauma.

    更新日期:2019-11-28
  • An immunoevasive strategy through clinically-relevant pan-cancer genomic and transcriptomic alterations of JAK-STAT signaling components
    Mol. Med. (IF 2.991) Pub Date : 2019-11-04
    Wai Hoong Chang; Alvina G. Lai

    Since its discovery almost three decades ago, the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway has paved the road for understanding inflammatory and immunity processes related to a wide range of human pathologies including cancer. Several studies have demonstrated the importance of JAK-STAT pathway components in regulating tumor initiation and metastatic progression, yet, the extent of how genetic alterations influence patient outcome is far from being understood. Focusing on 133 genes involved in JAK-STAT signaling, we investigated genomic, transcriptomic and clinical profiles of over 18,000 patients representing 21 diverse cancer types. We identified a core set of 28 putative gain- or loss-of-function JAK-STAT genes that correlated with survival outcomes using Cox proportional hazards regression and Kaplan-Meier analyses. Differential expression analyses between high- and low-expressing patient groups were performed to evaluate the consequences of JAK-STAT misexpression. We found that copy number alterations underpinning transcriptional dysregulation of JAK-STAT pathway genes differ within and between cancer types. Integrated analyses uniting genomic and transcriptomic datasets revealed a core set of JAK-STAT pathway genes that correlated with survival outcomes in brain, renal, lung and endometrial cancers. High JAK-STAT scores were associated with increased mortality rates in brain and renal cancers, but not in lung and endometrial cancers where hyperactive JAK-STAT signaling is a positive prognostic factor. Patients with aberrant JAK-STAT signaling demonstrated pan-cancer molecular features associated with misexpression of genes in other oncogenic pathways (Wnt, MAPK, TGF-β, PPAR and VEGF). Brain and renal tumors with hyperactive JAK-STAT signaling had increased regulatory T cell gene (Treg) expression. A combined model uniting JAK-STAT and Tregs allowed further delineation of risk groups where patients with high JAK-STAT and Treg scores consistently performed the worst. Providing a pan-cancer perspective of clinically-relevant JAK-STAT alterations, this study could serve as a framework for future research investigating anti-tumor immunity using combination therapy involving JAK-STAT and immune checkpoint inhibitors.

    更新日期:2019-11-28
  • Integrated transcriptomic analysis reveals hub genes involved in diagnosis and prognosis of pancreatic cancer
    Mol. Med. (IF 2.991) Pub Date : 2019-11-09
    Yang-Yang Zhou; Li-Ping Chen; Yi Zhang; Sun-Kuan Hu; Zhao-Jun Dong; Ming Wu; Qiu-Xiang Chen; Zhi-Zhi Zhuang; Xiao-Jing Du

    The hunt for the molecular markers with specificity and sensitivity has been a hot area for the tumor treatment. Due to the poor diagnosis and prognosis of pancreatic cancer (PC), the excision rate is often low, which makes it more urgent to find the ideal tumor markers. Robust Rank Aggreg (RRA) methods was firstly applied to identify the differentially expressed genes (DEGs) between PC tissues and normal tissues from GSE28735, GSE15471, GSE16515, and GSE101448. Among these DEGs, the highly correlated genes were clustered using WGCNA analysis. The co-expression networks and molecular complex detection (MCODE) Cytoscape app were then performed to find the sub-clusters and confirm 35 candidate genes. For these genes, least absolute shrinkage and selection operator (lasso) regression model was applied and validated to build a diagnostic risk score model. Cox proportional hazard regression analysis was used and validated to build a prognostic model. Based on integrated transcriptomic analysis, we identified a 19 gene module (SYCN, PNLIPRP1, CAP2, GNMT, MAT1A, ABAT, GPT2, ADHFE1, PHGDH, PSAT1, ERP27, PDIA2, MT1H, COMP, COL5A2, FN1, COL1A2, FAP and POSTN) as a specific predictive signature for the diagnosis of PC. Based on the two consideration, accuracy and feasibility, we simplified the diagnostic risk model as a four-gene model: 0.3034*log2(MAT1A)-0.1526*log2(MT1H) + 0.4645*log2(FN1) -0.2244*log2(FAP), log2(gene count). Besides, a four-hub gene module was also identified as prognostic model = − 1.400*log2(CEL) + 1.321*log2(CPA1) + 0.454*log2(POSTN) + 1.011*log2(PM20D1), log2(gene count). Integrated transcriptomic analysis identifies two four-hub gene modules as specific predictive signatures for the diagnosis and prognosis of PC, which may bring new sight for the clinical practice of PC.

    更新日期:2019-11-28
  • Association of radiation risk in the second and third generations with polymorphisms in the genes CYP1A1, CYP2E1, GSTP1 and changes in the thyroid
    Mol. Med. (IF 2.991) Pub Date : 2019-11-14
    Meruyert Massabayeva; Nailya Chaizhunusova; Nurlan Aukenov; Tolkyn Bulegenov; Bakytbek Apsalikov; Aigerim Shapihanova; Yersin Zhunussov

    To study the association of radiation risk in the 2nd –3rd generations with polymorphisms in the genes CYP1A1, CYP2E1, GSTP1 and changes in the thyroid. 5 polymorphic gene variants (rs1048943, rs4646421, rs2070676, rs3813867, rs1695) were studied in 399 people living in the East Kazakhstan region in this research. 248 people of the 2nd - 3rd generation lived in the territory with radiation exposure in Abai, Borodulikha areas, and 151 people the comparison group lived in Kurchum district without radiation exposure comparable in sex and age with control group. The results show that there is a significant association of rs1048943 in exposed and unexposed groups (p < 0.003), and the absence of association of rs4646421, rs2070676, rs3813867, rs1695 in the studied groups. The mean value of thyroxine in carriers of the AG + GG genotype of rs4646421 is significantly lower than in AA genotype carriers (p = 0.04); no significant changes were found in genotypes’ distribution with thyroid-stimulating hormone and anti-thyroid peroxidase indicators. Significant changes were in levels of anti-thyroid peroxidase between exposed and unexposed groups (p = 0.007). The thyroxine - thyroid-stimulating hormone levels were not significantly different in exposed and unexposed groups (p > 0.3). This study demonstrated the association of rs1048943 polymorphism with living in the radiation zone in the 2nd and 3rd generations for the first time. Thyroxine levels decrease was identified in the 2nd and 3rd generation residents of the exposed area, as well as a significant increase of anti-thyroid peroxidase occurs in individuals of the 2nd and 3rd generation living in areas with radiation exposure.

    更新日期:2019-11-28
  • Inhibition of phosphatidylinositol 3-kinase by PX-866 suppresses temozolomide-induced autophagy and promotes apoptosis in glioblastoma cells
    Mol. Med. (IF 2.991) Pub Date : 2019-11-14
    Bryan G. Harder; Sen Peng; Christopher P. Sereduk; Andrej M. Sodoma; Gaspar J. Kitange; Joseph C. Loftus; Jann N. Sarkaria; Nhan L. Tran

    Temozolomide (TMZ) is the most commonly used chemotherapeutic agent used to treat glioblastoma (GBM), which causes significant DNA damage to highly proliferative cells. Our observations have added to accumulating evidence that TMZ induces stress-responsive cellular programs known to promote cell survival, including autophagy. As such, targeting these survival pathways may represent new vulnerabilities of GBM after treatment with TMZ. Using the T98G human glioma cell line, we assessed the molecular signaling associated with TMZ treatment, the cellular consequences of using the pan-PI3K inhibitor PX-866, and performed clonogenic assays to determine the effect sequential treatment of TMZ and PX-866 had on colony formation. Additionally, we also use subcutaneous GBM patient derived xenograft (PDX) tumors to show relative LC3 protein expression and correlations between survival pathways and molecular markers which dictate clinical responsiveness to TMZ. Here, we report that TMZ can induce autophagic flux in T98G glioma cells. GBM patient-derived xenograft (PDX) tumors treated with TMZ also display an increase in the autophagosome marker LC3 II. Additionally, O6-methylguanine-DNA-methyltransferase (MGMT) expression correlates with PI3K/AKT activity, suggesting that patients with inherent resistance to TMZ (MGMT-high) would benefit from PI3K/AKT inhibitors in addition to TMZ. Accordingly, we have identified that the blood-brain barrier (BBB) penetrant pan-PI3K inhibitor, PX-866, is an early-stage inhibitor of autophagic flux, while maintaining its ability to inhibit PI3K/AKT signaling in glioma cells. Lastly, due to the induction of autophagic flux by TMZ, we provide evidence for sequential treatment of TMZ followed by PX-866, rather than combined co-treatment, as a means to shut down autophagy-induced survival in GBM cells and to enhance apoptosis. The understanding of how TMZ induces survival pathways, such as autophagy, may offer new therapeutic vulnerabilities and opportunities to use sequential inhibition of alternate pro-survival pathways that regulate autophagy. As such, identification of additional ways to inhibit TMZ-induced autophagy could enhance the efficacy of TMZ.

    更新日期:2019-11-28
  • Circulating tumor cells: a valuable marker of poor prognosis for advanced nasopharyngeal carcinoma
    Mol. Med. (IF 2.991) Pub Date : 2019-11-15
    Guoping Ou; Shan Xing; Jianpei Li; Lin Zhang; Shulin Chen

    To evaluate the prognostic value of circulating tumor cells (CTCs) in nasopharyngeal carcinoma (NPC). Cox’s proportional hazards regression models were used to identify whether CTCs was a poor prognostic factor for NPC. Chi-square tests were used to analyze and compare the distribution characteristics of CTCs in NPC. ROC curve was used to estimate the cut-off point of CTCs. Kaplan-Meier survival analyses were used to observe the prognostic value of CTCs alone and in combined with Epstein-Barr Virus DNA (EBV-DNA). CTCs was confirmed to be an independent risk factor for poor prognosis of NPC by Cox’s regression models that enrolled 370 NPC cases and took age, gender, EBV-DNA and CTCs as variables. The proportion of CTCs in stage IV NPC was statistically different from that in stage III; the cut-off point of CTCs between stage IV (288 cases) and stage III (70 cases) NPC estimated by ROC curve was 0.5. The prognosis of advanced NPC patients became worse with the increase of CTCs count. The combined detection of CTCs and EBV-DNA could better predict the prognosis of NPC compared with the single detection of EBV-DNA.

    更新日期:2019-11-28
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