We aimed to investigate the association of rheumatoid arthritis (RA) with interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) through a meta-analysis.
The case-control studies that investigated the association between RA and serum levels of IL-6 and TNF-α were retrieved strictly according to the inclusion and exclusion criteria. The statistical analysis was performed using STATA statistical software (Version 12.0, Stata Corporation, College Station, TX, USA).
Fourteen studies were enrolled in our meta-analysis, with a total of 890 patients with RA and 441 healthy people as the controls. The results of this meta-analysis revealed that the serum IL-6 and TNF-α levels of RA patients were significantly higher than in the controls, and this difference was statistically significant (IL-6: SMD=2.40, 95% CI=1.57~3.24, P<0.001; TNF-α: SMD=1.93, 95% CI=1.23~2.64, P<0.001). According to ethnic subgroup analysis, the serum IL-6 and TNF-α levels of RA patients were also significantly higher compared with the controls in Asians and Caucasians (IL-6: Asians: SMD=3.64, 95% CI=2.16~5.12, P<0.001; Caucasians: SMD=0.75, 95% CI=0.47~1.02, P<0.001; TNF-α: Asians: SMD=2.74, 95%CI=1.58~3.91, P<0.001; Caucasians: SMD=0.81, 95% CI=0.50~1.11, P<0.001).
IL-6 and TNF-α may play crucial roles in the activity and severity of RA.
Arthritis, Juvenile; Interleukin-6; Meta-Analysis
Adjuvant sufentanil could achieve effective spinal anesthesia with low dose of hyperbaric ropivacaine for cesarean delivery. Two previous studies had calculated the 50% effective dose (ED50) of intrathecal ropivacaine coadministered with sufentanil for cesarean delivery. However, the 95% effective dose (ED95) of intrathecal hyperbaric ropivacaine coadministered with sufentanil for cesarean delivery remains uncertain. This study determined the ED95 of intrathecal hyperbaric ropivacaine coadministered with sufentanil for cesarean delivery. 80 ASA physical status I or II parturients undergoing elective cesarean delivery were enrolled in this prospective, randomized, double-blind investigation. A combined spinal and epidural anesthesia was performed at the L3-L4 interspace. Patients received a dose of spinal ropivacaine coadministered with sufentanil 5 μg diluted to 3.0 ml with normal saline and 0.5 ml of 10% dextrose: 7.5 mg (n = 20), 9.0 mg (n = 20), 10.5 mg (n = 20), or 12 mg (n = 20). An effective dose was defined as a dose that provided bilateral sensory block to T7 within 10 min after intrathecal drug administration and required no epidural top-up for surgery to be completed. The ED50 and ED95 values for successful anesthesia were determined using a logistic regression model. The ED50 (95% confidence interval [CI]) for successful anesthesia was 8.4 (4.0-9.8) mg and the ED95 (95% CI) was 11.4 (9.7-13.9) mg. The results show that the ED95 of intrathecal hyperbaric ropivacaine coadministered with sufentanil 5 μg for cesarean delivery was 11.4 mg. The addition of sufentanil could significantly reduce the dosage of ropivacaine.
Cesarean delivery; ropivacaine; sufentanil; spinal anesthesia
The aim of the present study was to assess the effects of sprouty homolog 2 (SPRY2) gene regulation by miR-21 on the occurrence, development and tumor metastasis in multiple myeloma (MM). The miR-21 expression lentiviral vector (LV)-anti-miR-21 and a liposome transfection method were used to screen MM cell lines with stable silent SPRY2. Real-time quantitative polymerase chain reaction (PCR) and western blot analyses were used to detect SPRY2 expression and miR-21 protein expression levels. An MTT assay was used to assess cell proliferation. Flow cytometry was used for analysis of cell cycle. A scratch test/wound healing assay was used to detect the cell migration ability. A Transwell assay was used to detect the cell invasion ability. Real-time quantitative PCR and western blot analysis showed that in the MM cell lines with high endogenous miR-21 expression (RPMI8226 and KM3), SPRY2 expression was significantly lower. Conversely, in the U266 cell line with low endogenous miR-21 expression, SPRY2 expression was significantly higher, and the gray values of miR-21 and SPRY2 protein in the respective cell lines showed statistically significant differences (P<0.01). Following transfection of U266 cells, the expression of miR-21 in the U266/LV-anti-miR21 lentiviral multiplicity of infection (MOI) 20 group and -MOI 40 group decreased significantly compared with that in the untransfected U266 group (P<0.05). SPRY2 protein expression in U266 cells transfected with miR-21 mimics was significantly reduced compared with that in the non-transfected (untreated) group and the negative control-transfected group (P<0.01). An MTT assay showed that compared with the non-transfected and negative control groups, the cell growth rate as well as the proliferation rate were significantly decreased in the transfection group 48, 72 and 96 h after transfection (P<0.01). Flow cytometric analysis showed that 48 and 72 h after transfection of U266 cells with miR-21 mimics, the apoptotic rates were (24.7±1.97 and 38.6±1.56%) in the U266 group, (27.3±1.72 and 37.3±1.59%) in the siRNA group and (12.7±1.27 and 22.1±1.63%) in the U266/miR-21 group. Compared with the two control groups, the apoptotic rate in the U266/miR-21 group was significantly decreased and the G0/G1 phase cell population was significantly reduced (P<0.05). Scratch experiments showed that the cell migration ability was significantly reduced in the transfection group 24 and 48 h after transfection (P<0.05). A Transwell invasion assay confirmed that the number of U266 cells which migrated through a Matrigel-covered polyphosphate membrane significantly decreased in the transfection group 24 and 48 h after transfection. The cell-penetrating ability was also significantly decreased (P<0.05). In conclusion, the downregulation of SPRY2 gene expression mediated by miR-21 promotes the proliferation and invasion of MM cells in vitro, suggesting that miR-21 may be a novel potential molecular therapeutic target in the treatment of MM.
multiple myeloma; microRNA-21; sprouty homolog 2; proliferation; invasion
Mitochondrial production of reactive oxygen species is often considered an unavoidable consequence of aerobic metabolism and currently cannot be manipulated without perturbing oxidative phosphorylation. Antioxidants are widely used to suppress effects of reactive oxygen species after formation, but they can never fully prevent immediate effects at the sites of production. To identify site-selective inhibitors of mitochondrial superoxide/H2O2 production that do not interfere with mitochondrial energy metabolism, we developed a robust small-molecule screen and secondary profiling strategy. We describe the discovery and characterization of a compound (N-cyclohexyl-4-(4-nitrophenoxy)benzenesulfonamide; CN-POBS) that selectively inhibits superoxide/H2O2 production from the ubiquinone-binding site of complex I (site IQ) with no effects on superoxide/H2O2 production from other sites or on oxidative phosphorylation. Structure/activity studies identified a core structure that is important for potency and selectivity for site IQ. By employing CN-POBS in mitochondria respiring on NADH-generating substrates, we show that site IQ does not produce significant amounts of superoxide/H2O2 during forward electron transport on glutamate plus malate. Our screening platform promises to facilitate further discovery of direct modulators of mitochondrially-derived oxidative damage and advance our ability to understand and manipulate mitochondrial reactive oxygen species production in both normal and pathological conditions.
superoxide; hydrogen peroxide; antioxidant; glycerol 3-phosphate dehydrogenase; NADH:Q oxidoreductase; complex II; complex III; energy metabolism; respiratory complexes
The aim of the present study was to investigate the expression level of microRNA 21 (miR-21) in the peripheral blood of patients with multiple myeloma (MM) and to investigate the correlation between miR-21 and sprouty homolog 2 (SPRY2) gene expression levels in MM. A total of 30 patients with MM, 15 with monoclonal gammopathy of undetermined significance (MGUS) and 20 normal control (NC) outpatients were selected for the detection of miR-21 and SPRY2 expression using reverse transcription-quantitative polymerase chain reaction. In addition, western blot analysis was performed to detect the expression of miR-21 and SPRY2 in MM cell lines. The expression of miR-21 in U-266 cells following lipofectamine transfection of fluorescence-labeled miR-21 mimic/inhibitor was observed using a fluorescence microscope and the expression level of SPRY2 in the miR-21 mimic/inhibitor-transfected U-266 cells was detected using western blot analysis. The miR-21 expression level in the circulating serum of the MM patient group was significantly higher (P<0.01) than that of the MGUS and NC groups. The MM cell lines with high endogenous miR-21 expression exhibited an expression level of SPRY2 that was significantly lower than that in the MM cells with low endogenous miR-21 expression. The transfection efficiency of fluorescence-labeled miR-21 mimic/inhibitor was >90%. Compared with the miR-21 expression level in untreated U-266 cells (0.82±0.13), the expression level of miR-21 was increased by 120.2-fold in miR-21 mimic-transfected cells (98.6±14.2; P<0.001) and was decreased by 61.9% in the miR-21 inhibitor-transfected cells (0.37±0.06; P<0.05). The grayscale value of protein bands demonstrated that SPRY2 protein expression significantly decreased in miR-21 mimic-transfected U-266 cells compared with that in the inhibitor-transfected, siRNA-transfected and untreated cells (P<0.01). miR-21 may represent a negative regulator involved in the downregulation of SPRY2 in MM. miR-21 is closely associated with the pathogenesis, progression and prognosis of MM and may thus be used as an indicator of poor MM prognosis.
microRNA-21; multiple myeloma; sprouty homolog 2; regulation
In most cells, mitochondria are highly dynamic organelles that constantly fuse, divide and move. These processes allow mitochondria to redistribute in a cell and exchange contents among the mitochondrial population, and subsequently repair damaged mitochondria. However, most studies on mitochondrial dynamics have been performed on cultured cell lines and neurons, and little is known about whether mitochondria are dynamic organelles in vivo, especially in the highly specialized and differentiated adult skeletal muscle cells. Using mitochondrial matrix-targeted photoactivatable green fluorescent protein (mtPAGFP) and electroporation methods combined with confocal microscopy, we found that mitochondria are dynamic in skeletal muscle in vivo, which enables mitochondria exchange contents within the whole mitochondrial population through nanotunneling-mediated mitochondrial fusion. Mitochondrial network promotes rapid transfer of mtPAGFP within the cell. More importantly, the dynamic behavior was impaired in high-fat diet (HFD)-induced obese mice, accompanying with disturbed mitochondrial respiratory function and decreased ATP content in skeletal muscle. We further found that proteins controlling mitochondrial fusion MFN1 and MFN2 but not Opa1 were decreased and proteins governing mitochondrial fission Fis1 and Drp1 were increased in skeletal muscle of HFD-induced mice when compared to normal diet-fed mice. Altogether, we conclude that mitochondria are dynamic organelles in vivo in skeletal muscle, and it is essential in maintaining mitochondrial respiration and bioenergetics.
Mitochondrial sn-glycerol 3-phosphate dehydrogenase (mGPDH) is a ubiquinone-linked enzyme in the mitochondrial inner membrane best characterized as part of the glycerol phosphate shuttle that transfers reducing equivalents from cytosolic NADH into the mitochondrial electron transport chain. Despite the widespread expression of mGPDH and the availability of mGPDH-null mice, the physiological role of this enzyme remains poorly defined in many tissues, likely because of compensatory pathways for cytosolic regeneration of NAD+ and mechanisms for glycerol phosphate metabolism. Here we describe a novel class of cell-permeant small-molecule inhibitors of mGPDH (iGP) discovered through small-molecule screening. Structure-activity analysis identified a core benzimidazole-phenyl-succinamide structure as being essential to inhibition of mGPDH while modifications to the benzimidazole ring system modulated both potency and off-target effects. Live-cell imaging provided evidence that iGPs penetrate cellular membranes. Two compounds (iGP-1 and iGP-5) were characterized further to determine potency and selectivity and found to be mixed inhibitors with IC50 and Ki values between ∼1–15 µM. These novel mGPDH inhibitors are unique tools to investigate the role of glycerol 3-phosphate metabolism in both isolated and intact systems.
Wnt/β-catenin-mediated gene transcription plays important roles in a wide range of biological and pathophysiological processes including tumorigenesis where β-catenin-mediated transcription activity frequently elevates. TRABID, a deubiquitinase, was shown to have a positive Wnt/β-catenin-mediated gene transcription and hence holds a promise as a putative anti-cancer target.
In this study, we used a combination of structure based virtual screening and an in vitro deubiquitinase (DUB) assay to identify several small molecules that inhibit TRABID DUB activity. However, these inhibitors failed to show inhibitory effects on β-catenin-mediated gene transcription. In addition, expression of TRABID shRNAs, wildtype TRABID, or the DUB activity-deficient mutant showed little effects on β-catenin-mediated gene transcription.
TRABID may not be a critical component in canonical Wnt/β-catenin signal transduction or that a minute amount of this protein is sufficient for its role in regulating Wnt activity.
A second degree epidermal scald burn in mice elicits an inflammatory response mediated by natural IgM directed to non-muscle myosin with complement activation that results in ulceration and scarring. We find that such burn injury is associated with early mast cell (MC) degranulation and is absent in WBB6F1-KitW/KitWv mice which lack MCs in a context of other defects due to a mutation of the KIT receptor. To further address a MC role, we used transgenic strains with normal lineage development and a deficiency in a specific secretory granule component. Mouse strains lacking the MC-restricted chymase, mouse MC protease (mMCP)-4, or elastase, mMCP-5, show decreased injury following a second degree scald burn while mice lacking the MC-restricted tryptases, mMCP-6 and mMCP-7, or the MC-specific carboxypeptidase A3 activity are not protected. Histologic sections showed some disruption of the epidermis at the scald site in the protected strains suggesting the possibility of topical reconstitution of full injury. Topical application of recombinant mMCP-5 or human neutrophil elastase to the scalded area increases epidermal injury with subsequent ulceration and scarring, both clinically and morphologically, in mMCP-5-deficent mice. Restoration of injury requires that topical administration of recombinant mMCP-5 occurs within the first h post burn. Importantly, topical application of human MC chymase restores burn injury to scalded mMCP-4-deficient mice but not to mMCP-5-deficient mice revealing non-redundant actions for these two MC proteases in a model of innate inflammatory injury with remodeling.
Erythromycin, a hydrophobic antibiotic used to treat infectious diseases, is now gaining attention because of its anti-inflammatory effects and ability to inhibit osteoclasts formation. The aim of this study was to explore a cyclodextrin-erythromycin (CD-EM) complex for sustained treatment of orthopedic inflammation.
Methods and results
Erythromycin was reacted with β-cyclodextrin to form a nonhost-guest CD-EM complex using both kneading and stirring approaches. Physiochemical measurement data indicated that erythromycin and cyclodextrin formed a packing complex driven by intermolecular forces instead of a host-guest structure due to the limited space in the inner cavity of β-cyclodextrin. The CD-EM complex improved the stability of erythromycin in aqueous solution and had a longer duration of bactericidal activity than free erythromycin. Cytotoxicity and cell differentiation were evaluated in both murine MC3T3 preosteoblast cells and RAW 264.7 murine macrophage cells. The CD-EM complex was noncytotoxic and showed significant inhibition of osteoclast formation but had little effect on osteoblast viability and differentiation.
These attributes are especially important for the delivery of an adequate amount of erythromycin to the site of periprosthetic inflammation and reducing local inflammation in a sustained manner.
erythromycin; cyclodextrin; drug stability; bactericidal activity; osteoclastogenesis
To investigate the effect of magnesium sulfate and its interaction with the non-depolarizing muscle relaxant vecuronium at adult muscle-type acetylcholine receptors in vitro.
Adult muscle-type acetylcholine receptors were expressed in HEK293 cells. Drug-containing solution was applied via a gravity-driven perfusion system. The inward currents were activated by brief application of acetylcholine (ACh), and recorded using whole-cell voltage-clamp technique.
Magnesium sulfate (1–100 mmol/L) inhibited the inward currents induced ACh (10 μmol/L) in a concentration-dependent manner (IC50=29.2 mmol/L). The inhibition of magnesium sulfate was non-competitive. In contrast, vecuronium produced a potent inhibition on the adult muscle-type acetylcholine receptor (IC50=8.7 nmol/L) by competitive antagonism. Magnesium sulfate at the concentrations of 1, 3, and 6 mmol/L markedly enhanced the inhibition of vecuronium (10 nmol/L) on adult muscle-type acetylcholine receptors.
Clinical enhancement of vecuronium-induced muscle relaxation by magnesium sulfate can be attributed partly to synergism between magnesium sulfate and non-depolarizing muscle relaxants at adult muscle-type acetylcholine receptors.
magnesium sulfate; non-depolarizing muscle relaxant; vecuronium; adult muscle-type acetylcholine receptor; synergism
To investigate the changing resistance to nondepolarizing muscle relaxants (NDMRs) during the first month after denervation.
The denervated and innervated skeletal muscle cells were examined on days 1, 4, 7, 14, 21, and 28 after denervation. Individual denervated and innervated cells were prepared from the flexor digitorum brevis of the surgically denervated and contralateral hind feet, respectively. Nicotinic acetylcholine receptors (nAChRs) in the cells were activated with 30 μmol/L acetylcholine, either alone or in combination with various concentrations of vecuronium. Currents were recorded using a whole-cell patch-clamp technique.
The concentrations of vecuronium resulting in half-maximal inhibitory responses (IC50) increased 1.2- (P>0.05), 1.7-, 3.7-, 2.5-, 1.9-, and 1.8-fold (P<0.05) at Days 1, 4, 7, 14, 21, and 28 after denervation, respectively, compared to the innervated control. Resistance to vecuronium appeared at Day 4, peaked at Day 7, and declined at Day 14 after denervation. Nevertheless, IC50 values at Day 28 remained significantly higher than those for the innervated control, suggesting that the resistance to vecuronium had not disappeared at Day 28.
The NDMR doses required to achieve satisfactory clinical effects differ at different times after muscle denervation.
denervation; nondepolarizing muscle relaxants; nicotinic acetylcholine receptors; skeletal muscle cells; vecuronium
Morphological innovations that significantly enhance performance capacity may enable exploitation of new resources and invasion of new ecological niches. The invasion of land from the aquatic realm requires dramatic structural and physiological modifications to permit survival in a gravity-dominated, aerial environment. Most fishes are obligatorily aquatic, with amphibious fishes typically making slow-moving and short forays on to land.
Here I describe the behaviors and movements of a little known marine fish that moves extraordinarily rapidly on land. I found that the Pacific leaping blenny, Alticus arnoldorum, employs a tail-twisting movement on land, previously unreported in fishes. Focal point behavioral observations of Alticus show that they have largely abandoned the marine realm, feed and reproduce on land, and even defend terrestrial territories. Comparisons of these blennies' terrestrial kinematic and kinetic (i.e., force) measurements with those of less terrestrial sister genera show A. arnoldorum move with greater stability and locomotor control, and can move away more rapidly from impending threats.
My results demonstrate that axial tail twisting serves as a key innovation enabling invasion of a novel marine niche. This paper highlights the potential of using this system to address general evolutionary questions about water-land transitions and niche invasions.
To test the hypothesis that different magnitude of resistance of denervated skeletal muscle to nondepolarizing muscle relaxants (NDMRs) is related to their varying potencies at ɛ-AChR and γ-AChR.
Both innervated and denervated mouse muscle cells, and human embryonic kidney 293 (HEK293) cells expressing ɛ-AChR or γ-AChR were used. The effects of NDMRs on nAChR were explored using whole-cell patch clamp technique.
NDMRs vecuronium (VEC), atracurium (ATR) and rocuronium (ROC) produced reversible, dose-dependent inhibition on the currents induced by 30 μmol/L acetylcholine both in innervated and denervated skeletal muscle cells. Compared to those obtained in innervated skeletal muscle cells, denervation shifted the concentration-response curves rightward and significantly increased the 50% inhibitory concentration (IC50) values (VEC: from 11.2 to 39.2 nmol/L, P<0.01; ATR: from 24.4 to 129.0 nmol/L, P<0.01; ROC: from 37.9 to 101.4 nmol/L, P<0.01). In HEK293 cell expression system, ATR was less potent at γ-AChR than ɛ-AChR (IC50 values: 35.9 vs 22.3 nmol/L, P<0.01), VEC was equipotent at both receptor subtypes (IC50 values: 9.9 vs 10.2 nmol/L, P>0.05), while ROC was more potent at γ-AChR than ɛ-AChR (IC50 values: 22.3 vs 33.5 nmol/L, P<0.05).
Magnitude differences of resistance to different NDMRs caused by denervation are associated with distinct potencies of NDMRs at nAChR subtypes.
acetylcholine; atracurium; denervation; nicotinic acetylcholine receptor; rocuronium; nondepolarizing muscle relaxants; vecuronium
Angiostrongylus cantonensis; eosinopilic meningitis; Shenzhen; human; rates; snails; letter
AIM: To explore how to improve the immunogenicity of HBcAg CTL epitope based polypeptides and to trigger an HBV-specific HLA I-restricted CD8+ T cell response in vitro.
METHODS: A new panel of mimetic therapeutic peptides based on the immunodominant B cell epitope of HBV PreS2 18-24 region, the CTL epitope of HBcAg18-27 and the universal T helper epitope of tetanus toxoid (TT) 830-843 was designed using computerized molecular design method and synthesized by Merrifield’s solid-phase peptide synthesis. Their immunological properties of stimulating activation and proliferation of lymphocytes, of inducing TH1 polarization, CD8+ T cell magnification and HBV-specific CD8+ CTL mediated cytotoxicity were investigated in vitro using HLA-A2+ human peripheral blood mononuclear cells (PBMCs) from healthy donors and chronic hepatitis B patients.
RESULTS: Results demonstrated that the therapeutic polypeptides based on immunodominant HBcAg18-27 CTL, PreS2 B- and universal TH epitopes could stimulate the activation and proliferation of lymphocytes, induce specifically and effectively CD8+ T cell expansion and vigorous HBV-specific CTL-mediated cytotoxicity in human PBMCs.
CONCLUSION: It indicated that the introduction of immunodominant T helper plus B-epitopes with short and flexible linkers could dramatically improve the immunogenicity of short CTL epitopes in vitro.
AIM: To explore how to trigger an HLAI-restricted CD8+ T cell response to exogenously synthesized polypeptides in vivo.
METHODS: Three mimetic therapeutic polypeptides based on the immunodominant CTL epitope of HBcAg, the B- epitope of HBV PreS2 region and a common T helper sequence of tetanus toxoid were designed and synthesized with Merrifield’s solid-phase peptide synthesis method. Their immunological properties of inducing TH1 polarization, CD8+ HBV-specific CTL expansion and CD8+ T cell mediated cytotoxicity were investigated in HLA-A2 transgenic mice.
RESULTS: Results demonstrated that the mimetic polypeptides comprised of the immunodominant CTL, B-, and T helper epitopes could trigger specifically and effectively vigorous CD8+ HBV-specific CTL-mediated cytotoxicity and TH1 polarization of T cells in HLA-A2 transgenic mice.
CONCLUSION: A designed universal T helper plus B-epitopes with short and flexible linkers could dramatically improve the immunogenicity of CTL epitopes in vivo. And that the mimetic therapeutic peptides based on the reasonable match of the above CTL, B- and T helper epitopes could be a promising therapeutic peptide vaccine candidate against HBV infection.
AIM: To characterize the biochemical and immunological properties of an experimental ISCOMS vaccine prepared from a novel therapeutic polypeptide based on T cell epitopes of HBsAg, and a heptatis B-ISCOMS was prepared and investigated.
METHODS: An immunostimulating complexes (ISCOMS)-based vaccine containing a novel therapeutic hepatits B polypeptide was prepared by dialysis method, and its formation was visualized by electron microscopy and biochemically verified by SDS-polyacrylamide gel electrophoresis. Amount of the peptide within ISCOMS was determined by Bradford assay, and specific CTL response was detected by ELISPOT assay.
RESULTS: Typical cage-like structures of submicroparticle with a diameter of about 40 nm were observed by electron microscopy. Results from Bradford assay showed that the level of peptide incorporation was about 0.33 g•L⁻¹. At the paralleled position close to the sixth band of the molecular weight marker (3480 kDa) a clear band was shown in SDS-PAGE analysis, indicating successful incorporation of polypeptide into ISCOMS. It is suggested that ISCOMS delivery system could efficiently improve the immunogenicity of polypeptide and elicit specific immune responses in vivo by the results of ELISPOT assay, which showed that IFN-γ producing cells (specific CTL responses) were increased (spots of ISCOMS-treated group: 47 ± 5, n = 3; control group: 5 ± 2, n = 3).
CONCLUSION: ISCOMS-based hepatitis B polypeptide vaccine is successfully constructed and it induces a higher CTL response compared with short polypeptides vaccine in vivo.