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Hypoxia, the typical and conspicuous characteristic of most solid tumors, worsens the tumor invasiveness and metastasis. Here, we engineered a sequential ultrasound (US)/hypoxia-sensitive sonochemotherapeutic nanoprodrug by initially synthesizing the hypoxia-activated azo bond-containing camptothecin (CPT) prodrug (CPTsub2/sub-Azo) and then immobilizing it into the mesopores of sonosensitizer-integrated metal organic frameworks (MOF NPs). Upon entering the hypoxic tumor microenvironment (TME), the structure of CPTsub2/sub-Azo immobilized MOFs (denoted as MCA) was ruptured and the loaded nontoxic CPTsub2/sub-Azo prodrug was released from the MOF NPs. Under US actuation, this sonochemotherapeutic nanoprodrug not only promoted sonosensitizer-mediated sonodynamic therapy (SDT) via the conversion of oxygen into cytotoxic reactive oxygen species (ROS) but also aggravated hypoxia in the TME by elevating oxygen consumption. The exacerbated hypoxia in turn served as a positive amplifier to boost the activation of CPTsub2/sub-Azo, and the controllable release of toxic chemotherapeutic drug (CPT), and compensated the insufficient treatment efficacy of SDT.iIn vitro/iandiin vivo/ievaluations confirmed that sequential SDT and tumor hypoxia-activated sonochemotherapy promoted the utmost of tumor hypoxia and thereby contributed to the augmented antitumor efficacy, resulting in conspicuous apoptotic cell death and noteworthy tumor suppressioniin vivo/i. Our work provides a distinctive insight into the exploitation of the hypoxia-activated sonochemotherapeutic nanoprodrug that utilizes the hypoxic condition in TME, a side effect of SDT, to initiate chemotherapy, thus causing a significantly augmented treatment outcome compared to conventional SDT.

Most fast synaptic inhibitions in the mammalian brain are mediated by GABA

Most fast synaptic inhibitions in the mammalian brain are mediated by GABAA receptors (GABAARs). An appropriate level of GABAAR expression at the cell surface is essential for neurodevelopment and the efficacy of GABAergic synaptic transmission. We previously reported that brefeldin A-inhibited GDP/GTP exchange factor 1 (BIG1), a binding partner of GABAARs, plays an important role in trafficking GABAARs to the cell surface. However, its regulatory mechanisms remain unknown. In the present study, we identified a new cellular protein, 14-3-3ζ, which can interact with the β subunit of GABAARs and BIG1 both in vitro and in vivo and colocalizes in the soma, dendrites and axons of hippocampal neurons. Overexpression of 14-3-3ζ-WT increased the surface expression of BIG1 in dendrites and axons, as well as the binding of BIG1 with GABAAR. Depleted 14-3-3ζ with efficacious siRNA attenuated the interaction between BIG1 and GABAARs and resulted in significant decreases in the surface expression levels of BIG1 and GABAAR. GABAAR agonist treatment increased the expression levels of BIG1 and 14-3-3ζ on the surface, indicating that 14-3-3ζ is involved in regulating BIG1-mediated GABAAR surface expression. Depletion of BIG1 or 14-3-3ζ significantly decreased GABAAR expression at the cell surface and suppressed the GABA-gated influx of chloride ions. These data indicate that the combination of 14-3-3ζ and BIG1 is required for GABAAR membrane expression. Our results provide a potential promising therapeutic target for neurological disorders involving GABAergic synaptic transmission.

Tophus is a characteristic manifestation of advanced gout, the clinical significance of which is often underestimated. This study aimed to compare the difference of clinical and ultrasound features between gout patients with and without ultrasound-detected tophus and identify risk factors associated with the presence of ultrasonographic tophus in gout patients.A total of 85 gout patients were divided into tophaceous (n = 54) and non-tophaceous group (n = 31) according to the presence of ultrasound-detected tophus. All patients underwent ultrasound examination of the bilateral knee, ankle, and first metatarsophalangeal joint (MTP1). Clinical information and ultrasound findings were compared between the groups. A multivariate logistic regression analysis to determine possible risk factors is associated with the number of ultrasound-detected tophaceous joints.Older age, longer gout duration, higher gout flare frequency, lower estimated glomerular filtration rate (eGFR), and higher prevalence of hypertension, hyperlipidemia, and ultrasound manifestations including double contour sign (DCS) and erosion were observed in tophaceous patients from the univariate analysis. Multivariable logistic regression analysis showed that eGFR and disease duration were independently associated with the number of tophaceous joints. Lower eGFR and longer course duration were associated with a higher risk of tophi (B = -0.020, 0.141; P = 0.009, 0.010, respectively).The main factors that may influence the formation of tophi are disease duration and eGFR.Key Points• Lower eGFR and longer course duration are independent risk factors of tophi formation in gout patients.• The incidence of ultrasound manifestations including double contour sign (DCS) and erosion in patients with tophi were higher than those without tophi.

β-amyloid peptide (Aβ) deposition derived from sequential cleavage of the amyloid precursor protein (APP) through the amyloidogenic pathway is an important characteristic feature of Alzheimer's disease (AD). During this process, cellular trafficking plays a crucial role. A large Sec7-domain containing ADP-ribosylation factor guanine nucleotide exchange factor (ARF-GEF), Golgi brefeldin A resistance factor 1 (GBF1) has been reported to initiate the ADP-ribosylation factor (Arf) activation cascade at trans-Golgi network, which plays a crucial function at the endoplasmic reticulum-Golgi interface. In this study, we investigated the role of GBF1 in APP transmembrane transport and Aβ formation. Using APP/PS1 (presenilin 1) overexpressing transgenic mice, we demonstrate that GBF1 has upregulated the expression of APP, indicating a role for GBF1 in APP physiological process. Knocking down of GBF1 using small interfering has significantly increased the intracellular but not the surface expression of APP. In contrast, overexpression of wild-type (WT) and guanine nucleotide exchange factor (GEF) in the activated form but not the GEF deficient mutation induced continuous activation of GBF1, which subsequently increased the surface level of APP. Interestingly, inhibition of GBF1 by c(BFA) also impaired APP trafficking and induced endoplasmic reticulum (ER) stress in SH-SY5Y cells. Our results thus for identified the role of GBF1 in APP trafficking and cleavage, and provide evidence for GBF1 as a possible therapeutic target in AD.

Cochlear implantation is the first-line treatment for severe and profound hearing loss in children and adults. However, deaf patients with cochlear malformations or with cochlear nerve deficiencies are ineligible for cochlear implants. Meanwhile, the limited spatial selectivity and high risk of invasive craniotomy restrict the wide application of auditory brainstem implants. A noninvasive alternative strategy for safe and effective neuronal stimulation is urgently needed to address this issue. Because of its advantage in neural modulation over electrical stimulation, low-intensity ultrasound (US) is considered a safe modality for eliciting neural activity in the central auditory system. Although the neural modulation ability of low-intensity US has been demonstrated in the human primary somatosensory cortex and primary visual cortex, whether low-intensity US can directly activate auditory cortical neurons is still a topic of debate. To clarify the direct effects on auditory neurons, in the present study, we employed low-intensity US to stimulate auditory cortical neurons in vitro. Our data show that both low-frequency (0.8 MHz) and high-frequency (>27 MHz) US stimulation can elicit the inward current and action potentials in cultured neurons. c-Fos staining results indicate that low-intensity US is efficient for stimulating most neurons. Our study suggests that low-intensity US can excite auditory cortical neurons directly, implying that US-induced neural modulation can be a potential approach for activating the auditory cortex of deaf patients.

Background and purpose The balance between T helper 17 (Th17) cells and regulatory T (Treg) cells, plays a critical role in rheumatoid arthritis (RA). The differentiation of Th17 cells requires the activation of STAT3, which determines the balance of Th17/Treg. Here, we investigated the therapeutic effect of Cryptotanshinone (CTS) on collagen induced mouse arthritis and explored the underlying mechanisms. Experimental approach Arthritis was induced in DBA/1 mice with bovine collagen type II and complete Freund’s adjuvant. CTS was given at 20 mg kg −1 d −1 or 60 mg kg −1 d −1 by gavage for 6 weeks. The immuno-inflammation and joint destruction were evaluated and the balance of Th17/Treg was determined. STAT3 acetylation and phosphorylation were detected by western blotting, and the involvement of p300 was investigated by siRNA and plasmid overexpression. Key results CTS at a dose of 60 mg kg −1 d −1 ameliorated the inflammation and joint destruction in CIA mice. It improved Th17/Treg imbalance, and inhibited both acetylation and phosphorylation of STAT3. CTS reduced p300 expression and its binding to STAT3, but increased phosphorylated AMPK. Knockdown of p300 mimicked the inhibitory effect of CTS on STAT3 acetylation and phosphorylation, which could be partially rescued by overexpression of p300-WT, but not p300-dominant negative (DN) construct. Conclusion and implications Our study suggested that the anti-arthritis effects of CTS were attained through suppression of p300-mediated STAT3 acetylation. Our data suggest that CTS might be a potential immune modulator for RA treatment.

Matrix metalloproteinases (MMPs) plays a critical role in the degradation of extracellular matrix (ECM). Sorting nexin (SNX) 10 is a member of the SNX family, which functions in regulation of endosomal sorting and osteoclast activation, has been implicated to play an important role in the bone erosion of rheumatoid arthritis. In this study, we aimed to investigate the possible role of SNX10 on MMP9 secretion and the potential mechanism. By immunostaining and co-immunoprecipitation, we found that SNX10 was extensively co-localized with MMP9, indicating that SNX10 might participate in MMP9 trafficking. After knocking down SNX10 via siRNA, the secretion and activity of MMP9 was significantly reduced, but the amount of protein was increased. By contraries, over-expression of SNX10 could increase the secretion and activity levels. Deficiency of SNX10 impaired the differentiation and bone resorption function of osteoclast, with a low activity of MMP9 compared to WT one. In SNX10 knockout osteoclast, the phosphorylation levels of JNK, p38, and ERK were obviously down-regulated. Our results first identified the role of SNX10 in MMP9 trafficking and secretion, and provided an evidence for SNX10 as a possible therapeutic target for bone destructing disease. J. Cell. Biochem. 118: 4664-4671, 2017. © 2017 Wiley Periodicals, Inc.

As the first-line antidepressant drugs, selective serotonin reuptake inhibitors (SSRIs) have efficacy in controlling the symptoms of depression. However, adverse events such as anxiety and hearing disorders were usually observed in patients and even healthy volunteers during the initial phase of SSRI administration. Hearing disorders, including auditory hallucination and tinnitus, are not only highly comorbid with mental disorders but also acknowledged factors that induce psychiatric disorders. The pharmacological and neural mechanisms underlying SSRI-induced anxiety and hearing disorders are not clear. In particularly, the methods evaluating hearing disorders are not well established in animal models, limiting the pre-clinical research on its mechanism. In the present study, we examined the mismatch negativity (MMN), a cognitive component of auditory event-related potential (ERP), to evaluate the hearing process of auditory cortex in mice. Under the acute administration of citalopram, a widely used SSRI, the anxiety-related behaviors and reduced MMN were observed in mice. Serotonin transporter (SERT) is a potential target of SSRIs. The anxiety-related behaviors and reduced MMN were also observed in SERT knockout mice, implying the role of SERT in anxiety and hearing disorders induced by SSRIs. Meanwhile, the auditory brainstem response and initial components of auditory ERP were kept intact in SERT knockout mice, suggesting that hearing neural pathway is less affected by serotonergic system. Our study suggests that the SERT deficient mice might represent a useful animal model in the investigation of the anxiety and hearing disorders during the SSRI treatment.

Gentamicin, one of the most widely used aminoglycoside antibiotics, is known to have toxic effects on the inner ear. Taken up by cochlear hair cells and spiral ganglion neurons (SGNs), gentamicin induces the accumulation of reactive oxygen species (ROS) and initiates apoptosis or programmed cell death, resulting in a permanent and irreversible hearing loss. Since the survival of SGNs is specially required for cochlear implant, new procedures that prevent SGN cell loss are crucial to the success of cochlear implantation. ROS modulates the activity of the mammalian target of rapamycin (mTOR) signaling pathway, which mediates apoptosis or autophagy in cells of different organs. However, whether mTOR signaling plays an essential role in the inner ear and whether it is involved in the ototoxic side effects of gentamicin remain unclear. In the present study, we found that gentamicin induced apoptosis and cell loss of SGNs in vivo and significantly decreased the density of SGN and outgrowth of neurites in cultured SGN explants. The phosphorylation levels of ribosomal S6 kinase and elongation factor 4E binding protein 1, two critical kinases in the mTOR complex 1 (mTORC1) signaling pathway, were modulated by gentamicin application in the cochlea. Meanwhile, rapamycin, a specific inhibitor of mTORC1, was co-applied with gentamicin to verify the role of mTOR signaling. We observed that the density of SGN and outgrowth of neurites were significantly increased by rapamycin treatment. Our finding suggests that mTORC1 is hyperactivated in the gentamicin-induced degeneration of SGNs, and rapamycin promoted SGN survival and outgrowth of neurites.

Tumor hypoxia substantially lowers the treatment efficacy of oxygen-relevant therapeutic modalities because the production of reactive oxygen species in oxygen-relevant anticancer modalities is highly dependent on oxygen level in tumor tissues. Here a distinctive magnetothermodynamic anticancer strategy is developed that takes the advantage of oxygen-irrelevant free radicals produced from magnetothermal decomposable initiators for inducing cancer-cell apoptosis in vitro and tumor suppression in vivo. Free-radical nanogenerator is constructed through in situ engineering of a mesoporous silica coating on the surface of superparamagnetic Mn and Co-doped nanoparticles (MnFe

Purpose To investigate the value of quantitative parameters of contrast-enhanced ultrasound (CEUS) in the differentiation of subtypes of renal cell carcinoma (RCC) and angiomyolipoma (AML). Methods The quantitative characteristics of 341 RCCs and 88 AMLs were analyzed with quantitative software (SonoLiver). Quantitative analysis was conducted in the whole tumor (ROI tumor ) and the maximum enhanced area of the tumor (ROI max ), acquiring the parameters of maximum intensity (IMAX), rise time (RT), time to peak (TTP), mean transit time (mTT), and area under the curve (AUC), were derived and analyzed. The difference values between ROI max and normal renal cortex (ΔPar.s, including ΔIMAX, ΔRT, ΔTTP, ΔmTT, ΔAUC) were compared among renal histotypes. Results All time-related parameters (including RT, TTP and mTT) of ROI max were shorter than the corresponding parameters of ROI tumor in RCC subtypes (all p p > 0.05). There were significant differences of all ΔPar.s among RCC subtypes and AML (all p AML > pRCC = chRCC. ΔTTP showed AML = pRCC = chRCC > ccRCC, ΔRT and ΔmTT showed AML > pRCC = chRCC = ccRCC. ΔmTT could distinguish RCC from AML with the area under the ROC curve (AUC) of 0.86. The AUC of ΔIMAX and ΔAUC was 0.89 and 0.92 vs 0.85 and 0.85 for discriminating between pRCC (or chRCC) and AML vs ccRCC and AML. Conclusions Quantitative analysis of CEUS is a useful modality in AML and RCC subtypes’ differentiation, by using ΔmTT, ΔIMAX and ΔAUC.

To compare the diagnostic values of retraction phenomenon in the coronal planes and descriptors in the Breast Imaging Reporting and Data System-Ultrasound (BI-RADS-US) lexicon in differentiating benign and malignant breast masses using an automated breast volume scanner (ABVS).Two hundred and eight female patients with 237 pathologically proven breast masses (120 benign and 117 malignant) were included in this study. ABVS was performed for each mass after preoperative localization by conventional ultrasonography (US). Multivariate logistic regression analysis was performed to assess independent variables for malignancy prediction. Diagnostic performance was evaluated through the receiver operating characteristic (ROC) curve analysis.Retraction phenomenon (odds ratio [OR]: 76.70; 95% confidence interval [CI]: 12.55, 468.70; P0.001) was the strongest independent predictor for malignant masses, followed by microlobulated margins (OR: 55.87; 95% CI: 12.56, 248.44; P0.001), angular margins (OR: 36.44; 95% CI: 4.55, 292.06; P=0.001), calcifications (OR: 5.53; 95% CI: 1.34, 22.88; P=0.018,) and patient age (OR: 1.10; 95% CI: 1.03, 1.17; P=0.004). Mass shape, orientation, echo pattern, indistinct margins, spiculated margins, and mass size were not significantly associated with breast malignancy. Area under the ROC curve (Az) for microlobulated margins and retraction phenomenon was higher than that for other significant independent predictors. Az, sensitivity, and specificity were 0.877 (95% CI: 0.829, 0.926) and 0.838 (95% CI: 0.783, 0.892), 82.9% and 70.1%, and 92.5% and 98.3%, respectively, for microlobulated margins and retraction phenomenon.Retraction phenomenon and microlobulated margins have high diagnostic values in the differentiation of benign and malignant breast masses using an ABVS.

Purpose Papillary renal cell carcinoma (pRCC) and clear cell renal cell carcinoma (ccRCC) have different biological behaviours and imaging features. The role of contrast-enhanced ultrasound (CEUS) in differentiating these two carcinoma subtypes has not been comprehensively studied. Materials and methods Forty-eight patients with 49 pRCC lesions and 153 patients with 156 ccRCC lesions underwent preoperative conventional ultrasound (US) and CEUS. Among them, 91 patients (25 pRCCs and 66 ccRCCs) also underwent preoperative contrast-enhanced computed tomography (CECT) in our hospital. The characteristics of CEUS and CECT images for each patient imaged were analysed by each of two blinded observers. Results Images for five (5/25, 20%) pRCC patients demonstrated equivocal or no enhancement using CECT, while all lesions were enhanced using CEUS. From CEUS, images of pRCCs, when compared with ccRCC images, demonstrated significantly higher frequencies of slow wash-in (59.2% vs. 5.8%), fast wash-out (87.7% vs. 46.1%), and hypo-enhancement (57.1% vs. 7.1%) patterns, p p =0.007. For lesions with large diameters (>3cm), a higher percentage of pRCC images demonstrated homogeneous enhancement compared with ccRCC images. Using the combination of slow wash-in, fast wash-out, and hypoenhancement patterns at peak as criteria to differentiate pRCC from ccRCC, positive and negative predictive value, and sensitivity and specificity were found to be 86.7%, 86.9%, 53.1%, and 97.4%, respectively. Conclusions CEUS imaging features of slow-in, fast-out, and hypo-enhancement patterns may be useful for differentiating pRCC and ccRCC. In addition, CEUS may be helpful for diagnosing hypovascular renal lesions that demonstrate equivocal or no enhancement by CECT and, thus, for improving diagnostic confidence.

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to amplify the weak vibration of low-level sound signal. The morphology and electrophysiological property of outer hair cells (OHCs) develop in early postnatal ages. The maturation of outer hair cell may contribute to the development of the auditory system. However, the process of OHCs development is not well studied. This is partly because of the difficulty to measure their function by an electrophysiological approach. With the purpose of developing a simple method to address the above issue, here we describe a step-by-step protocol to study the function of OHCs in acutely dissociated cochlea from postnatal rats. With this method, we can evaluate the cochlear response to pure tone stimuli and examine the expression level and function of the motor protein prestin in OHCs. This method can also be used to investigate the inner hair cells (IHCs).

ObjectivePeriarticular and subchondral bone erosion in rheumatoid arthritis caused by osteoclast differentiation and activation is a critical index for diagnosis, therapy and monitoring of the disease. Sorting nexin (SNX) 10, a member of the SNX family which functions in regulation of endosomal sorting, has been implicated to play an important clinical role in malignant osteopetrosis. Here we studied the roles and precise mechanisms of SNX10 in the bone destruction of collagen-induced arthritis (CIA) mice.MethodsThe role of SNX10 in bone destruction was evaluated by a CIA mice model which was induced in male SNX10−/− mice and wild type littermates. The mechanism was explored in osteoclasts induced by receptor activator of nuclear factor κB ligand from bone marrow mononuclear cells of wild type and SNX10−/− mice.ResultsSNX10 knockout prevented bone loss and joint destruction in CIA mice with reduced serum levels of TNF-α, interleukin 1β and anticollagen IgG 2α antibody. SNX10 deficiency did not block osteoclastogenesis, but significantly impaired osteoclast maturation and bone-resorption function by disturbing the formation of actin belt. The production of TRAP, CtsK and MMP9 in SNX10−/− osteoclasts was significantly inhibited, and partially restored by SNX10 overexpression. We further demonstrated that the degradation of NFATc1 was accelerated in SNX10−/− osteoclasts causing an inhibition of integrin β3-Src-PYK2 signalling.ConclusionsOur study discloses a crucial role and novel mechanism for SNX10 in osteoclast function, and provides evidence for SNX10 as a promising novel therapeutic target for suppression of immune inflammation and bone erosion in rheumatoid arthritis.

The discovery of new therapeutic drugs with the ability of preventing inflammation and joint destruction with less adverse effects is extremely urgent for rheumatoid arthritis (RA). Cryptotanshinone (CTS), an active component isolated from the root of Salvia miltiorrhiza Bunge, has been reported to have antibacterial and antitumor effects. However, its effects on RA have not been clearly elucidated. Here, we investigated the therapeutic effect of CTS on type II collagen-induced arthritis (CIA) in rats and explored the underlying mechanisms. Our results revealed that CTS treatment efficaciously ameliorated inflammation and joint destruction of rats with CIA. Both in vivo and in vitro studies showed that CTS suppressed the production of proinflammatory cytokines including interleukin 1β, tumor necrosis factor alpha, and interleukin 17α production and downregulated the production and activity of matrix metalloproteinase 9. By receptor activator of nuclear factor kappa B (NF-κB) ligand–induced bone marrow macrophages, we observed that CTS could inhibit osteoclast differentiation, which is critic for joint destruction. Further studies on inflammatory signaling revealed that CTS could inhibit the degradation of inhibitor of NF-κB (IκB) α in vivo and in vitro, prevent the nuclear translocation of NF-κB p65 induced by lipopolysaccharide in a time- and dose-dependent manner. By electrophoretic mobility shift assay and luciferase reporter assay, we found that CTS distinctively inhibited the NF-κB DNA binding activity and NF-κB–dependent luciferase activity. These results indicate that the therapeutic effect of CTS on CIA is accomplished mainly through the inhibition of NF-κB signaling. Our findings provide the evidence to develop CTS as a potential therapeutic agent for patients with RA.

To retrospectively evaluate the dynamic enhancement pattern of contrast-enhanced ultrasound (CEUS) in intrahepatic cholangiocarcinoma (ICC) of varying sizes and hepatic backgrounds.CEUS was performed in 98 pathologically confirmed ICCs (n = 3930 mm, n = 5930 mm; n = 45 with cirrhosis and n = 53 with normal liver). The dynamic enhancement pattern of CEUS was retrospectively analyzed.In the arterial phase, heterogeneous hyper-enhancement was more frequent in ICCs with cirrhosis (21/45, 46.7% vs. 11/53, 20.8% in ICCs with normal liver, p = 0.009), while peripheral hyper-enhancement and hypo-enhancement were more common in ICCs with normal liver (14/53, 26.4%; 11/53, 20.8% vs. 2/45, 4.4%; 2/45, 4.4% in ICCs with cirrhosis, p = 0.005 and 0.033, respectively). There were no significant differences between portal and delayed phases. In ICCs30 mm, homogeneous hyper-enhancement was more frequently identified (27/39, 69.2% vs. 10/59, 16.9% in ICCs30 mm, p0.001), whereas in ICCs30 mm, heterogeneous, and peripheral hyper-enhancement were more commonly observed (26/59, 44.1% vs. 6/39, 15.4% in ICCs30 mm, p = 0.004, and 14/59, 23.7% vs. 2/39, 5.1% in ICCs30 mm, p = 0.023, respectively). The washout pattern in portal and delayed phases were not significantly different in ICCs with different sizes. 60.7% (17/28) ICCs30 mm and 85.2% (23/27) ICCs30 mm with cirrhosis, together with 66.7% (14/21) ICCs30 mm with normal liver displayed intense contrast agent uptake (homogeneous or heterogeneous hyper-enhancement) in arterial phase followed by washout in portal and delayed phase, which was much higher than that in ICCs30 mm with normal liver (34.4%, 11/32, p0.001,0.001 and =0.027, respectively).The CEUS dynamic enhancement pattern of ICC varies with size and hepatic background. The enhancement pattern is indistinguishable from hepatocellular carcinoma on CEUS in most ICCs with cirrhosis and in most ICCs30 mm with normal liver.

Reports suggest that silent information regulation 2 homolog 3 (SIRT3) protects cardiomyocytes from oxidative stress-mediated death. SIRT3, a mitochondrial protein, is an essential regulator of mitochondrial function, and this regulation is important in many cerebrovascular diseases, especially stroke. Here, we investigated the role of SIRT3 in ischemia-induced neuronal death due to oxygen-glucose deprivation (OGD) using an in vitro model of cerebral ischemia. We found that exposure of differentiated PC12 cells to OGD for 6 h caused a marked decrease in cell viability and up regulated SIRT3. SIRT3 knockdown using short interfering RNA (siRNA) exacerbated OGD-induced injury whereas application of recombinant SIRT3 protected against OGD-induced cell death. Pre-treatment of the cells in which the SIRT3 gene was knocked down with recombinant SIRT3 before OGD partially restored cell viability and concomitantly reduced lactate dehydrogenase (LDH) release and increased ATP generation in mitochondria. Recombinant SIRT3 treatment resulted in increased expression of peroxisome proliferator activated receptor (PPAR)-γ co-activator 1-α (PGC-1α) and manganese superoxide dismutase (MnSOD). After knockdown of PGC-1α or MnSOD, recombinant SIRT3 failed to protect against OGD-induced injury. We also found that the protein and mRNA expression of PGC-1α was down regulated following SIRT3 knockdown. The expression level of SIRT3 was reduced when the PGC-1α gene was knocked down. Both SIRT3 and PGC-1α knockdown led to reduced mitochondrial membrane potential (Δψ) and Ca 2+ transients, especially under OGD conditions. Thus, our data suggest that SIRT3 protects PC12 cells from hypoxic injury via a mechanism that may involve PGC-1α and MnSOD. SIRT3 and PGC-1α regulate each other under physiologic and OGD conditions, thereby partially protecting against hypoxia or ischemia.

To determine the contrast-enhanced ultrasonography (CEUS) characteristics of minimal fat renal angiomyolipoma (AML) (triphasic and epithelioid) and compare them to each other and to clear cell renal cell carcinoma (ccRCC) to explore their differential diagnostic clue.Qualitative and quantitative CEUS analyses were retrospectively conducted for epithelioid renal AMLs (EAMLs) (n = 15), triphasic minimal fat AMLs (TAMLs) (n = 25), and ccRCCs (n = 113). Enhancement patterns and features with CEUS were qualitatively evaluated. As for the quantitative parameters, rise times (RT), time to peak (TTP), and tumor-to-cortex enhancement ratio (TOC ratio) were compared among these renal tumor histotypes.No significant differences were detected on conventional ultrasound in the three histotypes of renal tumor. On qualitative CEUS analysis, centripetal enhancement in cortical phase (73.3% in EAMLs, 84.0% in TAMLs vs. 18.6% in ccRCCs, p0.001 for both), homogeneous peak enhancement (100.0% in both EAMLs and TAMLs vs. 43.4% in ccRCCs, p0.001 for both), and iso-enhancement in parenchyma phase (53.3% in AMLs, 52.0% in TAMLs vs. 26.5% in ccRCCs, p = 0.034 and 0.013, respectively) were valuable traits for differentiating EAMLs and TAMLs from ccRCCs. Furthermore, with quantitative analysis, RT and TTP were much shorter in ccRCCs than those in EAMLs and TAMLs. However, all these qualitative and quantitative characteristics made no significant difference between EAMLs and TAMLs. In the differential diagnosis of EAMLs from TAMLs, pseudocapsule sign was valuable (40.0% in EAMLs vs. 0.0% in TAMLs, p0.001), and TOC ratio was much higher in EAMLs (166.01 ± 64.47%) than that in TAMLs (93.74 ± 46.56%)(p0.001), though they did make overlaps with ccRCCs. With either heterogeneous peak enhancement or the presence of pseudocapsule or TOC ratio97.34% as the criteria to differentiate ccRCCs and EAMLs from TAMLs, the sensitivity and specificity were 80.0% and 87.5%, respectively.Qualitative and quantitative CEUS analyses are helpful in the differential diagnosis of ccRCCs, EAMLs, and TAMLs.

Aristolochia yunnanensis, known as Nan Mu Xiang in traditional Chinese medicine, has long been used to treat hypertension and chest pain. In this study, the effect of ethyl acetate extract of Nan Mu Xiang (NMX) on cardiac fibrosis was assessed in vitro by cultured adult rat cardiac fibroblasts with angiotensin II (AngII) stimulation, and in vivo by rats with abdominal aorta constriction (AAC). In cultured adult rat cardiac fibroblasts stimulated by AngII, NMX inhibited cardiac fibroblast proliferation, reduced the expression of fibronectin, α-smooth muscle actin (α-SMA), and transforming growth factor β (TGF-β) in a dose-dependent manner; and suppressed AngII-induced phosphorylation of extracellular signal-regulated kinase (ERK)1/2, C- rapidly accelerated fibrosarcoma (C-Raf), and small mother against decapentaplegic (Smad) 2. Similar results were also observed in AAC rats with intraperitoneal injection of NMX, which not only ameliorated myocardial fibrosis, but also improved cardiac function. The therapeutic effect of NMX on myocardial fibrosis is attributed mainly to the inhibition of ERK and the TGF-β/Smad signaling pathways. NMX may be a promising potential drug candidate for myocardial fibrosis.

The elongation of neuron is highly dependent on membrane trafficking. Brefeldin A (BFA)-inhibited guanine nucleotide-exchange protein 1 (BIG1) functions in the membrane trafficking between the Golgi apparatus and the plasma membrane. BFA, an uncompetitive inhibitor of BIG1 can inhibit neurite outgrowth and polarity development. In this study, we aimed to define the possible role of BIG1 in neurite development and to further investigate the potential mechanism. By immunostaining, we found that BIG1 was extensively colocalized with synaptophysin, a marker for synaptic vesicles in soma and partly in neurites. The amount of both protein and mRNA of BIG1 were up-regulated during rat brain development. BIG1 depletion significantly decreased the neurite length and inhibited the phosphorylation of phosphatidylinositide 3-kinase (PI3K) and protein kinase B (AKT). Inhibition of BIG1 guanine nucleotide-exchange factor (GEF) activity by BFA or overexpression of the dominant-negative BIG1 reduced PI3K and AKT phosphorylation, indicating regulatory effects of BIG1 on PI3K-AKT signaling pathway is dependent on its GEF activity. BIG1 siRNA or BFA treatment also significantly reduced extracellular signal-regulated kinase (ERK) phosphorylation. Overexpression of wild-type BIG1 significantly increased ERK phosphorylation, but the dominant-negative BIG1 had no effect on ERK phosphorylation, indicating the involvement of BIG1 in ERK signaling regulation may not be dependent on its GEF activity. Our result identified a novel function of BIG1 in neurite development. The newly recognized function integrates the function of BIG1 in membrane trafficking with the activation of PI3K-AKT and ERK signaling pathways which are critical in neurite development.

Malignant gliomas (MGs) are among the most aggressive types of cancers in the human brain. Frequent tumor recurrence caused by a lack of effective therapeutic approaches results in a poor prognosis. Signal transducer and activator of transcription 3 (STAT3), an oncogenic protein, is constitutively activated in MGs and predicts a poor clinical outcome. STAT3 therefore is considered to be a promising target for the treatment of MGs. Cryptotanshinone (CTS), the main bioactive compound from the root of Salvia miltiorrhiza Bunge, has been reported to have various pharmacological effects. However, little is known about its function in MG cells. In this study, we evaluated the effect of CTS on the proliferation of human glioma cell lines (T98G and U87). Our results revealed that CTS significantly suppresses glioma cell proliferation. The phosphorylation of STAT3 Tyr705, but not Ser727, was inhibited by CTS, and STAT3 nuclear translocation was attenuated. Overexpression of constitutively active mutant STAT3C reversed the inhibitory effect of CTS, while knockdown STAT3 showed a similar inhibitory effect as CTS treatment. Following the downregulation of STAT3-regulated proteins cyclinD1 and survivin, cell cycle progression significantly arrested in G1/G0 phase. These results indicate that CTS may be a potential antiproliferation agent for the treatment of MGs and that its mechanism may be related to the inhibition of STAT3 signaling.

Prestin is the motor protein expressed in the cochlear outer hair cells (OHCs) of mammalian inner ear. The electromotility of OHCs driven by prestin is responsible for the cochlear amplification which is required for normal hearing in adult animals. Postnatal expression of prestin and activity of OHCs may contribute to the maturation of hearing in rodents. However, the temporal and spatial expression of prestin in cochlea during the development is not well characterized. In the present study, we examined the expression and function of prestin from the OHCs in apical, middle, and basal turns of the cochleae of postnatal rats. Prestin first appeared at postnatal day 6 (P6) for basal turn, P7 in middle turn, and P9 for apical turn of cochlea. The expression level increased progressively over the next few days and by P14 reached the mature level for all three segments. By comparison with the time course of the development of auditory brainstem response for different frequencies, our data reveal that prestin expression synchronized with the hearing development. The present study suggests that the onset time of hearing may require the expression of prestin and is determined by the mature function of OHCs.

Peroxisome proliferator-activated receptor alpha (PPARα) has been implicated in the pathogenesis of cardiac hypertrophy, although its mechanism of action remains largely unknown. To determine the effect of PPARα activation on endothelin-1 (ET-1)-induced cardiomyocyte hypertrophy and explore its molecular mechanisms, we evaluated the interaction of PPARα with nuclear factor of activated T-cells c4 (NFATc4) in nuclei of cardiomyocytes from neonatal rats in primary culture. In ET-1-stimulated cardiomyocytes, data from electrophoretic mobility-shift assays (EMSA) and co-immunoprecipitation (co-IP) revealed that fenofibrate (Fen), a PPARα activator, in a concentration-dependent manner, enhanced the association of NFATc4 with PPARα and decreased its interaction with GATA-4, in promoter complexes involved in activation of the rat brain natriuretic peptide (rBNP) gene. Effects of PPARα overexpression were similar to those of its activation by Fen. PPARα depletion by small interfering RNA abolished inhibitory effects of Fen on NFATc4 binding to GATA-4 and the rBNP DNA. Quantitative RT-PCR and confocal microscopy confirmed inhibitory effects of PPARα activation on elevation of rBNP mRNA levels and ET-1-induced cardiomyocyte hypertrophy. Our results suggest that activated PPARα can compete with GATA-4 binding to NFATc4, thereby decreasing transactivation of NFATc4, and interfering with ET-1 induced cardiomyocyte hypertrophy.

We aimed to assess the difference of enhancement patterns among the three RCC subtypes with contrast-enhancement ultrasound (CEUS). Two hundreds cases of pathologically proved clear cell renal cell carcinomas (ccRCC), 58 papillary renal cell carcinomas (pRCC) and 51 chromophobe renal cell carcinomas (chRCC) underwent preoperative conventional ultrasound and CEUS. The wash-in and wash-out pattern, peak enhancement degree and homogeneity, and the presence of pseudocapsule were evaluated by two blinded observers respectively. The interreader agreement in the characterization of CEUS features between two observers was good (κ = 0.649-0.775). Compared with pRCCs and chRCCs, ccRCCs demonstrated higher frequency of simultaneous wash-in pattern, hyperenhancement and heterogeneity with necrotic areas. Most pRCCs and chRCCs manifested hypoenhancement, homogeneity, fast wash-out and presence of pseudocapsule. The only difference we obtained between pRCC and chRCC was the wash-in pattern, with slow wash-in in pRCC and simultaneous wash-in in chRCC. In small lesions with long diameter ≤ 3 cm, the majority of the three subtypes of RCC showed homogeneous enhancement and there was no difference among them. CEUS was a useful method to preoperatively differentiate the ccRCC from non-ccRCC subtypes. There were no distinguishing features identifid on CEUS that allowed reliable differentiation of pRCC from chRCC.

The purpose of this study was to compare MRI findings of solitary hypovascular hepatic nodules, benign and malignant, to identify their MRI characteristics.We retrospectively assessed solitary hypovascular hepatic nodules ≤ 3 cm in 135 patients, among them there were 55 malignant nodules [29 peripheral nodules of cholangiocarcinoma, PCC, and 26 hepatic metastases, HM] and 80 benign nodules [48 inflammatory myofibroblastic tumors, IMT, and 32 hepatic hemangioma, HG], proved by surgery, biopsy or follow-up imaging. Unenhanced and dynamic enhanced MRI findings of the 135 patients were analyzed retrospectively. Statistical analysis included Chi-square test or Fisher's exact test, and receiver operating characteristic (ROC) curve.There was significant difference (P0.05) between the malignant group and benign group in terms of location, margin, T2WI signal intensity, heterogeneity or homogeneity of the nodule, and type and degree of peritumoral and intratumoral enhancement. Area under the curve at the first film reading by three radiologists was 0.678 ± 0.047, 0.920 ± 0.022 at the second time, and there was a significant difference (Z = 5.22, P0.05) between them.Our data indicated that solitary hypovascular hepatic nodules show unenhanced and dynamic enhanced MRI features. Therefore, MR imaging combined with clinical and biochemical data does provide reliable information for a proper diagnosis of such hepatic lesions and differentiation of malignant from benign nodules.

The purpose of this study is to evaluate qualitative and quantitative analysis of contrast-enhanced ultrasound (CEUS) in differential diagnoses of hypo-echoic renal tumor histotypes.Our study cohort comprised 103 clear cell renal cell carcinomas (ccRCCs), 24 papillary renal cell carcinomas (pRCCs), 28 chromophobe renal cell carcinomas (cRCCs), and 34 angiomyolipomas (AMLs), hypo-echoic on ultrasound, and imaged between January 2011 and December 2013. Enhancement homogeneity and tumor-to-cortex intensity ratio (TOC ratio) were retrospectively analyzed.Overall, heterogeneous enhancement was more common in ccRCCs than AMLs, pRCCs, and cRCCs. TOC ratio showed the trend ccRCCAMLpRCC = cRCC. Similar trends were seen in tumors4 cm. Using heterogeneous enhancement or TOC ratio107.5% to differentiate ccRCC from other histotypes, the sensitivity, specificity, positive and negative predictive values were 93.1%, 74.5%, 84.8%, and 87.5%, respectively. Tumors4 cm exhibited considerable overlap in enhancement homogeneity among different histotypes. TOC ratios were similar between homo- and heterogeneously enhancing tumors for ccRCCs and for pRCCs and cRCCs, but higher in homogeneously enhancing than heterogeneously enhancing AMLs. In homo- and heterogeneously enhancing tumors, TOC ratios followed the trends ccRCCsAMLspRCCs = cRCCs and ccRCCsAMLs = pRCCs = cRCCs, respectively. With TOC ratio105.81% and72.37% to differentiate homo- and heterogeneously enhancing ccRCCs from other histotypes in tumors4 cm with same enhancement homogeneity, the sensitivity, specificity, positive and negative predictive values were 70.0%, 85.7%, 70.0%, 85.7%, and 91.7%, 94.4%, 95.7%, 89.5%, respectively.CEUS homogeneity and TOC ratio are helpful in differential diagnosis of hypo-echoic renal tumor histotypes. Diameter and enhancement homogeneity should be considered when deciding the diagnostic TOC ratio cutoff.

GABAA receptors (GABAARs) mediate the majority of fast synaptic inhibition. Trafficking regulation and protein-protein interactions that maintain the appropriate number of GABAARs at the cell surface are considered to be important mechanisms for controlling the strength of synaptic inhibition. Here, we report that BIG1, a brefeldin A (BFA)-inhibited guanine nucleotide-exchange factor (GEF) which has a known role in vesicle trafficking, is a new binding partner of GABAARs. Treatment of neurons with BFA, an uncompetitive inhibitor of BIG1 GEF activity, or depletion of BIG1 by small RNA interference (siRNA) significantly decreased GABAARs at the neuronal surface and suppressed GABA-gated influx of chloride ions. Over-expression of HA-tagged BIG1-E793K, a dominant-negative mutant, also significantly decreased GABAARs at the neuronal surface, but had no effect on the total amount of GABAARs. Inhibition of GABAAR endocytosis by muscimol increased both GABAARs and BIG1 at the neuronal surface in a time-dependent fashion, and this increase could be abolished by bicuculline. Finally, depletion of BIG1 by siRNA inhibited the muscimol-stimulated increase of GABAARs. Those data suggest an important function of BIG1 in trafficking of GABAARs to the cell surface through its GEF activity. Thus, we identify an important role of BIG1 in modulating GABA-gated Cl(-) influx through the regulation of cell surface expression of GABAARs.

Objective— Cell-surface localization and intracellular trafficking are essential for the function of ATP-binding cassette transporter A-1 (ABCA1). However, regulation of these activities is still largely unknown. Brefeldin A, an uncompetitive inhibitor of brefeldin A-inhibited guanine nucleotide-exchange proteins (BIGs), disturbs the intracellular distribution of ABCA1, and thus inhibits cholesterol efflux. This study aimed to define the possible roles of BIGs in regulating ABCA1 trafficking and cholesterol efflux, and further to explore the potential mechanism. Methods and Results— By vesicle immunoprecipitation, we found that BIG1 was associated with ABCA1 in vesicles preparation from rat liver. BIG1 depletion reduced surface ABCA1 on HepG2 cells, and inhibited by 60% cholesterol release. In contrast, BIG1 overexpression increased surface ABCA1 and cholesterol secretion. With partial restoration of BIG1 through overexpression in BIG1-depleted cells, surface ABCA1 was also restored. Biotinylation and glutathione cleavage revealed that BIG1 small interfering RNA dramatically decreased the internalization and recycling of ABCA1. This novel function of BIG1 was dependent on the guanine nucleotide-exchange activity and achieved through activation of ADP-ribosylation factor 1. Conclusion— BIG1, through its ability to activate ADP-ribosylation factor 1, regulates cell-surface levels and function of ABCA1, indicating a transcription-independent mechanism for controlling ABCA1 action.

Malignant gliomas represent one of the most aggressive types of cancers and their recurrence is closely linked to acquired therapeutic resistance. A combination of chemotherapy is considered a promising therapeutic model in overcoming therapeutic resistance and enhancing treatment efficacy. Herein, we show by colony formation, Hochest 33342 and TUNEL staining, as well as by flow cytometric analysis, that LY294002, a specific phosphatidylinositide-3-kinase (PI3K) inhibitor, enhanced significantly the sensitization of a traditional cytotoxic chemotherapeutic agent, tamoxifen-induced apoptosis in C6 glioma cells. Activation of PI3K signaling pathway by IGF-1 protected U251 cells from apoptosis induced by combination treatment of LY294002 and tamoxifen. Interference of PI3K signaling pathway by PI3K subunit P85 siRNA enhanced the sensitization of U251 glioma cells to tamoxifen -induced apoptosis. By Western blotting, we found that combination treatment showed lower levels of phosphorylated Akt(Ser473) and GSK-3β(Ser9) than a single treatment of LY294002. Further, we showed a significant decrease of nuclear β-catenin by combination treatment. In response to the inhibition of β-catenin signaling, mRNA and protein levels of Survivin and the other three antiapoptotic genes Bcl-2, Bcl-xL, and Mcl-1 were significantly decreased by combination treatment. Our results indicated that the synergistic cytotoxic effect of LY294002 and tamoxifen is achieved by the inhibition of GSK-3β/β-catenin signaling pathway.

Sixteen carbazole alkaloids from Clausena vestita D. D. Tao were extracted, and their anti-tumor activities were evaluated. Among the extracts, Clauszoline-I exhibited an obvious growth inhibitory activity against several cancer cell lines through its ability to induce cell cycle arrest in the S and G2/M phases. A dramatic morphologic change with decreased F-actin staining and RhoA activity was found in Clauszoline-I treated HepG2 cells, in which the phosphorylation of PKCδ (Ser643) was inhibited. Our results indicated that induction cell cycle arrest by Clauszoline-I might be achieved by decreasing the RhoA activity via the inhibition of PKCδ phosphorylation.

Aim of the study TBL-II is the water extract of an anti-arthritic Chinese herbal formula Tongbiling (TBL), which has been used to treat rheumatoid arthritis (RA) for many years. We herein aimed to confirm its anti-arthritic effect and explore the potential mechanism of action on collagen-induced arthritis (CIA) in mice. Materials and methods Four weeks after the first collagen immunization, mice were treated with TBL-II orally at a lower dose of 100 mg/kg/d and higher dose of 300 mg/kg/d for 2 or 8 weeks. The severity of arthritis was evaluated by symptoms, radiological scores and histological assessment. Levels of IL-1β, IL-6, TNFα and IgG2a type anti-collagen II (CII) antibody in serum were measured by ELISA. Competitive RT-PCR and immunohistochemical analysis were used to investigate MMP-2, -3, -9 mRNA and protein expression. Results Our results revealed treatment with higher dose of TBL-II for 2 weeks attenuated significantly acute inflammation, and decreased the amounts of IL-1β and TNFα in serum; treatment for 8 weeks could obviously suppress chronic inflammation, ameliorate cartilage and bone destruction, and reduce the levels of matrix metalloproteinase (MMP)-2, -3, -9 mRNA and protein expression in joints. The levels of IgG2a type anti-CII antibody in serum were significantly reduced by treatment with higher dose of TBL-II for either 2 or 8 weeks. In contrast, treatment with lower dose of TBL-II for 8 weeks had no effect on articular destruction. Conclusions Our results suggested that TBL-II at higher dose not only ameliorated symptoms but also modified disease of CIA. TBL-II would be a potent candidate as a novel botanical drug for further investigation.

Bladder cancer (BC) is very common and associated with significant morbidity and mortality, though the molecular underpinnings of its origination and progression remain poorly understood. In this study, we demonstrate that Prohibitin 1 (PHB) was overexpressed in human BC tissues and that PHB upregulation was associated with poor prognosis. We also found that PHB was necessary and sufficient for BC cell proliferation. Interestingly, the overexpressed PHB was primarily found within mitochondria, and we provide the first direct evidence that phosphorylation by Akt at Thr258 of PHB induces this mitochondrial localization. Inhibiton of Akt reverses these effects and inhibited the proliferation of BC cells. Finally, the phosphorylation of PHB was required for BC cell proliferation, further implicating the importance of the Akt in BC. Taken together, these findings identify the Akt/PHB signaling cascade as a novel mechanism of cancer cell proliferation and provide the scientific basis for the establishment of PHB as a new prognostic marker and treatment target for BC.

Objective: To evaluate the value of enhancement features and quantitative parameters of contrast-enhanced ultrasonography (CEUS) in differentiating solid hypoechoic renal angiomyolipomas (AMLs) from clear cell renal cell carcinomas (ccRCCs). Materials and Methods: We analyzed the enhancement features and quantitative parameters of CEUS in 174 hypoechoic renal masses (32 AMLs and 142 ccRCCs) included in the study. Results: Centripetal enhancement pattern was more common in AMLs than in ccRCCs on CEUS (71.9% vs. 23.2%, p < 0.001). At peak enhancement, all AMLs showed homogeneous enhancement (100% in AML, 27.5% in ccRCCs; p < 0.001). Quantitative analysis showed no significant difference between rise time and time to peak. Tumor-to-cortex (TOC) enhancement ratio in AMLs was significantly lower than that in ccRCCs (p < 0.001). The criteria of centripetal enhancement and homogeneous peak enhancement together with TOC ratio < 91.0% used to differentiate hypoechoic AMLs from ccRCCs resulted in a sensitivity and specificity of 68.9% and 95.8%, respectively. Conclusion: Both qualitative and quantitative analysis with CEUS are valuable in the differential diagnosis of hypoechoic renal AMLs from ccRCCs. Index terms: Angiomyolipoma; Renal cell carcinoma; Contrast-enhanced ultrasonography; Quantitative analysis; Minimal fat