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Xiaoyu Li,1,* Lin Wu,2,* Zhifang Yang,1 Yunyun Hu,1 Chun Zhou,1 Ri Ji1 1Department of Ultrasound, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, People’s Republic of China; 2Department of Geriatrics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, People’s Republic of China*These authors contributed equally to this workCorrespondence: Chun Zhou; Ri JiDepartment of Ultrasound, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, People’s Republic of ChinaTel/Fax +86 021 63846590; Tel +86 18717771587Email zc40343@rjh.com.cn; jiri_1980@163.comObjective: To evaluate the foot microcirculation in type 2 diabetes mellitus (T2DM) and impaired glucose tolerance patients (IGT) with contrast-enhanced ultrasound (CEUS).Methods: The study included 37 patients with T2DM but without diabetic foot (DM group), 15 patients with impaired glucose tolerance (IGT group) and 10 elderly males with normal fasting blood glucose (FBS) and negative glucose tolerance tests (control group). Color Doppler flow imaging (CDFI) and CEUS were performed on the right foot great toes for detecting the blood perfusion performance. CEUS images were recorded and parameters of CDFI and flow time-intensity curves (TICs) were analyzed by the Student’s t-test.Results: There was no significant difference in CDFI parameters pulse index and peak systolic blood flow velocity (PSV) among the three groups (P > 0.05). Compared with control group, CEUS images of IGT and DM groups showed lower microvascular density and were pale. Peak intensity (PI) and area under time-intensity curve (AUC) in control, IGT and DM groups were decreased gradually (PI 46.36± 10.96 vs 35.26± 11.65 vs 28.15± 7.94, P = 0.001, AUC 5.12± 1.02 vs 3.25± 1.60 vs 2.81± 1.20, P = 0.001). The arrival times (AT) and time to peak (TTP) tended to be increased with the extension of DM course, but the difference was not statistically significant (AT, P = 0.260, TTP, P = 0.481).Conclusion: CEUS, as a noninvasive and valuable technique, could detect the alterations in foot microcirculation of DM and IGT patients.Keywords: type 2 diabetes mellitus, impaired glucose tolerance, contrast-enhanced ultrasound, microcirculation

BACKGROUND: Chronic hepatitis B is the most common chronic liver disease in China. For patients with chronic hepatitis B, steatosis increases the risk of cirrhosis and hepatocellular carcinoma. This study aimed to analyze and compare the clinical value of a newly developed ultrasound attenuation parameter, liver steatosis analysis (LiSA), acquired by Hepatus (Mindray, China), and controlled attenuation parameter (CAP), a widely used ultrasound attenuation parameter acquired by FibroScan (Echosens, France), for grading liver steatosis in patients with chronic hepatitis B infection. METHODS: A total of 203 patients were divided into two groups according to liver fat content validated by liver biopsy: group 1 (liver fat content 0.7) for steatosis ≥10%, and the performance of LiSA was significantly better than CAP (AUC: 0.859 vs. 0.801, P=0.048). Using optimal cut-off points, LiSA had specificity and sensitivity of 96.23% and 76.08%, respectively, for the diagnosis of steatosis ≥10%, compared to 91.53% and 72.10%, respectively, for CAP. CONCLUSIONS: LiSA and CAP are extremely efficient tools for assessing liver steatosis, even at a low grade. Both parameters are non-invasive, inexpensive, and easy to use, and can provide immediate results with high sensitivity.

Renin-angiotensin system (RAS) signaling has been implicated in the development of cancer. The new RAS ACE2/Ang-(1-7)/Mas axis antagonizes the classical ACE/Ang II/AT1R axis. Ang-(1-7) has pleiotropic roles in lung cancer including suppressing proliferation, angiogenesis, and metastasis. This research was designed to investigate the effect of Ang-(1-7) on tumor-associated angiogenesis in DDP-resistant lung cancer cell lines. We first established acquired DDP-resistant cell lines A549 (A549-DDP) and LLC (LLC-DDP). We next performed RT-qPCR, western blot, ELISA, tube formation, microvessel density detection, immunohistochemistry, and tumor formation assays. The results showed that the mRNA and protein levels of RAS components and vascular endothelial growth factor A (VEGFa) were lessened in the A549/LLC-DDP+Ang-(1-7) group compared with the A549/LLC-DDP group. This effect could be blocked by the MAS receptor antagonist A779. The data revealed that Ang-(1-7) could perform its antiangiogenic function by PI3K/AKT and MAPK pathways. Furthermore, the impact of Ang-(1-7) on tumor-associated angiogenesis has been confirmed in lung cancer xenograft model with acquired DDP resistance. These results provide a theoretical basis for designing therapeutic strategies for targeting Ang-(1-7) in the treatment of NSCLC.

Objectives This systematic review aimed to evaluate the efficacy and safety of Tao-Hong Siwu Tang (TST) for the treatment of primary dysmenorrhea. Methods We searched four English databases (MEDLINE, EMBASE, Allied and Complementary Medicine Database, and Cochrane Central Register of Controlled Trials [CENTRAL, Cochrane Library]), three Chinese databases (China National Knowledge Infrastructure, Wanfang, and Chinese Science and Technology Periodical Database), two Korean databases (Oriental Medicine Advanced Searching Integrated System and Korean traditional Knowledge Portal), and one Japanese database (Citation Information by NII). All randomized controlled trials (RCTs) using TST or modified TST (MTST) were included. Three independent reviewers extracted the data, assessed the risk of bias according to the Cochrane criteria, and performed a meta-analysis. Results A total of 85 possibly relevant articles were identified, and five trials met our inclusion criteria. The meta-analysis showed a favorable effect of MTST compared to non-steroidal anti-inflammatory drugs (NSAIDs) (n = 486, risk ratio [RR] = 1.53, 95% confidence interval [95% CI] = 1.37–1.72, I2 = 39%). Among the included trials, one RCT showed superior effects of MTST on primary dysmenorrhea recurrence rate compared to NSAIDs (n = 246, RR = 0.31, 95% CI = 0.15–0.63, P = 0.001). Another RCT revealed a beneficial impact of oral contraceptives (OCs) used in combination with TST compared to OCs alone (n = 60, RR = 1.35, 95% CI = 1.02–1.79, P = 0.04). Conclusion This systematic review and meta-analysis provides moderate quality evidence for the superiority of MTST over NSAIDs as well as that of TST plus OCs over OCs in the treatment of primary dysmenorrhea.

Limb hemiplegia is one of the common sequelae of stroke. Physical therapy-based rehabilitation training can rapidly improve limb functioning and muscle strength. This study investigated the effect of physical therapy on the recovery of upper limb motor function by employing a literature search and meta-analysis.We searched Embase, The Cochrane Central Register of Controlled Trials library, Wiley online library, PubMed, Ovid, and Clinicaltrials.org for randomized controlled trial (RCT) studies and performed an electronic search with the keyword combinations 'physical therapy/rehabilitation''stroke/post-stroke''upper extremity/upper limb'. After screening the literature for inclusion criteria and assessing the risk of bias, Revman 5.4 software was used for the analysis and to obtain forest and funnel plots.A total of 15 RCTs with 1,081 patients were included in the quantitative analysis for this study. The meta-analysis results showed that compared with conventional therapy, physical therapy improved the upper limb Fugl-Meyer Assessment for Upper Extremity (FMA) scores in convalescent stroke patients (MD =7.27, 95% CI: 4.23-10.32, Z=4.68, P0.00001), and increased the Functional Independence Measure (FIM) scores (MD =18.82, 95% CI: 6.34-31.30, Z=2.96, P=0.003), the Action Research Arm Test (ARAT) scores (MD =8.84, 95% CI: 6.53-11.15, Z=7.50, P0.00001), and the Box and Block Test (BBT) scores (MD =6.11, 95% CI: 1.18-11.04, Z=2.43, P=0.02) of patients during the rehabilitation period.The use of physical therapy-based rehabilitation training in the recovery period for stroke patients with hemiplegia can improve upper limb movement ability, increase muscle strength, reduce limb pain, and improve the quality of life.

Background We investigated whether glycemic control affects the relation between endothelial dysfunction and coronary artery disease in patients with type 2 diabetes mellitus (T2DM). Methods In 102 type 2 diabetic patients with stable angina, endothelial function was evaluated using brachial artery flow-mediated dilation (FMD) with high-resolution ultrasound, and significant stenosis of major epicardial coronary arteries (≥ 50% diameter narrowing) and degree of coronary atherosclerosis (Gensini score and SYNTAX score) were determined. The status of glycemic control was assessed by blood concentration of glycated hemoglobin (HbA1c). Results The prevalence of significant coronary artery stenosis (67.9% vs. 37.0%, P = 0.002) and degree of coronary atherosclerosis (Gensini score: 48.99 ± 48.88 vs. 15.07 ± 21.03, P 0.05). Conclusion Poor glycemic control negatively influences the association of endothelial dysfunction and coronary artery disease in T2DM patients.

Damage to the sensory hair cells and the spiral ganglion neurons of the cochlea leads to deafness. Induced pluripotent stem cells (iPSCs) are a promising tool to regenerate the cells in the inner ear that have been affected by pathology or have been lost. To facilitate the clinical application of iPSCs, the reprogramming process should minimize the risk of introducing undesired genetic alterations while conferring the cells the capacity to differentiate into the desired cell type. Currently, reprogramming induced by synthetic mRNAs is considered to be one of the safest ways of inducing pluripotency, as the transgenes are transiently delivered into the cells without integrating into the genome. In this study, we explore the ability of integration-free human-induced pluripotent cell lines that were reprogrammed by mRNAs, to differentiate into otic progenitors and, subsequently, into hair cell and neuronal lineages. hiPSC lines were induced to differentiate by culturing them in the presence of fibroblast growth factors 3 and 10 (FGF3 and FGF10). Progenitors were identified by quantitative microscopy, based on the coexpression of otic markers PAX8, PAX2, FOXG1, and SOX2. Otic epithelial progenitors (OEPs) and otic neuroprogenitors (ONPs) were purified and allowed to differentiate further into hair cell-like cells and neurons. Lineages were characterised by immunocytochemistry and electrophysiology. Neuronal cells showed inward Na+ (INa) currents and outward (Ik) and inward K+ (IK1) currents while hair cell-like cells had inward IK1 and outward delayed rectifier K+ currents, characteristic of developing hair cells. We conclude that human-induced pluripotent cell lines that have been reprogrammed using nonintegrating mRNAs are capable to differentiate into otic cell types.

Objective. Inorganic polyphosphates (polyP) play a multitude of roles in mammalian biology. PolyP research is hindered by the lack of a simple and sensitive quantification method. The aim of this study was to develop a robust method for quantifying the low levels of polyP in mammalian tissue such as cartilage, which is rich in macromolecules that interfere with its determination. Design. Native and in vitro formed tissues were digested with proteinase K to release sequestrated polyP. The tissue digest was loaded on to silica spin columns, followed by elution of bound polyP and various treatments were assessed to minimize non-polyP fluorescence. The eluent was then quantified for polyP content using fluorometry based on DAPI (4′,6-diamidino-2-phenylindole) fluorescence shift occurring with polyP. Results. Proteinase K pretreatment reduced the inhibitory effect of proteins on polyP recovery. The eluent was contaminated with nucleic acids and glycosaminoglycans, which cause extraneous fluorescence signals. These were then effectively eliminated by nucleases treatment and addition of concentrated Tris buffer. PolyP levels were quantified and recovery ratio determined using samples spiked with a known amount of polyP. This silica spin column method was able to recover at least 80% of initially loaded polyP, and detect as little as 10−10 mol. Conclusions. This sensitive, reproducible, easy to do method of quantifying polyP will be a useful tool for investigation of polyP biology in mammalian cells and tissues. Although the protocol was developed for mammalian tissues, this method should be able to quantify polyP in most biological sources, including fluid samples such as blood and serum.

Bone morphogenetic proteins (BMPs) are growth factors that have important functions in cell proliferation, migration and differentiation. To date, BMP pathway activation has been found in multiple human tumors and is associated with enhanced malignant tumor growth and metastasis. BMP activity is tightly regulated by a family of soluble extracellular secreted BMP modulators. Twisted gastrulation BMP signaling modulator 1 (TWSG1) is a direct BMP regulator that is required for the full signaling activity of BMPs. However, the functions and mechanisms of TWSG1 in papillary thyroid cancer (PTC) metastasis have not been reported. TWSG1 expression was detected in 44 PTC tissues with lymph node metastasis (LNM) and 56 PTC tissues without LNM using quantitative real-time polymerase chain reaction (qRT-PCR). Gain- and loss-of-function approaches were used to assess the biological function of TWSG1 in PTC cells. Matrigel assays demonstrated the effect of tumor cell-derived TWSG1 on endothelial cell function. Our results showed that TWSG1 expression was significantly enhanced in PTC with LNM compared to that in PTC without LNM. TWSG1 knockdown inhibited migration, invasion and proliferation of PTC cells. Additionally, TWSG1 suppression impaired the tumor cell-induced endothelial cell sprout formation. We found that TWSG1 signaling may be transduced by the BMP target transcription factor inhibitor of DNA binding 1 (Id1) and matrix metalloproteinases (MMPs) 2 and 9. In conclusion, TWSG1 was highly expressed in metastasized PTC; tumor growth, migration and invasion were dependent on TWSG1, and it may be a new diagnostic and therapeutic target for PTC.

Background Multimodal contrast agents with low toxicity and targeted modification have opened up new possibilities for specific imaging of breast cancer and shown broad application prospects in biomedicine and great potential for clinical transformation. In this work, a potential multifunctional imaging agent was developed by doping Fe into hollow silica nanoparticles (HS-Fe NPs), followed by modification with specific anti-HER2 antibodies, enabling the NPs to have dual-mode ultrasound (US)–magnetic resonance (MR)-specific imaging capacity with low toxicity. Methods Anti-HER2 antibodies were conjugated to silane–polyethylene glycol (PEG)–COOH-modified HS-Fe (HS-Fe-PEG) NPs to produce HER2-targeted HS-Fe-PEG (HS-Fe-PEG-HER2) NPs. The toxicity of HS-Fe-PEG-HER2 NPs on targeted cells in vitro and blood and organ tissue of mice in vivo was investigated. Distribution in vivo was also studied. Confocal laser-scanning microscopy and flow cytometry were used to evaluate the targeting ability of HS-Fe-PEG-HER2 NPs in vitro. US and MR instruments were used for imaging both in vivo and in vitro. Results The obtained HS-Fe-PEG-HER2 NPs (average diameter 234.42±48.76 nm) exhibited good physical properties and biosafety. In solution, they showed obvious enhancement of the US signal and negative contrast in T2-weighted MR imaging. The binding rate of HS-Fe-PEG-HER2 NPs to targeted cells (SKBR3) was 78.97%±4.41% in vitro. US and MR imaging in vivo confirmed that the HS-Fe-PEG-HER2 NPs were delivered passively into the tumor region of SKBR3 and bound specifically to tumor cells. Target enhancement was better than untargeted and targeted competition groups. Conclusion HS-Fe-PEG-HER2 NPs have potential as a low-cytotoxicity and dual-mode US–MR-specific imaging agent.

Xiaoyu Li,* Shujun Xia,* Wei Zhou, Ri Ji, Weiwei Zhan Ultrasound Department, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China *These authors contributed equally to this work Background: Multimodal contrast agents with low toxicity and targeted modification have opened up new possibilities for specific imaging of breast cancer and shown broad application prospects in biomedicine and great potential for clinical transformation. In this work, a potential multifunctional imaging agent was developed by doping Fe into hollow silica nanoparticles (HS-Fe NPs), followed by modification with specific anti-HER2 antibodies, enabling the NPs to have dual-mode ultrasound (US)–magnetic resonance (MR)-specific imaging capacity with low toxicity.Methods: Anti-HER2 antibodies were conjugated to silane–polyethylene glycol (PEG)–COOH-modified HS-Fe (HS-Fe-PEG) NPs to produce HER2-targeted HS-Fe-PEG (HS-Fe-PEG-HER2) NPs. The toxicity of HS-Fe-PEG-HER2 NPs on targeted cells invitro and blood and organ tissue of mice invivo was investigated. Distribution invivo was also studied. Confocal laser-scanning microscopy and flow cytometry were used to evaluate the targeting ability of HS-Fe-PEG-HER2 NPs invitro. US and MR instruments were used for imaging both invivo and in vitro.Results: The obtained HS-Fe-PEG-HER2 NPs (average diameter 234.42±48.76 nm) exhibited good physical properties and biosafety. In solution, they showed obvious enhancement of the US signal and negative contrast in T2-weighted MR imaging. The binding rate of HS-Fe-PEG-HER2 NPs to targeted cells (SKBR3) was 78.97%±4.41% invitro. US and MR imaging invivo confirmed that the HS-Fe-PEG-HER2 NPs were delivered passively into the tumor region of SKBR3 and bound specifically to tumor cells. Target enhancement was better than untargeted and targeted competition groups.Conclusion: HS-Fe-PEG-HER2 NPs have potential as a low-cytotoxicity and dual-mode US–MR-specific imaging agent. Keywords: dual-mode, ultrasound imaging, magnetic resonance imaging, HER2, breast cancer

Background Dangguijagyag-san, also known as Dangguishaoyao-san in Chinese and Toki-shakuyakui-san in Japanese, has been frequently used to treat symptoms associated with dysmenorrhea. The purpose of this trial is to evaluate the efficacy and safety of the herbal medicine, Dangguijagyag-san, relative to those of active control, Gamisoyo-san, and a placebo control for primary dysmenorrhea. Methods This protocol details a randomized, double-blind, parallel-group, multi-center, investigator-initiated, controlled trial evaluating treatment of primary dysmenorrhea. Two hundred and forty participants will be randomly divided into one of three groups: 1) the Dangguijagyag-san experimental group (EG) (n = 105), 2) the Gamisoyo-san active control group (ACG) (n = 30), and 3) the placebo control group (PCG) (n = 105). The interventions will be administered for two menstrual cycles, and the follow-up will be carried out for the following six menstrual cycles. The primary outcomes are difference in response rates between the EG and the ACG (non-inferiority comparison) and difference in changes from baseline in average pain intensity measured by the visual analogue scale between the EG and PCG (superiority comparison). The secondary outcomes are pain scores derived from pain assessment tools (verbal multidimensional scoring system, retrospective symptom scale, and short form McGill pain questionnaire), dosage of analgesics, pattern diagnosis questionnaires, and short form 36 health survey. Adverse events and vital signs will be checked at every visit, and laboratory tests will be performed for safety evaluation. Discussion The results of this clinical trial will offer evidence for the efficacy and safety of Dangguijagyag-san for primary dysmenorrhea. Trial registration Clinical Research Information Service of Korea: KCT0003005

Development of functional theranostic platform for systemic contrast-enhanced diagnostic imaging and therapy is of great necessity in nanomedicine. However, synthesizing the biocompatible theranostic agents with enhanced merits in imaging and therapy via a facile and green way is still highly challenged. Here, we report a novel theranostic agent based on colloidal polydopamine/Au (PDA/Au) hollow spheres, which are synthesized with combined use of PDA chemistry and sacrificial template techniques. Colloidal polystyrene (PS) spheres serve as the templates with coatings of a PDA shell and Au nanoparticles in sequence, which are subsequently removed with trichloromethane, giving rise to colloidal PDA/Au hollow spheres. Colloidal PDA/Au hollow spheres exhibit excellent contrast enhancement for ultrasound imaging, and can serve as the near-infrared (NIR) photoabsorbers for the effective photothermal ablation of 4 T1 breast cancer cells in vitro with minor cytotoxicity to living cells. This method suggests a novel avenue for theranostic treatment in oncology.

Adrenomedullin (AM) is a peptide expressed in all body tissues, and its related receptors are increased in liver fibrosis. In this study, we evaluated the effect of AM deficiency on liver fibrogenesis induced by CCl 4 using AM heterozygous (HT) mice. The animals received a single injection of CCl 4 or olive oil for the acute experiment, and received CCl 4 or olive oil three times a week for 6 weeks for the chronic experiment. Fibrosis was accessed using histopathological analysis and the western blot. The AM HT mice showed mild pericentrilobular degeneration when compared to the AM wild type (WT) mice. In the acute experiment, there was no significant difference between the AM WT and AM HT mice. However, in the chronic experiment, the CCl 4 -treated AM HT mice showed more severe liver fibrosis than that of the CCl 4 -treated AM WT mice. The AST and ALT levels of the AM HT CCl 4 group were higher than those of the AM WT CCl 4 group. Additionally, the collagen deposition, α-SMA protein and TGF-β protein were increased in the AM HT CCl 4 group when compared to the AM WT CCl 4 group. The AM HT mice also exhibited severe lipid peroxidation through the GSH decrement. Taken together, our data suggest that AM deficiency increases the susceptibility to liver fibrosis induced by CCl 4 , indicating a novel therapeutic target for patients with liver fibrosis.

Background The dysregulation of long noncoding RNAs (lncRNAs) has been identified in a variety of cancers. An increasing number of studies have found the critical role of lncRNAs in the regulation of cellular processes, such as proliferation, invasion and differentiation. Long noncoding RNA papillary thyroid carcinoma susceptibility candidate 3 (PTCSC3) is a novel lncRNA that was primarily detected in papillary thyroid carcinoma. However, the biological function and molecular mechanism of lncRNA PTCSC3 in glioma are still unknown. Methods The expression level of lncRNA PTCSC3 in human microglia and glioma cell lines was examined using quantitative real-time polymerase chain reaction (qRT-PCR). The influence of lncRNA PTCSC3 on cell proliferation were studied using the cell counting kit-8, and cell cycle and apoptosis were analyzed by flow cytometry assays. The migration and invasion abilities were investigated by transwell and wound healing assays. The target genes of lncRNA PTCSC3 were explored by qRT-PCR, immunofluorescence and western blot. Results LncRNA PTCSC3 was significantly downregulated in glioma cell lines. The overexpression of lncRNA PTCSC3 suppressed proliferation and induced apoptosis in U87 and U251 cells. Additionally, the overexpression of lncRNA PTCSC3 inhibited the migration and invasion of U87 and U251 cells. Moreover, lncRNA PTCSC3 inhibited the epithelial-mesenchymal transition of U87 cells. The study also demonstrated that LRP6, as a receptor of the Wnt/β-catenin pathway, was a target of lncRNA PTCSC3. By evaluating the expression levels of Axin1, active β-catenin, c-myc, and cyclin D1, the study indicated that lncRNA PTCSC3 inhibited the activation of the Wnt/β-cateninpathway through targeting LRP6. Conclusions LncRNA PTCSC3 inhibits the proliferation and migration of glioma cells and suppresses Wnt/β-catenin signaling pathway by targeting LRP6. LncRNA PTCSC3 is a potential therapeutic target for treatment of glioma. Electronic supplementary material The online version of this article (doi:10.1186/s12883-017-0813-6) contains supplementary material, which is available to authorized users.

We observed the biphasic production of ethylene and reactive oxygen species (ROS) in susceptible tobacco (Nicotiana tabacum ‘Wisconsin 38’) plants after shoot inoculation with Phytophthora parasitica var nicotianae. The initial transient increase in ROS and ethylene at 1 and 3 h (phase I), respectively, was followed by a second massive increase at 48 and 72 h (phase II), respectively, after pathogen inoculation. This biphasic pattern of ROS production significantly differed from the hypersensitive response exhibited by cryptogein-treated wild-type tobacco plants. The biphasic increase in ROS production was mediated by both NADPH oxidase isoforms, respiratory burst oxidase homolog (Rboh) D and RbohF. Conversely, different 1-aminocyclopropane-1-carboxylic acid synthase members were involved in specific phases of ethylene production: NtACS4 in the first phase and NtACS1 in the second phase. Biphasic production of ROS was inhibited in transgenic antisense plant lines expressing 1-aminocyclopropane-1-carboxylic acid synthase/oxidase or ethylene-insensitive3 as well as in transgenic plants impaired in ROS production. All tested transgenic plants were more tolerant against P. parasitica var nicotianae infection as determined based on trypan blue staining and pathogen proliferation. Further, silencing of NtACS4 blocked the second massive increase in ROS production as well as pathogen progression. Pathogen tolerance was due to the inhibition of ROS and ethylene production, which further resulted in lower activation of ROS-detoxifying enzymes. Accordingly, the synergistic inhibition of the second phase of ROS and ethylene production had protective effects against pathogen-induced cell damage. We conclude that the levels of ethylene and ROS correlate with compatible P. parasitica proliferation in susceptible plants.

Department of Bio-engineering, College of Engineering, Hanyang University, Seoul 133-791, KoreaReceived February 7, 2011, Accepted April 26, 2011Matrix metalloproteinases (MMPs), a family of zinc-containing endopeptidases, participate in many normalprocesses such as embryonic development and wound repair, and in many pathological situations such ascancer, atherosclerosis,and arthritis. Peptidomimetic MMP inhibitors were designed and synthesized with N-formylhydroxylamine (retrohydroxamate) as a zinc-binding group and various side chains on the α, P1', andP2' positions. Using in vitro MMP assays with purified MMPs (MMP-1, MMP-2, MMP-3, MMP-9, and MMP-14) and fluorogenic peptide substrates, it was found that compounds 2d and 2g selectively inhibit gelatinases(MMP-2 and MMP-9) and interstitial collagenase (MMP-1). They also inhibited the chemo-invasion offibrosarcoma HT-1080 cells and tube formation of human umbilical vascular endothelial cells in a dose-dependent manner. Our results suggest that retrohydroxamate-based MMP inhibitors, especially compounds2d and 2g, have the potential to be used as therapeutic drugs for cancer and other MMP-related diseases. Key Words : Inhibitor, Invasion, Matrix metalloproteinase (MMP), N-Formylhydroxylamine, Retrohydrox-amateIntroductionMatrix metalloproteinases (MMPs) are endopeptidasesthat can cleave virtually any component of the extracellularmatrix. Under normal physiological conditions, the activitiesof MMPsare precisely regulated by the level of transcrip-tion, by activationof precursor zymogens, by inhibition ofendogenous inhibitors,and by interaction with specificmatrix components. Deregulation of MMP activity is involv-ed in many disastrous diseases such as tumor invasion,metastasis, atherosclerosis,aneurysms, arthritis, tissue ulcers,and fibrosis.

비생물학적 스트레스로 $H_2O_2$ 를 이용하여 산화적 스트레스와 고염분 스트레스를 처리한 후 ROS의 생성을 확인한 결과 스트레스 처리 후 30분에 일시적으로 1차 peak를 형성하였다가 거의 basal level까지 감소하고 다시 증가하여 3시간에 매우 다량의 2차 peak를 형성한 후 거의 basal level로 다시 낮아지는 biphasic 양상을 나타내게 된다. 따라서 ROS의 생성은 초기 30분 내에 일시적으로 발생한 Phase I의 ROS와 Phase II의 좀 더 장기적으로 다량의 고농도로 발생된 ROS의 생리적 역할이 다를 것으로 여겨진다. 본 논문에서는 스트레스 처리 시 생성되는 ROS를 확인한 후 ROS 생성 유전자인 RbohD와 RbohF 유전자 발현이 억제된 RbohD-AS, RbohF-AS 형질전환 식물체를 이용하여 실험을 수행하였다. 스트레스에 의해 생성되는 ROS의 생성을 억제시킴으로써 스트레스에 대한 ethylene 생성이 더 적은 것으로 나타났다. 또한, 이들 형질전환 식물체에서 ethylene 생성과 $H_2O_2$ 억제 효과를 확인하였으며 고염분 등의 스트레스에 대한 저항성은 ROS와 ethylene의 생성이 저하되어 나타난 것으로 판단된다. 산화적 스트레스와 고염분 스트레스에서 후기 ethylene이 다량으로 생성되는 시기, 즉 세포손상이 초래되는 후기에서 DNA fragmentation 분석을 통해서 ROS와 ethylene의 생성이 높은 식물체일수록 PCD가 높게 나타난 것으로 여겨지며, 이 과정에서 작용하는 유전자는 RbohD와 RbohF인 것으로 보이며, RbohD가 더 효과적으로 작용하는 것으로 생각된다. 따라서 스트레스에 반응하는 신호전달과정에서 초기에 ROS가 생성이 되고 후기에 ethylene이 다량으로 생성되어 결국 세포죽음에 이르게 하는 상호 synergism을 일으켜 반응을 나타내며, 이러한 반응 과정에서 RbohD와 RbohF 유전자 발현의 억제가 스트레스에 대한 식물체의 저항성을 높이는 것으로 사료된다. 【Although reactive oxygen species (ROS) are inevitable by-products of many redox reactions in eukaryotic cells, they play a crucial role as signaling molecules in many cellular processes for development and defense response to abiotic stresses. The biphasic ROS production which was peaked twice in a first transient phase and a second massive phase was occurred after treatment of abiotic stress such as oxidative stress, high salinity. This biphasic generation of ROS was followed by the biphasic production of stress hormone, ethylene. The mechanism of interactions between ROS and ethylene biosynthesis is studied in tobacco (Nicotiana tabaccum L.) plants under the abiotic stresses. The stress-induced ethylene production was significantly inhibited in RbohD-AS and RbohF-AS, in which antisense expression of NADPH oxidase genes was performed. The accumulation of ROS, which was determined by DAB and DCFH-DA staining, was significantly decreased after abiotic stresses in transgenic plants. The suppression of signaling with ethylene and ROS induced more tolerance in response to abiotic stress. The transgenic plants were more tolerant in MS medium supplemented with salinity stress in contrast with wild-type. Stress-induced cell damage determined by DNA fragmentation was decreased at phase II in those transgenic plants. Therefore, the first burst of ROS is more responsible for making a role as a signaling molecule during stress-induced response. These results suggested that ethylene and ROS act in a positive feedback cycle that results in mutual enhancement of ethylene and ROS production during stress-induced cell death.】

The aim of the study was to clarify the charac-teristics of fatigue status in Chinese adolescents. 1018 adolescents (490 males, 528 females) aged 12 or 15 years in Jixi city were enrolled. Between May and September 2009, fatigue status was investigated using a questionnaire devised by ourselves, which consisted of 4 physical and 4 mental fatigue items. These questions were scored from 0 to 3 based on the frequency of an item occurring, given as the average of the past one month, and the fatigue scores were calculated. Lifestyle issues such as daily sleep duration, feeling of refreshment in the morning after wake-up, extracurricular sports activity, and breakfast intake, and a physical examination such as height, weight, and waist circumference were also investigated. Physical and mental fatigue scores were significantly higher in subjects aged 15 yrs old than in those aged 12 yrs old in both genders. These scores in females were significantly higher than those in males at the age of 15 yrs. Multivariate regression analysis showed that total (physical plus mental) fatigue scores were associated with female gender and various lifestyles such as unrefreshed wake-up feeling, poor extracurricular sports activity, and more frequent skipping of breakfast. However, body mass index and waist circumference were not associated with total fatigue scores.

We aimed to compare the modulation of the autonomic nervous system (ANS) in women with polycystic ovary syndrome (PCOS) with that in healthy ovulatory women on the basis of heart rate variability (HRV), and to analyze the characteristics of the ANS in PCOS. In a retrospective chart review, HRV, body mass index, and physical examination data in women with PCOS and those with regular menstrual cycles were collected. Approval from the institutional review board (IRB) was obtained (IRB No. 2017-05-007-001) for this study. The mean outcomes were the values of HRV in the time [standard deviation of all normal R-R intervals (SDNN), the square root of the sum of the squares of the differences between the adjacent normal R-R intervals (rMSSD), and the mean heart rate turbulence (mean HRT)] and frequency [total power (TP), very-low-frequency power (VLF), low-frequency power (LF), normalized low-frequency power (LF norm), high-frequency power (HF), normalized high-frequency power (HF norm), and LF/HF ratio] domains. Differences between the 2 groups were analyzed by Mann-Whitney U test, using SPSS for Windows (version 22.0). There was no significant difference in the values of the time domain (SDNN, rMSSD, and mean HRT) between the groups. In the frequency domain, women with PCOS showed significantly higher LF (598.63 ± 94.38 vs 459.13 ± 163.64, P = .028), LF norm (48.64 ± 3.39 vs 36.49 ± 2.82, P = .009), and LF/HF ratio (1.49 ± 0.31 vs 0.73 ± 0.13, P = .009) than the control group. HF norm was significantly lower in the women with PCOS than in the controls (51.38 ± 3.39 vs 63.51 ± 2.82, P = .009). The TP, VLF, and HF showed no significant difference between the groups. The results of the present study indicated that PCOS is related to increased sympathetic modulation in HRV.

This systematic review aimed to evaluate the current evidence regarding the efficacy and safety of acupuncture on primary dysmenorrhea.Ten electronic databases were searched for relevant articles published before December 2017. This study included randomized controlled trials (RCTs) of women with primary dysmenorrhea; these RCTs compared acupuncture to no treatment, placebo, or medications, and measured menstrual pain intensity and its associated symptoms. Three independent reviewers participated in data extraction and assessment. The risk of bias in each article was assessed, and a meta-analysis was conducted according to the types of acupuncture. The results were expressed as mean difference (MD) or standardized mean difference (SMD) with 95% confidence intervals (CIs).This review included 60 RCTs; the meta-analysis included 49 RCTs. Most studies showed a low or unclear risk of bias. We found that compared to no treatment, manual acupuncture (MA) (SMD = -1.59, 95% CI [-2.12, -1.06]) and electro-acupuncture (EA) was more effective at reducing menstrual pain, and compared to nonsteroidal anti-inflammatory drugs (NSAIDs), MA (SMD = -0.63, 95% CI [-0.88, -0.37]) and warm acupuncture (WA) (SMD = -1.12, 95% CI [-1.81, -0.43]) were more effective at reducing menstrual pain. Some studies showed that the efficacy of acupuncture was maintained after a short-term follow-up.The results of this study suggest that acupuncture might reduce menstrual pain and associated symptoms more effectively compared to no treatment or NSAIDs, and the efficacy could be maintained during a short-term follow-up period. Despite limitations due to the low quality and methodological restrictions of the included studies, acupuncture might be used as an effective and safe treatment for females with primary dysmenorrhea.

Sudden deaths have occurred in feedlot cattle with marked necro-hemorrhagic enteritis of the jejunum, ileum and colon. Suckling beef calves are the most frequently affected. Over-consumption of large amounts of milk, inadequate colostrum intake, chilling and stress are conducive to the development of enterotoxemia. Enterotoxemia caused by Clostridium perfringens type D mostly occurs following a sudden change of diet, particularly to feeds made richer in order to grow the cattle to market weight in feedlots. During July 2006, sudden deaths of cattle occurred in the Youngcheon regional area of Gyeongbuk province. There were no significant clinical signs except anorexia, depression, intermittent diarrhea and mild respiratory failure. Histological findings revealed a prominent intranuclear inclusion as well as infiltration of the globular leukocytes in various organs including the heart, kidneys, liver, spleen and lymph nodes. Spleen and lymphatic tissues showed lymphatic necrosis and a starry sky appearance. In the submucosa of the small intestines, basophilic aggregation was detected with massive infiltration of the globular leukocytes and eosinophils. Gram staining for the tissue sections containing inclusions of the small intestines revealed a positive histochemical reaction. Taken together, we suggest that Clostridium perfringens type D-induced enterotoxemia is determined to be the cause of sudden death of feedlot cattle.

Senescence marker protein 30 (SMP30), an important aging marker molecule that is highly expressed in the liver, has been known to protect hepatocytes from apoptosis by the synthesis of vitamin C. To explore the function of SMP30 in liver fibrosis, the effect of SMP30 deficiency on liver fibrosis was investigated in SMP30 knockout (KO) mice. Moreover, the in vivo results were further confirmed by way of hepatic stellate cell (HSC) isolation. We demonstrated that carbon tetrachloride (CCl4)-induced liver fibrosis and the nuclear translocation of p-Smad2/3, the immediate downstream of transforming growth factor beta (TGF-β), were significantly inhibited in the liver of SMP30 KO mice compared with wildtype (WT) mice. We also confirmed that both WT and SMP30 KO HSCs did not express SMP30. Finally, we further confirmed that up-regulation of peroxisome proliferator-activated receptor-gamma (PPAR-γ) caused by a lack of vitamin C was the pivotal factor in the mechanisms for attenuated liver fibrosis of SMP30 KO mice, and feeding with vitamin C restored CCl4-induced liver fibrosis in SMP30 KO mice. Conclusion: Vitamin C deficiency by SMP30 depletion attenuated liver fibrosis by way of up-regulated PPAR-γ expression in SMP30 KO mice. Our results provide, for the first time, the possible mechanisms underlying inhibition of HSC activation associated with vitamin C and PPAR-γ up-regulation in liver fibrosis of SMP30 KO mice. (HEPATOLOGY 2010.)

We have generated iPSCs from peripheral blood mononuclear cells (PBMCs) of a healthy man using heat sensitive and non-integrative Sendai virus containing Sox2, Oct3/4, c-Myc and Klf4. Human GRX-MCiPS4F-A2 cell line was established and characterized through this study.

Gastric cancer, one of the most common malignancies worldwide, typically has a poor prognosis and poor survival rate. Previous studies have investigated the chemopreventive effect of celecoxib. In the present study, the SGC-7901 human gastric cancer cell line was utilized to examine the chemopreventive mechanisms of celecoxib. The inhibition of cell proliferation was determined using MTT assay, cell apoptosis was monitored by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) and flow cytometry, and cell ultrastructural changes were assessed via transmission electron microscopy. The mRNA expression of Akt, caspase-8 and -9 was examined using quantitative reverse-transcription-polymerase chain reaction (qRT-PCR) and p-Akt, procaspase-8 and -9 were analyzed via western blotting. The results showed that celecoxib inhibited the proliferation of SGC-7901 cells in a time- and dose-dependent manner. Additionally, celecoxib induced apoptosis as substantiated by typical apoptotic bodies, autophagosomes and an increased apoptotic rate. It was found that following celecoxib treatment, Akt mRNA expression was not significantly altered, and that p-Akt protein levels decreased in a time- and dose-dependent manner. Additionally, caspase-8 and -9 mRNA expression was significantly increased, while procaspase-8 and -9 protein expression decreased relative to the time- and dose-dependent effects. These results demonstrated that celecoxib induced apoptosis and autophagy of gastric cancer cells in vitro through the PI3K/Akt signaling pathway. Moreover, our findings suggested that celecoxib induces apoptosis in gastric cancer cells through the mitochondrial and death receptor pathways, providing additional understanding regarding the chemopreventive behaviors of celecoxib and its uses in cancer therapy.

A 4-year-old, male, dachshund was referred to a certain local veterinary hospital because of a soft and fluctuant swelling in the left upper cervical region. The swelling was surgically removed and appeared to be filled with bloody mucus. Grossly, the swelling was identified as salivary mucocele and showed small multifocal whitish ossified tissue on its surface. Microscopically, the wall of salivary mucocele appeared as granulation tissue surrounding mucin, which was composed of loose edematous and vascularized connective tissue containing chronic inflammatory cells such as lymphocytes, plasma cells and macrophages. Characteristically, present case had ossifying components formed by metaplastic spindle cells in the wall of salivary mucocele. Therefore, the present case was diagnosed as salivary mucocele with osseous metaplasia in a dog.

We trapped a rat (Rattus norvegicus) infected with Capillaria hepatica. At necropsy, grossly yellowish-white nodules (2-3 mm in diameter) were noted to be scattered on the liver's surface. Microscopically, granulomatous and fibrotic nodules that contained the eggs and/or adult worms of Capillaria hepatica were detected in the liver. Septal fibrosis was diffusely formed throughout the liver. There were a number of ED1-positive macrophages located in the sinusoids of the pseudolobules. On the double staining, myofibroblasts and mast cells were generally observed within the fibrous septa with the mast cells in close proximity to the myofibroblasts. We suggest that the interactions between macrophages, myofibroblasts and mast cells play a role in the septal fibrosis observed in rats infected by Capillaria hepatica.

A 3-year-old, female Spitz, was presented due to lack of response to therapies with a 6-month history of skin lesions characterized by diffuse erythema and scaling on the dorsal trunk. Physical examination revealed the dog was active and healthy. Skin culture isolated no fungus. Histological examination of skin biopsy specimens revealed interface dermatitis with hydropic degeneration of the basal layers, predominant plasmacytic perivascular accumulation in the dermis, and intensive plasma cell-rich interface mural folliculitis. Moderate CD3-positive lymphocytes infiltrated the superficial dermis. This report may provide unique information of canine discoid lupus erythematosus in an unusual breed with atypical cutaneous lesions.

Published data on the association between the cytotoxic T-lymphocyte-associated antigen-4 gene A49G polymorphism and the risk for Hashimoto's thyroiditis (HT) are inconclusive. A meta-analysis was performed to derive a more precise estimation. Published case-control studies in English or Chinese were identified. In total, 24 studies with 2295 cases and 4521 controls were investigated. A random-effect model was performed irrespective of between-study heterogeneity. Study quality was assessed in duplicate, and subgroup analyses were conducted by ethnicity or age. Overall, the 49G allele was associated with an increase in HT risk [odds ratio (OR) = 1.31; 95% confidence interval (95%CI) = 1.17-1.47; P < 0.00001]. In a subgroup analysis by ethnicity, comparison of allele 49G with 49A generated a 27% increased risk among East Asians (OR = 1.48; 95%CI = 1.24-1.76; P < 0.00001) and whites (OR = 1.27; 95%CI = 1.12-1.44; P = 0.0002). We also found an increased risk among adults (OR = 1.31; 95%CI = 1.17-1.47; P < 0.00001) but not among children (OR = 1.44; 95%CI = 0.75-2.79; P = 0.27), possibly owing to the small sample sizes in children. No publication biases were observed. This meta-analysis suggested that the cytotoxic T-lymphocyte-associated antigen-4 gene 49G allele was associated with an increased HT risk, especially in adults.

Lactobacillus paraplantarum KNUC25 strain was isolated from overfermented kimchi, a Korean traditional food. The strain had a broad antimicrobial activity spectrum, from gram-positive to gram-negative bacteria. The aim of this study was to evaluate the activity of L. paraplantarum KNUC25 against Helicobacter pylori strains. Judged by a disc agar diffusion method, the anti-H. pylori activity existed in the cell-free supernatants (CFSs) of KNUC25. The mean diameters of growth inhibition by 10, 30, and 60 microL of a 15-fold concentrated CFS per disc were 11.2, 17.7, and 23.7 mm, respectively. The neutralized CFS lost its anti-H. pylori activity, suggesting that acidic pH in CFS may be responsible for the anti-H. pylori activity. Adherence was determined by urease activity of H. pylori adhered to gastric epithelial cell line AGS cells after co-incubation of AGS cells with CFS and H. pylori strain ATCC43504 (s1m1vacA/cagA(+)), ATCC51932 (s2m2vacA/cagA(-)), or SS1 (s2m2vacA/cagA(+)) in vitro followed by three washes by means of centrifugation with saline. Adherence of ATCC43504 or SS1 to AGS cells was reduced by about 70% after a 30-minute incubation with 30 microL of a 15-fold concentrated KNUC25 CFS, whereas that of ATCC51932 to AGS cells was not. The results show KNUC25 CFS is effective in inhibiting the growth of H. pylori, which is related to pH and the adherence of cagA-positive H. pylori to gastric cells.

The recombinant vacuolating cytotoxin (rVacA) of Helicobacter pylori that retains native conformational epitopes was evaluated as a vaccine antigen for anti-H. pylori treatment.s1m1 vacA gene fraction encoding the mature VacA protein was expressed as a soluble protein in E. coli at low temperature. The efficacy of anti-rVacA antibody against VacA or H. pylori was assessed in vitro using AGS cells and in vivo using a murine model.The rabbit antisera against rVacA completely neutralized the vacuolating activity and partially inhibited the cell death induced by VacA in AGS cells. Oral immunization of C57BL/6 mice with rVacA plus CpG-oligodeoxynucleotide (ODN) as an ajuvant stimulated specific anti-VacA antibody and mucosal immune responses which correlated with decreased systemic immune responses and gastric urease activities (p0.05).The rVacA antigen possessing conformational epitopes may have potential as a vaccine component and may be useful in serological and histopathological analysis.

We describe 2 cases of malignant fibrous histiocytomas (MFHs) that spontaneously developed in young pet dogs. To classify these tumors, we applied a panel of antibodies (vimentin, desmin, alpha-SMA, and ED1) and Azan staining for collagen. The MFHs were most consistent with osteoclastlike giant and inflammatory cell types. The first case had positive staining for ED1 and vimentin, and given the osteoclast-like giant cells, calcification sites accompanying peripheral giant cell infiltrates. The latter case, the inflammatory cell type, exhibited a storiform-pleomorphic variant of neoplastic cells, including an ossifying matrix. MFHs are among the most highly aggressive tumors occurring in soft tissue sarcomas in elderly dogs; however, MFHs have been poorly studied from a diagnostic point of view. Herein, we describe the histologic and immunohistologic features of MFHs in detail, thus classifying the subtypes of these tumors.