The microsomal enzyme inducers phenobarbital (PB), pregnenolone-16 alpha-carbonitrile (PCN), 3-methylcholanthrene (3MC), and Aroclor 1254 (PCB) are known to induce thyroxine (T(4)) glucuronidation and reduce serum T(4) concentrations in rats. Also, microsomal enzyme inducers that increase serum TSH (i.e., PB and PCN) also increase thyroid follicular cell proliferation in rats. Little is known about the effects of these microsomal enzyme inducers on T(4) glucuronidation, serum thyroid hormone concentrations, serum TSH, and thyroid gland growth in mice. Therefore, we tested the hypothesis that microsomal enzyme inducers induce T(4) UDP-GT activity, resulting in reduced serum T(4) concentrations, as well as increased serum TSH and thyroid follicular cell proliferation in mice. B6C3F male mice were fed a control diet or a diet containing PB (600, 1200, 1800, or 2400 ppm), PCN (250, 500, 1000, or 2000 ppm), 3MC (62.5, 125, 250, or 500 ppm), or PCB (10, 30, 100, or 300 ppm) for 21 days. All four inducers increased liver weight and hepatic microsomal UDP-GT activity toward chloramphenicol, alpha-naphthol, and T(4). PB and PCB decreased serum total T(4), but PCN and 3MC did not. Serum thyroid stimulating hormone was markedly increased by PCN and 3MC treatments, and slightly increased by PB and PCB treatments. All four microsomal enzyme inducers dramatically increased thyroid follicular cell proliferation in mice. The findings suggest that PB, PCN, 3MC, and PCB disrupt thyroid hormone homeostasis in mice.

Upregulated gene 11 (URG11), a new gene upregulated by Hepatitis B Virus X protein (HBx), was previously shown to activate beta-catenin and promote hepatocellular growth and tumourigenesis. Although the oncogenic role of URG11 in the development of hepatocellular carcinoma has been well documented, its relevance to other human malignancies and the underlying molecular mechanisms remain largely unknown. Here we reported a novel function of URG11 to promote gastric cancer growth and metastasis. URG11 was found to be highly expressed in gastric cancer tissues compared with adjacent nontumourous ones by immunohistochemical staining and western blot. Knockdown of URG11 expression by small interfering RNA (siRNA) effectively attenuated the proliferation, anchorage-independent growth, invasiveness and metastatic potential of gastric cancer cells. URG11 inhibition led to decreased expression of beta-catenin and its nuclear accumulation in gastric cancer cells and extensive costaining between URG11 and beta-catenin was observed in gastric cancer tissues. Transient transfection assays with the beta-catenin promoter showed that it was inhibited by URG11-specific small inhibitory RNA. Moreover, suppression of endogenous URG11 expression results in decreased activation of beta-catenin/TCF and its downstream effector genes, cyclinD1 and membrane type 1 matrix metallopeptidase (MT1-MMP), which are known to be involved in cell proliferation and invasion, respectively. Taken together, our data suggest that URG11 contributes to gastric cancer growth and metastasis at least partially through activation of beta-catenin signalling pathway. These findings also propose a promising target for gene therapy in gastric cancer.

Protein-protein interactions play an essential role in the assembly of the macromolecular complexes that form functional networks and control cellular behavior. Elucidating principles of molecular recognition governing potentially complex interfaces is a challenging goal for structural and systems biology. Extensive studies of alpha-helical coiled coils have provided fundamental insight into the determinants of one seemingly tractable class of oligomeric protein interfaces. We report here that two different valine-containing mutants of the GCN4 leucine zipper that fold individually as four-stranded coiled coils associate preferentially in mixtures to form an antiparallel, heterotetrameric structure. X-ray crystallographic analysis reveals that the coinciding hydrophobic interfaces of the hetero- and homotetramers differ in detail, thereby controlling their partnering and structural specificity. Equilibrium disulfide exchange and thermal denaturation experiments show that the 50-fold preference for heterospecificity is determined by interfacial van der Waals interactions and hydrophobicity. Parallel studies of two alanine-containing variants confirm the above-mentioned interpretation of the basis and mechanism of this heterospecificity. Our results suggest that coiled-coil recognition is an inherently geometric process in which heterotypic interaction specificity derives from a complementarity of both shape and chemistry.

Cadmium (Cd) is a nephrotoxic industrial and environmental pollutant that causes a generalized dysfunction of the proximal tubule. Kim-1 is a transmembrane glycoprotein that is normally not detectable in non-injured kidney, but is up-regulated and shed into the urine during the early stages of Cd-induced proximal tubule injury. The objective of the present study was to examine the relationship between the Cd-induced increase in Kim-1 expression and the onset of necrotic and apoptotic cell death in the proximal tubule. Adult male Sprague-Dawley rats were treated with 0.6 mg (5.36 micromol) Cd/kg, subcutaneously, 5 days per week for up to 12 weeks. Urine samples were analyzed for levels of Kim-1 and the enzymatic markers of cell death, lactate dehydrogenase (LDH) and alpha-glutathione-S-transferase (alpha-GST). In addition, necrotic cells were specifically labeled by perfusing the kidneys in situ with ethidium homodimer using a procedure that has been recently developed and validated in the Prozialeck laboratory. Cryosections of the kidneys were also processed for the immunofluorescent visualization of Kim-1 and the identification of apoptotic cells by TUNEL labeling. Results showed that significant levels of Kim-1 began to appear in the urine after 6 weeks of Cd treatment, whereas the levels of total protein, alpha-GST and LDH were not increased until 8-12 weeks. Results of immunofluorescence labeling studies showed that after 6 weeks and 12 weeks, Kim-1 was expressed in the epithelial cells of the proximal tubule, but that there was no increase in the number of necrotic cells, and only a modest increase in the number of apoptotic cells at 12 weeks. These results indicate that the Cd-induced increase in Kim-1 expression occurs before the onset of necrosis and at a point where there is only a modest level of apoptosis in the proximal tubule.

Dmrt7, a member of the Dmrt family of genes, is required for spermatogenesis. However, promoter functions of the gene Dmrt7 remain unknown. We have cloned and characterized the proximal promoter region of the mouse Dmrt7 gene. Functional analysis of the 5' flanking region by sequential deletion mutations revealed crucial positive elements between -60 and +1, in which two highly conserved and tandem CCAAT boxes: the CCAAT box1 (-48/-44) and the CCAAT box2 (-7/-3) are located. Site-directed mutagenesis studies demonstrated that both CCAAT boxes are indispensable to the promoter activity. Electrophoretic mobility shift assays (EMSAs) and gel-supershift assays indicated that transcription factor NF-Y binds to the promoter. Chromatin immunoprecipitation (ChIP) analysis demonstrated that NF-Y interacts in vivo with the promoter of the Dmrt7 gene in testis. Co-transfection and reporter analysis showed that over-expression of NF-Ys increased transcription of the Dmrt7-luc gene whereas expression of a dominant-negative NF-Ya decreased the transcription. This suggests that NF-Y can activate the Dmrt7 promoter. These results provide evidence of a transcription regulatory mechanism that controls Dmrt7 gene expression in mouse testis.

Transient outward potassium currents (I(to)) are major early repolarization currents in shaping cardiac action potential (AP). Downregulation of I(to) contributes to AP configuration alteration in myocardial infarction (MI) and numerous other heart diseases. High-mobility group box 1 (HMGB1), a proinflammatory cytokine, has been reported to increase dramatically in the serum of patients with MI, participating in ischemia-reperfusion injury and recovery of post-infarction failing heart. This study investigated the possible role of HMGB1 in regulating cardiac I(to) and electrical stability. HMGB1 treatment for 24h significantly inhibited the current densities of heterologously expressed Kv4.3 and Kv4.2 in COS-7 cells and native I(to) in neonatal rat ventricular myocytes (NRVMs) in a dose-dependent manner. HMGB1 decreased the mRNA and protein levels of the I(to) alpha subunits Kv4.2 and Kv4.3 channels, but not the beta subunit KChIP2 and KCNE2 in NRVMs. The receptor binding domain (150-186 amino acid residues) responsible for receptor of advanced glycation end product (RAGE) binding similarly inhibited I(to)(,) while treatment with soluble RAGE that blocks binding of ligands to cell-surface RAGE partially restored I(to) current density and Kv4 protein expressions. Box A which possesses no proinflammatory activity of HMGB1 still remained part of the I(to) suppression effect. In addition to downregulating I(to), HMGB1 modestly inhibited L-type Ca(2+) current, but not I(K1). The AP duration (APD) was slightly prolonged by HMGB1 treatment. These results collectively establish HMGB1 as a novel pathological factor downregulating I(to) partially through HMGB1-RAGE interaction, providing new insights into the potential molecular mechanisms underlying the electrical remodeling in MI.

The importance of mechanical signals in normal and inflamed cartilage is well established. Chondrocytes respond to changes in the levels of proinflammatory cytokines and mechanical signals during inflammation. Cytokines like interleukin (IL)-1beta suppress homeostatic mechanisms and inhibit cartilage repair and cell proliferation. However, matrix synthesis and chondrocyte (AC) proliferation are upregulated by the physiological levels of mechanical forces. In this study, we investigated intracellular mechanisms underlying reparative actions of mechanical signals during inflammation.

We carried out an association study between GNB1L and three mental disorders (major depressive disorder, bipolar disorder, schizophrenia) in Chinese Han population. Among 1135 cases and 1135 controls, findings suggests that GNB1L is linked with bipolar disorder and schizophrenia and not with major depressive disorder.

Sperm-oocyte fusion is a critical step in fertilization, which requires a series of proteins from both spermatozoa and oocyte to mediate membrane adhesion and subsequent fusion. A rat spermatozoa membrane protein is endoplasmic reticulum protein 29 (ERp29), which significantly increases on the sperm surface as well as in the cytoplasm of epididymal epithelia from caput to cauda as the sperm undergo epididymal maturation. Moreover, ERp29 facilitates viral infection via mediating membrane penetration. We determined if in addition to promoting sperm maturation ERp29 may also play a role in facilitating gamete fusion during the fertilization process.

HF (heart failure) after MI (myocardial infarction) is a major cause of morbidity and mortality worldwide. Recent studies have shown that hydrogen sulfide (H2S) has cardioprotective effects. Hence, we aimed to elucidate the potential effects of H2S on HF after MI in rats. The HF model after MI was made by ligating the left anterior descending coronary artery. HF groups and sham-operated groups of rats were treated with vehicle, sodium hydrosulfide (NaHS) or PAG (propagylglycine). Equal volumes of saline, 3.136 mg · kg-1 · day-1 NaHS or 37.5 mg · kg-1 · day-1 PAG, were intraperitoneally injected into rats for 6 weeks after operation. Survival, lung-to-body weight ratio and left ventricular haemodynamic parameters were measured. The protein and gene expression of Bcl-2, Bax, caspase 3 and cytochrome c were analysed by Western blotting and RT-PCR (reverse transcription-PCR). TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) and EM (electron microscopy) were used to examine apoptosis of heart tissues. NaHS was found to improve the survival and lower the lung-to-body weight ratio. It increased the LVSP (left ventricular systolic pressure) and the maximum rate of pressure and decreased LVEDP (left ventricular end-diastolic pressure). Furthermore, NaHS promoted Bcl-2 protein and mRNA expression and demoted Bax, caspase 3 protein and mRNA expression in HF rats. We also showed that NaHS decreased the leakage of cytochrome c protein from the mitochondria to the cytoplasm. Histological observation by TUNEL and EM proved that NaHS inhibited cardiac apoptosis in HF hearts and improved mitochondrial derangements, but that PAG aggravated those indices. Hence, H2S has protective effects in HF rats.

Chronic pain conditions are difficult to treat and are major health problems. Bone marrow stromal cells (BMSCs) have generated considerable interest as a candidate for cell-based therapy. BMSCs are readily accessible and are easy to isolate and expand ex vivo. Clinical studies show that direct injection of BMSCs does not produce unwanted side effects and is well tolerated and safe. Here, we show that a single systemic (intravenous) or local injection (into the lesion site) of rat primary BMSCs reversed pain hypersensitivity in rats after injury and that the effect lasted until the conclusion of the study at 22 weeks. The pain hypersensitivity was rekindled by naloxone hydrochloride, an opioid receptor antagonist that acts peripherally and centrally, when tested at 1-5 weeks after BMSC infusion. In contrast, naloxone methiodide, a peripherally acting opioid receptor antagonist, only rekindled hyperalgesia in the first 3 weeks of BMSC treatment. Focal downregulation of brainstem mu opioid receptors by RNA interference (RNAi) reversed the effect of BMSCs, when RNAi was introduced at 5- but not 1-week after BMSC transplantation. Thus, BMSCs produced long-term relief of pain and this effect involved activation of peripheral and central opioid receptors in distinct time domains. The findings prompt studies to elucidate the cellular mechanisms of the BMSC-induced pain relieving effect and translate these observations into clinical settings.

Transplantations of olfactory ensheathing cells (OECs) have been reported to promote axonal regeneration and functional recovery after spinal cord injury, but have demonstrated limited growth promotion of rat rubrospinal axons after a cervical dorsolateral funiculus crush. Rubrospinal neurons undergo massive atrophy after cervical axotomy and show only transient expression of regeneration-associated genes. Cell body treatment with brain-derived neurotrophic factor (BDNF) prevents this atrophy, stimulates regeneration-associated gene expression and promotes regeneration of rubrospinal axons into peripheral nerve transplants. Here, we hypothesized that the failure of rubrospinal axons to regenerate through a bridge of OEC transplants was due to this weak intrinsic cell body response. Hence, we combined BDNF treatment of rubrospinal neurons with transplantation of highly enriched OECs derived from the nasal mucosa and assessed axonal regeneration as well as behavioral changes after a cervical dorsolateral funiculus crush. Each treatment alone as well as their combination prevented the dieback of the rubrospinal axons, but none of them promoted rubrospinal regeneration beyond the lesion/transplantation site. Motor performance in a food-pellet reaching test and forelimb usage during vertical exploration (cylinder test) were more impaired after combining transplantation of OECs with BDNF treatment. This impaired motor performance correlated with lowered sensory thresholds in animals receiving the combinatorial therapy - which were not seen with each treatment alone. Only this combinatorial treatment group showed enhanced sprouting of calcitonin gene-related peptide-positive axons rostral to the lesion site. Hence, some combinatorial treatments, such as OECs with BDNF, may have undesired effects in the injured spinal cord.

Genotypic methods for drug susceptibility testing of Mycobacterium tuberculosis are desirable to speed the diagnosis and proper therapy of tuberculosis (TB). However, the numbers of genes and polymorphisms implicated in resistance have proliferated, challenging diagnostic design. We developed a microfluidic TaqMan array card (TAC) that utilizes both sequence-specific probes and high-resolution melt analysis (HRM), providing two layers of detection of mutations. Twenty-seven primer pairs and 40 probes were designed to interrogate 3,200 base pairs of critical regions of the inhA, katG, rpoB, embB, rpsL, rrs, eis, gyrA, gyrB, and pncA genes. The method was evaluated on 230 clinical M. tuberculosis isolates from around the world, and it yielded 96.1% accuracy (2,431/2,530) in comparison to that of Sanger sequencing and 87% accuracy in comparison to that of the slow culture-based susceptibility testing. This TAC-HRM method integrates assays for 10 genes to yield fast, comprehensive, and accurate drug susceptibility results for the 9 major antibiotics used to treat TB and could be deployed to improve treatment outcomes.

Recent evidence has demonstrated that UBASH3A gene was associated with multiple autoimmune diseases. The aim of this study was to explore the association between UBASH3A gene single-nucleotide polymorphisms (SNPs) and systemic lupus erythematosus (SLE) in a Chinese Han population. Four UBASH3A polymorphisms (rs11203203, rs3788013, rs2277798, and rs1893592) were genotyped using the Fluidigm 192.24 Dynamic Array™ Integrated Fluidic Circuit (IFC). Data were analyzed by SPSS 11.5 software. A total of 792 SLE patients and 777 healthy controls were included in this study. The CC genotype and C allele of rs3788013 polymorphism were more frequent in the patient group than in controls (OR=1.583, 95% CI=1.095-2.287; OR=1.258, 95% CI=1.083-1.461, respectively). We also found a statistical significance under the recessive model (OR=1.298, 95% CI=1.049-1.607, p=0.017). The frequency of variant genotype AC of rs3788013 was associated with the phenotype of vasculitis (p=0.012). A statistically significant association was observed between UBASH3A rs1893592 C allele and skin rash, oral ulcer and arthritis (p<0.05). Moreover, we found that the genotype distribution of rs2277798 was significantly associated with hematuria in the SLE patients (p=0.003). However, UBASH3A rs11203203, rs2277798, and rs1893592 polymorphisms were not associated with the risk of SLE (p>0.05). The findings suggest that UBASH3A gene might contribute to SLE susceptibility and influence the clinical phenotype of the disease. Further studies are necessary to elucidate the exact role of UBASH3A gene in the pathogenesis of SLE.

Clinical practice guidelines (CPGs) play an important role in health care. The guideline development process should be precise and rigorous to ensure that the results are reproducible and not vague. To determine the quality of guidelines, the Appraisal of Guidelines and Research and Evaluation (AGREE) instrument was developed and introduced. The objective of this study is to assess the methodological quality of CPGs on pancreatic cancer. Five databases (included MEDLINE and EMBASE) and guideline websites were searched till April, 2014. The methodological quality of the guidelines was assessed by 4 authors independently using the AGREE II instrument. From 2526 citations, 21 relevant guidelines were included. The overall agreement among reviewers was moderate (intraclass correlation coefficient = 0.86, 95% confidence interval 0.64-0.96). The mean scores were moderate for the domains "scope and purpose" and "clarity of presentation"; however, they were low for the domains "stakeholder involvement" (31.22), "rigor of development", "applicability", and "editorial independence". These domain scores were lower when compared with international levels. There are 5 (23.81%) guidelines that described the systematic methods for searching. Moreover, only 5 (23.81%) guidelines reported that methodological expertise were included in the guideline developing teams. The quality and transparency of the development process and the consistency in the reporting of pancreatic cancer guidelines need to be improved. Many other methodological disadvantages were identified. In the future, pancreatic cancer CPGs should base on the best available evidence rigorously developed and reported. Greater efforts are needed to provide high-quality guidelines that serve as a useful and reliable tool for clinical decision making in this field.

Our previous studies demonstrated that xyloketal B, a novel marine compound with a unique chemical structure, has strong antioxidant actions and can protect against endothelial injury in different cell types cultured in vitro and model organisms in vivo. The oxidative endothelial dysfunction and decrease in nitric oxide (NO) bioavailability are critical for the development of atherosclerotic lesion. We thus examined whether xyloketal B had an influence on the atherosclerotic plaque area in apolipoprotein E-deficient (apoE-/-) mice fed a high-fat diet and investigated the underlying mechanisms. We found in our present study that the administration of xyloketal B dose-dependently decreased the atherosclerotic plaque area both in the aortic sinus and throughout the aorta in apoE-/- mice fed a high-fat diet. In addition, xyloketal B markedly reduced the levels of vascular oxidative stress, as well as improving the impaired endothelium integrity and NO-dependent aortic vasorelaxation in atherosclerotic mice. Moreover, xyloketal B significantly changed the phosphorylation levels of endothelial nitric oxide synthase (eNOS) and Akt without altering the expression of total eNOS and Akt in cultured human umbilical vein endothelial cells (HUVECs). Here, it increased eNOS phosphorylation at the positive regulatory site of Ser-1177, while inhibiting phosphorylation at the negative regulatory site of Thr-495. Taken together, these findings indicate that xyloketal B has dramatic anti-atherosclerotic effects in vivo, which is partly due to its antioxidant features and/or improvement of endothelial function.

Peripheral nerve injury is a common clinical problem. Nerve growth factor (NGF) promotes peripheral nerve regeneration, but its clinical applications are limited by several constraints. In this study, we found that the time-dependent expression profiles of eight let-7 family members in the injured nerve after sciatic nerve injury were roughly similar to each other. Let-7 microRNAs (miRNAs) significantly reduced cell proliferation and migration of primary Schwann cells (SCs) by directly targeting NGF and suppressing its protein translation. Following sciatic nerve injury, the temporal change in let-7 miRNA expression was negatively correlated with that in NGF expression. Inhibition of let-7 miRNAs increased NGF secretion by primary cultured SCs and enhanced axonal outgrowth from a coculture of primary SCs and dorsal root gangalion neurons. In vivo tests indicated that let-7 inhibition promoted SCs migration and axon outgrowth within a regenerative microenvironment. In addition, the inhibitory effect of let-7 miRNAs on SCs apoptosis might serve as an early stress response to nerve injury, but this effect seemed to be not mediated through a NGF-dependent pathway. Collectively, our results provide a new insight into let-7 miRNA regulation of peripheral nerve regeneration and suggest a potential therapy for repair of peripheral nerve injury.

Magnetic liposomes have been frequently used as nanocarriers for targeted drug delivery and magnetic resonance imaging in recent years. Despite great potentials, their morphological/structural instability in the physiological environment still remains an intractable challenge for clinical applications. In this study, stable hybrid liposomal cerasomes (ie, liposomes partially coated with silica) which can co-encapsulate Fe3O4 nanoparticles and the anticancer drug paclitaxel were developed using thin film hydration method. Compared with the drug loaded liposomes, the paclitaxel-loaded magnetic cerasomes (PLMCs) exhibited much higher storage stability and better sustained release behavior. Cellular uptake study showed that the utilization of an external magnetic field significantly facilitated the internalization of PLMCs into cancer cells, resulting in potentiated drug efficacy of killing tumor cells. The T2 relaxivity (r2) of our PLMCs was much higher than that of free Fe3O4 nanoparticles, suggesting increased sensitivity in T2-weighted imaging. Given its excellent biocompatibility also shown in the study, such dual functional PLMC is potentially a promising nanosystem for effective cancer diagnosis and therapy.

Previously, we conducted a randomized phase III trial of TPF (docetaxel, cisplatin, and 5-fluorouracil) induction chemotherapy in surgically managed locally advanced oral squamous cell carcinoma (OSCC) and found no improvement in overall survival. This study reports long-term follow-up results from our initial trial. All patients had clinical stage III or IVA locally advanced OSCC. In the experimental group, patients received two cycles of TPF induction chemotherapy (75mg/m2 docetaxel d1, 75mg/m2 cisplatin d1, and 750mg/m2/day 5-fluorouracil d1-5) followed by radical surgery and post-operative radiotherapy; in the control group, patients received upfront radical surgery and post-operative radiotherapy. The primary endpoint was overall survival. Among 256 enrolled patients with a median follow-up of 70 months, estimated 5-year overall survival, disease-free survival, locoregional recurrence-free survival, and distant metastasis-free survival rates were 61.1%, 52.7%, 55.2%, and 60.4%, respectively. There were no significant differences in survival rates between experimental and control groups. However, patients with favorable pathologic responses had improved outcomes compared to those with unfavorable pathologic responses and to those in the control group. Although TPF induction chemotherapy did not improve long-term survival compared to surgery upfront in patients with stage III and IVA OSCC, a favorable pathologic response after induction chemotherapy may be used as a major endpoint and prognosticator in future studies. Furthermore, the negative results observed in this trial may be represent type II error from an underpowered study. Future larger scale phase III trials are warranted to investigate whether a significant benefit exists for TPF induction chemotherapy in surgically managed OSCC.

Myocardial infarction (MI) is a major cause of death and disability worldwide. In the last decade, mesenchymal stem cells (MSCs) based cell therapy has emerged as a promising therapeutic strategy. Although great advance have been made using MSCs to treat MI, the low viability of transplanted MSCs severely limits the efficiency of MSCs therapy. Here, we show evidence that ex vivo pre-treatment with melatonin, an endogenous hormone with newly found anti-oxidative activity, could improve survival and function of adipose tissue derived MSCs (ADSCs) in vitro as well as in vivo. ADSCs with 5 μM melatonin pre-treatment for 24 hrs showed increased expression of the antioxidant enzyme catalase and Cu/Zn superoxide dismutase (SOD-1), as well as pro-angiogenic and mitogenic factors like insulin-like growth factor 1, basic fibroblast growth factor, hepatocyte growth factor (HGF), epidermal growth factor. Furthermore, melatonin pre-treatment protected MSCs from reactive oxygen species (ROS) induced apoptosis both directly by promoting anti-apoptosis kinases like p-Akt as well as blocking caspase cascade, and indirectly by restoring the ROS impaired cell adhesion. Using a rat model of MI, we found that melatonin pre-treatment enhanced the viability of engrafted ADSCs, and promoted their therapeutic potency. Hopefully, our results may shed light on the design of more effective therapeutic strategies treating MI by MSCs in clinic.