AIM: To explore the function of Nonstructural 5A (NS5A) protein of genotype 2a (JFH1) in the replication and infection of hepatitis C virus (HCV).
METHODS: Intergenotypic chimera FL-J6JFH/J4NS5A was constructed by inserting NS5A gene from 1b stain HC-J4 by the overlapping polymerase chain reaction (PCR) method and the restriction enzyme reaction. In vitro RNA transcripts of chimera, prototype J6JFH and negative control J6JFH1 (GND) were prepared and transfected into Huh-7.5 cells with liposomes. Immunofluorescence assay (IFA), fluorescence quantitative PCR and infection assay were performed to determine the protein expression and gene replication in Huh-7.5 cells.
RESULTS: The HCV RNA levels in FL-J6JFH/J4NS5A chimera RNA transfected cells were significantly lower than the wild type at any indicated time point (2.58 ± 5.97 × 106 vs 4.27 ± 1.72 × 104, P = 0.032). The maximal level of HCV RNA in chimera was 5.6 ± 1.8 × 104 GE/μg RNA at day 34 after transfection, while the wild type reached a peak level at day 13 which was 126 folds higher (70.65 ± 14.11 × 105 vs 0.56 ± 0.90 × 105, P = 0.028). HCV proteins could also be detected by IFA in chimera-transfected cells with an obviously low level. Infection assay showed that FL-J6JFH/J4NS5A chimera could produce infectious virus particles, ranging from 10 ± 5 ffu/mL to 78.3 ± 23.6 ffu/mL, while that of FL-J6JFH1 ranged from 5.8 ± 1.5 × 102 ffu/mL to 2.5 ± 1.4 × 104 ffu/mL.
CONCLUSION: JFH1 NS5A might play an important role in the robust replication of J6JFH1. The establishment of FL-J6JFH/J4NS5A provided a useful platform for studying the function of other proteins of HCV.
Hepatitis C virus; Nonstructural 5A; Chimera; Cell culture-produced virus; Replication; Infection
There are four cell lineages derived from intestinal stem cells that are located at the crypt and villus in the mammalian intestine the non-secretory absorptive enterocytes, and the secretory cells, which include mucous-secreting goblet cells, regulatory peptide-secreting enteroendocrine cells and antimicrobial peptide-secreting Paneth cells. Although fibroblast growth factor (Fgf) signaling is important for cell proliferation and differentiation in various tissues, its role in intestinal differentiation is less well understood.
We used a loss of function approach to investigate the importance of Fgf signaling in intestinal cell differentiation in zebrafish; abnormal differentiation of goblet cells was observed when Fgf signaling was inhibited using SU5402 or in the Tg(hsp70ldnfgfr1-EGFP) transgenic line. We identified Fgfr2c as an important receptor for cell differentiation. The number of goblet cells and enteroendocrine cells was reduced in fgfr2c morphants. In addition to secretory cells, enterocyte differentiation was also disrupted in fgfr2c morphants. Furthermore, proliferating cells were increased in the morphants. Interestingly, the loss of fgfr2c expression repressed secretory cell differentiation and increased cell proliferation in the mibta52b mutant that had defective Notch signaling.
In conclusion, we found that Fgfr2c signaling derived from mesenchymal cells is important for regulating the differentiation of zebrafish intestine epithelial cells by promoting cell cycle exit. The results of Fgfr2c knockdown in mibta52b mutants indicated that Fgfr2c signaling is required for intestinal cell differentiation. These findings provide new evidences that Fgf signaling is required for the differentiation of intestinal cells in the zebrafish developing gut.
The efficacy of bifunctional peptide inhibitor (BPI) in preventing blood-brain barrier (BBB) breakdown during onset of experimental autoimmune encephalomyelitis (EAE) and suppression of the disease was evaluated in mice. The mechanism that defines how BPI prevents the disease was investigated by measuring the in vitro cytokine production of splenocytes. Peptides were injected 5 to 11 days prior to induction of EAE, and the severity of the disease was monitored by a standard clinical scoring protocol and change in body weight. The BBB breakdown in diseased and treated mice was compared to that in normal control mice by determining deposition of gadolinium diethylenetriaminepentaacetate (Gd-DTPA) in the brain using magnetic resonance imaging (MRI). Mice treated with PLP-BPI showed no or low indication of EAE as well as normal increase in body weight. In contrast, mice treated with the control peptide or PBS showed a decrease in body weight and a high disease score. The diseased mice had high deposition of Gd-DTPA in the brain, indicating breakdown in the BBB. However, the deposition of Gd-DTPA in PLP-BPI-treated mice was similar to that in normal control mice. Thus, PLP-BPI can suppress EAE when administered as a peptide vaccine and maintain the integrity of the BBB.
Blood Brain Barrier; Experimental Autoimmune Encephalomyelitis; Bifunctional Peptide Inhibitor; Antigen Presenting Cell; T cell; Magnetic Resonance Imaging
Only two genome-wide association studies (GWAS) have been conducted to date to identify potential markers for total mortality after diagnosis of breast cancer. Here we report the identification of two SNPs associated with total mortality from a two-stage GWAS conducted among 6,110 Shanghai-resident Chinese women with TNM stage I-IV breast cancer. The discovery stage included 1,950 patients and evaluated 613,031 common SNPs. The top 49 associations were evaluated in an independent replication stage of 4,160 Shanghai breast cancer patients. A consistent and highly significant association with total mortality was documented for SNPs rs3784099 and rs9934948. SNP rs3784099, located in the RAD51L1 gene, was associated with total morality in both the discovery stage (P=1.44×10−8) and replication stage (P=0.06; P-combined=1.17×10−7). Adjusted hazard ratios (HR) for total mortality were 1.41 (95%CI=1.18–1.68) for the AG genotype and 2.64 (95%CI=1.74–4.03) for the AA genotype, when compared with the GG genotype. The variant C allele of rs9934948, located on chromosome 16, was associated with a similarly elevated risk of total mortality (P-combined: 5.75×10−6). We also observed this association among 1,145 breast cancer patients of European-ancestry from the Nurses’ Health Study (NHS; P=0.006); the association was highly significant in a combined analysis of NHS and Chinese data (P=1.39×10−7). Similar associations were observed for these two SNPs with breast cancer-specific mortality. This study provides strong evidence suggesting that the RAD51L1 gene and a chromosome 16 locus influence breast cancer prognosis.
breast cancer; survival; genome-wide association study; Asian population; RAD51L1 gene
We conducted a population-based cross-sectional study to examine gender differences in severity, management, and outcome among patients with acute biliary pancreatitis (ABP) because available data are insufficient and conflicting.
We analyzed 13,110 patients (50.6% male) with first-attack ABP from Taiwan’s National Health Insurance Research Database between 2000 and 2009. The primary outcome was hospital mortality. Secondary outcomes included the development of severe ABP and the provision of treatment measures. Gender difference was assessed using multivariable analyses with generalized estimating equations models.
The odds of gastrointestinal bleeding (adjusted odds ratio [aOR] 1.44, 95% confidence interval [CI] 1.18–1.76) and local complication (aOR 1.38, 95% CI 1.05–1.82) were 44% and 38% higher in men than in women, respectively. Compared with women, men had 24% higher odds of receiving total parenteral nutrition (aOR 1.24, 95% CI 1.00–1.52), but had 18% and 41% lower odds of receiving cholecystectomy (aOR 0.82, 95% CI 0.72–0.93) and hemodialysis (aOR 0.59, 95% CI 0.42–0.83), respectively. Hospital mortality was higher in men than in women (1.8% vs. 1.1%, p = 0.001). After adjustment for potential confounders, men had 81% higher odds of in-hospital death than women (aOR 1.81, 95% CI 1.15–2.86). Among patients with severe ABP, hospital mortality was 11.0% and 7.5% in men and women (p<0.001), respectively. The adjusted odds of death remained higher in men than in women with severe ABP (aOR 1.72, 95% CI 1.10–2.68).
Gender is an important determinant of outcome in patients with ABP and may affect their treatment measures.
The body proximate compositions were assessed in juvenile Coreius heterodon and C. guichenoti from the upstream of the Yangtze River. The migratory C. guichenoti has a higher lipid content (FAT) than the residential C. heterodon. FAT of C. guichenoti showed an interesting pattern of increase, where FAT increased up rapidly and then leveled off as body mass (M) increased above 6.5 g, suggesting that the lipid concentration reaches an upper limit of deposition. In both species, FAT of the smaller individuals was lower than protein content (PRO), but FAT increased more rapidly than PRO as the fish grew. This indicates that more energy was allocated to protein synthesis than lipid in the smaller fish, with an energy allocation shift from protein synthesis to lipid storage as the fish grew. Strong relationships between both FAT and energy content (E) and water content (WAT) were found in both species, suggesting strong predictive power for future application. However, different models for the two species should be used to predict FAT or E by WAT.
Body size; Water content; Lipid content; Energy content; Bronze gudgeon
Uncovering factors underlying the network formation is a long-standing challenge for data mining and network analysis. In particular, the microscopic organizing principles of directed networks are less understood than those of undirected networks. This article proposes a hypothesis named potential theory, which assumes that every directed link corresponds to a decrease of a unit potential and subgraphs with definable potential values for all nodes are preferred. Combining the potential theory with the clustering and homophily mechanisms, it is deduced that the Bi-fan structure consisting of 4 nodes and 4 directed links is the most favored local structure in directed networks. Our hypothesis receives strongly positive supports from extensive experiments on 15 directed networks drawn from disparate fields, as indicated by the most accurate and robust performance of Bi-fan predictor within the link prediction framework. In summary, our main contribution is twofold: (i) We propose a new mechanism for the local organization of directed networks; (ii) We design the corresponding link prediction algorithm, which can not only testify our hypothesis, but also find out direct applications in missing link prediction and friendship recommendation.
Sorafenib, a multi-tyrosine kinase inhibitor, is a standard treatment for advanced hepatocellular carcinoma (HCC). The present study was undertaken to determine whether the growth and metastasis of HCC were influenced in mice receiving sorafenib prior to implantation with tumors, and to investigate the in-vivo and in-vitro effect of sorafenib on natural killer (NK) cells. In sorafenib-pretreated BALB/c nu/nu mice and C57BL/6 mice, tumor growth was accelerated, mouse survival was decreased, and lung metastasis was increased. However, the depletion of NK1.1+ cells in C57BL/6 mice eliminated sorafenib-mediated pro-metastatic effects. Sorafenib significantly reduced the number of NK cells and inhibited reactivity of NK cells against tumor cells, in both tumor-bearing and tumor-free C57BL/6 mice. Sorafenib down-regulated the stimulatory receptor CD69 in NK cells of tumor-bearing mice, but not in tumor-free mice, and inhibited proliferation of NK92-MI cells, which is associated with the blocking of the PI3K/AKT pathway, and inhibited cytotoxicity of NK cells in response to tumor targets, which was due to impaired ERK phosphorylation. These results suggest immunotherapeutic approaches activating NK cells may enhance the therapeutic efficacy of sorafenib in HCC patients.
Hantaviruses are among the most important zoonotic pathogens of humans and the subject of heightened global attention. Despite the importance of hantaviruses for public health, there is no consensus on their evolutionary history and especially the frequency of virus-host co-divergence versus cross-species virus transmission. Documenting the extent of hantavirus biodiversity, and particularly their range of mammalian hosts, is critical to resolving this issue. Here, we describe four novel hantaviruses (Huangpi virus, Lianghe virus, Longquan virus, and Yakeshi virus) sampled from bats and shrews in China, and which are distinct from other known hantaviruses. Huangpi virus was found in Pipistrellus abramus, Lianghe virus in Anourosorex squamipes, Longquan virus in Rhinolophus affinis, Rhinolophus sinicus, and Rhinolophus monoceros, and Yakeshi virus in Sorex isodon, respectively. A phylogenetic analysis of the available diversity of hantaviruses reveals the existence of four phylogroups that infect a range of mammalian hosts, as well as the occurrence of ancient reassortment events between the phylogroups. Notably, the phylogenetic histories of the viruses are not always congruent with those of their hosts, suggesting that cross-species transmission has played a major role during hantavirus evolution and at all taxonomic levels, although we also noted some evidence for virus-host co-divergence. Our phylogenetic analysis also suggests that hantaviruses might have first appeared in Chiroptera (bats) or Soricomorpha (moles and shrews), before emerging in rodent species. Overall, these data indicate that bats are likely to be important natural reservoir hosts of hantaviruses.
Hantaviruses are important human pathogens, occasionally emerging from animal reservoirs. However, both the biodiversity of hantaviruses in nature, as well as the frequency with which they have jumped species barriers in the past, are unclear. Here, we describe four novel hantaviruses (Huangpi virus, Lianghe virus, Longquan virus, and Yakeshi virus) that were sampled from bats and shrews in China. These viruses are different from known hantaviruses, with each representing a novel species. An evolutionary analysis of all known hantaviruses including the novel viruses described here reveals the existence of four distinct phylogenetic groups of viruses that infect a range of mammalian hosts, and which have sometimes exchanged genes through segment reassortment. Our analysis also suggests that hantaviruses might have first appeared in bats or insectivores, before spreading to rodents, even though rodents are currently the best documented hosts of hantaviruses. Because the phylogenetic trees of the hantaviruses do not always match those of their mammalian hosts, we conclude that both host-jumping and co-divergence have played important roles in hantavirus evolution. Overall, our study shows that bats are likely to be important natural reservoir hosts of hantaviruses from which novel hantaviruses may emerge in the future.
There are two independent molecules in the asymmetric unit of the title compound, C12H9N5, in which the C—N(amine)—C angles differ slightly [129.63 (11) and 132.02 (11)°]. In each independent molecule, an intramolecular C—H⋯N hydrogen bond stabilizes the molecular structure, forming an S(6) ring motif. The independent molecules are linked via an N—H⋯N hydrogen bond. Further N—H⋯N and C—H⋯N hydrogen bonds connect the molecules into chains along c axis. Pairs of C—H⋯π interactions between the chains lead to sheets parallel to the b axis. These are linked by π–π interactions between the naphthyridine and pyrazine rings [centroid–centroid separations of 3.553 (8) Å] into a three-dimensional supramolecular network.
Type D (distressed) personality, defined by negative affectivity and social inhibition, is related to cardiovascular outcomes. Little is known about Type D in non-Western cultures. We examined the validity of this construct and its assessment in Taiwanese patients with coronary artery disease (CAD) and adults from the general population.
CAD patients (N = 87) and adults from the general population (N = 421) completed the 14-item Type D Scale- Taiwanese version (DS14-T), State-Trait Anxiety Inventory, Beck Depression Inventory-II, and Chinese Hostility Inventory Short-Form.
Based on the psychometric examination, item #3 of the original DS14, "I often talk to strangers" was replaced by "I don't like to have a lot of people around me" which comes from the “Withdrawal” facet of social inhibition of DS-24. The reliability of Type D assessment in Taiwan was good, with Cronbach’s α for negative affectivity and social inhibition of .86 and .79. Factor analyses confirmed the two-factor model of the Type D construct. The prevalence rate of Type D personality in Taiwan was 20% in CAD patients and 16% in the general population. Negative affectivity was positively associated with anxiety, depression and hostility, and social inhibition was positively associated with suppressive hostility and negatively associated with expressive hostility after controlling for the total hostility. Furthermore, Taiwanese individuals with a Type D personality displayed elevated levels of anxiety, depression and hostility.
The Type D construct and its assessment with the DS14-T is generalizable to an Asian setting, Taiwan. The DS14-T showed good psychometric properties, and the prevalence of Type D personality in Taiwan was similar to the prevalence rates in Western countries and Mainland China, and Type D was associated with anxiety, depression and hostility.
Type D personality; DS14-T; Coronary artery disease; Taiwan
We used the patch-clamp technique to study the effect of changing the external Ca2+ on the basolateral 50-pS K channel in the thick ascending limb (TAL) of rat kidney. Increasing the external Ca2+ concentration from 1 mM to 2 or 3 mM inhibited the basolateral 50 −pS K channels while decreasing external Ca2+ to 10 μM increased the 50-pS K channel activity. The effect of the external Ca2+ on the 50-pS K channels was observed only in cell-attached patches but not in excised patches. Moreover, the inhibitory effect of increasing external Ca2+ on the 50-pS K channels was absent in the presence of NPS2390, an antagonist of Ca2+-sensing receptor (CaSR), suggesting that the inhibitory effect of the external Ca2+ was the result of stimulation of the CaSR. Application of the membrane-permeable cAMP analogue increased the 50-pS K channel activity but did not block the effect of raising the external Ca2+ on the K channels. Neither inhibition of phospholipase A2 (PLA2) nor suppression of cytochrome P450-ω-hydroxylation-dependent metabolism of arachidonic acid was able to abolish the effect of raising the external Ca2+ on the 50-pS K channels. In contrast, inhibition of phospholipase C (PLC) or blocking protein kinase C (PKC) completely abolished the inhibition of the basolateral 50-pS K channels induced by raising the external Ca2+. We conclude that the external Ca2+ concentration plays an important role in the regulation of the basolateral K channel activity in the TAL and that the effect of the external Ca2+ is mediated by the CaSR which stimulates PLC-PKC pathways. The regulation of the basolateral K channels by the CaSR may be the mechanism by which extracellular Ca2+ level modulates the reabsorption of divalent cations.
External Ca2+; inwardly-rectifying K channel; phospholipase C; PKC
The aim of this study is to test whether the Cyp2c44 epoxygenase dependent metabolism of arachidonic acid (AA) prevents the hypertensive effect of a high K intake (HK) by inhibiting the ENaC activity. A HK intake elevated Cyp2c44 mRNA expression and 11,12-epoxyeicosatrienoic acid (11,12-EET) levels in the cortical collecting duct (CCD) in Cyp2c44(+/+) mice(wt). However, a HK intake failed to increase 11,12-EET formation in the CCDs of Cyp2c44(-/-) mice. Moreover, increasing K intake enhanced AA-induced inhibition of ENaC in the wt but not in Cyp2c44(-/-) mice. In contrast, 11,12-EET, a Cyp2c44 metabolite, inhibited ENaC in the wt and Cyp2c44(-/-) mice. The notion that Cyp2c44 is the epoxygenase responsible for mediating the inhibitory effects of AA on ENaC is further suggested by the observation that inhibiting Cyp-epoxygenase increased the whole-cell Na currents in principal cells of wt but not in Cyp2c44(-/-) mice. Feeding mice with a HK diet raised the systemic blood pressures of Cyp2c44(-/-) mice but was without an effect on wt mice. Moreover, application of amiloride abolished the HK-induced hypertension in Cyp2c44(-/-) mice. The HK-induced hypertension of Cyp2c44(-/-) mice was accompanied by decreasing 24-hr urinary Na excretion and increasing the plasma Na concentration, the effects were absent in wt mice. In contrast, disruption of the Cyp2c44 gene did not alter K excretion. We conclude that Cyp2c44 epoxygenase mediates the inhibitory effect of AA on ENaC and that Cyp2c44 functions as a HK-inducible anti-hypertensive enzyme responsible for inhibiting ENaC activity and Na absorption in the aldosterone-sensitive distal nephron (ASDN).
11,12-EET; Arachidonic acid; ENaC; hypertension; kidney
Super-resolution three-dimensional (3D) optical microscopy has incomparable advantages over other high-resolution microscopic technologies, such as electron microscopy and atomic force microscopy, in the study of biological molecules, pathways and events in live cells and tissues. We present a novel approach of structured illumination microscopy (SIM) by using a digital micromirror device (DMD) for fringe projection and a low-coherence LED light for illumination. The lateral resolution of 90 nm and the optical sectioning depth of 120 μm were achieved. The maximum acquisition speed for 3D imaging in the optical sectioning mode was 1.6×107 pixels/second, which was mainly limited by the sensitivity and speed of the CCD camera. In contrast to other SIM techniques, the DMD-based LED-illumination SIM is cost-effective, ease of multi-wavelength switchable and speckle-noise-free. The 2D super-resolution and 3D optical sectioning modalities can be easily switched and applied to either fluorescent or non-fluorescent specimens.
Insomnia is a widespread human health problem, but there currently are the limitations of conventional therapies available. Suanzaoren decoction (SZRD) is a well known classic Chinese herbal prescription for insomnia and has been treating people’s insomnia for more than thousand years. The objective of this study was to evaluate the efficacy and safety of SZRD for insomnia.
A systematic literature search was performed for 6 databases up to July of 2012 to identify randomized control trials (RCTs) involving SZRD for insomniac patients. The methodological quality of RCTs was assessed independently using the Cochrane Handbook for Systematic Reviews of Interventions.
Twelve RCTs with total of 1376 adult participants were identified. The methodological quality of all included trials are no more than 3/8 score. Majority of the RCTs concluded that SZRD was more significantly effective than benzodiazepines for treating insomnia. Despite these positive outcomes, there were many methodological shortcomings in the studies reviewed, including insufficient information about randomization generation and absence of allocation concealment, lack of blinding and no placebo control, absence of intention-to-treat analysis and lack of follow-ups, selective publishing and reporting, and small number of sample sizes. A number of clinical heterogeneity such as diagnosis, intervention, control, and outcome measures were also reviewed. Only 3 trials reported adverse events, whereas the other 9 trials did not provide the safety information.
Despite the apparent reported positive findings, there is insufficient evidence to support efficacy of SZRD for insomnia due to the poor methodological quality and the small number of trials of the included studies. SZRD seems generally safe, but is insufficient evidence to make conclusions on the safety because fewer studies reported the adverse events. Further large sample-size and well-designed RCTs are needed.
Insomnia; Suanzaoren decoction; Systematic review
Virus-specific cellular immune responses play a critical role in virus clearance during acute or chronic HBV infection. Currently, the commercially available HBV vaccine is combined with alum adjuvant, which stimulates mainly Th2 immune responses. Therefore, development of new therapeutic HBV vaccine adjuvants and immune strategies that also promote Th1 and CTL responses is urgently needed.
To improve the immunity induced by the novel HBSS1 HBV vaccine, we evaluated the ability of adjuvants, including alum, CpG and polyriboinosinic polyribocytidylic acid [poly(I:C)], to enhance the response when boosted with the recombinant adenoviral vector vaccine rAdSS1. The immune responses to different adjuvant combinations were assessed in C57BL/6 mice by enzyme-linked immunosorbent assay (ELISA), ELISpot and cytokine release assays. Among the combinations tested, a HBV protein particle vaccine with CpG/alum and poly(I:C)/alum priming combinations accelerated specific seroconversion and produced high antibody (anti-PreS1, anti-S antibody) titres with a Th1 bias. After boosting with recombinant adenoviral vector vaccine rAdSS1, both groups produced a strong multi-antigen (S and PreS1)-specific cellular immune response. HBSS1 immunisation with poly(I:C)/alum priming also generated high-level CD4+ and CD8+ T cell responses in terms of Th1 cytokines (IFN-γand IL-2).
The protein-vaccine HBSS1 with mixed poly(I:C)/alum adjuvant priming, followed by a rAdSS1 vaccine boost, maximises specific antibody and Th1-biased cellular immune responses. This regime might prove useful in the development of HBV therapeutic vaccines. Furthermore, this promising strategy might be applied to vaccines against other persistent infections, such as human immunodeficiency virus and tuberculosis.
We had reported that N-myc downstream–regulated gene (NDRG2) regulates colorectal cancer, breast cancer, clear cell renal cell carcinoma, pancreatic cancer, thyroid cancer and esophageal squamous cell proliferation, development, and apoptosis. The goal of this study was to determine the expression pattern of NDRG2 in human lung cancer and its correlation with prognosis. Immunohistochemistry, RT-PCR and western blot were used to explore the expression of NDRG2 in 185 human lung cancer patients. The correlation of NDRG2 expression with patients’ survival rate was assessed by Kaplan–Meier and Cox regression. Results showed that the expression level of NDRG2 was decreased in human lung cancer tissues, and NDRG2 was positively correlated with depth of invasion (P = 0.038), vascular invasion (P = 0.036), tumor grade (P = 0.039), and tumor size (P = 0.026). Both RT-PCR and Western blots demonstrated that NDRG2 mRNA and protein levels were lower in lung cancer compared to the adjacent normal tissue from the same individual, and NDRG2 level was negatively correlated with UICC stage. Additionally, survival time of lung cancer patients with high expression of NDRG2 was longer than those with low expression during the 5-year follow-up period (P = 0.001). Meanwhile, COX regression analysis indicated that low expression of NDRG2, ≥pT3, pM1, ≥pN1 and vascular invasion were independent, poor prognostic factors of lung cancer patients. These data showed that NDRG2 may play an important role in human lung cancer tumourigenesis, and NDRG2 might serve as a novel prognostic marker in human lung cancer.
N-myc downstream–regulated gene 2; Lung cancer; Prognosis; Immunohistochemistry; UICC
Covalent adduction of a NO moiety to cysteines (S-nitrosylation or SNO) is a major route for NO to directly regulate protein functions. In uterine artery endothelial cells (UAEC), estradiol-17β (E2) rapidly stimulated protein SNO that maximized within 10-30 min post-E2 exposure. E2-bovine serum albumin stimulated protein SNO similarly. Stimulation of SNO by both was blocked by ICI 182, 780, implicating mechanisms linked to specific estrogen receptors (ERs) localized on the plasma membrane. E2-induced protein SNO was attenuated by selective ERβ, but not ERα, antagonists. A specific ERβ but not ERα agonist was able to induce protein SNO. Overexpression of ERβ, but not ERα, significantly enhanced E2-induced SNO. Overexpression of both ERs increased basal SNO, but did not further enhance E2-stimulated SNO. E2-induced SNO was inhibited by N-nitro-L-arginine-methylester and specific endothelial NO synthase (eNOS) siRNA. Thus, estrogen-induced SNO is mediated by endogenous NO via eNOS and mainly ERβ in UAEC. We further analyzed the nitroso-proteomes by CyDye switch technique combined with two dimensional (2D) fluorescence difference gel electrophoresis. Numerous nitrosoprotein (spots) were visible on the 2D gel. Sixty spots were chosen and subjected to matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Among the 54 identified, 9 were novel SNO-proteins, 32 were increased, 8 were decreased, and the rest were unchanged by E2. Tandom MS identified Cys139 as a specific site for SNO in GAPDH. Pathway analysis of basal and estrogen-responsive nitroso-proteomes suggested that SNO regulates diverse protein functions, directly implicating SNO as a novel mechanism for estrogen to regulate uterine endothelial function and thus uterine vasodilatation.
Estrogen; nitric oxide; S-nitrosylation; proteomics; uterine artery endothelial cells
Neuropathic pain (NP) is an intractable clinical problem without satisfactory treatments. However, certain natural products have been revealed as effective therapeutic agents for the management of pain states. In this study, we used the spinal nerve ligation (SNL) pain model to investigate the antinociceptive effect of triptolide (T10), a major active component of the traditional Chinese herb Tripterygium wilfordii Hook F. Intrathecal T10 inhibited the mechanical nociceptive response induced by SNL without interfering with motor performance. Additionally, the anti-nociceptive effect of T10 was associated with the inhibition of the activation of spinal astrocytes. Furthermore, intrathecal administration of T10 attenuated SNL-induced janus kinase (JAK) signal transducers and activators of transcription 3 (STAT3) signalling pathway activation and inhibited the upregulation of proinflammatory cytokines, such as interleukin-6, interleukin-1 beta, and tumour necrosis factor-α, in dorsal horn astrocytes. Moreover, NR2B-containing spinal N-methyl D-aspartate receptor (NMDAR) was subsequently inhibited. Above all, T10 can alleviate SNL-induced NP via inhibiting the neuroinflammation in the spinal dorsal horn. The anti-inflammation effect of T10 may be related with the suppression of spinal astrocytic JAK-STAT3 activation. Our results suggest that T10 may be a promising drug for the treatment of NP.
Pepcase is a gene encoding phosphoenolpyruvate carboxylase that exists in bacteria, archaea and plants,playing an important role in plant metabolism and development. Most plants have two or more pepcase genes belonging to two gene sub-families, while only one gene exists in other organisms. Previous research categorized one plant pepcase gene as plant-type pepcase (PTPC) while the other as bacteria-type pepcase (BTPC) because of its similarity with the pepcase gene found in bacteria. Phylogenetic reconstruction showed that PTPC is the ancestral lineage of plant pepcase, and that all bacteria, protistpepcase and BTPC in plants are derived from a lineage of pepcase closely related with PTPC in algae. However, their phylogeny contradicts the species tree and traditional chronology of organism evolution. Because the diversification of bacteria occurred much earlier than the origin of plants, presumably all bacterialpepcase derived from the ancestral PTPC of algal plants after divergingfrom the ancestor of vascular plant PTPC. To solve this contradiction, we reconstructed the phylogeny of pepcase gene family. Our result showed that both PTPC and BTPC are derived from an ancestral lineage of gamma-proteobacteriapepcases, possibly via an ancient inter-kingdom horizontal gene transfer (HGT) from bacteria to the eukaryotic common ancestor of plants, protists and cellular slime mold. Our phylogenetic analysis also found 48other pepcase genes originated from inter-kingdom HGTs. These results imply that inter-kingdom HGTs played important roles in the evolution of the pepcase gene family and furthermore that HGTsare a more frequent evolutionary event than previouslythought.
A series of salicylate-based compounds were designed and synthesized based on the simple function group replacement from our previously reported catechol-containing inhibitors of methionine aminopeptidase (MetAP). Some of these salicylate derivatives showed similar potency and metalloform selectivity, and some showed considerable antibacterial activity. These findings are consistent with our previous conclusion that Fe(II) is the likely metal used by MetAP in bacterial cells and provide new lead structures that can be further developed as novel antibacterial agents.
salicylic acid; metalloenzyme; inhibition; antibacterial; drug discovery
The neural crest is a stem cell-like population exclusive to vertebrates that gives rise to many different cell types including chondrocytes, neurons and melanocytes. Arising from the neural plate border at the intersection of Wnt and Bmp signaling pathways, the complexity of neural crest gene regulatory networks has made the earliest steps of induction difficult to elucidate. Here, we report that tfap2a and foxd3 participate in neural crest induction and are necessary and sufficient for this process to proceed. Double mutant tfap2a (mont blanc, mob) and foxd3 (mother superior, mos) mob;mos zebrafish embryos completely lack all neural crest-derived tissues. Moreover, tfap2a and foxd3 are expressed during gastrulation prior to neural crest induction in distinct, complementary, domains; tfap2a is expressed in the ventral non-neural ectoderm and foxd3 in the dorsal mesendoderm and ectoderm. We further show that Bmp signaling is expanded in mob;mos embryos while expression of dkk1, a Wnt signaling inhibitor, is increased and canonical Wnt targets are suppressed. These changes in Bmp and Wnt signaling result in specific perturbations of neural crest induction rather than general defects in neural plate border or dorso-ventral patterning. foxd3 overexpression, on the other hand, enhances the ability of tfap2a to ectopically induce neural crest around the neural plate, overriding the normal neural plate border limit of the early neural crest territory. Although loss of either tfap2 or foxd2 alters Bmp and Wnt signaling patterns, only their combined inactivation sufficiently alters these signaling gradients to abort neural crest induction. Collectively, our results indicate that tfap2a and foxd3, in addition to their respective roles in the differentiation of neural crest derivatives, also jointly maintain the balance of Bmp and Wnt signaling in order to delineate the neural crest induction domain.
Neural crest induction; Stem cells; Tfap2a; Foxd3; zebrafish
Methionine aminopeptidase (MetAP) carries out an essential function of protein N-terminal processing in many bacteria and is a promising target to develop novel antitubercular agents. Natural bengamides potently inhibit proliferation of mammalian cells by targeting MetAP enzymes, and the X-ray structure of human type 2 MetAP in complex with a bengamide derivative revealed the key interactions at the active site. By preserving the interactions with the conserved residues inside the binding pocket while exploring the differences between bacterial and human MetAPs around the binding pocket, seven bengamide derivatives were synthesized and evaluated for inhibition of MtMetAP1a and MtMetAP1c in different metalloforms, inhibition of growth of M. tuberculosis in replicating and non-replicating states, and inhibition of growth of human K562 cells. Potent inhibition of MtMetAP1a and MtMetAP1c and modest growth inhibition of M. tuberculosis were observed for some of these derivatives. X-ray structures of MtMetAP1c in complex with two of the derivatives provided the valuable structural information for improvement of these inhibitors for potency and selectivity.
antibacterial; drug discovery; enzyme inhibition; hydrolases; metalloenzymes
Cognitive decline is a debilitating feature of most neurodegenerative diseases of the central nervous system, including Alzheimer’s disease (AD)1. The causes leading to such impairment are only poorly understood and effective treatments are slow to emerge2. Here, we show that cognitive capacities in the neurodegenerating brain are constrained by an epigenetic blockade of gene transcription that is potentially reversible. This blockade is mediated by histone deacetylase (HDAC) 2, which is increased by AD-related neurotoxic insults in vitro, in two mouse models of neurodegeneration, and in AD patients. HDAC2 associates with and reduces the histone acetylation of genes important for learning and memory, which show a concomitant decrease in expression. Importantly, reversing the buildup of HDAC2 by shRNA-mediated knockdown unlocks the repression of these genes, re-instates structural and synaptic plasticity, and abolishes neurodegeneration-associated memory impairments. These findings advocate for the development of HDAC2-selective inhibitors, and suggest that cognitive capacities following neurodegeneration are not entirely lost, but merely impaired by this epigenetic blockade.
Promotion of endothelial normalization restores tumor oxygenation and obstructs tumor cells invasion, intravasation, and metastasis. We therefore investigated whether a vasoactive drug, tanshinone IIA, could inhibit metastasis by inducing vascular normalization after palliative resection (PR) of hepatocellular carcinoma (HCC).
A liver orthotopic double-tumor xenograft model in nude mouse was established by implantation of HCCLM3 (high metastatic potential) and HepG2 tumor cells. After removal of one tumor by PR, the effects of tanshinone IIA administration on metastasis, tumor vascularization, and survival were evaluated. Tube formation was examined in mouse tumor-derived endothelial cells (TECs) treated with tanshinone IIA.
PR significantly accelerated residual hepatoma metastases. Tanshinone IIA did not inhibit growth of single-xenotransplanted tumors, but it did reduce the occurrence of metastases. Moreover, it inhibited PR-enhanced metastases and, more importantly, prolonged host survival. Tanshinone IIA alleviated residual tumor hypoxia and suppressed epithelial-mesenchymal transition (EMT) in vivo; however, it did not downregulate hypoxia-inducible factor 1α (HIF-1α) or reverse EMT of tumor cells under hypoxic conditions in vitro. Tanshinone IIA directly strengthened tube formation of TECs, associated with vascular endothelial cell growth factor receptor 1/platelet derived growth factor receptor (VEGFR1/PDGFR) upregulation. Although the microvessel density (MVD) of residual tumor tissue increased after PR, the microvessel integrity (MVI) was still low. While tanshinone IIA did not inhibit MVD, it did dramatically increase MVI, leading to vascular normalization.
Our results demonstrate that tanshinone IIA can inhibit the enhanced HCC metastasis associated with PR. Inhibition results from promoting VEGFR1/PDGFR-related vascular normalization. This application demonstrates the potential clinical benefit of preventing postsurgical recurrence.
Tanshinone IIA; Vascular normalization; Palliative resection; Hepatocellular carcinoma; Metastasis