Herein we test the following hypotheses: (1) High-risk Human Papillomavirus (HR-HPV) may be involved in the etiology of mucoepidermoid carcinoma (MEC), and (2) The detection rate of HR-HPV in MEC has been increasing over time. Ninety-eight archival MEC specimens from three institutions spanning three decades were studied for HPV16/18 E6/E7 transcripts. RNA was extracted from formalin-fixed paraffin embedded specimens and HPV16/18 E6/E7 expression assessed by nested reverse transcription polymerase chain reaction (RT-PCR). A subset of MEC were also studied for MECT1-MAML2 fusion transcripts by nested RT-PCR and amplicon sequencing. The HPV expression data was validated by immunofluorescence (IF) with monoclonal HPV16/18 E6 antibody, PCR with the GP5+/6+ consensus primers, and sequencing of RT-PCR amplicons. HPV genome was localized by in-situ hybridization with the Ventana Inform HPVIII Family 16 probe. P16INK4a overexpression and aberrant p53 expression were assessed by immunohistochemistry. HPV16 E6/E7 transcripts were demonstrated in (29/98) 30 % of MEC by RT-PCR. HPV18 E6/E7 transcripts were demonstrated in 13/98 (13 %) of MEC by RT-PCR. Seven of 98 tumors (7 %) demonstrated both HPV16/18. No significant association was found between HPV status and gender, age, and tumor site. All 13 HPV18+ MEC were diagnosed between 2001 and 2010, whereas 45 MEC diagnosed from 1977 to 2000 were negative for HPV18 (p = 0.002). By contrast, there was no significant difference with respect to HPV16 detection and date of diagnosis. All MEC that were positive for E6 protein were also HPV16/18 positive by RT-PCR. Sequencing a subset of RT-PCR amplicons confirmed HPV type- and region-specific sequences. PCR using GP5+/6+ consensus primers demonstrated HPV status concordance in 9 of 10 cases. DNA degradation was present in the last case; the RT-PCR amplicons were sequenced from this case which confirmed the presence of HPV type- and region-specific sequences. Strong (+4/+4) and diffuse (>50 %) nuclear and cytoplasmic p16 expression was seen in 64 % of MEC in the glandular regions, and 18 % of MEC in the solid, squamoid regions. No correlation was seen between p16 expression and HPV status. Twenty-nine MEC (22 HPV+ and 7 HPV-negative) were selected for further evaluation for p53 expression. Strong aberrant nuclear p53 expression was present in only 2/22 HPV + MEC (9 %, both Grade 3); no HPV-negative MEC demonstrated aberrant p53 expression. MECT1-MAML2 fusion transcripts were demonstrated in 23/37 (62 %) MEC. No significant association was found between the presence of the MECT1-MAML2 fusion transcripts and tumor grade, HPV status, gender, era of diagnosis (2000 and earlier vs. 2001–2010) or tumor site. We demonstrate for the first time that transcriptionally active HPV16/18 is common to MEC. These findings were validated by demonstrating concordant results by separate PCR with consensus primers, and/or confirming the presence of HPV type- and region-specific sequences in the RT-PCR amplicons. We also visualized E6 viral oncoprotein and HPV genome within tumor cells. HR-HPV is thus potentially implicated in the pathogenesis of MEC. The frequency of HPV18 detection is significantly increased in MEC diagnosed after 2001, whereas we found no differences in the HPV16 detection rates per era of diagnosis.
Mucoepidermoid carcinoma; Human Papillomavirus; HPV16/18; E6
To evaluate the safety and efficacy of robotic gastrectomy versus open gastrectomy for gastric cancer.
A comprehensive search of PubMed, EMBASE, Cochrane Library, and Web of Knowledge was performed. Systematic review was carried out to identify studies comparing robotic gastrectomy and open gastrectomy in gastric cancer. Intraoperative and postoperative outcomes were also analyzed to evaluate the safety and efficacy of the surgery. A fixed effects model or a random effects model was utilized according to the heterogeneity.
Four studies involving 5780 patients with 520 (9.00%) cases of robotic gastrectomy and 5260 (91.00%) cases of open gastrectomy were included in this meta-analysis. Compared to open gastrectomy, robotic gastrectomy has a significantly longer operation time (weighted mean differences (WMD) =92.37, 95% confidence interval (CI): 55.63 to 129.12, P<0.00001), lower blood loss (WMD: -126.08, 95% CI: -189.02 to -63.13, P<0.0001), and shorter hospital stay (WMD = -2.87; 95% CI: -4.17 to -1.56; P<0.0001). No statistical difference was noted based on the rate of overall postoperative complication, wound infection, bleeding, number of harvested lymph nodes, anastomotic leakage and postoperative mortality rate.
The results of this meta-analysis suggest that robotic gastrectomy is a better alternative technique to open gastrectomy for gastric cancer. However, more prospective, well-designed, multicenter, randomized controlled trials are necessary to further evaluate the safety and efficacy as well as the long-term outcome.
Higher-order structure of chromatin is essential for chromosome segregation and repetitive DNA stability. Monopolin recruits condensin to organize centromere DNA irrespective of the number of kinetochore–microtubule attachments. In addition, the role of monopolin in stabilizing repeat tracts observed in budding yeast is conserved in Candida albicans.
The establishment and maintenance of higher-order structure at centromeres is essential for accurate chromosome segregation. The monopolin complex is thought to cross-link multiple kinetochore complexes to prevent merotelic attachments that result in chromosome missegregation. This model is based on structural analysis and the requirement that monopolin execute mitotic and meiotic chromosome segregation in Schizosaccharomyces pombe, which has more than one kinetochore–microtubule attachment/centromere, and co-orient sister chromatids in meiosis I in Saccharomyces cerevisiae. Recent data from S. pombe suggest an alternative possibility: that the recruitment of condensin is the primary function of monopolin. Here we test these models using the yeast Candida albicans. C. albicans cells lacking monopolin exhibit defects in chromosome segregation, increased distance between centromeres, and decreased stability of several types of repeat DNA. Of note, changing kinetochore–microtubule copy number from one to more than one kinetochore–microtubule/centromere does not alter the requirement for monopolin. Furthermore, monopolin recruits condensin to C. albicans centromeres, and overexpression of condensin suppresses chromosome segregation defects in strains lacking monopolin. We propose that the key function of monopolin is to recruit condensin in order to promote the assembly of higher-order structure at centromere and repetitive DNA.
Changes in cell homeostasis, or cell ‘stress’, are thought to tax the ability of the Hsp90 chaperone to facilitate an array of processes critical for genome maintenance. Here, we review the current understanding of how Hsp90 chaperone machinery ensures the function of proteins important for DNA repair, recombination and chromosome segregation. We discuss the idea that cell ‘stress’ can overload Hsp90, resulting in genomic instability that may have important implications for stress adaptation and selection. The importance of Hsp90 in genome maintenance and its limited capacity to buffer the proteome may underlie the initiation or progression of diseases such as cancer.
Hsp90; chaperone; cell stress; genome instability; adaptation
The normal intrauterine fluid environment is essential for embryo implantation. In hydrosalpinx patients, the implantation and pregnancy rates are markedly decreased after IVF–embryo transfer, while salpingectomy could significantly improve the pregnancy rates. The leakage of hydrosalpinx fluid into the endometrial cavity was supposed to be the major cause for impaired fertility. However, the underlying mechanisms of hydrosalpinx fluids on implantation and ongoing pregnancy were not fully understood and remain controversial regarding its toxicity. In present study, by infusing different volume of non-toxic fluid (0.9% saline) into uterine lumen before embryo implantation in mice (Day4 08:30), we found that while the embryos were not “flushed out” from the uteri, the timing of implantation was deferred and normal intrauterine distribution (embryo spacing) was disrupted. The abnormal implantation at early pregnancy further lead to embryo growth retardation, miscarriage and increased pregnancy loss, which is similar to the adverse effects observed in hydrosalpinx patients undergoing IVF-ET. We further examined uterine receptivity related gene expression reported to be involved in human hydrosalpinx (Lif, Hoxa10, Integrin α(v) and β(3)). The results showed that expression of integrin α(v) and β(3) were increased in the fluid infused mouse uteri, implicating a compensatory effect to cope with the excessive fluid environment. Our data suggested that the adverse effects of excessive non-toxic luminal fluid on pregnancy are primarily due to the mechanical interference for normal timing and location of embryo apposition, which might be the major cause of decreased implantation rate in IVF-ET patients with hydrosalpinx.
The ginseng family (Araliaceae) includes a number of economically important plant species. Previously phylogenetic studies circumscribed three major clades within the core ginseng plant family, yet the internal relationships of each major group have been poorly resolved perhaps due to rapid radiation of these lineages. Recent studies have shown that phyogenomics based on chloroplast genomes provides a viable way to resolve complex relationships.
We report the complete nucleotide sequences of five Araliaceae chloroplast genomes using next-generation sequencing technology. The five chloroplast genomes are 156,333–156,459 bp in length including a pair of inverted repeats (25,551–26,108 bp) separated by the large single-copy (86,028–86,566 bp) and small single-copy (18,021–19,117 bp) regions. Each chloroplast genome contains the same 114 unique genes consisting of 30 transfer RNA genes, four ribosomal RNA genes, and 80 protein coding genes. Gene size, content, and order, AT content, and IR/SC boundary structure are similar among all Araliaceae chloroplast genomes. A total of 140 repeats were identified in the five chloroplast genomes with palindromic repeat as the most common type. Phylogenomic analyses using parsimony, likelihood, and Bayesian inference based on the complete chloroplast genomes strongly supported the monophyly of the Asian Palmate group and the Aralia-Panax group. Furthermore, the relationships among the sampled taxa within the Asian Palmate group were well resolved. Twenty-six DNA markers with the percentage of variable sites higher than 5% were identified, which may be useful for phylogenetic studies of Araliaceae.
The chloroplast genomes of Araliaceae are highly conserved in all aspects of genome features. The large-scale phylogenomic data based on the complete chloroplast DNA sequences is shown to be effective for the phylogenetic reconstruction of Araliaceae.
Objective: To investigate the correlation between CD133-positive non-small cell lung cancer (NSCLC) and clinicopathological features and its impact on survival. Methods: A search in the Pubmed, Embase and Wanfang databases (up to July 15, 2013) was performed. Only articles in which CD133 antigen was detected in situ localization by immunohistochemical staining were included. This meta-analysis was done using RevMan 5.2 software. Outcomes included overall survival and various clinicopathological features. Results: A total of 1004 NSCLC patients from 11 studies were included. Meta-analysis showed that CD133 expression patients had a significant worse 5-year overall survival compared to the low expression ones (RR = 3.19, 95% CI: 2.05-4.98, P<0.0001 fixed random). With respect to clinicopathological features, CD133 expression by IHC method was closely correlated with tumor T stage (OR = 0.91, 95% CI: 0.59-1.39, P = 0.67 fixed-effect) and tumor grade (OR = 1.20, 95% CI: 0.80-1.79, P = 0.37 fixed-effect). Conclusion: CD133-positive NSCLC patients had worse prognosis, and was associated with common clinicopathological poor prognostic factors.
Non-small cell lung cancer; cancer stem cells; CD133; prognosis
Tau protein is implicated in the pathogenesis of neurodegenerative disorders such as tauopathies including Alzheimer disease, and Tau fibrillization is thought to be related to neuronal toxicity. Physiological inhibitors of Tau fibrillization hold promise for developing new strategies for treatment of Alzheimer disease. Because protein disulfide isomerase (PDI) is both an enzyme and a chaperone, and implicated in neuroprotection against Alzheimer disease, we want to know whether PDI can prevent Tau fibrillization. In this study, we have investigated the interaction between PDI and Tau protein and the effect of PDI on Tau fibrillization.
As evidenced by co-immunoprecipitation and confocal laser scanning microscopy, human PDI interacts and co-locates with some endogenous human Tau on the endoplasmic reticulum of undifferentiated SH-SY5Y neuroblastoma cells. The results from isothermal titration calorimetry show that one full-length human PDI binds to one full-length human Tau (or human Tau fragment Tau244–372) monomer with moderate, micromolar affinity at physiological pH and near physiological ionic strength. As revealed by thioflavin T binding assays, Sarkosyl-insoluble SDS-PAGE, and transmission electron microscopy, full-length human PDI remarkably inhibits both steps of nucleation and elongation of Tau244–372 fibrillization in a concentration-dependent manner. Furthermore, we find that two molecules of the a-domain of human PDI interact with one Tau244–372 molecule with sub-micromolar affinity, and inhibit both steps of nucleation and elongation of Tau244–372 fibrillization more strongly than full-length human PDI.
We demonstrate for the first time that human PDI binds to Tau protein mainly through its thioredoxin-like catalytic domain a, forming a 1∶1 complex and preventing Tau misfolding. Our findings suggest that PDI could act as a physiological inhibitor of Tau fibrillization, and have applications for developing novel strategies for treatment and early diagnosis of Alzheimer disease.
Affinity chromatography is a separation technique that has become increasingly important in work with biological samples and pharmaceutical agents. This method is based on the use of a biologically-related agent as a stationary phase to selectively retain analytes or to study biological interactions. This review discusses the basic principles behind affinity chromatography and examines recent developments that have occurred in the use of this method for biomedical and pharmaceutical analysis. Techniques based on traditional affinity supports are discussed, but an emphasis is placed on methods in which affinity columns are used as part of HPLC systems or in combination with other analytical methods. General formats for affinity chromatography that are considered include step elution schemes, weak affinity chromatography, affinity extraction and affinity depletion. Specific separation techniques that are examined include lectin affinity chromatography, boronate affinity chromatography, immunoaffinity chromatography, and immobilized metal ion affinity chromatography. Approaches for the study of biological interactions by affinity chromatography are also presented, such as the measurement of equilibrium constants, rate constants, or competition and displacement effects. In addition, related developments in the use of immobilized enzyme reactors, molecularly imprinted polymers, dye ligands and aptamers are briefly considered.
Affinity chromatography; Lectin affinity chromatography; Boronate affinity chromatography; Immunoaffinity chromatography; Immobilized metal ion affinity chromatography
Although Asian Indian (people of Indian subcontinent descent) men are shown to have higher total and truncal body fat as well as greater insulin resistance compared to white men matched for total body fat and age, data in women are not conclusive. The objective of this study was to compare total and regional fat distribution and insulin sensitivity between healthy young premenopausal Asian Indian and white women of similar body mass index (BMI). Twenty Asian Indian women (65% immigrants and 35% first generation living in Dallas) and 31 white women of similar age and BMI [age 24±3 vs. 25±4; BMI 22±4 vs. 23±5; mean±standard deviation (SD) in Asian Indian and white, respectively] without diabetes were evaluated with anthropometric measurements, underwater weighing for percentage of total body fat mass, magnetic resonance imaging of whole abdomen for measurement of abdominal subcutaneous and intraperitoneal fat mass, and euglycemic–hyperinsulinemic clamp study for measurement of insulin sensitivity. There were no differences in waist or hip circumference, total body subcutaneous abdominal or intraperitoneal fat mass, fasting plasma glucose, and insulin levels between Asian Indian women and white women. The peripheral glucose disposal rate (Rd) during hyperinsulinemic–euglycemic clamp was found to be almost identical in the two study groups (median value of 6.9 and 6.8 mg/min per kg of body weight, for Asian Indians and whites, respectively). For similar total or regional fat content, the glucose disposal rate was comparable in the two study groups. In conclusion, we demonstrate that young Asian Indian women do not have excess abdominal or intraperitoneal fat or insulin resistance for similar BMI compared to white women of European descent.
Despite its wide existence, the adaptive role of aneuploidy (the abnormal state of having unequal number of different chromosomes) has been a subject of debate. Cellular aneuploidy has been associated with enhanced resistance to stress, whereas on the organismal level it is detrimental to multi-cellular species. Certain aneuploid karyotypes are deleterious for specific environments, but karyotype diversity in a population potentiates adaptive evolution. To reconcile these paradoxical observations, this review distinguishes the role of aneuploidy in cellular versus organismal evolution. Further, it proposes a population genetics perspective to examine the behavior of aneuploidy on a populational versus individual level. By altering the copy number of a significant portion of the genome, aneuploidy introduces large phenotypic leap that enables small cell populations to explore a wide phenotypic landscape, from which adaptive traits can be selected. The production of chromosome number variation can be further increased by stress- or mutation-induced chromosomal instability, fueling rapid cellular adaptation.
Gene-modified cell vaccines are the best way to achieve the immunotherapy for all types of acute leukemia. In this study, the recombinant eukaryotic expression vector (pDisplay-HSP70) of heat shock protein 70 (HSP70) of Bacille Calmette-Guérin (BCG) was constructed by amplifying the whole BCG HSP70 gene using polymerase chain reaction (PCR) and sub-cloning into the polyclone endonuclease sites in pDisplay. Then the HL-60 cell vaccine expressing the protein onto the cell surface was prepared by lipofectamine transfection and its anti-tumor effect and mechanism were further studied. Results showed that the fragment of BCG HSP70 was consistent with Mycobacterium tuberculosis HSP70 gene published in GeneBank. DNA sequencing showed that the recombinant vector was correctly constructed and named pDisplay-HSP70. After BCG HSP70 gene transfection, the yellow-green fluorescence on the HL-60 cells surface was observed under a fluorescence microscope. The immunogenicity of HSP70-transfected HL-60 cells exhibited upregulated proliferation of lymphocytes, increased cytokine secretion (IFN-γ) and enhanced killing activity. These results suggested that gene transfection of BCG HSP70 could significantly enhance the immunogenicity of HL-60 cells. It may be used as a suitable candidate gene-modified cell vaccine for cancer immunotherapy.
BCG; Heat shock protein 70; gene transfection; HL-60; cancer vaccine
Achyranthes bidentata, a Chinese medicinal herb, is reported to be neuroprotective. However, its role in cardioprotection remains largely unknown. Our present study aimed to investigate the effects of Achyranthes bidentata polypeptides (ABPP) preconditioning on myocardial ischemia/reperfusion (MI/R) injury and to test the possible mechanisms. Rats were treated with ABPP (10 mg/kg/d, i.p.) or saline once daily for one week. Afterward, all the animals were subjected to 30 min of myocardial ischemia followed by 4 h of reperfusion. ABPP preconditioning for one week significantly improved cardiac function following MI/R. Meanwhile, ABPP reduced infarct size, plasma creatine kinase (CK)/lactate dehydrogenase (LDH) activities and myocardial apoptosis at the end of reperfusion in rat hearts. Moreover, ABPP preconditioning significantly inhibited superoxide generation, gp91phox expression, malonaldialdehyde formation and enhanced superoxide dismutase activity in I/R hearts. Furthermore, ABPP treatment inhibited PTEN expression and increased Akt phosphorylation in I/R rat heart. PI3K inhibitor wortmannin blocked Akt activation, and abolished ABPP-stimulated anti-oxidant effect and cardioprotection. Our study demonstrated for the first time that ABPP reduces oxidative stress and exerts cardioprotection against MI/R injury in rats. Inhibition of PTEN and activation of Akt may contribute to the anti-oxidant capacity and cardioprotection of ABPP.
Achyranthes bidentata polypeptides; oxidative stress; myocardial ischemia/reperfusion; apoptosis
Tumor microenviroment is characteristic of inflammation, ischemia and starvation of nutrient. TNF-α, which is an extraordinarily pleiotropic cytokine, could be an endogenous tumor promoter in some tumor types. The basic objective of this study was to investigate the effects of TNF-α on the cell viability and apoptosis of hepatocellular carcinoma cells under serum starvation, and to identify the molecular mechanisms involved.
For this purpose, five different concentrations of TNF-α and two different serum settings (serum-cultured and serum-deprived) were used to investigate the effects of TNF-α on the cell viability and apoptosis of Hep3B and SMMC-7721 cells.
TNF-α (10 ng/ml) attenuated serum starvation-induced apoptosis of hepatocellular carcinoma cells, and autophagy conferred this process. BAY11-7082, a specific inhibitor of NF-κB, reversed the suppression of serum starvation-induced apoptosis by TNF-α. Moreover, TNF-α-induced NF-κB transactivation was suppressed by autophagy inhibitor 3-MA. In addition, TNF-α up-regulated Ferritin heavy chain (FHC) transiently by NF-κB activation and FHC levels were correlated with the TNF-α-induced protection against serum starvation-mediated apoptosis of hepatocellular carcinoma cells. Furthermore, FHC-mediated inhibition of apoptosis depended on suppressing ROS accumulation.
Our findings suggested that autophagy conferred the TNF-α protection against serum starvation-mediated apoptosis of hepatocellular carcinoma cells, the mechanism involved with the activation of the TNF-α/ NF-κB /FHC signaling pathway.
TNF-α; Starvation; NF-κB; Ferritin heavy chain; Autophagy; Hepatocellular carcinoma
Ribosomal S6 Kinase 2 (RSK2) is a member of the p90RSK family of serine/threonine kinases, which are widely expressed and respond to many growth factors, peptide hormones, and neurotransmitters. Loss-of function mutations in the RPS6KA3 gene, which encodes the RSK2 protein, have been implicated in Coffin-Lowry Syndrome (CLS), an X-linked mental retardation disorder associated with cognitive deficits and behavioral impairments. However, the cellular and molecular mechanisms underlying this neurological disorder are not known. Recent evidence suggests that defective DNA damage signaling might be associated with neurological disorders, but the role of RSK2 in the DNA damage pathway remains to be elucidated. Here, we show that Adriamycin-induced DNA damage leads to the phosphorylation of RSK2 at Ser227 and Thr577 in the chromatin fraction, promotes RSK2 nuclear translocation, and enhances RSK2 and Atm interactions in the nuclear fraction. Furthermore, using RSK2 knockout mouse fibroblasts and RSK2-deficient cells from CLS patients, we demonstrate that ablation of RSK2 impairs the phosphorylation of Atm at Ser1981 and the phosphorylation of p53 at Ser18 (mouse) or Ser15 (human) in response to genotoxic stress. We also show that RSK2 affects p53-mediated downstream cellular events in response to DNA damage, that RSK2 knockout relieves cell cycle arrest at the G2/M phase, and that an increased number of γH2AX foci, which are associated with defects in DNA repair, are present in RSK2-deficient cells. Taken together, our findings demonstrated that RSK2 plays an important role in the DNA damage pathway that maintains genomic stability by mediating cell cycle progression and DNA repair.
Peach (Prunus persica (L.) Batsch) is one of the most important model fruits in the Rosaceae family. Native to the west of China, where peach has been domesticated for more than 4,000 years, its cultivation spread from China to Persia, Mediterranean countries and to America. Chinese peach has had a major impact on international peach breeding programs due to its high genetic diversity. In this research, we used 48 highly polymorphic SSRs, distributed over the peach genome, to investigate the difference in genetic diversity, and linkage disequilibrium (LD) among Chinese cultivars, and North American and European cultivars, and the evolution of current peach cultivars.
In total, 588 alleles were obtained with 48 SSRs on 653 peach accessions, giving an average of 12.25 alleles per locus. In general, the average value of observed heterozygosity (0.47) was lower than the expected heterozygosity (0.60). The separate analysis of groups of accessions according to their origin or reproductive strategies showed greater variability in Oriental cultivars, mainly due to the high level of heterozygosity in Chinese landraces. Genetic distance analysis clustered the cultivars into two main groups: one included four wild related Prunus, and the other included most of the Oriental and Occidental landraces and breeding cultivars. STRUCTURE analysis assigned 469 accessions to three subpopulations: Oriental (234), Occidental (174), and Landraces (61). Nested STRUCTURE analysis divided the Oriental subpopulation into two different subpopulations: ‘Yu Lu’ and ‘Hakuho’. The Occidental breeding subpopulation was also subdivided into nectarine and peach subpopulations. Linkage disequilibrium (LD) analysis in each of these subpopulations showed that the percentage of linked (r2 > 0.1) intra-chromosome comparisons ranged between 14% and 47%. LD decayed faster in Oriental (1,196 Kbp) than in Occidental (2,687 Kbp) samples. In the ‘Yu Lu’ subpopulation there was considerable LD extension while no variation of LD with physical distance was observed in the landraces. From the first STRUCTURE result, LG1 had the greatest proportion of alleles in LD within all three subpopulations.
Our study demonstrates a high level of genetic diversity and relatively fast decay of LD in the Oriental peach breeding program. Inclusion of Chinese landraces will have a greater effect on increasing genetic diversity in Occidental breeding programs. Fingerprinting with genotype data for all 658 cultivars will be used for accession management in different germplasms. A higher density of markers are needed for association mapping in Oriental germplasm due to the low extension of LD. Population structure and evaluation of LD provides valuable information for GWAS experiment design in peach.
Failing cardiomyocytes exhibit decreased efficiency of excitation-contraction (E-C) coupling. The down-regulation of junctophilin-2 (JP2), a protein anchoring the sarcoplasmic reticulum (SR) to T-tubules (TTs), has been identified as a major mechanism underlying the defective E-C coupling. However, the regulatory mechanism of JP2 remains unknown.
To determine whether microRNAs regulate JP2 expression.
Methods and Results
Bioinformatic analysis predicted two potential binding sites of miR-24 in the 3′-untranslated regions of JP2 mRNA. Luciferase assays confirmed that miR-24 suppressed JP2 expression by binding to either of these sites. In the aortic stenosis model, miR-24 was up-regulated in failing cardiomyocytes. Adenovirus-directed over-expression of miR-24 in cardiomyocytes decreased JP2 expression and reduced Ca2+ transient amplitude and E-C coupling gain.
MiR-24-mediated suppression of JP2 expression provides a novel molecular mechanism for E-C coupling regulation in heart cells, and suggests a new target against heart failure.
myocardial contractility; excitation-contraction coupling; heart failure; calcium signaling; heart failure
Meiosis I chromosome migration is biphasic, with an early, slow phase requiring Fmn2 and a later, fast and highly directed phase requiring the Arp2/3 complex.
Polar body extrusion during oocyte maturation is critically dependent on asymmetric positioning of the meiotic spindle, which is established through migration of the meiosis I (MI) spindle/chromosomes from the oocyte interior to a subcortical location. In this study, we show that MI chromosome migration is biphasic and driven by consecutive actin-based pushing forces regulated by two actin nucleators, Fmn2, a formin family protein, and the Arp2/3 complex. Fmn2 was recruited to endoplasmic reticulum structures surrounding the MI spindle, where it nucleated actin filaments to initiate an initially slow and poorly directed motion of the spindle away from the cell center. A fast and highly directed second migration phase was driven by actin-mediated cytoplasmic streaming and occurred as the chromosomes reach a sufficient proximity to the cortex to activate the Arp2/3 complex. We propose that decisive symmetry breaking in mouse oocytes results from Fmn2-mediated perturbation of spindle position and the positive feedback loop between chromosome signal-induced Arp2/3 activation and Arp2/3-orchestrated cytoplasmic streaming that transports the chromosomes.
As the main source of extracellular matrix proteins in tumor stroma, hepatic stellate cells (HSCs) have a great impact on biological behaviors of hepatocellular carcinoma (HCC). In the present study, we have investigated a mechanism whereby HSCs modulate the chemoresistance of hepatoma cells. We used human HSC line lx-2 and chemotherapeutic agent cisplatin to investigate their effects on human HCC cell line Hep3B. The results showed that cisplatin resistance in Hep3B cells was enhanced with LX-2 CM (cultured medium) exposure in vitro as well as co-injection with LX-2 cells in null mice. Meanwhile, in presence of LX-2 CM, Hep3B cells underwent epithelial to mesenchymal transition (EMT) and upregulation of cancer stem cell (CSC) -like properties. Besides, LX-2 cells synthesized and secreted hepatic growth factor (HGF) into the CM. HGF receptor tyrosine kinase mesenchymal–epithelial transition factor (Met) was activated in Hep3B cells after LX-2 CM exposure. The HGF level of LX-2 CM could be effectively reduced by using HGF neutralizing antibody. Furthermore, depletion of HGF in LX-2 CM abolished its effects on activation of Met as well as promotion of the EMT, CSC-like features and cisplatin resistance in Hep3B cells. Collectively, secreting HGF into tumor milieu, HSCs may decrease hepatoma cells sensitization to chemotherapeutic agents by promoting EMT and CSC-like features via HGF/Met signaling.
The contraction of a heart cell is controlled by Ca2+-induced Ca2+ release between L-type Ca2+ channels (LCCs) in the cell membrane/T-tubules (TTs) and ryanodine receptors (RyRs) in the junctional sarcoplasmic reticulum (SR). During heart failure, LCC–RyR signalling becomes defective. The purpose of the present study was to reveal the ultrastructural mechanism underlying the defective LCC–RyR signalling and contractility.
Methods and results
In rat models of heart failure produced by transverse aortic constriction surgery, stereological analysis of transmission electron microscopic images showed that the volume density and the surface area of junctional SRs and those of SR-coupled TTs were both decreased in failing heart cells. The TT–SR junctions were displaced or missing from the Z-line areas. Moreover, the spatial span of individual TT–SR junctions was markedly reduced in failing heart cells. Numerical simulation and junctophilin-2 knockdown experiments demonstrated that the decrease in junction size (and thereby the constitutive LCC and RyR numbers) led to a scattered delay of Ca2+ release activation.
The shrinking and eventual absence of TT–SR junctions are important mechanisms underlying the desynchronized and inhomogeneous Ca2+ release and the decreased contractile strength in heart failure. Maintaining the nanoscopic integrity of TT–SR junctions thus represents a therapeutic strategy against heart failure and related cardiomyopathies.
Heart failure; Ultrastructure; Calcium channel; Excitation–contraction coupling
Transient ischemic attack (TIA) is usually defined as a neurologic ischemic disorder without permanent cerebral infarction. Studies have showed that patients with TIA can have lasting cognitive functional impairment. Inherent brain activity in the resting state is spatially organized in a set of specific coherent patterns named resting state networks (RSNs), which epitomize the functional architecture of memory, language, attention, visual, auditory and somato-motor networks. Here, we aimed to detect differences in RSNs between TIA patients and healthy controls (HCs).
Twenty one TIA patients suffered an ischemic event and 21 matched HCs were enrolled in the study. All subjects were investigated using cognitive tests, psychiatric tests and functional magnetic resonance imaging (fMRI). Independent component analysis (ICA) was adopted to acquire the eight brain RSNs. Then one-sample t-tests were calculated in each group to gather the spatial maps of each RSNs, followed by second level analysis to investigate statistical differences on RSNs between twenty one TIA patients and 21 controls. Furthermore, a correlation analysis was performed to explore the relationship between functional connectivity (FC) and cognitive and psychiatric scales in TIA group.
Compared with the controls, TIA patients exhibited both decreased and increased functional connectivity in default mode network (DMN) and self-referential network (SRN), and decreased functional connectivity in dorsal attention network (DAN), central-executive network (CEN), core network (CN), somato-motor network (SMN), visual network (VN) and auditory network (AN). There was no correlation between neuropsychological scores and functional connectivity in regions of RSNs.
We observed selective impairments of RSN intrinsic FC in TIA patients, whose all eight RSNs had aberrant functional connectivity. These changes indicate that TIA is a disease with widely abnormal brain networks. Our results might put forward a novel way to look into neuro-pathophysiological mechanisms in TIA patients.
Spontaneous symmetry breaking leading to polarization of the cell is a key step initiating many morphogenetic processes. In addition to experimental studies model-based theoretical description helps to understand the conditions and limitations of this process. Such description is limited usually to linear stability analysis supplied by the numerical simulations to establish the dependence of the polarization dynamics on the model parameters. Here we describe application of a powerful weakly nonlinear analysis method to a minimalistic model characterized by the conservation of mass of the protein governing the polarization dynamics.
To compare the long-term immunogenicity and seroprotection rates in healthy children following 23 years of vaccination with 10 μg or 20 μg doses of plasma-derived hepatitis B vaccine, we revisited all participants from our previous randomized controlled trial. At year 23, 81 participants were tested for HBV serological markers and HBV-DNA, and a booster dose was given to those with anti-HBs titer < 10 mIU/mL. After eliminating the interference of a Year 11 booster dose and vaccines received outside of the trial, around 50% of participants still maintained anti-HBs titers ≥ 10 mIU/mL in both 10 μg and 20 μg groups (p > 0.05). The peak immune response of vaccination (anti-HBs antibody levels at 12 mo after 1st vaccine dose) and Year 11 anti-HBs levels were significantly associated with Year 23 seroprotection rates. Most of the participants in both groups, regardless of their prior immune status, developed a rapid and robust anamnestic antibody response after the booster dose at year 23. No case of clinically significant HBV infection was observed during the entire study period of 23 y with only one transient HBsAg seroconversion in 10 μg vaccine group. We concluded that seroprotection provided by 10μg or 20 μg doses of hepatitis B vaccine persists for 23 years in more than half of vaccinated individuals in highly HBV-endemic areas, irrespective of 10 μg or 20 μg vaccine doses. Future studies with larger sample sizes comparing long-term efficacy of various doses of plasma-derived and recombinant HBV vaccines are recommended.
Anamnestic response; HBV; Hepatitis B; PDV; Vaccine; anti-HBs; clinical trial; immune response; long-term; plasma-derived vaccine; vaccine intervention study
Pineapples, or self-organized, Taxol-stabilized microtubule assemblies, reveal the richness of self-organizing mechanisms that operate on assembled microtubules during cell division and provide a biochemically tractable system for investigating these mechanisms during meiosis and cytokinesis.
Previous study of self-organization of Taxol-stabilized microtubules into asters in Xenopus meiotic extracts revealed motor-dependent organizational mechanisms in the spindle. We revisit this approach using clarified cytosol with glycogen added back to supply energy and reducing equivalents. We added probes for NUMA and Aurora B to reveal microtubule polarity. Taxol and dimethyl sulfoxide promote rapid polymerization of microtubules that slowly self-organize into assemblies with a characteristic morphology consisting of paired lines or open circles of parallel bundles. Minus ends align in NUMA-containing foci on the outside, and plus ends in Aurora B–containing foci on the inside. Assemblies have a well-defined width that depends on initial assembly conditions, but microtubules within them have a broad length distribution. Electron microscopy shows that plus-end foci are coated with electron-dense material and resemble similar foci in monopolar midzones in cells. Functional tests show that two key spindle assembly factors, dynein and kinesin-5, act during assembly as they do in spindles, whereas two key midzone assembly factors, Aurora B and Kif4, act as they do in midzones. These data reveal the richness of self-organizing mechanisms that operate on microtubules after they polymerize in meiotic cytoplasm and provide a biochemically tractable system for investigating plus-end organization in midzones.
Some syphilis patients remain in a serologically active state after the recommended therapy. We currently know too little about the characteristics of this serological response.
We conducted a cohort study using the clinical database from Zhongshan Hospital, Medical College of Xiamen. In total, 1,327 HIV-negative patients with primary, secondary, latent, and tertiary syphilis were enrolled. Bivariate and multivariate analyses were utilised to identify factors associated with a serological cure and serofast state in syphilis patients one year after therapy. Chi-square tests were used to determine the differences in the serological cure rate across different therapy time points.
One year after the recommended therapy, 870 patients achieved a serological cure, and 457 patients (34.4%) remained in the serofast state. The serological cure rate increased only within the first 6 months. The bivariate analysis indicated that male or younger patients had a higher likelihood of a serological cure than female or older patients. Having a baseline titre ≤1∶2 or ≥1∶64 was associated with an increased likelihood of a serological cure. The serological cure rate decreased for the different disease stages in the order of primary, secondary, latent, and tertiary syphilis. A distinction should be drawn between early and late syphilis. The multivariate analysis indicated that a serological cure was significantly associated with the disease phase, gender, age, and baseline rapid plasma reagin (RPR) titre.
The serofast state is common in clinical work. After one year of the recommended therapy, quite a few syphilis patients remained RPR positive. The primary endpoint of the study indicated that disease phase, gender, age and baseline RPR titre were crucial factors associated with a serological cure.