AIM: To investigate the antiproliferative activity of cinobufacini on human hepatocellular carcinoma HepG2 cells and the possible mechanism of its action.
METHODS: HepG2 cells were treated with different concentrations of cinobufacini. Cell viability was measured by methylthiazolyl tetrazolium (MTT) assay. Cell cycle distribution was analyzed by flow cytometry (FCM). Cytoskeletal and nuclear alterations were observed by fluorescein isothiocyanate-phalloidin and DAPI staining under a laser scanning confocal microscope. Changes in morphology and ultrastructure of cells were detected by atomic force microscopy (AFM) at the nanoscale level.
RESULTS: MTT assay indicated that cinobufacini significantly inhibited the viability of HepG2 cells in a dose-dependent manner. With the concentration of cinobufacini increasing from 0 to 0.10 mg/mL, the cell viability decreased from 74.9% ± 2.7% to 49.41% ± 2.2% and 39.24% ± 2.1% (P < 0.05). FCM analysis demonstrated cell cycle arrest at S phase induced by cinobufacini. The immunofluorescence studies of cytoskeletal and nuclear morphology showed that after cinobufacini treatment, the regular reorganization of actin filaments in HepG2 cells become chaotic, while the nuclei were not damaged seriously. Additionally, high-resolution AFM imaging revealed that cell morphology and ultrastructure changed a lot after treatment with cinobufacini. It appeared as significant shrinkage and deep pores in the cell membrane, with larger particles and a rougher cell surface.
CONCLUSION: Cinobufacini inhibits the viability of HepG2 cells via cytoskeletal destruction and cell membrane toxicity.
Cinobufacini; Cell viability; Atomic force microscopy; HepG2 cells; Hepatocarcinoma
Brassinosteroid (BR) regulates plant development by activating the transcription factor BRASSINAZOLE RESISTANT1 (BZR1), which activates and represses different target genes to switch cellular programs. The mechanisms that determine BZR1’s transcriptional activities remain largely unknown. Here we show that BZR1 represses target genes by recruiting the Groucho/TUP1-like transcriptional corepressor TOPLESS (TPL). Specific deletion or mutation of an evolutionarily conserved ERF-ASSOCIATED AMPHIPHILIC REPRESSION (EAR) motif at the C-terminus abolishes BZR1’s abilities to regulate gene expression and cell elongation, but these defects are rescued by TPL fusion to the EAR motif-mutated BZR1. The EAR motif in BZR1 mediates recruitment of TPL to BZR1-repressed promoters. A triple tpl mutant (tpl;tpr1;tpr4) shows reduced BR sensitivity and suppresses the gain-of-function bzr1-1D mutant phenotype. BR repression of gene expression also requires histone deacetylases that interact with TPL. Our study demonstrates key roles of the EAR motif and TPL in BR regulation of gene expression and plant growth.
The p53 tumor suppressor protein coordinates the cellular responses to a broad range of cellular stresses, leading to DNA repair, cell cycle arrest or apoptosis. The stability of p53 is essential for its tumor suppressor function, which is tightly controlled by ubiquitin-dependent degradation primarily through its negative regulator murine double minute 2 (Mdm2). To better understand the regulation of p53, we tested the interaction between p53 and USP11 using co-immunoprecipitation. The results show that USP11, an ubiquitin-specific protease, forms specific complexes with p53 and stabilizes p53 by deubiquitinating it. Moreover, down-regulation of USP11 dramatically attenuated p53 induction in response to DNA damage stress. These findings reveal that USP11 is a novel regulator of p53, which is required for p53 activation in response to DNA damage.
p53; USP11; Deubiquitination; Stability
Methanobactin (Mb) is a copper-binding peptide that appears to function as an agent for copper sequestration and uptake in methanotrophs. Mb can also bind and reduce Au(III) to Au(0). In this paper, Au/Al2O3 catalysts prepared by a novel incipient wetness-Mb-mediated bioreduction method were used for glucose oxidation. The catalysts were characterized, and the analysis revealed that very small gold nanoparticles with a particle size <4 nm were prepared by the incipient wetness-Mb-mediated bioreduction method, even at 1.0% Au loading (w/w). The influence of Au loading, calcination temperature and calcination time on the specific activity of Au/Al2O3 catalysts was systematically investigated. Experimental results showed that decomposing the Mb molecules properly by calcinations can enhance the specific activity of Au/Al2O3 catalysts, though they acted as reductant and protective agents during the catalyst preparation. Au/Al2O3 catalysts synthesized by the method exhibited optimum specific activity under operational synthesis conditions of Au loading of 1.0 wt % and calcined at 450 °C for 2 h. The catalysts were reused eight times, without a significant decrease in specific activity. To our knowledge, this is the first attempt at the preparation of Au/Al2O3 catalysts by Mb-mediated in situ synthesis of gold nanoparticles.
methanobactin; bioreduction; glucose oxidation; gold nanoparticles; Au/Al2O3 catalysts
Monolayer transition metal dichalcogenides (TMDs) and their van der Waals heterostructures have been experimentally and theoretically demonstrated as potential candidates for photovoltaic and optoelectronic devices due to the suitable bandgap and excellent light absorption. In this work, we report the observation of photodiode behavior in (both n- and p- type) silicon/monolayer MoS2 vertical heterostructures. The photocurrent and photoresponsivity of heterostructures photodiodes were dependent both on the incident light wavelength and power density, and the highest photoresponsivity of 7.2 A/W was achieved in n-Si/monolayer MoS2 vertical heterostructures photodiodes. Compared with n-Si/MoS2 heterostructures, the photoresponsivity of p-Si/MoS2 heterostructure was much lower. Kelvin probe microscope (KFM) results demonstrated the more efficient separation of photogenerated excitons in n-Si/MoS2 than that in p-Si/MoS2. Coupling KFM results with band alignments of (p-, n-) Si/MoS2 heterostructures, the origins of photodiode-like phenomena of p-Si/MoS2 and n-Si/MoS2 have been unveiled, that is intrinsic built-in electric field in p-n junction, and modulated barrier height and width at the interface in n-n junction. Our work may benefit to the deep understanding of the integration of two-dimensional materials with more conventional three-dimensional semiconductors, and then contribute to the developments in the area of van der Waals heterostructures.
Long non-coding RNAs (lncRNAs) have been regarded as the primary genetic regulators of several important biological processes. However, the biological functions of lncRNAs in radiation-induced lung damage remain largely unknown. The present study aimed to investigate the potential effects of lncRNAs on radiation-induced lung injury (RILI). Female C57BL/6 mice were exposed to 12 Gy single doses of total body irradiation (TBI). LncRNA microarray screening was conducted at 24 h post-irradiation (IR) to investigate the differentially-expressed lncRNAs during RILI. Following the subsequent bioinformatics analysis and reverse transcription-polymerase chain reaction (RT-PCR) validation, one of the verified differentially-expressed long intergenic radiation-responsive ncRNAs (LIRRs), LIRR1, was selected for further functional study. The normal human bronchial epithelial BEAS-2B cell line was used as the cell model. The recombinant eukaryotic expression vector for the lncRNA was designed, constructed and transfected using lipofectamine. RT-PCR, clonogenic and flow cytometry assays, immunofluorescence detection and western blot analysis were performed to reveal the role of the lncRNA in the radiosensitivity regulation of the RILI target cells. In lung tissues 24 h after 12 Gy TBI, six of the identified differentially-expressed LIRRs near the coding genes were validated using quantitative (q)PCR. The upregulation of two LIRRs was observed and confirmed using qPCR. LIRR1 was chosen for further functional study. Following the stable transfection of LIRR1, identified through G418 screening, increased radiosensitivity, evident cell cycle G1 phase arrest and increased γ-H2AX foci formation were observed in the bronchial epithelial BEAS-2B cell line subsequent to IR. LIRR1 overexpression also led to decreased expression of the KU70, KU80 and RAD50 DNA repair proteins, marked activation of p53, decreased mouse double minute 2 homolog (MDM2) expression, and substantially induced p21 and suppressed cyclin-dependent kinase 2 in BEAS-2B following IR. Subsequent to the use of Pifithrin-α, a specific inhibitor of p53 activation, increased MDM2 expression was observed in the LIRR1-overexpressing cells, suggesting that LIRR1 could mediate the DNA damage response (DDR) signaling in a p53-dependent manner. The present study provides a novel mechanism for RILI, using the concept of lncRNAs.
long non-coding RNA; radiation-induced lung injury; radiosensitivity; cell cycle; DNA damage response; p53
AIM: To identify an appropriate therapeutic regimen for using aconite cake-separated moxibustion to treat diarrhea-predominant irritable bowel syndrome (D-IBS).
METHODS: A factorial design was employed to examine the two factors of moxibustion frequency and number of cones. The two tested frequencies were three or six moxibustion sessions per week, and the two tested doses were one or two cones per treatment. A total of 166 D-IBS patients were randomly divided into four treatment groups, which included each combination of the examined frequencies and doses. The bilateral Tianshu acupoints (ST25) and the Qihai acupoint (RN6) were selected for aconite cake-separated moxibustion. Each patient received two courses of treatment, and each course had a duration of 2 wk. For each group, the scores on the Birmingham irritable bowel syndrome (IBS) symptom questionnaire, the IBS Quality of Life scale, the Self-Rating Depression Scale (SDS), the Self-Rating Anxiety Scale (SAS), the Hamilton Depression (HAMD) scale, and the Hamilton Anxiety (HAMA) scale were determined before treatment, after the first course of treatment, and after the second course of treatment.
RESULTS: The symptom, quality of life, SDS, SAS, HAMD, and HAMA scores of the patients in all 4 aconite cake-separated moxibustion groups were significantly lower after the first and second courses of treatment than before treatment (P < 0.001 for all). The symptom, quality of life, SDS, SAS, HAMD, and HAMA scores of the patients in all four aconite cake-separated moxibustion groups were significantly lower after the second course of treatment than after the first course of treatment (P < 0.001 for all). Between-group comparisons after the second course of treatment revealed that the symptom scores for group 1 (1 cone, 3 treatments/wk) and group 3 (2 cones, 3 treatments/wk) were significantly lower than that for group 2 (1 cone, 6 treatments/wk) (5.55 ± 5.05 vs 10.45 ± 6.61, P < 0.001; 5.65 ± 4.00 vs 10.45 ± 6.61, P < 0.001). Regarding the two levels of the two examined factors for aconite cake-separated moxibustion, after the first course of treatment, the changes in HAMA scores were significantly different for the two tested moxibustion frequencies (P = 0.011), with greater changes for the “6 treatments/wk” groups than for the “3 treatments/wk” groups; in addition, there were interaction effects between the number of cones and moxibustion frequency (P = 0.028). After the second course of treatment, changes in symptom scores for the 2 tested moxibustion frequencies were significantly different (P = 0.002), with greater changes for the “3 treatments/wk” groups than for the “6 treatments/wk” groups.
CONCLUSION: An aconite cake-separated moxibustion treatment regimen of 3 treatments/wk and 1 cone/treatment appears to produce better therapeutic effects for D-IBS compared with the other tested regimens.
Diarrhea-predominant irritable bowel syndrome; Aconite cake-separated moxibustion; Factorial design; Moxibustion quantity; Clinical research
Lung cancer, particularly non-small-cell lung cancer (NSCLC) is the leading cause of cancer mortality. Chemotherapy combined dendritic cells co-cultured with cytokine-induced killer cells (DC-CIK) immunotherapy has been applied in advanced NSCLC patients' treatment, but couldn't provide consistent beneficial results. Therefore, it is necessary to evaluate the efficiency and safety of combination therapy to promote the application.
A literature search for randomized controlled trials of NSCLC was conducted in PubMed database. Before meta-analysis was performed, studies were evaluated heterogeneity. Pooled risk ratios (RRs) were estimated and 95% confidence intervals (CIs) were calculated using a fixed-effect model. Sensitivity analysis was also performed.
Six eligible trials were enrolled. Efficiency and safety of chemotherapy followed by DC-CIK immunotherapy (experimental group) and chemotherapy alone (control group) were compared. 1-year overall survival (OS) (P = 0.02) and progression free survival (PFS) (P = 0.005) in the experimental group were significantly increased compared with the control. Disease control rate (DCR) (P = 0.006) rose significantly in experimental group. However, no significant differences between the two groups were observed in 2-year OS (P = 0.21), 2-year PFS (P = 0.10), overall response rate (ORR) (P = 0.76) and partial response (PR) (P = 0.22). Temporary fever, anemia, leukopenia and nausea were the four major adverse events (AEs) treated by chemotherapy. The incidence of anemia, leukopenia and nausea in the experimental group was obviously lower than the control group. Temporary fever rate was higher in experimental group than that in the control, but could be alleviated by taking sufficient rest.
Chemotherapy combined with DC-CIK immunotherapy showed superiority in DCR, 1-year OS and PFS, and no more AEs appeared, however, there was no significant improvement in ORR, PR, 2-year OS and PFS. As a whole, the combination therapy is safer but modest in efficacy for advanced NSCLC patients.
Background: The increasing use of the transversus abdominis plane (TAP) block, as a form of pain relief after laparoscopic surgery, warrants evaluation of its effectiveness, when compared with other analgesic techniques. Methods: We searched online databases of MEDLINE, EMBASE, Google scholar and The Cochrane Database of Systematic Review. Mean differences (MD) were formulated for continuous data; odds ratios (OR) were calculated for dichotomous data. Results were produced with a random effects model with 95% confidence intervals (CI). Results: 14 trials with a total of 905 patients were included for the analysis, TAP block resulted in significantly less postoperative analgesic consumption at 24 h (MD = -25.46, 95% CI [-32.22, -18.69], P < 0.00001), and less number of patients requiring analgesic postoperatively (OR = 0.16, 95% CI 0.03-0.87, P = 0.03). Meanwhile, pain sores were significantly different at 2 h (MD = -1.55, 95% CI [-2.50, -0.59], P < 0.00001), a borderline difference between the groups seen at 6 hours ( MD = -1.13, 95% CI [-1.69, -0.56], P = 0.05), and there was not affect pain at 24 h (MD = -0.33, 95% CI [-0.08, 0.15], P = 0.14) with TAP block groups compared with the groups without TAP block. There was a significant difference in postoperative nausea and vomiting (random effects model: OR = 2.04, 95% CI [1.19-3.48], P = 0.34). Conclusion: TAP block would result in less analgesic consumption, less requirement of analgesic, and less pain at 2 h and slightly at 6 h but at 24 h after laparoscopic surgery in comparison with usual care alone or placebo block. In addition TAP block can increase the incidence of postoperative nausea and vomiting.
TAP block; transversus abdominis plane block; laparoscopic surgery; meta-analysis
As a kind of medicine which can also be used as food, Alpinia oxyphylla Miq. has a long clinical history in China. A variety of studies demonstrated the significant neuroprotective activity effects of chloroform (CF) extract from the fruits of Alpinia oxyphylla. In order to further elucidate the possible mechanisms of CF extract which mainly contains sesquiterpenes with neuroprotection on the cognitive ability, mice were injected with Aβ1−42 and later with CF in this study. The results showed that the long-term treatment of CF enhanced the cognitive performances in behavior tests, increased activities of glutathione peroxidase (GSH-px) and decreased the level of malondialdehyde (MDA), acetylcholinesterase (AChE), and amyloid-β (Aβ), and reversed the activation of microglia, degeneration of neuronal acidophilia, and nuclear condensation in the cortex and hippocampus. These results demonstrate that CF ameliorates learning and memory deficits by attenuating oxidative stress and regulating the activation of microglia and degeneration of neuronal acidophilia to reinforce cholinergic functions.
The fungus Harpophora oryzae is a close relative of the pathogen Magnaporthe oryzae and a beneficial endosymbiont of wild rice. Here, we show that H. oryzae evolved from a pathogenic ancestor. The overall genomic structures of H. and M. oryzae were found to be similar. However, during interactions with rice, the expression of 11.7% of all genes showed opposing trends in the two fungi, suggesting differences in gene regulation. Moreover, infection patterns, triggering of host defense responses, signal transduction and nutritional preferences exhibited remarkable differentiation between the two fungi. In addition, the H. oryzae genome was found to contain thousands of loci of transposon-like elements, which led to the disruption of 929 genes. Our results indicate that the gain or loss of orphan genes, DNA duplications, gene family expansions and the frequent translocation of transposon-like elements have been important factors in the evolution of this endosymbiont from a pathogenic ancestor.
How to promote physical activity is an important public health problem that is attracting increasing attention. Although the application of environmental approaches is believed to promote resident walking, there remains insufficient evidence of the effectiveness of these interventions.
This study employed direct observation and questionnaires. Observations were performed on each Tuesday, Thursday, Saturday, and Sunday from April 13th to May 16th. Fourteen trained observers observed six community walking paths, and an additional walking path in a park. The trained observers filled out 2388 observation forms in the field, including 228 forms rating the permanent environment, and 1080 forms assessing the current environment and counting the number of walkers. A total of 1800 questionnaireswere administered to community residents.
The results of both observation and questionnaires showed good association regarding the characteristics of walking path users (for observation, female = 54.4%; for questionnaire interviews, female, OR = 1.441), and the environmental features associated with walking path utilization (for observation, positive associations were observed between the utilization index and observational environmental variables; for questionnaire interviews, roads and aesthetics were important, OR = 1.044). There were positive associations between path use and time, a preference for brisk walking, and the observed current and permanent environmental variables. Female participants were more likely to use walking paths than males (OR = 1.441, 95% confidence interval [CI] 1.126–1.846). BMI and traffic hazard safety were significantly negatively associated with walking path use (OR = 0.948, 95% CI 0.915–0.981, and OR = 0.933, 95% CI 0.887–0.981, respectively). Roads, aesthetics, and knowledge of physical activity were significantly positively correlated with use of walking paths (OR = 1.044, 95% CI 1.017–1.072, and OR = 1.175, 95% CI 1.043–1.323). Participants that resided further than 1 km from the park were less likely to use walking paths (OR = 0.703, 95% CI 0.530–0.933). Gender-specific associations were also found.
Both perceived and objective environmental factors were associated with walking path use. Data suggested that the permanent and current conditions of the paths might influence walking path utilization, and that gender-specific promotion strategies should be considered.
Walking path utilization; Direct observation; Perceived and observed environment; Chinese residents
As the major mechanism of plant growth and morphogenesis, cell elongation is controlled by many hormonal and environmental signals. How these signals are coordinated at the molecular level to ensure coherent cellular responses remains unclear. In this study, we illustrate a molecular circuit that integrates all major growth-regulating signals, including auxin, brassinosteroid, gibberellin, light, and temperature. Analyses of genome-wide targets, genetic and biochemical interactions demonstrate that the auxin-response factor ARF6, the light/temperature-regulated transcription factor PIF4, and the brassinosteroid-signaling transcription factor BZR1, interact with each other and cooperatively regulate large numbers of common target genes, but their DNA-binding activities are blocked by the gibberellin-inactivated repressor RGA. In addition, a tripartite HLH/bHLH module feedback regulates PIFs and additional bHLH factors that interact with ARF6, and thereby modulates auxin sensitivity according to developmental and environmental cues. Our results demonstrate a central growth-regulation circuit that integrates hormonal, environmental, and developmental controls of cell elongation in Arabidopsis hypocotyl.
Plants can grow by making more cells or by increasing the size of these existing cells. Plant growth is carefully controlled, but it must be able to respond to changes in the plant's environment.
Many different plant hormones and various signals from the environment—such as light and temperature—influence how and when a plant grows. The different signals that affect cell growth typically act via distinct pathways that change which genes are switched on or off inside the cells. However, the ways in which these different signals are coordinated by plants are not fully understood.
Now, Oh et al. have looked at the genes that are switched on and off in response to all the major signals that regulate the growth of the first stem to emerge from the seed of Arabidopsis, a small flowering plant that is widely studied by plant biologists. Oh et al. found that the proteins that change gene expression in response to hormones or the environment bind to each other. These proteins, which are collectively called transcription factors, were also revealed to cooperate to regulate the expression of hundreds of genes: transcription factors have not been seen to behave in this way in plants before.
By discovering a central mechanism that coordinates the different signals that control plant growth, these findings may guide future efforts to boost the yields of food crops and plants that are grown to make biofuels.
auxin; Arabidopsis; crosstalk; brassinosteroid; light; transcription factors; Arabidopsis
A 5′ truncated snake venom metalloproteinase was identified from a cDNA library constructed from venom glands of an eastern diamondback rattlesnake (Crotalus adamanteus). The 5′-rapid amplification of cDNA ends (RACE) was used to obtain the 1865 bp full-length cDNA sequence of a snake venom metalloproteinase (CamVMPII). CamVMPII encodes an open reading frame of 488 amino acids, which includes a signal peptide, a pro-domain, a metalloproteinase domain, a spacer, and an RGD-disintegrin domain. The predicted amino acid sequence of CamVMPII showed a 91%, 90%, 83%, and 82% sequence homology to the P-II class enzymes of C. adamanteus metalloproteinase 2, C. atrox CaVMP-II, Gloydius halys agkistin, and Protobothrops jerdonii jerdonitin, respectively. Disintegrins are potent inhibitors of both platelet aggregation and integrin-dependent cell adhesion. Therefore, the disintegrin domain (Cam-dis) of CamVMPII was amplified by PCR, cloned into a pET-43.1a vector, and expressed in Escherichia coli BL21. Affinity purified recombinantly modified Cam-dis (r-Cam-dis) with a yield of 8.5 mg/L culture medium was cleaved from the fusion tags by enterokinase cleavage. r-Cam-dis was further purified by two-step chromatography consisting of HiTrap™ Benzamidine FF column, followed by Talon Metal affinity column with a final yield of 1 mg/L culture. r-Cam-dis was able to inhibit all three processes of platelet thrombus formation including platelet adhesion with an estimated IC50 of 1 nM, collagen- and ADP-induced platelet aggregation with the estimated IC50s of 18 and 6 nM, respectively, and platelet function on clot retraction. It is a potent anti-platelet inhibitor, which should be further investigated for drug discovery to treat stroke patients or patients with thrombotic disorders.
Snake venom metalloproteinase; Disintegrin; Crotalus adamanteus; cDNA library; Platelet activity
Snake venom disintegrins inhibit platelet aggregation and have anti-cancer activities. In this study, we report the cloning, expression, and functional activities of a recombinant disintegrin, r-viridistatin 2 (GenBank ID: JQ071899), from the Prairie rattlesnake. r-Viridistatin 2 was tested for anti-invasive and anti-adhesive activities against six different cancer cell lines (human urinary bladder carcinoma (T24), human fibrosarcoma (HT-1080), human skin melanoma (SK-Mel-28), human colorectal adenocarcinoma (CaCo-2), human breast adenocarcinoma (MDA-MB-231) and murine skin melanoma (B16F10)). r-Viridistatin 2 shares 96% and 64% amino acid identity with two other Prairie rattlesnake medium-sized disintegrins, viridin and viridistatin, respectively. r-Viridistatin 2 was able to inhibit adhesion of T24, SK-MEL-28, HT-1080, CaCo-2 and MDA-MB-231 to various extracellular matrix proteins with different affinities. r-Viridistatin 2 decreased the ability of T24 and SK-MEL-28 cells to migrate by 62 and 96% respectively, after 24 h of incubation and the invasion of T24, SK-MEL-28, HT-1080 and MDA-MB-231 cells were inhibited by 80, 85, 65 and 64% respectively, through a reconstituted basement membrane using a modified Boyden chamber. Finally, r-viridistatin 2 effectively inhibited lung colonization of murine melanoma cells in BALB/c mice by 71%, suggesting that r-viridistatin 2 could be a potent anti-cancer agent in vivo.
Disintegrin; Recombinant snake toxins; Cancer cell lines; Anti-metastatic activity; Crotalus viridis viridis
Mitochondrial oxidative phosphorylation is the major source of energy in cardiac muscle. In the streptozotocin-induced diabetic (STZ-DM) mice, myocardial oxidative phosphorylation was perturbated and oxidative phosphorylation complex V (ATP synthase) activity was significantly reduced. To determine the independent effects of hyperglycemia and insulin deficiency on the changes of myocardial complex V, we used phlorizin (Ph) to normalize blood glucose in the diabetic mice. Ph treatment did not improve myocardial complex V activity in the STZ-DM mice, whereas insulin treatment normalized myocardial complex V activity in the diabetic mice. Therefore, the reduction of complex V activity was caused by insulin deficiency and not by hyperglycemia in STZ-DM myocardium. Acute insulin stimulation induced phosphorylation of Akt and translocation of Akt to mitochondria in myocardium. Translocation of phospho-Akt to mitochondria was enhanced in the STZ-DM mice and was blunted in the diet-induced diabetic mice. In parallel, insulin activation of complex V was enhanced in the STZ-DM myocardium and suppressed in the diet-induced diabetic myocardium. In vivo inhibition of Akt blocked insulin stimulation of phospho-Akt translocation and blunted activation of complex V. Insulin-activated Akt translocation to mitochondria in cardiac muscle is a novel paradigm that may have important implications on myocardial bioenergetics.
Akt translocation; Mitochondria; Diabetes; Cardiac muscle; Oxidative phosphorylation; Insulin
Insulin can translocate Akt to mitochondria in cardiac muscle. The goals of this study were to define sub-mitochondrial localization of the translocated Akt, to dissect the effects of insulin on Akt isoform translocation, and to determine the direct effect of mitochondrial Akt activation on Complex V activity in normal and diabetic myocardium. The translocated Akt sequentially localized to the mitochondrial intermembrane space, inner membrane, and matrix. To confirm Akt translocation, in vitro import assay showed rapid entry of Akt into mitochondria. Akt isoforms were differentially regulated by insulin stimulation, only Akt1 translocated into mitochondria. In the insulin-resistant Type 2 diabetes model, Akt1 translocation was blunted. Mitochondrial activation of Akt1 increased Complex V activity by 24% in normal myocardium in vivo and restored Complex V activity in diabetic myocardium. Basal mitochondrial Complex V activity was lower by 22% in the Akt1−/− myocardium. Insulin-stimulated Complex V activity was not impaired in the Akt1−/− myocardium, due to compensatory translocation of Akt2 to mitochondria. Akt1 is the primary isoform that relayed insulin signaling to mitochondria and modulated mitochondrial Complex V activity. Activation of mitochondrial Akt1 enhanced ATP production and increased phosphocreatine in cardiac muscle cells. Dysregulation of this signal pathway might impair mitochondrial bioenergetics in diabetic myocardium.
The pine tip moth, Rhyacionia leptotubula (Lepidoptera: Tortricidae) is one of the most destructive forestry pests in Yunnan Province, China. Despite its importance, less is known regarding all aspects of this pest. Understanding the genetic information of it is essential for exploring the specific traits at the molecular level. Thus, we here sequenced the transcriptome of R. leptotubula with high-throughput Illumina sequencing.
In a single run, more than 60 million sequencing reads were generated. De novo assembling was performed to generate a collection of 46,910 unigenes with mean length of 642 bp. Based on Blastx search with an E-value cut-off of 10−5, 22,581 unigenes showed significant similarities to known proteins from National Center for Biotechnology Information (NCBI) non-redundant (Nr) protein database. Of these annotated unigenes, 10,360, 6,937 and 13,894 were assigned to Gene Ontology (GO), Clusters of Orthologous Group (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, respectively. A total of 5,926 unigenes were annotated with domain similarity derived functional information, of which 55 and 39 unigenes respectively encoding the insecticide resistance related enzymes, cytochrome P450 and carboxylesterase. Using the transcriptome data, 47 unigenes belonging to the typical “stress” genes of heat shock protein (Hsp) family were retrieved. Furthermore, 1,450 simple sequence repeats (SSRs) were detected; 3.09% of the unigenes contained SSRs. Large numbers of SSR primer pairs were designed and out of randomly verified primer pairs 80% were successfully yielded amplicons.
A large of putative R. leptotubula transcript sequences has been obtained from the deep sequencing, which extensively increases the comprehensive and integrated genomic resources of this pest. This large-scale transcriptome dataset will be an important information platform for promoting our investigation of the molecular mechanisms from various aspects in this species.
Preparation of gold nanoparticles with a narrow size distribution has enormous importance in nanotechnology. Methanobactin (Mb) is a copper-binding small peptide that appears to function as an agent for copper sequestration and uptake in methanotrophs. Mb can also bind and catalytically reduce Au (III) to Au (0). In this study, we demonstrate a facile Mb-mediated one-step synthetic route to prepare monodispersed gold nanoparticles. Continuous reduction of Au (III) by Mb can be achieved by using hydroquinone as the reducing agent. The gold nanoparticles have been characterized by UV-visible spectroscopy. The formation and the surface plasmon resonance properties of the gold nanoparticles are highly dependent on the ratio of Au (III) to Mb in solution. X-ray photoelectron spectroscopy (XPS), fluorescence spectra and Fourier transform-infrared spectroscopy (FT-IR) spectra suggest that Mb molecules catalytically reduce Au (III) to Au (0) with the concomitant production of gold nanoparticles, and then, Mb statically adsorbed onto the surface of gold nanoparticles to form an Mb-gold nanoparticles assembly. This avoids secondary nucleation. The formed gold nanoparticles have been demonstrated to be monodispersed and uniform by transmission electron microscopy (TEM) images. Analysis of these particles shows an average size of 14.9 nm with a standard deviation of 1.1 nm. The gold nanoparticles are extremely stable and can resist aggregation, even after several months.
methanobactin; methanotrophs; gold nanoparticles; hydroquinone; monodisperse
Classical swine fever (CSF) caused by CSF virus (CSFV) is a highly contagious disease of pigs. The RNA helicases retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA-5) are differentially involved in the detection of various RNA viruses. In present study, we investigated the roles of RIG-I and MDA-5 in eliciting antiviral and inflammatory responses to CSFV shimen strain in Porcine alveolar macrophages (PAMs).
CSFV Shimen strain was used as challenge virus in this study and PAMs were cultured in vitro. Interferon regulatory factor (IRF)-3 and nuclear factor-kappa B (NF-κB) translocation was detected using immunofluorescent staining; RIG-I, MDA5, interferon promoter-stimulating factor 1 (IPS-1), IRF-3 and NF-κB expression was measured by Western Blotting; Interferon beta (IFN-β), IFN-α, interleukin-1beta (IL-1β), IL-6 and tumor necrosis factor (TNF-α) expression was tested by Enzyme-linked immunosorbent assays (ELISA) and shRNA-mediated knockdown of MDA5 or RIG-I was performed.
The findings suggested that the initial response to CSFV infection resulted in the higher expression of RIG-I and MDA5 leading to the activation of IPS-1, IRF-3 and NF-κB in a dose-dependent manner. Evaluation of IFN-α, IFN-β, IL-1β, IL-6 or TNF-α expressed by PAMs showed significant differences between infected and uninfected cells. CSFV infected cells induced to express high levels of IFN-α, IFN-β, IL-1β, IL-6 and TNF-α in a dose-dependent way within 24 h post-infection (hpi). At the same time, CSFV improved the nuclear translocation of IRF-3 and NF-κB. We also directly compared and assessed the roles of RIG-I and MDA5 in triggering innate immune actions during CSFV infection through shRNA-mediated knockdown of MDA5 or RIG-I. We found that, compared to the control, the production of IFN-α, IFN-β, IL-1β, IL-6 and TNF-α in response to CSFV infection was heavily reduced in RIG-I knockdown cells while it was moderately decreased in MDA5 knockdown cells. PAMs derived from knockdown of both RIG-I and MDA5 almost failed to produce IFNs and inflammatory cytokines.
It indicates that CSFV can be recognized by both RIG-I and MDA5 to initiate the RIG-I signaling pathway to trigger innate defenses against infection.
RIG-I; MDA5; CSF; CSFV; IRF-3; NF-κB; Interferon; Inflammatory response; PAM
The selection criteria for patients with hepatocellular carcinoma (HCC) to undergo liver transplantation should accurately predict posttransplant recurrence while not denying potential beneficiaries. In the present study, we attempted to identify risk factors associated with posttransplant recurrence and to expand the selection criteria.
Patients and Methods
Adult patients with HCC who underwent liver transplantation between November 2004 and September 2012 at our centre were recruited into the current study (N = 241). Clinical and pathological data were retrospectively reviewed. Patients who died during the perioperative period or died of non-recurrence causes were excluded from this study (N = 25). All potential risk factors were analysed using uni- and multi-variate analyses.
Sixty-one recipients of 216 qualified patients suffered from recurrence. Similar recurrence-free and long-term survival rates were observed between living donor liver transplant recipients (N = 60) and deceased donor liver transplant recipients (N = 156). Total tumour volume (TTV) and preoperative percentage of lymphocytes (L%) were two independent risk factors in the multivariate analysis. We propose a prognostic score model based on these two risk factors. Patients within our criteria achieved a similar recurrence-free survival to patients within the Milan criteria. Seventy-one patients who were beyond the Milan criteria but within our criteria also had comparable survival to patients within the Milan criteria.
TTV and L% are two risk factors that contribute to posttransplant recurrence. Selection criteria based on these two factors, which are proposed by our study, expanded the Milan criteria without increasing the risk of posttransplant recurrence.
Hyperpolarization-activated currents (Ih) mediated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels modulate excitability of myelinated A− and Ah-type visceral ganglion neurons (VGN). Whether alterations in Ih underlie the previously reported reduction of excitability of myelinated Ah-type VGNs following ovariectomy (OVX) has remained unclear. Here we used the intact nodose ganglion preparation in conjunction with electrophysiological approaches to examine the role of Ih remodeling in altering Ah-type neuron excitability following ovariectomy in adult rats. Ah-type neurons were identified based on their afferent conduction velocity. Ah-type neurons in nodose ganglia from non-OVX rats exhibited a voltage ‘sag’ as well as ‘rebound’ action potentials immediately following hyperpolarizing current injections, which both were suppressed by the Ih blocker ZD7288. Repetitive spike activity induced afterhyperpolarizations lasting several hundreds of milliseconds (termed post-excitatory membrane hyperpolarizations, PEMHs), which were significantly reduced by ZD7288, suggesting that they resulted from transient deactivation of Ih during the preceding spike trains. Ovariectomy reduced whole-cell Ih density, caused a hyperpolarizing shift of the voltage-dependence of Ih activation, and slowed Ih activation. OVX-induced Ih remodeling was accompanied by a flattening of the stimulus frequency/response curve and loss of PEMHs. Also, HCN1 mRNA levels were reduced by ∼30% in nodose ganglia from OVX rats compared with their non-OVX counterparts. Acute exposure of nodose ganglia to 17beta-estradiol partly restored Ih density and accelerated Ih activation in Ah-type cells. In conclusion, Ih plays a significant role in modulating the excitability of myelinated Ah-type VGNs in adult female rats.
Advanced chronic kidney disease (CKD) is associated with impaired exercise capacity, skeletal muscle dysfunction, and oxidative stress. Mitochondria are the primary source for energy production and generation of reactive oxygen species (ROS). Mitochondrial state 3 respiration, mitochondrial complex I enzyme activity, and tissue porin/actin ratio were determined in the gastrocnemius muscle of male SD rats 14 weeks after 5/6 nephrectomy (CKD) or sham-operation (control). The CKD group exhibited azotemia, hypertension, significant reduction (-39%) of state 3 mitochondrial respiration, and a significant increase in the mitochondrial complex I enzyme activity. The latter is the first step in oxidative phosphorylation, a process linked to production of ROS. These abnormalities were associated with a significant reduction in muscle porin/β actin ratio denoting substantial reduction of mitochondrial mass in skeletal muscle of animals with CKD. CKD results in impaired mitochondrial respiration, reduced muscle mitochondrial mass, depressed energy production and increased ROS generation in the skeletal muscle. These events can simultaneously contribute to the reduction of exercise capacity and oxidative stress in CKD.
End stage renal disease; oxidative stress; muscle weakness; exercise capacity; uremia
Plant growth is coordinately regulated by environmental and hormonal signals. Brassinosteroid (BR) plays essential roles in growth regulation by light and temperature, but the interactions between BR and these environmental signals remain poorly understood at the molecular level. Here, we show that direct interaction between the dark- and heat-activated transcription factor phytochrome-interacting factor4 (PIF4) and the BR-activated transcription factor BZR1 integrates the hormonal and environmental signals. BZR1 and PIF4 interact with each other in vitro and in vivo, bind to nearly two thousand common target genes, and synergistically regulate many of these target genes, including the PRE family HLH factors required for promoting cell elongation. Genetic analysis indicates that BZR1 and PIFs are interdependent in promoting cell elongation in response to BR, darkness, or heat. These results show that the BZR1-PIF4 interaction controls a core transcription network, allowing plant growth co-regulation by the steroid and environmental signals.
To evaluate the development of diabetic neuropathy, the current study examined changes in peripheral axonal function. Nerve excitability techniques were undertaken in 108 type 2 diabetic patients with nerve conduction studies (NCS), HbA1c levels, and total neuropathy score (TNS). Patients were categorized into two cohorts: patients with diabetes without neuropathy (DWN group [n = 56]) and patients with diabetes with neuropathy (DN group [n = 52]) and further into severity grade 0 (TNS 0–1 [n = 35]), grade 1 (TNS 2–8 [n = 42]), and grade 2/3 (TNS 9–24 [n = 31]). Results revealed that the DWN group had a significantly increased threshold, prolonged latency, and changes in excitability parameters compared with age-matched control subjects. Patients with neuropathy demonstrated significant alteration in recovery cycle parameters and depolarizing threshold electrotonus. Within the DWN cohort, there were significant correlations between HbA1c level and latency and subexcitability, whereas the estimated glomerular filtration rate correlated with superexcitability in patients with neuropathy. Furthermore, excitability parameters became progressively more abnormal with increasing clinical severity. These results suggest a spectrum of excitability abnormalities in patients with diabetes and that early axonal dysfunction may be detected prior to the development of neuropathy. As progressive changes in excitability parameters correlated to neuropathy severity, excitability testing may provide a biomarker of the early development and severity of diabetic neuropathy, providing insights into the pathophysiological mechanisms producing axonal dysfunction.