Enhanced G2/M Arrest,Caspase Related Apoptosis and Reduced E-Cadherin Dependent Intercellular Adhesion by Trabectedin in Prostate Cancer Stem Cells

Trabectedin (Yondelis, ET-743) is a marine-derived tetrahydroisoquinoline alkaloid. It is originally derived from the Caribbean marine tunicate Ecteinascidia turbinata and currently produced synthetically. Trabectedin is active against a variety of tumor cell lines growing in culture. The present study focused on the effect of trabectedin in cell proliferation, cell cycle progression, apoptosis and spheroid formation in prostate cancer stem cells (CSCs). Cluster of differentiation (CD) 133^+high/CD44^+high prostate CSCs were isolated from the DU145 and PC-3 human prostate cancer cell line through flow cytometry. We studied the growth-inhibitory effects of trabectedin and its molecular mechanisms on human prostate CSCs and non-CSCs. DU-145 and PC-3 CSCs were treated with 0.1, 1, 10 and 100 nM trabectedin for 24, 48 and 72 h and the growth inhibition rates were examined using the sphere-forming assay. Annexin-V assay and immunofluorescence analyses were performed for the detection of the cell death. Concentration-dependent effects of trabectedin on the cell cycle were also evaluated. The cells were exposed to the different doses of trabectedin for 24, 48 and 72 h to evaluate the effect of trabectedin on the number and diameter of spheroids. According to the results, trabectedin induced cytotoxicity and apoptosis at the IC₅₀ dose, resulting in a significant increase expression of caspase-3, caspase-8, caspase-9, p53 and decrease expression of bcl-2 in dose-dependent manner. Cell cycle analyses revealed that trabectedin induces dose-dependent G2/M-phase cell cycle arrest, particularly at high-dose treatments. Three-dimensional culture studies showed that trabectedin reduced the number and diameter of spheroids of DU145 and PC3 CSCs. Furthermore, we have found that trabectedin disrupted cell-cell interactions via E-cadherin in prostasphere of DU-145 and PC-3 CSCs. Our results showed that trabectedin inhibits cellular proliferation and accelerates apoptotic events in prostate CSCs; and may be a potential effective therapeutic agent against prostate cancer.     

Optimizing Exchange Transfusion for Severe Unconjugated Hyperbilirubinemia: Studies in the Gunn Rat

Background

Severe unconjugated hyperbilirubinemia carries the risk of neurotoxicity. Phototherapy (PT) and exchange transfusion (ET) are cornerstones in the treatment of unconjugated hyperbilirubinemia. Studies to improve ET efficacy have been hampered by the low application of ET in humans and by the lack of an in vivo model. The absence of an appropriate animal model has also prevented to determine the efficacy of adjunct or alternative treatment options such as albumin (Alb) administration.

Aim

To establish an in vivo model for ET and to determine the most effective treatment (combination) of ET, PT and Alb administration.

Methods

Gunn rats received either PT, PT+Alb, ET, ET+PT, ET+PT+Alb or sham operation (each n = 7). ET was performed via the right jugular vein in ∼20 min. PT (18 µW/cm²/nm) was started after ET or at T₀. Albumin i.p. injections (2.5 g/kg) were given after ET or before starting PT. Plasma unconjugated bilirubin (UCB), plasma free bilirubin (Bf), and brain bilirubin concentrations were determined.

Results

We performed ET in 21 Gunn rats with 100% survival. At T₁, ET was profoundly more effective in decreasing both UCB −44%, p<0.01) and Bf −81%, p<0.05) than either PT or PT+Alb. After 48 h, the combination of ET+PT+Alb showed the strongest hypobilirubinemic effect (−54% compared to ET).

Conclusions

We optimized ET for severe unconjugated hyperbilirubinemia in the Gunn rat model. Our data indicate that ET is the most effective treatment option, in the acute as well as the follow-up situation.     

北太平洋两个柔鱼群体角质颚形态及生长特征

根据2011年5—11月我国鱿钓船在北太平洋海域(150°E—176°W)采集的渔获物样本,对柔鱼两个群体各项角质颚外部形态和生长进行分析。结果表明,角质颚参数值雌性均大于雄性,冬春生群体雌雄参数值间差异要大于秋生群体;角质颚各项参数值中,上头盖长(Upper Hood length,UHL)、上脊突长(Upper Crest length,UCL)、下脊突长(Lower crest length,LCL)、下翼长(Lower Wing length,LWL)与胴长、体重分别呈线性和指数关系(P0.01)。主成分分析认为,秋生群体雌、雄个体角质颚第一主成分因子均为UCL/ML,第二主成分因子均为UWL/ML,冬春生群体雌、雄个体角质颚第一主成分因子分别ULWL/ML和LLWL/ML,第二主成分分别与URW/ML和LRL/ML,这些这成分因子代表了角质颚水平和垂直方向上的生长特征。两个群体的雌性个体角质颚形态上存在显著差异,但雄性个体间差异不显著。方差分析(ANOVA)表明,2个群体不同胴长组间的角质颚形态均存在着显著差异(P0.01);LSD法分析认为,除了秋生群体雌性个体上头盖长、上喙长、下头盖长、下喙宽在胴长组250 mm和250—300 mm间不存在差异外,其他组之间均有着显著差异(P0.01)。同一群体不同性腺成熟度等级间的柔鱼角质颚形态存在显著差异(P0.01),但性成熟度为Ⅰ期和Ⅱ期时2个群体角质颚各项形态指标均存在显著差异(P0.01),而性成熟度Ⅲ期时则不存在差异(P0.05)。研究认为,不同群体的柔鱼角质颚形态及生长特征均存在着一定差异。     

Cladosporol A triggers apoptosis sensitivity by ROS-mediated autophagic flux in human breast cancer cells

Background

Endophytes have proven to be an invaluable resource of chemically diverse secondary metabolites that act as excellent lead compounds for anticancer drug discovery. Here we report the promising cytotoxic effects of Cladosporol A (HPLC purified >98%) isolated from endophytic fungus Cladosporium cladosporioides collected from Datura innoxia. Cladosporol A was subjected to in vitro cytotoxicity assay against NCI60 panel of human cancer cells using MTT assay. We further investigated the molecular mechanism(s) of Cladosporol A induced cell death in human breast (MCF-7) cancer cells. Mechanistically early events of cell death were studied using DAPI, Annexin V-FITC staining assay. Furthermore, immunofluorescence studies were carried to see the involvement of intrinsic pathway leading to mitochondrial dysfunction, cytochrome c release, Bax/Bcl-2 regulation and flowcytometrically measured membrane potential loss of mitochondria in human breast (MCF-7) cancer cells after Cladosporol A treatment. The interplay between apoptosis and autophagy was studied by microtubule dynamics, expression of pro-apoptotic protein p21 and autophagic markers monodansylcadaverine staining and LC3b expression.

Results

Among NCI60 human cancer cell line panel Cladosporol A showed least IC₅₀ value against human breast (MCF-7) cancer cells. The early events of apoptosis were characterized by phosphatidylserine exposure. It disrupts microtubule dynamics and also induces expression of pro-apoptotic protein p21. Moreover treatment of Cladosporol A significantly induced MMP loss, release of cytochrome c, Bcl-2 down regulation, Bax upregulation as well as increased monodansylcadaverine (MDC) staining and leads to LC3-I to LC3-II conversion.

Conclusion

Our experimental data suggests that Cladosporol A depolymerize microtubules, sensitize programmed cell death via ROS mediated autophagic flux leading to mitophagic cell death.

Graphical abstract

The proposed mechanism of Cladosporol A -triggered apoptotic as well as autophagic death of human breast cancer (MCF-7) cells. The figure shows that Cladosporol A induced apoptosis through ROS mediated mitochondrial pathway and increased p21 protein expression in MCF-7 cells in vitro.

Open image in new window

     

Efficacy of marbofloxacin against respiratory infections of lambs

The trial was carried out in a sheep flock with mixed Mannheimia haemolytica/Mycoplasma spp. respiratory infection; 60 lambs were included therein and allocated as follows: lambs in group MH were subcutaneously injected with marbofloxacin (3.0 mg/kg bodyweight) on two occasions, once daily 2 days apart – lambs in group ML3 were given marbofloxacin (2.0 mg/kg) on three occasion, once daily for three consecutive days – lambs in group ML2 were given marbofloxacin (2.0 mg/kg) on two occasions, 2 days apart – lambs in group T were subcutaneously injected with tilmicosin (15 mg/kg) on two occasions, 4 days apart – lambs in group C were untreated controls. The lambs were monitored before and after treatment and the clinical findings were scored; they were euthanatised 42 days after treatment for pathological examination of the lungs. Groups were similar at the start of a field trial with respect to all parameters (general clinical condition, presence of nasal discharge, presence of ophthalmic discharge, results of lung auscultation) evaluated (P > 0.05). After treatment, no systemic or local adverse reactions were observed in any lamb. Forty-two days after treatment, the median general clinical assessment score of groups MH, ML3 and T was 0, that of group ML2 was 1 and that of group C lambs was 2 (P < 0.01). Treatment with marbofloxacin was associated with improved clinical scores; the results were similar for all evaluations 14 days after treatment and subsequently. Clinical cure rate 42 days after treatment was 100, 100, 42, 100 and 0% for group MH, ML3, ML2, T and C lambs, respectively (P < 0.01). After controlling for initial weight, treatment was found to have a significant effect on carcass weight (P < 0.01). Pairwise differences in lung lesion scores between any of the four treated groups and the untreated controls were significant (P < 0.02). It is concluded that marbofloxacin is effective against respiratory infections of lambs at a dose rate of 3.0 mg/kg bodyweight given on two occasions, once daily 2 days apart or at a dose rate of 2.0 mg/kg given on three occasions for three consecutive days.     

Network Properties of Robust Immunity in Plants

Two modes of plant immunity against biotrophic pathogens, Effector Triggered Immunity (ETI) and Pattern-Triggered Immunity (PTI), are triggered by recognition of pathogen effectors and Microbe-Associated Molecular Patterns (MAMPs), respectively. Although the jasmonic acid (JA)/ethylene (ET) and salicylic acid (SA) signaling sectors are generally antagonistic and important for immunity against necrotrophic and biotrophic pathogens, respectively, their precise roles and interactions in ETI and PTI have not been clear. We constructed an Arabidopsis dde2/ein2/pad4/sid2-quadruple mutant. DDE2, EIN2, and SID2 are essential components of the JA, ET, and SA sectors, respectively. The pad4 mutation affects the SA sector and a poorly characterized sector. Although the ETI triggered by the bacterial effector AvrRpt2 (AvrRpt2-ETI) and the PTI triggered by the bacterial MAMP flg22 (flg22-PTI) were largely intact in plants with mutations in any one of these genes, they were mostly abolished in the quadruple mutant. For the purposes of this study, AvrRpt2-ETI and flg22-PTI were measured as relative growth of Pseudomonas syringae bacteria within leaves. Immunity to the necrotrophic fungal pathogen Alternaria brassicicola was also severely compromised in the quadruple mutant. Quantitative measurements of the immunity levels in all combinatorial mutants and wild type allowed us to estimate the effects of the wild-type genes and their interactions on the immunity by fitting a mixed general linear model. This signaling allocation analysis showed that, contrary to current ideas, each of the JA, ET, and SA signaling sectors can positively contribute to immunity against both biotrophic and necrotrophic pathogens. The analysis also revealed that while flg22-PTI and AvrRpt2-ETI use a highly overlapping signaling network, the way they use the common network is very different: synergistic relationships among the signaling sectors are evident in PTI, which may amplify the signal; compensatory relationships among the sectors dominate in ETI, explaining the robustness of ETI against genetic and pathogenic perturbations.     

Effects of nanowollastonite on biological resistance of particleboard made from wood chips and chicken feather against Antrodia vaillantii

The effect of wollastonite nanofibers (NW) on biological resistance of particleboard, made from wood chips and chicken-feather fibers (CF), against Antrodia vaillantii was studied. 10% of NW, as well as 5 and 10% of CF, were applied to the particleboard-matrix based on the dry weight of wood chips. Then, as a complimentary study, 10% wood fibers were also added to the wood chips; totaling, 10 mixing treatments. Specimens were prepared and mass loss (ML) values were measured in accordance with the EN 113 specifications. Results indicated that the highest ML (37%) occurred in the control treatment without NW- or CF-content; the lowest ML was seen in panels with 10% of NW, CF, and wood fibers (2.5%). NW substantially decreased ML in all mixing ratios. CF also showed a significant decreasing effect on ML. No significant correlation was found between ML values with any of physical and mechanical properties; however, high significant correlations were found between most of the mechanical properties. It is concluded that NW may be considered an effective filler in wood-composite industry to increase the durability against fungal attack; however, more studies should first be conducted on different fungi and wood and wood-composite materials to finalize this conclusion.     

Microarray and Proteomic Analyses of Myeloproliferative Neoplasms with a Highlight on the mTOR Signaling Pathway

The gene and protein expression profiles in myeloproliferative neoplasms (MPNs) may reveal gene and protein markers of a potential clinical relevance in diagnosis, treatment and prediction of response to therapy. Using cDNA microarray analysis of 25,100 unique genes, we studied the gene expression profile of CD34⁺ cells and granulocytes obtained from peripheral blood of subjects with essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF). The microarray analyses of the CD34⁺ cells and granulocytes were performed from 20 de novo MPN subjects: JAK2 positive ET, PV, PMF subjects, and JAK2 negative ET/PMF subjects. The granulocytes for proteomic studies were pooled in 4 groups: PV with JAK2 mutant allele burden above 80%, ET with JAK2 mutation, PMF with JAK2 mutation and ET/PMF with no JAK2 mutation. The number of differentially regulated genes was about two fold larger in CD34⁺ cells compared to granulocytes. Thirty-six genes (including RUNX1, TNFRSF19) were persistently highly expressed, while 42 genes (including FOXD4, PDE4A) were underexpressed both in CD34⁺ cells and granulocytes. Using proteomic studies, significant up-regulation was observed for MAPK and PI3K/AKT signaling regulators that control myeloid cell apoptosis and proliferation: RAC2, MNDA, S100A8/9, CORO1A, and GNAI2. When the status of the mTOR signaling pathway related genes was analyzed, PI3K/AKT regulators were preferentially up-regulated in CD34⁺ cells of MPNs, with down-regulated major components of the protein complex EIF4F. Molecular profiling of CD34⁺ cells and granulocytes of MPN determined gene expression patterns beyond their recognized function in disease pathogenesis that included dominant up-regulation of PI3K/AKT signaling.     

Extracellular vesicles from Trichinella spiralis: Proteomic analysis and protective immunity

Trichinella spiralis (T. spiralis) derived extracellular vesicles (EVs) have been proposed to play a key role in regulating the host immune responses. In this study, we provided the first investigation of EVs proteomics released by T. spiralis muscle larvae (ML). T. spiralis ML EVs (Ts-ML-EVs) were successfully isolated and characterized by transmission electron microscopy (TEM) and western blotting. Using liquid chromatograph mass spectrometer (LC-MS/MS) analysis, we identified 753 proteins in the Ts-ML-EVs proteome and annotated by gene ontology (GO). These proteins were enriched in different categories by GO, kyoto encyclopedia of genes and genomes (KEGG) and domain analysis. GO enrichment analysis indicated association of protein deglutathionylation, lysosomal lumen and serine-type endopeptidase inhibitor activity with proteins which may be helpful during parasite-host interaction. Moreover, KEGG enrichment analysis revealed involvement of Ts-ML-EVs proteins in other glycan degradation, complement and coagulation cascades, proteasome and various metabolism pathways. In addition, BALB/c mice were immunized by subcutaneous injection of purified Ts-ML-EVs. Ts-ML-EVs group demonstrated a 23.4% reduction in adult worms and a 43.7% reduction in ML after parasite challenge. Cellular and humoral immune responses induced by Ts-ML-EVs were detected, including the levels of specific antibodies (IgG, IgM, IgE, IgG1 and IgG2a) as well as cytokines (IL-12, IFN-γ, IL-4 and IL-10) in serum. The results showed that Ts-ML-EVs could induce a Th1/Th2 mixed immune response with Th2 predominant. This study revealed a potential role of Ts-ML-EVs in T. spiralis biology, particularly in the interaction with host. This work provided a critical step to against T. spiralis infection based on Ts-ML-EVs.     

Design and synthesis of new substituted spirooxindoles as potential inhibitors of the MDM2–p53 interaction

The designed compounds, 4a–p, were synthesized using a simple and smooth method with an asymmetric 1,3-dipolar reaction as the key step. The chemical structures for all synthesized compounds were elucidated and confirmed by spectral analysis. The molecular complexity and the absolute stereochemistry of 4b and 4e designed analogs were determined by X-ray crystallographic analysis. The anticancer activities of the synthesized compounds were tested against colon (HCT-116), prostate (PC-3), and hepatocellular (HepG-2) cancer cell lines. Molecular modeling revealed that the compound 4d binds through hydrophobic–hydrophobic interactions with the essential amino acids (LEU: 57, GLY: 58, ILE: 61, and HIS: 96) in the p53-binding cleft, as a standard p53-MDM2 inhibitor (6SJ). The mechanism underlying the anticancer activity of compound 4d was further evaluated, and the study showed that compound 4d inhibited colony formation, cell migration, arrested cancer cell growth at G2/M, and induced apoptosis through intrinsic and extrinsic pathways. Transactivation of p53 was confirmed by flow cytometry, where compound 4d increased the level of activated p53 and induced mRNA levels of cell cycle inhibitor, p21.     

阿根廷滑柔鱼两个群体间耳石和角质颚的形态差异

头足类硬组织具有稳定的形态特征、良好的信息储存以及抗腐蚀性等特点。根据2007年2—5月和2010年1—3月我国鱿钓船采集的阿根廷滑柔鱼样品,提取出625对耳石和787对角质颚,测量耳石10个形态数据和角质颚12个形态数据,通过除以胴长(Mantle Length,ML)校正后,对南巴塔哥尼亚群体(South Patagonic Stock,SPS)和布宜诺斯艾利斯-巴塔哥尼亚群体(Bonaerensis-Northpatagonic Stock,BNS)形态差异进行分析,并建立不同群体的判别函数。结果表明,BNS群体耳石和角质颚外形参数雌性大于雄性,而SPS群体则为雄性大于雌性。均数差异性检验认为,同一群体不同性别的耳石总长(Total StatolithLength,TSL)、最大宽度(Maximum Width,MW)、侧区长(Lateral Dome Length,LDL)、翼区长(Wing Length,WL)、翼区宽(WingWidth,WW)存在显著差异(P<0.05),同性别不同群体的MW、背侧区长(Dorsal Lateral Length,DLL)、吻侧区长(RostrumLateral Length,RLL)和WW存在显著差异(P<0.05)。而同一群体不同性别间角质颚的上头盖长(Upper Hood length,UHL)、上脊突长(Upper Crest length,UCL)、上喙长(Upper Rostrum length,URL)、上喙宽(Upper rostrum width,URW)、上侧壁长(UpperLateral wall length,ULWL)、下喙长(Lower Rostrum length,LRL)存在显著差异(P<0.01),同一性别不同群体角质颚的下头盖长(Lower Hood length,LHL)、下脊突长(Lower crest length,LCL)、LRL、下喙宽(Lower Rostrum width,LRW)、下侧壁长(LowerLateral wall length,LLWL)、下翼长(Lower Wing length,LWL)存在显著差异(P<0.01)。耳石形态参数经主成分分析,认为BNS群体雌雄的主成分因子主要集中在TSL/ML、DLL/ML、RW/ML和MW/ML,SPS群体主要集中于TSL/ML、RW/ML、WW/ML和DDL/ML;角质颚形态参数经主成分分析,认为BNS群体主成分因子主要集中在UHL/ML、UCL/ML、ULWL/ML和LRW/ML,SPS群体主要集中在UHL/ML、UCL/ML、ULWL/ML、URL/ML、LWL/ML和LRL/ML。利用角质颚和耳石对两群体样本分性别建立了判别函数,判别正确率均高于60%,所划分群体在部分形态指标上差异明显,具有一定的可信度。今后应加强样本采集个体大小和时间跨度,以更好的分析其群体变化规律。     

Electron transfer patterns of the di-heme protein cytochrome c4 from Pseudomonas stutzeri

We report kinetic data for the two-step electron transfer (ET) oxidation and reduction of the two-domain di-heme redox protein Pseudomonas stutzeri cytochrome (cyt) c₄ by [Co(bipy)₃]^2+/3+ (bipy = 2,2′-bipyridine). Following earlier reports, the data accord with both bi- and tri-exponential kinetics. A complete kinetic scheme includes both “cooperative” intermolecular ET between each heme group and the external reaction partner, and intramolecular ET between the two heme groups. A new data analysis scheme shows unequivocally that two-ET oxidation and reduction of P. stutzeri cyt c₄ is entirely dominated by intermolecular ET between the heme groups and the external reaction partner in the ms time range, with virtually no contribution from intramolecular interheme ET in this time range. This is in striking contrast to two-ET electrochemical oxidation or reduction of P. stutzeri cyt c₄ for which fast, ms to sub-ms intramolecular interheme ET is a crucial step. The rate constant dependence on the solvent viscosity has disclosed strong coupling to both a (set of) frictionally damped solvent/protein nuclear modes and intramolecular friction-less “ballistic” modes, indicative of notable protein structural mobility in the overall two-ET process. We suggest that conformational protein mobility blocks intramolecular interheme ET in bulk homogeneous solution but triggers opening of this gated ET channel in the electrochemical environment or in the membrane environment of natural respiratory cyt c₄ function.     

Hcn1 Is a Tremorgenic Genetic Component in a Rat Model of Essential Tremor

Genetic factors are thought to play a major role in the etiology of essential tremor (ET); however, few genetic changes that induce ET have been identified to date. In the present study, to find genes responsible for the development of ET, we employed a rat model system consisting of a tremulous mutant strain, TRM/Kyo (TRM), and its substrain TRMR/Kyo (TRMR). The TRM rat is homozygous for the tremor (tm) mutation and shows spontaneous tremors resembling human ET. The TRMR rat also carries a homozygous tm mutation but shows no tremor, leading us to hypothesize that TRM rats carry one or more genes implicated in the development of ET in addition to the tm mutation. We used a positional cloning approach and found a missense mutation (c. 1061 C>T, p. A354V) in the hyperpolarization-activated cyclic nucleotide-gated 1 channel (Hcn1) gene. The A354V HCN1 failed to conduct hyperpolarization-activated currents in vitro, implicating it as a loss-of-function mutation. Blocking HCN1 channels with ZD7288 in vivo evoked kinetic tremors in nontremulous TRMR rats. We also found neuronal activation of the inferior olive (IO) in both ZD7288-treated TRMR and non-treated TRM rats and a reduced incidence of tremor in the IO-lesioned TRM rats, suggesting a critical role of the IO in tremorgenesis. A rat strain carrying the A354V mutation alone on a genetic background identical to that of the TRM rats showed no tremor. Together, these data indicate that body tremors emerge when the two mutant loci, tm and Hcn1^A354V, are combined in a rat model of ET. In this model, HCN1 channels play an important role in the tremorgenesis of ET. We propose that oligogenic, most probably digenic, inheritance is responsible for the genetic heterogeneity of ET.     

Application of a multi-cylinder evapotranspirometer method for evapotranspiration measurements in wetlands

This paper introduces a multi-cylinder evapotranspirometer method, which can directly measure evapotranspiration (ET) from emergent plants in different species and states as well as simultaneously measure evaporation (E_W) from an open water surface. Values of daily ET from three contrasting reed (Phragmites australis) stands, with different leaf area indexes (LAI), were obtained through in situ measurements of the Baiyangdian wetland using this method during the growing seasons in 2008 and 2009. The results showed that the ET rate of the reed belt was very high, even exceeding 20 mm d⁻¹ under extreme weather conditions. Depending on the LAI change, the annual ET from the different reed canopies ranged from 970 to 2035 mm, whereas the ET/E_W ratios ranged from 2.05 to 3.98. Accuracy analysis results showed that the errors of the measurement from this method were no more than 2 mm. The relative errors of the measurement were correspondingly from 0.04% to 0.33%. It is indicated that the accuracy of our measurement is good enough for the requirements of the ET measurement.