Tube and Pelle are essential components in Drosophila Toll signaling pathway. In this study, we characterized a pair of crustacean homologs of Tube and Pelle in Scylla paramamosain, namely, SpTube and SpPelle, and analyzed their immune functions. The full-length cDNA of SpTube had 2052 bp with a 1578 bp open reading frame (ORF) encoding a protein with 525 aa. A death domain (DD) and a kinase domain were predicted in the deduced protein. The full-length cDNA of SpPelle had 3825 bp with a 3420 bp ORF encoding a protein with 1140 aa. The protein contained a DD and a kinase domain. Two conserved repeat motifs previously called Tube repeat motifs present only in insect Tube or Tube-like sequences were found between these two domains. Alignments and structure predictions demonstrated that SpTubeDD and SpPelleDD significantly differed in sequence and 3D structure. Similar to TubeDD, SpTubeDD contained three common conserved residues (R, K, and R) on one surface that may mediate SpMyD88 binding and two common residues (A and A) on the other surface that may contribute to Pelle binding. By contrast, SpPelleDD lacked similar conservative residues. SpTube, insect Tube-like kinases, and human IRAK4 were found to be RD kinases with an RD dipeptide in the kinase domain. SpPelle, Pelle, insect Pelle-like kinases, and human IRAK1 were found to be non-RD kinases lacking an RD dipeptide. Both SpTube and SpPelle were highly expressed in hemocytes, gills, and hepatopancreas. Upon challenge, SpTube and SpPele were significantly increased in hemocytes by Gram-negative or Gram-positive bacteria, whereas only SpPelle was elevated by White Spot Syndrome Virus. The pull-down assay showed that SpTube can bind to both SpMyD88 and SpPelle. These results suggest that SpTube, SpPelle, and SpMyD88 may form a trimeric complex involved in the immunity of mud crabs against both Gram-negative and Gram-positive bacteria.
Polo-like kinase 1 (PLK1) is a serine/threonine protein kinase and plays a critical role in mitosis. PLK1 has also been regarded as a valuable target for cancer treatment, and several PLK1 inhibitors are currently undergoing clinical investigations. In this study, our data show that the expression level of PLK1 is upregulated in human pancreatic cancer cells. Molecular modeling studies indicate that DMTC inhibits PLK1 activity through competitive displacement of ATP from its binding pocket. Our data further show that DMTC suppresses the proliferation of pancreatic cancer cells and induces the formation of multinucleated cells, ultimately resulting in apoptosis. In addition, combination index analysis demonstrates that DMTC acts synergistically with the chemotherapeutic drug gemcitabine in inhibiting the proliferation of pancreatic cancer cells. These results thus suggest a potential of using PLK1 inhibitors for the treatment of pancreatic cancer.
PLK1; PLK1 inhibitor; cell proliferation and apoptosis; gemcitabine; pancreatic cancer
Thrombotic disease is a leading cause of death and disability worldwide. The development of magnetic resonance molecular imaging provides potential promise for early disease diagnosis. In this study, we explore the preparation and characterization of gadolinium (Gd)-loaded poly (lactic-co-glycolic acid) (PLGA) particles surface modified with the Arg-Gly-Asp-Ser (RGDS) peptide for the detection of thrombus. PLGA was employed as the carrier-delivery system, and a double emulsion solvent-evaporation method (water in oil in water) was used to prepare PLGA particles encapsulating the magnetic resonance contrast agent Gd diethylenetriaminepentaacetic acid (DTPA). To synthesize the Gd-PLGA/chitosan (CS)-RGDS particles, carbodiimide-mediated amide bond formation was used to graft the RGDS peptide to CS to form a CS-RGDS film that coated the surface of the PLGA particles. Blank PLGA, Gd-PLGA, and Gd-PLGA/CS particles were fabricated using the same water in oil in water method. Our results indicated that the RGDS peptide successfully coated the surface of the Gd-PLGA/CS-RGDS particles. The particles had a regular shape, smooth surface, relatively uniform size, and did not aggregate. The high electron density of the Gd-loaded particles and a translucent film around the particles coated with the CS and CS-RGDS films could be observed by transmission electron microscopy. In vitro experiments demonstrated that the Gd-PLGA/CS-RGDS particles could target thrombi and could be imaged using a clinical magnetic resonance scanner. Compared with the Gd-DTPA solution, the longitudinal relaxation time of the Gd-loaded particles was slightly longer, and as the Gd-load concentration increased, the longitudinal relaxation time values decreased. These results suggest the potential of the Gd-PLGA/CS-RGDS particles for the sensitive and specific detection of thrombus at the molecular level.
poly (lactic-co-glycolic acid); Arg-Gly-Asp-Ser peptide; magnetic resonance imaging; thrombus; particle
RNA-sequencing is a powerful tool in studying RNomics. However, the highly abundance of ribosomal RNAs (rRNA) and transfer RNA (tRNA) have predominated in the sequencing reads, thereby hindering the study of lowly expressed genes. Therefore, rRNA depletion prior to sequencing is often performed in order to preserve the subtle alteration in gene expression especially those at relatively low expression levels. One of the commercially available methods is to use DNA or RNA probes to hybridize to the target RNAs. However, there is always a concern with the non-specific binding and unintended removal of messenger RNA (mRNA) when the same set of probes is applied to different organisms. The degree of such unintended mRNA removal varies among organisms due to organism-specific genomic variation. We developed a computer-based method to design probes to deplete rRNA in an organism-specific manner. Based on the computation results, biotinylated-RNA-probes were produced by in vitro transcription and were used to perform rRNA depletion with subtractive hybridization. We demonstrated that the designed probes of 16S rRNAs and 23S rRNAs can efficiently remove rRNAs from Mycobacterium smegmatis. In comparison with a commercial subtractive hybridization-based rRNA removal kit, using organism-specific probes is better in preserving the RNA integrity and abundance. We believe the computer-based design approach can be used as a generic method in preparing RNA of any organisms for next-generation sequencing, particularly for the transcriptome analysis of microbes.
Microbial biomass phosphorus (MBP) is one of the most active forms of phosphorus (P) in soils. MBP plays an important role in the biogeochemical P cycle. To explore MBP distribution and its relationship with other factors, the MBP and rhizosphere soil P concentrations and fractions in six vegetation zones on the eastern slope of Gongga Mountain in SW China were investigated. The MBP distribution followed a parabolic pattern with altitude and the concentration was highest in the subalpine dark coniferous forest (SDC) zone, which was approximately 3500 m above sea level (asl). Below 3500 m asl, the MBP distribution was controlled by precipitation and vegetation type. In addition, temperature, precipitation and vegetation type controlled the MBP distribution at elevations above 3500 m asl. No specific distribution pattern was determined regarding rhizosphere soil P fractions. However, MBP was significantly correlated with the unavailable P fraction in the rhizosphere rather than with the available P fraction. This result suggests that the relationships between the rhizosphere soil P fractions and the MBP depend on time. The microbial biomass element ratios were relatively consistent on the eastern slope of Gongga Mountain. However, variations in the microbial biomass element rations were observed in six of the vegetation zones. The mean C:N:P ratio was 9.0∶1.3∶1. Overall, vegetation type resulted in the observed fluctuations of the microbial biomass element ratio.
Endothelial tubular morphogenesis relies on an exquisite interplay of microtubule dynamics and actin remodeling to propel directed cell migration. Recently, the dynamicity and integrity of microtubules have been implicated in the trafficking and efficient translation of the mRNA for HIF-1α (hypoxia-inducible factor), the master regulator of tumor angiogenesis. Thus, microtubule-disrupting agents that perturb the HIF-1α axis and neovascularization cascade are attractive anticancer drug candidates. Here we show that EM011 (9-bromonoscapine), a microtubule-modulating agent, inhibits a spectrum of angiogenic events by interfering with endothelial cell invasion, migration and proliferation. Employing green-fluorescent transgenic zebrafish, we found that EM011 not only inhibited vasculogenesis but also disrupted preexisting vasculature. Mechanistically, EM011 caused proteasome-dependent, VHL-independent HIF-1α degradation and repressed expression of HIF-1α downstream targets, namely VEGF and survivin. Furthermore, EM011 inhibited membrane ruffling and impeded formation of filopodia, lamellipodia and stress fibers, which are critical for cell migration. These events were associated with a drug-mediated decrease in activation of Rho GTPases- RhoA, Cdc42 and Rac1, and correlated with a loss in the geometric precision of centrosome reorientation in the direction of movement. This is the first report to describe a previously unrecognized, antiangiogenic property of a noscapinoid, EM011, and provides evidence for novel anticancer strategies recruited by microtubule-modulating drugs.
We have previously discovered the naturally occurring antitussive alkaloid noscapine as a tubulin-binding agent that attenuates microtubule dynamics and arrests mammalian cells at mitosis via activation of the c-Jun NH2-terminal kinase pathway. It is well established that the p53 protein plays a crucial role in the control of tumor cell response to chemotherapeutic agents and DNA-damaging agents; however, the relationship between p53-driven genes and drug sensitivity remains controversial. In this study, we compared chemosensitivity, cell cycle distribution, and apoptosis on noscapine treatment in four cell lines derived from the colorectal carcinoma HCT116 cells: p53+/+ (p53-wt), p53−/− (p53-null), p21−/− (p21-null), and BAX−/− (BAX-null). Using these isogenic variants, we investigated the roles of p53, BAX, and p21 in the cellular response to treatment with noscapine. Our results show that noscapine treatment increases the expression of p53 over time in cells with wild-type p53 status. This increase in p53 is associated with an increased apoptotic BAX/Bcl-2 ratio consistent with increased sensitivity of these cells to apoptotic stimuli. Conversely, loss of p53 and p21 alleles had a counter effect on both BAX and Bcl-2 expression and the p53-null and p21-null cells were significantly resistant to the antiproliferative and apoptotic effects of noscapine. All but the p53-null cells displayed p53 protein accumulation in a time-dependent manner on noscapine treatment. Interestingly, despite increased levels of p53, p21-null cells were resistant to apoptosis, suggesting a proapoptotic role of p21 and implying that p53 is a necessary but not sufficient condition for noscapine-mediated apoptosis.
Dengue virus (DENV) infection is the most important arthropod- borne viral disease in human, but antiviral therapy and approved vaccines remain unavailable due to antibody-dependent enhancement (ADE) phenomenon. Many studies showed that pre-membrane (prM)-specific antibodies do not efficiently neutralize DENV infection but potently promote ADE infection. However, most of the binding epitopes of these antibodies remain unknown.
In the present study, we characterized a DENV cross-reactive monoclonal antibody (mAb), 4D10, that neutralized poorly but potently enhanced infection of four standard DENV serotypes and immature DENV (imDENV) over a broad range of concentration. In addition, the epitope of 4D10 was successfully mapped to amino acid residues 14 to18 of DENV1-4 prM protein using a phage-displayed peptide library and comprehensive bioinformatics analysis. We found that the epitope was DENV serocomplex cross-reactive and showed to be highly immunogenic in Balb/c mice. Furthermore, antibody against epitope peptide PL10, like 4D10, showed broad cross-reactivity and weak neutralizing activtity with four standard DENV serotypes and imDENV but significantly promoted ADE infection. These results suggested 4D10 and anti-PL10 sera were infection-enhancing antibodies and PL10 was infection-enhancing epitope.
We mapped the epitope of 4D10 to amino acid residues 14 to18 of DENV1-4 prM and found that this epitope was infection-enhancing. These findings may provide significant implications for future vaccine design and facilitate understanding the pathogenesis of DENV infection.
Dengue virus; prM protein; Epitope; Phage display peptide library; Antibody-dependent enhancement
The present study aimed to determine the association between levels of the gender hormones, follicle-stimulating hormone (FSH), luteinizing hormone (LH), progesterone (P) and prolactin (PRL), and two breast cancer molecular markers, human epidermal growth factor receptor 2 (Her-2) and Ki67, in post-menopausal patients with breast cancer. A retrospective study of the serum hormone levels of FSH, LH, P and PRL and the expression status of Her-2 and Ki67 was performed using 187 post-menopausal females with breast cancer. Her-2+ breast cancer patients exhibited higher serum FSH levels compared with Her-2− patients (69.47±3.219 vs. 58.56±1.516 IU/l). The patients with high Ki67 expression [immunohistochemistry (IHC), 3+] displayed higher FSH (72.51±4.616 vs. 60.53±1.476 IU/l) and LH (32.33±1.916 vs. 26.98±0.8852 IU/l) levels than those with lower Ki67 expression. No correlation was identified between the FSH, LH, P and PRL hormone levels, tumor stages and lymphovascular invasion (LVI). In conclusion, a higher serum FSH level was identified in Her-2+ post-menopausal patients with breast cancer. Higher serum FSH and LH levels were also observed in breast cancer patients with high Ki67 expression. FSH and LH may function in the progression of breast cancer.
breast cancer; human epidermal growth factor receptor 2; Ki67; follicle-stimulating hormone; post-menopausal
Both inhibitory and activating forms of natural killer (NK) cell receptors are found in mammals. The activating receptors play a direct role in the recognition of virally infected or transformed cells and transduce activating signals into the cell by partnering with an adaptor protein, which contains a cytoplasmic activation motif. Activating NK receptors encoded by the mammalian leukocyte receptor complex (e.g., killer cell immunoglobulin-like receptors) and the natural killer complex (e.g., Ly49s) partner with the adaptor protein DAP12, whereas NK receptors encoded in the CD94/NKG2 complex partner with the adaptor protein DAP10. Novel immune-type receptors (NITRs) found in bony fish share several common features with immunoglobulin-type NK receptors. Nitr9 is a putative activating receptor in zebrafish that induces cytotoxicity within the context of human NK cells. One isoform of Nitr9, Nitr9L, is shown here to preferentially partner with a zebrafish ortholog of Dap12. Cross-linking the Nitr9L–Dap12 complex results in activation of the phosphytidylinositol 3-kinase→AKT→extracellular signal-regulated kinase pathway suggesting that the DAP12-based activating pathway is conserved between bony fish and mammals.
Natural cytotoxicity; DAP10; ERK; AKT; DAP12; NITR
The Tat protein of HIV-1 has several well-known properties, such as nucleocytoplasmic trafficking, transactivation of transcription, interaction with tubulin, regulation of mitotic progression, and induction of apoptosis. Previous studies have identified a couple of lysine residues in Tat that are essential for its functions. In order to analyze the functions of all the lysine residues in Tat, we mutated them individually to alanine, glutamine, and arginine. Through systematic analysis of the lysine mutants, we discovered several previously unidentified characteristics of Tat. We found that lysine acetylation could modulate the subcellular localization of Tat, in addition to the regulation of its transactivation activity. Our data also revealed that lysine mutations had distinct effects on microtubule assembly and Tat binding to bromodomain proteins. By correlation analysis, we further found that the effects of Tat on apoptosis and mitotic progression were not entirely attributed to its effect on microtubule assembly. Our findings suggest that Tat may regulate diverse cellular activities through binding to different proteins and that the acetylation of distinct lysine residues in Tat may modulate its interaction with various partners.
The purpose of this study is to investigate the prevalence rate and risk factors of depression in outpatients who were diagnosed with PE. Therefore, between September 2009 and September 2011, 1801 outpatients at andrology clinics were enrolled and consented to participate in our survey by completed a verbal questionnaire. It included the following: (1) demographic data (e.g., age, body mass index), (2) PE duration, medical history, and sexual history, (3) self-estimated intravaginal ejaculatory latency times, (4) the Zung Self-rating Depression Scale (SDS), and (5) the National Institute of Health Chronic Prostatitis Symptom Index (NIH-CPSI) and (6) the International Index of Erectile Function (IIEF-5). The results showed that a total of 1,206 patients were diagnosed with PE. The prevalence rate of depression in these PE patients was 26.78%. Depression was associated with PE duration, NIH-CPSI score, and IIEF-5 score. Risk factors for depression specifically included PE durations for 13–24, 25–60, or ≥61 months, CPSI scores of 15–30 or ≥31, and IIEF-5 scores <22. These findings suggested that several associated factors (PE duration, CPSI scores, and IIEF-5 scores) were the risk factors of depression in men with PE.
The nature of source rocks of basaltic magmas plays a fundamental role in understanding the composition, structure and evolution of the solid earth. However, identification of source lithology of basalts remains uncertainty. Using a parameterization of multi-decadal melting experiments on a variety of peridotite and pyroxenite, we show here that a parameter called FC3MS value (FeO/CaO-3*MgO/SiO2, all in wt%) can identify most pyroxenite-derived basalts. The continental oceanic island basalt-like volcanic rocks (MgO>7.5%) (C-OIB) in eastern China and Mongolia are too high in the FC3MS value to be derived from peridotite source. The majority of the C-OIB in phase diagrams are equilibrium with garnet and clinopyroxene, indicating that garnet pyroxenite is the dominant source lithology. Our results demonstrate that many reputed evolved low magnesian C-OIBs in fact represent primary pyroxenite melts, suggesting that many previous geological and petrological interpretations of basalts based on the single peridotite model need to be reconsidered.
The palliative therapy effect by docetaxel for CRPC patients makes it urgent to improve the therapy. It was suggested that PI3K and androgen receptor-directed combination therapy may be effective for prostate cancer (PCa) patients PTEN negative. However, for those patients PTEN positive, the mechanism of anti-apoptosis survival of cancer cells is not yet well defined. Amplification of AURKA has been detected in 5% of PCa. In this work, Du145, a PTEN positive PCa cell model, was employed to investigate the role of aurora kinase a (AURKA) on cell growth. Inhibition of AURKA expression by shRNA markedly reduced prostate cancer cell viability. Furthermore, we demonstrate that AURKA inhibition induced a remarkable downregulation of AKT activity and Bax induction. Moreover, specific inhibition of the activity of AURKA, but not other aurora family members, by small molecular chemical inhibitors induced significant cell killing effects. Notably, AURKA inhibition sensitized prostate cancer cells to docetaxel treatment. Our work suggests that AURKA-directed monotherapy or combination therapy with docetaxel could be a potent treatment for PCa patients in future.
Prostate cancer; AURKA; p53; docetaxel; castration-resistant prostate cancer; aurora kinases
The present study was conducted to determine whether the activation of TRESK in the dorsal root ganglion (DRG) by the TRESK gene recombinant adenovirus vector inhibits the capsaicin-evoked substance P (SP) release using a radioimmunoassay. TRESK is an outwardly rectifying K+ current channel that contributes to the resting potential and is the most important background potassium channel in DRG. Previous studies have shown that neuropathic pain (NP) is closely related to the regulation of certain potassium channels in DRG neurons, while DRG-released SP is important in the peripheral mechanism of NP. In the present study, the TRESK gene adenovirus vector significantly enhanced the TRESK mRNA and protein of the cultured rat DRG neurons. Radioimmunoassay analysis revealed that the capsaicin-mediated SP release was significantly inhibited by the TRESK gene recombinant adenovirus vector in rat DRG neurons. These findings suggest that TRESK plays a role in adjusting the release of SP in DRG, which is related to NP.
dorsal root ganglion neuron; TRESK; subtance P release
HIV protease inhibitors (PI) are core components of Highly Active Antiretroviral Therapy (HAART), the most effective treatment for HIV infection currently available. However, HIV PIs have now been linked to lipodystrophy and dyslipidemia, which are major risk factors for cardiovascular disease and metabolic syndrome. Our previous studies have shown that HIV PIs activate endoplasmic reticulum (ER) stress and disrupt lipid metabolism in hepatocytes and macrophages. Yet, little is known on how HIV PIs disrupt lipid metabolism in adipocytes, a major cell type involved in the pathogenesis of metabolic syndrome.
Methodology and Principal Findings
Cultured and primary mouse adipocytes and human adipocytes were used to examine the effect of frequently used HIV PIs in the clinic, lopinavir/ritonavir, on adipocyte differentiation and further identify the underlying molecular mechanism of HIV PI-induced dysregulation of lipid metabolism in adipocytes. The results indicated that lopinavir alone or in combination with ritonavir, significantly activated the ER stress response, inhibited cell differentiation, and induced cell apoptosis in adipocytes. In addition, HIV PI-induced ER stress was closely linked to inhibition of autophagy activity. We also identified through the use of primary adipocytes of CHOP−/− mice that CHOP, the major transcriptional factor of the ER stress signaling pathway, is involved in lopinavir/ritonavir-induced inhibition of cell differentiation in adipocytes. In addition, lopinavir/ritonavir-induced ER stress appears to be associated with inhibition of autophagy activity in adipocytes.
Conclusion and Significance
Activation of ER stress and impairment of autophagy activity are involved in HIV PI-induced dysregulation of lipid metabolism in adipocytes. The key components of ER stress and autophagy signaling pathways are potential therapeutic targets for HIV PI-induced metabolic side effects in HIV patients.
Continuous exposure of breast cancer cells to adriamycin induces high expression of P-gp and multiple drug resistance. However, the biochemical process and the underlying mechanisms for the gradually induced resistance are not clear. To explore the underlying mechanism and evaluate the anti-tumor effect and resistance of adriamycin, the drug-sensitive MCF-7S and the drug-resistant MCF-7Adr breast cancer cells were used and treated with adriamycin, and the intracellular metabolites were profiled using gas chromatography mass spectrometry. Principal components analysis of the data revealed that the two cell lines showed distinctly different metabolic responses to adriamycin. Adriamycin exposure significantly altered metabolic pattern of MCF-7S cells, which gradually became similar to the pattern of MCF-7Adr, indicating that metabolic shifts were involved in adriamycin resistance. Many intracellular metabolites involved in various metabolic pathways were significantly modulated by adriamycin treatment in the drug-sensitive MCF-7S cells, but were much less affected in the drug-resistant MCF-7Adr cells. Adriamycin treatment markedly depressed the biosynthesis of proteins, purines, pyrimidines and glutathione, and glycolysis, while it enhanced glycerol metabolism of MCF-7S cells. The elevated glycerol metabolism and down-regulated glutathione biosynthesis suggested an increased reactive oxygen species (ROS) generation and a weakened ability to balance ROS, respectively. Further studies revealed that adriamycin increased ROS and up-regulated P-gp in MCF-7S cells, which could be reversed by N-acetylcysteine treatment. It is suggested that adriamycin resistance is involved in slowed metabolism and aggravated oxidative stress. Assessment of cellular metabolomics and metabolic markers may be used to evaluate anti-tumor effects and to screen for candidate anti-tumor agents.
Electronic supplementary material
The online version of this article (doi:10.1007/s11306-013-0517-x) contains supplementary material, which is available to authorized users.
Cellular metabolomics; Adriamycin; Breast cancer MCF-7 cell line; Drug resistance; Reactive oxygen species; Biomarkers
An investigation of terrestrial bryophyte species diversity and community structure along an altitudinal gradient from 2,001 to 4,221 m a.s.l. in Gongga Mountain in Sichuan, China was carried out in June 2010. Factors which might affect bryophyte species composition and diversity, including climate, elevation, slope, depth of litter, vegetation type, soil pH and soil Eh, were examined to understand the altitudinal feature of bryophyte distribution. A total of 14 representative elevations were chosen along an altitudinal gradient, with study sites at each elevation chosen according to habitat type (forests, grasslands) and accessibility. At each elevation, three 100 m × 2 m transects that are 50 m apart were set along the contour line, and three 50 cm × 50 cm quadrats were set along each transect at an interval of 30 m. Species diversity, cover, biomass, and thickness of terrestrial bryophytes were examined. A total of 165 species, including 42 liverworts and 123 mosses, are recorded in Gongga mountain. Ground bryophyte species richness does not show any clear elevation trend. The terrestrial bryophyte cover increases with elevation. The terrestrial bryophyte biomass and thickness display a clear humped relationship with the elevation, with the maximum around 3,758 m. At this altitude, biomass is 700.3 g m−2 and the maximum thickness is 8 cm. Bryophyte distribution is primarily associated with the depth of litter, the air temperature and the precipitation. Further studies are necessary to include other epiphytes types and vascular vegetation in a larger altitudinal range.
Gemcitabine is among the most efficacious and widely used antimetabolite agents. Its molecular targets are ribonucleotide reductase M1 (RRM1) and elongating DNA. Acquired and de novo resistance as a result of RRM1 overexpression are major obstacles to therapeutic efficacy. We deployed a synthetic lethality screen to investigate if knockdown of 87 selected protein kinases by siRNA could overcome RRM1-dependent gemcitabine resistance in high and low RRM1-expressing model systems. The models included genetically RRM1-modified lung and breast cancer cell lines, cell lines with gemcitabine-induced RRM1 overexpression, and a series of naturally gemcitabine-resistant cell lines. Lead molecular targets were validated by determination of differential gemcitabine activity using cell lines with and without target knock down, and by assessing synergistic activity between gemcitabine and an inhibitor of the lead target. CHK1 was identified has the kinase with the most significant and robust interaction, and it was validated using AZD7762, a small-molecule ATP-competitive inhibitor of CHK1 activation. Synergism between CHK1 inhibition and RRM1-dependent gemcitabine efficacy was observed in cells with high RRM1 levels, while antagonism was observed in cells with low RRM1 levels. In addition, four cell lines with natural gemcitabine resistance demonstrated improved gemcitabine efficacy after CHK1 inhibition. In tumor specimens from 187 patients with non-small-cell lung cancer, total CHK1 and RRM1 in situ protein levels were significantly (p = 0.003) and inversely correlated. We conclude that inhibition of CHK1 may have its greatest clinical utility in malignancies where gemcitabine resistance is a result of elevated RRM1 levels. We also conclude that CHK1 inhibition in tumors with low RRM1 levels may be detrimental to gemcitabine efficacy.
Rho family guanosine triphosphatases (GTPases), such as RhoA, Cdc42, and Rac1, play a fundamental role in various cellular processes. The activation of Rho proteins is catalyzed by guanine nucleotide-exchange factors (GEFs), which promote the exchange of GDP for GTP. The precise mechanisms regulating the activation of Rho proteins are not fully understood. Herein, we demonstrate that RhoA activity is regulated by cylindromatosis (CYLD), a deubiquitinase harboring multiple functions. In addition, we find that RhoA-mediated cytoskeletal rearrangement, chromosome separation, and cell polarization are altered in CYLD-depleted cells. Mechanistically, CYLD does not interact with RhoA; instead, it interacts with and deubiquitinates leukemia-associated RhoGEF (LARG). Our data further show that CYLD-mediated deubiquitination of LARG enhances its ability to stimulate the GDP/GTP exchange on RhoA. These data thus identify LARG as a new substrate of CYLD and provide novel insights into the regulation of RhoA activation. Our results also suggest that the LARG-RhoA signaling pathway may play a role in diverse CYLD-mediated cellular events.
R2 retrotransposable elements exclusively insert into the tandemly repeated rRNA genes, the rDNA loci, of their animal hosts. R2 elements form stable long-term associations with their host, in which all individuals in a population contain many potentially active copies, but only a fraction of these individuals show active R2 retrotransposition. Previous studies have found that R2 RNA transcripts are processed from a 28S co-transcript and that the likelihood of R2-inserted units being transcribed is dependent upon their distribution within the rDNA locus. Here we analyze the rDNA locus and R2 elements from nearly 100 R2-active and R2-inactive individuals from natural populations of Drosophila simulans. Along with previous findings concerning the structure and expression of the rDNA loci, these data were incorporated into computer simulations to model the crossover events that give rise to the concerted evolution of the rRNA genes. The simulations that best reproduce the population data assume that only about 40 rDNA units out of the over 200 total units are actively transcribed and that these transcribed units are clustered in a single region of the locus. In the model, the host establishes this transcription domain at each generation in the region with the fewest R2 insertions. Only if the host cannot avoid R2 insertions within this 40-unit domain are R2 elements active in that generation. The simulations also require that most crossover events in the locus occur in the transcription domain in order to explain the empirical observation that R2 elements are seldom duplicated by crossover events. Thus the key to the long-term stability of R2 elements is the stochastic nature of the crossover events within the rDNA locus, and the inevitable expansions and contractions that introduce and remove R2-inserted units from the transcriptionally active domain.
Selfish transposable elements survive in eukaryotic genomes despite the elaborate mechanisms developed by the hosts to limit their activity. One accessible system that simplifies the complex interactions between element and host involves the R2 elements, which exclusively insert in the tandemly arranged rRNA genes. R2 exhibits remarkable stability in animal lineages even though each insertion inactivates one rRNA gene. Here we determine the size of the rDNA locus and R2 number in natural isolates of Drosophila simulans. Combined with previous data concerning the expression and regulation of R2, we develop a detailed population genetic model for rRNA gene and R2 evolution that duplicates all properties of the rRNA loci in natural populations. Critical components of the model are that only a contiguous 40 unit array of rRNA gene units are needed for transcription, that R2 elements are active only when present in this transcription domain, and that most of the crossovers in the rDNA loci occur in this domain. These results suggest that the key to the long-term survival of R2 is the redistribution of rDNA units in the locus brought about by the crossovers that maintain sequence identity in all rDNA units.
Microgels with two interpenetrating polymer networks of poly-N-isopropylacrylamide and poly-acrylic acid (PNIPAM-IPN-PAAc) were synthesized using a seed method. The IPN microgels in water have an average hydrodynamic radius of about 85 nm at 21 °C, measured by dynamic light scattering method. The atomic force microscope image showed that the particles were much smaller after they were dried but remain their spherical shape. The storage and loss moduli G' and G' ' of dispersions of IPN microgels were measured in the linear stress regime as functions of temperature and frequency at various polymer concentrations using a stress-controlled rheometer. For dispersions with high polymer concentration (3.5 and 6.0 wt%) and at high temperatures (34 and 38 °C), the samples behave as viscoelastic solids and the storage modulus was larger than the loss modulus over the entire frequency range. The loss tangent was measured at various frequencies as a function of temperature. The gelation temperature was determined to be 33 °C at the point where a frequency-independent value of the loss tangent was first observed.
Using an animal implantation model, the biocompatibility and drug release properties of the IPN microgl dispersion were evaluated. Fluorescein as a model drug was mixed into an aqueous microgel dispersion at ambient temperature. This drug loaded liquid was then injected subcutaneously in Balb/C mice from Taconic Farms. The test results have shown that the IPN microgels were biocompatible in this acute implantation model and the presence of gelled microgel dispersion substantially slowed the release of fluorescein.
Inflammatory diseases are associated with the accumulation of activated inflammatory cells, particularly polymorphonuclear neutrophils (PMN), which release reactive oxygen species (ROS) to eradicate foreign bodies and microorganisms. To assess the location and extent of localized inflammatory responses, L-012, a highly-sensitive chemiluminescence probe, was employed to non-invasively monitor the production of ROS. We find that L-012-associated chemiluminescence imaging can be used to identify and to quantify the extent of inflammatory responses. Furthermore, regardless of differences among animal models, there is a good linear relationship between chemiluminescence intensity and PMN numbers surrounding inflamed tissue. Depletion of PMN substantially diminished L-012-associated chemiluminescence in vivo. Finally, L-012-associated chemiluminescence imaging was found to be a powerful tool for assessing implant-mediated inflammatory responses by measuring chemiluminescent intensities at the implantation sites. These results support the use of L-012 for monitoring the kinetics of inflammatory responses in vivo via the detection and quantification of ROS production.
Reactive oxygen species; non-invasive imaging; Inflammatory responses; Biomaterials; Chemiluminescence
Angiomatous nasal polyps (ANPs), also known as angiectatic polyps, have rarely been reported in the literature. ANPs are characterized by extensive vascular proliferation and ectasia. ANPs can grow rapidly and exhibit aggressive clinical behavior that could simulate malignancy preoperatively, and they are easily confused with other diseases. In the present study, we analyzed the correlation between the computed tomography (CT) findings of nasal angiomatous polyps and their pathological features.
We evaluated CT findings and pathological features of 31 surgically proven ANPs.
The study population included 16 males and 15 females aged between 27 and 81 years (mean age, 53.5 years). On CT, the masses were heterogeneous; they had a soft tissue density and filled the maxillary and/or nasal cavities. Calcifications were found in 2 of the 31 cases. The lesions showed a clear boundary (15/31). The low-density shading on CT was related to the inflammatory, necrotic, and cystic changes, and the high-density shading on CT was related to hemorrhagic areas of the mass. On contrast-enhanced CT, the center of the lesions was non-enhanced with peripheral intensification due to occlusion or compression of feeder vessels of the polyp center, and the inflammatory cells and neovascularization around the edge of the mass. The most common site of maxillary wall erosion was the medial wall (21/31), followed by the posterior lateral wall (3/31), upper wall (2/31), and septum (3/31). Of these, the nasal cavity and/or maxillary sinus were enlarged in 28 cases. These findings were associated with the chronic progress of nasal angiomatous changes.
CT of ANPs may demonstrate benign bone changes associated with the lesions and may also reflect the fact that ANPs do not invade peripheral soft tissue. CT demonstrated these lesions consistently and provided information useful for surgical planning.
Ferritin, an iron homeostasis protein, has important functions in transition and storage of toxic metal ions. In this study, the full-length cDNA of ferritin was isolated from Dendrorhynchus zhejiangensis by cDNA library and RACE approaches. The higher similarity and conserved motifs for ferritin were also identified in worm counterparts, indicating that it belonged to a new member of ferritin family. The temporal expression of worm ferritin in haemocytes was analyzed by RT-PCR, and revealed the ferritin could be induced by Cd2+, Pb2+ and Fe2+. The heavy metal binding activity of recombinant ferritin was further elucidated by atomic force microscopy (AFM). It was observed that the ferritin protein could form a chain of beads with different size against three metals exposure, and the largest one with 35∼40 nm in height was identified in the Cd2+ challenge group. Our results indicated that worm ferritin was a promising candidate for heavy metals detoxification.