Immunological functions of heat shock proteins (HSPs) have long been recognized. In this study we aimed to efficiently purify HSP70 from renal cell carcinoma and test it as a tumor antigen for pulsing dendritic cells in vitro. HSP70 was purified from renal cell carcinoma specimens by serial column chromatography on Con A-sepharose, PD-10, ADP-agarose and DEAE-cellulose, and finally subjected to fast protein liquid chromatography (FPLC). Dendritic cells derived from the adherent fraction of peripheral blood mononuclear cells were cultured in the presence of IL-4 and GM-CSF and exposed to tumor HSP70. After 24 hours, dendritic cells were phenotypically characterized by flow cytometry. T cells obtained from the non-adherent fraction of peripheral blood mononuclear cells were then co-cultured with HSP70-pulsed dendritic cells and after 3 days T cell cytotoxicity towards primary cultured renal cell carcinoma cells was examined by Cell Counting Kit-8 assay. Dendritic cells pulsed in vitro with tumor-derived HSP70 expressed higher levels of CD83, CD80, CD86 and HLA-DR maturation markers than those pulsed with tumor cell lysate and comparable to that of dendritic cells pulsed with tumor cell lysate plus TNF-?. Concomitantly, cytotoxic T-lymphocytes induced by HSP70-pulsed dendritic cells presented the highest cytotoxic activity. There were no significant differences when using homologous or autologous HSP70 as the tumor antigen. HSP70 can be efficiently purified by chromatography and induces in vitro dendritic cell maturation in the absence of TNF-?. Conspecific HSP70 may effectively be used as a tumor antigen to pulse dendritic cells in vitro.
RNA editing increases transcriptome diversity through post-transcriptional modifications of RNA. Adenosine deaminases that act on RNA (ADARs) catalyze the adenosine-to-inosine (A-to-I) conversion, the most common type of RNA editing in higher eukaryotes. C. elegans has two ADARs, ADR-1 and ADR-2, but their functions remain unclear. Here, we profiled the RNA editomes of C. elegans at different developmental stages of the wild type and ADAR mutants. We developed a new computational pipeline with a 'bisulfite-seq-mapping-like' step and achieved a 3-fold increase in identification sensitivity. 99.5% of the 47,660 A-to-I editing sites were found in clusters. Of the 3,080 editing clusters, 65.7% overlapped with DNA transposons in noncoding regions and 73.7% could form hairpin structures. The numbers of editing sites and clusters were highest at the L1 and embryonic stages. The editing frequency of a cluster positively correlated with its number of editing sites within it. Intriguingly, for 80% of the clusters with ten or more editing sites, almost all expressed transcripts were edited. Deletion of adr-1 reduced the editing frequency but not the number of editing clusters, whereas deletion of adr-2 nearly abolished RNA editing, indicating a modulating role of ADR-1 and an essential role of ADR-2 in A-to-I editing. Quantitative proteomics analysis showed that adr-2 mutant worms altered the abundance of proteins involved in aging and lifespan regulation. Consistent with this finding, we observed that worms lacking RNA editing were short-lived. Taken together, our results revealed a sophisticated landscape of RNA editing and distinct modes of action of different ADARs.
To analyze the diversity of both Bacteroides and Clostridium in patients with primary gout and the difference from that of normal individuals. And to investigate the relationship between the primary gout and the intestinal flora. Fecal samples of 90 cases with the primary gout and 94 cases normal comparison group were selected, together with the cases that match the filter criteria. The DNA is extracted from the feces. 16S rRNA specific primers of both Bacteroides and Clostridium were adopted for the PCR amplification. The molecular fingerprints of Bacteroides and Clostridium in both the primary gout group and the normal control group were obtained through DGGE and subjected for further analysis on both the diversity and the similarity. Compared with normal individuals, the number of bands and Shannon-Weaver (H') of Bacteroides in patients with primary gout was not reduced, but significantly decreased in Clostridium. Furthermore, the intra-group and inter-group similarity of both Bacteroides and Clostridium were lower. The primary gout has caused the structural change of both Bacteroides and Clostridium, inducing the low similarity, especially for Clostridium. It has statistic significance. The gut predominant flora may play an important role in the development of primary gout.
E2F1 and FOXO3 are two transcription factors that have been shown to participate in cellular senescence. Previous report reveals that E2F1 enhanced cellular senescence in human fibroblast cells, while FOXO transcription factors play against senescence by regulation reactive oxygen species scavenging proteins. However, their functional interplay has been unclear. Here we use E2F1 knockout Murine Embryonic Fibroblasts (MEFs), knockdown RNAi constructs, and ectopic expression of E2F1 to show that it functions by negatively regulating FOXO3. E2F1 attenuates FOXO3-mediated expression of MnSOD and Catalase without affecting FOXO3 protein stability, subcellular localization, or phosphorylation by Akt. We mapped the interaction between E2F1 and FOXO3 to a region including the DNA binding domain of E2F1 and the C-terminal transcription-activation domain of FOXO3. We propose that E2F1 inhibits FOXO3-dependent transcription by directly binding FOXO3 in the nucleus and preventing activation of its target genes. Moreover, knockdown of the C. elegans E2F1 ortholog efl-1 significantly extends lifespan in a manner that requires the activity of the C. elegans FOXO gene daf-16. We conclude that there is an evolutionarily conserved signaling connection between E2F1 and FOXO3, which regulates cellular senescence and aging by regulating the activity of FOXO3. We speculate that drugs and/or therapies that inhibit this physical interaction might be good candidates for reducing cellular senescence and increasing longevity.
We study theoretically four-wave parametric amplification arising from the nonlinear optical response of hybrid molecules composed of semiconductor quantum dots and metallic nanoparticles. It is shown that highly efficient four-wave parametric amplification can be achieved by adjusting the frequency and intensity of the pump field and the distance between the quantum dot and the metallic nanoparticle. Specifically, the induced probe-wave gain is tunable in a large range from 1 to 1.43 × 105. This gain reaches its maximum at the position of three-photon resonance. Our findings hold great promise for developing four-wave parametric oscillators.
One of the goals of tumor immunotherapy is to generate immune cells with potent anti-tumor activity through in vitro techniques using peripheral blood collected from patients. However, cancer patients generally have poor immunological function. Thus using patient T cells, which have reduced in vitro proliferative capabilities and less tumor cell killing activity to generate lymphokine-activated killer (LAK) cells, fails to achieve optimal clinical efficacy. Interleukin-2 (IL-2) is a potent activating cytokine for both T cells and natural killer cells. Thus, this study aimed to identify optimal donors for allogeneic LAK cell immunotherapy based on single nucleotide polymorphisms (SNP) in the IL-2 and IL-2R genes. IL-2 and IL-2R SNPs were analyzed using HRM- PCR. LAK cells were derived from peripheral blood mononuclear cells by culturing with IL-2. The frequency and tumor-killing activity of LAK cells in each group were analyzed by flow cytometry and tumor cell killing assays, respectively. Regarding polymorphisms at IL-2-330 (rs2069762) T/G, LAK cells from GG donors had significantly greater proliferation, tumor-killing activity, and IFN-? production than LAK cells from TT donors (P<0.05). Regarding polymorphisms at IL-2R rs2104286 A/G, LAK cell proliferation and tumor cell killing were significantly greater in LAK cells from AA donors than GG donors (P<0.05). These data suggest that either IL- 2-330(rs2069762)T/G GG donors or IL-2R rs2104286 A/G AA donors are excellent candidates for allogeneic LAK cell immunotherapy.
Structure-specific nucleases play crucial roles in many DNA repair pathways. They must be precisely controlled to ensure optimal repair outcomes; however, mechanisms of their regulation are not fully understood. Here, we report a fission yeast protein, Pxd1, that binds to and regulates two structure-specific nucleases: Rad16XPF-Swi10ERCC1 and Dna2-Cdc24. Strikingly, Pxd1 influences the activities of these two nucleases in opposite ways: It activates the 3' endonuclease activity of Rad16-Swi10 but inhibits the RPA-mediated activation of the 5' endonuclease activity of Dna2. Pxd1 is required for Rad16-Swi10 to function in single-strand annealing, mating-type switching, and the removal of Top1-DNA adducts. Meanwhile, Pxd1 attenuates DNA end resection mediated by the Rqh1-Dna2 pathway. Disabling the Dna2-inhibitory activity of Pxd1 results in enhanced use of a break-distal repeat sequence in single-strand annealing and a greater loss of genetic information. We propose that Pxd1 promotes proper DNA repair by differentially regulating two structure-specific nucleases.
Nosocomial bacterial meningitis requires timely treatment, but what is difficult is the prompt and accurate diagnosis of this disease. The aim of this study was to assess the potential role of decoy receptor 3 (DcR3) levels in the differentiation of bacterial meningitis from non-bacterial meningitis. A total of 123 patients were recruited in this study, among them 80 patients being with bacterial meningitis and 43 patients with non-bacterial meningitis. Bacterial meningitis was confirmed by bacterial culture of cerebrospinal fluid (CSF) culture and enzyme-linked immunosorbent assay (ELISA) was used to detect the level of DcR3 in CSF. CSF levels of DcR3 were statistically significant between patients with bacterial meningitis and those with non-bacterial meningitis (p < 0.001). A total of 48.75% of patients with bacterial meningitis received antibiotic >24 h before CSF sampling, which was much higher than that of non-bacterial meningitis. CSF leucocyte count yielded the highest diagnostic value, with an area under the receiver operating characteristic curve (ROC) of 0.928, followed by DcR3. At a critical value of 0.201 ng/mL for DcR3, the sensitivity and specificity were 78.75% and 81.40% respectively. DcR3 in CSF may be a valuable predictor for differentiating patients with bacterial meningitis from those with non-bacterial meningitis. Further studies are needed for the validation of this study.
It is well known that the addition of Ag into Pd can promote the selectivity of acetylene hydrogenation to ethylene, and early theoretical studies focus on ideal single crystal model catalysts, so it is worth studying relatively realistic catalyst models, such as metal oxide supported PdAg systems. In this work, the reaction mechanisms for acetylene selective hydrogenation on the anatase TiO2(101) supported PdaAgb (a + b = 4) cluster are studied by density functional theory calculations with a Hubbard U correction. The results show that Ag addition to the Pd4 cluster reduces the interaction between the PdAg cluster and the support, and the possible reason is that the amount of electron transfer from the TiO2 support to the PdAg cluster decreases with increasing number of Ag atoms. Consequently the adsorption energies of acetylene and ethylene would become smaller on the anatase supported PdAg cluster as compared to that on the anatase supported Pd4 cluster, and this may help to enhance the selectivity of ethylene formation. Moreover, the reaction kinetics study of acetylene hydrogenation on anatase TiO2(101) supported PdAg cluster shows that the activation energy of the hydrogenation step is higher on the PdAg cluster than that on the pure Pd4 cluster, and thus reduces its catalytic activity. Importantly, the present calculation results suggested that the selectivity of ethylene formation, which is defined as the energy difference between the adsorption energy of ethylene and the barrier for its further hydrogenation, varies with the ratio of Pd/Ag in the PdAg cluster: the Pd3Ag system shows relatively low selectivity compared to that of the pure Pd4 cluster, whereas Pd2Ag2/PdAg3 displays higher selectivity than that of the pure Pd4 cluster. Furthermore, our present results demonstrated that the anatase support plays a key role in the acetylene hydrogenation processes: on one hand, it reduces the reaction activity of acetylene hydrogenation processes compared to the Pd2Ag2/Pd(111) and Pd2Ag2 clusters; on the other hand, it enhances the selectivity of ethylene due to its lower desorption energy. It was also found that the carbon species inside the Pd2Ag2 cluster has little effect on the catalytic selectivity towards ethylene formation, whereas the hydrogenation catalytic activity is enhanced significantly. Finally the role of the Pd2Ag2-anatase interface on the catalytic properties of acetylene hydrogenation was studied, and it was found that the interface can increase the activity of acetylene hydrogenation but the selectivity is not improved.
In relative protein abundance determination from peptide intensities recorded in full mass scans, a major complication that affects quantitation accuracy is signal interference from coeluting ions of similar m/z values. Here, we present pQuant, a quantitation software tool that solves this problem. pQuant detects interference signals, identifies for each peptide a pair of least interfered isotopic chromatograms: one for the light and one for the heavy isotope-labeled peptide. On the basis of these isotopic pairs, pQuant calculates the relative heavy/light peptide ratios along with their 99.75% confidence intervals (CIs). From the peptides ratios and their CIs, pQuant estimates the protein ratios and associated CIs by kernel density estimation. We tested pQuant, Census and MaxQuant on data sets obtained from mixtures (at varying mixing ratios from 10:1 to 1:10) of light- and heavy-SILAC labeled HeLa cells or (14)N- and (15)N-labeled Escherichia coli cells. pQuant quantitated more peptides with better accuracy than Census and MaxQuant in all 14 data sets. On the SILAC data sets, the nonquantified "NaN" (not a number) ratios generated by Census, MaxQuant, and pQuant accounted for 2.5-10.7%, 1.8-2.7%, and 0.01-0.5% of all ratios, respectively. On the (14)N/(15)N data sets, which cannot be quantified by MaxQuant, Census and pQuant produced 0.9-10.0% and 0.3-2.9% NaN ratios, respectively. Excluding these NaN results, the standard deviations of the numerical ratios calculated by Census or MaxQuant are 30-100% larger than those by pQuant. These results show that pQuant outperforms Census and MaxQuant in SILAC and (15)N-based quantitation.
As a core factor in S-RNase-based gametophytic self-incompatibility (GSI), the SCF (SKP1-Cullin1-F-box-Rbx1) complex (including pollen determinant SLF, S-locus-F-box) functions as an E3 ubiquitin ligase on non-self S-RNase. The SCF complex is formed by SKP1 bridging between SLF, CUL1, and Rbx1; however, it is not known whether an SCF complex lacking SKP1 can mediate the ubiquitination of S-RNase. Three SKP1-like genes from pollen were cloned based on the structural features of the SLF-interacting-SKP1-like (SSK) gene and the 'Golden Delicious' apple genome. These genes have a motif of five amino acids following the standard 'WAFE' at the C terminal and, in addition, contain eight sheets and two helices. All three genes were expressed exclusively in pollen. In the yeast two-hybrid and pull-down assays only one was found to interact with MdSFBB and MdCUL1, suggesting it is the SLF-interacting SKP1-like gene in apple which was named MdSSK1. In vitro experiments using MdSSK1, S2-MdSFBB1 (S2-Malus domestica S-locus-F-box brother) and MdCUL1 proteins incubated with S 2-RNase and ubiquitin revealed that the SCF complex ubiquitinylates S-RNase in vitro, while MdSBP1 (Malus domestica S-RNase binding protein 1) could not functionally replace MdSSK1 in the SCF complex in ubiquitinylating S-RNase. According to the above experiments, MdSBP1 is probably the only factor responsible for recognition with S-RNase, while not a component of the SCF complex, and an SCF complex containing MdSSK1 is required for mediating the ubiquitination of S-RNase.
Human cytomegalovirus (HCMV) infections are the leading cause of viral induced birth defects, affecting the central nervous system (CNS) primarily. Fetal CNS is especially vulnerable to CMV induced injury. As HCMV permissive cells, astrocytes are responsible for major glutamate transport and regulate extracellular levels of glutamate avoiding its accumulation which is implicated in neurodegenerative disorders. In this study, highly purified astrocytes isolated from human first trimester aborted fetal brain were infected with HCMV AD169, glutamate uptake function was detected by (3)H labeling technic, and the expression level alterations of glutamate transporters (GLAST, GLT-1), glutamine synthetase (GS) and its activity were also investigated. Protein kinases C (PKC) inhibitor treatment was to identify whether PKC signalling involved in regulating glutamate uptake, protein expression of GLAST, GLT-1, GS and GS activity. Results indicated HCMV AD169 infection could modulate glutamate uptake, expression levels of GLAST, GLT-1, GS and it activity through PKC signalling, suggesting a great susceptibility of human fetal astrocytes to HCMV infection, which significantly alters the uptake and metabolism of an important excitatory amino acid, glutamate, may be a potential mechanism for HCMV associated neurological disease, and an effective therapeutic target in neural diseases.
Limonium sinense (Girard) Kuntze is a traditional Chinese folk medicine used for the treatment of fever, hemorrhage, hepatitis and other disorders. Recently, it was found that the crude polysaccharides from L. sinense (LSP) has significant anti-tumor activity. However, research on the isolation and identification of anti-tumor polysaccharide fractions from LSP has not yet been reported. In this study, three polysaccharides LSP11, LSP21, LSP31 were isolated and purified from LSP by using DEAE-52 cellulose column and Sephadex G-100 column chromatography. It was found that LSP21 exhibited the most significant inhibitory effect on the growth of HepG2 cells in vitro. Further research showed that LSP21 inhibited the growth of HepG2 cells in a dose-dependent manner and could induce cell body shrinkage, chromatin condensation, and reduction in the number of tumor cells with normal morphology which suggested that its cytotoxicity on tumor cell might be related to both inhibition on cell proliferation and inducement of cell death. Finally, the structural characteristics of LSP21 were analyzed by high performance liquid chromatography (HPLC) and gas chromatography (GC). The results showed that LSP21 is a heteropolysaccharide with an average molecular weight of 1.31×10(6) Da and consists of glucose, galactose and mannose in the ratio of 1.77:1:2.38.
Colloids often crystallize into polymorphic structures, which are only separated by marginal differences in free energy. Due to this fact, the face-centred cubic and hexagonal close-packed hard-sphere morphologies, for example, usually crystallize simultaneously from a supersaturated solution. The resulting lack of long-range order in these polymorphic structures has been a significant barrier to the widespread application of these crystals in, for instance, photonic bandgap materials. Here, we report a simple method to stabilize one out of two competing polymorphs by exploiting the fact that they have significantly different spatial distributions of voids. We use a variety of polymeric additives whose geometries can be tuned such that their entropy loss, which is related to crystal void symmetries, is different in the two competing polymorphs. This, in turn, controls which polymorph is most thermodynamically stable, providing a generalizable means to stabilize a selected crystal polymorph from a suite of competing structures.
Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a molecular chaperone involved in multidrug resistance and antiapoptosis in some human tumors, but its regulatory mechanisms have not been revealed in esophageal squamous cell carcinoma (ESCC). In this study, 138 specimens of ESCC were analyzed. TRAP1 was overexpressed in ESCC, particularly in poorly differentiated tumors. To further explore the molecular regulatory mechanism, we constructed specific small interfering RNA-expressing vectors targeting Trap1, and knocked down Trap1 expression in the esophageal cancer cell lines ECA109 and EC9706. Knockdown of Trap1 induced increases in reactive oxygen species and mitochondrial depolarization, which have been proposed as critical regulators of apoptosis. The cell cycle was arrested in G2/M phase, and in vitro inhibition of cell proliferation was confirmed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide and bromodeoxyuridine assays. Furthermore, re-expression of TRAP1 in Trap1 small interfering RNA-transfected ESCC cells restored cell proliferation and cell apoptosis. Bioluminescence of subcutaneously xenografted ESCC tumor cells demonstrated significant inhibition of in vivo tumor growth by Trap1 knockdown. This study shows that TRAP1 was overexpressed in most patients with ESCC, and caused an increase in antiapoptosis potency. TRAP1 may be regarded as a target in ESCC biotherapy.
We find that a stacked pair of graphene ribbon arrays with a lateral displacement can excite plasmon waveguide mode in the gap between ribbons, as well as surface plasmon mode on graphene ribbon surface. When the resonance wavelengthes of plasmon waveguide mode and surface plasmon mode are close to each other, there is a strong electromagnetic interaction between the two modes, and then they contribute together to transmission dip. The plasmon waveguide mode resonance can be manipulated by the lateral displacement and longitudinal interval between arrays due to their influence on the manner and strength of electromagnetic coupling between two arrays. The findings expand our understanding of electromagnetic resonances in graphene-ribbon array structure and may affect further engineering of nanoplasmonic devices and metamaterials.
Many existing studies have demonstrated that pituitary tumor transforming gene (PTTG) expression may contribute to the development of pituitary adenomas (PAs), but individually published studies showed inconclusive results. This meta-analysis aimed to derive a more precise estimation of the relationships of PTTG expression with tumor invasiveness and microvessel density of pituitary adenomas.
Premixed insulin regimens are commonly used for the treatment of patients with type-2 diabetes mellitus (T2DM). However, limited data are available regarding next-step therapy options in cases where premixed insulin fails to provide adequate glycemic control. This 20-week observational study of everyday clinical practice evaluated the efficacy, safety and treatment satisfaction of insulin glargine plus oral anti-diabetic drugs (OADs) in T2DM patients previously treated with premixed insulin.
Self-incompatibility (SI) is a reproductive isolation mechanism in flowering plants. Plants in the Solanaceae, Rosaceae and Plantaginaceae belong to the gametophytic self-incompatibility type. S-RNase, which is encoded by a female-specific gene located at the S locus, degrades RNA in the pollen tube and causes SI. Recent studies have provided evidence that S-RNase is transported non-selectively into the pollen tube, but have not specified how this transportation is accomplished. We show here that the apple (Malus domestica) MdABCF protein, which belongs to group F of the ABC transporter family, assists in transportation of S-RNase into the pollen tube. MdABCF is located in the pollen tube membrane and interacts with S-RNase. S-RNase was unable to enter the pollen tube when MdABCF was silenced by antisense oligonucleotide transfection. Our results show that MdABCF assists in transportation of either self or non-self S-RNase into the pollen tube. Moreover, MdABCF coordinates with the cytoskeleton to transport S-RNase. Blockage of S-RNase transport disrupts self-incompatibility in this system.
Patients with temporal lobe epilepsy (TLE) often suffer from comorbid psychiatric diagnoses such as depression, anxiety, or impaired cognitive performance. Endocannabinoid (eCB) signaling is a key regulator of synaptic neurotransmission and has been implicated in the mechanisms of epilepsy as well as several mood disorders and cognitive impairments.
S-RNase is the female determinant of gametophytic self-incompatibility in apple and is usually considered to be the reason for rejection of pollen. In this study, we investigated the role of microtubules (MTs) in internalization of S-RNases by pollen tubes cultured in vitro. The results showed that S-RNase was imported into the pollen tube where it inhibits pollen tube growth, and that S-RNase is co-localized with the Golgi vesicle during the internalization process. Moreover, MT depolymerization is observed following accumulation of S-RNases in the pollen cytosol. On the other hand, S-RNase was prevented from entering the pollen tube when the pollen was treated with the actin filament (AF) inhibitor latrunculin A (LatA), the MT inhibitor oryzalin, or the MT stabilizer taxol at subtoxic concentrations. These hindered the construction of the MT, with pollen tubes capable of growth under these conditions. Pollen tubes showed improved growth in self-pollinated styles that were pre-treated with taxol. This suggests that cytoskeleton antagonists can prevent S-RNase-mediated inhibition of pollen tubes in vivo by blocking S-RNase internalization. These results suggest that an intact and dynamic cytoskeleton is required for the in vitro internalization of S-RNase, as shown by the effects of various cytoskeleton inhibitors. S-RNase internalization takes place via a membrane/cytoskeleton-based Golgi vesicle system, which can also affect self-incompatibility in apple.
High fluorodeoxyglucose (FDG) uptake by human carcinomas, including head and neck cancers, is associated with a poor prognosis. Glucose transporter-1 (Glut-1) is believed to be an intrinsic marker of hypoxia in malignant tumors. The expression of hypoxia-inducible factor-1? (HIF-1?) and correlated target genes, including Glut-1, is regulated by the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) pathway. However, it remains unclear whether the PI3K/Akt signaling pathway is involved in regulating FDG uptake directly. In the present study, 24 consecutive patients with laryngeal carcinoma were examined pre-operatively and the standardized uptake values (SUV) of the laryngeal carcinomas were determined. Glut-1, HIF-1?, PI3K and phosphorylated-Akt (p-Akt) expression was detected by immunohistochemical staining of paraffin sections from the tumor specimens. Associations among SUVmax, Glut-1, HIF-1?, PI3K and p-Akt protein expression and the other clinical parameters were analyzed. The univariate analyses revealed a significantly shorter survival time along with higher HIF-1? (P=0.018) and PI3K (P=0.008) expression, but the survival time was not significantly correlated with Glut-1 or p-Akt expression. The multivariate analysis demonstrated that higher SUVmax (P=0.043) and PI3K expression (P=0.012) were significantly correlated with a poor survival time. Spearman's rank analysis showed significant correlations between SUVmax and HIF-1? (r=0.577; P=0.003), PI3K (r=1.0; P<0.0001) and p-Akt (r=0.577; P=0.003) expression. PI3K was associated with poorly- and moderately-differentiated laryngeal carcinoma (P=0.012). In conclusion, a high SUVmax indicates a poor prognosis for laryngeal carcinoma. Also, a high SUVmax may be associated with the increased expression of Glut-1, HIF-1?, PI3K and p-Akt.
It has been theorized for decades that mitochondria act as the biological clock of ageing, but the evidence is incomplete. Here we show a strong coupling between mitochondrial function and ageing by in vivo visualization of the mitochondrial flash (mitoflash), a frequency-coded optical readout reflecting free-radical production and energy metabolism at the single-mitochondrion level. Mitoflash activity in Caenorhabditis elegans pharyngeal muscles peaked on adult day 3 during active reproduction and on day 9 when animals started to die off. A plethora of genetic mutations and environmental factors inversely modified the lifespan and the day-3 mitoflash frequency. Even within an isogenic population, the day-3 mitoflash frequency was negatively correlated with the lifespan of individual animals. Furthermore, enhanced activity of the glyoxylate cycle contributed to the decreased day-3 mitoflash frequency and the longevity of daf-2 mutant animals. These results demonstrate that the day-3 mitoflash frequency is a powerful predictor of C.?elegans lifespan across genetic, environmental and stochastic factors. They also support the notion that the rate of ageing, although adjustable in later life, has been set to a considerable degree before reproduction ceases.
mTORC1 plays critical roles in the regulation of protein synthesis, growth, and proliferation in response to nutrients, growth factors, and energy conditions. One of the substrates of mTORC1 is 4E-BP1, whose phosphorylation by mTORC1 reverses its inhibitory action on eIF4E, resulting in the promotion of protein synthesis. Raptor in mTOR complex 1 is believed to recruit 4E-BP1, facilitating phosphorylation of 4E-BP1 by the kinase mTOR. We applied chemical cross-linking coupled with mass spectrometry analysis to gain insight into interactions between mTORC1 and 4E-BP1. Using the cross-linking reagent bis[sulfosuccinimidyl] suberate, we showed that Raptor can be cross-linked with 4E-BP1. Mass spectrometric analysis of cross-linked Raptor-4E-BP1 led to the identification of several cross-linked peptide pairs. Compilation of these peptides revealed that the most N-terminal Raptor N-terminal conserved domain (in particular residues from 89 to 180) of Raptor is the major site of interaction with 4E-BP1. On 4E-BP1, we found that cross-links with Raptor were clustered in the central region (amino acid residues 56-72) we call RCR (Raptor cross-linking region). Intramolecular cross-links of Raptor suggest the presence of two structured regions of Raptor: one in the N-terminal region and the other in the C-terminal region. In support of the idea that the Raptor N-terminal conserved domain and the 4E-BP1 central region are closely located, we found that peptides that encompass the RCR of 4E-BP1 inhibit cross-linking and interaction of 4E-BP1 with Raptor. Furthermore, mutations of residues in the RCR decrease the ability of 4E-BP1 to serve as a substrate for mTORC1 in vitro and in vivo.
The goal of the work described here was to evaluate the role of virtual touch tissue quantification (VTQ) combined with urinary ?2-microglobulin (?2-MG) measurement in the early diagnosis of gouty kidney damage. Two hundred fifty-nine patients with gouty kidney damage and 200 healthy control subjects were tested. The shear wave velocity (SWV) of the renal parenchyma and sinus as determined with VTQ and the urinary ?2-MG level of the two groups were analyzed. Although there were no significant differences in age, body mass index, creatinine level and blood urea nitrogen between the two groups (all p's > 0.05), the aforementioned parameters were higher in the group with gouty kidney damage than in the control group. Urinary ?2-MG levels of the patients with kidney damage were significantly higher than those of the control subjects (t = 6.38, p < 0.01). The SWV of the renal parenchyma was higher than that of the sinus in both groups. Compared with controls, patients with kidney damage had significantly increased renal parenchyma and sinus SWVs (all p-values < 0.05). Urinary ?2-MG level was positively linearly correlated with the SWV of renal parenchyma in patients with kidney damage (r = 0.442, p < 0.0001). However, there was no correlation between urinary ?2-MG level and the SWV of the sinus in patients with kidney damage (r = 0). In the control group, there was no correlation between urinary ?2-MG level and the SWV of the renal parenchyma or sinus. The elasticity of the kidney as determined with VTQ, combined with the urinary ?2-MG level, may be helpful in the early diagnosis of gouty kidney damage.
Cannabinoid type 1 receptor (CB1R), which is traditionally located on axon terminals, plays an important role in the pathology of epilepsy and neurodegenerative diseases by modulating synaptic transmission. Using the pilocarpine model of chronic spontaneous recurrent seizures, which mimics the main features of mesial temporal lobe epilepsy (TLE) with hippocampal sclerosis (HS) in humans, we examined the expression of CB1R in hippocampal astrocytes of epileptic rats. Furthermore, we also examined the expression of astrocytic CB1R in the resected hippocampi from patients with medically refractory mesial TLE. Using immunofluorescent double labeling, we found increased expression of astrocytic CB1R in hippocampi of epileptic rats, whereas expression of astrocytic CB1R was not detectable in hippocampi of saline treated animals. Furthermore, CB1R was also found in some astrocytes in sclerotic hippocampi in a subset of patients with intractable mesial TLE. Detection with immune electron microscopy showed that the expression of CB1R was increased in astrocytes of epileptic rats and modest levels of CB1R were also found on the astrocytic membrane of sclerotic hippocampi. These results suggest that increased expression of astrocytic CB1R in sclerotic hippocampi might be involved in the cellular basis of the effects of cannabinoids on epilepsy.
To evaluate the molecular mechanism of fluoroquinolones resistance in Mycoplasma hominis (MH) clinical strains isolated from urogenital specimens. 15 MH clinical isolates with different phenotypes of resistance to fluoroquinolones antibiotics were screened for mutations in the quinolone resistance-determining regions (QRDRs) of DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE) in comparison with the reference strain PG21, which is susceptible to fluoroquinolones antibiotics. 15 MH isolates with three kinds of quinolone resistance phenotypes were obtained. Thirteen out of these quinolone-resistant isolates were found to carry nucleotide substitutions in either gyrA or parC. There were no alterations in gyrB and no mutations were found in the isolates with a phenotype of resistance to Ofloxacin (OFX), intermediate resistant to Levofloxacin (LVX) and Sparfloxacin (SFX), and those susceptible to all three tested antibiotics. The molecular mechanism of fluoroquinolone resistance in clinical isolates of MH was reported in this study. The single amino acid mutation in ParC of MH may relate to the resistance to OFX and LVX and the high-level resistance to fluoroquinolones for MH is likely associated with mutations in both DNA gyrase and the ParC subunit of topoisomerase IV.
Under favorable conditions, Caenorhabditis elegans larvae grow into reproductive adults after a series of molting cycles. When environmental conditions are harsh, they arrest as dauer larvae. Dafachronic acid (DA), a C. elegans steroid hormone, is required for reproductive development. Here, we report a mass spectrometry (MS) method for absolute quantitation of DA in C. elegans. The extraction of DA from C. elegans was optimized to achieve a recovery rate of greater than 83%. The MS sensitivity to DA increased 100-fold after carboxyl group derivatization with 2-picolylamine. High-resolution selected ion monitoring (HR-SIM) on a Q-Orbitrap mass spectrometer Q Exactive outperformed targeted-MS2 on the same instrument and selected reaction monitoring (SRM) on a triple-quadrupole mass spectrometer TSQ Quantum Discovery. With a limit of quantification as low as 1 pg of DA, the HR-SIM method enables absolute quantification of endogenous DA during the reproductive development of C. elegans. We found that in wild-type (WT) worms, DA increases from 0.04 ± 0.02 ng/mg protein in the L1 larval stage to 1.21 ± 0.67 ng/mg protein in the L2 larval stage and decreases again after the L3 stage. In comparison, four genetic mutants that have a constitutive dauer-formation phenotype due to disrupted insulin, TGF-?, or cGMP signaling all have a very low DA level in the L2 stage (below 15% of the WT). These mutants are able to escape the dauer fate and most of them grow into fertile adults when supplied with exogenous DA. Therefore, a DA spike in the L2 stage is critical for the reproductive development of C. elegans.
Upregulation of sphingosine kinase 1 (SPHK1) protein has been reported to be associated with a poor prognosis in a variety of malignant tumors. However, the role of SPHK1 in bladder cancer (BC) has not been thoroughly elucidated. The purpose of this study was to assess SPHK1 expression and to explore its contribution to BC. Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was conducted to detect SPHK1 mRNA expression in 37 pairs of fresh-frozen BC tissues and corresponding noncancerous tissues. Results showed that SPHK1 mRNA expression level in BC tissues was significantly higher than that in corresponding noncancerous tissues. To investigate the association between SPHK1 protein expression and clinicopathological characteristics of BC, immunohistochemistry (IHC) was performed in 153 archived paraffin-embedded BC samples. Interestingly, high SPHK1 expression was significantly associated with histologic grade (P?=?0.045) and tumor stage (P?0.001) of patients with BC. The Kaplan-Meier survival curve showed that patients with high SPHK1 expression had significantly reduced overall 5-year survival rates (P?0.001). Multivariate Cox regression analysis further suggested that the increased expression of SPHK1 was an independent poor prognostic factor for this disease. In conclusion, our data offer the convincing evidence for the first time that the increased expression of SPHK1 may be involved in the pathogenesis and progression of BC. SPHK1 might be a potential marker to predict the prognosis in BC.
Macroautophagy (autophagy) is crucial for cell survival during starvation and plays important roles in animal development and human diseases. Molecular understanding of autophagy has mainly come from the budding yeast Saccharomyces cerevisiae, and it remains unclear to what extent the mechanisms are the same in other organisms. Here, through screening the mating phenotype of a genome-wide deletion collection of the fission yeast Schizosaccharomyces pombe, we obtained a comprehensive catalog of autophagy genes in this highly tractable organism, including genes encoding three heretofore unidentified core Atg proteins, Atg10, Atg14, and Atg16, and two novel factors, Ctl1 and Fsc1. We systematically examined the subcellular localization of fission yeast autophagy factors for the first time and characterized the phenotypes of their mutants, thereby uncovering both similarities and differences between the two yeasts. Unlike budding yeast, all three Atg18/WIPI proteins in fission yeast are essential for autophagy, and we found that they play different roles, with Atg18a uniquely required for the targeting of the Atg12-Atg5·Atg16 complex. Our investigation of the two novel factors revealed unforeseen autophagy mechanisms. The choline transporter-like protein Ctl1 interacts with Atg9 and is required for autophagosome formation. The fasciclin domain protein Fsc1 localizes to the vacuole membrane and is required for autophagosome-vacuole fusion but not other vacuolar fusion events. Our study sheds new light on the evolutionary diversity of the autophagy machinery and establishes the fission yeast as a useful model for dissecting the mechanisms of autophagy.
MicroRNA (miRNA) has emerged as an important regulator of gene expression in plants. 146 miRNAs were identified from apple (Malus domestica cv. Golden Delicious) by bioinformatic analysis and RNA library sequencing. From these, 135 were conserved and 11 were novel miRNAs. Target analysis predicted one of the novel miRNAs, Md-miRLn11 (Malus domestica microRNA Ln11), targeted an apple nucleotide-binding site (NBS)-leucine-rich repeat (LRR) class protein coding gene (Md-NBS). 5 RACE assay confirmed the ability of Md-miRLn11 to cleave Md-NBS at the 11-12-nt position. Analysis of the expression of Md-miRLn11 and Md-NBS during the optimum invasion period in 40 apple varieties showed that the expression of Md-NBS gene in resistant varieties is higher than in susceptible varieties, with an inverse pattern for Md-miRLn11. Seedlings from the resistant apple variety JiGuan were used to carry out an Agrobacterium infiltration assay, and then inoculated with the apple leaf spot disease. The result showed a clear decline of disease resistance in JiGuan apples. In contrast, the susceptible variety FuJi infiltrated with the Md-NBS gene showed a significant increase in disease resistance. Based on the above results, we propose that Md-miRLn11 regulates Md-NBS gene expression in particular under the condition of pathogen infection, and that the Md-miRLn11 targeting P-loop site may regulate many NBS-LRR protein class genes in woody plants.
We demonstrate that the self-assembly of spherical nanoparticles (NPs), grafted isotropically with polymeric ligands, into anisotropic structures is a manifestation of the fluctuations inherent in small number statistics. Computer simulations show that the organization of ligand atoms around an individual NP is not spatially isotropic for small numbers of grafts and ligand monomers. This inherent, spatially asymmetric ligand distribution causes the effective, two-body inter-NP potential to have a strong orientational dependence, which reproduces the anisotropic assembly observed ubiquitously for these systems. In contrast, ignoring this angular dependence does not permit us to capture NP self-assembly. This idea of fluctuation-driven behavior should be broadly relevant, and, for example, it should be important for the assembly of ligand-decorated quantum dots into arrays.
Raman spectroscopy is a valuable and complementary tool for studying geometrically frustrated magnetic systems due to the intrinsic spin-phonon coupling. Here, we report on a Raman spectroscopic study of the geometrically frustrated spin 1/2 antiferromagnet microcrystalline clinoatacamite Cu2(OH)3Cl, focusing on the anomalous transition into the intermediate phase at T(c1) = 18.1 K. By measuring the temperature-dependent (295-4 K) full spectral profiles and main representative modes in spectral regions from 4000 to 95 cm(-1), we observed probable signatures of successive magnetic transitions near T(c1) = 18 K and T(c2) = 6.4 K in the Raman band frequencies and peak widths of the representative modes. Further, we observed a pronounced Raman spectroscopy background featuring a broad continuum at all temperatures. A quantitative analysis reveals that spin fluctuations may exist on a picosecond time scale in the intermediate phase. The short time scale falls out of the ?SR time window; therefore, in the intermediate phase, the ?SR study as reported in (2005 Phys. Rev. Lett. 95 057201) apparently only probed the local field of the ordered spins but overlooked the quickly fluctuating ones. This is likely to give a reasonable explanation of the fact that only a small entropy release occurs at T(c1) = 18 K although a long-range order is formed.
An N-type four level system where two coupling fields interact with two separate optical transitions was constructed in the present paper. By discussing the behaviors of probing field absorption profiles under the effect of different Rabi frequencies of two coupling fields, EIT, Mollow and Autler-Townes doublet can be seen and mutual transformation between them can be obtained. Multiple transition channels in the system were found and the results show that the system can be divided into several subsystems according to the transition channels. Quantum interference between different transition channels can be realized through different dividing methods, so three nonlinear effects with different generating conditions and physical nature can be seen in the system.
As a negative regulator of P53, MDM2 plays an important role in carcinogenesis; a polymorphism in its promoter region. SNP309 T>G, is known to increase the expression of MDM2, thus being considered related to higher susceptibility to neoplasia. However, no agreement has been achieved regarding its effects on gastric cancer.
Transmission of human cytomegalovirus (HCMV) to the fetus is the most common type of intrauterine infection; the mechanism of HCMV pathogenesis in the developing central nervous system remains unclear. Thrombospondins 1 and 2 (TSP1, TSP2) produced by immature astrocytes are critical for fetal synaptogenesis. To examine the effect of HCMV on fetal astrocytes, human fetal astrocytes were isolated and cultured with HCMV AD169. Cells were harvested at different time points. Protein and mRNA expressions of TSP1 and TSP2 were determined using RT-qPCR, western blotting analysis, and enzyme-linked immunosorbent assay. The results showed that HCMV infection induced time-dependent decreases in mRNA and protein expressions of both TSP1 and TSP2 in astrocytes. Flow cytometry was used to detect apoptosis of HCMV-infected astrocytes, and the result indicated that there was no linkage between cell apoptosis and the decrease in TSP1 and TSP2 expressions induced by HCMV infection. When ganciclovir treatment was performed on HCMV-infected astrocytes, results showed that ganciclovir treatment inhibited the reduction of TSP1 and TSP2 expression in astrocytes. In the further study, pEGFP-N3-IE1 was transfected into astrocytes to identify that it was not IE1 but active viral replication that was essential in the continuous decrease of TSP1 and TSP2 expressions in HCMV-infected astrocytes.
To observe the effect of electroacupuncture (EA) of auricular concha region (ACR), "Zusanli" (ST 36) and "Sanyinjiao"(SP 6) on abnormal skin barrier and color in streptozotocin (STZ)-induced type 2 diabetes mellitus (T 2 DM) rats so as to find a better therapeutic method.
Varieties of the European pear (Pyrus communis) can produce trees with both red- and green-skinned fruits, such as the Max Red Bartlett (MRB) variety, although little is known about the mechanism behind this differential pigmentation. In this study, we investigated the pigmentation of MRB and its green-skinned sport (MRB-G). The results suggest that a reduction in anthocyanin concentration causes the MRB-G sport. Transcript levels of PcUFGT (for UDP-glucose:flavonoid 3-O-glucosyltransferase), the key structural gene in anthocyanin biosynthesis, paralleled the change of anthocyanin concentration in both MRB and MRB-G fruit. We cloned the PcMYB10 gene, a transcription factor associated with the promoter of PcUFGT. An investigation of the 2-kb region upstream of the ATG translation start site of PcMYB10 showed the regions -604 to -911 bp and -1,218 to -1,649 bp to be highly methylated. A comparison of the PcMYB10 promoter methylation level between the MRB and MRB-G forms indicated a correlation between hypermethylation and the green-skin phenotype. An Agrobacterium tumefaciens infiltration assay was conducted on young MRB fruits by using a plasmid constructed to silence endogenous PcMYB10 via DNA methylation. The infiltrated fruits showed blocked anthocyanin biosynthesis, higher methylation of the PcMYB10 promoter, and lower expression of PcMYB10 and PcUFGT. We suggest that the methylation level of PcMYB10 is associated with the formation of the green-skinned sport in the MRB pear. The potential mechanism behind the regulation of anthocyanin biosynthesis is discussed.
To explore the related pathogenesis of degenerative aortic valvular disease by observing the histopathological changes of aortic valves from patients with aortic degenerative stenosis and compare the results with those controls with normal aortic valves.
Regulation of actomyosin dynamics by post-transcriptional modifications in cytoplasmic actin is still poorly understood. Here we demonstrate that dioxygenase ALKBH4-mediated demethylation of a monomethylated site in actin (K84me1) regulates actin-myosin interaction and actomyosin-dependent processes such as cytokinesis and cell migration. ALKBH4-deficient cells display elevated K84me1 levels. Non-muscle myosin II only interacts with unmethylated actin and its proper recruitment to and interaction with actin depend on ALKBH4. ALKBH4 co-localizes with the actomyosin-based contractile ring and midbody via association with methylated actin. ALKBH4-mediated regulation of actomyosin dynamics is completely dependent on its catalytic activity. Disorganization of cleavage furrow components and multinucleation associated with ALKBH4 deficiency can all be restored by reconstitution with wild-type but not catalytically inactive ALKBH4. Similar to actin and myosin knock-out mice, homozygous Alkbh4 mutant mice display early embryonic lethality. These findings imply that ALKBH4-dependent actin demethylation regulates actomyosin function by promoting actin-non-muscle myosin II interaction.
As a master regulator of jasmonic acid (JA)-signaled plant immune responses, the basic helix-loop-helix (bHLH) Leu zipper transcription factor MYC2 differentially regulates different subsets of JA-responsive genes through distinct mechanisms. However, how MYC2 itself is regulated at the protein level remains unknown. Here, we show that proteolysis of MYC2 plays a positive role in regulating the transcription of its target genes. We discovered a 12-amino-acid element in the transcription activation domain (TAD) of MYC2 that is required for both the proteolysis and the transcriptional activity of MYC2. Interestingly, MYC2 phosphorylation at residue Thr328, which facilitates its turnover, is also required for the MYC2 function to regulate gene transcription. Together, these results reveal that phosphorylation-coupled turnover of MYC2 stimulates its transcription activity. Our results exemplify that, as with animals, plants employ an "activation by destruction" mechanism to fine-tune their transcriptome to adapt to their ever-changing environment.
The aim of the present study was to investigate the association between genetic variants in 17 tagSNPs of the NLRP3 gene and the susceptibility to primary gouty arthritis. A genotype-phenotype analysis of 480 primary gout and 480 control patients was performed. Samples from all the patients were collected from The Affiliated Hospital of Medical College (Qingdao, China). Seventeen tagSNPs of the NLRP3 gene were amplified using polymerase chain reaction (PCR) and MassARRAY technology was used for single nucleotide polymorphism (SNP) genotyping. The genetic frequency of rs7512998 was significantly different between the gout and control patients (P<0.05), whereas no significant differences were identified for the remaining SNPs. The 17 SNPs conformed to the Hardy-Weinberg equilibrium (HWE) in the control group (P>0.05). The haplotype association among the 17 SNPs of the NLRP3 gene indicated that no individual SNP was significantly associated with primary gouty arthritis. CTATCAGCGCCCAGTGC was the most common haplotype in the case and control groups, with a frequency of 0.224 and 0.243, respectively. However, the odds ratios (ORs) of the 8 haplotypes were not identified to be significantly associated with gouty arthritis (P>0.05 for all the 8 haplotypes). To the best of our knowledge, this is the first study to investigate the association between SNPs of the NLRP3 gene and the risk of primary gouty arthritis, although no significant association was identified. Further clinical studies and functional analysis are required to explore the potential associations between NLRP3 gene polymorphisms and the risk of primary gouty arthritis.
The insulin-like signaling pathway maintains a relatively short wild-type lifespan in Caenorhabditis elegans by phosphorylating and inactivating DAF-16, the ortholog of the FOXO transcription factors of mammalian cells. DAF-16 is phosphorylated by the AKT kinases, preventing its nuclear translocation. Calcineurin (PP2B phosphatase) also limits the lifespan of C. elegans, but the mechanism through which it does so is unknown. Herein, we show that TAX-6•CNB-1 and UNC-43, the C. elegans Calcineurin and Ca(2+)/calmodulin-dependent kinase type II (CAMKII) orthologs, respectively, also regulate lifespan through DAF-16. Moreover, UNC-43 regulates DAF-16 in response to various stress conditions, including starvation, heat or oxidative stress, and cooperatively contributes to lifespan regulation by insulin signaling. However, unlike insulin signaling, UNC-43 phosphorylates and activates DAF-16, thus promoting its nuclear localization. The phosphorylation of DAF-16 at S286 by UNC-43 is removed by TAX-6•CNB-1, leading to DAF-16 inactivation. Mammalian FOXO3 is also regulated by CAMKIIA and Calcineurin. DOI:http://dx.doi.org/10.7554/eLife.00518.001.
C. elegans PUD-1 and PUD-2, two proteins up-regulated in daf-2(loss-of-function) (PUD), are homologous 17-kD proteins with a large abundance increase in long-lived daf-2 mutant animals of reduced insulin signaling. In this study, we show that both PUD-1 and PUD-2 are abundantly expressed in the intestine and hypodermis, and form a heterodimer. We have solved their crystal structure to 1.9-Å resolution and found that both proteins adopt similar ?-sandwich folds in the V-shaped dimer. In contrast, their homologs PUD-3, PUD-4, PUDL-1 and PUDL-2 are all monomeric proteins with distinct expression patterns in C. elegans. Thus, the PUD-1/PUD-2 heterodimer probably has a function distinct from their family members. Neither overexpression nor deletion of pud-1 and pud-2 affected the lifespan of WT or daf-2 mutant animals, suggesting that their induction in daf-2 worms does not contribute to longevity. Curiously, deletion of pud-1 and pud-2 was associated with a protective effect against paralysis induced by the amyloid ?-peptide (1-42), which further enhanced the protection conferred by daf-2(RNAi) against A?.
Following our previous work on the solvent-response of uncharged diblock copolymer brushes (D. Meng and Q. Wang, J. Chem. Phys. 130, 134904 (2009)), we have performed continuum self-consistent field calculations to study the response (i.e., changes of brush height and surface-layer composition) of diblock brushes with only one block charged to various external stimuli, including solvent selectivity, solution pH, ionic strength, and applied electric field. Our study revealed complex interplay among various stimuli, which needs to be well understood in order to design smart surfaces from charged diblock brushes.
To assess the association between a C421A single nucleotide polymorphism (SNP) in exon 5 of ATP-binding cassette, sub-family G (WHITE), member 2 (ABCG2) gene and susceptibility of primary gout in Han Chinese males.
At room temperature, the mid-infrared spectra of geometrically frustrated natural atacamite (hydroxyl copper chloride, beta-Cu2(OH)3Cl) in the range of 4 000-400 cm(-1) were measured by FTIR spectrometers, and meanwhile its Raman spectrum in the range of 4 000-95 cm(-1) was obtained by Jobin Yvon LabRAM HR800 Raman spectrometer. According to its crystal structure parameters, the authors confirmed the characteristic peaks of sample 4 000-2 500-1 000 cm(-1) in the functional group region and 1 000-550-200-95 cm(-1) in the fingerprint region, and also explored its microscopic origin Five distinct regions were assigned: the hydroxyl stretching vibration v(O-H) determined by the overall environment around the hydroxyl group; the overtones generated by the sum or multiplication of fundamental frequencies of hydroxyl bending vibration; the hydroxyl bending vibration modes delta(O-H) of the combination of delta(Cu-O-H) and delta(O-H...HCl); the vibration modes of strongly bonded planar CuO4 units; the vibration modes of weakly bonded linear-triatomic chain Cl-Cu-O/Cl. The bands were assigned in accordance with its crystal structure parameters, which is more reasonable to establish the relationship between its molecular structure and its respective spectral properties.
Functional nuclear medicine imaging techniques have become particularly important in the diagnosis of osteomyelitis. The aim of our study was to perform a meta-analysis to obtain a reliable estimate of the diagnostic performance of fluorodeoxyglucose-positron emission tomography (FDG-PET), three-phase bone scintigraphy, leukocyte scintigraphy, and monoclonal antigranulocyte antibody (MOAB) scintigraphy in the assessment of suspected osteomyelitis and to perform pairwise comparisons of the diagnostic accuracy between these different imaging modalities.
Spinocerebellar ataxia type 10 (SCA10) is an autosomal dominant neurologic disorder, whose symptoms include cerebellar ataxia and epilepsy. The disease is caused by ATTCT expansion in the ATXN10 gene, which encodes the Ataxin-10 protein. Here we identified polo-like kinase 1 (Plk1) as one of Ataxin-10s binding partners. We show that epitope-tagged Ataxin-10 and Plk1 coimmunoprecipitate, and Plk1 phosphorylates Ataxin-10 at S77 and T82 in vitro. Knockdown of ATXN10 with siRNA in HeLa cells results in cytokinesis defects-multinucleation, which are rescued by wild-type Ataxin-10, but not the phosphor-deficient 2A mutant. Phosphorylation-specific antibodies towards pS77 detect specific signals at the midbody. Like the knockdown, overexpression of the 2A mutant generates multinucleated cells and the 2A mutant shows decreased interaction with the Plk1 polo-box domain. In addition, we found that Ataxin-10 is ubiquitinated, and is subject to proteasome-dependent degradation, which is delayed in the 2A mutant. We propose a model in which Plk1 phosphorylation of Ataxin-10 influences its degradation and cytokinesis, which may provide mechanistic insight to SCA10s pathogenesis.
The genomic material of hepatitis B virus (HBV) is confined within a fenestrated nucleocapsid consisting of 240 identical copies of the capsid protein, which has a rigid core and a positively charged and highly flexible C-terminal domain (CTD). Although previous mutagenesis studies have demonstrated the importance of the CTD in viral RNA packaging and reverse transcription, the microscopic structure of the CTD and its interaction with encapsidated nucleic acids at various stages of viral maturation remain poorly understood. Here, we present a theoretical analysis of the radial distributions of the CTD chains and nucleic acids in the hepatitis B virus nucleocapsid at the beginning and final stages of viral reverse transcription based on classical density functional theory and a coarse-gained model for the pertinent biomolecules. We find that a significant portion of the CTD is exposed at the surface of the RNA-containing immature nucleocapsid and that the CTD is mostly confined within the DNA-containing mature nucleocapsid. Large accumulation of cations is predicted inside both immature and mature nucleocapsids. The theoretical results provide new insights into the molecular mechanism of CTD regulation of viral reverse transcription and nucleocapsid trafficking during various stages of the viral replication processes.
In order to investigate the special role of HOXB4 in expansion and self renewal of hematopoietic stem cells, the cDNA of HOXB4 was extracted and cloned from umbilical cord blood mononuclear cells by using RT-PCR. Then the eukaryotic expression bicistronic plasmid vector pIRES2-EGFP/HOXB4 was designed and constructed after cutting HOXB4 and pIRES2-EGFP respectively by restriction enzyme EcoRI and BamHI. The recombinant plasmid was delivered into competent cells of Escherichia coli. The successful construction of plasmid was confirmed by the identification of endonuclease cutting and sequencing. The results showed that the HOXB4 cDNA was cloned successfully from umbilical cord blood mononuclear cells and the recombinant eukaryotic expression bicistronic plasmid vector was constructed, and then introduced it into 293T cells successfully. It is concluded that a pIRES2-EGFP/HoxB1 eukaryotic expression bicistronic plasmid vector has been constructed successfully, which results provide a useful material basis for exploration of HoxB4 function in the proliferation and differentiation of hematopoietic cells.
We theoretically demonstrate the coupling between the unit cells and the interaction between constituents within each cell in metamaterials consisting of stacked split ring resonator arrays which are embedded in a homogeneous dielectric. It is found that the resonant frequency due to plasmon hybridization depends on the symmetry of resonance modes. Both for the first and third order plasmon resonances, we show that the resonances at lower frequency are not sensitive to the variation of lattice density, while the resonances at higher frequency rely on the coupling between cells due to the symmetric distribution of current. The underlying physics is qualitatively interpreted according to the quasistatic electric and magnetic dipole coupling model combined by the calculated field distributions.
Nanoparticles delivery of oligonucleotides represents a potential approach for cancer treatment. However, most of the experiments were based on established cancer cell lines and may not reflect the original solid tumor in vivo. Both, tumor microenvironment and tumor cell biological properties in the tumor can influence the delivery efficiency of oligonucleotides. Therefore, it is important to understand the effect of nanoparticles delivery of oligonucleotides on tumor response in intact tissue architecture of individual tumors. We used freshly isolated human tumor tissue slices and primary lung cancer cells from non-small cell lung cancer patients to evaluate this nanocarrier system. Chitosan-coated poly(lactide-co-glycolide) (PLGA) nanoparticles were used to form oligonucleotide-nanoparticle-complexes (nanoplexes) with antisense 2-O-methyl-RNA (OMR) that can inhibit telomerase activity by binding to the RNA component of telomerase. OMR cellular uptake was strongly enhanced by nanoplexes mediated delivery in both, primary cells and tissue slices. More than 80% of primary cancer cells and 50% of cells in tissue slices showed OMR uptake. Telomerase activity was inhibited by approximately 45% in primary cancer cells and about 40% in tissue slices. Nanoplexes could penetrate into tumor tissue without influencing tissue architecture and the delivered OMR was able to inhibit telomerase activity with relatively low cytotoxicity.
The DNA fragment detection focusing technique has further enhanced the sensitivity and information of DNA targets. The DNA fragment detection method was established by capillary electrophoresis with laser-induced fluorescence detection and restriction endonuclease chromatographic fingerprinting (CE-LIF-REF) in our experiment. The silica capillary column was coated with short linear polyarclarylamide (SLPA) using nongel sieving technology. The excision product of various restricted enzymes of DNA fragments was obtained by REF with the molecular biology software Primer Premier 5. The PBR322/BsuRI DNA marker was used to establish the optimization method. The markers were focused electrophoretically and detected by CE-LIF. The results demonstrate that the CE-LIF-REF with SLPA can improve separation, sensitivity and speed of analysis. This technique may be applied to analysis of the excision product of various restricted enzymes of prokaryotic plasmid (pIRES2), eukaryote plasmid (pcDNA3.1) and the PCR product of codon 248 region of gastric cancer tissue. The results suggest that this method could very sensitively separate the excision products of various restricted enzymes at a much better resolution than the traditional agarose electrophoresis.
Cell growth can be suppressed by stressful environments, but the role of stress pathways in this process is largely unknown. Here we show that a cascade of p38? mitogen-activated protein kinase (MAPK) and p38-regulated/activated kinase (PRAK) plays a role in energy-starvation-induced suppression of mammalian target of rapamycin (mTOR), and that energy starvation activates the p38?-PRAK cascade. Depletion of p38? or PRAK diminishes the suppression of mTOR complex 1 (mTORC1) and reduction of cell size induced by energy starvation. We show that p38?-PRAK operates independently of the known mTORC1 inactivation pathways--phosphorylation of tuberous sclerosis protein 2 (TSC2) and Raptor by AMP-activated protein kinase (AMPK)--and surprisingly, that PRAK directly regulates Ras homologue enriched in brain (Rheb), a key component of the mTORC1 pathway, by phosphorylation. Phosphorylation of Rheb at Ser 130 by PRAK impairs the nucleotide-binding ability of Rheb and inhibits Rheb-mediated mTORC1 activation. The direct regulation of Rheb by PRAK integrates a stress pathway with the mTORC1 pathway in response to energy depletion.
Pseudohypoparathyroidism type Ia (PHP Ia) is defined as a series of disorders characterized by multihormone resistance in end-organs and Albright hereditary osteodystrophy (AHO) phenotype. PHP Ia is caused by heterozygous inactivating mutations in GNAS, which encodes the stimulatory G-protein alpha subunit (Gsa). A patient with typical clinical manifestations of pseudohypoparathyroidism (PHP) (round face, short stature, centripetal obesity, brachydactyly, and multi-hormone resistance: parathyroid hormone (PTH), thyroid-stimulating hormone (TSH), and gonadotropins) presented at our center. The sequence of the GNAS gene from the patient and her families revealed a novel missense mutation (Y318H) in the proband and her mother. An in vitro Gsa functional study showed that Gsa function was significantly impaired. These results stress the importance of GNAS gene investigation.
Opening up a band gap in graphene holds a crucial significance in the realization of graphene-based electronics. Doping with organic molecules to alter the electronic properties of graphene is perceived as an effective band gap engineering approach. Using the tight binding model, we examined the band gap opening of monolayer graphene due to the adsorption of pyrene molecules on both of its sides. It was found that the breakdown of the sublattice symmetry in pyrene-dispersed graphene leads to a band gap of ?10 meV.
Nerve growth factor (NGF) is mainly secreted by the neuroglia cells, which can exert biological effect through its receptors on the specific target cell surface. NGF is closely related to neurocyte growth, differentiation and apoptosis. As a neurotropic virus, HSV-1 an easily lead to neurocyte, neuroglia cells death or apoptosis. In this study, the U251 human glioma cells were chosen as target cells to study the change of NGF and its receptors in the apoptosis process of HSV-1 infection. Our results showed that U251 cells were permissive to HSV-1 replication. In the apoptosis process of HSV-1 infected U251 cells, the expression of both NGF and P75NTR increased and then decreased, while the expression of TrkA decreased gradually. These result indicated that HSV-1 was able to induce the abnormal expression of NGF and its receptors in U251 cells.
Mutations in superoxide dismutase 1 (SOD1) cause familial ALS. Mutant SOD1 preferentially associates with the cytoplasmic face of mitochondria from spinal cords of rats and mice expressing SOD1 mutations. Two-dimensional gels and multidimensional liquid chromatography, in combination with tandem mass spectrometry, revealed 33 proteins that were increased and 21 proteins that were decreased in SOD1(G93A) rat spinal cord mitochondria compared with SOD1(WT) spinal cord mitochondria. Analysis of this group of proteins revealed a higher-than-expected proportion involved in complex I and protein import pathways. Direct import assays revealed a 30% decrease in protein import only in spinal cord mitochondria, despite an increase in the mitochondrial import components TOM20, TOM22, and TOM40. Recombinant SOD1(G93A) or SOD1(G85R), but not SOD1(WT) or a Parkinsons disease-causing, misfolded ?-synuclein(E46K) mutant, decreased protein import by >50% in nontransgenic mitochondria from spinal cord, but not from liver. Thus, altered mitochondrial protein content accompanied by selective decreases in protein import into spinal cord mitochondria comprises part of the mitochondrial damage arising from mutant SOD1.
In recent years, electron transfer dissociation (ETD) has enjoyed widespread applications from sequencing of peptides with or without post-translational modifications to top-down analysis of intact proteins. However, peptide identification rates from ETD spectra compare poorly with those from collision induced dissociation (CID) spectra, especially for doubly charged precursors. This is in part due to an insufficient understanding of the characteristics of ETD and consequently a failure of database search engines to make use of the rich information contained in the ETD spectra. In this study, we statistically characterized ETD fragmentation patterns from a collection of 461?440 spectra and subsequently implemented our findings into pFind, a database search engine developed earlier for CID data. From ETD spectra of doubly charged precursors, pFind 2.1 identified 63-122% more unique peptides than Mascot 2.2 under the same 1% false discovery rate. For higher charged peptides as well as phosphopeptides, pFind 2.1 also consistently obtained more identifications. Of the features built into pFind 2.1, the following two greatly enhanced its performance: (1) refined automatic detection and removal of high-intensity peaks belonging to the precursor, charge-reduced precursor, or related neutral loss species, whose presence often set spectral matching askew; (2) a thorough consideration of hydrogen-rearranged fragment ions such as z + H and c - H for peptide precursors of different charge states. Our study has revealed that different charge states of precursors result in different hydrogen rearrangement patterns. For a fragment ion, its propensity of gaining or losing a hydrogen depends on (1) the ion type (c or z) and (2) the size of the fragment relative to the precursor, and both dependencies are affected by (3) the charge state of the precursor. In addition, we discovered ETD characteristics that are unique for certain types of amino acids (AAs), such as a prominent neutral loss of SCH(2)CONH(2) (90.0014 Da) from z ions with a carbamidomethylated cysteine at the N-terminus and a neutral loss of histidine side chain C(4)N(2)H(5) (81.0453 Da) from precursor ions containing histidine. The comprehensive list of ETD characteristics summarized in this paper should be valuable for automated database search, de novo peptide sequencing, and manual spectral validation.
The diagnosis of patients with fever of unknown origin (FUO) remains a challenging medical problem for internal medicine. A reliable estimate of the diagnostic performance of FDG-PET and FDG-PET/CT in the assessment of FUO unidentified by conventional workup has never been systematically assessed, and present systematic review was aimed at this issue.
While fever of unknown origin (FUO) remains a challenging problem in clinical practice, fluorine-18 fluorodeoxyglucose ((18)F-FDG) positron emission tomography/computed tomography (PET/CT) has been considered helpful in diagnosing its cause. The present study is set to evaluate the diagnostic value of PET/CT for patients with FUO.
An important task of aging research is to find genes that regulate lifespan. However, identification of genes related to longevity (referred to as longevity genes hereafter) through web-lab experiments such as genetic screens is a tedious and labor-intensive activity. Developing an algorithm to predict longevity genes should facilitate aging research. In this paper, we systematically analyzed properties of longevity genes in Caenorhabditis elegans and found that, when compared to genes not yet known to be involved in longevity, known longevity genes display the following features: (i) longer genomic sequences and protein sequences, (ii) a stronger tendency to co-express with other genes during a transition from dauer state (an extremely long lifespan) to non-dauer state (a normal lifespan), (iii) significant enrichment in certain functions and RNAi phenotypes, (iv) higher sequence conservation, and (v) higher in several network topological features such as degrees in a functional interaction network. Based on these features, we developed an algorithm to predict longevity genes in C. elegans and obtained 243 novel longevity genes with a precision rate of 0.85. Some of the predicted genes have been validated by published articles or wet lab experiments. The contribution of each feature to the predicted results was also evaluated.
We propose a compound metallic grating with perpendicular cuts in each slit and investigate its optical transmission property theoretically. The odd and even waveguide modes exhibit different behaviors when the cuts are set asymmetrically in the slits. Particularly, it is shown that the transmission dips of transmission spectrum for this compound periodic structure can be realized alternately by shifting the position of cuts in the slit. The effect of cut size on the phase resonances in the proposed metallic grating is also identified, and the underlying physics is discussed by the simulated field and phase maps.
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