The methyltransferase enzyme (MTase), which catalyzes the transfer of a methyl group from S-adenosyl-methionine (AdoMet) to viral RNA, and generates S-adenosyl-homocysteine (AdoHcy) as a by-product, is essential for the life cycle of many significant human pathogen flaviviruses. Here we investigated inhibition of the flavivirus MTase by several AdoHcy-derivatives. Unexpectedly we found that AdoHcy itself barely inhibits the flavivirus MTase activities, even at high concentrations. AdoHcy was also shown to not inhibit virus growth in cell-culture. Binding studies confirmed that AdoHcy has a much lower binding affinity for the MTase than either the AdoMet co-factor, or the natural AdoMet analog inhibitor sinefungin (SIN). While AdoMet is a positively charged molecule, SIN is similar to AdoHcy in being uncharged, and only has an additional amine group that can make extra electrostatic contacts with the MTase. Molecular Mechanics Poisson-Boltzmann Sovation Area analysis on AdoHcy and SIN binding to the MTase suggests that the stronger binding of SIN may not be directly due to interactions of this amine group, but due to distributed differences in SIN binding resulting from its presence. The results suggest that better MTase inhibitors could be designed by using SIN as a scaffold rather than AdoHcy.
Although HCO3− is known to be required for early embryo development, its exact role remains elusive. Here we report that HCO3− acts as an environmental cue in regulating miR-125b expression through CFTR-mediated influx during preimplantation embryo development. The results show that the effect of HCO3− on preimplantation embryo development can be suppressed by interfering the function of a HCO3−-conducting channel, CFTR, by a specific inhibitor or gene knockout. Removal of extracellular HCO3− or inhibition of CFTR reduces miR-125b expression in 2 cell-stage mouse embryos. Knockdown of miR-125b mimics the effect of HCO3− removal and CFTR inhibition, while injection of miR-125b precursor reverses it. Downregulation of miR-125b upregulates p53 cascade in both human and mouse embryos. The activation of miR-125b is shown to be mediated by sAC/PKA-dependent nuclear shuttling of NF-κB. These results have revealed a critical role of CFTR in signal transduction linking the environmental HCO3− to activation of miR-125b during preimplantation embryo development and indicated the importance of ion channels in regulation of miRNAs.
embryo; HCO3−; miR-125b; CFTR; sAC
Depression is a highly debilitating and widely distributed illness in the general population. Geranylgeranylacetone (GGA), a non-toxic anti-ulcer drug, has been reported to have protective effects in the central nervous system. The aim of this study was to determine the antidepressant effect of GGA in a chronic mild stress (CMS) model of depression. We confirmed that CMS in rats caused a reduction in locomotor activity and an increase in the levels of monoamine oxidase-A (MAO-A) and caspase-3 in the hippocampus. GGA treatment reversed stress-induced alterations in locomotor activity and target levels of MAO-A and caspase-3. In addition, GGA treatment induced heat shock protein 70 (Hsp70) expression in the hippocampus. These findings suggest that GGA possesses an antidepressant activity in a CMS model of depression. The activity of GGA in the relief of depression may be mediated via the induction of Hsp70 expression to suppress MAO-A expression and the apoptosis cascade.
depression; geranylgeranylacetone; heat shock protein 70; apoptosis; monoamine oxidase
China experienced several large measles outbreaks in the past two decades, and a series of enhanced control measures were implemented to achieve the goal of measles elimination. Molecular epidemiologic surveillance of wild-type measles viruses (MeV) provides valuable information about the viral transmission patterns. Since 1993, virologic surveillnace has confirmed that a single endemic genotype H1 viruses have been predominantly circulating in China. A component of molecular surveillance is to monitor the genetic characteristics of the hemagglutinin (H) gene of MeV, the major target for virus neutralizing antibodies.
Analysis of the sequences of the complete H gene from 56 representative wild-type MeV strains circulating in China during 1993–2009 showed that the H gene sequences were clustered into 2 groups, cluster 1 and cluster 2. Cluster1 strains were the most frequently detected cluster and had a widespread distribution in China after 2000. The predicted amino acid sequences of the H protein were relatively conserved at most of the functionally significant amino acid positions. However, most of the genotype H1 cluster1 viruses had an amino acid substitution (Ser240Asn), which removed a predicted N-linked glycosylation site. In addition, the substitution of Pro397Leu in the hemagglutinin noose epitope (HNE) was identified in 23 of 56 strains. The evolutionary rate of the H gene of the genotype H1 viruses was estimated to be approximately 0.76×10−3 substitutions per site per year, and the ratio of dN to dS (dN/dS) was <1 indicating the absence of selective pressure.
Although H genes of the genotype H1 strains were conserved and not subjected to selective pressure, several amino acid substitutions were observed in functionally important positions. Therefore the antigenic and genetic properties of H genes of wild-type MeVs should be monitored as part of routine molecular surveillance for measles in China.
Epigallocatechin gallate (EGCG), the major polyphenol in green tea, acutely stimulates production of nitric oxide (NO) from vascular endothelium to reduce hypertension, and improve endothelial dysfunction in SHR rats. Herein, we explored additional mechanisms whereby EGCG may mediate beneficial cardiovascular actions. When compared with vehicle-treated controls, EGCG treatment (2.5 μM, 8 h) of human aortic endothelial cells (HAEC) caused a ~3-fold increase in hemeoxygenase-1 (HO-1) mRNA and protein with comparable increases in HO-1 activity. This was unaffected by pre-treatment of cells with wortmannin, LY294002, PD98059, or L-NAME (PI 3-kinase, MEK, and NO synthase inhibitors, respectively). Pre-treatment of HAEC with SB203580 (p38 MAPK inhibitor) or siRNA knockdown of p38 MAPK completely blocked EGCG-stimulated induction of HO-1. EGCG treatment also inhibited TNF-α-stimulated expression of VCAM-1 and decreased adhesion of monocytes to HAEC. siRNA knockdown of HO-1, p38 MAPK, or Nrf-2 blocked these inhibitory actions of EGCG. In HAEC transiently transfected with a human HO-1 promoter luciferase reporter (or an isolated Nrf-2 responsive region), luciferase activity increased in response to EGCG. This was inhibitable by SB203580 pre-treatment. EGCG-stimulated expression of HO-1 and Nrf-2 was blocked by siRNA knockdown of Nrf-2 or p38 MAPK. Finally, liver from mice chronically treated with EGCG had increased HO-1 and decreased VCAM-1 expression. Thus, in vascular endothelium, EGCG requires p38 MAPK to increase expression of Nrf-2 that drives expression of HO-1 resulting in increased HO-1 activity. Increased HO-1 expression may underlie anti-inflammatory actions of EGCG in vascular endothelium that may help mediate beneficial cardiovascular actions of green tea.
Epigallocatechin gallate (EGCG); hemeoxygenase-1; p38 kinase; nuclear factor-E2-related factor-2; endothelium
Huntington's disease (HD) is a neurodegenerative disease caused by the expansion of a CAG repeat in the Huntingtin (HTT) gene. Abnormal regulation of the cyclic AMP (cAMP)/protein kinase A (PKA) pathway occurs during HD progression. Here we found that lower PKA activity was associated with proteasome impairment in the striatum for two HD mouse models (R6/2 and N171-82Q) and in mutant HTT (mHTT)-expressing striatal cells. Because PKA regulatory subunits (PKA-Rs) are proteasome substrates, the mHTT-evoked proteasome impairment caused accumulation of PKA-Rs and subsequently inhibited PKA activity. Conversely, activation of PKA enhanced the phosphorylation of Rpt6 (a component of the proteasome), rescued the impaired proteasome activity, and reduced mHTT aggregates. The dominant-negative Rpt6 mutant (Rpt6S120A) blocked the ability of a cAMP-elevating reagent to enhance proteasome activity, whereas the phosphomimetic Rpt6 mutant (Rpt6S120D) increased proteasome activity, reduced HTT aggregates, and ameliorated motor impairment. Collectively, our data demonstrated that positive feedback regulation between PKA and the proteasome is critical for HD pathogenesis.
Crocodilians are diving reptiles that can hold their breath under water for long periods of time and are crepuscular animals with excellent sensory abilities. They comprise a sister lineage of birds and have no sex chromosome. Here we report the genome sequence of the endangered Chinese alligator (Alligator sinensis) and describe its unique features. The next-generation sequencing generated 314 Gb of raw sequence, yielding a genome size of 2.3 Gb. A total of 22 200 genes were predicted in Alligator sinensis using a de novo, homology- and RNA-based combined model. The genetic basis of long-diving behavior includes duplication of the bicarbonate-binding hemoglobin gene, co-functioning of routine phosphate-binding and special bicarbonate-binding oxygen transport, and positively selected energy metabolism, ammonium bicarbonate excretion and cardiac muscle contraction. Further, we elucidated the robust Alligator sinensis sensory system, including a significantly expanded olfactory receptor repertoire, rapidly evolving nerve-related cellular components and visual perception, and positive selection of the night vision-related opsin and sound detection-associated otopetrin. We also discovered a well-developed immune system with a considerable number of lineage-specific antigen-presentation genes for adaptive immunity as well as expansion of the tripartite motif-containing C-type lectin and butyrophilin genes for innate immunity and expression of antibacterial peptides. Multifluorescence in situ hybridization showed that alligator chromosome 3, which encodes DMRT1, exhibits significant synteny with chicken chromosome Z. Finally, population history analysis indicated population admixture 0.60-1.05 million years ago, when the Qinghai-Tibetan Plateau was uplifted.
diving hypoxia; oxidative phosphorylation; olfaction; immunity; FISH; SNP
To compare the bacterial flora in palpebral conjunctiva of xerophthalmia seniors of Tibetan, Yi and Han, and analyze the differences and similarities of the bacteria.
The test subjects were selected from 2 Tibetan, 2 Yi and 3 Han populated places, respectively. Total 222 seniors (444 eyes) with dry eye were examined. Secretion was collected from the palpebral conjunctiva of the subjects and then inoculated onto a blood agar plate. After 48h of incubation, the bacteria were examined for the differences and similarities between different ethnics.
There was no significant difference (P>0.05) of Gram stain characterization, dominant bacteria and number of the bacterial species present in oxrophthalmia patients among Tibetan, Yi and Han nationalities. The bacteria presented in all groups include staphylococcus epidermidis, corynebacterium, micrococcus luteu, intracellular bacteria sphingomonas, pseudomonas aeruginosa. The bacteria detected from the two of three ethnic groups were staphylococcus aureus, staphylococcus haemolyticus, escherichia coli, kytococcus sedentarius, streptococcus angina, micrococcus lylae, and staphylococcus heads. The incidence rate of bacteria-associated dry eye in Tibetan population was significantly lower than that of Han and Yi population.
There is no significant difference in the bacteria flora of palpebral conjunctiva observed among dry eye elder populations of Tibetan, Yi and Han people. All of staphylococcus epidermidis, corynebacterium, micrococcus luteu, intracellular bacteria sphingomonas, pseudomonas aeruginosa, staphylococcus aureus, staphylococcus haemolyticus, escherichia coli, kytococcus sedentarius, streptococcus angina, micrococcus lylae and staphylococcus heads are common bacteria flora of the three nationalities inhibiting in this area.
conjunctiva; bacteria; dry eye; Tibetan; Yi ethnic
The objective of this study was to examine, first, the relationship of having a rural vs. urban background with suicidal ideation in Chinese college students, and second, whether a potential relationship was mediated by depression.
A survey was conducted among 1,145 undergraduate students at a university in China. Suicidal ideation and depressive symptoms were measured by the revised Hopkins’ Symptom checklist (SCL-90-R). Associations between rural vs. urban background, depression and suicidal ideation were estimated by multivariable linear regression-based β coefficients, logistic regression-based odds ratios (ORs), and corresponding 95% confidence intervals (CIs). The magnitude of indirect effect and bias-corrected 95% CIs were obtained through bootstrap techniques.
Rural background was positively associated with depression, which was in turn associated with suicidal ideation. The OR for rural status and suicidal ideation equaled 2.15 (95% CI = 1.36–3.41). This OR was slightly, though significantly (p<0.05) attenuated by additional adjustment for depressive symptoms (OR = 1.99, 95% CI = 1.15–3.44).
Having a rural background is a determinant of suicidal ideation in Chinese college students. Depression may only marginally mediate this association.
There are conflicting reports as to the association between smoking, radiotherapy, diabetes and osteoporosis and the risk of dental implant failure. We undertook a meta-analysis to evaluate the association between smoking, radiotherapy, diabetes and osteoporosis and the risk of dental implant failure.
A comprehensive research on MEDLINE and EMBASE, up to January 2013, was conducted to identify potential studies. References of relevant studies were also searched. Screening, data extraction and quality assessment were conducted independently and in duplicate. A random-effects meta-analysis was used to pool estimates of relative risks (RRs) with 95% confidence intervals (CIs).
A total of 51 studies were identified in this meta-analysis, with more than 40,000 dental implants placed under risk-threatening conditions. The pooled RRs showed a direct association between smoking (n = 33; RR = 1.92; 95% CI, 1.67–2.21) and radiotherapy (n = 16; RR = 2.28; 95% CI, 1.49–3.51) and the risk of dental implant failure, whereas no inverse impact of diabetes (n = 5; RR = 0.90; 95% CI, 0.62–1.32) on the risk of dental implant failure was found. The influence of osteoporosis on the risk of dental implant failure was direct but not significant (n = 4; RR = 1.09; 95% CI, 0.79–1.52). The subgroup analysis indicated no influence of study design, geographical location, length of follow-up, sample size, or mean age of recruited patients.
Smoking and radiotherapy were associated with an increased risk of dental implant failure. The relationship between diabetes and osteoporosis and the risk of implant failure warrant further study.
Mucosal tissues are the primary route of transmission for most respiratory and sexually transmitted diseases, including human immunodeficiency virus (HIV). There is epidemiological evidence that genital mucosal inflammation leads to enhanced HIV type 1 (HIV-1) transmission. The objective of this study was to assess the influence of periodontal inflammation on oral HIV transmission using a nonhuman primate model of teeth ligature-induced periodontitis. Simian immunodeficiency virus (SIV) was nontraumatically applied to the gingiva after moderate gingivitis was identified through clinical and immunologic analyses (presence of inflammatory cytokines). Overall oral SIV infection rates were similar in the gingivitis-induced and control groups (5 infections following 12 SIV administrations for each), although more macaques were infected with multiple viral variants in the gingivitis group. SIV infection also affected the levels of antiviral and inflammatory cytokines in the gingival crevicular fluid, and a synergistic effect was observed, with alpha interferon and interferon-inducible protein 10 undergoing significant elevations following SIV infection in macaques with gingivitis compared to controls. These increases in antiviral and inflammatory immune modulators in the SIV-infected gingivitis macaques could also be observed in blood plasma, although the effects at both compartments were generally restricted to the acute phase of the infection. In conclusion, while moderate gingivitis was not associated with increased susceptibility to oral SIV infection, it resulted in elevated levels of cytokines in the oral mucosa and plasma of the SIV-infected macaques. These findings suggest a synergy between mucosal inflammation and SIV infection, creating an immune milieu that impacts the early stages of the SIV infection with potential implications for long-term pathogenesis.
Glaucoma is characterized by retinal ganglion cell (RGC) death and frequently associated with elevated IOP. How RGCs degenerate before death is little understood, so we sought to investigate RGC degeneration in a mouse model of ocular hypertension.
A laser-induced mouse model of chronic ocular hypertension mimicked human high-tension glaucoma. Immunohistochemistry was used to characterize overall RGC loss and an optomotor behavioral test to measure corresponding changes in visual capacity. Changes in RGC functional properties were characterized by a large-scale multielectrode array (MEA). The transgenic Thy-1-YFP mouse line, in which a small number of RGCs are labeled with yellow fluorescent protein (YFP), permitted investigation of whether subtypes of RGCs or RGCs from particular retinal areas were differentially vulnerable to elevated IOP.
Sustained IOP elevation in mice was achieved by laser photocoagulation. We confirmed RGC loss and decreased visual acuity in ocular hypertensive mice. Furthermore, these mice had fewer visually responsive cells with smaller receptive field sizes compared to controls. We demonstrated that RGC dendritic shrinkage started from the vertical axis of hypertensive eyes and that mono-laminated ON cells were more susceptible to IOP elevation than bi-laminated ON-OFF cells. Moreover, a subgroup of ON RGCs labeled by the SMI-32 antibody exhibited significant dendritic atrophy in the superior quadrant of the hypertensive eyes.
RGC degeneration depends on subtype and location in hypertensive eyes. This study introduces a valuable model to investigate how the structural and functional degeneration of RGCs leads to visual impairments.
Dendritic degeneration of retinal ganglion cells depends on cell type and location in a mouse model of laser-induced chronic ocular hypertension.
Objective: To investigate the expression of apolipoprotein D (ApoD) and androgen receptor (AR), two proteins related to E-3M2H secretion, in the apocrine sweat gland of patients with axillary osmidrosis (AO) and healthy subjects, and to explore the cause of abnormal ApoD expression in these patients. Methods: Samples were collected from healthy controls (n=4) and AO patients (n=10). Immunohistochemistry, real-time PCR and western blot assay were performed to measure the mRNA and protein expression of ApoD and AR. In vitro sweat gland cells were treated with androgen to explore the AR signals in regulation of ApoD expression and the role of JNK1 signaling pathway in the ApoD expression. Results: There was significant difference in the expression of ApoD and AR between AO patients and healthy controls. The ApoD expression in AO patients was 2-fold higher than that in healthy controls and the AR expression in AO patients was also markedly increased when compared with healthy controls. Moreover, the activation of JNK1 increased in AO patients. Androgen can increase the ApoD expression in healthy subjects accompanied bu JNK1 activation. Inhibition of JNK1 activation may reduce the ApoD expression in AO patients and the androgen induced ApopD expression. Conclusion: The increase ApoD expression is closely related to the AR signaling pathway. JNK1 activation is a major cause of increased ApoD expression in AO patients and the androgen induced ApopD expression. To inhibit the JNK1 activation may suppress the endogenous ApoD expression in AO patients and the androgen induced ApopD expression.
Axillary osmidrosis; apolipoprotein D; c-Jun N-terminal kinase; expression regulation
Epilepsy surgery has improved over the last decade, but non-seizure-free outcome remains at 10%–40% in temporal lobe epilepsy (TLE) and 40%–60% in extratemporal lobe epilepsy (ETLE). This paper reports a complex multifocal case. With a normal magnetic resonance imaging (MRI) result and nonlocalizing electroencephalography (EEG) findings (bilateral TLE and ETLE, with more interictal epileptiform discharges [IEDs] in the right frontal and temporal regions), a presurgical EEG-functional MRI (fMRI) was performed before the intraoperative intracranial EEG (icEEG) monitoring (icEEG with right hemispheric coverage). Our previous EEG-fMRI analysis results (IEDs in the left hemisphere alone) were contradictory to the EEG and icEEG findings (IEDs in the right frontal and temporal regions). Thus, the EEG-fMRI data were reanalyzed with newly identified IED onsets and different fMRI model options. The reanalyzed EEG-fMRI findings were largely concordant with those of EEG and icEEG, and the failure of our previous EEG-fMRI analysis may lie in the inaccurate identification of IEDs and wrong usage of model options. The right frontal and temporal regions were resected in surgery, and dual pathology (hippocampus sclerosis and focal cortical dysplasia in the extrahippocampal region) was found. The patient became seizure-free for 3 months, but his seizures restarted after antiepileptic drugs (AEDs) were stopped. The seizures were not well controlled after resuming AEDs. Postsurgical EEGs indicated that ictal spikes in the right frontal and temporal regions reduced, while those in the left hemisphere became prominent. This case suggested that (1) EEG-fMRI is valuable in presurgical evaluation, but requires caution; and (2) the intact seizure focus in the remaining brain may cause the non-seizure-free outcome.
EEG-fMRI; focus localization; presurgical evaluation; epilepsy surgery
The placental hormone leptin has important functions in fetal and neonatal growth, and prevents depressed respiration in leptin-deficient mice. The effect of leptin on respiratory distress suffered by low birth weight and premature infants has been studied. However, it is unclear how leptin enhances lung maturity in the fetus and ameliorates neonatal respiratory distress. In the present study, we found that antenatal treatment with leptin for 2 d significantly enhanced the relative alveolus area and improved the maturity of fetal lungs in a rat model of fetal growth restriction (FGR). Mean birth weight and lung wet weight were higher in the leptin-treated group than in the PBS-treated group, indicating promotion of fetal growth. Leptin upregulated the intracellular expression and extracellular secretion of surfactant protein (SP) A in type-II alveolar epithelial cells (AECs) in vivo and in vitro. Dual positive effects of leptin were found on protein expression and transcriptional activity of thyroid transcription factor-1 (TTF-1), a nuclear transcription essential for branching morphogenesis of the lung and expression of SP-A in type-II AECs. Knockdown of TTF-1 by RNA interference indicated that TTF-1 may play a vital role in leptin-induced SP-A expression. These results suggest that leptin may have great therapeutic potential for the treatment of FGR, and leptin-mediated SP-A induction and lung maturity of the fetus are TTF-1 dependent.
To evaluate the correlation of total lymphocyte count (TLC) and CD4 cell count and the suitability of TLC as a surrogate marker for CD4 cell count of HIV-infected patients in China.
Usefulness of TLC as a surrogate marker for a CD4 cell count <350 cells/mm3 for HIV-positive patients in China was evaluated by 977 pairs of TLC and CD4 cell count from 977 outpatients. The result was then validated by a literature review which was conducted on 9 relevant articles. Further investigation using the 977 pairs of TLC and CD4 cell count data was done to determine a TLC threshold for predicting a CD4 cell count <500 cells/mm3. Correlation and receiver operating characteristic (ROC) analysis were performed for both CD4 cell counts, and the sensitivity and specificity were computed.
Good correlation was noted between TLC and CD4 count (r = 0.60, 95% CI, 0.56–0.64). TLC obtained a relatively high diagnostic performance (area under ROC curve, 0.80) for predicting a CD4 cell count <350 cells/mm3, with a sensitivity of 0.65 (95% CI, 0.61–0.68) and a specificity of 0.80 (95% CI, 0.75–0.85) at the TLC threshold of 1570 cells/mm3. The literature review suggested that for a CD4 cell count <350 cells/mm3, the optimal TLC threshold was 1500 cells/mm3, which was similar to the figure presented in this observational study. As for predicting a CD4 cell count <500 cells/mm3, TLC obtained a high diagnostic performance (area under ROC curve, 0.82) as well with a sensitivity of 0.70 (95% CI, 0.67–0.73) and a specificity of 0.80 (95% CI, 0.73–0.87).
When considering the antiretroviral therapy for HIV-infected Chinese individuals, total lymphocyte count can be considered as an inexpensive and easily available surrogate marker for predicting two clinically important thresholds of CD4 count of 350 cells/mm3 and 500 cells/mm3.
A new strain of canine distemper virus, strain HX, has been isolated from piglets in China, and its complete genome has been sequenced and analyzed. Phylogenetic analysis suggests that HX belongs to the 2d genotype cluster and it is highly prevalent in China.
Microalgal lipid is one of the most promising feedstocks for biodiesel production. Chlorella appears to be a particularly good option, and nitrogen (N) starvation is an efficient environmental pressure used to increase lipid accumulation in Chlorella cells. The effects of N starvation of an oil-producing wild microalga, Chlorella sorokiniana C3, on lipid accumulation were investigated using thin layer chromatography (TLC), confocal laser scanning microscopy (CLSM) and flow cytometry (FCM). The results showed that N starvation resulted in lipid accumulation in C. sorokiniana C3 cells, oil droplet (OD) formation and significant lipid accumulation in cells were detected after 2 d and 8 d of N starvation, respectively. During OD formation, reduced photosynthetic rate, respiration rate and photochemistry efficiency accompanied by increased damage to PSII were observed, demonstrated by chlorophyll (Chl) fluorescence, 77K fluorescence and oxygen evolution tests. In the mean time the rate of cyclic electron transportation increased correspondingly to produce more ATP for triacylglycerols (TAGs) synthesis. And 0.5 d was found to be the turning point for the early stress response and acclimation of cells to N starvation. Increased level of membrane peroxidation was also observed during OD formation, and superoxide dismutase (SOD), peroxide dismutase (POD) and catalase (CAT) enzyme activity assays suggested impaired reactive oxygen species (ROS) scavenging ability. Significant neutral lipid accumulation was also observed by artificial oxidative stress induced by H2O2 treatment. These results suggested coupled neutral lipid accumulation and oxidative stress during N starvation in C. sorokiniana C3.
The 1H-15N 2D NMR correlation spectrum of the widely studied FK506-binding protein FKBP12 (FK506-binding protein of 12 kDa) contains previously unreported peak doublings for at least 31 residues that arise from a minor conformational state (12% of total) which exchanges with the major conformation with a time constant of 3.0 s at 43°C. The largest differences in chemical shift occur for the 80′s loop that forms critical recognition interactions with many of the protein partners for the FKBP family. The residues exhibiting doubling extend into the adjacent strands of the β-sheet, across the active site to the α-helix and into the 50′s loop. Each of the seven proline residues adopts a trans-peptide linkage in both the major and minor conformations, indicating that this slow transition is not the result of prolyl isomerization. Many of the residues exhibiting resonance doubling also participate in conformational line-broadening transition(s) that occur ~105-fold more rapidly, proposed previously to arise from a single global process. The 1.70 Å (1 Å=0.1 nm) resolution X-ray structure of the H87V variant is strikingly similar to that of FKBP12, yet this substitution quenches the slow conformational transition throughout the protein while quenching the line-broadening transition for residues near the 80′s loop. Line-broadening was also decreased for the residues in the α-helix and 50′s loop, whereas line-broadening in the 40′s loop was unaffected. The K44V mutation selectively reduces the line-broadening in the 40′s loop, verifying that at least three distinct conformational transitions underlie the line-broadening processes of FKBP12.
conformational dynamics; FK506-binding protein of 12 kDa (FKBP12); NMR; slow exchange; X-ray structure; CPMG, Carr–Purcell–Meiboom–Gill; CT-HSQC, constant-time heteronuclear single-quantum coherence; FKBP12, FK506-binding protein of 12 kDa; mTOR, mammalian target of rapamycin; NOE, nuclear Overhauser effect; RyR, ryanodine receptor; TCEP, tris-(2-carboxyethyl)phosphine
Chondrosarcoma is a primary malignant bone cancer, with a potent capacity to invade locally and cause distant metastasis; it has a poor prognosis and shows a predilection for metastasis to the lungs. Brain derived neurotrophic factor (BDNF) is a small-molecule protein from the neurotrophin family of growth factors that is associated with the disease status and outcomes of cancers. However, the effect of BDNF on migration activity in human chondrosarcoma cells is mostly unknown. Here, we found that human chondrosarcoma tissues showed significant expression of BDNF, which was higher than that in normal cartilage and primary chondrocytes. We also found that BDNF increased the migration and expression of β5 integrin in human chondrosarcoma cells. In addition, knockdown of BDNF expression markedly inhibited migratory activity. BDNF-mediated migration and β5 integrin up-regulation were attenuated by antibody, inhibitor, or siRNA against the TrkB receptor. Pretreatment of chondrosarcoma cells with PI3K, Akt, and NF-κB inhibitors or mutants also abolished BDNF-promoted migration and integrin expression. The PI3K, Akt, and NF-κB signaling pathway was activated after BDNF treatment. Taken together, our results indicate that BDNF enhances the migration of chondrosarcoma by increasing β5 integrin expression through a signal transduction pathway that involves the TrkB receptor, PI3K, Akt, and NF-κB. BDNF thus represents a promising new target for treating chondrosarcoma metastasis.
Dioscorea tuber phytoextracts can confer immunomodulatory activities ex vivo and improve regeneration of bone marrow cells in vivo. In present study, we evaluated specific Dioscorea phytoextracts for use ex vivo as a bone-marrow-derived dendritic cell- (DC-) based vaccine adjuvant for cancer immunotherapy. Fractionated Dioscorea extracts (DsII) were assayed for their effect on maturation and functions of DC ex vivo and antimelanoma activity of DC-based vaccine in vivo. The phytoextract from 50–75% ethanol-precipitated fraction of Dioscorea alata var. purpurea Tainung no. 5 tuber, designated as DsII-TN5, showed a strong augmentation of tumor cell lysate- (TCL-) loaded DC-mediated activation of T-cell proliferation. DsII-TN5 stimulated the expression of CD40, CD80, CD86, and IL-1β in TCL-loaded DCs and downregulated the expression of TGF-β1. DC vaccines prepared by a specific schema (TCL (2 h) + LPS (22 h)) showed the strongest antitumor activity. DsII-TN5 as a DC vaccine adjuvant showed strong antimelanoma activity and reduced myeloid-derived suppressor cell (MDSC) population in tested mice. DsII-TN5 can also activate DCs to enhance Th1- and Th17-related cytokine expressions. Biochemical analysis showed that DsII-TN5 consists mainly of polysaccharides containing a high level (53%) of mannose residues. We suggest that DsII-TN5 may have potential for future application as a potent, cost-effective adjuvant for DC-based cancer vaccines.
Lifestyle plays an important role in the development of diabetic retinopathy. The lifestyle in Guangzhou is different from other cities in China as the Cantonese prefer eating rice porridge, but not spicy foods. The objectives of this study were to investigate the prevalence and determinants of diabetic retinopathy in a high-risk population of Guangzhou.
Subjects (619 totals) aged over 45 years old, without known diabetes were recruited from five randomly selected Guangzhou communities in 2009–2010. All participants were invited to complete the Finnish Diabetes Risk Score (FINDRISC) questionnaire. Subjects with FINDRISC score ≥ 9 were included in the study, and underwent an investigation of demographic data, a standardized physical examination, ocular fundus examination, and laboratory analyses. The minimum criterion for diagnosis of diabetic retinopathy was the presence of at least one microaneurysm.
Of 619 subjects, 208 eligible subjects (122 women) with FINDRISC score ≥ 9 were included in the study. The mean age was 69.2 ± 8.5 years. Diabetic retinopathy was detected in 31 subjects, and the prevalence of diabetic retinopathy in subjects with high risk for diabetes was 14.9%. In binary logistic regression analysis, risk factors associated with diabetic retinopathy were history of impaired glucose regulation [odds ratio (OR), 7.194; 95% confidence interval (CI): 1.083, 47.810], higher hemoglobin A1c (HbA1c; OR, 2.912; 95% CI: 1.009, 8.402), higher two-hour postprandial plasma glucose level (OR, 1.014; 95% CI: 1.003, 1.025), and presence of microalbuminuria (OR, 5.387; 95% CI: 1.255, 23.129).
Diabetic retinopathy was prevalent in a high-risk Chinese population from Guangzhou. Histories of impaired glucose regulation and microalbuminuria were strong risk factors for diabetic retinopathy.
Finnish diabetes risk score; People with high risk for diabetes; Diabetic retinopathy; Prevalence; Risk factors
The effect of acetic acid on hepatic lipid metabolism in ruminants differs significantly from that in monogastric animals. Therefore, the aim of this study was to investigate the regulation mechanism of acetic acid on the hepatic lipid metabolism in dairy cows. The AMP-activated protein kinase (AMPK) signaling pathway plays a key role in regulating hepatic lipid metabolism. In vitro, bovine hepatocytes were cultured and treated with different concentrations of sodium acetate (neutralized acetic acid) and BML-275 (an AMPKα inhibitor). Acetic acid consumed a large amount of ATP, resulting in an increase in AMPKα phosphorylation. The increase in AMPKα phosphorylation increased the expression and transcriptional activity of peroxisome proliferator-activated receptor α, which upregulated the expression of lipid oxidation genes, thereby increasing lipid oxidation in bovine hepatocytes. Furthermore, elevated AMPKα phosphorylation reduced the expression and transcriptional activity of the sterol regulatory element-binding protein 1c and the carbohydrate responsive element-binding protein, which reduced the expression of lipogenic genes, thereby decreasing lipid biosynthesis in bovine hepatocytes. In addition, activated AMPKα inhibited the activity of acetyl-CoA carboxylase. Consequently, the triglyceride content in the acetate-treated hepatocytes was significantly decreased. These results indicate that acetic acid activates the AMPKα signaling pathway to increase lipid oxidation and decrease lipid synthesis in bovine hepatocytes, thereby reducing liver fat accumulation in dairy cows.
Mitochondrial dysfunction in pathogenic fungi or model yeast causes altered susceptibilities to antifungal drugs. Here we have characterized the role of mitochondrial complex I (CI) of Candida albicans in antifungal susceptibility. Inhibitors of CI to CV, except for CII, increased the susceptibility of both patient and lab isolates, even those with a resistance phenotype. In addition, in a C. albicans library of 12 CI null mutants, 10 displayed hypersusceptibility to fluconazole and were severely growth inhibited on glycerol, implying a role for each gene in cell respiration. We chose two other hypersusceptible null mutants of C. albicans, the goa1Δ and ndh51Δ mutants, for transcriptional profiling by RNA-Seq. Goa1p is required for CI activity, while Ndh51p is a CI subunit. RNA-Seq revealed that both the ndh51Δ mutant and especially the goa1Δ mutant had significant downregulation of transporter genes, including CDR1 and CDR2, which encode efflux proteins. In the goa1Δ mutant, we noted the downregulation of genes required for the biogenesis and replication of peroxisomes, as well as metabolic pathways assigned to peroxisomes such as β-oxidation of fatty acids, glyoxylate bypass, and acetyl coenzyme A (acetyl-CoA) transferases that are known to shuttle acetyl-CoA between peroxisomes and mitochondria. The transcriptome profile of the ndh51Δ mutant did not include downregulation of peroxisome genes but had, instead, extensive downregulation of the ergosterol synthesis gene family. Our data establish that cell energy is required for azole susceptibility and that downregulation of efflux genes may be an outcome of that dysfunction. However, there are mutant-specific changes that may also increase the susceptibility of both of these C. albicans mutants to azoles.
To determine the prevalence and distribution of human enteroviruses (HEVs) among healthy children in Shenzhen, China.
Clinical specimens were obtained from 320 healthy children under 5 years old in Shenzhen, China from 2010 to 2011. The specimens were evaluated using real-time PCR and cell cultures. The positive specimens were further tested using reverse transcription-seminested PCR (RT-snPCR). Molecular typing and phylogenetic analysis were based on the sequence determined.
Among the 320 samples, 34 were tested positive for HEVs (10.6%) and 22 different serotypes were identified using RT-snPCR. PV1 and PV2 were also detected. The predominant serotype observed was EV71 (17.6%), followed by CV-B4 (14.7%). HEV-B was detected most frequently, with an overall prevalence of 47.1%. HEV-A and HEV-C were found in 32.3% and 20.6% of the samples, respectively. No HEV-D was identified. Molecular phylogeny indicated that all EV71 strains were of C4 genotype.
Although a variety of HEVs was detected in healthy children, HEV-B was relatively more prevalent than other HEV species. Considering HEV-A is more prevalent than HEV-B among patients with hand-foot-mouth disease, additional long-term surveillance of HEV is warranted in both asymptomatic and symptomatic populations.