The 2H2 monoclonal antibody recognizes the precursor peptide on immature dengue virus and might therefore be a useful tool for investigating the conformational change that occurs when the immature virus enters an acidic environment. During dengue virus maturation, spiky, immature, noninfectious virions change their structure to form smooth-surfaced particles in the slightly acidic environment of the trans-Golgi network, thereby allowing cellular furin to cleave the precursor-membrane proteins. The dengue virions become fully infectious when they release the cleaved precursor peptide upon reaching the neutral-pH environment of the extracellular space. Here we report on the cryo-electron microscopy structures of the immature virus complexed with the 2H2 antigen binding fragments (Fab) at different concentrations and under various pH conditions. At neutral pH and a high concentration of Fab molecules, three Fab molecules bind to three precursor-membrane proteins on each spike of the immature virus. However, at a low concentration of Fab molecules and pH 7.0, only two Fab molecules bind to each spike. Changing to a slightly acidic pH caused no detectable change of structure for the sample with a high Fab concentration but caused severe structural damage to the low-concentration sample. Therefore, the 2H2 Fab inhibits the maturation process of immature dengue virus when Fab molecules are present at a high concentration, because the three Fab molecules on each spike hold the precursor-membrane molecules together, thereby inhibiting the normal conformational change that occurs during maturation.
Chronic low-grade inflammation has long been recognized as the central link between obesity and type 2 diabetes (T2D). The novel subset of T helper (Th) cells, Th22, plays an emerging role in chronic inflammation. We investigated the potential association between Th22 and the pathogenesis of obesity and T2D.
Ninety T2D inpatients (T2D group), 30 healthy participants with BMI ranged from 19 to 23.9 kg/m2 (CTL group) and 30 metabolically healthy obese controls with BMI ≥ 30 kg/m2 (MHO group) were employed in our study. Peripheral frequencies of Th22 and Th1 and Th17 cells were determined by flow cytometry based on their specific cytokine patterns. Cytokine levels in fresh plasma were quantified by ELISA.
Compared to that in CTL group (1.18±0.06%, n = 28), peripheral frequency of Th22 cells was significantly increased in MHO group (1.88±0.10%, n = 30) and in T2D group (2.247±0.10%, n = 89). There was a consistent notable increase in plasma interleukin (IL)-22 of T2D patients [47.56 (30.55–76.89) pg/mL] as compared with that of MHO group [36.65 (29.52–55.70) pg/ml; *P<0.0001] and CTLs [36.33 (31.93–40.62) pg/mL; *P<0.0001]. Furthermore, other than Th1/Th17, previously frequently described participants in obesity and T2D, there was a strong correlation between Th22 frequency and the homeostasis model of assessment for insulin resistance index (r = 0.6771, *P<0.0001) and HOMA for β-cell function (r = −0.7264, *P<0.0001).
There were increased Th22 frequencies and IL-22 levels in obesity and T2D. Elevated Th22 and IL-22 also aided in the differentiation of MHO from T2D patients. The notable correlation implied that Th22 might play a more determinant role in both insulin resistance and β-cell impairment.
The phase variation (reversible on-off switching) of the type 1 fimbrial adhesin of Escherichia coli involves a DNA inversion catalyzed by FimB (switching in either direction) or FimE (on-to-off switching). Here, we demonstrate that RfaH activates expression of a FimB-LacZ protein fusion while having a modest inhibitory effect on a comparable fimB-lacZ operon construct and on a FimE-LacZ protein fusion, indicating that RfaH selectively controls fimB expression at the posttranscriptional level. Further work demonstrates that loss of RfaH enables small RNA (sRNA) MicA inhibition of fimB expression even in the absence of exogenous inducing stress. This effect is explained by induction of σE, and hence MicA, in the absence of RfaH. Additional work confirms that the procaine-dependent induction of micA requires OmpR, as reported previously (A. Coornaert et al., Mol. Microbiol. 76:467–479, 2010, doi:10.1111/j.1365-2958.2010.07115.x), but also demonstrates that RfaH inhibition of fimB transcription is enhanced by procaine independently of OmpR. While the effect of procaine on fimB transcription is shown to be independent of RcsB, it was found to require SlyA, another known regulator of fimB transcription. These results demonstrate a complex role for RfaH as a regulator of fimB expression.
Extracellular adenosine triphosphate (eATP) transduces purinergic signal and plays an important regulatory role in many biological processes, including tumor cell growth and cell death. A large amount of eATP exists in the fast-growing tumor center and inflammatory tumor microenvironment. Tumor cells could acquire anoikis resistance and anchorage independence in tumor microenvironment and further cause metastatic lesion. Whether such a high amount of eATP has any effect on the anchored and non-anchored tumor cells in tumor microenvironment has not been elucidated and is investigated in this study. Our data showed that autophagy helped hepatoma cells to maintain survival under the treatment of no more than 1 mM of eATP. Only when eATP concentration reached a relatively high level (2.5 mM), cell organelle could not be further maintained by autophagy, and apoptosis and cell death occurred. In hepatoma cells under treatment of 2.5 mM of eATP, an AMP-activated protein kinase (AMPK) pathway was dramatically activated while mTOR signaling pathway was suppressed in coordination with apoptosis. Further investigation showed that the AMPK/mTOR axis played a key role in tipping the balance between autophagy-mediated cell survival and apoptosis-induced cell death under the treatment of eATP. This work provides evidence to explain how hepatoma cells escape from eATP-induced cytotoxicity as well as offers an important clue to consider effective manipulation of cancer.
Apoptosis; Autophagy; Anoikis; Extracellular ATP; AMPK; mTOR
Purpose. To investigate the effect of hypothermia on 96 hr neurological outcome and survival by quantitatively characterizing early postresuscitation EEG in a rat model of cardiac arrest. Materials and Methods. In twenty male Sprague-Dawley rats, cardiac arrest was induced through high frequency transesophageal cardiac pacing. Cardiopulmonary resuscitation was initiated after 5 mins untreated arrest. Immediately after resuscitation, animals were randomized to either 2 hrs of hypothermia (N = 10) or normothermia (N = 10). EEG, ECG, aortic pressure, and core temperature were continuously recorded for 6 hrs. Neurological outcome was evaluated daily during the 96 hrs postresuscitation period. Results. No differences in the baseline measurements and resuscitation outcome were observed between groups. However, 96 hr neurological deficit score (204 ± 255 versus 500 ± 0, P = 0.005) and survival (6/10 versus 0/10, P = 0.011) were significantly better in the hypothermic group. Quantitative analysis of early postresuscitation EEG revealed that burst frequency and spectrum entropy were greatly improved in the hypothermic group and correlated with 96 hr neurological outcome and survival. Conclusion. The improved burst frequency during burst suppression period and preserved spectrum entropy after restoration of continuous background EEG activity for animals treated with hypothermia predicted favorable neurological outcome and survival in this rat model of cardiac arrest.
Phosphorylation of viral proteins plays important roles in the influenza A virus (IAV) life cycle. By using mass spectrometry, we identified tyrosine 132 (Y132) as a phosphorylation site of the matrix protein (M1) of the influenza virus A/WSN/1933(H1N1). Phosphorylation at this site is essential to the process of virus replication by controlling the nuclear import of M1. We further demonstrated that the phosphorylated tyrosine is crucial for the binding of M1 to the nuclear import factor importin-α1, since any substitutions at this site severely reduce this protein-protein interaction and damage the importin-α1-mediated nuclear import of M1. Additionally, the tyrosine phosphorylation which leads to the nuclear import of M1 is blocked by a Janus kinase inhibitor. The present study reveals a pivotal role of this tyrosine phosphorylation in the intracellular transportation of M1, which controls the process of viral replication.
In the present study, we investigated the anti-inflammatory activity of water-soluble polysaccharide of Agaricus blazei Murill (WSP-AbM) on ovariectomized osteopenic rats. The rats were administered orally WSP-AbM (200 mg/kg BW) for 8 weeks. Subsequent serum maleic dialdehyde (MDA) level, total antioxidant status (TAOS), nuclear factor kappa B (NF-κB) level, polymorphonuclear (PMN) cells level, interleukin-1β (IL-1β) level, inducible nitric oxide synthase (iNOS) level, tumor necrosis factor-α (TNF-α) level, adhesion molecule (ICAM-1), and cyclooxygenase-2 (COX-2) were determined by enzyme linked immunosorbent assay (ELISA) and immunohistochemistry, respectively. WSP-AbM administration markedly (P < 0.05) decreased serum IL-1β and TNF-α levels and the expressions of ICAM-1, COX-2, and iNOS NF-κB compared with OVX rats. WSP-AbM administration alsomarkedly (P < 0.05) decreased PMN infiltration. In conclusion, we observed that WSP-AbM supplementation had anti-inflammatory effects in a model of osteoporosis disease.
Multi-marker methods for genetic association analysis can be performed for common and low frequency SNPs to improve power. Regression models are an intuitive way to formulate multi-marker tests. In previous studies we evaluated regression-based multi-marker tests for common SNPs, and through identification of bins consisting of correlated SNPs, developed a multi-bin linear combination (MLC) test that is a compromise between a 1 df linear combination test and a multi-df global test. Bins of SNPs in high linkage disequilibrium (LD) are identified, and a linear combination of individual SNP statistics is constructed within each bin. Then association with the phenotype is represented by an overall statistic with df as many or few as the number of bins. In this report we evaluate multi-marker tests for SNPs that occur at low frequencies. There are many linear and quadratic multi-marker tests that are suitable for common or low frequency variant analysis. We compared the performance of the MLC tests with various linear and quadratic statistics in joint or marginal regressions. For these comparisons, we performed a simulation study of genotypes and quantitative traits for 85 genes with many low frequency SNPs based on HapMap Phase III. We compared the tests using (1) set of all SNPs in a gene, (2) set of common SNPs in a gene (MAF ≥ 5%), (3) set of low frequency SNPs (1% ≤ MAF < 5%). For different trait models based on low frequency causal SNPs, we found that combined analysis using all SNPs including common and low frequency SNPs is a good and robust choice whereas using common SNPs alone or low frequency SNP alone can lose power. MLC tests performed well in combined analysis except where two low frequency causal SNPs with opposing effects are positively correlated. Overall, across different sets of analysis, the joint regression Wald test showed consistently good performance whereas other statistics including the ones based on marginal regression had lower power for some situations.
genetic association analysis; multi-marker association analysis; rare variant analysis; common variant analysis; multi-bin multi-marker tests; generalized Wald test; minimum p-value test; indirect association
Background and Purpose
Although endovascular therapy (ET) is increasingly used in patients with moderate to severe acute ischemic stroke, its efficacy and safety remains controversial. We performed a meta-analysis aiming to compare the benefits and safety of endovascular treatment and intravenous thrombolysis in the treatment of acute ischemic stroke.
We systematically searched PubMed, Embase, Science direct and Springer unitil July, 2013. The primary outcomes included good outcome (mRS ≤ 2) and excellent outcome (mRS ≤ 1) at 90 days or at trial end point. Secondary outcomes were occurrence of symptomatic hemorrhage and all-cause mortality.
Using a prespecified search strategy, 5 RCTs with 1106 patients comparing ET and intravenous thrombolysis (IVT) were included in the meta-analysis. ET and IVT were associated with similar good (43.06% vs 41.78%; OR=1.14; 95% CI, 0.77 to 1.69; P=0.52;) and excellent (30.43% vs 30.42%; OR=1.05; 95% CI, 0.80 to 1.38; P=0.72;) outcome. For additional end points, ET was not associated with increased occurrence of symptomatic hemorrhage (6.25% vs. 6.22%; OR=1.03; 95% CI, 0.62 to 1.69; P=0.91;), or all-cause mortality (18.45% vs. 17.35%; OR=1.00; 95% CI, 0.73 to 1.39; P=0.99;).
Formal meta-analysis indicates that there are similar safety outcomes and functional independence with endovascular therapy and intravenous thrombolysis for acute ischemic stroke.
Adipose tissue has a central role in the regulation of energy balance and homoeostasis. There are two main types of adipose tissue: WAT (white adipose tissue) and BAT (brown adipose tissue). WAT from certain depots, in response to appropriate stimuli, can undergo a process known as browning where it takes on characteristics of BAT, notably the induction of UCP1 (uncoupling protein 1) expression and the presence of multilocular lipid droplets and multiple mitochondria. How browning is regulated is an intense topic of investigation as it has the potential to tilt the energy balance from storage to expenditure, a strategy that holds promise to combat the growing epidemic of obesity and metabolic syndrome. This review focuses on the transcriptional regulators as well as various proteins and secreted mediators that have been shown to play a role in browning. Emphasis is on describing how many of these factors exert their effects by regulating the three main transcriptional regulators of classical BAT development, namely PRDM16 (PR domain containing 16), PPARγ (peroxisome proliferator-activated receptor γ) and PGC-1α (peroxisome proliferator-activated receptor γ coactivator 1α), which have been shown to be the key nodes in the regulation of inducible brown fat.
beige fat; browning; peroxisome-proliferator-activated receptor γ (PPARγ); peroxisome-proliferator-activated receptor γ coactivator 1-α (PGC-1α); PR domain containing 16 (PRDM16); white adipocytes; 4E-BP1, eukaryotic translation initiation factor 4E binding protein 1; ANP, atrial natriuretic peptide; ATF2, activating transcription factor 2; BAT, brown adipose tissue; BMP7, bone morphogenetic protein 7; C/EBP, CCAAT/enhancer-binding protein; Cidea, cell death-inducing DFFA-like effector a; CNP, cardiac natriuretic peptide; COX2, cyclo-oxygenase 2; CtBP, C-terminal-binding protein; EBF2, early B cell factor-2; eIF4E, eukaryotic translation initiation factor 4E; Elovl3, elongation of very long chain fatty acids (FEN1/Elo2, SUR4/Elo3, yeast)-like 3; FGF21, fibroblast growth factor 21; FoxC2, forkhead box protein C2; MAPK, mitogen-activated protein kinase; miRNA, microRNA; Myf5, myogenic factor 5; NPRC, natriuretic peptide receptor C; PGC-1α, peroxisome-proliferator-activated receptor γ coactivator 1α; PGI2, prostacyclin; PKA, protein kinase A (=cAMP-dependent protein kinase); PKG, protein kinase G (=cGMP-dependent protein kinase); PPARγ, peroxisome-proliferator-activated receptor gamma; PRDM16, PR domain containing 16; pRb, retinoblastoma protein; SRC-1, steroid receptor coactivator-1; SVF, stromal vascular fraction; TBX15, T-box 15; TFAM, mitochondrial transcription factor A; TIF2, transcriptional intermediary factor-2; TRPV4, transient receptor potential vanilloid 4; UCP1, uncoupling protein 1; WAT, white adipose tissue
Visualization of the blood vessels can provide valuable morphological information for diagnosis and therapy strategies for cardiovascular disease. Intravascular ultrasound (IVUS) is able to delineate internal structures of vessel wall with fine spatial resolution. However, the developed IVUS is insufficient to identify the fibrous cap thickness and tissue composition of atherosclerotic lesions. Novel imaging strategies have been proposed, such as increasing the center frequency of ultrasound or using a modulated excitation technique to improve the accuracy of diagnosis. Dual-mode tomography combining IVUS with optical tomography has also been developed to determine tissue morphology and characteristics. The implementation of these new imaging methods requires an open system that allows users to customize the system for various studies. This paper presents the development of an IVUS system that has open structures to support various imaging strategies. The system design is based on electronic components and printed circuit board, and provides reconfigurable hardware implementation, programmable image processing algorithms, flexible imaging control, and raw RF data acquisition. In addition, the proposed IVUS system utilized a miniaturized ultrasound transducer constructed using PMN-PT single crystal for better piezoelectric constant and electromechanical coupling coefficient than traditional lead zirconate titanate (PZT) ceramics. Testing results showed that the IVUS system could offer a minimum detectable signal of 25 μV, allowing a 51 dB dynamic range at 47 dB gain, with a frequency range from 20 to 80 MHz. Finally, phantom imaging, in vitro IVUS vessel imaging, and multimodality imaging with photoacoustics were conducted to demonstrate the performance of the open system.
Deletion of ribosomal protein L32 genes resulted in a nonsexual flocculation of fission yeast. Nonsexual flocculation also occurred when two other ribosomal protein genes, rpl21-2 and rpl9-2, were deleted. However, deletion of two nonribosomal protein genes, mpg and fbp, did not cause flocculation. Overall transcript levels of rpl32 in rpl32-1Δ and rpl32-2Δ cells were reduced by 35.9% and 46.9%, respectively, and overall ribosome levels in rpl32-1Δ and rpl32-2Δ cells dropped 31.1% and 27.8%, respectively, compared to wild-type cells. Interestingly, ribosome protein expression levels and ribosome levels were also reduced greatly in sexually flocculating diploid YHL6381/WT (h+/h−) cells compared to a mixture of YHL6381 (h+) and WT (h−) nonflocculating haploid cells. Transcriptome analysis indicated that the reduction of ribosomal levels in sexual flocculating cells was caused by more-extensive suppression of ribosomal biosynthesis gene expression, while the reduction of ribosomal levels caused by deleting ribosomal protein genes in nonsexual flocculating cells was due to an imbalance between ribosomal proteins. We propose that once the reduction of ribosomal levels is below a certain threshold value, flocculation is triggered.
Zebrafish can fully regenerate their myocardium after ventricular resection without evidence of scars. This extraordinary regenerative ability provides an excellent model system to study the activation of the regenerative potential for human heart tissue. In addition to the morphology, it is vital to understand the cardiac function of zebrafish. To characterize adult zebrafish cardiac function, an ultrasound biomicroscope (UBM) was customized for real-time imaging of the zebrafish heart (about 1 mm in diameter) at a resolution of around 37 µm. Moreover, we developed an image segmentation algorithm to track the cardiac boundary and measure the dynamic size of the zebrafish heart for further quantification of zebrafish cardiac function. The effectiveness and accuracy of the proposed segmentation algorithm were verified on a tissue-mimicking phantom and in vivo zebrafish echocardiography. The quantitative evaluation demonstrated that the accuracy of the proposed algorithm is comparable to the manual delineation by experts.
Micro-ultrasound is an invaluable imaging tool for many clinical and preclinical applications requiring high resolution (approximately several tens of micrometers). Imaging systems for micro-ultrasound, including single-element imaging systems and linear-array imaging systems, have been developed extensively in recent years. Single-element systems are cheaper, but linear-array systems give much better image quality at a higher expense. Annular-array-based systems provide a third alternative, striking a balance between image quality and expense. This paper presents the development of a novel programmable and real-time annular-array imaging platform for micro-ultrasound. It supports multi-channel dynamic beamforming techniques for large-depth-of-field imaging. The major image processing algorithms were achieved by a novel field-programmable gate array technology for high speed and flexibility. Real-time imaging was achieved by fast processing algorithms and high-speed data transfer interface. The platform utilizes a printed circuit board scheme incorporating state-of-the-art electronics for compactness and cost effectiveness. Extensive tests including hardware, algorithms, wire phantom, and tissue mimicking phantom measurements were conducted to demonstrate good performance of the platform. The calculated contrast-to-noise ratio (CNR) of the tissue phantom measurements were higher than 1.2 in the range of 3.8 to 8.7 mm imaging depth. The platform supported more than 25 images per second for real-time image acquisition. The depth-of-field had about 2.5-fold improvement compared to single-element transducer imaging.
Next generation sequencing has dramatically increased our ability to localize disease-causing variants by providing base-pair level information at costs increasingly feasible for the large sample sizes required to detect complex-trait associations. Yet, identification of causal variants within an established region of association remains a challenge. Counter-intuitively, certain factors that increase power to detect an associated region can decrease power to localize the causal variant. First, combining GWAS with imputation or low coverage sequencing to achieve the large sample sizes required for high power can have the unintended effect of producing differential genotyping error among SNPs. This tends to bias the relative evidence for association toward better genotyped SNPs. Second, re-use of GWAS data for fine-mapping exploits previous findings to ensure genome-wide significance in GWAS-associated regions. However, using GWAS findings to inform fine-mapping analysis can bias evidence away from the causal SNP toward the tag SNP and SNPs in high LD with the tag. Together these factors can reduce power to localize the causal SNP by more than half. Other strategies commonly employed to increase power to detect association, namely increasing sample size and using higher density genotyping arrays, can, in certain common scenarios, actually exacerbate these effects and further decrease power to localize causal variants. We develop a re-ranking procedure that accounts for these adverse effects and substantially improves the accuracy of causal SNP identification, often doubling the probability that the causal SNP is top-ranked. Application to the NCI BPC3 aggressive prostate cancer GWAS with imputation meta-analysis identified a new top SNP at 2 of 3 associated loci and several additional possible causal SNPs at these loci that may have otherwise been overlooked. This method is simple to implement using R scripts provided on the author's website.
As next-generation sequencing (NGS) costs continue to fall and genome-wide association study (GWAS) platform coverage improves, the human genetics community is positioned to identify potentially causal variants. However, current NGS or imputation-based studies of either the whole genome or regions previously identified by GWAS have not yet been very successful in identifying causal variants. A major hurdle is the development of methods to distinguish disease-causing variants from their highly-correlated proxies within an associated region. We show that various common factors, such as differential sequencing or imputation accuracy rates and linkage disequilibrium patterns, with or without GWAS-informed region selection, can substantially decrease the probability of identifying the correct causal SNP, often by more than half. We then describe a novel and easy-to-implement re-ranking procedure that can double the probability that the causal SNP is top-ranked in many settings. Application to the NCI Breast and Prostate Cancer (BPC3) Cohort Consortium aggressive prostate cancer data identified new top SNPs within two associated loci previously established via GWAS, as well as several additional possible causal SNPs that had been previously overlooked.
The Tat protein of HIV-1 has several well-known properties, such as nucleocytoplasmic trafficking, transactivation of transcription, interaction with tubulin, regulation of mitotic progression, and induction of apoptosis. Previous studies have identified a couple of lysine residues in Tat that are essential for its functions. In order to analyze the functions of all the lysine residues in Tat, we mutated them individually to alanine, glutamine, and arginine. Through systematic analysis of the lysine mutants, we discovered several previously unidentified characteristics of Tat. We found that lysine acetylation could modulate the subcellular localization of Tat, in addition to the regulation of its transactivation activity. Our data also revealed that lysine mutations had distinct effects on microtubule assembly and Tat binding to bromodomain proteins. By correlation analysis, we further found that the effects of Tat on apoptosis and mitotic progression were not entirely attributed to its effect on microtubule assembly. Our findings suggest that Tat may regulate diverse cellular activities through binding to different proteins and that the acetylation of distinct lysine residues in Tat may modulate its interaction with various partners.
Alzheimer's disease (AD) is a progressive neurodegenerative disorder that accounts for the major cause of dementia, and the increasing worldwide prevalence of AD is a major public health concern. Increasing epidemiological studies suggest that diet and nutrition might be important modifiable risk factors for AD. Dietary supplementation of antioxidants, B vitamins, polyphenols, and polyunsaturated fatty acids are beneficial to AD, and consumptions of fish, fruits, vegetables, coffee, and light-to-moderate alcohol reduce the risk of AD. However, many of the results from randomized controlled trials are contradictory to that of epidemiological studies. Dietary patterns summarizing an overall diet are gaining momentum in recent years. Adherence to a healthy diet, the Japanese diet, and the Mediterranean diet is associated with a lower risk of AD. This paper will focus on the evidence linking many nutrients, foods, and dietary patterns to AD.
We used a whole blood assay to characterize the immune system’s response following cardiopulmonary bypass (CPB) in children to identify the risk for postoperative infections. We assessed the impact of CPB on histone methylation as a potential mechanism for altering gene expression necessary for the immune system’s capacity to defend against infections.
We prospectively enrolled patients <18 years old undergoing heart surgery requiring CPB at C.S. Mott Children’s Hospital. Blood was obtained from patients prior to CPB, on CPB and on postoperative days 1, 3 and 5. Ex vivo LPS-induced TNF-α production measured the capacity of the immune system. Serum cytokines were measured using a multiplex assay. Chromatin Immunoprecipitation (ChIP) to detect histone modifications at the interleukin (IL)-10 promoter was performed on circulating mononuclear cells from a subgroup of patients.
We enrolled 92 patients and postoperative day 1 samples identified a subpopulation of immunocompetent patients at low risk for infections with a specificity of 93% (C.I. 83–98%) and a negative predictive value of 88% (C.I. 77–95%; p=0.006). Patients classified as immunoparalyzed had serum IL-10 levels 2.4 fold higher than the immunocompetent group (mean 14.3 ± 18.3 pg/ml vs. 6.0 ± 5.0 pg/ml, p=0.01). In a subgroup of patients, we identified a greater percent of the “gene on” epigenetic signature, H3K4me3, associated with the IL-10 promoter following CPB.
Our data demonstrate that immunophenotyping patients after CPB can predict their risk of developing postoperative infections. Novel mechanistic data suggest that CPB impacts epigenetic alterations in IL-10 gene regulation.
Cardiopulmonary Bypass; Immunoparalysis; Post-translational histone modification; Interleukin-10; Epigenetics; Sepsis
Background: Congenital heart disease (CHD) is one of the most common human birth defects. The etiology and pathogenesis of CHD are complex and involve several genes as well as multiple changes in signaling pathways. The aim of this study was to identify potential pathological mutations in the Homeobox C9 (Hoxc9) gene in 350 Chinese children with CHD to further understand the etiology of CHD. Method: Sequence analysis of the Hoxc9 gene in 350 nonsyndromic patients with CHD Result: We did not identify any nonsynonymous variants in the coding regions of Hoxc9 in the patients with CHD. We found one synonymous variant c.C564T (p. his188his) in one ventricular septal defect patient. We also identified four previously reported polymorphisms (rs56368105, rs12817092, rs34079606, and rs2241820) in CHD. Conclusions: We did not find any diagnostic alterations in the coding regions of Hoxc9 among the patients with CHD. Nevertheless, to our knowledge, this is the first study of Hoxc9 in nonsyndromic CHD and has expanded our overall knowledge of the etiology of this disease.
Over 40 missense mutations in the human SCN1A sodium channel gene are linked to an epilepsy syndrome termed genetic epilepsy with febrile seizures plus (GEFS+). Inheritance of GEFS+ is dominant but the underlying cellular mechanisms remain poorly understood. Here we report knock-in of a GEFS+ SCN1A mutation (K1270T) into the Drosophila sodium channel gene, para, causes a semi-dominant temperature-induced seizure phenotype. Electrophysiological studies of GABAergic interneurons in the brains of adult GEFS+ flies reveal a novel cellular mechanism underlying heat-induced seizures: the deactivation threshold for persistent sodium currents reversibly shifts to a more negative voltage when the temperature is elevated. This leads to sustained depolarizations in GABAergic neurons and reduced inhibitory activity in the central nervous system. Further, our data indicate a natural temperature-dependent shift in sodium current deactivation (exacerbated by mutation) may contribute to febrile seizures in GEFS+ and perhaps normal individuals.
Fission yeast cells express Rpl32-2 highly while Rpl32-1 lowly in log phase; in contrast, expression of Rpl32-1 raises and reaches a peak level while Rpl32-2 is downregulated to a low basic level when cells enter into stationary phase. Overexpression of Rpl32-1 inhibits cell growth while overexpression of Rpl32-2 does not. Deleting rpl32-2 impairs cell growth more severely than deleting rpl32-1 does. Cell growth impaired by deleting either paralog can be rescued completely by reintroducing rpl32-2, but only partly by rpl32-1. Overexpression of Rpl32-1 inhibits cell division, yielding 4c DNA and multiple septa, while overexpressed Rpl32-2 promotes it. Transcriptomics analysis proved that Rpl32 paralogs regulate expression of a subset of genes related with cell division and stress response in a distinctive way. This functional difference of the two paralogs is due to their difference of 95th amino acid residue. The significance of a competitive inhibition between Rpl32 paralogs on their expression is discussed.
Chicken interferon α (ChIFN-α) and ChIFN-β are type I IFNs that are important antiviral cytokines in the innate immune system. In the present study, we identified the virus-induced expression of ChIFN-α and ChIFN-β in chicken fibroblast DF-1 cells and systematically evaluated the antiviral activities of recombinant ChIFN-α and ChIFN-β by cytopathic-effect (CPE) inhibition assays. We found that ChIFN-α exhibited stronger antiviral activity than ChIFN-β in terms of inhibiting the replication of vesicular stomatitis virus, Newcastle disease virus and avian influenza virus, respectively. To elucidate the mechanism of differential antiviral activities between the two ChIFNs, we measured the relative mRNA levels of IFN-stimulated genes (ISGs) in IFN-treated DF-1 cells by real-time PCR. ChIFN-α displayed greater induction potency than ChIFN-β on several ISGs encoding antiviral proteins and MHC-I, whereas ChIFN-α was less potent than ChIFN-β for inducing ISGs involved in signaling pathways. In conclusion, ChIFN-α and ChIFN-β presented differential induction potency on various sets of ISGs, and the stronger antiviral activity of ChIFN-α is likely attributed to the greater expression levels of downstream antiviral ISGs.
Previously, it has been shown that GPI proteins are required for cell wall synthesis and organization in Aspergillus fumigatus, a human opportunistic pathogen causing life-threatening invasive aspergillosis (IA) in immunocompromised patients. Blocking GPI anchor synthesis leads to severe phenotypes such as cell wall defects, increased cell death, and attenuated virulence. However, the mechanism by which these phenotypes are induced is unclear. To gain insight into global effects of GPI anchoring in A. fumigatus, in this study a conditional expression mutant was constructed and a genome wide transcriptome analysis was carried out. Our results suggested that suppression of GPI anchor synthesis mainly led to activation of phosphatidylinositol (PtdIns) signaling and ER stress. Biochemical and morphological evidence showed that autophagy was induced in response to suppression of the GPI anchor synthesis, and also an increased necroptosis was observed. Based on our results, we propose that activation of PtdIns3K and increased cytosolic Ca2+, which was induced by both ER stress and PtdIns signaling, acted as the main effectors to induce autophagy and possible necroptosis.
The efficacy of radiation therapy for lung cancer is limited by radiation-induced lung toxicity (RILT). Although tumor necrosis factor-alpha (TNF-α) signaling plays a critical role in RILT, the molecular regulators of radiation-induced TNF-α production remain unknown. We investigated the role of a major TNF-α regulator, Tristetraprolin (TTP), in radiation-induced TNF-α production by macrophages. For in vitro studies we irradiated (4 Gy) either a mouse lung macrophage cell line, MH-S or macrophages isolated from TTP knockout mice, and studied the effects of radiation on TTP and TNF-α levels. To study the in vivo relevance, mouse lungs were irradiated with a single dose (15 Gy) and assessed at varying times for TTP alterations. Irradiation of MH-S cells caused TTP to undergo an inhibitory phosphorylation at Ser-178 and proteasome-mediated degradation, which resulted in increased TNF-α mRNA stabilization and secretion. Similarly, MH-S cells treated with TTP siRNA or macrophages isolated from ttp (−/−) mice had higher basal levels of TNF-α, which was increased minimally after irradiation. Conversely, cells overexpressing TTP mutants defective in undergoing phosphorylation released significantly lower levels of TNF-α. Inhibition of p38, a known kinase for TTP, by either siRNA or a small molecule inhibitor abrogated radiation-induced TNF-α release by MH-S cells. Lung irradiation induced TTPSer178 phosphorylation and protein degradation and a simultaneous increase in TNF-α production in C57BL/6 mice starting 24 h post-radiation. In conclusion, irradiation of lung macrophages causes TTP inactivation via p38-mediated phosphorylation and proteasome-mediated degradation, leading to TNF-α production. These findings suggest that agents capable of blocking TTP phosphorylation or stabilizing TTP after irradiation could decrease RILT.
Rho family guanosine triphosphatases (GTPases), such as RhoA, Cdc42, and Rac1, play a fundamental role in various cellular processes. The activation of Rho proteins is catalyzed by guanine nucleotide-exchange factors (GEFs), which promote the exchange of GDP for GTP. The precise mechanisms regulating the activation of Rho proteins are not fully understood. Herein, we demonstrate that RhoA activity is regulated by cylindromatosis (CYLD), a deubiquitinase harboring multiple functions. In addition, we find that RhoA-mediated cytoskeletal rearrangement, chromosome separation, and cell polarization are altered in CYLD-depleted cells. Mechanistically, CYLD does not interact with RhoA; instead, it interacts with and deubiquitinates leukemia-associated RhoGEF (LARG). Our data further show that CYLD-mediated deubiquitination of LARG enhances its ability to stimulate the GDP/GTP exchange on RhoA. These data thus identify LARG as a new substrate of CYLD and provide novel insights into the regulation of RhoA activation. Our results also suggest that the LARG-RhoA signaling pathway may play a role in diverse CYLD-mediated cellular events.