Here we report the examination of two convenient strategies, the use of a D-amino acid residue or a glycoside segment, for increasing the proteolytic resistance of supramolecular hydrogelators based on small peptides. Our results show that the introduction of D-amino acid or glycoside to the peptides significantly increases the resistance of the hydrogelators against proteinase K, a powerful endopeptidase. The insertion of D-amino acid in the peptide backbone, however, results relatively low storage moduli of the hydrogels, likely due to the disruption of the superstructures of the molecular assembly. In contrast, the introduction of a glycoside to the C-terminal of peptide enhances the biostability of the hydrogelators without the significant decrease of the storage moduli of the hydrogels. This work suggests that the inclusion of a simple glycogen in hydrogelators is a useful approach to increase their biostability, and the gained understanding from the work may ultimately lead to development of hydrogels of functional peptides for biomedical applications that require long-term biostability.
Rat strains differ dramatically in their susceptibility to mammary carcinogenesis. On the assumption that susceptibility genes are conserved across mammalian species and hence inform human carcinogenesis, numerous investigators have used genetic linkage studies in rats to identify genes responsible for differential susceptibility to carcinogenesis. Using a genetic backcross between the resistant Copenhagen (Cop) and susceptible Fischer 344 (F344) strains, we mapped a novel mammary carcinoma susceptibility (Mcs30) locus to the centromeric region on chromosome 12 (LOD score of ∼8.6 at the D12Rat59 marker). The Mcs30 locus comprises approximately 12 Mbp on the long arm of rat RNO12 whose synteny is conserved on human chromosome 13q12 to 13q13. After analyzing numerous genes comprising this locus, we identified Fry, the rat ortholog of the furry gene of Drosophila melanogaster, as a candidate Mcs gene. We cloned and determined the complete nucleotide sequence of the 13 kbp Fry mRNA. Sequence analysis indicated that the Fry gene was highly conserved across evolution, with 90% similarity of the predicted amino acid sequence among eutherian mammals. Comparison of the Fry sequence in the Cop and F344 strains identified two non-synonymous single nucleotide polymorphisms (SNPs), one of which creates a putative, de novo phosphorylation site. Further analysis showed that the expression of the Fry gene is reduced in a majority of rat mammary tumors. Our results also suggested that FRY activity was reduced in human breast carcinoma cell lines as a result of reduced levels or mutation. This study is the first to identify the Fry gene as a candidate Mcs gene. Our data suggest that the SNPs within the Fry gene contribute to the genetic susceptibility of the F344 rat strain to mammary carcinogenesis. These results provide the foundation for analyzing the role of the human FRY gene in cancer susceptibility and progression.
Here we report the first example of the use of supramolecular hydrogels to discover the protein targets of aggregates of small molecules.
Genetic studies have demonstrated very high heritability for Alzheimer's disease (AD) risk in humans; however, these genetic contributions have proven extremely challenging to map in large studies of AD patients. Processing of the amyloid precursor protein (APP) to produce amyloid-beta (Aβ) peptide is increasingly believed to be of central importance in AD pathogenesis. Intriguingly, mice from the C57BL/6J and DBA2/J inbred strains carrying the R1.40 APP transgene produce identical levels of unprocessed APP, but demonstrate significant, heritable differences in Aβ levels. To identify specific loci responsible for the observed genetic control of Aβ metabolism in this model system, we have performed a whole-genome quantitative trait locus (QTL) mapping experiment on a total of 516 animals from a C57BL/6JxDBA/2J intercross using a dense set of SNP genetic markers. Our studies have identified three loci on mouse chromosomes 1, 2, and 7 showing significant or suggestive associations with brain Aβ levels, several of which contain regions syntenic to previous reports of linkage in human AD.
APP; beta-amyloid; Abeta; Alzheimer; genetics; QTL; mouse; intercross
Here we report the generation of a novel class of supramolecular hydrogelators based on the integration of nucleobase, Arg-Gly-Asp (RGD) peptides, and glucosamine in a single molecule. These novel small molecule hydrogelators self-assemble in water to form stable supramolecular nanofibers/hydrogels and exhibit useful biostability. This approach provides a new opportunity for systematic exploration of the self-assembly of small biomolecules by varying any individual segment to generate a large array of supramolecular hydrogels for biological functions and for biomedical applications.
This study examines the regulating effect of Sonic Hedgehog (Shh) on the permeability of the blood-brain barrier (BBB) in cerebral ischemia. By employing permanent middle cerebral artery occlusion (pMCAO) model, we find that Shh significantly decreases brain edema and preserves BBB permeability. Moreover, Shh increases zonula occludens-1 (ZO-1), occludin and angiopiotetin-1 (Ang-1) expression in the ischemic penumbra. Blockage of Shh with cyclopamine abolishes the effects of Shh on brain edema, BBB permeability and ZO-1, occludin, Ang-1 expression. Primary brain microvessel endothelial cells (BMECs) and astrocytes were pre-treated with Shh, cyclopamine, Ang-1-neutralizing antibody, and subjected to oxygen-glucose deprivation (OGD). Results show that the Ang-1 protein level in the culture medium of Shh-treated astrocytes is significantly higher. Shh also increased ZO-1, occludin and Ang-1 expression in BMECs, while cyclopamine and Ang-1-neutralizing antibody inhibited the effects of Shh on the ZO-1 and occludin expression, respectively. This study suggests that, under ischemic insults, Shh triggers Ang-1 production predominantly in astrocytes, and the secreted Ang-1 acts on BMECs, thereby upregulating ZO-1 and occludin to repair the tight junction and ameliorate the brain edema and BBB leakage.
A novel facultative psychrotroph (strain CBS-1), which accumulates poly-β-hydroxybutyrate (PHB), was isolated from soil samples taken from Changbai Mountain, China. Phylogenetic analysis based on 16S rRNA sequence data and Biolog analysis identified strain CBS-1 as Pseudomonas mandelii. Transmission electron micrographs revealed abundant electron-transparent intracellular granules. 1H-nuclear magnetic resonance analysis revealed that the granules were composed of PHB. P. mandelii CBS-1 grew optimally at 20°C. When cultured aerobically for 48 h with sucrose as the sole carbon source, strain CBS-1 yielded a maximum cell density of 29.3 g/L cell dry weight and synthesized 22.3 g/L of PHB. The ability of strain CBS-1 to grow at a low temperature and rapidly synthesize high levels of PHB may reduce the costs of industrial PHB production.
PHB; Pseudomonas mandelii CBS-1; Facultative psychrotrophs; 1H- nuclear magnetic resonance; Fermentation
The aim of this study was to assess the in vitro release kinetics of antituberculosis drug-loaded nanoparticles (NPs) using a “modified” cylindrical apparatus fitted with a regenerated cellulose membrane attached to a standard dissolution apparatus (modifiedcylinder method). The model drugs that were used were rifampicin (RIF) and moxifloxacin hydrochloride (MX). Gelatin and polybutyl cyanoacrylate (PBCA) NPs were evaluated as the nanocarriers, respectively. The dissolution and release kinetics of the drugs from loaded NPs were studied in different media using the modified cylinder method and dialysis bag technique was used as the control technique. The results showed that use of the modified cylinder method resulted in different release profiles associated with unique release mechanisms for the nanocarrier systems investigated. The modified cylinder method also permitted discrimination between forced and normal in vitro release of the model drugs from gelatin NPs in the presence or absence of enzymatic degradation. The use of dialysis bag technique resulted in an inability to differentiate between the mechanisms of drug release from the NPs in these cases. This approach offers an effective tool to investigate in vitro release of RIF and MX from NPs, which further indicate that this technique can be used for performance testing of nanosized carrier systems.
To evaluate the therapeutic benefit of 3D-image-guided high-dose-rate intracavitary brachytherapy (3D-image-guided HDR-BT) used as a salvage treatment of intensity modulated radiation therapy (IMRT) in patients with locally persistent nasopharyngeal carcinoma (NPC).
Thirty-two patients with locally persistent NPC after full dose of IMRT were evaluated retrospectively. 3D-image-guided HDR-BT treatment plan was performed on a 3D treatment planning system (PLATO BPS 14.2). The median dose of 16 Gy was delivered to the 100% isodose line of the Gross Tumor Volume.
The whole procedure was well tolerated under local anesthesia. The actuarial 5-y local control rate for 3D-image-guided HDR-BT was 93.8%, patients with early-T stage at initial diagnosis had 100% local control rate. The 5-y actuarial progression-free survival and distant metastasis-free survival rate were 78.1%, 87.5%. One patient developed and died of lung metastases. The 5-y actuarial overall survival rate was 96.9%.
Our results showed that 3D-image-guided HDR-BT would provide excellent local control as a salvage therapeutic modality to IMRT for patients with locally persistent disease at initial diagnosis of early-T stage NPC.
Nasopharyngeal carcinoma; Intensity-modulated radiotherapy; Persistent disease; 3D-image-guided HDR Brachytherapy; Local tumor control
To examine whether serum uric acid (SUA) is associated with 2-hour postload glucose (2-h PG) in Chinese with impaired fasting plasma glucose (IFG) and/or HbA1c (IA1C).
Research Design and Methods
Anthropometric and biochemical examinations, such as SUA concentration, were performed in 3763 individuals from all the villages in Baqiao County, China. A 75-g oral glucose tolerance test (OGTT) was conducted in 1197 Chinese with prediabetes as having IFG (110≤ fasting plasma glucose [FPG] <126 mg/dl and HbA1c <6.5%), IA1C (5.7% ≤ HbA1c <6.5% and FPG <126 mg/dl), or both.
The present study included 1197 participants with IFG and/or IA1C (mean age 56.5±10.3 years; 50.6% men). In multivariate linear regression, after adjustment for gender, age, smoking and drinking, body mass index (BMI), systolic and diastolic blood pressure (SBP, DBP), lipid profiles, logarithmic transformed C-reactive protein (log-CRP), estimated glomerular filtration rate (e-GFR), FPG and HbA1c, with a 1-mg/dl increment of SUA, 2-h PG increased by 5.04±0.72 (P<0.001), 3.06±1.08 (P = 0.001), 5.40±1.26 (P<0.001), and 2.34±2.16 mg/dl (P = 0.056) in all participants, in participants with normal glucose tolerance (NGT), with impaired glucose tolerance (IGT), and with 2-h newly diagnosed diabetes (2-h NDM, with 2-h PG ≥200 mg/dl), respectively. In both men and women, 2-h PG increased progressively and significantly from the lower to the upper SUA tertiles (P<0.001). Moreover, in multivariate logistic regression, 1-standard deviation (SD; 1.53 mg/dl) increment of SUA was significantly associated with a 36% higher risk for 2-h NDM (Odds ratio [CI 95%]: 1.36 [1.09–1.99]; P = 0.03).
SUA is significantly associated with 2-h PG in Chinese with IFG and/or IA1C.
Microbacterium sp. 4N2-2, isolated from a wastewater treatment plant, converts the antibacterial fluoroquinolone norfloxacin to N-acetylnorfloxacin and three other metabolites. Because N-acetylation results in loss of antibacterial activity, identification of the enzyme responsible is important for understanding fluoroquinolone resistance. The enzyme was identified as glutamine synthetase (GS); N-acetylnorfloxacin was produced only under conditions associated with GS expression. The GS gene (glnA) was cloned, and the protein (53 kDa) was heterologously expressed and isolated. Optimal conditions and biochemical properties (Km and Vmax) of purified GS were characterized; the purified enzyme was inhibited by Mn2+, Mg2+, ATP, and ADP. The contribution of GS to norfloxacin resistance was shown by using a norfloxacin-sensitive Escherichia coli strain carrying glnA derived from Microbacterium sp. 4N2-2. The GS of Microbacterium sp. 4N2-2 was shown to act as an N-acetyltransferase for norfloxacin, which produced low-level norfloxacin resistance. Structural and docking analysis identified potential binding sites for norfloxacin at the ADP binding site and for acetyl coenzyme A (acetyl-CoA) at a cleft in GS. The results suggest that environmental bacteria whose enzymes modify fluoroquinolones may be able to survive in the presence of low fluoroquinolone concentrations.
A total of 110 strains of Klebsiella pneumoniae were used to optimize pulsed-field gel electrophoresis (PFGE) for subtyping of K. pneumoniae. For optimization of electrophoresis parameters (EPs) of XbaI-PFGE, 11 isolates were analyzed with XbaI digestion using three EPs. The EP of a switch time of 6 to 36 s for 18.5 h gave clearest patterns and was declared the optimal EP for XbaI PFGE of K. pneumoniae. By software analysis and pilot study, AvrII was chosen as another PFGE enzyme. Both XbaI- and AvrII-PFGE gave D-values higher than 0.99 for 69 K. pneumoniae isolated from different sources. Our results also showed good typeability, reproducibility of both XbaI- and AvrII-PFGE for K. pneumoniae subtyping. Furthermore, the established PFGE method also had good discriminatory power to distinguish outbreak K. pneumoniae strains and a high degree of consistency with multilocus sequence typing method. A rapid PFGE protocol was established here, which could be used for genotyping and other researches of K. pneumoniae.
Klebsiella pneumoniae; pulsed-field gel electrophoresis; molecular subtyping
Chronic growth hormone (GH) therapy has been shown to cause insulin resistance, but the mechanism remains unknown. PTEN, a tumor suppressor gene, is a major negative regulator of insulin signaling. In this study, we explored the effect of chronic GH on insulin signaling in the context of PTEN function. Balb/c healthy mice were given recombinant human or bovine GH intraperitoneally for 3 weeks. We found that phosphorylation of Akt was significantly decreased in chronic GH group and the expression of PTEN was significantly increased. We further examined this effect in the streptozotocin-induced Type I diabetic mouse model, in which endogenous insulin secretion was disrupted. Insulin/PI3K/Akt signaling was impaired. However, different from the observation in healthy mice, the expression of PTEN did not increase. Similarly, PTEN expression did not significantly increase in chronic GH-treated mice with hypoinsulinemia induced by prolonged fasting. We conducted in-vitro experiments in HepG2 cells to validate our in-vivo findings. Long-term exposure to GH caused similar resistance of insulin/PI3K/Akt signaling in HepG2 cells; and over-expression of PTEN enhanced the impairment of insulin signaling. On the other hand, disabling the PTEN gene by transfecting the mutant PTEN construct C124S or siPTEN, disrupted the chronic GH induced insulin resistance. Our data demonstrate that PTEN plays an important role in chronic-GH-induced insulin resistance. These findings may have implication in other pathological insulin resistance.
Programmed cell death (PCD) is a genetically-based cell death mechanism with vital roles in eukaryotes. Although there is limited consensus on similar death mode programs in prokaryotes, emerging evidence suggest that PCD events are operative. Here we present cell death events in a cyanobacterium living endophytically in the fern Azolla microphylla, suggestive of PCD. This symbiosis is characterized by some unique traits such as a synchronized development, a vertical transfer of the cyanobacterium between plant generations, and a highly eroding cyanobacterial genome. A combination of methods was used to identify cell death modes in the cyanobacterium. Light- and electron microscopy analyses showed that the proportion of cells undergoing cell death peaked at 53.6% (average 20%) of the total cell population, depending on the cell type and host developmental stage. Biochemical markers used for early and late programmed cell death events related to apoptosis (Annexin V-EGFP and TUNEL staining assays), together with visualization of cytoskeleton alterations (FITC-phalloidin staining), showed that all cyanobacterial cell categories were affected by cell death. Transmission electron microscopy revealed four modes of cell death: apoptotic-like, autophagic-like, necrotic-like and autolytic-like. Abiotic stresses further enhanced cell death in a dose and time dependent manner. The data also suggest that dynamic changes in the peptidoglycan cell wall layer and in the cytoskeleton distribution patterns may act as markers for the various cell death modes. The presence of a metacaspase homolog (domain p20) further suggests that the death modes are genetically programmed. It is therefore concluded that multiple, likely genetically programmed, cell death modes exist in cyanobacteria, a finding that may be connected with the evolution of cell death in the plant kingdom.
Akt kinase plays a central role in cell growth, metabolism and tumorigenesis. Although TRAF6 E3 ligase orchestrates IGF-1-mediated Akt ubiquitination and activation, it is unclear whether TRAF6 is involved in Akt activation by other growth factor receptors as well. Here we show that Akt ubiquitination is also induced by activation of ErbB receptors; unexpectedly, Skp2 SCF complex, but not TRAF6, is a critical E3 ligase for ErbB receptor-mediated Akt ubiquitination and membrane recruitment. Interestingly, Skp2 deficiency impairs Akt activation, Glut1 expression, glucose uptake and glycolysis, and breast cancer progression in various tumor models. Moreover, Skp2 overexpression correlates with Akt activation, breast cancer metastasis, and serves as a marker for poor prognosis in Her2-positive patients. Finally, we showed that Skp2 silencing sensitizes Her2-overexpressing tumors to Herceptin treatment. Our study suggests that distinct E3 ligases are utilized by diverse growth factors for Akt ubiquitination and activation.
Acute lung injury (ALI) is considered to be the major cause of respiratory failure in critically ill patients. Clinical studies have found that in patients with sepsis and after hemorrhage, the elevated level of high mobility group box-1(HMGB-1) in their circulation is highly associated with ALI, but the underlying mechanism remains unclear. Extracellular HMGB-1 has cytokine-like properties and can bind to Toll-like Receptor-4 (TLR4), which was reported to play an important role in the pathogenesis of ALI. The aim of this study was to determine whether HMGB-1 directly contributes to ALI and whether TLR4 signaling pathway is involved in this process.
Recombinant human HMGB-1 (rhHMGB-1) was used to induce ALI in male Sprague-Dawley rats. Lung specimens were collected 2 h after HMGB-1 treatment. The levels of TNF-α, IL-1β, TLR4 protein, and TLR4 mRNA in lungs as well as pathological changes of lung tissue were assessed. In cell studies, the alveolar macrophage cell line, NR8383, was collected 24 h after rhHMGB-1 treatment and the levels of TNF-α and IL-1β in cultured medium as well as TLR4 protein and mRNA levels in the cell were examined. TLR4-shRNA-lentivirus was used to inhibit TLR4 expression, and a neutralizing anti-HMGB1 antibody was used to neutralize rhHMGB-1 both in vitro and in vivo.
Features of lung injury and significant elevation of IL-1β and TNF-α levels were found in lungs of rhHMGB-1-treated animals. Cultured NR8383 cells were activated by rhHMGB-1 treatment and resulted in the release of IL-1β and TNF-α. TLR4 expression was greatly up-regulated by rhHMGB-1. Inhibition of TLR4 or neutralization of HMGB1 with a specific antibody also attenuated the inflammatory response induced by HMGB-1 both in vivo and in vitro.
HMGB-1 can activate alveolar macrophages to produce proinflammatory cytokines and induce ALI through a mechanism that relies on TLR-4.
The Mre11/Rad50/NBS1 (MRN) complex is thought to be a critical sensor that detects damaged DNA and recruits ATM to DNA foci for activation. However, it remains to be established how the MRN complex regulates ATM recruitment to the DNA foci during DNA double-strand breaks (DSBs). Here we show that Skp2 E3 ligase is a key component for the MRN complex-mediated ATM activation in response to DSBs. Skp2 interacts with NBS1 and triggers K63-linked ubiquitination of NBS1 upon DSBs, which is critical for the interaction of NBS1 with ATM, thereby facilitating ATM recruitment to the DNA foci for activation. Finally, we show that Skp2 deficiency exhibits a defect in homologous recombination (HR) repair, thereby increasing IR sensitivity. Our results provide molecular insights into how Skp2 and the MRN complex coordinate to activate ATM, and identify Skp2-mediatetd NBS1 ubiquitination as a vital event for ATM activation in response to DNA damage.
Functional genome annotation is important for studies of dynamic genetic architectures, revealing critical developmental pathways, and facilitating understanding of disease and evolution. Characterization of the transcriptome has revealed many active genes with spatio-temporal regulation and evolutionary significance. However, expression studies have often been limited to gene or exon-based microarrays, EST sequencing, or small amounts of cDNA sequencing on next generation sequencing (NGS) platforms. These studies have often been limited by technology, depth of sequencing, and a lack of sufficient controls for comparison. Very few RNA sequencing standards or expected measures exist to help in the quantification of gene or splice form expression.
We present the results of an ongoing large-scale ABRF study of RNA-Seq. The goals of this ABRF-NGS study are to evaluate the performance of NGS platforms and to identify optimal methods and best practices. The study includes five ABRF Research Groups and over 20 core facility laboratories. To assess the detection of expression-based molecular signatures using RNA-Seq and to study the sources of possible site-to-site variance in results, we performed sequencing on five NGS platforms using standardized RNA samples with synthetic RNA spike-ins. The platforms included Illumina (HiSeq 2000/2500 and MiSeq), Roche 454 GS FLX, Life Technologies (Ion PGM and Proton), and PacBio.
We observed high correlation of RNA-Seq results within sites, but “site effect” was the largest variance factor outside of biological sources. Additionally, we observed that the “bioinformatics noise” of aligners and annotations contributed substantial variance, underscoring the need for data provenance for long-term studies. As part of this study, we are evaluating many of the popular current RNA and DNA sequence alignment tools and accessing how they deal with issues such as multiple splicing events, multiple type and number of variants, read length, and providing efficient computational analysis time.
Increased aortic stiffness is an acknowledged predictor and cause of cardiovascular disease. The sources and mechanisms of vascular stiffness are not well understood, although the extracellular matrix (ECM) has been assumed to be a major component. We tested here the hypothesis that the focal adhesions (FAs) connecting the cortical cytoskeleton of vascular smooth muscle cells (VSMCs) to the matrix in the aortic wall are a component of aortic stiffness and that this component is dynamically regulated. First, we examined a model system in which magnetic tweezers could be used to monitor cellular cortical stiffness, serum-starved A7r5 aortic smooth muscle cells. Lysophosphatidic acid (LPA), an activator of myosin that increases cell contractility, increased cortical stiffness. A small molecule inhibitor of Src-dependent FA recycling, PP2, was found to significantly inhibit LPA-induced increases in cortical stiffness, as well as tension-induced increases in FA size. To directly test the applicability of these results to force and stiffness development at the level of vascular tissue, we monitored mouse aorta ring stiffness with small sinusoidal length oscillations during agonist-induced contraction. The alpha-agonist phenylephrine, which also increases myosin activation and contractility, increased tissue stress and stiffness in a PP2- and FAK inhibitor 14-attenuated manner. Subsequent phosphotyrosine screening and follow-up with phosphosite-specific antibodies confirmed that the effects of PP2 and FAK inhibitor 14 in vascular tissue involve FA proteins, including FAK, CAS, and paxillin. Thus, in the present study we identify, for the first time, the FA of the VSMC, in particular the FAK-Src signaling complex, as a significant subcellular regulator of aortic stiffness and stress.
Schistosoma japonicum (S. japonicum) is an extremely harmful pathogen, which infects humans and causes severe public health problems. To date, no effective therapeutic drugs for this pathogen are available. In this study, we designed and constructed three hammerhead ribozymes targeting the eggshell protein gene of S. japonicum (SjESG). The cleavage activities of these three ribozymes were determined using cleavage experiments. The in vitro cleavage results showed that among the three synthesized ribozymes (Rz1, Rz2 and Rz3), Rz1 and Rz3 cleaved their target RNAs effectively. However, Rz2 did not cleave its target RNA detectably. The putative therapeutic roles of these three ribozymes to inhibit the reproduction of S. japonicum in mice were studied in vivo. Compared with the negative controls, Rz1 and Rz3 treatments resulted in increased levels of IFN-γ but decreased levels of IL-4 in mice. Rz2 affected levels of IFN-γ and IL-4 to degrees similar with those caused by the vector controls. In addition, Rz1 and Rz3 reduced the amounts of adult worms and eggs in the livers of mice more extensively than Rz2 and the vector controls. Altogether, these results suggest a correlation between the in vitro cleavage abilities of Rz1 and Rz3 and their roles in reproduction inhibition of S. japonicum.
Schistosoma japonicum; eggshell protein gene; hammerhead ribozyme; in vitro cleavage; anti-reproduction
The asymmetric unit of the title Pb-based coordination polymer, [Pb2(C24H16N2O8)(H2O)2]n, consists of one PbII cation, half of a 4,4′-(1,4-phenylene)bis(2,6-dimethylpyridine-3,5-dicarboxylate (L
4−) ligand and one coordinating water molecule. The centers of the benzene ring of the ligand and the four-membered Pb/O/Pb/O ring are located on centers of inversion. The PbII ion is coordinated in form of a distorted polyhedron by seven O atoms from four separate L
4− ligands and by one water O atom. The PbO7 polyhedra share O atoms, forming infinite zigzag [PbO4(H2O)]n chains along  that are bridged by L
4− ligands, forming a two-dimensional coordination network parallel to (001). O—H⋯O hydrogen bonds involving the water molecule are observed.
Histone modification has emerged as a very important mechanism regulating the transcriptional status of the genome. Insulin-like growth factor 2 (IGF2) is a peptide hormone controlling various cellular processes, including proliferation and apoptosis. H19 gene is closely linked to IGF2 gene, and IGF2 and H19 are reciprocally regulated imprinted genes. The epigenetic signature of H19 promoter (hypermethylation) on the paternal allele plays a vital role in allowing the expression of the paternal allele of IGF2.46 Our previous studies demonstrate that butyrate regulates the expression of IGF2 as well as genes encoding IGF Binding proteins. To obtain further understanding of histone modification and its regulatory potentials in controlling IGF2/H19 gene expression, we investigated the histone modification status of some key histones associated with the expression of IGF2/H19 genes in bovine cells using RNA-seq in combination with Chip-seq technology. A high-resolution map of the major chromatin modification at the IGF2/H19 locus induced by butyrate was constructed to illustrate the fundamental association of the chromatin modification landscape that may play a role in the activation of the IGF2 gene. High-definition epigenomic landscape mapping revealed that IGF2 and H19 have distinct chromatin modification patterns at their coding and promoter regions, such as TSSs and TTSs. Moreover, the correlation between the differentially methylated regions (DMRs) of IGF2/H19 locus and histone modification (acetylation and methylation) indicated that epigenetic signatures/markers of DNA methylation, histone methylation and histone acetylation were differentially distributed on the expressed IGF2 and silenced H19 genes. Our evidence also suggests that butyrate-induced regional changes of histone acetylation statusin the upstream regulation domain of H19 may be related to the reduced expression of H19 and strong activation of IGF2. Our results provided insights into the mechanism of butyrate-induced loss of imprinting (LOI) of IGF2 and regulation of gene expression by histone modification.
bovine; butyrate; ChIP-seq; chromatin; histone modification; IGF2
Toll-like receptor 9 (TLR9) recognises unmethylated CpG DNA and activates a signalling cascade, leading to the production of inflammatory cytokines such as TNF-α, IL-1, IL-6 and IL-12 via the adaptor protein MyD88. However, the specific sequence and structural requirements of the CpG DNA for the recognition of and binding to TLR9 are unknown. Moreover, the 3D structures of TLR9 and the TLR9-ODN complex have not been determined. In this study, we propose a reliable model of the interaction of the TLR9 ECD with CpG ODN using bioinformatics tools.
The three-dimensional structures of two TLR9 ECD-CpG ODN complexes were constructed using a homology modelling and docking strategy. Based on the models of these complexes, the TLR9 ECD-CpG ODN interaction patterns were calculated. The results showed that the interface between the human TLR9 and the CpG ODN molecule is geometrically complementary. The computed molecular interactions indicated that LRR11 is the main region of TLR9 that binds to CpG ODN and that five positively charged residues within LRR11 are involved in the binding of the TLR9 ECD to the CpG ODN. Observations in the close-up view of these interactions indicated that these five positively charged residues contribute differently to the binding region within the TLR9 ECD-CpG ODN complex. 337Arg and 338Lys reside in the binding sites of ODN, forming hydrogen bonds and direct contacts with the CpG ODN, whereas 347Lys, 348Arg, and 353His do not directly contact the CpG ODN. These results are in agreement with previously reported experimental data.
In this study, we present two structural models for the human and mouse TLR9 ECD in a complex with CpG ODN. Some features predicted by this model are consistent with previously reported experimental data. This complex model may lead to a better understanding of the function of TLR9 and its interaction with CpG ODN and will improve our understanding of TLR9-ligand interaction in general.
The aim of this study is to evaluate carotid atherosclerosis in patients of type 2 diabetes mellitus with microalbuminuria (MA) by high-frequency ultrasonography. Two hundred and fifty patients of type 2 diabetes mellitus were divided into two groups according to urinary albumin excretion rate (UAER): normoalbuminuria group (130 cases) and microalbuminuria group (120 cases). The intimal-medial thickness (IMT) and the atherosclerotic plaques of carotid artery were observed in both groups by high-frequency ultrasound. Fasting blood glucose (FBG), hemoglobin A1c, and lipid profiles were measured. The values of IMT of microalbuminuria group were significantly higher than those of normoalbuminuria group (P < 0.05). In univariate analysis, IMT was positively and significantly associated with age (r = 0.265, P < 0.05), waist circumference (r = 0.263, P < 0.05), body mass index (r = 0.285, P < 0.05), systolic blood pressure (r = 0.276, P < 0.05), UAER (r = 0.359, P < 0.05), HbA1c (r = 0.462, P < 0.05) and, duration of diabetes (r = 0.370, P < 0.05). In multivariate linear regression analysis, UAER and HbA1c were independent predictors of IMT (P < 0.05 for all). In the two groups, the rate of soft plaques was higher than that of dense plaques and calcified plaques. In conclusion, there is a significant association between microalbuminuria and IMT which is regarded as the early sign of carotid atherosclerosis in type 2 diabetic patients.
Polyploidization can precede the development of aneuploidy in cancer. Polyploidization in megakaryocytes (Mk), in contrast, is a highly controlled developmental process critical for efficient platelet production via unknown mechanisms. Using primary cells, we demonstrate that the guanine exchange factors GEF-H1 and ECT2, which are often overexpressed in cancer and are essential for RhoA activation during cytokinesis, must be downregulated for Mk polyploidization. The first (2N-to-4N) endomitotic cycle requires GEF-H1 downregulation while subsequent cycles (>4N) require ECT2 downregulation. Exogenous expression of both GEF-H1 and ECT2 prevents endomitosis, resulting in proliferation of 2N Mk. Furthermore, we have shown that the mechanism by which polyploidization is prevented in Mk lacking Mkl1, which is mutated in megakaryocytic leukemia, is via elevated GEF-H1 expression; shRNA-mediated GEF-H1 knockdown alone rescues this ploidy defect. These mechanistic insights enhance our understanding of normal versus malignant megakaryocytopoiesis, as well as aberrant mitosis in aneuploid cancers.