Chronic infection with hepatitis B virus (HBV) is associated with the majority of cases of liver cirrhosis (LC) in China. Although liver biopsy is the reference method for evaluation of cirrhosis, it is an invasive procedure with inherent risk. The aim of this study is to discover novel noninvasive specific serum biomarkers for the diagnosis of HBV-induced LC. We performed bead fractionation/MALDI-TOF MS analysis on sera from patients with LC. Thirteen feature peaks which had optimal discriminatory performance were obtained by using support-vector-machine-(SVM-) based strategy. Based on the previous results, five supervised machine learning methods were employed to construct classifiers that discriminated proteomic spectra of patients with HBV-induced LC from those of controls. Here, we describe two novel methods for prediction of HBV-induced LC, termed LC-NB and LC-MLP, respectively. We obtained a sensitivity of 90.9%, a specificity of 94.9%, and overall accuracy of 93.8% on an independent test set. Comparisons with the existing methods showed that LC-NB and LC-MLP held better accuracy. Our study suggests that potential serum biomarkers can be determined for discriminating LC and non-LC cohorts by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. These two classifiers could be used for clinical practice in HBV-induced LC assessment.
Several CC-motif chemokine ligands (CCLs) can block HIV-1 binding sites on CC-motif chemokine receptor 5 (CCR5) and inhibit viral entry. We studied single nucleotide polymorphisms (SNPs) in genes encoding three CCR5 ligands [CCL3 (MIP-1α), CCL4 (MIP-1β), and CCL5 (RANTES)] along with an adjacent gene encoding a CCR2 ligand [CCL2 (MCP-1)] to identify candidate markers for HIV-1 infection and pathogenesis. Analyses of 567 HIV-1 serodiscordant Zambian couples revealed that rs5029410C (in CCL3 intron 2) was associated with lower viral load (VL) in seroconverters, adjusted for gender and age (regression β=−0.57 log10, P=4×10−6). In addition, rs34171309A in CCL3 exon 3 was associated with increased risk of HIV-1 acquisition in exposed seronegatives (hazard ratio=1.52, P=0.006 when adjusted for donor VL and genital ulcer/inflammation). The CCL3 exon 3 SNP, encoding a conservative Glu-to-Asp substitution, and five neighboring SNPs in tight linkage disequilibrium all showed similar associations with HIV-1 acquisition. How these multiple CCL3 SNPs may alter the occurrence or course of HIV-1 infection remains to be determined.
HIV-1 transmission; CCL2; CCL3; CCL4; CCL5; SNP
Bioinformatic tools and databases for glycobiology and glycomics research are playing increasingly important roles in functional studies. However, to verify hypotheses generated by computational glycomics with empirical functional assays is only an emerging field. In this study, we predicted glycan epitopes expressed by a cancer-derived mucin, MUC1, by computational glycomics. MUC1 is expressed by tumor cells with a deficiency in glycosylation. Although numerous diagnostic reagents and cancer vaccines have been designed based on abnormally glycosylated MUC1 sequences, the glycan and peptide sequences responsible for immune responses in vivo are poorly understood. The immunogenicity of synthetic MUC1 glycopeptides bearing Tn or sialyl-Tn antigens have been studied in mouse models, while authentic glyco-epitopes expressed by tumor cells remain unclear. To examine the immunogenicity of authentic cancer derived MUC1 glyco-epitopes, we expressed membrane bound forms of MUC1 tandem repeats in Jurkat, a mutant cancer cell line deficient of mucin-type core-1 β1-3 galactosyltransferase activity, and immunized mice with cancer cells expressing authentic MUC1 glyco-epitopes. Antibody responses to individual glyco-epitopes were determined by chemically synthesized candidate MUC1 glycopeptides predicted through computational glycomics. Monoclonal antibodies can be generated toward chemically synthesized glycopeptide sequences. With RPAPGS(Tn)TAPPAHG as an example, a monoclonal antibody 16A, showed 25-fold higher binding to glycosylated peptide (EC50=9.278±1.059 ng/ml) compared to its non-glycosylated form (EC50=247.3±16.29 ng/ml) as measured by ELISA experiments with plate-bound peptides. A library of monoclonal antibodies toward authentic MUC1 glycopeptide epitopes may be a valuable tool for studying glycan and peptide sequences in cancer, as well as reagents for diagnosis and therapy.
computational glycomics; MUC1; glycopeptide; monoclonal antibodies; cancer; immunopathogenesis; immunotherapy
Mobile robot operators must make rapid decisions based on information about the robot’s surrounding environment. This means that terrain modeling and photorealistic visualization are required for the remote operation of mobile robots. We have produced a voxel map and textured mesh from the 2D and 3D datasets collected by a robot’s array of sensors, but some upper parts of objects are beyond the sensors’ measurements and these parts are missing in the terrain reconstruction result. This result is an incomplete terrain model. To solve this problem, we present a new ground segmentation method to detect non-ground data in the reconstructed voxel map. Our method uses height histograms to estimate the ground height range, and a Gibbs-Markov random field model to refine the segmentation results. To reconstruct a complete terrain model of the 3D environment, we develop a 3D boundary estimation method for non-ground objects. We apply a boundary detection technique to the 2D image, before estimating and refining the actual height values of the non-ground vertices in the reconstructed textured mesh. Our proposed methods were tested in an outdoor environment in which trees and buildings were not completely sensed. Our results show that the time required for ground segmentation is faster than that for data sensing, which is necessary for a real-time approach. In addition, those parts of objects that were not sensed are accurately recovered to retrieve their real-world appearances.
terrain reconstruction; 3D ground segmentation; 3D boundary estimation; height histogram; Gibbs-Markov Random Field
Bioinformatic tools and databases for glycobiology and glycomics research are playing increasingly important roles in functional studies. However, to verify hypotheses generated by computational glycomics with empirical functional assays is only an emerging field. In this study, we predicted glycan epitopes expressed by a cancer-derived mucin, MUC1, by computational glycomics. MUC1 is expressed by tumor cells with a deficiency in glycosylation. Although numerous diagnostic reagents and cancer vaccines have been designed based on abnormally glycosylated MUC1 sequences, the glycan and peptide sequences responsible for immune responses in vivo are poorly understood. The immunogenicity of synthetic MUC1 glycopeptides bearing Tn or sialyl-Tn antigens have been studied in mouse models, while authentic glyco-epitopes expressed by tumor cells remain unclear. To examine the immunogenicity of authentic cancer derived MUC1 glyco-epitopes, we expressed membrane bound forms of MUC1 tandem repeats in Jurkat, a mutant cancer cell line deficient of mucin-type core-1 β1–3 galactosyltransferase activity, and immunized mice with cancer cells expressing authentic MUC1 glyco-epitopes. Antibody responses to individual glyco-epitopes were determined by chemically synthesized candidate MUC1 glycopeptides predicted through computational glycomics. Monoclonal antibodies can be generated toward chemically synthesized glycopeptide sequences. With RPAPGS(Tn)TAPPAHG as an example, a monoclonal antibody 16A, showed 25-fold higher binding to glycosylated peptide (EC50=9.278±1.059 ng/ml) compared to its non-glycosylated form (EC50=247.3±16.29 ng/ml) as measured by ELISA experiments with plate-bound peptides. A library of monoclonal antibodies toward authentic MUC1 glycopeptide epitopes may be a valuable tool for studying glycan and peptide sequences in cancer, as well as reagents for diagnosis and therapy.
computational glycomics; MUC1; glycopeptide; monoclonal antibodies; cancer; immunopathogenesis; immunotherapy
Much recent research effort in traumatic brain injury (TBI) has been devoted to the discovery of a reliable biomarker correlating with severity of injury. Currently, no consensus has been reached regarding a representative marker for traumatic brain injury. In this study, we explored the potential of epithelial/endothelial tyrosine kinase (Etk) as a novel marker for TBI.
TBI was induced in Sprague Dawley (SD) rats by controlled cortical impact. Brain tissue samples were analyzed by Western blot, Q-PCR, and immunofluorescence staining using various markers including glial fibrillary acidic protein, and epithelial/endothelial tyrosine kinase (Etk). Results show increased Etk expression with increased number and severity of impacts. Expression increased 2.36 to 7-fold relative to trauma severity. Significant upregulation of Etk appeared at 1 hour after injury. The expression level of Etk was inversely correlated with distance from injury site. Etk and trauma/inflammation related markers increased post-TBI, while other tyrosine kinases did not.
The observed correlation between Etk level and the number of impacts, the severity of impact, and the time course after impact, as well as its inverse correlation with distance away from injury site, support the potential of Etk as a possible indicator of trauma severity.
To evaluate the stability and heterogeneity of cytokine and chemokine profiles in 80 youth with and without HIV-1 infection, we tested plasma samples at repeated visits without antiretroviral therapy. Among nine analytes that were quantified using multiplexing assays, interleukin 10 (IL-10), IL-18, and soluble CD30 persistently showed a positive correlation with HIV-1 viral load (Spearman ρ = 0.40–0.59, p < 0.01 for all). A negative correlation with CD4+ T cell counts (ρ = −0.40 to −0.60, p < 0.01 for all) was also persistent for the three analytes. Analyses restricted to 48 AIDS-free youth (96 visits) yielded similar findings, as did multivariable models in which race, sex, age, body mass index, and time interval between visits were treated as covariates. These relationships reflected two novel features observed for all three analytes. First, their presence in plasma was relatively stable between visits (ρ = 0.50–0.90, p < 0.03), regardless of HIV-1 infection status. Second, pairwise correlation was strong and persistent in HIV-1-seropositive youth (ρ = 0.40–0.59, p < 0.01), but not in HIV-1, seronegatives (p > 0.13). Additional analytes, especially eotaxin/CCL11 and SDF-1β/CXCL12, had no correlation with HIV-1-related outcomes despite their stability between visits. Overall, circulating IL-10, IL-18, and soluble CD30 could partially track unfavorable responses to HIV-1 infection in youth. These markers of persistent immune activation are individually and collectively indicative of HIV-1 pathogenesis.
We have previously reported that rhomboid domain containing 1 (RHBDD1), a mammalian rhomboid protease highly expressed in the testis, can cleave the Bcl-2 protein Bik. In this study, we identified a multi-pass transmembrane protein, tumor suppressor activated pathway-6 (TSAP6) as a potential substrate of RHBDD1. RHBDD1 was found to induce the proteolysis of TSAP6 in a dose- and activity-dependent manner. The cleavage of TSAP6 was not restricted to its glycosylated form and occurred in three different regions. In addition, mass spectrometry and mutagenesis analyses both indicated that the major cleavage site laid in the C-terminal of the third transmembrane domain of TSAP6. A somatic cell knock-in approach was used to genetically inactivate the endogenous RHBDD1 in HCT116 and RKO colon cancer cells. Exosome secretion was significantly elevated when RHBDD1 was inactivated in the two cells lines. The increased exosome secretion was verfied through the detection of certain exosomal components, including Tsg101, Tf-R, FasL and Trail. In addition, the elevation of exosome secretion by RHBDD1 inactivation was reduced when TSAP6 was knocked down, indicating that the role of RHBDD1 in regulating exosomal trafficking is very likely to be TSAP6-dependent. We found that the increase in FasL and Trail increased exosome-induced apoptosis in Jurkat cells. Taken together, our findings suggest that RHBDD1 is involved in the regulation of a nonclassical exosomal secretion pathway through the restriction of TSAP6.
Diploid Saccharomyes cerevisae strains lacking the RAD52 gene required for homologous recombination have a very high rate of chromosome loss. Two of four isolates subcultured ∼20 times (∼500 cell divisions) became haploid. These strains were capable of mating with wild-type haploids to produce diploid progeny capable of undergoing meiosis to produce four viable spores.
Hypermethylation of the glutathione S-transferase π 1 (GSTP1) gene promoter region has been reported to be a potential biomarker to distinguish hepatocellular carcinoma (HCC) from other liver diseases. However, reports regarding how specific a marker it is have ranged from 100% to 0%. We hypothesized that, to a large extent, the variation of specificity depends on the location of the CpG sites analyzed. To test this hypothesis, we compared the methylation status of the GSTP1 promoter region of the DNA isolated from HCC, cirrhosis, hepatitis, and normal liver tissues by bisulfite–PCR sequencing. We found that the 5′ region of the position −48 nt from the transcription start site of the GSTP1 gene is selectively methylated in HCC, whereas the 3′ region is methylated in all liver tissues examined, including normal liver and the HCC tissue. Interestingly, when DNA derived from fetal liver and 11 nonhepatic normal tissue was also examined by bisulfite-PCR sequencing, we found that methylation of the 3′ region of the promoter appeared to be liver-specific. A methylation-specific PCR assay targeting the 5′ region of the promoter was developed and used to quantify the methylated GSTP1 gene in various diseased liver tissues including HCC. When we used an assay targeting the 3′ region, we found that the methylation of the 5′-end of the GSTP1 promoter was significantly more specific than that of the 3′-end (97.1% vs. 60%, p<0.0001 by Fisher's exact test) for distinguishing HCC (n = 120) from hepatitis (n = 35) and cirrhosis (n = 35). Encouragingly, 33.8% of the AFP-negative HCC contained the methylated GSTP1 gene. This study clearly demonstrates the importance of the location of CpG site methylation for HCC specificity and how liver-specific DNA methylation should be considered when an epigenetic DNA marker is studied for detection of HCC.
Mesenchymal stem cells (MSCs) are a potential source of material for the generation of tissue-engineered cardiac grafts because of their ability to transdifferentiate into cardiomyocytes after chemical treatments or co-culture with cardiomyocytes. Cardiomyocytes in the body are subjected to cyclic strain induced by the rhythmic heart beating. Whether cyclic strain could regulate rat bone marrow derived MSC (rBMSC) differentiation into cardiomyocyte-like lineage was investigated in this study. A stretching device was used to generate the cyclic strain for rBMSCs. Cardiomyogenic differentiation was evaluated using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR), immunocytochemistry and western-blotting. The results demonstrated that appropriate cyclic strain treatment alone could induce cardiomyogenic differentiation of rBMSCs, as confirmed by the expression of cardiomyocyte-related markers at both mRNA and protein levels. Furthermore, rBMSCs exposed to the strain stimulation expressed cardiomyocyte-related markers at a higher level than the shear stimulation. In addition, when rBMSCs were exposed to both strain and 5-azacytidine (5-aza), expression levels of cardiomyocyte-related markers significantly increased to a degree suggestive of a synergistic interaction. These results suggest that cyclic strain is an important mechanical stimulus affecting the cardiomyogenic differentiation of rBMSCs. This provides a new avenue for mechanistic studies of stem cell differentiation and a new approach to obtain more committed differentiated cells.
We achieved photoacoustic ophthalmoscopy (PAOM) imaging of the retina with near-infrared (NIR) light illumination. A PAOM imaging system with dual-wavelength illumination at 1064 nm and 532 nm was built. We compared in vivo imaging results of both albino and pigmented rat eyes at the two wavelengths. The results show that the bulk optical absorption of the retinal pigment epithelium (RPE) is only slightly higher than that of the retinal vessels at 532 nm while it becomes more than an order of magnitude higher than that of the retinal vessels at 1064 nm. These studies suggest that although visible light illumination is suitable for imaging both the retinal vessels and the RPE, NIR light illumination, being more comfortable to the eye, is better suited for RPE melanin related investigations and diagnoses.
(110.5120) Photoacoustic imaging; (170.4470) Ophthalmology
Host genetic variation, particularly within the human leukocyte antigen (HLA) loci, reportedly mediates heterogeneity in immune response to certain vaccines; however, no large study of genetic determinants of anthrax vaccine response has been described. We searched for associations between the IgG antibody to protective antigen (AbPA) response to Anthrax Vaccine Adsorbed (AVA) in humans and polymorphisms at HLA class I (HLA-A, -B, and -C) and class II (HLA-DRB1, -DQA1, -DQB1, -DPB1) loci. The study included 794 European-Americans and 200 African-Americans participating in a 43-month, double-blind, placebo-controlled, clinical trial of AVA (clinicaltrials.gov identifier NCT00119067). Among European-Americans, genes from tightly linked HLA-DRB1-DQA1-DQB1 haplotypes displayed significant overall associations with longitudinal variation in AbPA levels at 4, 8, 26, and 30 weeks from baseline in response to vaccination with 3 or 4 doses of AVA (global p=6.53×10−4). In particular, carriage of the DRB1-DQA1-DQB1 haplotypes *1501-*0102-*0602 (p=1.17×10−5), *0101-*0101-*0501 (p=0.009), and *0102-*0101-*0501 (p=0.006) was associated with significantlylower AbPA levels. In carriers of two copies of these haplotypes, lower AbPA levels persisted following subsequent vaccinations. No significant associations were observed amongst African-Americans or for any HLA class I allele/haplotype. Further studies will be required to replicate these findings and to explore the role of host genetic variation outside of the HLA region.
Anthrax vaccines; Bacillus anthracis; Bacterial vaccines; Vaccination; HLA Antigens
As part of an ongoing study of early human immunodeficiency virus type 1 (HIV-1) infection in sub-Saharan African countries, we have identified 134 seroconverters (SCs) with distinct acute-phase (peak) and early chronic-phase (set-point) viremias. SCs with class I human leukocyte antigen (HLA) variants B*44 and B*57 had much lower peak viral loads (VLs) than SCs without these variants (adjusted linear regression beta values of −1.08 ± 0.26 log10 [mean ± standard error] and −0.83 ± 0.27 log10, respectively; P < 0.005 for both), after accounting for several nongenetic factors, including gender, age at estimated date of infection, duration of infection, and country of origin. These findings were confirmed by alternative models in which major viral subtypes (A1, C, and others) in the same SCs replaced country of origin as a covariate (P ≤ 0.03). Both B*44 and B*57 were also highly favorable (P ≤ 0.03) in analyses of set-point VLs. Moreover, B*44 was associated with relatively high CD4+ T-cell counts during early chronic infection (P = 0.02). Thus, at least two common HLA-B variants showed strong influences on acute-phase as well as early chronic-phase VL, regardless of the infecting viral subtype. If confirmed, the identification of B*44 as another favorable marker in primary HIV-1 infection should help dissect mechanisms of early immune protection against HIV-1 infection.
AIM: To investigate the effects of ESC-3 isolated from crocodile bile on the growth and apoptosis induction of human cholangiocarcinoma cells.
METHODS: ESC-3 was isolated from crocodile bile by Sephadex LH-20 and RP-18 reversed-phase column. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was conducted to determine the effects of ESC-3 on the proliferation of human cholangiocarcinoma cell lines (QBC939, Sk-ChA-1 and MZ-ChA-1). Giemsa staining, Hoechst 33258 and acridine orange/ethidium bromide staining showed the morphological changes of Mz-ChA-1 cells exposed to ESC-3 at different concentrations. Flow cytometry with regular propidium iodide (PI) staining was performed to analyze the cell cycle distribution of Mz-ChA-1 cells and to assess apoptosis by annexin v-fluorescein isothiocyanate (V-FITC)/PI staining. Rh123 staining was used to detect the alteration of mitochondrial membrane potential (ΔΨm). The protein levels of Bax, Bcl-2, Cdk2, cytochrome c and caspase-3 were further confirmed by Western blotting.
RESULTS: ESC-3 significantly inhibited the growth of three human cholangiocarcinoma cell lines and arrested Mz-ChA-1 cell cycle at G0/G1 phase. Mz-ChA-1 cells showed typical apoptotic morphological changes after treated with ESC-3 (10 μg/mL) for 48 h. Cell death assay indicated that Mz-ChA-1 cells underwent apoptosis in a dose-dependent manner induced by ESC-3. In addition, ESC-3 treatment could downregulate the protein level of Bcl-2 and upregulate the Bax, leading to the increase in the ratio of Bax to Bcl-2 in Mz-ChA-1 cells. Meanwhile, cytochrome c was released from the mitochondria into the cytosol, which subsequently initiated the activation of caspase-3. All these events were associated with the collapse of the mitochondrial membrane potential.
CONCLUSION: ESC-3, the active ingredient of crocodile bile, induced apoptosis in Mz-ChA-1 cells through the mitochondria-dependent pathway and may be a potential chemotherapeutic drug for the treatment of cholangiocarcinoma.
Crocodylus siamensis bile; Cholangiocarcinoma; Antiproliferation; Apoptosis; Mitochondria
The higher order chromatin structure has recently been revealed as a critical new layer of gene transcriptional control. Changes in higher order chromatin structures were shown to correlate with the availability of transcriptional factors and/or MAR (matrix attachment region) binding proteins, which tether genomic DNA to the nuclear matrix. How posttranslational modification to these protein organizers may affect higher order chromatin structure still pending experimental investigation. The type III histone deacetylase silent mating type information regulator 2, S. cerevisiae, homolog 1 (SIRT1) participates in many physiological processes through targeting both histone and transcriptional factors. We show that MAR binding protein SATB1, which mediates chromatin looping in cytokine, MHC-I and β-globin gene loci, as a new type of SIRT1 substrate. SIRT1 expression increased accompanying erythroid differentiation and the strengthening of β-globin cluster higher order chromatin structure, while knockdown of SIRT1 in erythroid k562 cells weakened the long-range interaction between two SATB1 binding sites in the β-globin locus, MARHS2 and MARε. We also show that SIRT1 activity significantly affects ε-globin gene expression in a SATB1-dependent manner and that knockdown of SIRT1 largely blocks ε-globin gene activation during erythroid differentiation. Our work proposes that SIRT1 orchestrates changes in higher order chromatin structure during erythropoiesis, and reveals the dynamic higher order chromatin structure regulation at posttranslational modification level.
Terrain classification allows a mobile robot to create an annotated map of its local environment from the three-dimensional (3D) and two-dimensional (2D) datasets collected by its array of sensors, including a GPS receiver, gyroscope, video camera, and range sensor. However, parts of objects that are outside the measurement range of the range sensor will not be detected. To overcome this problem, this paper describes an edge estimation method for complete scene recovery and complete terrain reconstruction. Here, the Gibbs-Markov random field is used to segment the ground from 2D videos and 3D point clouds. Further, a masking method is proposed to classify buildings and trees in a terrain mesh.
mobile robot; terrain reconstruction; multisensor integration; Gibbs-MRF; classification
When studying the altered expression of genes associated with cartilage regeneration by quantitative real-time RT-PCR (RT-qPCR), reference genes with highly stable expression during different stages of chondrocyte developmental are necessary to normalize gene expression accurately. Until now, no reports evaluating expression changes of commonly used reference genes in rabbit articular cartilage have been published. In this study, defects were made in rabbit articular cartilage, with or without insulin-like growth factor 1 (IGF-1) treatment, to create different chondrocyte living environments. The stability and intensity of the expressions of the candidate reference genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S Ribosomal RNA (18S rRNA), cyclophilin (CYP), hypoxanthine phosphoribosyl transferase (HPRT1), and β-2-microglobulin (B2M) were evaluated. The data were analyzed by geNorm and NormFinder. B2M and 18S rRNA were identified to be suitable reference genes for rabbit cartilage tissues.
reference gene; quantitative real-time PCR; cartilage tissue; geNorm; NormFinder
Erythromycin, a hydrophobic antibiotic used to treat infectious diseases, is now gaining attention because of its anti-inflammatory effects and ability to inhibit osteoclasts formation. The aim of this study was to explore a cyclodextrin-erythromycin (CD-EM) complex for sustained treatment of orthopedic inflammation.
Methods and results
Erythromycin was reacted with β-cyclodextrin to form a nonhost-guest CD-EM complex using both kneading and stirring approaches. Physiochemical measurement data indicated that erythromycin and cyclodextrin formed a packing complex driven by intermolecular forces instead of a host-guest structure due to the limited space in the inner cavity of β-cyclodextrin. The CD-EM complex improved the stability of erythromycin in aqueous solution and had a longer duration of bactericidal activity than free erythromycin. Cytotoxicity and cell differentiation were evaluated in both murine MC3T3 preosteoblast cells and RAW 264.7 murine macrophage cells. The CD-EM complex was noncytotoxic and showed significant inhibition of osteoclast formation but had little effect on osteoblast viability and differentiation.
These attributes are especially important for the delivery of an adequate amount of erythromycin to the site of periprosthetic inflammation and reducing local inflammation in a sustained manner.
erythromycin; cyclodextrin; drug stability; bactericidal activity; osteoclastogenesis
Hypermethylation of the promoter of the tumor suppressor gene, adenomatous polyposis coli (APC), occurs in various malignancies, including hepatocellular carcinoma (HCC). However, reports on the specificity of the methylation of the APC gene for HCC have varied. To gain insight into how these variations occur, bisulfite PCR sequencing was performed to analyze the methylation status of both sense and antisense strands of the APC gene in samples of HCC tissue, matched adjacent non-HCC liver tissue, hepatitis, cirrhosis, and normal liver tissues. DNA derived from fetal liver and 12 nonhepatic normal tissue was also examined. These experiments revealed liver-specific, antisense strand-biased CpG methylation of the APC gene and suggested that, although methylation of the antisense strand of the APC gene exists in normal liver and other non-HCC disease liver tissue, methylation of the sense strand of the APC gene occurs predominantly in HCC. To determine the effect of the DNA strand on the specificity of the methylated APC gene as a biomarker for HCC detection, quantitative methylation-specific PCR assays for sense and antisense strand DNA were developed and performed on DNA isolated from HCC (n = 58), matched adjacent non-HCC (n = 58), cirrhosis (n = 41), and hepatitis (n = 39). Receiver operating characteristic curves were constructed. With the cutoff value set at the limit of detection, the specificity of sense and antisense strand methylation was 84% and 43%, respectively, and sensitivity was 67.2% and 72.4%, respectively. This result demonstrated that the identity of the methylated DNA strand impacted the specificity of APC for HCC detection. Interestingly, methylation of the sense strand of APC occurred in 40% of HCCs from patients with serum AFP levels less than 20 ng/mL, suggesting a potential role for APC as a biomarker to complement AFP in HCC screening.
A hallmark of human immunodeficiency virus type 1 (HIV-1) pathogenesis is the rapid loss of CD4 T cells leading to generalized immune dysfunction, including an exhausted CD8 T cell phenotype. Understanding the necessary factors that govern the functional quality and protective potential of antiviral T cell responses would facilitate rational vaccine design and improve therapeutic strategies to combat persistent infections. Mouse models of chronic viral infection demonstrate that interleukin-21 (IL-21), produced primarily by CD4 T cells, is required for the generation and maintenance of functionally competent CD8 T cells and viral containment. We reasoned that preserved IL-21 production during HIV-1 infection would be associated with enhanced CD8 T cell function, allowing improved viral control. Here we analyzed the ability of CD4 and CD8 T cells to produce several cytokines in addition to IL-21 ex vivo following stimulation with overlapping HIV-1 peptides. Both CD4 and CD8 T cells were able to produce IL-21 in response to HIV-1 infection, with the latter cell type more closely associated with viral control. Furthermore, IL-21-producing HIV-1-specific CD4 T cells (compared to those producing other cytokines) were the best indicator of functional CD8 T cells. Our results demonstrate that HIV-1-specific IL-21-producing CD8 T cells are induced following primary infection and enriched in elite controllers, suggesting a critical role for these cells in the maintenance of viremia control.
To demonstrate drug/polymer nanoparticles can increase the rate and extent of oral absorption of a low-solubility, high-permeability drug.
Amorphous drug/polymer nanoparticles containing celecoxib were prepared using ethyl cellulose and either sodium caseinate or bile salt. Nanoparticles were characterized using dynamic light scattering, transmission and scanning electron microscopy, and differential scanning calorimetry. Drug release and resuspension studies were performed using high-performance liquid chromatography. Pharmacokinetic studies were performed in dogs and humans.
A physical model is presented describing the nanoparticle state of matter and release performance. Nanoparticles dosed orally in aqueous suspensions provided higher systemic exposure and faster attainment of peak plasma concentrations than commercial capsules, with median time to maximum drug concentration (Tmax) of 0.75 h in humans for nanoparticles vs. 3 h for commercial capsules. Nanoparticles released celecoxib rapidly and provided higher dissolved-drug concentrations than micronized crystalline drug. Nanoparticle suspensions are stable for several days and can be spray-dried to form dry powders that resuspend in water.
Drug/polymer nanoparticles are well suited for providing rapid oral absorption and increased bioavailability of BCS Class II drugs.
bioavailability; celecoxib; ethyl cellulose; nanoparticles; rapid absorption
Acquisition of human immunodeficiency virus type 1 (HIV-1) infection is mediated by a combination of characteristics of the infectious and the susceptible member of a transmission pair, including human behavioral and genetic factors, as well as viral fitness and tropism. Here we report on the impact of established and potential new HLA class I determinants of heterosexual HIV-1 acquisition in the HIV-1-exposed seronegative (HESN) partners of serodiscordant Zambian couples.
We assessed the relationships of behavioral and clinically documented risk factors, index partner viral load, and host genetic markers to HIV-1 transmission among 568 cohabiting couples followed for at least nine months. We genotyped subjects for three classical HLA class I genes known to influence immune control of HIV-1 infection. From 1995 to December 2006, 240 HESNs seroconverted and 328 remained seronegative. In Cox proportional hazards models, HLA-A*68:02 and the B*42-C*17 haplotype in HESN partners were significantly and independently associated with faster HIV-1 acquisition (relative hazards = 1.57 and 1.55; p = 0.007 and 0.013, respectively) after controlling for other previously established contributing factors in the index partner (viral load and specific class I alleles), in the HESN partner (age, gender), or in the couple (behavioral and clinical risk score). Few if any previously implicated class I markers were associated here with the rate of acquiring infection.
A few HLA class I markers showed modest effects on acquisition of HIV-1 subtype C infection in HESN partners of discordant Zambian couples. However, the striking disparity between those few markers and the more numerous, different markers found to determine HIV-1 disease course makes it highly unlikely that, whatever the influence of class I variation on the rate of infection, the mechanism mediating that phenomenon is identical to that involved in disease control.
Pancreatoblastoma is a rare primary pancreatic neoplasm of children that may arise in any portion of the pancreas. We report a case of a 3-yr-old boy who presented to with abdominal pain our hospital and a progressive bulge in his right abdomen. Biochemical evaluation and serum levels of tumoral markers were within reference limits. On the computed tomography, two tumors were found. One located in the head of the pancreas; however, a laparotomy revealed that the head of pancreas was compressed but normal. The other was in the left abdomen near the spleen and the tail of the pancreas. The diagnosis of two synchronous pancreatoblastoma originating from the omentum was confirmed by pathology. Therefore, a pancreatoblastoma should be considered when a large well-defined, lobulated, and heterogeneous mass is identified in the pancreas of children. In addition, an ectopic pancreatoblastoma should be considered when identified within or near the ectopic pancreatic tissue.
Pancreatic Neoplasms; Pancreatoblastoma; Ectopic; CT; Child
SH2B1 is a key regulator of body weight in mammals. Here we identified dSH2B as the Drosophila homolog of SH2B1. dSH2B bound to Chico and directly promoted insulin-like signaling. Disruption of dSH2B decreased insulin-like signaling and somatic growth in flies. dSH2B deficiency also increased hemolymph carbohydrate levels, whole body lipid levels, lifespan, and resistance to starvation and oxidative stress. Systemic overexpression of dSH2B resulted in opposite phenotypes. dSH2B overexpression in fat body decreased lipid and glucose levels, whereas neuron-specific overexpression of dSH2B decreased oxidative resistance and lifespan. Genetic deletion of SH2B1 also resulted in growth retardation, obesity, and type 2 diabetes in mice; surprisingly, lifespan and oxidative resistance were reduced in SH2B1 null mice. These data suggest that dSH2B regulation of insulin-like signaling, growth, and metabolism is conserved in SH2B1, whereas dSH2B regulation of oxidative stress and longevity may be conserved in other SH2B family members.