The purpose of this study was to determine the relationship between insulin resistance, obesity and serum prostate-specific antigen (PSA) levels in healthy men with serum PSA level below 4 ng mL−1. The men included in the study cohort were 11 827 healthy male employees of the Korea Hydro and Nuclear Power Co., LTD who had undergone medical checkups including fasting glucose, fasting insulin and serum PSA between January 2003 and December 2008. Insulin resistance was calculated by homeostasis model assessment (HOMA [fasting glucose × fasting insulin]/22.5) and quantitative insulin sensitivity check index (QUICKI; 1/[log (fasting insulin) + log (fasting glucose)]). Age-adjusted body mass index (BMI) was significantly increased according to increasing quartile of insulin resistance as determined by HOMA and QUICKI, respectively, in analysis of variance (ANOVA) test and Duncan's multiple comparison test (P < 0.001), but age-adjusted serum PSA concentration was significantly decreased according to increasing quartile of insulin resistance as determined by HOMA and QUICKI (P < 0.001). Age, BMI, insulin resistance by HOMA or QUICKI were significantly independent variables to serum PSA level in a multivariate linear regression analysis (P < 0.001). Insulin resistance was a significant independent variable to serum PSA level along with BMI. Insulin resistance and BMI were negatively correlated with serum PSA level in healthy men. Insulin resistance was positively correlated with BMI.
body mass index; insulin resistance; metabolic syndrome X; obesity; prostate-specific antigen
Long-term memory requires transcriptional regulation by a combination of positive and negative transcription factors. Aplysia activating factor (ApAF) is known to be a positive transcription factor that forms heterodimers with ApC/EBP and ApCREB2. How these heterodimers are regulated and how they participate in the consolidation of long-term facilitation (LTF) has not, however, been characterized. We found that the functional activation of ApAF required phosphorylation of ApAF by PKA on Ser-266. In addition, ApAF lowered the threshold of LTF by forming a heterodimer with ApCREB2. Moreover, once activated by PKA, the ApAF–ApC/EBP heterodimer transactivates enhancer response element–containing genes and can induce LTF in the absence of CRE- and CREB-mediated gene expression. Collectively, these results suggest that PKA-activated ApAF–ApC/EBP heterodimer is a core downstream effector of ApCREB in the consolidation of LTF.
Eucommia ulmoides Oliver is a natural product widely used as a dietary supplement and medicinal plant. Here, we examined the potential regulatory effects of Eucommia ulmoides Oliver extracts (EUE) on hepatic dyslipidemia and its related mechanisms by in vitro and in vivo studies. EUE and its two active constituents, aucubin and geniposide, inhibited palmitate-induced endoplasmic reticulum (ER) stress, reducing hepatic lipid accumulation through secretion of apolipoprotein B and associated triglycerides and cholesterol in human HepG2 hepatocytes. To determine how EUE diminishes the ER stress response, lysosomal and proteasomal protein degradation activities were analyzed. Although proteasomal activity was not affected, lysosomal enzyme activities including V-ATPase were significantly increased by EUE as well as aucubin and geniposide in HepG2 cells. Treatment with the V-ATPase inhibitor, bafilomycin, reversed the inhibition of ER stress, secretion of apolipoprotein B, and hepatic lipid accumulation induced by EUE or its component, aucubin or geniposide. In addition, EUE was determined to regulate hepatic dyslipidemia by enhancing lysosomal activity and to regulate ER stress in rats fed a high-fat diet. Together, these results suggest that EUE and its active components enhance lysosomal activity, resulting in decreased ER stress and hepatic dyslipidemia.
The anti-apoptotic protein Bax inhibitor-1 (BI-1) is a regulator of apoptosis linked to endoplasmic reticulum (ER) stress. It has been hypothesized that BI-1 protects against neuron degenerative diseases. In this study, BI-1−/− mice showed increased vulnerability to chronic mild stress accompanied by alterations in the size and morphology of the hippocampi, enhanced ROS accumulation and an ER stress response compared with BI-1+/+ mice. BI-1−/− mice exposed to chronic mild stress showed significant activation of monoamine oxidase A (MAO-A), but not MAO-B, compared with BI-1+/+ mice. To examine the involvement of BI-1 in the Ca2+-sensitive MAO activity, thapsigargin-induced Ca2+ release and MAO activity were analyzed in neuronal cells overexpressing BI-1. The in vitro study showed that BI-1 regulates Ca2+ release and related MAO-A activity. This study indicates an endogenous protective role of BI-1 under conditions of chronic mild stress that is primarily mediated through Ca2+-associated MAO-A regulation.
Colorectal cancer is the third leading cause of cancer-related mortality in the world. The main cause of death of colorectal cancer is hepatic metastases which can be treated using isolated hepatic perfusion (IHP), allowing treatment of colorectal metastasis with various methods. In this study we present a novel potent multimodality strategy comprising humanized death receptor 4 (DR4) antibody mapatumumab (Mapa) in combination with oxaliplatin and hyperthermia to treat human colon cancer cells. Oxaliplatin and hyperthermia sensitized colon cancer cells to Mapa in the mitochondrial dependent apoptotic pathway and increased reactive oxygen species production, leading to Bcl-xL phosphorylation at Serine 62 in a c-Jun N-terminal kinase (JNK)-dependent manner. Overexpression of Bcl-xL reduced the efficacy of the multimodality treatment, while phosphorylation of Bcl-xL decreased its anti-apoptotic activity. The multimodality treatment dissociated Bcl-xL from Bax, allowing Bax oligomerization to induce cytochrome c release from mitochondria. In addition, the multimodality treatment significantly inhibited colorectal cancer xenografts’ tumor growth. The successful outcome of this study will support the application of multimodality strategy to colorectal hepatic metastases.
Mapatumumab; Hyperthermia; Oxaliplatin; Mitochondria-dependent pathway
Cell-tracking methods with molecular-imaging modality can monitor the biodistribution of cells. In this study, the direct-labeling method with 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (64Cu-PTSM), indirect cell-labeling methods with herpes simplex virus type 1-thymidine kinase (HSV1-tk)-mediated 124I-2′-fluoro-2′-deoxy-1-β-d-arabinofuranosyl-5-iodouracil (124I-FIAU) were comparatively investigated in vitro and in vivo for tracking of human chronic myelogenous leukemia cells. K562-TL was established by retroviral transduction of the HSV1-tk and firefly luciferase gene in the K562 cell. K562-TL cells were labeled with 64Cu-PTSM or 124I-FIAU. Cell labeling efficiency, viability, and radiolabels retention were compared in vitro. The biodistribution of radiolabeled K562-TL cells with each radiolabel and small-animal positron emission tomography imaging were performed. Additionally, in vivo and ex vivo bioluminescence imaging (BLI) and tissue reverse transcriptase–polymerase chain reaction (RT-PCR) analysis were used for confirming those results. K562-TL cells were efficiently labeled with both radiolabels. The radiolabel retention (%) of 124I-FIAU (95.2%±1.1%) was fourfold higher than 64Cu-PTSM (23.6%±0.7%) at 24 hours postlabeling. Viability of radiolabeled cells was statistically nonsignificant between 124I-FIAU and 64Cu-PTSM. The radioactivity of each radiolabeled cells was predominantly accumulated in the lungs and liver at 2 hours. Both the radioactivity of 64Cu-PTSM- and 124I-FIAU-labeled cells was highly accumulated in the liver at 24 hours. However, the radioactivity of 124I-FIAU-labeled cells was markedly decreased from the body at 24 hours. The K562-TL cells were dominantly localized in the lungs and liver, which also verified by BLI and RT-PCR analysis at 2 and 24 hours postinjection. The 64Cu-PTSM-labeled cell-tracking method is more efficient than 124I-FIAU-labeled cell tracking, because of markedly decrease of radioactivity and fast efflux of 124I-FIAU in vivo. In spite of a high labeling yield and radiolabel retention of 124I-FIAU in vitro, the in vivo cell-tracking method using 64Cu-PTSM could be a useful method to evaluate the distribution and targeting of various cell types, especially, stem cells and immune cells.
gene transfer; molecular imaging; PET
During ligament balancing for severe medial contracture in varus knee total knee arthroplasty (TKA), complete distal release of the medial collateral ligament (MCL) or a medial epicondylar osteotomy can be necessary if a large amount of correction is needed.
This study retrospectively reviewed 9 cases of complete distal release of the MCL and 11 cases of medial epicondylar osteotomy which were used to correct severe medial contracture. The mean follow-up periods were 46.5 months (range, 36 to 78 months) and 39.8 months (range, 32 to 65 months), respectively.
There were no significant differences in the clinical results between the two groups. However, the valgus stress radiograph revealed significant differences in medial instability. In complete distal release of the MCL, some stability was obtained by repair and bracing but the medial instability could not be removed completely.
Medial epicondylar osteotomy for a varus deformity in TKA could provide constant medial stability and be a useful ligament balancing technique.
Varus knee; Total knee arthroplasty; Medial epicondylar osteotomy
There is little consensus on how to optimally reconstruct the posterior cruciate ligament (PCL) and the natural history of injured PCL is also unclear. The graft material (autograft vs. allograft), the type of tibial fixation (tibial inlay vs. transtibial tunnel), the femoral tunnel position within the femoral footprint (isometric, central, or eccentric), and the number of bundles in the reconstruction (1 bundle vs. 2 bundles) are among the many decisions that a surgeon must make in a PCL reconstruction. In addition, there is a paucity of information on rehabilitation after reconstruction of the PCL and posterolateral structures. This article focused on the conflicting issues regarding the PCL, and the scientific rationales behind some critical points are discussed.
Knee; Posterior cruciate ligament; Biomechanics; Outcome; Rehabilitation
The disease septoria leaf blotch of wheat, caused by fungal pathogen Septoria tritici, is of worldwide concern. The fungus exhibits a hemibiotrophic lifestyle, with a long symptomless, biotrophic phase followed by a sudden transition to necrotrophy associated with host necrosis. Little is known about the systematic interaction between fungal pathogenicity and host responses at specific growth stages and the factors triggering the transition. In order to gain some insights into global transcriptome alterations in both host and pathogen during the two phases of the compatible interaction, disease transition was monitored using pathogenesis-related gene markers and H2O2 signature prior to RNA-Seq. Transcriptome analysis revealed that the slow symptomless growth was accompanied by minor metabolic responses and slightly suppressed defences in the host, whereas necrotrophic growth was associated with enhanced host responses involving energy metabolism, transport, signalling, defence and oxidative stress as well as a decrease in photosynthesis. The fungus expresses distinct classes of stage-specific genes encoding potential effectors, probably first suppressing plant defence responses/facilitating the symptomless growth and later triggering life style transition and inducing host necrosis/facilitating the necrotrophic growth. Transport, signalling, anti-oxidative stress mechanisms and apoplastic nutrient acquisition play important roles in the entire infection process of S. tritici. Our findings uncover systematic S. tritici-induced expression profiles of wheat related to specific fungal infection strategies and provide a transcriptome resource for studying both hosts and pathogens in plant-Dothideomycete interactions.
AIM: To evaluate the clinical characteristics of nonvariceal upper gastrointestinal hemorrhage (NGIH) in patients with chronic kidney disease (CKD).
METHODS: From 2003 to 2010, a total of 72 CKD patients (male n = 52, 72.2%; female n = 20, 27.8%) who had undergone endoscopic treatments for NGIH were retrospectively identified. Clinical findings, endoscopic features, prognosis, rebleeding risk factors, and mortality-related factors were evaluated. The characteristics of the patients and rebleeding-related data were recorded for the following variables: gender, age, alcohol use and smoking history, past hemorrhage history, endoscopic findings (the cause, location, and size of the hemorrhage and the hemorrhagic state), therapeutic options for endoscopy, endoscopist experience, clinical outcomes, and mortality.
RESULTS: The average size of the hemorrhagic site was 13.7 ± 10.2 mm, and the most common hemorrhagic site in the stomach was the antrum (n = 21, 43.8%). The most frequent method of hemostasis was combination therapy (n = 32, 44.4%). The incidence of rebleeding was 37.5% (n = 27), and 16.7% (n = 12) of patients expired due to hemorrhage. In a multivariate analysis of the risk factors for rebleeding, alcoholism (OR = 11.19, P = 0.02), the experience of endoscopists (OR = 0.56, P = 0.03), and combination endoscopic therapy (OR = 0.06, P = 0.01) compared with monotherapy were significantly related to rebleeding after endoscopic therapy. In a risk analysis of mortality after endoscopic therapy, only rebleeding was related to mortality (OR = 7.1, P = 0.02).
CONCLUSION: Intensive combined endoscopic treatments by experienced endoscopists are necessary for the treatment of NGIH in patients with CKD, especially when a patient is an alcoholic.
Chronic kidney diseases; Gastrointestinal hemorrhage; Endoscopy; Peptic ulcer; Alcoholics
Kaposi sarcoma (KS), the most common cancer in HIV-positive individuals, is caused by endothelial transformation mediated by the KS herpes virus (KSHV)-encoded G-protein coupled receptor (vGPCR). Infection of blood vascular endothelial cells (BECs) by KSHV reactivates an otherwise silenced embryonic program of lymphatic differentiation. Thus, KS tumors express numerous lymphatic endothelial cell (LEC)-signature genes. A key unanswered question is how lymphatic reprogramming by the virus promotes tumorigenesis leading to KS formation. In this study, we present evidence that this process creates an environment needed to license the oncogenic activity of vGPCR. We found that the G-protein regulator RGS4 is an inhibitor of vGPCR that is expressed in BECs, but not in LECs. RGS4 was downregulated by the master regulator of LEC differentiation PROX1, which is upregulated by KSHV and directs KSHV-induced lymphatic reprogramming. Moreover, we found that KSHV upregulates the nuclear receptor LRH1, which physically interacts with PROX1 and synergizes with it to mediate repression of RGS4 expression. Mechanistic investigations revealed that RGS4 reduced vGPCR-enhanced cell proliferation, migration, VEGF expression and Akt activation and to suppress tumor formation induced by vGPCR. Our findings resolve long-standing questions about the pathological impact of KSHV-induced reprogramming of host cell identity, and they offer biological and mechanistic insights supporting the hypothesis that a lymphatic microenvironment is more favorable for KS tumorigenesis.
Kaposi sarcoma; lymphatic reprogramming; RGS4; viral GPCR; endothelial cells
Caldanaerobacter yonseiensis is a strictly anaerobic, thermophilic, spore-forming bacterium, which was isolated from a geothermal hot stream in Indonesia. This bacterium utilizes xylose and produces a variety of proteases. Here, we report the draft genome sequence of C. yonseiensis, which reveals insights into the pentose phosphate pathway and protein degradation metabolism in thermophilic microorganisms.
Muscle satellite cells (MSCs) represent a devoted stem cell population that is responsible for postnatal muscle growth and skeletal muscle regeneration. An important characteristic of MSCs is that they encompass multi potential mesenchymal stem cell activity and are able to differentiate into myocytes and adipocytes. To achieve a global view of the genes differentially expressed in MSCs, myotube formed-cells (MFCs) and adipocyte-like cells (ALCs), we performed large-scale EST sequencing of normalized cDNA libraries developed from bovine MSCs.
A total of 24,192 clones were assembled into 3,333 clusters, 5,517 singletons and 3,842contigs. Functional annotation of these unigenes revealed that a large portion of the differentially expressed genes are involved in cellular and signaling processes. Database for Annotation, Visualization and Integrated Discovery (DAVID) functional analysis of three subsets of highly expressed gene lists (MSC233, MFC258, and ALC248) highlighted some common and unique biological processes among MSC, MFC and ALC. Additionally, genes that may be specific to MSC, MFC and ALC are reported here, and the role of dimethylarginine
dimethylaminohydrolase2 (DDAH2) during myogenesis and hemoglobin
alpha2 (HBA2) during transdifferentiation in C2C12 were assayed as a case study. DDAH2 was up-regulated during myognesis and knockdown of DDAH2 by siRNA significantly decreased myogenin (MYOG) expression corresponding with the slight change in cell morphology. In contrast, HBA2 was up-regulated during ALC formation and resulted in decreased intracellular lipid accumulation and CD36 mRNA expression upon knockdown assay.
In this study, a large number of EST sequences were generated from the MSC, MFC and ALC. Overall, the collection of ESTs generated in this study provides a starting point for the identification of novel genes involved in MFC and ALC formation, which in turn offers a fundamental resource to enable better understanding of the mechanism of muscle differentiation and transdifferentiation.
The overproduction, purification, crystallization and preliminary X-ray analysis of the MST2 SARAH domain are described.
The SARAH domain at the C-terminus of human MST2 (residues 436–484) was overproduced and purified using an Escherichia coli expression system. The purified domain was crystallized using the hanging-drop vapour-diffusion technique. Two crystal forms were obtained. The crystals belonged to space group P2, with unit-cell parameters a = 62.0, b = 119.2, c = 62.0 Å, α = 90.0, β = 90.5, γ = 90.0°, or to space group P6122, with unit-cell parameters a = 54.5, b = 54.5, c = 303.1 Å. These crystals diffracted to 2.7 and 3.0 Å resolution, respectively.
MST2 SARAH domain; Hippo signalling
Clarithromycin, amoxicillin, metronidazole, tetracycline, and levofloxacin have been commonly used for the eradication of Helicobacter pylori. We compared the change in antibiotic resistance of H. pylori strains during two separate periods and investigated the effect of antibiotic resistance on H. pylori eradication.
H. pylori strains were isolated from 71 patients between 2009 and 2010 and from 94 patients between 2011 and 2012. The distribution of minimal inhibitory concentration (MIC) of 5 antibiotics was assessed using the agar dilution method, and H. pylori eradication based on the antimicrobial susceptibility of the isolates was investigated retrospectively.
Antibiotic resistance rate against clarithromycin, amoxicillin, tetracycline, metronidazole, and levofloxacin for the 2009-2010 isolates were 7.0% (5/71), 2.8% (2/71), 0% (0/71), 45.1% (32/71), and 26.8% (19/71), respectively, and for the 2011-2012 isolates were 16.0% (15/94), 2.1% (2/94), 0% (0/94), 56.3% (53/94), and 22.3% (21/94), respectively. Multi-drug resistance for 2 or more antibiotics increased slightly from 16.9% (12/71) in the 2009-2010 isolates to 23.4% (22/94) in the 2011-2012 isolates. In follow-up testing of 66 patients, first-line treatment successfully eradicated H. pylori in 50 patients (75.8%) and failed in 4 of 7 patients (57.1%) in a clarithromycin-resistant and amoxicillin-susceptible group.
We observed an increase in resistance to clarithromycin and an overall increase in multi-drug resistance during the 2 study periods. The effectiveness of the eradication regimen was low with combinations of clarithromycin and amoxicillin, particularly in the clarithromycin-resistant group. Thus, eradication of H. pylori depends upon periodic monitoring of antimicrobial susceptibility.
Helicobacter pylori; Antibiotic resistance; Eradication
Lapatinib, a dual inhibitor of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) tyrosine kinases, has shown promising results as a growth inhibitor of HER2-positive cancer cells in vitro. However, similar to other EGFR-targeting drugs, acquired resistance to lapatinib by HER2-positive cancer cells remains a major clinical challenge. To elucidate resistance mechanisms to EGFR/HER2-targeting agents, we performed a systematic quantitative comparison of the phosphoproteome of lapatinib-resistant (LR) human gastric cancer cells (SNU216-LR) versus parental cells (SNU216) using a titanium dioxide (TiO2) phosphopeptide enrichment method and analysis with a Q-Exactive hybrid quadrupole-Orbitrap mass spectrometer. Biological network analysis of differentially expressed phosphoproteins revealed apparent constitutive activation of the MET-axis phosphatidylinositide 3-kinase (PI3K)/α-serine/threonine-protein kinase (AKT) and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathways in SNU216-LR. Inhibition of the PI3K/AKT and MAPK/ERK signaling pathways in SNU216-LR also leads to cell cycle arrest, confirming the biological network analysis. Lapatinib sensitivity was restored when cells were treated with several molecular targeting agents in combination with lapatinib. Thus, by integrating phosphoproteomic data, protein networks and effects of signaling pathway modulation on cell proliferation, we found that SNU216-LR maintains constitutive activation of the PI3K/AKT and MAPK/ERK pathways in a MET-dependent manner. These findings suggest that pathway activation is a key compensatory intracellular phospho-signaling event that may govern gastric cancer cell resistance to drug treatment.
drug resistance; HER2-positive gastric cancer; lapatinib; phosphoproteins; Q-Exactive; therapeutic targets
Peptidoglycan recognition proteins (PGRPs) are a family of innate immune molecules that recognize bacterial peptidoglycan. PGRP-LE, a member of the PGRP family, selectively binds to diaminopimelic acid (DAP)-type peptidoglycan to activate both the immune deficiency (Imd) and proPhenoloxidase (proPO) pathways in insects. A PGRP-LE-dependent induction of autophagy to control Listeria monocytogenes has also been reported. We identified and partially characterized a novel PGRP-LE homologue, from Tenebrio molitor and analyzed its functional role in the survival of the insect against infection by a DAP-type PGN containing intracellular pathogen, L. monocytogenes. The cDNA is comprised of an open reading frame (ORF) of 990 bp and encodes a polypeptide of 329 residues. TmPGRP-LE contains one PGRP domain, but lacks critical residues for amidase activity. Quantitative RT-PCR analysis showed a broad constitutive expression of the transcript at various stages of development spanning from larva to adult. RNAi mediated knockdown of the transcripts, followed by a challenge with L. monocytogenes, showed a significant reduction in survival rate of the larvae, suggesting a putative role of TmPGRP-LE in sensing and control of L. monocytogenes infection in T. molitor. These results implicate PGRP-LE as a defense protein necessary for survival of T. molitor against infection by L. monocytogenes.
peptidoglycan recognition protein; Tenebrio molitor; Listeria monocytogenes; PGRP domain; RNA interference
The BRAF point mutation is the most common genetic event in papillary thyroid carcinoma (PTC), occurring in 29–69% of such tumors. The V600E mutation accounts for up to 95% of all BRAF mutations. Therefore, the majority of diagnostic assays have been developed to detect only the V600E mutation of the BRAF gene. A peptide nucleic-acid (PNA)-clamp quantitative polymerase chain reaction (qPCR) was developed to detect the V600E mutation and other mutations in the BRAF gene. In this study, a 3-bp deletion mutation (c.1799_ 1801delTGA) was detected in a subject with a PTC by PNA clamp qPCR, in contrast with the results of allele-specific (AS)-PCR. The mutant allele was not detected by AS-PCR, but was detected using PNA-clamp PCR. The atypical 3-bp deletion mutation (c.1799_1801delTGA) was identified by confirmatory PCR combined with sequencing. The conversion of codons 600 (GTG) and 601 (AAA) into a single codon (GAA) resulted in the insertion of a glutamic acid residue into the activation segment of the B-raf protein (p.V600_K601delinsE). In cases where PTC is highly suspected but no mutation is detected by AS-PCR specific for V600E, PNA clamp qPCR, which is complementary to other sequencing methods, should be performed in order to detect other mutations in the BRAF gene.
BRAF; papillary thyroid carcinoma; peptide nucleic acid; sequencing
Primary renal glucosuria (PRG; OMIM #233100) is characterized by persistent glucosuria due to a reduction in the renal tubular reabsorption of glucose in the presence of a normal concentration of serum glucose and the absence of any other impairment of tubular function. The SLC5A2 gene is the causative gene, which codes for the low-affinity sodium/glucose co-transporter SGLT2. In the present study, the case of a patient with PRG associated with a novel mutation of the SLC5A2 gene is reported. The patient visited hospital for the evaluation of glucosuria in the absence of hyperglycemia, a condition that had been present for >20 years. The patient showed a fasting blood sugar level of 104 mg/dl, a 2-h postprandial sugar level of 101 mg/dl, a sodium level of 144 mmol/l, a potassium level of 3.7 mmol/l and a chloride level of 106 mmol/l in serum. Urine chemistry revealed that the amount of glucose excreted was 10.8 g/1.73 m2/24 h; however, the levels of the other parameters were unremarkable. Polymerase chain reaction (PCR) sequencing analysis of the SLC5A2 gene from the patient revealed a novel 1 bp deletion mutation, which altered the coding sequence of exon 10 in the transmembrane domain (c.1162delG; Ala388ProfsX48), suggesting an autosomal dominant inheritance pattern. This study identified a novel mutation in the SLC5A2 gene related to a benign clinical characteristic and suggests that the molecular diagnosis of the SLC5A2 gene may be useful for diagnosing renal glucosuria in patients and for deciding intervention measures for their family members.
renal glucosuria; SLC5A2; SGLT2; mutation; Korea
Regular immunosuppressant drug monitoring is important for maintaining the drug concentrations of organ recipients within the therapeutic range. The standardized liquid chromatography-tandem mass spectrometry (LC-TMS) technique has been used for the accurate analysis of immunosuppressive drugs. In the present study, the performance of the recently developed high-throughput, rapid ultra-performance liquid chromatography combined with tandem mass spectrometry (UPLC-TMS) method was validated for the simultaneous measurement of cyclosporin A and tacrolimus in whole blood. The method of measuring cyclosporin A and tacrolimus using UPLC-TMS was established and the precision, limit of detection (LOD), limit of quantitation (LOQ) and matrix effect were validated. In addition, the performance of UPLC-TMS was compared with that of a chemiluminescence immunoassay (CLIA) in >3,400 clinical specimens. The UPLC-TMS revealed a within-run and between-run precision of <8% and showed a bias of <5%. The LOD and LOQ were 2.0 and 2.5 ng/ml for cyclosporin A, and 0.3 and 0.4 ng/ml for tacrolimus, respectively. Interference from the matrix was not observed. The CLIA measurements of cyclosporin A and tacrolimus showed correlations corresponding with the formulae: Concentration(CLIA) = 1.18 × UPLC-TMS – 5.85; [95% CI: proportional, 1.16–1.19; constant, −6.86–(−4.81)] and Concentration(CLIA) = 1.14 × UPLC-TMS – 0.38; [(95% CI: proportional, 1.13–1.14; constant, −0.35–(−0.43)], respectively. The majority of results were higher for the immunoassay than for the UPLC-TMS. The newly developed rapid UPLC-TMS method was suitable for use with a large therapeutic concentration range of the analyzed immunosuppressive drugs. Sample preparation was simple and it was possible to detect several immunosuppressants simultaneously, thus significantly lowering the cost of analysis. In conclusion, this method may contribute to improved accuracy and may be preferred to immunoassays for the routine clinical measurement of immunosuppressive drug concentrations in whole blood.
cyclosporin A; tacrolimus; ultra-performance liquid chromatography; tandem mass spectrometry; chemiluminescence immunoassay
CD63, a member of the tetraspanin membrane protein family, plays a pivotal role in cell growth, motility, signal transduction, host-pathogen interactions and cancer. In this work, the cDNA encoding CD63 homologue (TmCD63) was cloned from larvae of a coleopteran beetle, Tenebrio molitor. The cDNA is comprised of an open reading frame of 705 bp, encoding putative protein of 235 amino acid residues. In silico analysis shows that the protein has four putative transmembrane domains and one large extracellular loop. The characteristic “Cys-Cys-Gly” motif and “Cys188” residues are highly conserved in the large extracellular loop. Phylogenetic analysis of TmCD63 revealed that they belong to the insect cluster with 50%–56% identity. Analysis of spatial expression patterns demonstrated that TmCD63 mRNA is mainly expressed in gut and Malphigian tubules of larvae and the testis of the adult. Developmental expression patterns of CD63 mRNA showed that TmCD63 transcripts are detected in late larval, pupal and adult stages. Interestingly, TmCD63 transcripts are upregulated to the maximum level of 4.5 fold, in response to DAP-type peptidoglycan during the first 6 h, although other immune elicitors also caused significant increase to the transcript level at later time-points. These results suggest that CD63 might contribute to T. molitor immune response against various microbial pathogens.
molecular cloning; CD63; TM4SF; expression analysis; Tenebrio molitor; immune elicitors
Vitamin D is an essential component of bone and mineral metabolism; its deficiency causes growth retardation and skeletal deformities in children and osteomalacia and osteoporosis in adults. Hypovitaminosis D (vitamin D insufficiency or deficiency) is observed not only in adults but also in infants, children, and adolescents. Previous studies suggest that sufficient serum vitamin D levels should be maintained in order to enhance normal calcification of the growth plate and bone mineralization. Moreover, emerging evidence supports an association between 25-hydroxyvitamin D (25[OH]D) levels and immune function, respiratory diseases, obesity, metabolic syndrome, insulin resistance, infection, allergy, cancers, and cardiovascular diseases in pediatric and adolescent populations. The risk factors for vitamin D insufficiency or deficiency in the pediatric population are season (winter), insufficient time spent outdoors, ethnicity (non-white), older age, more advanced stage of puberty, obesity, low milk consumption, low socioeconomic status, and female gender. It is recommended that all infants, children, and adolescents have a minimum daily intake of 400 IU (10 µg) of vitamin D. Since the vitamin D status of the newborn is highly related to maternal vitamin D levels, optimal vitamin D levels in the mother during pregnancy should be maintained. In conclusion, given the important role of vitamin D in childhood health, more time spent in outdoor activity (for sunlight exposure) and vitamin D supplementation may be necessary for optimal health in infants, children, and adolescents.
Vitamin D; Vitamin D deficiency; Child; Adolescent; Health
Primary pulmonary lymphoepithelioma-like carcinoma (LELC) is rare, with a more favorable prognosis compared with that of other types of non-small cell lung cancers. Herein, we describe an interesting case of primary pulmonary LELC confirmed postoperatively, which had been initially diagnosed as poorly differentiated adenocarcinoma. We suggest that despite the rarity of pulmonary LELC, it should be included as one of the differential diagnoses for lung malignancies. Physicians should consider taking a larger biopsy, especially when histologic examination shows undifferentiated nature.
Carcinoma, Non-Small-Cell Lung; Lung Neoplasms; Adenocarcinoma
STAT3 regulates CD4+ T cell survival and differentiation. However, its effects on CD8+ T cells are not well understood. Here, we show that in comparison to WT CD8+ T cells, STAT3-deficient CD8+ T cells exhibit a preactivated memory-like phenotype, produce more IL-2, proliferate faster, and are more sensitive to activation-induced cell death (AICD). The enhanced proliferation and sensitivity to AICD correlated with downregulation of class-O forkhead transcription factors (FoxO1, FoxO3A), p21waf1, p27KIP1, Bcl-2, OX-40, and upregulation of FasL, Bax, and Bad. We examined whether STAT3-deficient CD8+ T cells can mount effective response during herpes simplex virus (HSV-1) infection and experimental autoimmune uveitis (EAU). Compared to WT mice, HSV-1-infected STAT3-deficient mice (STAT3KO) produced less IFN-γ and virus-specific KLRG-1+ CD8+ T cells. STAT3KO mice are also resistant to EAU and produced less IL-17-producing Tc17 cells. Resistance of STAT3KO to EAU correlated with marked expansion of IL-10-producing regulatory CD8+ T cells (CD8-Treg) implicated in recovery from autoimmune encephalomyelitis. Thus, increases of IL-6-induced STAT3 activation observed during inflammation may inhibit expansion of CD8-Tregs, thereby impeding recovery from uveitis. These results suggest that STAT3 is a potential therapeutic target for upregulating CD8+ T cell-mediated responses to viruses and suggest the successful therapeutic targeting of STAT3 as treatment for uveitis, derived, in part, from promoting CD8-Treg expansion.
Brain edema after ischemic brain injury is a key determinant of morbidity and mortality. Aquaporin-4 (AQP4) plays an important role in water transport in the central nervous system and is highly expressed in brain astrocytes. However, the AQP4 regulatory mechanisms are poorly understood. In this study, we investigated whether mitogen-activated protein kinases (MAPKs), which are involved in changes in osmolality, might mediate AQP4 expression in models of rat cortical astrocytes after ischemia. Increased levels of AQP4 in primary cultured astrocytes subjected to oxygen-glucose deprivation (OGD) and 2 h of reoxygenation were observed, after which they immediately decreased at 0 h of reoxygenation. Astrocytes exposed to OGD injury had significantly increased phosphorylation of three kinds of MAPKs. Treatment with SB203580, a selective p38 MAPK inhibitor, or SP600125, a selective c-Jun N-terminal kinase inhibitor, significantly attenuated the return of AQP4 to its normal level, and SB203580, but not SP600125, significantly decreased cell death. In an in vivo study, AQP4 expression was upregulated 1–3 days after reperfusion, which was consistent with the time course of p38 phosphorylation and activation, and decreased by the p38 inhibition after transient middle cerebral artery occlusion (MCAO). These results suggest that p38 MAPK may regulate AQP4 expression in cortical astrocytes after ischemic injury.
aquaporin-4; astrocyte; brain edema; ischemia; mitogen-activated protein kinase