Atherothrombotic complications are important causes of morbidity and mortality in diabetic patients. Diabetes has been considered to be a prothrombotic status. Several factors contribute to the prothrombotic condition, such as increasing coagulation, impaired fibrinolysis, endothelial dysfunction, and platelet hyperreactivity. Among the factors that contribute to the prothrombotic status in diabetes, altered platelet function plays a crucial role. Although understanding platelet function abnormalities in diabetes still remains as a challenge, more attention should be focused on platelet function for effective management and the prediction of atherothrombotic events in diabetic patients. This review will provide an overview on the current status of knowledge of platelet function abnormalities and clinical marker of platelet hyperreactivity in patients with diabetes.
Blood platelets; Diabetes mellitus; Hyperreactivity; Marker
Several studies have suggested that elevated levels of hemoglobin A1c (HbA1c) are associated with cardiovascular disease (CVD) in nondiabetic individuals. However, it is unclear whether HbA1c levels can serve as a simple screening marker for increased CVD risk in nondiabetic individuals. Our objective was to evaluate the relationship between HbA1c levels and CVD risk using the Framingham risk score (FRS) in older, apparently healthy nondiabetic Korean adults.
We retrospectively studied 2,879 Korean adults between the ages of 40 and 79 who underwent voluntary health check-ups at the Health Promotion Center of our hospital from July 2009 to June 2011. Subjects were subdivided based on their HbA1c levels into four groups: tertiles within the HbA1c normal tolerance range and a group for subjects with an increased risk for diabetes (IRD).
The mean FRS for the upper tertile (9.6±3.8) group was significantly higher than that of the middle tertile (8.4±4.0) and lower tertile (7.6±3.8) groups. In addition, FRS was highest in the IRD group (10.5±3.7). Multiple linear regression analysis demonstrated that HbA1c levels exhibited a significant positive correlation with FRS when adjusted for confounding variables in all subjects (β±standard error [SE], 0.018±0.002; R2, 0.131), women (β±SE, 0.023±0.003; R2, 0.170), and men (β±SE, 0.016±0.004; R2, 0.109).
HbA1c levels were positively correlated with FRS in older, apparently healthy nondiabetic Korean adults. We propose that HbA1c levels may reflect CVD risk in nondiabetic individuals.
Hemoglobin A, glycosylated; Framingham risk score; Nondiabetic individuals
To analyze the effect of short-term supportive temporary partial enteral nutrition therapy for treating severe pediatric Crohn’s disease (CD).
We conducted a prospective, open-label study in pediatric patients with CD (n=78) from January 2007 to December 2011. The CD patients were divided into three groups according to disease severity (mild, moderate, and severe). Seventeen patients with severe CD received short-term partial enteral nutrition (SPEN) in addition to their general diet for 4 weeks after the induction of remission with medical treatment. This SPEN group was further divided into two groups by age (<13 years, ≥13 years). Nutritional parameters and Pediatric Crohn’s Disease Activity Index scores were analyzed at the initial enrollment and following 1 year of treatment for all groups.
Nutritional status improved substantially after 1 year of treatment in the severe CD group. Nutritional status in the SPEN group improved considerably more than that in the non-SPEN group. Additionally, the <13-year-old group demonstrated better nutritional status improvement than the ≥13-year-old group.
SPEN may be effective in pediatric patients with severe CD for improving nutritional status and moderating disease severity.
Crohn disease; Younger age; Partial enteral nutrition; Disease activity; Nutritional status
The objective of this study was to evaluate the validity of fine needle aspiration biopsy (FNAB) according to ultrasonography (US) characteristics in thyroid nodules 4 cm and larger.
We retrospectively reviewed the cases of 263 patients who underwent thyroid surgery for thyroid nodules larger than 4 cm between January 2001 and December 2010.
The sensitivity of US-FNAB was significantly higher in nodules with calcifications (micro- or macro-) than those without (97.9% vs. 87.% P<0.05). The accuracy of US-FNAB was higher in large thyroid nodules with US features suspicious of malignancy, such as a solid component, ill-defined margin, hypoechogenicity or marked hypoechogenicity, or any calcifications (micro- or macro-) compared to thyroid nodules with none of these features. Furthermore, the accuracy improved as the number of these features increased. The overall false negative rate (FNR) was 11.9%. The FNR of thyroid nodules that appeared benign on US, such as mixed nodules (7.7%) or nodules without calcification (9.8%), trended toward being lower than that of solid nodules (17.9%) or nodules with any microcalcification or macrocalcification (33.3%). In nodules without suspicious features of malignancy, the FNR of US-FNAB was 0% (0/15).
We suggest individualized strategies for large thyroid nodules according to US features. Patients with benign FNAB can be followed in the absence of any malignant features in US. However, if patients exhibit any suspicious features, potential false negative results of FNAB should be kept in mind and surgery may be considered.
Biopsy, fine-needle; Validity; Reliability; Ultrasonography; Thyroid nodule
Background and Purpose
Activation of hepatic stellate cells (HSCs) is a crucial step in the pathogenesis of hepatic fibrosis. Histone deacetylase (HDAC) is an attractive target in liver fibrosis because it plays a key role in gene expression and cell differentiation. We have developed a HDAC inhibitor, N-hydroxy-7-(2-naphthylthio)heptanomide (HNHA), and investigated the anti-fibrotic activity of HNHA in vitro and in vivo.
We investigated the anti-fibrotic effect of HNHA on mouse and human HSC activation in vitro and in the liver of bile duct-ligated (BDL) rats in vivo using cell proliferation assays, cell cycle analysis, biochemical assay, immunohistochemistry and Western blots. Liver pathology was assessed with histochemical techniques.
HNHA inhibited proliferation and arrested the cell cycle via p21 induction in HSCs. In addition, HNHA induced apoptosis of HSCs, which was correlated with reduced COX-2 expression, NF-κB activation and cell death signals. HNHA restored liver function and decreased the accumulation of extracellular matrix in the liver via suppression of HSC activation in BDL rats in vivo. HNHA administration also increased survival in BDL rats.
Conclusions and Implications
HNHA improved liver function, suppressed liver fibrosis and increased survival of BDL rats, accompanied by reduction of cell growth, activation and survival of HSCs. These findings show that HNHA may be a potent anti-fibrosis agent against hepatic fibrosis because of its multi-targeted inhibition of HSC activity in vivo and in vitro.
One of the most remarkable chromatin remodeling processes occurs during spermiogenesis, the post-meiotic phase of sperm development during which histones are replaced with sperm-specific protamines to repackage the genome into the highly compact chromatin structure of mature sperm. Here we identify Chromodomain helicase DNA binding protein 5 (Chd5) as a master regulator of the histone-to-protamine chromatin remodeling process. Chd5 deficiency leads to defective sperm chromatin compaction and male infertility in mice, mirroring the observation of low CHD5 expression in testes of infertile men. Chd5 orchestrates a cascade of molecular events required for histone removal and replacement, including histone 4 (H4) hyperacetylation, histone variant expression, nucleosome eviction, and DNA damage repair. Chd5 deficiency also perturbs expression of transition proteins (Tnp1/Tnp2) and protamines (Prm1/2). These findings define Chd5 as a multi-faceted mediator of histone-to-protamine replacement and depict the cascade of molecular events underlying chromatin remodeling during this process of extensive chromatin remodeling.
We have previously reported that interleukin-1 (IL-1) receptor-associated kinase (IRAK1) is essential for Epstein-Barr virus (EBV) latent infection membrane protein 1 (LMP1)-induced p65/RelA serine 536 phosphorylation and NF-κB activation but not for IκB kinase α (IKKα) or IKKβ activation (Y. J. Song, K. Y. Jen, V. Soni, E. Kieff, and E. Cahir-McFarland, Proc. Natl. Acad. Sci. U. S. A. 103:2689–2694, 2006, doi:10.1073/pnas.0511096103). Since the kinase activity of IRAK1 is not required for LMP1-induced NF-κB activation, IRAK1 is proposed to function as a scaffold protein to recruit a p65/RelA serine 536 kinase(s) to enhance NF-κB-dependent transcriptional activity. We now report that Ca2+/calmodulin-dependent protein kinase II (CaMKII) interacts with IRAK1 and is critical for LMP1-induced p65/RelA serine 536 phosphorylation and NF-κB activation. CaMKII bound the death domain of IRAK1 and directly phosphorylated p65/RelA at serine 536 in vitro. Downregulation of CaMKII activity or expression significantly reduced LMP1-induced p65/RelA serine 536 phosphorylation and NF-κB activation. Furthermore, LMP1-induced CaMKII activation and p65/RelA serine 536 phosphorylation were significantly reduced in IRAK1 knockout (KO) mouse embryonic fibroblasts (MEFs). Thus, IRAK1 may recruit and activate CaMKII, which phosphorylates p65/RelA serine 536 to enhance the transactivation potential of NF-κB in LMP1-induced NF-κB activation pathway.
Recently, patient‐tailored statin therapy was proven effective for achieving target low‐density lipoprotein (LDL) cholesterol levels. It is unclear, however, whether this therapeutic modality would be effective for atherogenic lipid profiles and inflammation in patients with type 2 diabetes.
Materials and Methods
The present study was an 8‐week, multicenter, single‐step titration trial of patient‐tailored atorvastatin therapy (10, 20 and 40 mg) according to baseline LDL cholesterol levels in 440 patients with type 2 diabetes. We measured the LDL particle size by polyacrylamide gel electrophoresis, and used high‐sensitivity C‐reactive protein (hsCRP) and adiponectin as surrogate markers of inflammation.
In the intention‐to‐treat analysis, 91% of the patients achieved their LDL cholesterol targets (<2.6 mmol/L) at week 8. There were significant reductions at week 8 in total cholesterol, triglycerides, non‐high‐density lipoprotein cholesterol (HDL) cholesterol, and the total cholesterol:HDL cholesterol ratio compared with the baseline values for all of the doses. The mean LDL particle size was significantly increased, and the small, dense LDL cholesterol levels were decreased in a dose‐dependent manner over the study period. In addition, the hsCRP levels were decreased in those high‐risk patients with baseline hsCRP levels over 3 mg/L (P < 0.001), and the adiponectin levels tended to increase with all of the doses (P = 0.004) at 8 weeks.
Patient‐tailored atorvastatin therapy based on LDL cholesterol at baseline was effective in ameliorating atherogenic LDL particle size and inflammation, in addition to achieving the target LDL cholesterol level without an undesirable effect on glycemic control in patients with type 2 diabetes. This trial was registered with ClinicalTrials.gov (no. NCT01239849).
Atorvastatin; Low‐density lipoprotein cholesterol; Type 2 diabetes mellitus
This phase II clinical trial was conducted to compare the immunogenicity and safety of a newly developed tetanus-reduced diphtheria (Td) vaccine (GC1107-T5.0 and GC1107-T7.5) and control vaccine. This study was also performed to select the proper dose of tetanus toxoid in the new Td vaccines. Healthy adolescents aged between 11 and 12 yr participated in this study. A total of 130 subjects (44 GC1107-T5.0, 42 GC1107-T7.5 and 44 control vaccine) completed a single dose of vaccination. Blood samples were collected from the subjects before and 4 weeks after the vaccination. In this study, all subjects (100%) in both GC1107-T5.0 and GC1107-T7.5 groups showed seroprotective antibody levels (≥ 0.1 U/mL) against diphtheria or tetanus toxoids. After the vaccination, the geometric mean titer (GMT) against diphtheria was significantly higher in Group GC1107-T5.0 (6.53) and GC1107-T7.5 (6.11) than in the control group (3.96). The GMT against tetanus was 18.6 in Group GC1107-T5.0, 19.94 in GC1107-T7.5 and 19.01 in the control group after the vaccination. In this study, the rates of local adverse reactions were 67.3% and 59.1% in GC1107-T5.0 and GC1107-7.5, respectively. No significant differences in the number of adverse reactions, prevalence and degree of severity of the solicited and unsolicited adverse reactions were observed among the three groups. Thus, both newly developed Td vaccines appear to be safe and show good immunogenicity. GC1107-T5.0, which contains relatively small amounts of tetanus toxoid, has been selected for a phase III clinical trial.
Tetanus-Reduced Diphtheria (Td) Vaccine; Immunogenicity; Safety
Meckel's diverticulum (MD) has various clinical manifestations, and diagnosis or selectection of proper diagnostic tools is not easy. This study was conducted in order to assess the clinical differences of MD diagnosed by scintigraphic and non-scintigraphic methods and to find the proper diagnostic tools.
We conducted a retrospective review ofthe clinical, surgical, radiologic, and pathologic findings of 34 children with symptomatic MD, who were admitted to Gachon University Gil Medical Center, Inha University Hospital, and The Catholic University of Korea, Incheon St. Mary's Hospital between January 2000 and December 2012. The patients were evaluated according to scintigraphic (12 cases; group 1) and non-scintigraphic (22 cases; group 2) diagnosis.
The male to female ratio was 7.5 : 1. The most frequent chief complaint was lower gastrointestinal (GI) bleeding in group 1 and nonspecific abdominal pain in group 2, respectively. The most frequent pre-operative diagnosis was MD in both groups. Red blood cell (RBC) index was significantly lower in group 1. MD was located at 7 cm to 85 cm from the ileocecal valve. Four patients in group 1 had ectopic gastric tissues causing lower GI bleeding. The most frequent treatment modality was diverticulectomy in group 1 and ileal resection in group 2, respectively.
To diagnose MD might be delayed unless proper diagnostic tools are considered. It is important to understand indications of scintigraphic and non-scintigraphic methods according to clinical and hematologic features of MD. Scintigraphy would be weighed in patients with anemia as well as GI symptoms.
Meckel's diverticulum; Scintigraphy; Meckel's scan; Diagnosis; Child; Radionuclide imaging
Somatic reprogramming induced by defined transcription factors is a low efficiency process that is enhanced by p53 deficiency 1-5. To date, p21 is the only p53 target shown to contribute to p53 repression of iPSC (induced pluripotent stem cell) generation 1, 3, suggesting additional p53 targets may regulate this process. Here, we demonstrated that mir-34 microRNAs (miRNAs), particularly miR-34a, exhibit p53-dependent induction during reprogramming. mir-34a deficiency in mice significantly increased reprogramming efficiency and kinetics, with miR-34a and p21 cooperatively regulating somatic reprogramming downstream of p53. Unlike p53 deficiency, which enhances reprogramming at the expense of iPSC pluripotency, genetic ablation of mir-34a promoted iPSC generation without compromising self-renewal and differentiation. Suppression of reprogramming by miR-34a was due, at least in part, to repression of pluripotency genes, including Nanog, Sox2 and Mycn (N-Myc). This post-transcriptional gene repression by miR-34a also regulated iPSC differentiation kinetics. miR-34b and c similarly repressed reprogramming; and all three mir-34 miRNAs acted cooperatively in this process. Taken together, our findings identified mir-34 miRNAs as novel p53 targets that play an essential role in restraining somatic reprogramming.
The generation of induced pluripotent stem cells (iPSCs) often results in aberrant epigenetic silencing of the imprinted Dlk1-Dio3 gene cluster, which compromises the cells’ ability to generate entirely iPSC-derived adult mice (“all-iPSC mice”). Here, we show that reprogramming in the presence of ascorbic acid attenuates hypermethylation of Dlk1-Dio3 by enabling a chromatin configuration that interferes with binding of the de novo DNA methyltransferase Dnmt3a. This allowed us to generate all-iPSC mice from mature B cells, which have thus far failed to support the development of exclusively iPSC-derived postnatal animals. Our data demonstrate that transcription factor-mediated reprogramming can endow a defined, terminally differentiated cell type with a developmental potential equivalent to that of embryonic stem cells. More generally, these findings indicate that culture conditions during cellular reprogramming can strongly influence the epigenetic and biological properties of resultant iPSCs.
In this study, 22 new betulinic acid (BA) derivatives were synthesized and tested for their inhibition of the chymotrypsin-like activity of 20S proteasome. From the SAR study, we concluded that the C-3 and C-30 positions are the pharmacophores for increasing the proteasome inhibition effects, and larger lipophilic or aromatic side chains are favored at these positions. Among the BA derivatives tested, compounds 13, 20, and 21 showed the best proteasome inhibition activity with IC50 values of 1.42, 1.56, and 1.80 µM, respectively, which are three- to four-fold more potent than the proteasome inhibition controls LLM-F and lactacystin.
The purpose of the study is to determine the effectiveness and safety of nicardipine infusion for controlling blood pressure in patients with subarachnoid hemorrhage (SAH).
We prospectively evaluated 52 patients with SAH and treated with nicardipine infusion for blood pressure control in a 29 months period. The mean blood pressure of pre-injection, bolus injection and continuous injection period were compared. This study evaluated the effectiveness of nicardipine for each Fisher grade, for different dose of continuous nicardipine infusion, and for the subgroups of systolic blood pressure.
The blood pressure measurement showed that the mean systolic blood pressure / diastolic blood pressure (SBP/DBP) in continuous injection period (120.9/63.0 mmHg) was significantly lower than pre-injection period (145.6/80.3 mmHg) and bolus injection period (134.2/71.3 mmHg), and these were statistically significant (p < 0.001). In each subgroups of Fisher grade and different dose, SBP/DBP also decreased after the use of nicardipine. These were statistically significant (p < 0.05), but there was no significant difference in effectiveness between subgroups (p > 0.05). Furthermore, controlling blood pressure was more effective when injecting higher dose of nicardipine in higher SBP group rather than injecting lower dose in lower SBP group, and it also was statistically significant (p < 0.05). During the infusion, hypotension and cardiogenic problems were transiently combined in five cases. However, patients recovered without any complications.
Nicardipine is an effective and safe agent for controlling acutely elevated blood pressure after SAH. A more systemic study with larger patients population will provide significant results and will bring solid evidence on effectiveness of nicardipine in SAH.
Nicardipine; Hypertension; Subarachnoid hemorrhage; Aneurysm
RNA interference is a powerful tool for studying gene function, however, the reproducible generation of RNAi transgenic mice remains a significant limitation. By combining optimized fluorescence-coupled miR30-based shRNAs with high efficiency ES cell targeting, we developed a fast, scalable pipeline for the production of shRNA transgenic mice. Using this system, we generated eight tet-regulated shRNA transgenic lines targeting Firefly and Renilla luciferases, Oct4 and tumor suppressors p53, p16INK4a, p19ARF and APC and demonstrate potent gene silencing and GFP-tracked knockdown in a broad range of tissues in vivo. Further, using an shRNA targeting APC, we illustrate how this approach can identify predicted phenotypes and also unknown functions for a well-studied gene. In addition, through regulated gene silencing we validate APC/Wnt and p19ARF as potential therapeutic targets in T cell acute lymphoblastic leukemia/lymphoma and lung adenocarcinoma, respectively. This system provides a cost-effective and scalable platform for the production of RNAi transgenic mice targeting any mammalian gene.
The present sutdy aimed to determine whether olmesartan, an angiotensin II (Ang II) type 1 (AT1) receptor blocker, can influence the CA release from the isolated perfused model of the rat adrenal medulla. Olmesartan (5~50 µM) perfused into an adrenal vein for 90 min produced dose- and time-dependent inhibition of the CA secretory responses evoked by ACh (5.32 mM), high K+ (56 mM, a direct membrane-depolarizer), DMPP (100 µM) and McN-A-343 (100 µM). Olmesartan did not affect basal CA secretion. Also, in adrenal glands loaded with olmesartan (15 µM), the CA secretory responses evoked by Bay-K-8644 (10 µM, an activator of voltage-dependent L-type Ca2+ channels), cyclopiazonic acid (10 µM, an inhibitor of cytoplasmic Ca2+ -ATPase), veratridine (100 µM, an activator of voltage-dependent Na+ channels), and Ang II (100 nM) were markedly inhibited. However, at high concentrations (150~300 µM), olmesartan rather enhanced the ACh-evoked CA secretion. Taken together, these results show that olmesartan at low concentrations inhibits the CA secretion evoked by cholinergic stimulation (both nicotininc and muscarinic receptors) as well as by direct membrane depolarization from the rat adrenal medulla, but at high concentrations it rather potentiates the ACh-evoked CA secretion. It seems that olmesartan has a dual action, acting as both agonist and antagonist at nicotinic receptors of the isolated perfused rat adrenal medulla, which might be dependent on the concentration. It is also thought that this inhibitory effect of olmesartan may be mediated by blocking the influx of both Na+ and Ca2+ into the rat adrenomedullary chromaffin cells as well as by inhibiting the Ca2+ release from the cytoplasmic calcium store, which is thought to be relevant to the AT1 receptor blockade, in addition to its enhancement on the CA secreton.
Olmesartan; Catecholamine secretion; Adrenal medulla; AT1 receptor blockade
Thymidine is an important precursor in the production of various antiviral drugs, including azidothymidine for the treatment of AIDS. Since thymidine-containing nucleotides are synthesized only by the de novo pathway during DNA synthesis, it is not easy to produce a large amount of thymidine biologically. In order to develop a host strain to produce thymidine, thymidine phosphorylase, thymidine kinase, and uridine phosphorylase genes were deleted from an Escherichia coli BL21 strain to develop BLdtu. Since the genes coding for the enzymes related to the nucleotide salvage pathway were disrupted, BLdtu was unable to utilize thymidine or thymine, and thymidine degradation activity was completely abrogated. We additionally expressed T4 thymidylate synthase, T4 nucleotide diphosphate reductase, bacteriophage PBS2 TMP phosphohydrolase, E. coli dCTP deaminase, and E. coli uridine kinase in the BLdtu strain to develop a thymidine-producing strain (BLdtu24). BLdtu24 produced 649.3 mg liter−1 of thymidine in a 7-liter batch fermenter for 24 h, and neither thymine nor uridine was detected. However, the dUTP/dTTP ratio was increased in BLdtu24, which could lead to increased double-strand breakages and eventually to cell deaths during fermentation. To enhance thymidine production and to prevent cell deaths during fermentation, we disrupted a gene (encoding uracil-DNA N-glycosylase) involved in DNA excision repair to suppress the consumption of dTTP and developed BLdtug24. Compared with the thymidine production in BLdtu24, the thymidine production in BLdtug24 was increased by ∼1.2-fold (740.3 mg liter−1). Here, we show that a thymidine-producing strain with a relatively high yield can be developed using a metabolic engineering approach.
“Protaminobacter rubrum” sucrose isomerase (SI) catalyzes the isomerization of sucrose to isomaltulose and trehalulose. SI catalyzes the hydrolysis of the glycosidic bond with retention of the anomeric configuration via a mechanism that involves a covalent glycosyl enzyme intermediate. It possesses a 325RLDRD329 motif, which is highly conserved and plays an important role in fructose binding. The predicted three-dimensional active-site structure of SI was superimposed on and compared with those of other α-glucosidases in family 13. We identified two Arg residues that may play important roles in SI-substrate binding with weak ionic strength. Mutations at Arg325 and Arg328 in the fructose-binding site reduced isomaltulose production and slightly increased trehalulose production. In addition, the perturbed interactions between the mutated residues and fructose at the fructose-binding site seemed to have altered the binding affinity of the site, where glucose could now bind and be utilized as a second substrate for isomaltose production. From eight mutant enzymes designed based on structural analysis, the R325Q mutant enzyme exhibiting high relative activity for isomaltose production was selected. We recorded 40.0% relative activity at 15% (wt/vol) additive glucose with no temperature shift; the maximum isomaltose concentration and production yield were 57.9 g liter−1 and 0.55 g of isomaltose/g of sucrose, respectively. Furthermore, isomaltose production increased with temperature but decreased at a temperature of >35°C. Maximum isomaltose production (75.7 g liter−1) was recorded at 35°C, and its yield for the consumed sucrose was 0.61 g g−1 with the addition of 15% (wt/vol) glucose. The relative activity for isomaltose production increased progressively with temperature and reached 45.9% under the same conditions.
Interleukin-18 (IL-18) plays pivotal roles in linking inflammatory immune responses and tumor progression and metastasis, yet the manner in which this occurs remains to be sufficiently clarified. Here we report that hypoxia induces the transcription and secretion of IL-18, which subsequently induces the expression of hypoxia-inducible factor-1α (HIF-1α). Mechanistically, IL-18 induces HIF-1α through the activity of the GTPase Rac1, which inducibly associates with the IL-18 receptor β (IL-18Rβ) subunit, via a PI3K-AKT-NF-κB–dependent pathway. Importantly, the knockdown of the IL-18Rβ subunit inhibited IL-18–driven tumor cell metastasis. Collectively, these findings demonstrate a feed-forward pathway in HIF-1α–mediated tumor progression, in which the induction of IL-18 by hypoxia or inflammatory cells augments the expression of both HIF-1α and tumor cell metastasis.