Evaluation of In Vivo Prepared Albumin-Drug Conjugate Using Immunoprecipitation Linked LC-MS Assay and Its Application to Mouse Pharmacokinetic Study.

There have been many attempts in pharmaceutical industries and academia to improve the pharmacokinetic characteristics of anti-tumor small-molecule drugs by conjugating them with large molecules, such as monoclonal antibodies, called ADCs. In this context, albumin, one of the most abundant proteins in the blood, has also been proposed as a large molecule to be conjugated with anti-cancer small-molecule drugs. The half-life of albumin is 3 weeks in humans, and its distribution to tumors is higher than in normal tissues. However, few studies have been conducted for the in vivo prepared albumin-drug conjugates, possibly due to the lack of robust bioanalytical methods, which are critical for evaluating the ADME/PK properties of in vivo prepared albumin-drug conjugates. In this study, we developed a bioanalytical method of the albumin-conjugated MAC glucuronide phenol linked SN-38 ((2S,3S,4S,5R,6S)-6-(4-(((((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b] quinolin-9-yl)oxy)methyl)(2 (methylsulfonyl)ethyl)carbamoyl)oxy)methyl)-2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-methylpropanamido)acetamido)phenoxy)-3,4,5-trihydroxytetra-hydro-2H-pyran-2-carboxylic acid) as a proof-of-concept. This method is based on immunoprecipitation using magnetic beads and the quantification of albumin-conjugated drug concentration using LC-qTOF/MS in mouse plasma. Finally, the developed method was applied to the in vivo intravenous (IV) mouse pharmacokinetic study of MAC glucuronide phenol-linked SN-38.

Quantitative Analysis of Daporinad (FK866) and Its In Vitro and In Vivo Metabolite Identification Using Liquid Chromatography-Quadrupole-Time-of-Flight Mass Spectrometry.

Daporinad (FK866) is one of the highly specific inhibitors of nicotinamide phosphoribosyl transferase (NAMPT) and known to have its unique mechanism of action that induces the tumor cell apoptosis. In this study, a simple and sensitive liquid chromatography-quadrupole-time-of-flight-mass spectrometric (LC-qTOF-MS) assay has been developed for the evaluation of drug metabolism and pharmacokinetics (DMPK) properties of Daporinad in mice. A simple protein precipitation method using acetonitrile (ACN) was used for the sample preparation and the pre-treated samples were separated by a C18 column. The calibration curve was evaluated in the range of 1.02~2220 ng/mL and the quadratic regression (weighted 1/concentration2) was used for the best fit of the curve with a correlation coefficient ≥ 0.99. The qualification run met the acceptance criteria of ±25% accuracy and precision values for QC samples. The dilution integrity was verified for 5, 10 and 30-fold dilution and the accuracy and precision of the dilution QC samples were also satisfactory within ±25% of the nominal values. The stability results indicated that Daporinad was stable for the following conditions: short-term (4 h), long-term (2 weeks), freeze/thaw (three cycles). This qualified method was successfully applied to intravenous (IV) pharmacokinetic (PK) studies of Daporinad in mice at doses of 5, 10 and 30 mg/kg. As a result, it showed a linear PK tendency in the dose range from 5 to 10 mg/kg, but a non-linear PK tendency in the dose of 30 mg/kg. In addition, in vitro and in vivo metabolite identification (Met ID) studies were conducted to understand the PK properties of Daporinad and the results showed that a total of 25 metabolites were identified as ten different types of metabolism in our experimental conditions. In conclusion, the LC-qTOF-MS assay was successfully developed for the quantification of Daporinad in mouse plasma as well as for its in vitro and in vivo metabolite identification.

Assessment of Pharmacokinetics and Metabolism Profiles of SCH 58261 in Rats Using Liquid Chromatography-Mass Spectrometric Method.

5-Amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo(4,3-e)-1,2,4-triazolo(1,5-c) pyrimidine (SCH 58261) is one of the new chemical entities that has been developed as an adenosine A2A receptor antagonist. Although SCH 58261 has been reported to be beneficial, there is little information about SCH 58261 from a drug metabolism or pharmacokinetics perspective. This study describes the metabolism and pharmacokinetic properties of SCH 58261 in order to understand its behaviors in vivo. Rats were used as the in vivo model species. First, an LC-MS/MS method was developed for the determination of SCH 58261 in rat plasma. A GastroPlus™ simulation, in vitro microsomal metabolic stability, and bile duct-cannulated studies were also performed to understand its pharmacokinetic profile. The parameter sensitivity analysis of GastroPlus™ was used to examine the factors that influence exposure when the drug is orally administered. The factors are as follows: permeability, systemic clearance, renal clearance, and liver first-pass effect. In vitro microsomal metabolic stability indicates how much the drug is metabolized. The extrapolated hepatic clearance value of SCH 58261 was 39.97 mL/min/kg, indicating that the drug is greatly affected by hepatic metabolism. In vitro microsomal metabolite identification studies revealed that metabolites produce oxidized and ketone-formed metabolites via metabolic enzymes in the liver. The bile duct-cannulated rat study, after oral administration of SCH 58261, showed that a significant amount of the drug was excreted in feces. These results imply that the drug is not absorbed well in the body after oral administration. Taken together, SCH 58261 showed quite a low bioavailability when administered orally and this was likely due to significantly limited absorption, as well as high metabolism in vivo.

In Vitro, In Silico, and In Vivo Assessments of Pharmacokinetic Properties of ZM241385.

Parkinson's disease is one of the most common neurodegenerative diseases. Adenosine regulates the response to other neurotransmitters in the brain regions related to motor function. In the several subtypes of adenosine receptors, especially, adenosine 2A receptors (A2ARs) are involved in neurodegenerative conditions. ZM241385 is one of the selective non-xanthine A2AR antagonists with high affinity in the nanomolar range. This study describes the in vitro and in vivo pharmacokinetic properties of ZM241385 in rats. A liquid chromatography-quadrupole time-of-flight mass spectrometric (LC-qToF MS) method was developed for the determination of ZM241385 in rat plasma. In vivo IV administration studies showed that ZM241385 was rapidly eliminated in rats. However, the result of in vitro metabolic stability studies showed that ZM241385 had moderate clearance, suggesting that there is an extra clearance pathway in addition to hepatic clearance. In addition, in vivo PO administration studies demonstrated that ZM241385 had low exposure in rats. The results of semi-mass balance studies and the in silico PBPK modeling studies suggested that the low bioavailability of ZM241385 after oral administration in rats was due to the metabolism and by liver, kidney, and gut.

Analysis of bone marrow supernatant neutrophil gelatinase-associated lipocalin and hematological parameters in hematological malignancy.

BACKGROUND: Neutrophil gelatinase-associated lipocalin (NGAL) is a urine biomarker related to acute renal injury. Whereas several studies have evaluated NGAL levels in hematological malignancy, using peripheral blood (PB). Recently, bone marrow (BM) NGAL level was reported to be higher than PB NGAL level in individuals with hematological malignancy, suggesting that BM NGAL would reflect BM microenvironment better than PB NGAL. We measured BM NGAL levels in patients with hematological malignancy, comparing those with NGAL levels in normal BM. We evaluated the association of BM NGAL with hematological parameters including neutrophil counts. METHODS: BM samples were collected from 107 patients who underwent BM examination. Immunoassays were used to assess NGAL levels. Data on hematological parameters were collected from medical records. Intergroup comparisons were performed using the Kruskal-Wallis H test and Pearson chi-square test. Single and multiple regression analyses were performed to analyze the relationships. RESULTS: The independent factors that affected the BM NGAL level were neutrophil counts and BM band neutrophil%, while neutrophil count was the main influencing factor. The acute myeloid leukemia (n = 18) and myelodysplastic syndrome (n = 25) groups showed statistically lower BM NGAL levels than patients with normal BM. The myeloproliferative neoplasm group (n = 34) showed higher BM NGAL levels than patients with normal BM, but this difference was not statistically significant. Neutrophil counts and BM band neutrophil% showed intergroup patterns similar to those of BM NGAL levels. CONCLUSION: BM NGAL was related to neutrophil count and BM band neutrophil%, showing different levels according to hematological malignant disease entities.

Multi-Platform Omics Analysis for Identification of Molecular Characteristics and Therapeutic Targets of Uveal Melanoma.

Currently, there is no effective treatment for metastatic uveal melanoma (UVM). Here, we aimed to identify the mechanism involving intrinsic chemoresistance of metastatic UVM and the relevant therapeutic targets for UVM. We analyzed cohorts of 80 and 67 patients with primary UVM and skin cutaneous melanoma (SKCM), respectively, using The Cancer Genome Atlas dataset. Mutational burdens identified by whole exome sequencing were significantly lower in UVM than in SKCM patients. COSMIC mutational signature analysis identified that most of the mutations in UVM patients (>90%) were associated with spontaneous deamination of 5-methylcytosine or defective mismatch repair. Transcriptome analysis revealed that the MYC signature was more enriched in UVM patients, as compared to SKCM patients. Fifty-nine (73.8%) of 80 UVM patients showed gains in MYC copy number, and a high MYC copy number was associated with aggressive clinicopathological features of tumors and poor survival. Kinome-wide siRNA library screening identified several therapeutic targets, reported as synthetic lethal targets for MYC-addicted cancers. Notably, UVM cell lines showed high susceptibility to a WEE1 inhibitor (MK-1775; adavosertib) at a clinically tolerable dose. Overall, our study identified high MYC activity in UVM, and suggested G2/M checkpoint inhibitors as effective therapeutic targets for UVM.

Comparative Effectiveness of Abdominal versus Laparoscopic Radical Hysterectomy for Cervical Cancer in the Postdissemination Era.

PURPOSE: Despite the benefits of minimally invasive surgery for cervical cancer, there are a lack of randomized trials comparing laparoscopic radical hysterectomy and abdominal radical hysterectomy. We compared morbidity, cost of care, and survival between abdominal radical hysterectomy and laparoscopic radical hysterectomy for cervical cancer. MATERIALS AND METHODS: We used the Korean nationwide database to identify women with cervical cancer who underwent radical hysterectomy from January 1, 2011 to December 31, 2014. Patients who underwent abdominal radical hysterectomy were compared to those who underwent laparoscopic radical hysterectomy. Perioperative morbidity, the use of adjuvant therapy, and survival were evaluated after propensity score balancing. RESULTS: We identified 6,335 patients, including 3,235 who underwent abdominal radical hysterectomy and 3,100 who underwent laparoscopic radical hysterectomy. The use of laparoscopic radical hysterectomy increased from 46.1% in 2011 to 51.8% in 2014. Patients who were younger, had a more recent year of diagnosis, and were treated in the metropolitan area were more likely to undergo a laparoscopic procedure (p < 0.001). Compared to abdominal radical hysterectomy, laparoscopic radical hysterectomy was associated with lower rates of complication, fewertransfusions, a shorter hospital stay, less adjuvant therapy, and reduced total medical costs (p < 0.001). Laparoscopic surgery was associated with a better overall survival than abdominal operation (hazard ratio, 0.74; 95% confidence interval, 0.64 to 0.85). CONCLUSION: In the postdissemination era, laparoscopic radical hysterectomy was associated with more favorable morbidity profiles, a lower cost of care, and comparable survival than abdominal radical hysterectomy.

Epstein-Barr Virus Fusion with Epithelial Cells Triggered by gB Is Restricted by a gL Glycosylation Site.

Epstein-Barr virus (EBV) entry into epithelial cells is mediated by the conserved core fusion machinery, composed of the fusogen gB and the receptor-binding complex gH/gL. The heterodimeric gH/gL complex binds to the EBV epithelial cell receptor or gp42, which binds to the B-cell receptor, triggering gB-mediated fusion of the virion envelope with cellular membranes. Our previous study found that the gL glycosylation mutant N69L/S71V had an epithelial cell-specific hyperfusogenic phenotype. To study the influence of this gL mutant on the initiation and kinetics of gB-driven epithelial cell fusion, we established a virus-free split-green fluorescent protein cell-cell fusion assay that enables real-time measurements of membrane fusion using live cells. The gL_N69L/S71V mutant had a large increase in epithelial cell fusion activity of up to 300% greater than that of wild-type gL starting at early time points. The hyperfusogenicity of the gL mutant was not a result of alterations in complex formation with gH or alterations in cellular localization. Moreover, the hyperfusogenic phenotype of the gL mutant correlated with the formation of enlarged syncytia. In summary, our present findings highlight an important role of gL in the kinetics of gB-mediated epithelial cell fusion, adding to previous findings indicating a direct interaction between gL and gB in EBV membrane fusion.IMPORTANCE EBV predominantly infects epithelial cells and B lymphocytes, which are the cells of origin for the EBV-associated malignancies Hodgkin and Burkitt lymphoma as well as nasopharyngeal carcinoma. Contrary to the other key players of the core fusion machinery, gL has the most elusive role during EBV-induced membrane fusion. We found that the glycosylation site N69/S71 of gL is involved in restricting epithelial cell fusion activity, strongly correlating with syncytium size. Interestingly, our data showed that the gL glycosylation mutant increases the fusion activity of the hyperfusogenic gB mutants, indicating that this gL mutant and the gB mutants target different steps during fusion. Our studies on how gL and gB work together to modulate epithelial cell fusion kinetics are essential to understand the highly tuned tropism of EBV for epithelial cells and B lymphocytes and may result in novel strategies for therapies preventing viral entry into target host cells. Finally, making our results of particular interest is the absence of gL syncytial mutants in other herpesviruses.

Next-generation sequencing of BRCA1/2 in breast cancer patients: potential effects on clinical decision-making using rapid, high-accuracy genetic results.

PURPOSE: We evaluated the clinical role of rapid next-generation sequencing (NGS) for identifying BRCA1/2 mutations compared to traditional Sanger sequencing. METHODS: Twenty-four paired samples from 12 patients were analyzed in this prospective study to compare the performance of NGS to the Sanger method. Both NGS and Sanger sequencing were performed in 2 different laboratories using blood samples from patients with breast cancer. We then analyzed the accuracy of NGS in terms of variant calling and determining concordance rates of BRCA1/2 mutation detection. RESULTS: The overall concordance rate of BRCA1/2 mutation identification was 100%. Variants of unknown significance (VUS) were reported in two cases of BRCA1 and 3 cases of BRCA2 after Sanger sequencing, whereas NGS reported only 1 case of BRCA1 VUS, likely due to differences in reference databases used for mutation identification. The median turnaround time of Sanger sequencing was 22 days (range, 14-26 days), while the median time of NGS was only 6 days (range, 3-21 days). CONCLUSION: NGS yielded comparably accurate results to Sanger sequencing and in a much shorter time with respect to BRCA1/2 mutation identification. The shorter turnaround time and higher accuracy of NGS may help clinicians make more timely and informed decisions regarding surgery or neoadjuvant chemotherapy in patients with breast cancer.

The Evolutionarily Conserved LIM Homeodomain Protein LIM-4/LHX6 Specifies the Terminal Identity of a Cholinergic and Peptidergic C. elegans Sensory/Inter/Motor Neuron-Type.

The expression of specific transcription factors determines the differentiated features of postmitotic neurons. However, the mechanism by which specific molecules determine neuronal cell fate and the extent to which the functions of transcription factors are conserved in evolution are not fully understood. In C. elegans, the cholinergic and peptidergic SMB sensory/inter/motor neurons innervate muscle quadrants in the head and control the amplitude of sinusoidal movement. Here we show that the LIM homeobox protein LIM-4 determines neuronal characteristics of the SMB neurons. In lim-4 mutant animals, expression of terminal differentiation genes, such as the cholinergic gene battery and the flp-12 neuropeptide gene, is completely abolished and thus the function of the SMB neurons is compromised. LIM-4 activity promotes SMB identity by directly regulating the expression of the SMB marker genes via a distinct cis-regulatory motif. Two human LIM-4 orthologs, LHX6 and LHX8, functionally substitute for LIM-4 in C. elegans. Furthermore, C. elegans LIM-4 or human LHX6 can induce cholinergic and peptidergic characteristics in the human neuronal cell lines. Our results indicate that the evolutionarily conserved LIM-4/LHX6 homeodomain proteins function in generation of precise neuronal subtypes.

Critical role of Rgs19 in mouse embryonic stem cell proliferation and differentiation.

Mouse embryonic stem cells (ESCs) are self-renewing, pluripotent, and have the ability to differentiate into the three germ layers required to form all embryonic tissues. These properties are maintained by both intrinsic and extrinsic factors. Many studies have contributed to the understanding of the molecular signal transduction required for pluripotency and controlled differentiation. Such an understanding is important in the potential application of stem cells to cell therapy for disease, and thus there is an interest in understanding the cell cycle regulation, pluripotency, and differentiation of ESCs. The regulator of G protein signaling (RGS) family consists of over 20 members. Rgs19, one such protein, specifically interacts with Gαi to enhance its GTPase activity. Growth factor receptors use Gi proteins for signal transduction, and Rgs19 may thus be involved in the regulation of cell proliferation. In a previous gain-of-function study, Rgs19 overexpression was found to enhance proliferation in various cell types. Our data demonstrate a role for Rgs19 in the regulation of ESC differentiation. Based on the presence of Rgs19 in ESCs, the morphological and molecular properties of wild-type and Rgs19 +/- ESCs during LIF withdrawal, in vitro differentiation, and teratoma formation were compared. Our findings provide insight for the first time into the mechanisms involved in Rgs19 regulation of mouse ESC proliferation and differentiation.

Over-expression of Roquin aggravates T cell mediated hepatitis in transgenic mice using T cell specific promoter.

Chronic hepatitis is a major cause of liver cancer, so earlier treatment of hepatitis might be reducing liver cancer incidence. Hepatitis can be induced in mice by treatment with Concanavalin A (Con A); the resulting liver injury causes significant CD4(+) T cell activation and infiltration. In these T cells, Roquin, a ring-type E3 ubiquitin ligase, is activated. To investigate the role of Roquin, we examined Con A-induced liver injury and T cell infiltration in transgenic (Tg) mice overexpressing Roquin specifically in T cells. In Roquin Tg mice, Con A treatment caused greater increases in both the levels of liver injury enzymes and liver tissue apoptosis, as revealed by TUNEL and H&E staining, than wild type (WT) mice. Further, Roquin Tg mice respond to Con A treatment with greater increases in the T cell population, particularly Th17 cells, though Treg cell counts are lower. Roquin overexpression also enhances increases in pro-inflammatory cytokines, including IFN-γ, TNF-α and IL-6, upon liver injury. Furthermore, Roquin regulates the immune response and apoptosis in Con A induced hepatitis via STATs, Bax and Bcl2. These findings suggest that over-expression of Roquin exacerbates T-cell mediated hepatitis.

Gestational loss and growth restriction by angiogenic defects in placental growth factor transgenic mice.

OBJECTIVE: Angiogenesis is an important biological process during development, reproduction, and in immune responses. Placental growth factor (PlGF) is a member of vascular endothelial growth factor that is critical for angiogenesis and vasculogenesis. We generated transgenic mice overexpressing PlGF in specifically T cells using the human CD2-promoter to investigate the effects of PlGF overexpression. APPROACH AND RESULTS: Transgenic mice were difficult to obtain owing to high lethality; for this reason, we investigated why gestational loss occurred in these transgenic mice. Here, we report that placenta detachment and inhibition of angiogenesis occurred in PlGF transgenic mice during the gestational period. Moreover, even when transgenic mice were born, their growth was restricted. CONCLUSIONS: Conclusively, PlGF overexpression prevents angiogenesis by inhibiting Braf, extracellular signal-regulated kinase activation, and downregulation of HIF-1α in the mouse placenta. Furthermore, it affected regulatory T cells, which are important for maintenance of pregnancy.

Overexpression of Jazf1 reduces body weight gain and regulates lipid metabolism in high fat diet.

Jazf1 is a 27 kDa nuclear protein containing three putative zinc finger motifs that is associated with diabetes mellitus and prostate cancer; however, little is known about the role that this gene plays in regulation of metabolism. Recent evidence indicates that Jazf1 transcription factors bind to the nuclear orphan receptor TR4. This receptor regulates PEPCK, the key enzyme involved in gluconeogenesis. To elucidate Jazf1's role in metabolism, we fed a 60% fat diet for up to 15 weeks. In Jazf1 overexpression mice, weight gain was found to be significantly decreased. The expression of Jazf1 in the liver also suppressed lipid accumulation and decreased droplet size. These results suggest that Jazf1 plays a critical role in the regulation of lipid homeostasis. Finally, Jazf1 may provide a new therapeutic target in the management of obesity and diabetes.

Immunophenotypic characterization and quantification of neoplastic bone marrow plasma cells by multiparametric flow cytometry and its clinical significance in Korean myeloma patients.

Multiparametric flow cytometry (MFC) allows discrimination between normal and neoplastic plasma cells (NeoPCs) within the bone marrow plasma cell (BMPC) compartment. This study sought to characterize immunophenotypes and quantitate the proportion of NeoPCs in BMPCs to diagnose plasma cell myeoma (PCM) and evaluate the prognostic impact of this method. We analyzed the MFC data of the bone marrow aspirates of 76 patients with PCM and 33 patients with reactive plasmacytosis. MFC analysis was performed using three combinations: CD38/CD138/-/CD45; CD56/CD20/CD138/CD19; and CD27/CD28/CD138/CD117. The plasma cells of patients with reactive plasmacytosis demonstrated normal immunophenotypic patterns. Aberrant marker expression was observed in NeoPCs, with negative CD19 expression observed in 100% of cases, CD56+ in 73.7%, CD117+ in 15.2%, CD27- in 10.5%, CD20+ in 9.2%, and CD28+ in 1.3%. In PCM patients, more than 20% of NeoPCs/BMPCs were significantly associated with factors suggestive of poor clinical outcomes. Patients who were CD27- or CD56+/CD27-, demonstrated shorter overall survival than patients of other CD56/CD27 combinations. Our results support the clinical value of immunophenotyping and quantifying NeoPCs in PCM patients. This strategy could help to reveal poor prognostic categories and delineate surrogate markers for risk stratification in PCM patients.

Application of BRAF, NRAS, KRAS mutations as markers for the detection of papillary thyroid cancer from FNAB specimens by pyrosequencing analysis.

BACKGROUND: BRAFV600E, the most common BRAF gene mutation, is detected in approximately 50% of sporadic papillary thyroid carcinoma (PTC) and may be associated with triggering tumorigenesis of PTC. The aim of our study was to discover additional mutations to increase the diagnostic performance of molecular tests in screening for thyroid cancer from fine needle aspiration biopsy (FNAB) specimens. METHODS: DNA was extracted from 120 freshly obtained FNAB specimens selected according to cytopathology grades of the Bethesda system. A conventional BRAF V600E test was carried out with real-time PCR, and further mutation screening for BRAF mutations in codons 464, 466, 469, NRAS and KRAS codons 12/13 and 61 was done by pyrosequencing. Histopathology reports were reviewed for those who underwent thyroidectomy (n=83). RESULTS: The real-time PCR method detected 45 BRAF V600E- positive cases whereas pyrosequencing detected 30 cases. Additional BRAF (n=4), NRAS (n=11) and KRAS (n=3) mutations were detected in 17 cases (one overlapping BRAF and NRAS mutation). Among 11 NRAS-mutated cases, eight were confirmed as PTC and one as FVPTC on histopathology reports. Five PTC-confirmed cases with BRAF V600E mutation showed additional mutations, all of which were NRAS mutations. DISCUSSION: Despite the higher sensitivity of real-time PCR for detecting BRAFV600E mutations, pyrosequencing easily detected additional point mutations. NRAS mutations were the most prevalently identified additional mutations and were highly associated with malignancy. In conclusion, our findings demonstrate that additional mutations identified by pyrosequencing may help in the pre-operative process in determining the possibility of malignancy and further studies on the occurrence of simultaneous mutations of BRAF, KRAS and NRAS may be warranted.

Study of peripheral BRAF(V600E) mutation as a possible novel marker for papillary thyroid carcinomas.

BACKGROUND: The BRAF(V600E) mutation can be detected peripherally in the serum of patients with thyroid cancer. The purpose of this study was to establish the value of detecting the peripheral BRAF(V600E) mutation as a serum tumor marker in this population. METHODS: In this study, we obtained 94 serum samples from patients with papillary thyroid cancer positive for the BRAF(V600E) mutation in the tumor itself. The serum samples were analyzed for BRAF(V600E) mutation using real-time polymerase chain reaction (PCR). RESULTS: Sixty-seven patients (71.3%) had papillary thyroid microcarcinoma and 26 patients (27.7%) had underlying lymphocytic thyroiditis. Forty-three patients (45.7%) were found to have stage III or stage IV thyroid cancer. None of the patients had a detectable serum BRAF(V600E) mutation. CONCLUSION: We were unable to identify peripheral BRAF(V600E) mutations in patients with papillary thyroid cancer using real-time PCR. Further studies will be needed to validate our results using various diagnostic methods.

Assessment of lifestyle effects on the levels of free oxygen radicals in the korean population.

BACKGROUND: As many studies revealed that oxidative stress due to the imbalance of reactive oxygen species (ROS) and antioxidant capacity is related with pathologic processes such as cardiovascular diseases, diabetes, as well as aging and obesity, the relationship between lifestyle and oxidative stress has recently gained much medical attention. However, little information exists on the effects of lifestyle on ROS in Korea. In this study, we investigated the effects of lifestyle on free oxygen radical levels in men and women in Korea. METHODS: A total of 138 adults participated in this study from September 2007 to June 2010 at a health promotion center and department of family medicine. Information on the lifestyle of each participant was obtained by questionnaire. Biochemical markers and a free oxygen radical test (FORT) were also measured. RESULTS: The average age was 47.28 ± 10.85 years and 79.7% were male. High sensitivity C-reactive protein (hs-CRP; r = 0.418, P = 0.012), triglycerides (r = -0.243, P = 0.008), hemoglobin (r = -0.445, P < 0.001), total protein (r = 0.210, P = 0.036), creatinine (r = -0.294, P = 0.001), fruit intake per day (P = 0.047), and smoking (P = 0.003) were related to the FORT levels in univariate analysis. Multiple linear regression analysis showed that hs-CRP (P = 0.039) was an independent predictor of serum FORT values. This statistical model can explain 78% of the variance in FORT values. CONCLUSION: This result suggests that hs-CRP showed a statistically significant positive association with FORT values. Further studies on the relationship between lifestyle and antioxidant capacity as well as ROS seem to be warranted to evaluate the overall effect of oxidative stress.