Galli Gigerii Endothelium Corneum(GGEC), the dried gizzard membrane of Gallus gallus domesticus is a Chinese medicinal material commonly used for digestion. However, due to the particularity of texture and composition, its active ingre-dients have not been clarified so far, and there is also a lack of quality evaluation indicators. In this study, UPLC-Q-TOF-MS was used to analyze the chemical components from the water extract of GGEC, and ten nucleosides were identified for the first time. HPLC fingerprints of the water extracts of GGEC were established and the content of seven nucleosides was determined. The fingerprint similarities of 40 batches of GGEC samples ranged from 0.765 to 0.959, indicating that there were great differences among the GGEC products processed with different methods. In addition, SPSS 22.0 and SIMCA 14.1 were used for hierarchical cluster analysis(HCA) and principal component analysis(PCA) on the 19 common peaks of the HPLC fingerprints of GGEC, and the 40 batches of samples were divided into three categories: raw GGEC, fried GGEC and vinegar-processed GGEC. Eight differential components in GGEC were marked by orthogonal partial least squares discrimination analysis(OPLS-DA), two of which were adenine and thymine. The results of content determination showed that the total content of the seven nucleosides in raw GGEC, fried GGEC and vinegar-processed GGEC were 182.5-416.8, 205.3-368.7, and 194.2-283.0 μg·g~(-1), respectively. There were significant differences in the content of hypoxanthine, thymine and thymidine among the GGEC products processed with different methods(P<0.05), which were graded in the order of fried GGEC>vinegar-processed GGEC>raw GGEC. This suggested that the content of hypoxanthine, thymine and thymidine tended to increase during the frying process, and the variation range might be related to the degree of heat exposure. The established methods in this study were simple and reproducible, and could be used for qualitative and quantitative analysis of GGEC and its processed pro-ducts. This study also provided reference for the establishment of quality standards of GGEC with chemical components as control index.
Nucleosides ; Drugs, Chinese Herbal/chemistry* ; Chromatography, High Pressure Liquid ; Acetic Acid ; Thymine ; Thymidine ; Water ; Hypoxanthines

OBJECTIVE: To investigate the mechanism of nucleolin (NCL) involved in lymphoma proliferation by regulating thymidine kinase 1 (TK1). METHODS: Twenty-three patients with diffuse large B-cell lymphoma (DLBCL) were selected and divided into initial treatment group (14 cases) and relapsed/refractory group (9 cases). Serum TK1 and C23 protein in peripheral blood mononuclear cells were detected. Cell models of CA46-NCL-KD (CA46-NCL-knockdown) and CA46-NCL-KNC (CA46-NCL-knockdown negative control) were established by lentivirus vector mediated transfection in Burkitt lymphoma cell line CA46. The half maximal inhibitory concentration (IC₅₀) of CA46-NCL-KD, CA46-NCL-KNC, and CA46 to adriamycin were detected by cell proliferation assay (MTS). The expression of NCL mRNA and protein in CA46-NCL-KD and CA46-NCL-KNC cells were dectected by Q-PCR and Western blot, respectively. The cell cycle of CA46-NCL-KD, CA46-NCL-KNC, and CA46 cells were detected by flow cytometry. The expression of TK1 protein in CA46-NCL-KD and CA46-NCL-KNC cells was detected by an enhanced chemiluminescence (ECL) dot blot assay. RESULTS: The level of serum TK1 in the initial treatment group was 0.43(0-30-1.01) pmol/L, which was lower than 10.56(2.19-14.99) pmol/L in the relapsed/refractory group (P<0-01), and the relative expression level of NCL protein in peripheral blood was also significantly lower. The IC₅₀ of CA46-C23-KD cells to adriamycin was (0.147±0.02) μg/ml, which was significantly lower than (0.301±0.04) μg/ml of CA46-C23-KNC cells and (0.338±0.05) μg/ml of CA46 cells (P<0.05). Compared with CA46-NCL-KNC cells, the expression of NCL mRNA and protein, TK1 protein decreased in CA46-NCL-KD cells, and the proportion of S phase and G₂/M phase also decreased, while G0/G1 phase increased in cell cycle. CONCLUSION The increased expression of NCL in DLBCL and CA46 cells indicates low sensitivity to drug. NCL may participate in regulation of lymphoma proliferation by affecting TK1 expression, thereby affecting the drug sensitivity.
Humans ; Leukocytes, Mononuclear/metabolism* ; Apoptosis ; Cell Line, Tumor ; Lymphoma ; Thymidine Kinase/pharmacology* ; Doxorubicin/pharmacology* ; Cell Division ; RNA, Messenger/genetics*

PURPOSE: To study the efficacy of capecitabine or S-1 plus oxaliplatin (CAPOX or SOX) for treating thymidine phosphorylase (TP)- or dihydropyrimidine dehydrogenase (DPD)-positive advanced gastric cancer.MATERIALS AND METHODS: Eighty-six patients with stage IIIC to IV gastric cancer were assessed for TP and DPD expression by immunohistochemistry. The association between CAPOX or SOX efficacy and TP/DPD expression was retrospectively analyzed.RESULTS: There were no significant differences in the objective remission rate (ORR, 52.27% vs. 47.62%; P>0.05), disease control rate (72.73% vs. 73.81%, P>0.05), progression-free survival (hazard ratio [HR], 1.119; 95% confidence interval [CI], 0.739–1.741; P=0.586), and overall survival (OS; HR, 0.855; 95% CI, 0.481–1.511; P=0.588) between CAPOX and SOX. A higher number of stage IV patients showed TP positivity, while DPD-positive patients predominantly showed intestinal type of gastric cancer. In TP-positive patients, the ORRs associated with CAPOX and SOX treatments were 57.14% and 38.10%, respectively; OS was better with CAPOX than with SOX (HR, 0.447; 95% CI, 0.179–0.978; P=0.046). Among DPD-positive patients, the SOX treatment-associated ORR (60.87%) was significantly higher than the CAPOX treatment-associated ORR (43.48%). Furthermore, SOX treatment resulted in better OS than did CAPOX treatment (HR, 2.020; 95% CI, 1.019–4.837; P=0.049).CONCLUSIONS: No significant difference in clinical efficacy was found between CAPOX and SOX. TP-positive patients might respond better to CAPOX while DPD-positive patients may respond better to SOX. Our findings might serve as a guide for personalized chemotherapy for gastric cancer.
Capecitabine ; Dihydrouracil Dehydrogenase (NADP) ; Disease-Free Survival ; Drug Therapy ; Humans ; Immunohistochemistry ; Retrospective Studies ; Stomach Neoplasms ; Thymidine Phosphorylase ; Thymidine ; Treatment Outcome

Herein, we report the results of the 2018 survey on the external quality assessment (EQA) scheme for the Transfusion Medicine Program (TMP) in Korea. The proficiency testing specimens were prepared at Ajou University Hospital and were sent to the participants biannually. The average accuracy rates for ten different test items on the regular survey were as follows: ABO typing, 99.5%–99.8% (N=841); RhD typing, 99.8%–100.0% (N=827); crossmatching, 89.4%–99.6% (N=708); ABO subtyping, 94.2% and 94.4% (N=53); Rh CcEe antigen testing, all 100.0% (N=51); weak D test, 80.9% (N=207) for the first trial and not graded for the second trial; antibody screening, 99.7%–100.0% (N=304); direct antiglobulin test (DAT) using a polyspecific reagent, 98.9%–100.0% (N=264); DAT using an immunoglobulin-G monospecific reagent, all 100.0% (N=66); DAT using a C3d monospecific reagent, 97.0%–100.0% (N=67); antibody identification, 98.4%–100.0% (N=127); and ABO antibody titration, 84.6%–100.0% (N=73). There were approximately 10.5% more participants in the 2018 EQA scheme than that in 2017. Excellent survey results were obtained in the 2018 EQA scheme compared with those in 2017, except for the weak D test. The 2018 EQA scheme for the TMP should be helpful for improving the quality of the participating laboratories.
Coombs Test ; Korea ; Laboratory Proficiency Testing ; Mass Screening ; Quality Improvement ; Thymidine Monophosphate ; Transfusion Medicine

Here, we have reported results of the surveys on the external quality assessment scheme (EQA) of the Transfusion Medicine Program (TMP) in Korea that were carried out in 2017. The proficiency testing specimens were prepared at Ajou University Hospital, and sent to the participants biannually. The average accuracy rates (N=the number of participants) for ten different test items on the regular survey were as follows: ABO typing, 99.1%–99.9% (N=714); RhD typing, 99.3%–100.0% (N=695); crossmatching, 88.9%–98.5% (N=618); ABO subtyping, 80.7% and 96.0% (N=51); Rh CcEe antigen testing, 98.8%–100.0% (N=51); weak D test, 99.3% and 100.0 (N=150); antibody screening, 98.6%–100.0% (N=295); direct antiglobulin test (DAT) using a poly-specific reagent, 99.2%–100.0 (N=256); DAT using an immunoglobulin-G monospecific reagent, all 100.0% (N=68); DAT using a C3d-monospecific reagent, 83.6%–100.0% (N=72); antibody identification, 88.7%–99.2% (N=123); and ABO Ab titration, 84.6%–100.0% (N=73). The number of participants for the EQA for TMP in 2017 was much higher than that in 2016. Except for the case of ABO subtyping, excellent survey results for the 2017 EQA for TMP were obtained, compared to those in 2016. Thus, the EQA for TMP in 2017 should be helpful for improving the quality of the participating laboratories.
Coombs Test ; Korea* ; Laboratory Proficiency Testing ; Mass Screening ; Quality Improvement ; Thymidine Monophosphate ; Transfusion Medicine*

Post-traumatic stress disorder (PTSD) is a trauma-induced psychiatric disorder characterized by impaired fear extermination, hyperarousal, and anxiety that may involve the release of monoamines in the fear circuit. The reported pharmacological properties of tetramethylpyrazine (TMP) include anti-cancer, anti-diabetic, anti-atherosclerotic, and neuropsychiatric activities. However, the anxiolytic-like effects of TMP and its mechanism of action in PTSD are unclear. This study measured several anxiety-related behavioral responses to examine the effects of TMP on symptoms of anxiety in rats after single prolonged stress (SPS) exposure by reversing the serotonin (5-HT) and hypothalamic-pituitary-adrenal (HPA) axis dysfunction. Rats were given TMP (10, 20, or 40 mg/kg, i.p.) for 14 days after SPS exposure. Administration of TMP significantly reduced grooming behavior, increased the time spent and number of visits to the open arm in the elevated plus maze test, and significantly increased the number of central zone crossings in the open field test. TMP administration significantly reduced the freezing response to contextual fear conditioning and significantly restored the neurochemical abnormalities and the SPS-induced decrease in 5-HT tissue levels in the prefrontal cortex and hippocampus. The increased 5-HT concentration during TMP treatment might be partially attribute to the tryptophan and 5-hydroxyindoleacetic acid mRNA level expression in the hippocampus of rats with PTSD. These findings support a role for reducing the altered serotonergic transmission in rats with PTSD. TMP simultaneously attenuated the HPA axis dysfunction. Therefore, TMP may be useful for developing an agent for treating psychiatric disorders, such those observed in patients with PTSD.
Animals ; Anxiety ; Arm ; Freezing ; Grooming ; Hippocampus ; Humans ; Models, Animal* ; Prefrontal Cortex ; Rats* ; RNA, Messenger ; Serotonin ; Stress Disorders, Post-Traumatic* ; Thymidine Monophosphate ; Tryptophan

Targeted point mutagenesis through homologous recombination has been widely used in genetic studies and holds considerable promise for repairing disease-causing mutations in patients. However, problems such as mosaicism and low mutagenesis efficiency continue to pose challenges to clinical application of such approaches. Recently, a base editor (BE) system built on cytidine (C) deaminase and CRISPR/Cas9 technology was developed as an alternative method for targeted point mutagenesis in plant, yeast, and human cells. Base editors convert C in the deamination window to thymidine (T) efficiently, however, it remains unclear whether targeted base editing in mouse embryos is feasible. In this report, we generated a modified high-fidelity version of base editor 2 (HF2-BE2), and investigated its base editing efficacy in mouse embryos. We found that HF2-BE2 could convert C to T efficiently, with up to 100% biallelic mutation efficiency in mouse embryos. Unlike BE3, HF2-BE2 could convert C to T on both the target and non-target strand, expanding the editing scope of base editors. Surprisingly, we found HF2-BE2 could also deaminate C that was proximal to the gRNA-binding region. Taken together, our work demonstrates the feasibility of generating point mutations in mouse by base editing, and underscores the need to carefully optimize base editing systems in order to eliminate proximal-site deamination.
APOBEC-1 Deaminase ; genetics ; metabolism ; Animals ; Bacterial Proteins ; genetics ; metabolism ; Base Sequence ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cytidine ; genetics ; metabolism ; Embryo Transfer ; Embryo, Mammalian ; Endonucleases ; genetics ; metabolism ; Gene Editing ; methods ; HEK293 Cells ; High-Throughput Nucleotide Sequencing ; Humans ; Mice ; Mice, Inbred C57BL ; Microinjections ; Plasmids ; chemistry ; metabolism ; Point Mutation ; RNA, Guide ; genetics ; metabolism ; Thymidine ; genetics ; metabolism ; Zygote ; growth & development ; metabolism ; transplantation

OBJECTIVETo observe the efficacy and safety of chemotherapy regimens oxaliplatin combined with capecitabine (CAPOX) or oxaliplatin combined with tegafur, gimeracil and oteracil potassium capsules (S-1)(SOX), and to investigate the value of expression of thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) proteins in tumor tissue for predicting the efficacy of CAPOX and SOX regimens in advanced gastric cancer patients.

METHODSA total of 107 newly-diagnosed, stage Ⅲc/Ⅳ gastric cancer patients (no surgical indication, ECOG performance scores 0-2 and expected survival time ≥3 months) were recruited with 101 patients evaluated. The patients were randomly divided into two groups. One was study group in which the patients received CAPOX regimen. The other was control group received SOX regimen. Each patient received four cycles, at least two cycles chemotherapy every three weeks and followed up until death or lost. Tumor biopsies were obtained by gastroscopy for immunohistochemical examination of the expression of TP and DPD proteins before chemotherapy. Response rate (ORR), overall survival (OS) and time to tumor progression (TTP) of the patients were assessed.

RESULTSThe objective response rate (ORR) of the study and control groups was 49.0% (5/51) vs. 46.0% (23/50), respectively (P>0.05). The overall survival (OS) was 357.36±24.69 days in the study group and 349.87±22.63 days in the control group, and the time-to-progression (TTP) was 216.75±19.32 days in the study group and 220.54±18.47 days in the control group (P>0.05 for both). Stratified analysis showed that the ORR of TP-positive patients in the study group was significantly higher than that in the control group (72.0 % vs. 41.7 %, P=0.032). There was no significant difference in ORR between the TP-negative patients in the study and control groups (26.9% vs. 50.0%, P=0.087), while the ORR of DPD-positive patients in the control group was significantly higher than that of the study group (51.9% vs. 34.6%, P=0.046). There was no significant difference in the ORR between DPD-negative patients in the study and control groups (64.0% vs. 39.1%, P=0.084). The follow-up showed that the OS (378.42±22.56 days) and TTP (271.77±24.92 days) in the TP-positive patients of the study group were significantly longer than those of the control group (OS: 326.57±19.84 days, and TTP: 229.13±22.68 days)( P<0.05). The OS was 371.25±23.97 days and TTP was 264.66±21.36 days in the DPD-positive patients of control group, significantly longer than those of the study group (OS: 334.73±21.47days, and TTP: 208.58±20.70 days) (P<0.05). But there was no significant difference in the OS and TTP between the TP- and DPD-negative patients in the two groups (P>0.05). In respect of adverse events, both the rates of hematological and non-hematological toxicities were low and similar between the two groups (P>0.05), and well-tolerated by the patients.

CONCLUSIONSBoth CAPOX and SOX regimens are effective chemotherapeutic protocols in treatment of patients with advanced gastric cancer. The expression levels of TP and DPD in tumor tissue can be used as a predictive factor for the efficacy of capecitabine or tegafur, gimeracil and oteracil potassium capsules combined with oxaliplatin regimens. CAPOX chemotherapy regimen is more suitable for the TP-positive gastric cancer patients, and SOX regimen is more suitable for the DPS-positive gastric cancer patients.

Antineoplastic Agents ; adverse effects ; therapeutic use ; Antineoplastic Combined Chemotherapy Protocols ; adverse effects ; therapeutic use ; Capecitabine ; administration & dosage ; adverse effects ; Capsules ; Dihydrouracil Dehydrogenase (NADP) ; metabolism ; Disease Progression ; Humans ; Neoplasm Proteins ; metabolism ; Organoplatinum Compounds ; administration & dosage ; adverse effects ; Oxonic Acid ; administration & dosage ; adverse effects ; Pyridines ; administration & dosage ; adverse effects ; Stomach Neoplasms ; drug therapy ; metabolism ; mortality ; pathology ; Tegafur ; administration & dosage ; adverse effects ; Thymidine Phosphorylase ; metabolism

BACKGROUNDAs the clinical value of cytokeratin-19 (CK19) and thymidine kinase-1 (TK1) in advanced gastrointestinal cancer remains controversial, we investigated their expression and clinical significance in this disease.

METHODSA total of 171 advanced gastrointestinal cancer patients were prospectively enrolled in this study. The mRNA level of CK19 was detected using quantitative real-time reverse transcription-polymerase chain reaction (PCR) in all patients, along with a control group of fifty healthy individuals. Furthermore, detection of TK1 protein was carried out in 96 patients using a chemiluminescence dot blot assay. The primary endpoint was overall survival (OS) time.

RESULTSPositive CK19 mRNA expression was detected in 74 (43.3%) of the 171 patients and positive TK1 expression was detected in 66 (68.8%) of the 96 patients. Furthermore, of the 96 patients, 36 (37.5%) were positive for both TK1 protein and CK19 mRNA, 30 (31.3%) were negative for TK1 protein, and 15 (15.6%) were negative for TK1 protein and positive for CK19 mRNA. The results indicated that patients who were positive for CK19 mRNA expression had significantly shorter OS times than those who were negative for it (median OS 7.7 vs. 9.7 months, respectively; P = 0.02). Moreover, patients who were positive for CK19 mRNA and TK1 protein expression had shorter OS times (median OS 6.1 months) than those who were positive for CK19 mRNA and negative for TK1 protein expression (median OS 9.1 months; P = 0.028). Positive CK19 mRNA expression was significantly associated with shorter OS in the univariate analysis (P = 0.027). Based on a multivariate Cox regression analysis, CK19 mRNA together with TK1 protein expression (P = 0.024) was an independent predictor for OS in gastrointestinal cancer patients.

CONCLUSIONSOur results suggest that positive expression of CK19 mRNA and TK1 protein is closely correlated with poor prognosis in advanced gastrointestinal cancer. Furthermore, both CK19 and TK1 are possible gastrointestinal cancer biomarkers.

Adult ; Aged ; Aged, 80 and over ; Biomarkers, Tumor ; blood ; genetics ; Female ; Gastrointestinal Neoplasms ; blood ; genetics ; mortality ; Humans ; Keratin-19 ; blood ; genetics ; Male ; Middle Aged ; Prospective Studies ; Thymidine Kinase ; blood ; genetics

BACKGROUND AND OBJECTIVES: The aim of this study was to investigate how fibrinogen-based collagen fleece (Tachocomb®) graft myringoplasty (FCGM), performed under microscopic guidance, improves both hearing and tympanic membrane tissue repair in patients with traumatic tympanic membrane perforation (TMP). SUBJECTS AND METHODS: Between August 2009 and March 2015, a total of 52 patients with traumatic TMP visited the department of otorhinolaryngology at a secondary medical center. Twenty-nine of these underwent FCGM under microscopic guidance in our outpatient clinic. For each patient, we recorded the location and size of the perforation, the time elapsed from the onset of TMP until the myringoplasty, and the hearing level both before and after myringoplasty. RESULTS: The TMP closed completely in all cases (29 of 29 patients). After myringoplasty, the postoperative air-bone gap (ABG) differed significantly from the preoperative ABG. Three of the 29 patients (10.3%) experienced complications. Specifically, 2 presented with otorrhea after FCGM, but conservative management led to improvement without recurrence of perforation. One patient showed delayed facial palsy 1 week after the procedure. The condition of this patient also improved and the palsy was not permanent. CONCLUSIONS: FCGM may be an effective treatment option in case of traumatic TMP. The procedure requires no hospitalization, and can be used to avoid traditional tympanoplasty.
Ambulatory Care Facilities ; Collagen* ; Facial Paralysis ; Hearing ; Hearing Loss, Conductive ; Hospitalization ; Humans ; Myringoplasty* ; Otolaryngology ; Paralysis ; Recurrence ; Thymidine Monophosphate ; Transplants* ; Tympanic Membrane Perforation* ; Tympanic Membrane* ; Tympanoplasty