Comparing IOLM700 TK, Berdahl and Hardten astigmatism fix calculator and Barrett Rx formula in managing residual astigmatism due to toric intraocular lens misalignment

Purpose: To compare the accuracy in astigmatism reduction by using IOLM 700 steep total keratometry (TK) axis, Berdahl and Hardten astigmatism fix, and Barrett Rx formula following misaligned toric intraocular lens (IOL). Methods: Ten patients with residual refractive astigmatism due to misalignment following toric IOL implantation were included in this retrospective study. They were analyzed at days 4, 7/8, and 10/11 following primary cataract surgery on the platform of Berdahl and Hardten astigmatism fix, Barrett Rx formula, and IOLM 700 to determine the optimum axis of repositioning, and underwent IOL realignment on the steep TK axis of IOLM 700 assisted by the Callisto eye. The final outcome parameters were subjective refraction and orientation of toric IOL assessed 22 ± 1 days following repositioning surgery. These parameters were fed in the Barrett Rx formula and its vector analysis graph was utilized to determine the predicted ideal axis with the least residual astigmatism and the estimated residual astigmatism if the toric IOL was realigned according to the axis suggested by Berdahl and Hardten astigmatism fix and Barrett Rx formula. Results: Realigning the toric IOL on IOLM 700 steep TK axis along with the Callisto eye reduces the residual refractive astigmatism significantly (P = 0.003) from 2.00 ± 0.78 D to 0.18 ± 0.12 D (90.5 ± 7.6%) in comparison to the estimated 0.57 ± 0.31 D (68.4 ± 21.9%) by Berdahl and Hardten astigmatism fix and 0.61 ± 0.33 D (66.4 ± 23.5%) by Barrett Rx formula. Conclusion: Realigning the misaligned toric IOL on the IOLM 700 steep TK axis gives a better reduction in the residual refractive astigmatism in comparison to Berdahl and Hardten astigmatism fix and Barrett Rx formula

Analysis of Customer Requirements and Technical Requirements for Inpatients at Bhayangkara Hospital, Kendari City

@#Introduction: The number of inpatient visiting the Bhayangkara Level III Hospital in Kendari City in 2016 was 2322 people, in 2017 there were 3611 people, in 2018 there were 3488 people and from January to November 2019 there were 4243 people. Methods: This research uses descriptive analysis method with a quantitative approach. This research is a case study of the service quality of the Bhayangkara Level III Kendari Hospital. In this study, the sample was taken using simple random sampling technique, in which each element was selected randomly. Results: There are 25 customer requirements for services in the Inpatient Installation of Bhayangkara Hospital, Kendari City which can be categorized into 8 dimensions of service quality according to Brown with priority order based on the level of importance (Tke), namely: 1) Safety Dimensions (98.6%); 2) Dimensions of Interpersonal Relations (98.6%); 3) Dimensions of Continuity (97.1%); 4) Dimension of Effectiveness (97.1%); 5) Efficiency Dimension (97.1%); 6) Dimension of Convenience (97.1%); 7) Dimensions of Access to Services (92.9%) and 8) Dimensions of Officer Competence (92.9%). Conclusion: According to the results of this research it is found that nurses did not take special time to communicate with patients. The officers were warm to patients, doctors always heard complaints and stories of patients, pharmacy officers always prayed for patients to get well soon.

Anti HSV-1 effect of camptothecin by inhibiting HSV-1 replication genes / 中草药

Objective To study the antiviral effect and mechanism of camptothecin against HSV-1 in vitro. Methods Adding different concentration of camptothecin to the Vero cells infected by HSV-1, the measure of cytopathologic effects (CPE) was taken to evaluate the result of antiviral effect and mechanism of camptothecin. The median toxic concentration (TC50), median inhibitory concentration (IC50), and therapeutic index (TI) of camptothecin were calculated, and the affection of camptothecin on HSV-1 mRNA expression was examined through the method of RT-qPCR. Results TC50 of camptothecin was 2 153.44 nmol/L, IC50 was 43.01 nmol/L, and TI was 50.07. The results of RT-qPCR showed that camptothecin can significantly inhibit the gene transcription of HSV-1, UL12, UL42, UL54, and TK, and its inhibitory effect was in dose-dependence relationship with the concentration of camptothecin. Conclusion Camptothecin has an obvious antiviral effect on HSV-1 and can inhibit the transcription of HSV-1 genes such as UL12, UL42, UL54, and TK.

Differential proteomics analysis on hydroquinone-induced cytotoxicity in TK6 cells / 中国职业医学

OBJECTIVE: To study the effect of hydroquinone on the protein expression on human lymphoblastoid cell TK6,and to explore the molecular mechanism of hydroquinone-induced cellular response. METHODS: The TK6 cells were treated with 20. 0 μmol/L of hydroquinone for 24. 0 hours. Total protein was extracted by protein lysis buffer and quantified. The proteins were separated by two-dimensional gel electroporthressis. After image analysis,the difference in electrophoresis was selected for enzymatic hydrolysis. The mass spectrometry identification was performed using matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry. The TK6 cells were treated with hydroquinone at a concentration of 0. 0,5. 0,10. 0 and 20. 0 μmol/L for 24. 0 hours,and total protein was extracted. The expression of heat shock protein 70( HSP70) and ubiquitin-binding enzyme 2( UBE2N) of which were identified by mass spectrometry were assayed by western blot. RESULTS: A total of 48 differential expression protein spots were detected after hydroquinone treatment,and the mass spectrometry identified 30 differentially expressed proteins with up-or down-regulation. These proteins were related to oxidative stress,mitochondrial energy metabolism,cytoskeleton,cell cycle,DNA damage repair,and so on. The relative expression levels of HSP70 and UBE2 N in TK6 cells of 5. 0,10. 0,20. 0 μmol/L hydroquinone group were higher than those of 0. 0 μmol/L hydroquinone group( P < 0. 05),which was consistent with the mass spectrometry results. CONCLUSION: Hydroquinone can induce cytotoxicity in TK6 cells through oxidative stress,which induces the change of mitochondrial energy metabolism and DNA damage repair.

Net case-control study on relationship between base level of Cyfra21-1,SCCA,TK1 and lung cancer prognosis / 中国免疫学杂志

Objective:To investigate the relationship between base level of Cyfra21-1,SCCA,TK1 and lung cancer prognosis.Methods: A nested case-control study was conducted.721 lung cancer cases who had no distant metastasis were recruited baseline population from January 2010 to January 2013.About 2 years follow-up,364 cases of death or brain (or multiple) metastasis were identified as case group, and the other 357 cases were included in the control group.The level of serum Cyfra21-1,SCCA,TK1 was detected.The relationship between base level of Cyfra21-1,SCCA,TK1 and lung cancer prognosis were analyzed.Results: The age in the case group was (59.3±10.1),and the control group was (59.0±9.9).There were obvious differences in body mass index,smoking index,pathological type,clinical stage,lymph node metastasis and with chronic diseases between case group and control group(P0.05).There were differences in the base level of Cyfra21-1,SCCA,TK1;and there were differences in Cyfra21-1,SCCA,TK1 distribution between case group and control group(P0.05) in patients with different stages of lung cancer.There were differences in the base levels of Cyfra21-1 and SCCA(P0.05) in patients with different pathological types of lung cancer.Logistic regression analysis results showed that the OR value of SCCA,TK1 with lung cancer prognosis were respective 7.235(1.674-14.613),5.009(0.973-10.778),5.816(0.879-16.235).Conclusion: The baseline level of Cyfra21-1 can reflect the prognosis of lung cancer patients,while SCCA,TK1 not.

Diagnosis Value of ROC Curve Analysis on TK1 and DNA Ploidy for Patients with Acute Myeloid Leukemia / 现代检验医学杂志

Objective To discuss the value of serum thymidine kinase 1 and DNA ploidy for the diagnosis of patients with acute myeloid lecukemia.Methods Determined TK1 and DI in 20 healthy people,6 patients with benign proliferate in hematological system and 66 patients with acute myeloid lecukemia by chemiluminescence detection technique and flow cytometry.Nonparametric comparisons among three group were done by rank sum test.ROC curve was used to determine the AUC and the diagnosis value serum thymidine kinase 1 and DNA.Results As showed by peripheral blood results,the TK1 (x2 =36.877,P＜0.001) and DI (x2=4.040,P＜0.05) had statistically difference among healthy people group,patients with benign proliferate in hematological system group and AML group.The normal control group compared with the AML group,TK1 (Z=-6.073,P＜0.001)and DI (Z=-2.012,P =0.044) had statistically difference;The normal control group compared with the benign proliferate patients,TK1 (Z=-1.234,P =0.169) and DI (Z=-1.084,P =0.278) had no statistically difference.The benign proliferate patients and that with AML patients,TK1 (Z=2.177,P=0.036) had statistically difference,but DI (Z=-1.801,P=0.061) had no statistically difference.The TK1 and DI area under the ROC curve were 0.950 (P＜0.001) and 0.638 (P=0.063),best cut-off were 1.73 pmol/L and 0.98,sensitivity were 0.95,0.78,and specifity were 0.88,0.39.Conclusion Serum TK1 and DI is a important diagnostic marker of early for AML patients,TK1 have a better diagnostic performance than DI significantly.

Expression of TK1 in benign and malignant tumors / 中华内分泌外科杂志

Objective To investigate the expression of thymidine kinase 1 (TK1) in benign and malignant tumors.Methods From Jan.2013 to May.2015,180 female patients with breast diseases aging 16 to 79 years,including 112 cases of breast cancer (breast cancer group),and 68 cases of breast fibroadenoma (breast fibroadenoma group) were collected.The mean age of the patients was 47 years,with a median age of 43 years.We collected all the patients' fasting venous blood,detected the level of TK1,compared their differences of expression levels between patients with fibroadenoma and breast cancer,and the differences in serum TK1 levels in patients with different pathological characteristics.Results The level of TK1 in breast cancer patients was significantly higher than that in patients with fibroadenoma,and the difference was statistically significant (P＜0.01).In breast cancer group,the tumor size was not correlated with TK1 expression of (P＞0.05).The level of serum TK1 in patients with advanced TNM stage was higher than that in patients with early TNM stage (P=0.038).The level of serum TK1 in patients with advanced lymph node stage was higher than that in early lymph node stage (P=0.048).The level of TK1 in M1 patients was higher than that in M0 patients and the difference had statistical significance (P=0.018).Conclusion TK1,as a new marker of cell proliferation index,can accurately reflect the proliferation of tumor cells in human body,which can be used as an important index to monitor the degree of tumor proliferation and identify benign and malignant tumors.

Clinical analysis of patients with pleural effusion of unknown causes examined by flexi-rigid thoracoscopy (25 cases) / 中国内镜杂志

Objective To investigate the value of flexi-rigid thoracoscopy in pleural effusion of unknown causes and the correlation with CEA, TK1 and ADA. Methods The clinical data and results of CEA, TK1 and ADA of 25 patients were retrospective analyzed in our department from 2015 January to November 2015. These patients accepted the examination of flexi-rigid thoracoscopy with pleural effusion of unknown causes. Results In the 25 patients with pleural effusion of unknown causes, definite diagnosis was made in 22 cases (88.00 %), of which 9 cases were malignant pleural effusion (36.00 %), 11 cases were tuberculous pleural effusion (44.00 %), 2 cases were inflammatory pleural effusion (8.00 %), 3 cases were undetermined (12.00 %). The positive rate of TK1 and CEA in malignant group was significantly higher than that in the tuberculosis group and inflammatory group, the positive rate of ADA in the tuberculosis group was significantly higher than that in the malignant group and inflammatory group. Conclusion Flexi-rigid medical thoracoscopy examination is an effective and safe method for diagnosis of unexplained pleural effusion with high exact diagnosis rate, less trauma and less complication. Combination with CEA, TK1 and ADA are helpful to improve diagnostic rate of pleural effusion of unknown causes.

Clinical significance of serum TK1 levels for patients with non-small-cell lung cancer undergoing chemotherapy / 实用医学杂志

Objective To investigate the dynamic change and its clinical significance of serum thymidine kinase 1 (STK1) in patients with non-small-cell lung cancer (NSCLC) when undergoing 4 cycles of chemotherapy. Methods We detected STK1 levels of 59 patients with NSCLC throughout 4 cycles of chemotherapy using Enhanced Chemiluminescence Western Blot and analyze its relationship with chemotherapy responses . Results STK1 levels with different chemotherapy regimens had no significant difference. STKK1 levels in patients with effective response were significantly lower after 4 cycles of chemotherapy. STK1 levels in patients with effective response were significantly lower than those in non-responders throughout 4 cycles of chemotherapy. The positive rates of STK1 in those with effective response were lower than those in non-responders after the last two cycles of chemotherapy. STK1 levels between lung squamous carcinoma and lung adenocarcinoma had no significant difference. Conclusion The detection of the changes of serum TK1 in patients with NSCLC undergoing chemotherapy is useful in evaluating the effect of chemotherapy and the later therapeutic schedule.

TK2-related mitochondrial DNA depletion syndrome:two cases report and review of literature / 中华实用儿科临床杂志

Objective To study the clinical characteristics,muscle pathological features,diagnosis and prognosis of TK2-related mitochondrial DNA depletion syndrome(MDS).Methods Clinical and laboratory data of 2 cases of TK2-related myopathic MDS were reported.And data of previously reported 58 TK2-related MDS cases were reviewed.Results Total 60 patients consisted of 35 male and 25 female.The age of onset ranged from the birth to the age of 74 years old,and 54 of the patients were attacked at the age younger than 3 years old.Muscle weakness and hypotonia were detected in all patients,with 40 patients(including the newly diagnosed 2 cases) manifested as pure myopathic form,and 20 patients with other multiple organs involvement.Serum creatine kinase was mildly increased (211-6 500 IU/L) in 53 patients.Elevated serum lactic acid level (2.3-12.0 mmol/L)was observed in 24 patients.Muscle biopsy was available from 55 patients,and ragged red fibers and/or cytochrome C oxidase (COX)-negative fibers were detected in 48 out of them.Nine out of 11 patients received electronic microscope study showed proliferation of abnormal mitochondria.Respiratory chain enzymatic activities in skeletal muscle were reduced in 31 out of 33 patients.Marked mtDNA content reduction was observed in 36 out of 41 patients (4％-25％ of age-and tissue-matched controls).A total of 42 TK2 mutations were found in 60 patients,including 2 novel mutations c.923A ＞ G and c.619-2A ＞ T in this study.Conclusions The most common clinical manifestations of TK2-related MDS are severely,rapidly progressing myopathy with infantile or early childhood onset.As the detection rate of characteristic pathologic features in muscle is high,muscle biopsy is important for the diagnosis of TK2-related MDS.

Application Value of Combined Detection of PGⅠ,PGⅡ,TK1, TSGF,CEA and CA7 2 4 in Diagnosis of Gastric Cancer / 现代检验医学杂志

Objective To investigate the serum levels of PGⅠ,PGⅡ,TK1,TSGF and CEA,CA724 in gastric cancer and eval-uate the application value of combined detection the above tumor markers in diagnosis of gastric cancer.Methods The serum levels of TSGF were measured in 94 patients with gastric cancer and 85 healthy control by rate method.PGⅠ,PGⅡ,TK1 and CEA,CA724 were detected by electrochemiluminescence method.Results PGⅠand PGⅠ/PGⅡwere lower than healthy control in serum of patients with gastric cancer (both P0.05),and other tumor markers were all higher than healthy control (all P<0.05).The sensitivity of PGⅠ,PGⅠ/PGⅡ were better than TK1,CEA and CA724 (all P<0.05),the specificity of PGⅠ/PGⅡ,CEA were better than TSGF (both P<0.05),the accuracy of PGⅠ,PGⅠ/PGⅡ were better than CA724 and TSGF alone (all P<0.05).When combined TSGF,TK1 and PGⅠ/PGⅡ,PGⅠ,the sensitivity was better than combined PGⅠ/PGⅡand PGⅠ alone (P<0.05).Then when added CEA, CA724,this sensitivity improved up to 82.98%.Although the combined detection would show a lower specificity,it still keep high to 84.71%.Combined detection improved the accuracy in diagnosis of gastric cancer,up to 83.80%.In this re-search,There was no difference in sensitivity,specificity and accuracy between the group of PGⅠ+PGⅠ/PGⅡ+TSGF+TK1+CEA and the group of PGⅠ+PGⅠ/PGⅡ+TSGF+TK1+CEA+CA724.Conclusion Compared with CEA and CA724 popular used in clinic,PGⅠ/PGⅡand PGⅠshowed a better application value.The group of PGⅠ+PGⅠ/PGⅡ+TSGF+TK1+CEA showed the best sensitivity.Combined detection of serum levels of PGⅠ,PGⅠ/PGⅡ,TSGF,TK1 ,CEA can significantly raise the sensitivity and accuracy in diagnosis of gastric cancer.

The monitoring analysis of perioperative tumor markers in non-small cell lung cancer of 55 cases / 安徽医科大学学报

Objective To investigate the change and effect of operation on TK1,CEA, SCC-Ag and CYFRA21-1 level in non-small cell lung cancer patients. Methods From January 2013 to August 2013, 55 patients with NSCLC and 29 patients with benign lesions were recruited. The tumor indexes including TK1,CEA, SCC-Ag and CYFRA21-1 were measured by electrical chemiluminescence assay. Results ① Serum TK1, CEA, SCC-Ag and CYFRA21-1 level and the positive rates in NSCLC patients during preoperative period were higher than the benign disease control group ( P0. 05 ) . Serum CEA , SCC-Ag and CYFRA21-1 level of postoperative one week and one month in NSCLC patients were significantly lower than the level before the operation ( P0. 05 ) , serum TK1 and SCC-Ag level of the postoperative one week showed statistically significant difference compared with benign disease control group ( P0. 05 ) .③The preoperative level of TK1 and CEA in adenocarcinoma were higher than that in squamous carcinoma ( P0. 05 ) . The preoperative level of serum TK1 in NSCLC patients negatively correlated with the degree of differentiation of lung cancer, the difference was statistically significant ( P<0. 05 ) . Conclusion The perioperative level of tumor markers in NSCLC may be useful in monitoring diagnosis and differential diagnosis of NSCLC, especially in analyzing the invisible tumor burden of NSCLC patients. The perioperative level of tumor markers in NSCLC is related with the surgical effect and tissue types, and serum TK1 and CEA are more suitable for the evaluation of patients with lung adenocarcinoma. There is some value in the clinical applications.

Application of mitochondrial toxicity markers during highly active antiretroviral therapy / 中华传染病杂志

Objective To explore whether the mitochondrial toxicity markers of peripheral blood mononuclear cells (PBMC)are of significance in monitoring mitochondrial toxicity during highly active antiretroviral therapy (HAART)in patients with acquired immune deficiency syndrome (AIDS).Methods Mitochondrial DNA (mtDNA),mitochondrial thymidine kinase (TK2 )and p53-inducible ribonucleotide reductase small subunit 2 (p53R2 )were selected as mitochondrial toxicity markers.The expression changes of theses markers of PBMC in 22 AIDS patients were detected by real time quantitative polymerase chain reaction (q-PCR)at baseline,48 weeks and 96 weeks after initiation of the treatment. All the patients received stavudine/zidovudine and lamivudine as the mainstay of the HAART regimen. Independent-samples t test was used.Results The relative expression level of mtDNA in patients before HAART was 3.27 ± 0.94,and decreased to 2.16±0.85 at week 48 and 1 .66±0.66 at week 96, respectively.The differences were both significant compared with the level prior to the treatment (t =-3.90,P <0.01 and t =-6.29,P <0.01 ,respectively).The relative expression level of TK2 before HAART was 0.37 ±0.13,and increased to 1 .01 ±0.25 at week 48 and 2.13 ±0.61 at week 96 of the treatment.After pairwise comparisons of the three pairs of data (pre-HAART vs week 48 of the treatment,pre-HAART vs week 96 of the treatment and week 48 vs week 96 of the treatment),the differences were all significant (t = 10.77,8.00 and 3.56,respectively;all P < 0.01 ).The relative expression level of p53R2 was 0.86±0.39 before HAART,but gradually increased to 2.36 ±1 .14 and 7.73±0.65 ,respectively,at week 48 and week 96 of the treatment.The differences in p53R2 levels among three groups after pairwise comparison were all significant (t=3.27,12.26 and 13.25,respectively;all P < 0.01 ).Conclusions The expression levels of mtDNA,TK2 and p53R2 in PBMC could change significantly during HAART in AIDS patients,which might be used as indexes for monitoring mitochondrial toxicity.

Expression of serum TK1 in human breast tumor and its clinical significance / 中国癌症杂志

Background and purpose: The position of thymidine kinase 1 (TK1) expression during cell division is in the cytoplasm. It is a catalytic enzyme to convert deoxythymidine into thymidylate. It is the key enzyme of pyrimidine salvage pathway. The aim of this study was to analyze the serum expression level of TK1 in patients with breast cancer, and explore the application of serum TK1 test in clinical assessments of diagnosis, treatment and prognosis for breast cancer. Methods: Patient data were collected from the patients admitted in Comprehensive Breast Health Center at Rui Jin Hospital. Chemiluminesence dot blot assay was used to detect serum TK1 levels in 145 breast cancer patients and 55 patients with breast ifbroadenoma. The correlations of serum TK1 levels with breast tumor biological behavior was further studied. Results:Serum TK1 expression levels was signiifcantly increased in breast cancer patients [(2.749±0.122)pmol/L] when compared to breast fibroadenoma patients[(1.319±0.126)pmol/L, P0.05), different tumor grades (P=0.453) and different tumor size (P=0.908). Preoperative imaging results including breast ultrasound, breast mammography and breast magnetic resonance were analyzed by assessments of BI-RADS category, and serum TK1 levels in patients with different BI-RADS categories were studied. Serum TK1 levels in patients with breast ultrasound BI-RADS categories 4C-6 were signiifcantly higher than those with category 0-4B (P0.05). Most patients were followed up in our outpatient department for about 2 years. No progression-free survival differences were found in 2years. Conclusion:Serum TK1 test might be a potential tool for screening, prognosis determination and effect evaluations of targeted therapy in breast carcinoma.

Vesicular Stomatitis Virus G Glycoprotein and ATRA Enhanced Bystander Killing of Chemoresistant Leukemic Cells by Herpes Simplex Virus Thymidine Kinase/Ganciclovir

Refractoriness of acute myeloid leukemia (AML) cells to chemotherapeutics represents a major clinical barrier. Suicide gene therapy for cancer has been attractive but with limited clinical efficacy. In this study, we investigated the potential application of herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) based system to inhibit chemoresistant AML cells. We first generated Ara-C resistant K562 cells and doxorubicin-resistant THP-1 cells. We found that the HSV-TK/GCV anticancer system suppressed drug resistant leukemic cells in culture. Chemoresistant AML cell lines displayed similar sensitivity to HSV-TK/GCV. Moreover, HSV-TK/GCV killing of leukemic cells was augmented to a mild but significant extent by all-trans retinoic acid (ATRA) with concomitant upregulation of Connexin 43, a major component of gap junctions. Interestingly, HSV-TK/GCV killing was enhanced by expression of vesicular stomatitis virus G glycoprotein (VSV-G), a fusogenic membrane protein, which also increased leukemic cell fusion. Co-culture resistant cells expressing HSV-TK and cells stably transduced with VSV-G showed that expression of VSV-G could promote the bystander killing effect of HSV-TK/GCV. Furthermore, combination of HSV-TK/GCV with VSV-G plus ATRA produced more pronounced antileukemia effect. These results suggest that the HSV-TK/GCV system in combination with fusogenic membrane proteins and/or ATRA could provide a strategy to mitigate the chemoresistance of AML.

A thymidine kinase-deleted bovine herpesvirus 5 establishes latent infection but reactivates poorly in a sheep model / Recombinante do herpesvírus bovino tipo 5 com deleção na timidina quinase estabelece infecção latente porém reativa ineficientemente em ovinos

The ability of thymidine kinase (tk)-deleted recombinant bovine herpesvirus 5 (BoHV-5tkΔ) to establish and reactivate latent infection was investigated in lambs. During acute infection, the recombinant virus replicated moderately in the nasal mucosa, yet to lower titers than the parental strain. At day 40 post-infection (pi), latent viral DNA was detected in trigeminal ganglia (TG) of all lambs in both groups. However, the amount of recombinant viral DNA in TGs was lower (9.7-fold less) than that of the parental virus as determined by quantitative real time PCR. Thus, tk deletion had no apparent effect on the frequency of latent infection but reduced colonization of TG. Upon dexamethasone (Dx) administration at day 40 pi, lambs inoculated with parental virus shed infectious virus in nasal secretions, contrasting with lack of infectivity in secretions of lambs inoculated with the recombinant virus. Nevertheless, some nasal swabs from the recombinant virus group were positive for viral DNA by PCR, indicating low levels of reactivation. Thus, BoHV-5 TK activity is not required for establishment of latency, but seems critical for efficient virus reactivation upon Dx treatment.

A capacidade de um recombinante do herpesvírus bovino tipo 5 com deleção no gene da timidina quinase (BoHV-5tk∆) em estabelecer e reativar infecção latente foi investigada em cordeiros. Durante a infecção aguda, o vírus recombinante replicou na mucosa nasal em títulos moderados, porém menores do que os da cepa parental. Aos 40 dias pós-infecção (pi) DNA viral latente foi detectado no gânglio trigêmeo (TG) de todos os cordeiros em ambos os grupos. No entanto, a quantidade de DNA do vírus recombinante nos TGs foi 9,7 vezes menor do que do vírus parental, segundo determinação por PCR em tempo real. Assim, a deleção do gene tk (timidina quinase) não produziu efeito aparente sobre a frequência da infecção latente, porém reduziu a colonização do TG. Após a administração de dexametasona (Dx) no dia 40pi, os cordeiros inoculados com o vírus parental excretaram partículas virais infecciosas, contrastando com a falta de infectividade nas secreções nasais dos animais inoculados com o vírus recombinante. Entretanto, alguns suabes nasais dos cordeiros do grupo do vírus recombinante foram positivos para o DNA viral por PCR, indicando baixos níveis de reativação. Assim, a atividade da enzima timidina quinase não é requerida para o estabelecimento de latência pelo BoHV-5, mas parece fundamental para reativação eficiente da infecção latente após tratamento com Dx.

Non-Cytotoxic Property and DNA Protective Activity against H2O2 and UVC of Thai GAC Fruit Extracts in Human TK6 Cells.

Momordica cochinchinensis (Lour.) fruit of Cucurbitaceae family is indigenous vegetable and fruit to Southeast Asia. It has been generally known as “GAC” whereas in Thailand it is called “Fhuck-khow”. Regarding its richness in anti-oxidants particularly lycopene and beta-carotene, GAC is named "fruit from heaven" and believed to promote longevity, health and vitality. In present study, GAC extracts were prepared from pulp (PU), skin (SK) and seed membrane (SM) by 50%, 95% ethanol and water (W). MTT assay was done in TK6 cells to determine their cytotoxicity property. The non-cytotoxic extracts were further evaluated for the DNA protective activity against H2O2 and UVC using high alkaline (pH >13) comet assay. TK6 cells (1x105 cells/ml) were pre-incubated with GAC extracts (25, 50 and 100μg/ml) for 24 h prior to an exposure to 50 μM H2O2 and a 254-nm UVC germicidal lamp for DNA damage induction. We found that H2O2-induced DNA damage was suppressed by 30- 60 % when cells were pre-treated with PU, SK and SM extracts at all concentrations used. Regarding test condition used, UVC clearly induced DNA damage in TK6 cells greater that that by H2O2. The protective effect of GAC extracts against UVC was found by 20-30%. Our present results suggest that GAC possesses DNA protective ability against H2O2 and UVC. The degree of anti-oxidative damage activity in TK6 cells of GAC extracts is SK95>SMW>SK50.

A bovine herpesvirus 5 recombinant defective in the thymidine kinase (TK) gene and a double mutant lacking TK and the glycoprotein E gene are fully attenuated for rabbits

Bovine herpesvirus 5 (BoHV-5), the agent of herpetic meningoencephalitis in cattle, is an important pathogen of cattle in South America and several efforts have been made to produce safer and more effective vaccines. In the present study, we investigated in rabbits the virulence of three recombinant viruses constructed from a neurovirulent Brazilian BoHV-5 strain (SV507/99). The recombinants are defective in glycoprotein E (BoHV-5gEÄ), thymidine kinase (BoHV-5TKÄ) and both proteins (BoHV-5gEÄTKÄ). Rabbits inoculated with the parental virus (N = 8) developed neurological disease and died or were euthanized in extremis between days 7 and 13 post-infection (pi). Infectivity was detected in several areas of their brains. Three of 8 rabbits inoculated with the recombinant BoHV-5gEÄ developed neurological signs between days 10 and 15 pi and were also euthanized. A more restricted virus distribution was detected in the brain of these animals. Rabbits inoculated with the recombinants BoHV-5TKÄ (N = 8) or BoHV-5gEÄTKÄ (N = 8) remained healthy throughout the experiment in spite of variable levels of virus replication in the nose. Dexamethasone (Dx) administration to rabbits inoculated with the three recombinants at day 42 pi did not result in viral reactivation, as demonstrated by absence of virus shedding and/or increase in virus neutralizing titers. Nevertheless, viral DNA was detected in the trigeminal ganglia or olfactory bulbs of all animals at day 28 post-Dx, demonstrating they were latently infected. These results show that recombinants BoHV-5TKÄ and BoHV-5gEÄTKÄ are attenuated for rabbits and constitute potential vaccine candidates upon the confirmation of this phenotype in cattle.

Construction and identification of prostate-specific double gene expression vector pIRES-PSMAe/p-TKCx43 / 中华泌尿外科杂志

Objective To construct the prostate-specific double gene expression vector pIRESPSMAe/p-TK-Cx43 and establish the foundation for experimental prostate cancer gene therapy research. Methods Cx43 gene was amplified and cloned into pMD19-T Simple vector. HSV-TK gene was then synthesized and cloned into multiple clone site (MCS) A of the eukaryotie vector plRES. The new plasmid was named plRES-TK: PSMAe/p was obtained and cloned into plRES-TK by replacing CMV promoter. The new plasmid was named plRES-PSMAe/p-TK; Fourth, Cx43 gene was cloned into the MCS B of pIRES-PSMAe/p-TK and the new plasmid was named pIRES-PSMAe/p-TK-Cx43.This plasmid was identified by double digestion with Sal Ⅰ/Not Ⅰ and sequenced; Finally, LNCaP cells were transfected by the plasmid plRES-PSMAe/p-TK-Cx43 and the mRNAs expression of HSV-TK gene and Cx43 gene was tested by RT-PCR. Results The plasmids synthesized in this experiment were double digested respectively and the specific bands of the inserted genes were confirmed by RTPCR. plRES-PSMAe/p-TK-Cx43 was in line with the expected design by DNA sequencing. The mRNAs of TK gene and Cx43 gene were expressed and successfully confirmed by RT-PCR after LNCaP cells transfected with pIRES-PSMAe/p-TK-Cx43. Conclusion Double gene expression vector pIRES-PSMAe/p-TK-Cx43 containing HSV-TK gene and Cx43 gene is constructed successfully.

Study on the relationship between TK gene regulated by enhanced suicide gene vector and telomerase activity in nasopharyngeal carcinoma cells / 临床耳鼻咽喉头颈外科杂志

Objective:To explore the relationship between TK gene expression regulated by enhanced suicide gene vector and telomerase activity in nasopharyngeal carcinoma cells.Method:The reformed reconstructed enhanced vector, pGL3-basic-EGFP-TK-hTRETp-CMV enhancer, and hTERT mono-promoter vector, pGL3-basic-EGFP-TK-hTRETp(as controls), were transfected into telomerase(+) nasopharyngeal carcinoma 5-8F cell lines,telomerase(+) human breast cancer MCF-7 cell lines and telomerase(-) normal vascular endothelium cell lines respectively. TK gene green fluorescent protein was observed by fluorescence microscope. The expression of TK gene mRNA was measured by the real-time fluorescent quantified PCR and the telomerase activity was determined by the method of TRAP argentation in maligment tumour cells pre- and post-transfected by enhanced vector . Meanwhile the relationship beteewn TK and telomerase was analyzed.Result:①A strong TK gene fluorescent show and TK mRNA expression were displayed after the enhanced suicide gene vector was transfected into nasopharyngeal carcinoma 5-8F cell lines and human breast cancer MCF-7 cell line, which were more stronger than those of mono-promoter group,pGL3-basic-EGFP-TK-hTRETp,and ECV cells transfected by enhanced suicide gene vector. Meanwhile,real-time fluorescent quantified PCR showed that the A value of enhanced vector group was higher than that of controls. ②Telomerase activity after transfection of enhanced vector and GCV was lower than those before by the method of TRAP argentation in nasopharyngeal carcinoma cell lines,but no change in normal control cells after transfection of enhanced vector and GCV.③ After adding GCV, the obvious inhibitory effect of tumour cells growth induced by pGL3-basic-EGFP-TK-hTRETp-CMV enhancer were observed in nasopharyngeal carcinoma 5-8F cell lines and human breast cancer MCF-7 cell line, which was higher than those of mono-promoter, pGL3-basic-EGFP-TK-hTRETp,pGL3-basic-EGFP3 and blank controls, but without inhibitory effect in ECV cells transfected by enhanced vector. Conclusion:TK gene expression is regulated by hTERT promoter and CMV enhancer, and then the telomerase activity is reduced and the cancer cells are specifically killed.But it is unclear how the telomerase are down-regulated by TK gene.