Differential expression of enzymes in thymidylate biosynthesis in zebrafish at different developmental stages: implications for dtymk mutation-caused neurodegenerative disorders.

BACKGROUND: Deoxythymidine triphosphate (dTTP) is an essential building block of DNA, and defects in enzymes involved in dTTP synthesis cause neurodegenerative disorders. For instance, mutations in DTYMK, the gene coding for thymidylate kinase (TMPK), cause severe microcephaly in human. However, the mechanism behind this is not well-understood. Here we used the zebrafish model and studied (i) TMPK, an enzyme required for both the de novo and the salvage pathways of dTTP synthesis, and (ii) thymidine kinases (TK) of the salvage pathway in order to understand their role in neuropathology. RESULTS: Our findings reveal that maternal-stored dNTPs are only sufficient for 6 cell division cycles, and the levels of dNTPs are inversely correlated to cell cycle length during early embryogenesis. TMPK and TK activities are prominent in the cytosol of embryos, larvae and adult fish and brain contains the highest TMPK activity. During early development, TMPK activity increased gradually from 6 hpf and a profound increase was observed at 72 hpf, and TMPK activity reached its maximal level at 96 hpf, and remained at high level until 144 hpf. The expression of dtymk encoded Dtymk protein correlated to its mRNA expression and neuronal development but not to the TMPK activity detected. However, despite the high TMPK activity detected at later stages of development, the Dtymk protein was undetectable. Furthermore, the TMPK enzyme detected at later stages showed similar biochemical properties as the Dtymk enzyme but was not recognized by the Dtymk specific antibody. CONCLUSIONS: Our results suggest that active dNTP synthesis in early embryogenesis is vital and that Dtymk is essential for neurodevelopment, which is supported by a recent study of dtymk knockout zebrafish with neurological disorder and lethal outcomes. Furthermore, there is a novel TMPK-like enzyme expressed at later stages of development.

DTYMK is essential for genome integrity and neuronal survival.

Nucleotide metabolism is a complex pathway regulating crucial cellular processes such as nucleic acid synthesis, DNA repair and proliferation. This study shows that impairment of the biosynthesis of one of the building blocks of DNA, dTTP, causes a severe, early-onset neurodegenerative disease. Here, we describe two unrelated children with bi-allelic variants in DTYMK, encoding dTMPK, which catalyzes the penultimate step in dTTP biosynthesis. The affected children show severe microcephaly and growth retardation with minimal neurodevelopment. Brain imaging revealed severe cerebral atrophy and disappearance of the basal ganglia. In cells of affected individuals, dTMPK enzyme activity was minimal, along with impaired DNA replication. In addition, we generated dtymk mutant zebrafish that replicate this phenotype of microcephaly, neuronal cell death and early lethality. An increase of ribonucleotide incorporation in the genome as well as impaired responses to DNA damage were observed in dtymk mutant zebrafish, providing novel pathophysiological insights. It is highly remarkable that this deficiency is viable as an essential component for DNA cannot be generated, since the metabolic pathway for dTTP synthesis is completely blocked. In summary, by combining genetic and biochemical approaches in multiple models we identified loss-of-function of DTYMK as the cause of a severe postnatal neurodegenerative disease and highlight the essential nature of dTTP synthesis in the maintenance of genome stability and neuronal survival.

Biochemical Characterizations of Human TMPK Mutations Identified in Patients with Severe Microcephaly: Single Amino Acid Substitutions Impair Dimerization and Abolish Their Catalytic Activity.

Deoxythymidylate kinase (TMPK) is a key enzyme in the synthesis of deoxythymidine triphosphate (dTTP). Four TMPK variants (P81L, A99T, D128N, and a frameshift) have been identified in human patients who suffered from severe neurodegenerative diseases. However, the impact of these mutations on TMPK function has not been clarified. Here we show that in fibroblasts derived from a patient, the P81L and D128N mutations led to a complete loss of TMPK activity in mitochondria and extremely low and unstable TMPK activity in cytosol. Despite the lack of TMPK activity, the patient-derived fibroblasts apparently grew normal. To investigate the impact of the mutations on the enzyme function, the mutant TMPKs were expressed, purified, and characterized. The wild-type TMPK mainly exists as a dimer with high substrate binding affinity, that is, low K M value and high catalytic efficiency, that is, k cat/K M. In contrast, all mutants were present as monomers with dramatically reduced substrate binding affinity and catalytic efficiencies. Based on the human TMPK structure, none of the mutated amino acids interacted directly with the substrates. By structural analysis, we could explain why the respective amino acid substitutions could drastically alter the enzyme structure and catalytic function. In conclusion, TMPK mutations identified in patients represent loss of function mutations but surprisingly the proliferation rate of the patient-derived fibroblasts was normal, suggesting the existence of an alternative and hitherto unknown compensatory TMPK-like enzyme for dTTP synthesis. Further studies of the TMPK enzymes will help to elucidate the role of TMPK in neuropathology.

Study of P50 sensory gating deficit in schizophrenic patients with violent and aggressive behaviors / 四川精神卫生

ObjectiveTo assess the specificity of P50 auditory-evoked potential in schizophrenic patients with violent and aggressive behaviors， so as to provide objective biological markers for predicting violent behaviors of schizophrenic patients. MethodsA total of135 schizophrenic patients who met the diagnostic criteria of the International Classification of Diseases， tenth edition （ICD-10） were divided into aggressive group （n=70） and non-aggressive group （n=65） according to the assessment results of the Modified Overt Aggression Scale （MOAS）， meantime， another 60 healthy individuals matched for age and gender were set as healthy group. Then the P50 auditory-evoked potentials of all selected individuals were measured using EP/EMG system （MEB-9200， Nihon Kohden， Japan）. ResultsAmp S2 of the aggressive group was significantly higher than those of the non-aggressive group and healthy control group， with statistical differences ［（9.86±6.04）μV vs. （7.06±3.88）μV， P=0.004； （9.86±6.04）μV vs. （7.82±3.87）μV， P=0.031］. The proportion of S2/S1 ratio ≥0.5 was 72.88%， 43.86% and 30.00% in aggressive group， non-aggressive group and healthy group， which was the highest in aggressive group， with statistical differences （P<0.01）. The amplitude difference of P50 （S1-S2） of the aggressive group was lower than those of the non-aggressive group and the healthy control group， the differences were of statistical significance ［（4.35±9.39）μV vs.（9.89±8.48）μV， P=0.001； （4.35±9.39）μV vs.（13.42±9.81）μV， P<0.01］. ConclusionThe violent and aggressive behaviors in schizophrenic patients may be related to the sensory gating deficit.

Identification of a novel thymidylate kinase activity.

Thymidylate kinase (TMPK, EC2.7.4.9) is the enzyme that converts deoxythymidine monophosphate (dTMP) to deoxythymidine diphosphate (dTDP) in the synthesis of dTTP, an essential building block of DNA. To date, there is only one gene (TYMK) known to encode TMPK in mammalian cells. In this study, we investigated the distribution of TMPK activity and protein in subcellular fractions by using activity measurements and by using a specific antibody against TYMK-encoded TMPK (canonical TMPK). TMPK activity was detected in all subcellular fractions, of which the mitochondrial outer membrane contained the highest activity. High levels of canonical TMPK protein were detected in the cytosolic fraction, whereas low levels were found in the nuclear and mitochondrial matrix fractions. Strikingly, despite the detection of high TMPK activity in the mitochondrial outer membrane, canonical TMPK protein was not detected in this fraction. These results suggest that the TMPK activity detected in the outer membrane fraction may originate from a novel dTMP kinase, distinct from the canonical TYMK.

Effects of laryngeal mask airway combined with lung-protective ventilation on postoperative pulmonary complications in patients undergoing open abdominal surgery with general anesthesia / 中华麻醉学杂志

Objective To investigate the effects of laryngeal mask airway combined with lung-protective ventilation on postoperative pulmonary complications in patients undergoing open abdominal surgery with general anesthesia.Methods Eighty-seven patients of both sexes,aged 18-64 yr,of American Society of Anesthesiologists physical status Ⅱ or Ⅲ,scheduled for elective open abdominal surgery under general anesthesia,were randomly divided into 2 groups:endotracheal intubation plus routine ventilation group (group IC,n =43) and laryngeal mask airway plus protective ventilation group (group LP,n =44).In group IC,single lumen endotracheal tube was inserted and the patients were mechanically ventilated,with inhaled pure oxygen 2 L/min,tidal volume 8-10 ml/kg,respiratory rate 8-12 breaths/min,inspiratory/expiratory ratio 1 ∶ 2.In group LP,the ProSeal laryngeal mask airway was inserted and lung-protective ventilation was performed,with inhaled pure oxygen 2 L/min,tidal volume 6-8 ml/kg,respiratory rate 12-16 breaths/min,inspiratory/expiratory ratio 1 ∶ 2,positive end-expiratory pressure 5 cm H2O,and recruitment maneuvers was performed manually every 30 min during operation.End-tidal pressure of carbon dioxide was maintained at 35-45 mmHg.The development of pulmonary complications such as pneumonia,pleural effusion,pulmonary atelectasis,pneumothorax,bronchospasm and acute respiratory failure was recorded within 7 days after surgery.Results Pulmonary atelectasis,pneumothorax or acute respiratory failure was not found in the two groups.Compared with group IC,no significant change was found in the incidence of pneumonia,pleural effusion and bronchospasm after operation (P＞0.05),and the total incidence of postoperative pulmonary complications was significantly decreased in group LP (P＜0.05).Conclusion Compared with endotracheal intubation and conventional ventilation,laryngeal mask airway combined with lung-protective ventilation can reduce postoperative pulmonary complications in the patients undergoing open abdominal surgery with general anesthesia.

Clinical research of percutaneous vertebroplasty combined with strontium-89 on patients with spinal malignant tumors / 重庆医学

Objective This study aims to investigate the clinical value of percutaneous vertebroplasty (PVP)combined with strontium-89 (89 Sr)on patients with spinal malignant tumor.Methods Seventy four patients who were diagnosed as spinal malig-nant tumor were divided into two groups randomly.37 patients of the control group were treated by PVP only,and 37 patients of joint group were treated by PVP and 89 Sr together.Then the clinical effect of the joint group and control group was compared after treatment.Results After treatment,the quality of life,ratio of pain relief,clinical effect and survival time in joint group were dis-tinctly better than control group (P <0.05).Conclusion PVP combined with 89 Sr can be an effective and safe treatment for pa-tients with spinal malignant tumor.

Brain structural imaging study in schizophrenia patients with violent aggressive behaviors / 中华行为医学与脑科学杂志

Objective To review recent neuroimaging studies of aggressive/violent behaviors in schizophrenia and related the neural circuits of aggressive behaviors,tocusing on structure magnetic resonance imaging (MRI) method,published from 2000 to 2014.Methods A systematic literature search was conducted using articles from PsycINFO,Embase,Pubmed and Cnki pnblished between 2000 and 2014,we usedaggressive, violent, schizophrenia, structure M RI as the key words,to explore the relationship between schizophrenia of aggressive/violent behaviors and the related neural circuits.Results Of the 18 studies included in the analysis,mostly were case-control studies,two were written in Chinese.The neuroimaging literature on aggression in schizophrenia revealed the related grey matter volume of brain areas which contained the frontal lobe,temporal lobe,amygdala,hippocampus,cerebelum and caudate nucleus.Conclusion Findings from the current reviews suggest that the occurrence of violent/aggressive behaviors of schizophrenia were related with serval neural circuits as the frontal-temporal cognitive circuit,Papez circuit,cerebellum advaneed cognitive circuit and caudate nucleus-reward seeking cireuits.However,the precise violent/aggressive neural mechanism has vet to be studied further.

Value of imaging findings score in valuing the activity of Crohn disease / 中华放射学杂志

Objective To evaluate the value of multi-slice CT enterography(MSCTE),capsule endoscopy(CE) and contrast-enhanced ultrasound(CEUS) findings score in the assessment of Crohn disease activity,prospectively.Methods One hundred and eight patients were diagnosed with CD by endoscopy combined with clinical or pathological results,and all of the patients' Crohn disease activity index(CDAI) were greater than or equal to 150.MSCTE were performed in all the patients.At the same time,48 and 60 patients of them were voluntary to accept CEUS and CE examination,respectively.The imaging score of the three different techniques was calculated.Patients' CDAI were calculated combined with the C-reactive protein(CRP) content and clinical feature.To analyze the correlation among the imaging score patients' CDAI,and CRP content,respectively using the Pearson method.Results The mean score of MSCTE,CEUS and CE were(7.6±2.7),(11.5 ±4.3) and(12.8±8.2),respectively.The patients' mean CDAI and CRP content were(296.1±93.5) and(23.7±5.2) mg/L,respectively.It was high correlation between MSCTE score and CDAI (r=0.916,P＜0.01),but it was moderate between CEUS(r=0.752,P＜0.01),CE(r=0.707,P＜0.01) score and CDAI.There was no evident correlation between MSCTE(r=0.167,P＞0.05),CEUS(r=0.200,P＞0.05) score and CRP content excepting the CE result(r=0.467,P＜0.01).Conclusions There was a good correlation between the MSCTE score and CDAI.We could use MSCTE findings score to assess the activity of CD patients prospectively.

A cross-sectional study on physicaly violent behavior among youth males in Chengdu, Sichuan province / 中华流行病学杂志

Objective To investigate the prevalence of physically violent behavior among 18-34 years old males in Chengdu.Methods 2 151 males aged 18-34 years was recruited in Chengdu,using a multistage stratified random sampling method."Men' s health and modem lifestyles survey questionnaire" was adopted to collect information on demography,physical violent behavior,attitude and reactions to violence.Results Self-reported prevalence of physical violent behavior since 15 years of age among those 18-34 year-old males was 51.8％.17.8％ of the young males reported having violent behavior in the past 5 years,but the prevalence declined progressively with age (P＜0.01).People who were religious or remained single had higher prevalence rates than those non-religious,married or having regular sex partners (P＜0.01).Prevalence seemed relatively high in students or workers at service/commercial fields,with rates as 24.5％ and 20.3％.Conclusion Data from our study suggested that physical violence was an important problem among young males in Chengdu.Factors as being young,single,divorced or widowed were associated with self-reported violent behavior.People who were religious,being students or working at the service/commercial fields seemed to be under high risk of carrying physical violence.

Current situation and relevant risk factors on sexual violence in 18-34 year-olds men in Chengdu / 中华流行病学杂志

Objective To understand the current situation and relevant factors on sexual violence in 18-34 year-olds men in Chengdu,in order to provide data for setting up prevention and reduction programs on sexual violence and other related problems.Methods By multi-stage random sampling,young men who were 18-34 years old in Chengdu were interviewed and screened.Questionnaire and multivariate logistic regression models were used to assess risk factors on sexual violence.Results The overall prevalence of sexual violence was 17.8％,with factors as child exposure to parent violence/family conflict (P＜0.01),child physical abuse (P＜0.01),alcohol abuse (P＜0.01),anxiety (P＜0.01),disharmony marriage (divorce/ widowed/separation) (P＜0.01),marriage status (P＜0.05),depression (P＜0.05) as risk factors of sexual violence.Conclusion The status on sexual violence among young men in Chengdu should not be overlooked and interventions for high-risk populations should be taken urqently.

Study on the event-related potentials P300 of schizophrenic patients with aggressive or violent behaviors / 中华行为医学与脑科学杂志

ObjectiveTo explore the electrophysiological mechanisms of the schizophrenic patients with aggressive or violent behaviors.MethodsAccess the aggressive behaviors of schizophrenic patients being treated in hospitals or clinics with the revised MOAS in accordance with the ICD-10 diagnostic criteria,and sort the qualified patients into two groups:the group of aggressive or violent schizophrenia (Aggressive Group,n=70) and the group of non-aggressive or non-violent schizophrenia ( Non-Aggressive Group,n =65 ) ; 60 age- and gendermatched healthy people were collected as Healthy Group.P300 tests were carried out on patients in these three groups with the MEB-9200 Nicolet Bravo Instrument by the Nihon Kohden Corporation.Results ( 1 ) latency P3a of the Aggressive Group on Cz point exceeded that of the Non-Aggressive Group (P =0.01 ),and that of the Non-Aggressive Group exceeded that of the Healthy Group.All these disparities were of statistical significance (P ＜0.01 ).Latency P3a of the Aggressive Group on Fz point exceeds that of the Non-Aggressive Group,and that of the Non-Aggressive Group exceeded that of Healthy Group.All these disparities are also of stafistical significance (P＜0.01).(2)N2' amplitude of the Aggressive Group on Cz point was higher than those of the Non-Aggressive Group and the Healthy Group.This disparity was of statistical significance(P ＜ 0.05 ) and the disparity between the Non-Aggressive Group and the Healthy Group did not have statistical significance (P =0.985 ).ConclusionCharacteristic electrophysiological changes exist in the event-related potentials P300 of schizophrenic patients with aggressive or violent behaviors.