OPM-MEG Measuring Phase Synchronization on Source Time Series: Application in Rhythmic Median Nerve Stimulation.

The magnetoencephalogram (MEG) based on array optically pumped magnetometers (OPMs) has the potential of replacing conventional cryogenic superconducting quantum interference device. Phase synchronization is a common method for measuring brain oscillations and functional connectivity. Verifying the feasibility and fidelity of OPM-MEG in measuring phase synchronization will help its widespread application in the study of aforementioned neural mechanisms. The analysis method on source-level time series can weaken the influence of instantaneous field spread effect. In this paper, the OPM-MEG was used for measuring the evoked responses of 20Hz rhythmic and arrhythmic median nerve stimulation, and the inter-trial phase synchronization (ITPS) and inter-reginal phase synchronization (IRPS) of primary somatosensory cortex (SI) and secondary somatosensory cortex (SII) were analysed. The results find that under rhythmic condition, the evoked responses of SI and SII show continuous oscillations and the effect of resetting phase. The values of ITPS and IRPS significantly increase at the stimulation frequency of 20Hz and its harmonic of 40Hz, whereas the arrhythmic stimulation does not exhibit this phenomenon. Moreover, in the initial stage of stimulation, the ITPS and IRPS values are significantly higher at Mu rhythm in the rhythmic condition compared to arrhythmic. In conclusion, the results demonstrate the ability of OPM-MEG in measuring phase pattern and functional connectivity on source-level, and may also prove beneficial for the study on the mechanism of rhythmic stimulation therapy for rehabilitation.

Adjuvant icotinib versus observation in patients with completely resected EGFR-mutated stage IB NSCLC (GASTO1003, CORIN): a randomised, open-label, phase 2 trial.

Background: This phase 2 trial aimed to compare adjuvant icotinib with observation in patients with epidermal growth factor receptor (EGFR) mutation-positive resected stage IB non-small cell lung cancer (NSCLC). Methods: We performed a randomised, open-label, phase 2 trial from May 1, 2015 to December 29, 2020 at Sun Yat-sen University Cancer Center in China. Patients with completely resected, EGFR-mutant, stage IB (the 7th edition of TNM staging) NSCLC without adjuvant chemotherapy were randomised (1:1) to receive adjuvant therapy with icotinib (125 mg, three times daily) for 12 months or to undergo observation until disease progression or intolerable toxicity occurred. The primary endpoint was 3-year disease-free survival (DFS). CORIN (GASTO1003) was registered with Clinicaltrials.gov, with the number NCT02264210. Findings: A total of 128 patients were randomised, with 63 patients in the icotinib group and 65 patients in the observation group. The median duration of follow-up was 39.9 months. The three-year DFS was significantly higher in the icotinib group (96.1%, 95% confidence interval [CI], 91.3-99.9) than in the observation group (84.0%, 95% CI, 75.1-92.9; P = 0.041). The DFS was significantly longer in the icotinib group than in the observation group, with a hazard ratio (HR) of 0.23 (95% CI, 0.07-0.81; P = 0.013). The OS data were immature, with three deaths in the observation arm. In the icotinib group, adverse events (AEs) of any grade were reported in 49 patients (77.8%), and grade 3 or greater AEs occurred in four patients (6.3%). No treatment-related deaths occurred. Interpretation: Our findings suggested that adjuvant icotinib improved the 3-year DFS in patients with completely resected EGFR-mutated stage IB NSCLC with a manageable safety profile. Funding: This study was sponsored by Betta Pharmaceutical Co., Ltd.

A retrospective cohort study of the relationship between waist circumference and the risk of hyperuricemia in the occupational population / 中华健康管理学杂志

Objective:To investigate the relationship between waist circumference and hyperuricemia in occupational population in Changsha city.Methods:Based on a retrospective cohort design, a total of 1 197 employees from 70 organizations who received 4 or more years of continuous physical examinations in Xiangya hospital from January 1, 2014 to December 31, 2018 were included in this study. The physical examination data of the year 2014 were set as baseline data, while the data between January 1, 2015 and December 31, 2018 were used as follow-up data. According to interquartile range of the waist circumference, the subjects was divided into four groups: the first quartile ( Q1),<77 cm for men and <68 cm for women; the second quartile ( Q2), 77 cm ≤ and<82 cm for men, 68 cm ≤ and <73 cm for women; the third quartile ( Q3), 82 cm ≤ and <87 cm for men, 73 cm ≤and <78 cm for women; the fourth quartile ( Q4), ≥87 cm for men, ≥78 cm for women. Among them, Q1 was set as the control group, and Q2, Q3 and Q4 as the exposed groups. Three models were established for the total population, men and women, respectively. The confounding factors were not adjusted in model Ⅰ. The model Ⅱ was adjusted for age, gender and body mass index (the male or female population were not adjusted for sex). Confounders including age, gender, body mass index, hypertension, fasting glucose, blood creatinine, triacylglycerol, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol were adjusted for model Ⅲ. Cox regression analysis was used to compare the hazard ratio ( HR), adjusted hazardratio (a HR) and their 95% CIs for the development of hyperuricemia in the subjects with different waist circumference over the 4 years. Results:Total of 1 197 subjects were followed-up for (2.05±1.18) years and 2 448 person-years. A total of 208 cases of hyperuricemia were identified in the total population during the 4 years (45 women/163 men), with a cumulative incidence of 17.4% (6.4% in women/33.3% in men) and an incidence density of 84.9/1 000 person-years (31.8/1 000 person-years in women, 157.6/1 000 person-years in men). And 626, 609, 629, and 584 person-years were followed-up in the 4 groups, respectively; with 15, 30, 59, and 104 cases of hyperuricemia occurred during 4 years, respectively. The cumulative incidence rates of hyperuricemia in the 4 yearswas 5.5%, 9.2%, 20.8% and 32.8%, respectively; and the incidence densities was 24.0/1 000 person-years, 49.3/1 000 person-years, 93.8/1 000 person-years and 178.1/1 000 person-years, respectively. Compared with that in the Q1 group, the risk of hyperuricemia was increased in the Q4 group, with a HR (95% CI) of 2.70 (1.81 to 4.04), P<0.05. After adjusted for confounding factors in the total population, the a HR (95% CI) of hyperuricemia was 2.12 (1.39 to 3.24), P<0.05. This risk remained when stratified by gender and adjusted for confounding factors. Compared with the Q1 group, the a HR (95% CI) of hyperuricemia in the Q4 group was 1.91 (1.18 to 3.09) for the male population and 2.93 (1.14 to 7.56) for the female population, respectively (both P<0.05). Conclusion:Among the occupational population, the risk of hyperuricemia increases with increase of waist circumference.

Relationship between serum uric acid level and hyperglycemia: a prospective cohort study / 中华健康管理学杂志

Objective:To investigate the correlation between serum uric acid level and hyperglycemia.Methods:A medical examination cohort of the staff of our hospital was constructed. From February 1 st, 2011, to December 31 st, 2011, 3 937 staff members without hyperglycemia were selected, and baseline data were collected through a questionnaire survey, physical examination, measurement of blood lipid and blood glucose, assessment of kidney function, and other laboratory tests. The subjects were followed up during the annual physical examination for 7 years, from January 1 st, 2012, to December 31 st, 2018. They were divided into four groups according to serum uric acid level: uric acid<360 μmol/L, 360≤uric acid<420 μmol/L, 420≤uric acid<480 μmol/L, and uric acid≥ 480 μmol/L. With the occurrence of hyperglycemia as the outcome indicator; uric acid level as the observation index; uric acid<360 μmol/L as the control group; and gender, age, body mass index, smoking, hypertension, dyslipidemia as confounding factors, Cox regression was performed before and after adjusting confounding factors to analyze the relationship between different uric acid levels and the incidence of hyperglycemia in the entire sample, in the male staff, and in the female staff. Results:The 7-year cumulative incidence of hyperglycemia in the four groups were 15.7%, 34.0%, 38.8%, and 43.8%, respectively ( Z=148.94, P<0.01). In the male staff, the 7-year cumulative incidence rates in the four groups were 23.4%, 29.9%, 34.7%, and 35.8%, respectively ( Z=11.17, P<0.01). In the female staff, the 7-year cumulative incidence rates in the four groups were 14.2%, 42.5%, 52.2%, and 65.0%, respectively ( Z=141.84, P<0.01. After adjusting for gender, age, body mass index, smoking, hypertension, and dyslipidemia, the risk of hyperglycemia in the 360≤uric acid<420 μmol/L, 420≤uric acid<480 μmol/L, and uric acid≥ 480 μmol/L groups were 1.73 (1.39-2.15), 1.86 (1.42-2.45), and 1.95 (1.34-2.85) times higher than that in the control group (all P<0.05). Among female staff, the risk of hyperglycemia in the 360≤uric acid<420 μmol/L, 420≤uric acid<480 μmol/L, and uric acid≥ 480 μmol/L groups were 2.18 (1.62-2.94), 3.41 (2.24-5.20), and 3.02 (1.69-5.40) times, respectively, and were also higher than those in the control group (all P<0.01). Conclusion:With the increase of serum uric acid level, the risk of hyperglycemia in medical staff increases, which is mainly manifested in female staff.

Association between admission diagnosis and immunotherapy outcome in patients with autoimmune epilepsy / 中南大学学报(医学版)

To evaluate the impact of admission diagnosis on seizure outcome in patients with autoimmune epilepsy (AE).
 Methods: We conducted a retrospective study on 40 AE patients at Department of Neurology, Xiangya Hospital, Central South University from Jan. 1st, 2017 to Nov. 21st, 2018. According to their admission diagnosis, these patients were further assigned into 2 groups: An AE diagnosed group and an AE undiagnosed group. Demographic data, clinical characteristics, cerebrospinal fluid (CSF), electroencephalogram, and brain imaging were compared between the 2 groups. Favorable seizure outcome was defined as >50% reduction of seizure frequency at the first follow-up. The impact of admission diagnosis on seizure outcome of AE patients was analyzed.
 Results: The ages of 40 AE patients were (33.23±16.41) years, comprising 19 males and 21 females. No significant difference was found between the AE diagnosed group and the AE undiagnosed group in gender, age, central nervous system-specific Ab profile, CSF, and brain imaging. Duration of symptom onset to Ab detection was significantly longer in the AE undiagnosed group than that in the AE diagnosed group (P<0.01). Duration of symptom onset to immunotherapy was also significantly longer in the AE undiagnosed group than that in the AE diagnosed group (P<0.001). There were more patients with favorable seizure outcome in the AE diagnosed group than the AE undiagnosed group (P<0.05).
 Conclusion: Admission diagnosis for patients with AE is associated with seizure outcome after immunotherapy. For adult-onset epilepsy or epilepsy with unknown etiology, the diagnosis of AE should be considered. Early diagnosis of AE and prompt initiation of immunosuppressive treatment are crucial to increase the likelihood of achieving favorable seizure outcome.

The Electrochemical Performance of Silicon Nanoparticles in Concentrated Electrolyte.

Silicon is a promising material for anodes in energy-storage devices. However, excessive growth of a solid-electrolyte interphase (SEI) caused by the severe volume change during the (de)lithiation processes leads to dramatic capacity fading. Here, we report a super-concentrated electrolyte composed of lithium bis(fluorosulfonyl)imide (LiFSI) and propylene carbonate (PC) with a molar ratio of 1:2 to improve the cycling performance of silicon nanoparticles (SiNPs). The SiNP electrode shows a remarkably improved cycling performance with an initial delithiation capacity of approximately 3000 mAh g-1 and a capacity of approximately 2000 mAh g-1 after 100 cycles, exhibiting about 6.8 times higher capacity than the cells with dilute electrolyte LiFSI-(PC)8 . Raman spectra reveal that most of the PC solvent and FSI anions are complexed by Li+ to form a specific solution structure like a fluid polymeric network. The reduction of FSI anions starts to play an important role owing to the increased concentration of contact ion pairs (CIPs) or aggregates (AGGs), which contribute to the formation of a more mechanically robust and chemically stable complex SEI layer. The complex SEI layer can effectively suppress the morphology evolution of silicon particles and self-limit the excessive growth, which mitigates the crack propagation of the silicon electrode and the deterioration of the kinetics. This study will provide a new direction for screening cycling-stable electrolytes for silicon-based electrodes.

Simultaneous determination of 19 bioactive components of different types in Xintiantai I by UHPLC-ESI-MS/MS / 中草药

Objective To establish an effective UHPLC-ESI-MS/MS method for the simultaneous determination of 19 active components (ginsenoside Rg1, Rb1, Rd, berberine, epiberberine, jatrorrhizine, palmatine, columbamine, coptisine, evodiamine, rutaecarpine, dehydroevodiamine, limonin, hyperin, curcumin, demethoxycurcumin, bisdemethoxycurcumin, echinacoside, and verbascoside) of different types in Xintiantai I (XI), and provide a comprehensive and efficient quality control method for traditional Chinese medicine (TCM). Methods The analysis was performed on an Agilent 1290 system with a Agilent Zorbax Eclipse Plus C18 column (150 mm × 4.6 mm, 3.5 μm) at a flow rate of 0.4 mL/min using acetonitrile and 0.1% formic acid aqueous solution as mobile phase. Mass spectrometric detection was performed on multiple reaction monitoring (MRM) in positive and negative ionization mode. The contents of 19 active components in XI were determined by monitoring the specific ions of each component. Results The 19 active components were accurately determined in 15 min and had the good linearity (r2 > 0.999) within the linear ranges. The average recovery rates of ginsenoside Rg1, Rb1, Rd, berberine, epiberberine, jatrorrhizine, palmatine, columbamine, coptisine, evodiamine, rutaecarpine, dehydroevodiamine, limonin, hyperin, curcumin, demethoxycurcumin, bisdemethoxycurcumin, echinacoside, and verbascoside were 94.80%, 96.78%, 95.59%, 96.88%, 97.74%, 100.08%, 96.27%, 100.25%, 98.32%, 97.16%, 95.60%, 95.28%, 96.81%, 95.22%, 96.85%, 95.31%, 93.86%, 94.79%, and 95.20%, respectively; The contents of three batches XI of the 19 components were in the ranges of 2.28-2.49, 0.82-0.90, 1.22-1.32, 14.44-15.50, 3.71-3.99, 3.26-3.49, 3.09-3.33, 4.39-4.72, 4.56-4.92, 0.52-0.57, 0.30-0.33, 4.46-4.76, 3.02-3.24, 2.59-2.76, 6.03-6.38, 1.47-1.58, 1.90-2.08, 3.40-3.88, and 1.53-1.74 mg/g, respectively. Conclusion The developed UHPLC-ESI-MS/MS-MRM method is fast, sensitive, and reproducible for TCM quality control. It can be used for the quality control of XI, which also provides reference for TCM quality research.

Ethyl 6-(4-fluoro-phen-yl)-4-hy-droxy-2-oxo-4-trifluoro-meth-yl-1,3-diazinane-5-carboxyl-ate monohydrate.

The asymmetric unit of the title compound, C(14)H(14)F(4)N(2)O(4)·H(2)O, contains two crystallographically independent organic mol-ecules and two water mol-ecules. The two 1,3-diazinane rings adopt a half-chair conformation and the dihedral angles between their mean planes and those of the benzene rings are 75.65 (4)° and 49.41 (3)° in the two mol-ecules. The crystal structure is stabilized by inter-molecular O-H⋯O and N-H⋯O hydrogen bonds.

(E)-1-(4-Methyl-phen-yl)-3-[(1-phenyl-ethyl-idene)amino]-thio-urea.

In the title compound, C(16)H(17)N(3)S, the amino-thio-urea unit is nearly planar (r.m.s. deviation = 0.0425 Å), and is twisted with respect to the tolyl and phenyl rings by 57.84 (7) and 15.88 (14)°, respectively; the tolyl and phenyl rings are twisted by 65.64 (11)° to each other. Inter-molecular N-H⋯S and weak C-H⋯S hydrogen bonds are present in the crystal structure.

Ethyl 6-[4-(dimethyl-amino)phen-yl]-4-hydr-oxy-2-oxo-4-(trifluoro-methyl)-hexa-hydro-pyrimidine-5-carboxyl-ate.

The title compound, C(16)H(20)F(3)N(3)O(4), was prepared by reaction of 4-(dimethyl-amino)benzaldehyde, ethyl 4,4,4-trifluoro-3-oxo-butanoate and urea. In the title mol-ecule, the pyrimidine ring adopts a half-chair conformation and there is an intra-molecular hydrogen bond (O-H⋯O). The crystal structure is stabilized by two types inter-molecular hydrogen bonds (N-H⋯O and N-H⋯N).

rac-Ethyl 4-hy-droxy-6-(2-hy-droxy-phen-yl)-2-oxo-4-(trifluoro-methyl)per-hydro-pyrimidine-5-carboxyl-ate.

In the title compound, C(14)H(15)F(3)N(2)O(5), prepared by reaction of 2-hy-droxy-benzaldehyde, ethyl 4,4,4-trifluoro-3-oxobutano-ate and urea, the tetra-pyrimidine ring adopts a half-chair conformation. The crystal structure is stabilized by five inter-molecular hydrogen bonds, three O-H⋯O and two N-H⋯O, giving cyclic dimers (through three hydrogen bonds) which are further extended into a two-dimensional network.

Epidemiological characteristics and related influencing factors on Japanese encephalitis in Hainan province / 中华流行病学杂志

Objective To understand the epidemiological characteristics and the influencing factors of Japanese encephalitis (Abbreviated JE) at different periods in Hainan province, and to provide evidence for JE control strategies. Methods Data on JE had been collected since 1952 and related incidence, mortality, fatality rates of JE and the rate of JE vaccination were calculated for four periods: the natural epidemic period (1952-1978), period for inactivated JE vaccine promotion (1979-1986), national immunization program period (1987-1990) and the promotion of live-attenuated JE vaccine period (1991-2007). History on the immunity of JE and data from the monitoring programs on cases and insect vectors were also analyzed. Results The average annual incidence in natural epidemic period was 7.12/100 000, which was also the highest among the 4 periods with the mortality rate as 0.89/100 000. The average annual incidence was 0.11/100 000 which was the lowest while the mortality rate was 0.01/100 000 in the period when live-attenuated JE vaccination was under promotion. Since the establishment of JE surveillance system was set up in Hainan province in 2002, the specimen collection rate of JE reported suspected cases had reached 92.8%, while the incidence of confirmed JE cases fluctuated between 0.05/100 000 and 0.2/100 000. Of the confirmed cases, 86% did not have clear history of JE vaccination. The annual average protective rate reached 95.69% in 2004-2007. The density of insect vectors was high in Hainan province. The infection rate of JE in host animal swine, kept rising from April to October, and in some surveillance points, the rate reached 99.3%. The density of insect vector plotted in trans-curve with the infection of host animals. Conclusion The application of live-attenuated JE vaccine was found to have had significant protective effect on JE in susceptible children. It helped reducing the incidence and controlling the epidemics of JE. It appears feasible to include live-attenuated JE vaccine into the expanded immunization program.

Loss of heterozygosity analysis of microsatellites on multiple chromosome regions in dysplasia and squamous cell carcinoma of esophagus / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate the molecular alterations related to the carcinogenesis of esophageal squamous cell carcinoma (SCC), and also to find some molecular markers for the early detection of this cancer.</p><p><b>METHODS</b>The resected tumor specimens and dysplasia tissues from 34 patients with esophageal cancer as well as their matched blood DNAs were analyzed for loss of heterozygosity (LOH) at 16 microsatellites by using PCR and fluorescence-based DNA sequencing technology. Mild and moderate dysplasia was classified as light-grade dysplasia (LGD), and severe dysplasia as high-grade dysplasia (HGD). The frequencies of LOH at 16 microsatellites were compared between tissue specimens with different histological diagnosis.</p><p><b>RESULTS</b>The total frequency of LOH for 16 microsatellites increased significantly in more severe lesions. There was significant difference in the frequency of LOH among LGD and HGD as well as SCC. A total of eight loci (D3S1597, D3S2452, D3S1285, D4S174, D5S2501, D9S125, D13S153 and D17S786) presented LOH in LGD samples. A reversion from LOH to retain of heterozygosity was observed at loci D3S2452, D4S174, D9S125 and D17S261 respectively when compared HGD with SCC samples obtained from 4 patients.</p><p><b>CONCLUSIONS</b>An accumulation of molecular alterations would be needed during the carcinogenesis of esophageal cancer. LOH analysis at some specific loci would be helpful for the early detection of esophageal cancer. The genetic heterogeneity possibly exists in the tumorigenesis of esophageal cancer.</p>

Study on the probability of hepatitis B virus infection at public service places / 中华流行病学杂志

Objective To provide data for the control and prevention of hepatitis B and HBV surface antigen(HBsAg)status among the appliances and practitioners working in the public service places.Methods 63 beauty parlors,barber shops and bathing centers selected under stratified randomization sampling method and 682 workers were investigated through questionnaire.HBsAg from the appliances of the public service places and employee was detected by RIA.Results Two main sanitizing modes that including alcohol cleaning(34.60%)and ultraviolet light disinfection(30.79%)were used.The rates of testing on HBsAg among the appliances were 2.13% at the public service places,and were 0.63%,2.67% and 3.70% in large-.medium-and small-sized appliances respectively.The rate of testing on HBsAg on large-,medium-and small-sized appliances were statistically different(χ2=6.68,P＜0.05).The positive rates of HBsAg on the appliances of beauty parlors,barbering shops and footbath inns were 2.97%,0.61% and 3.42% respectively.People working in different service sites had different rates of HBsAg:those who worked at the‘acne needle'and the forceps were 5.13% and 4.17%.The positive rate of HBsAg among the workers in the public service places was 7.13%.The rates of HBsAg among the workers in large-,medium-and small-sized public service places were 7.34%,8.33% and 2.94% respectively.The rates of HBsAg among the workers in beauty parlors,barbering shops,footbath inns and bathing centers were 9.01%,6.37%,4.35% and 7.29% respectively.HBsAg positive rates were different among the workers working at different service sites:13.33% at tattoo business.12.68% in pedicures workers and 8.03% in massagists.Conclusion It is important to improve the sanitizing management of the appliances used in the public service places and to improve the knowledge,attitude,as well as practice of vaccination on hepatitis B among those populations.

Study on human papillomavirus infection and loss of heterozygosity of microsatellite in esophageal cancer / 中华流行病学杂志

<p><b>OBJECTIVE</b>To investigate the relationship between the loss of heterozygosity(LOH) at 14 microsatellites in esophageal cancer and human papillomavirus (HPV) infection.</p><p><b>METHODS</b>HPV-16,18 DNA was examined in 112 tumor specimens using fluorescence quantitative PCR. 112 tumor specimens and their matched blood DNAs were analyzed for LOH at 14 microsatellites by PCR and fluorescence-based DNA sequencing technology. The frequencies of LOH at 14 microsatellites were compared between HPV positive and negative specimens.</p><p><b>RESULTS</b>High frequency of LOH was observed among chromosome arms 3p, 9p, 13q, 17p and 17q. The frequency of LOH was significantly higher at loci D13S260 and D6S497 in HPV positive specimens, comparing with HPV negative ones.</p><p><b>CONCLUSION</b>The findings regarding loci with allele loss indicated that widespread chromosome instability might have existed in esophageal cancer. HPV positive specimens with higher frequency of LOH than negative ones at locus D13S260 and D6S497 suggesting that the target of HPV in esophageal cancer might serve as candidate genes at these two loci. In addition,this result also indicated that HPV might be a high-risk factor for esophageal cancer in Sichuan area with a high incidence of this cancer.</p>

Construction of FANCA mutant protein from Fanconi anemia patient and analysis of its function / 中华血液学杂志

<p><b>OBJECTIVE</b>To study FANCA protein expression in Fanconi anemia patient's (FA) cells and explore its function.</p><p><b>METHODS</b>FANCA protein expression was analyzed in 3 lymphoblast cell lines derived from 3 cases of type A FA (FA-A) patients using Western blot. Nucleus and cytoplasm localization of FANCA protein was analyzed in one case of FA-A which contained a truncated FANCA (exon 5 deletion). The FANCA mutant was constructed from the same patient and its interaction with FANCG was evaluated by mammalian two-hybrid (M2H) assay.</p><p><b>RESULTS</b>FANCA protein was not detected in the 3 FA-A patients by rabbit anti-human MoAb, but a truncated FANCA protein was detected in 1 of them by mouse anti-human MoAb. The truncated FANCA could not transport from cytoplasm into nucleus. The disease-associated FANCA mutant was defective in binding to FANCG in M2H system.</p><p><b>CONCLUSIONS</b>FANCA proteins are defective in the 3 FA-A patients. Disfunction of disease-associated FANCA mutant proved to be the pathogenic mutations in FANCA gene. Exon 5 of FANCA gene was involved in the interaction between FANCA and FANCG.</p>

Utility of acetyldithio-CoA in detecting the influence of active site residues on substrate enolization by 3-hydroxyl-3-methylglutaryl-CoA synthase.

Hydroxymethylglutaryl-CoA synthase-catalyzed condensation of acetyl-CoA with acetoacetyl-CoA requires enolization/carbanion formation from the acetyl C-2 methyl group prior to formation of a new carbon-carbon bond. Acetyldithio-CoA, a readily enolizable analog of acetyl-CoA, was an effective competitive inhibitor of avian hydroxymethylglutaryl-CoA synthase (Ki = 28 microm). In the absence of cosubstrate, enzyme catalyzed the enolization/proton exchange from the C-2 methyl group of acetyldithio-CoA. Mutant enzymes that exhibited impaired formation of the covalent acetyl-S-enzyme reaction intermediate exhibited diminished (D159A and D203A) or undetectable (C129S) rates of enolization of acetyldithio-CoA. The results suggest that covalent thioacetylation of protein, which has not been detected previously for other enzymes that enolize this analog, occurs with hydroxymethylglutaryl-CoA synthase. Enzyme catalyzed the transfer of the thioacetyl group of this analog to 3'-dephospho-CoA suggesting the intermediacy of a covalent thioacetyl-S-enzyme species, which appears to be important for proton abstraction from C-2 of the thioacetyl group. Avian enzyme glutamate 95 is crucial to substrate condensation to form a new carboncarbon bond. Mutations of this invariant residue (avian enzyme E95A and E95Q; Staphylococcus aureus enzyme E79Q) correlated with diminished ability to catalyze enolization of acetyldithio-CoA. Enolization by E95Q was not stimulated in the presence of acetoacetyl-CoA. These observations suggest either a direct (proton abstraction) or indirect (solvent polarization) role for this active site glutamate.

[Study on gene knock-out in Mycobacterium BCG].

OBJECTIVE: To establish the methodology of plasmid for gene knock-out in Mycobacterium BCG. METHODS: We designed two pairs of primers for amplification of MDP1 gene and inserted two fragments into pKO plasmid, and then the recombinant plasmid for MDP1 gene knock-out was obtained, and named pKO-MDP1. Gene exchange took place within the genome of BCG after pKO-MDP1 plasmid was transformed into Mycobacterium BCG. The strain of Mycobacterium BCG with MDP1 gene knock-out was selected, and the curve of growth rate was studied. RESULTS: The target strain was that of the positive strain by two step PCR and one step sucrose counter selection, without growth in culture media with hygomycin. The value of A(600) per 12 hours was detected for sixteen days. A "S" shaped growth curve was detected. However, there was no significant difference in the growth rates between the wild type Mycobacterium BCG strain and the gene knock-out Mycobacterium BCG strain. CONCLUSIONS: The plasmid of pKO was a useful tool for gene knock-out in Mycobacteria. MDP1 maybe one of the factors influencing the growth rate, but it was not the only one.