Methanotrophs use methane as the sole carbon and energy source, which cause slow growth, low cell density and hinder its industrial applications. One promising solution is to heterologously express methane monooxygenase (MMO) in other host cells that can be easily cultivated at high cell density. We systematically exploited the possibility of functional expression of pMMO by choosing different promoters and different host cells. The results showed that the recombinants could oxidize methane to methanol. In particular, ethanol could also be detected in the oxidized products, but the enzyme activity was instable, implying that some changes of pMMO expressed in the host cells might have occurred. In addition, SDS-PAGE analysis showed that many recombinants could express the subunits of pMMO, but the enzyme activity could not be detected. In conclusion, correct fold of pMMO in the host cells is important for its functional expression.
Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gene Expression Regulation, Enzymologic ; Genetic Vectors ; genetics ; Methane ; metabolism ; Methanococcaceae ; enzymology ; Methanol ; metabolism ; Oxygenases ; biosynthesis ; genetics ; Promoter Regions, Genetic ; Protein Folding ; Recombinant Proteins ; biosynthesis ; genetics

Amphetamine-type stimulants (ATS) is the most widespread narcotics in the 21st century. The methamphetamine's intoxication mechanism, psychological dependence, drug resistance and therapeutic drug development are the hot spots in current research. Establishment of animal model with methamphetamine poisoning is the basic for the relative studies, the normalization and standardization of the animal model settles the foundation for methamphetamine's further research. This article reviews the animal model of methamphetamine poisoning in China and abroad, the brief history of the acute, subacute and chronic animal model of methamphetamine poisoning, as well as the principles and methods of the animal model establishment and its evaluation criteria. The necessity, significance and its scientific expansion of performing experimental research on the methamphetamine poisoning animal model are also discussed.
Amphetamine-Related Disorders/psychology* ; Animals ; Behavior, Animal/drug effects* ; Central Nervous System Stimulants/poisoning* ; Cerebral Cortex/drug effects* ; Disease Models, Animal ; Forensic Toxicology ; Humans ; Injections, Intraperitoneal ; Methamphetamine/poisoning* ; Stereotyped Behavior/drug effects*

Objective:To clarify the effects of bortezomib combined with or without siramesine on the proliferation of multiple myeloma cell lines, the expression changes of transcription factor EBC (TFEB) nuclear translocation and the level of autophagy, and to provide basis for further exploring the regulation mechanism of transcription factor TFEB on autophagy.Methods:The multiple myeloma cell lines RPMI8226 and U266 were cultured in vitro, and the multiple myeloma cells were treated with a certain concentration of bortezomib and siramesine. The changes of cell proliferation inhibition were detected by CCK-8 method. Real time PCR and Western blot were used to detect the relative expression of TFEB, autophagy-related factor LC3B, Beclin1, p62, LAMP1 mRNA and protein.Results:As the concentration of bortezomib increased and the duration of action increased, the proliferation inhibition rates of the two cell lines gradually increased ( P<0.05) . The combination of the two drugs has a synergistic inhibitory effect on the proliferation of the above-mentioned multiple myeloma cell lines ( P<0.05) . In the blank control group, single drug group, and combination drug group, the relative expression of TFEB mRNA and protein in the cytoplasm decreased sequentially ( P<0.05) , and the relative expression of TFEB mRNA and protein in the nucleus increased sequentially ( P<0.05) . The relative expression of autophagy-related factors LC3B, Beclin1, LAMP1 mRNA and protein increased sequentially, and the relative expression of p62 mRNA and protein decreased sequentially ( P<0.05) . Conclusion:Bortezomib and siramesine can synergistically inhibit the growth of multiple myeloma cells, which is related to the increased autophagy expression in multiple myeloma cell lines and the expression of TFEB with nuclear translocation is also enhanced.

BACKGROUNDEpidermic studies have suggested a pathophysiological link between obstructive sleep apnea hypopnea syndrome (OSAHS) and atherosclerosis (AS); for which carotid intima-media thickness (IMT) has been considered as an early marker. The pathogenesis by which OSAHS can induce AS has not been elucidated. This study was conducted to investigate the association among plasma interleukin-18 (IL-18) levels, carotid IMT and the severity of OSAHS.

METHODSBased on the apnea hypopnea index (AHI) during sleep monitored by polysomnography, 52 male patients with OSAHS were recruited as the OSAHS group which was further divided into mild OSAHS (n = 16), moderate OSAHS (n = 18), and severe OSAHS (n = 18) subgroups. Eighteen healthy subjects were selected as the control group. Of all OSAHS patients, 20 with moderate-to-severe OSAHS underwent continuous positive airway pressure (CPAP) treatment for 90 days. HDL5000 color Doppler ultrasonography was used to measure carotid IMT. Plasma IL-18 levels were measured by ELISA.

RESULTSCompared with the plasma IL-18 levels in the control group ((250.27 +/- 76.48) pg/ml), there was a significant increase in the mild OSAHS subgroup ((352.08 +/- 76.32) pg/ml), the moderate subgroup ((600.17 +/- 83.91) pg/ml), and the severe OSAHS subgroup ((9797.64 +/- 109.83) pg/ml) (all P < 0.01). Moreover, there was a significant difference in plasma IL-18 levels among the three OSAHS subgroups (P < 0.01). Carotid IMT was significantly greater in the severe OSAHS subgroup than in the mild OSAHS subgroup (P < 0.01). Before CPAP treatment, plasma IL-18 levels were positively correlated with carotid IMT (r = 0.486, P < 0.001) and with AHI (r = 0.865, P < 0.001). On day 90 of CPAP treatment, plasma IL-18 levels were significantly declined but carotid IMT was not changed significantly.

CONCLUSIONSIn untreated OSAHS patients carotid IMT and plasma IL-18 were positively correlated and were significantly higher than in normal controls; the elevation of plasma IL-18 levels was correlated with the severity of OSAHS. Inflammatory response associated with OSAHS may be related to the development of AS. By improving AHI, miniSaO(2), and reducing plasma IL-18 levels, CPAP treatment may slow down or prevent the development of AS in OSAHS patients.

Adult ; Carotid Arteries ; pathology ; Electrocardiography ; Electroencephalography ; Enzyme-Linked Immunosorbent Assay ; Humans ; Interleukin-18 ; blood ; Male ; Middle Aged ; Sleep Apnea, Obstructive ; blood ; diagnosis ; pathology ; therapy ; Tunica Intima ; pathology

OBJECTIVETo summarize experience of using screws and cement to rebuild tibial bone defect in primary total knee arthroplasty (TKA) and to discuss the relationship between the number of required screws and the severity of tibial bone defects.

METHODSFrom July 2009 to May 2015, 34 patients (40 knees) with varus knees underwent TKA, and the screw and cement technique was used to rebuild medial tibia plateau during operation. There were 8 males (8 knees) and 26 females (32 knees), and the average age was (65.00 +/- 7.25) years old (ranged,55 to 82 years old). One to 6 screws were used in each case. Extension stems were used in 2 cases (4 and 5 screws was used respectively). The area percentages of the bone defects measured as defect area/tibia plateau area, depth of each defect, the number of screws needed in each case, were all used to determine the relationship between the number of screws and the area percentage in certain depth of bone defect by statistic methods, as well as the relationship between screw number and defect depth.

RESULTSAll the patients were followed up and the average duration was 24 months (ranged, 1 to 72 months). The average preoperative HSS score was 43.33 +/- 6.11 (ranged, 32 to 51 scores). Whereas the average postoperative HSS score was 92.15 +/- 4.64 (ranged,83 to 96 scores). The preoperative individual scores including pain, function, activity, nuscle strength, flexion deformity and stability were all improved compared with preoperation,and the differences were statistically significant. All the patients received normal alignment postoperatively, femoraltibial angle was improved from (167.00 +/- 6.39) degrees preoperatively to (175.00 +/- 2.69) degrees postoperatively, the tibial angle was improved from (78.09 +/- 4.51) degrees preoperatively to (88.75 +/- 1.24) degrees postoperatively. Both area percentage and depth of bone defect in a fitting Ologistic model had a significant statistical relationship with the screw number, and a rectangular coordinate system could be formed according to the relationship.

CONCLUSIONScrews and cement technique is a simple, safe and convenient method to rebuild tibial bone defects in primary TKA and its short-term and midterm effect are both reliable. During opera- tion, according to the rectangular coordinate system, the screw number needed in the operation can be inferred form th area and depth of tibia defect, which could have a guiding function in surgery.

Aged ; Aged, 80 and over ; Arthroplasty, Replacement, Knee ; instrumentation ; methods ; Bone Screws ; utilization ; Female ; Fracture Fixation, Internal ; Humans ; Knee Injuries ; surgery ; Knee Joint ; surgery ; Male ; Middle Aged ; Tibia ; surgery

Acetylcholine ; metabolism ; Animals ; Arcuate Nucleus of Hypothalamus ; metabolism ; Dopamine ; metabolism ; Epinephrine ; metabolism ; Female ; Neurotransmitter Agents ; metabolism ; Norepinephrine ; metabolism ; Physical Conditioning, Animal ; Rats ; Rats, Sprague-Dawley ; Synaptic Transmission

Objective: To discuss the application principle in tuina manipulation for lumbar intervertebral disc herniation (LIDH) in Chinese literatures published in recent 30 years. Methods: The three major Chinese databases, Wanfang Academic Journal Full-text Database (Wanfang), Chongqing VIP Database (CQVIP) and China National Knowledge Infrastructure (CNKI), were searched to collect the studies of tuina manipulations in treatment of LIDH published in recent 30 years. Clustering analysis was applied to analyze the top 20 tuina manipulations for LIDH. Results: The top 20 most frequently used manipulations for LIDH were Gun-rolling, Rou-kneading, Dian-digital pressing, oblique Ban-pulling, An-pressing, Tanbo-plucking, Bashen-pulling and extending, horizontal Tui-pushing, Na-grasping, Anrou-pressing and kneading, Dou-shaking, Yao-rocking, Ca-scrubbing, Pai-patting, post-extension Ban-pulling, Mo-rubbing, Zhen-vibrating, Nie-pinching, fist-back Ji-tapping, and dorsal Shen-extending methods. The involved manipulations can be divided into two categories by the treated body areas. One category is applied to the soft tissues, including Gun-rolling, Rou-kneading, Dian-digital pressing, An-pressing, Tanbo-plucking, horizontal Tui-pushing, Na-grasping, Anrou-pressing and kneading, Ca-scrubbing, Pai-patting, Mo-rubbing, Zhen-vibrating, Nie-pinching, and fist-back Ji-tapping methods. The other category is applied to bones and joints, including oblique Ban-pulling, Bashen-pulling and extending, Dou-shaking, Yao-rocking, post-extension Ban-pulling, and dorsal Shen-extending methods. Conclusion: Based on the treated body area, the tuina manipulations applied to treat LIDH are predominated by the ones performed on soft tissues, assisted by those on bones and joints. From the way of force exertion, the involved manipulations are majorly the swinging methods, followed by squeezing and pressing ones. The manipulations applied to bones and joints are predominated by the Ban-pulling ones, followed by the Bashen-pulling and extending ones.

Objective To investigate the effect of telbivudine in HBeAg positive chronic hepatitis B patients.Methods The clinical data of 120 patients with HBeAg positive chronic hepatitis B treated with lamivudine and telbivudine in our hospital were collected.The HBV DNA level and HBeAg seroconversion rate of lamivudine group and telbivudine group at the 4th, 8th, 12th, 24th and 48th week were compared as well as differ-ences of adverse reactions during treatment.Results The result showed that there were no statistical differences between the groups at the 4th, 8th and 12 th week ( P>0.05 ).Compared with the lamivudine group , the HBV-DNA level in telbivudine group was significantly lower while E an-tigen seroconversion rate was significantly higher at the 24 th and 48 th week , the difference between groups were statistically significant ( P <0.05 ).The incidence of adverse reactions between the two groups had no statistical difference ( P>0.05 ).In telbivudine group , the HBeAg seroconversion rate of patients under the age of 40 was 49% at the 48 th week of treatment while it was 9.5%in patients over 40 , the difference between two groups was statistically significant ( P<0.05 ).Conclusion Petients under the age of 40 with HBeAg positive chronic hepatitis B treated with telbivudine has a higher HBeAg seroconversion rate and mild adverse reactions.

Objective To establish a HPLC method for the determination of paraquat in human plasma which can be used in clinical practice.Methods The plasma samples were processed with WCX solid phase extraction column and were determinated by RP-HPLC.The HPLC determination was conducted on a Diamonsil C18 column (250 mm × 4.6 mm,5 μm)with the mobile phase consisted of 0.1 mol · L-1 phosphate buffer (containing 80 mmol · L-1 sodium heptanesulfonate,pH 3.0)-acetonitrile (80:20),and the flow rate was 1.0 mL · min-1.The detection wavelength was set at 258 nm,and the column temperature was 25 ℃.Results The plasma concentration of paraquat had good linearity over the range of 20-5000 ng· mL-1 (r =0.999 9).The limit of detection for the paraquat was 20 ng· mL-1.The relative recovery rates of paraquat were 90.06％-104.50％.The absolute recovery rates were 97.66％-99.11％,respectively.The intra-day and inter-day precision (RSD) were all less than 4.00％.The paraquat plasma sample was all stable at the conditions of maintaining at 4 ℃ for 8 h,after freeze thaw cycle and keeping at-40 ℃ for 21 days.The method was applied to the clinical determination of paraquat and the calculation of severityindex of paraquatpoison (SIPP),which was helpful in the clinical treatment of patients with paraquat poison and to predict the prognosis.Conclusion A rapid,sensitive and accurate RP-HPLC method was established,which can be used in the determination of paraquat in human plasma and predict the effect of clinical treatment of patients with paraquat poison.