Controlled expression of lysis gene E by a mutant of the promoter pL of the thermo-inducible λcI857-pL system.

AIMS: To identify a lambda promoter pL mutant that could extend the thermal stability of the thermo-inducible λcI857-pR/pL system and to evaluate the effects of the modified system for the controlled expression of lysis gene E during the production of bacterial ghosts (BGs). METHODS AND RESULTS: The promoter pL mutant was identified by random mutagenesis and site-directed mutagenesis. The results showed that a T â†’ 35C mutation in the pL promoter was responsible for the phenotype alteration. Under the same induction conditions, the lysis rates of the modified lytic system on Escherichia coli and Salmonella enteritidis were significantly lower than that of the control, while the lysis rates of Escherichia coli with the thermo-inducible lytic system were significantly higher than that of S. enteritidis with the corresponding plasmid (P < 0·05). CONCLUSIONS: Increasing the heat stability of the thermo-inducible lytic systems decreased lysis efficiency during the production of BGs. There exist differences in the lysis efficiency of thermo-inducible lytic systems between different bacterial strains. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings enrich current knowledge about modifications to thermo-inducible systems and provide a reference for the application of these modified systems for the production of BGs and controlled gene expression in bacteria.

[Effects of tranexamic acid on the blood conservation and the long-term prognosis in pediatric patients undergoing repair for tetralogy of fallot].

Objective: To evaluate the perioperative tranexamic acid (TXA) on blood conservation in pediatric patients undergoing complete repair for tetralogy of fallot (TOF) and its impact on short-term or long-term adverse event and mortality. Methods: The study was a retrospective cohort study. From January 2009 to December 2010, 386 consecutive patients aged from 31 days to 8 years old, ASA physical status Ⅱ or Ⅲ, receiving primary complete repair for TOF in Fuwai Hospital were enrolled in the study. They were divided into two groups: the control group (n=161) and the TXA group (n=225), according to whether TXA was used during the operation. Patients and their families were followed up by telephone in the 8th-year after surgery. The amount of perioperative blood loss, allogeneic transfusion, short-term or long-term adverse event and mortality were recorded and analyzed. Results: The patients in the TXA group were associated with significant decreased 12 h and total postoperative blood loss compared with the control group [(7.8±0.3) ml/kg vs (8.8±0.3) ml/kg, t=2.412, P<0.05; and (14.0±0.6) ml/kg vs (17.0±0.7) ml/kg, t=3.141, P<0.05]. There were no significant differences in both the volume and incidence of red blood cell, plasma, and platelet transfusion, postoperatively (P>0.05). There were no significant differences in the incidence of reoperation for bleeding and prolonged mechanical ventilation, ICU stay, postoperative hospital length of stay, the short-term and long-term incidence of seizure, stroke, renal failure, deep venous thrombosis, pulmonary embolism and death between the two groups(P>0.05). Conclusion: TXA can decrease postoperative blood loss, but has little impact on the allogeneic blood transfusion, as well as the short-term or long-term adverse event and mortality in pediatric patients undergoing complete repair for TOF.

[A 3.0 T MRI study on the alterations of the volume and morphology of fifteen subcortical nucleus in patients with early post-stroke depression].

Objective: To investigate the alterations of the volumes and 3D shapes of fifteen subcortical nucleus in patients with post-stroke depression (PSD) and to explore the pathogenesis regularity and mechanism of early PSD. Methods: From 2015 to 2017, a total of 28 patients with PSD and 18 stroke patients without depression (PSND), 13 patients with depression (De) and 11 cases of healthy volunteers (NC) were enrolled to perform 3.0 T high resolution MRI.Computer automatic segmentation and vertex analysis were used to segment and measure the volume of bilateral nucleus accumbens, caudate nucleus, putamen, globus pallidus, thalamus, hippocampus, mygdale and brainstem. Results: The volume of bilateral nucleus accumbens and bilateral thalamus, left pallidum were different among groups with statistical difference (P<0.05). The nucleus volume of the PSD group was (415±128) mm(3) (L-Nac)/(303±90) mm(3) (R-Nac), (7 590±867) mm(3) (L-Th)/(7 459±905) mm(3) (R-Th), (1 675±328) mm(3) (L-Pa), which was smaller than that of PSND group (433±100) mm(3) /(307±88) mm(3), (7 999±961) mm(3) /(7 753± 955) mm(3), (1 790±286) mm(3) and other groups.The nuclei with significantly statistical differences between inter-group were found in following: between PSD group and NC group, right accumbens and bilateral thalamus (P<0.01); between PSD group and De group, right accumbens and right thalamus (P<0.001), left accumbens, left pallidum and left thalamus (P<0.01); between PSND group and NC group, right accumbens (P<0.05); between PSND group and De group, right accumbens (P<0.001), left accumbens and right thalamus (P<0.05). Significant differences in morphology changes of nuclei (P<0.05) by F test mainly located on the top and tail of right accumbens, the anterior and middle body of right caudate nucleus, the most part of bilateral thalamus, the ventromedial body of bilateral hippocampus, the anterior and body of left caudate nucleus, especially in left thalamus. Conclusion: PSD has abnormal volume and morphological structure of subcortical nuclei, which supports the role of subcortical structures changes in the pathophysiology and pathogenesis of early PSD.

Accumulation of antibiotics and heavy metals in meat duck deep litter and their role in persistence of antibiotic-resistant Escherichia coli in different flocks on one duck farm.

Meat duck deep litter is considered to be an ideal environment for the evolution of bacterial antibiotic resistance if it is under poor management. The aim of this study was to characterize the accumulation of antibiotics and heavy metals in the deep litter and their role in the persistence of antibiotic resistance of Escherichia coli, and evaluate the service life of the deep litter. Samples were collected from initial, middle, and final stages of deep litter within 3 barns (zero, 4, and 8 rounds of meat duck fattening, d 34) and 9 flocks, with known consumption of antibiotics in the controlled trail. The feed and litter levels of consumed antibiotics and heavy metals were measured. E. coli (n = 147) was isolated and typed by Eric-PCR and the phylogenetic grouping technique, while minimal inhibitory concentrations of antibiotics and heavy metals were measured. This study confirmed the continuous accumulation of doxycycline and many heavy metals in the deep litter. The population of resistant certain bacteria to doxycycline (16 mg/L, 100 mg/L) or ofloxacin (8 g/mL, 50 g/mL) increased in the used deep litter (rounds 4 and 8). E. coli isolated from the 3 stages of sampling were highly resistant to ampicillin, tetracycline, florfenicol, and doxycycline. Increased resistance to ceftiofur, enrofloxacin, ofloxacin, and gentamicin were seen in the isolates from the final stage of deep litter. In addition, the percentage of isolates tolerant to zinc, copper, and cadmium and the numbers of Group-B2 isolates all increased in the used deep litter, and the isolates of each stage belonged predominantly to commensal groups. The antibiotic resistance of isolates with identical Eric-PCR patterns had improved from round 4 to 8, and differences still existed in the resistance profiles of isolates with identical Eric-PCR patterns from different barns of the same round. This study concluded that deep litter could be suitable for the evolution of bacterial antibiotic-resistance under conditions of continuous usage or accumulation of antibiotics and heavy metals without proper management.

Prevalence and risk factors of radiographic vertebral fractures in elderly Chinese men and women: results of Mr. OS (Hong Kong) and Ms. OS (Hong Kong) studies.

UNLABELLED: This study investigated the prevalence of radiographic vertebral fractures using Genant's semiquantitative (SQ) scoring system in elderly Chinese men (n = 2,000; mean age, 72.4 years) and women (n = 2,000; mean age, 72.6 years). Vertebral deformities had similar prevalence in elderly men (14.9 %) and women (16.5 %). Majority of the deformities in men were mild (9.9 %, grade = 1). The prevalence of vertebral fractures (grade ≥ 2) was 5.0 % among men and 12.1 % among women. INTRODUCTION: Vertebral fracture is a serious consequence of osteoporosis and is often under-diagnosed. Researches on different ethnicities and territories to estimate the prevalence of vertebral fractures and to identify the risk factors are necessary. METHODS: Mr. OS (Hong Kong) and Ms. OS (Hong Kong) represent the first large-scale cohort studies ever conducted on bone health in elderly Chinese men (n = 2,000) and women (n = 2,000). The current study investigated the prevalence of radiographic vertebral fractures in these subjects using Genant's SQ scoring system and identified risk factors for vertebral fractures. RESULTS: The radiographs of all men (mean age, 72.4 years) and women (mean age, 72.6 years) were obtained. Six hundred twenty-seven subjects (15.7 %) had at least one vertebral deformity (SQ grade ≥ 1), including 297 men (14.9 %) and 330 women (16.5 %, p = 0.151). Three hundred forty-two participants (8.6 %) were defined as having at least one vertebra fracture (SQ grade ≥ 2), consisted of 100 men (5.0 %) and 242 women (12.1 %, p < 0.001). Older age, lower bone mineral density, lower physical activity, lower grip strength, fracture history, and low back pain were significantly associated with higher vertebral fracture rate for both men and women. CONCLUSION: Vertebral deformity had similar prevalence in older men and women, while vertebral fracture was more common in women. Majority of deformities in men was mild. The vertebral deformity prevalence of women from this study is similar to previous reports of other East Asian women and Latin American women.

A novel action of alzheimer's amyloid beta-protein (Abeta): oligomeric Abeta promotes lipid release.

Interactions between amyloid beta-protein (Abeta) and lipids have been suggested to play important roles in the pathogenesis of Alzheimer's disease. However, the molecular mechanism underlying these interactions has not been fully understood. We examined the effect of Abeta on lipid metabolism in cultured neurons and astrocytes and found that oligomeric Abeta, but not monomeric or fibrillar Abeta, promoted lipid release from both types of cells in a dose- and time-dependent manner. The main components of lipids released after the addition of Abeta were cholesterol, phospholipids, and monosialoganglioside (GM1). Density-gradient and electron microscopic analyses of the conditioned media demonstrated that these Abeta and lipids formed particles and were recovered from the fractions at densities of approximately 1.08-1.18 g/ml, which were similar to those of high-density lipoprotein (HDL) generated by apolipoproteins. The lipid release mediated by Abeta was abolished by concomitant treatment with Congo red and the PKC inhibitor, H7, whereas it was not inhibited with N-acetyl-l-cysteine. These Abeta-lipid particles were not internalized into neurons, whereas HDL-like particles produced by apolipoprotein E were internalized. Our findings indicate that oligomeric Abeta promotes lipid release from neuronal membrane, which may lead to the disruption of neuronal lipid homeostasis and the loss of neuronal function.

A case of cardiomyopathy showing progression from the hypertrophic to the dilated form: association of Mt8348A-->G mutation in the mitochondrial tRNA(Lys) gene with severe ultrastructural alterations of mitochondria in cardiomyocytes.

This report describes a case of cardiomyopathy with a novel point mutation of mitochondrial DNA coding lysine tRNA in association with severe ultrastructural alterations of the mitochondria in the cardiomyocytes. Abnormalities of energy production and/or abnormal protein synthesis because of the mutation of mitochondrial DNA may have played an important role in the pathogenesis of this case, which showed severe cardiomyocyte degeneration and deterioration from hypertrophic cardiomyopathy to severe dilated cardiomyopathy.

Antiatherogenic mitochondrial genotype in patients with type 2 diabetes.

OBJECTIVE: To evaluate the significance of a longevity-associated mitochondrial genotype (Mt5178A) derived from a C --> A transversion at nucleotide position 5178 of mitochondrial DNA, which causes a Leu-to-Met substitution within the NADH dehydrogenase subunit 2 gene, in type 2 diabetic subjects. RESEARCH DESIGN AND METHODS: Mt5178 typing was done by polymerase chain reaction-restriction fragment-length polymorphism with the restriction enzyme AluI in 1,148 type 2 diabetic Japanese subjects, and the results were compared with the clinical characteristics. Then, the association of Mt5178 type with early atherosclerotic changes of the bilateral carotid arteries on ultrasonography was assessed in 412 diabetic subjects randomly selected from the original 1,148 type 2 diabetic subjects, while maintaining the same frequency of Mt5178A and Mt5178C. RESULTS: The frequency of Mt5178A in the type 2 diabetic subjects (454 of 1,148; 40%) was not different from that previously found in healthy blood donors (114 of 252; 45%). Clinical characteristics regarding diabetes were not significantly different between the Mt5178A group (n = 454) and the Mt5178C group (n = 694). However, the mean intima-media thickness (IMT) at six sites in the bilateral carotid arteries was significantly smaller in the Mrt5178A group than in the Mt5178C group (0.906 +/- 0.018 vs. 0.995 +/- 0.021 mm, mean +/- SEM, P = 0.022), and the Mt5178 type was significantly correlated with both the mean IMT and the presence of plaque on multiple regression analysis and discriminant analysis. CONCLUSIONS: The Mt5178A genotype may be unrelated to the etiology of type 2 diabetes. However, Mt5178A seems to have an antiatherogenic effect, at least in type 2 diabetic individuals.

Site-specific phosphorylation of tau accompanied by activation of mitogen-activated protein kinase (MAPK) in brains of Niemann-Pick type C mice.

Niemann-Pick type C (NPC) disease is characterized by an accumulation of cholesterol in most tissues and progressive neurodegeneration with the formation of neurofibrillary tangles. Neurofibrillary tangles are composed of paired helical filaments (PHF), a major component of which is the hyperphosphorylated tau. In this study we used NPC heterozygous and NPC homozygous mouse brains to investigate the molecular mechanism responsible for tauopathy in NPC. Immunoblot analysis using anti-tau antibodies (Tau-1, PHF-1, AT-180, and AT-100) revealed site-specific phosphorylation of tau at Ser-396 and Ser-404 in the brains of NPC homozygous mice. Mitogen-activated protein kinase, a potential serine kinase known to phosphorylate tau, was activated, whereas other serine kinases such as glycogen synthase kinase-3beta and cyclin-dependent kinase 5 were inactive. Morphological examination demonstrated that a number of neurons, the perikarya of which strongly immunostained with PHF-1, exhibited polymorphorous cytoplasmic inclusion bodies and multi-concentric lamellar-like bodies. Importantly, the accumulation of intracellular cholesterol in NPC mouse brains was determined to be a function of age. From these results we conclude that abnormal cholesterol metabolism due to the genetic mutation in NPC1 may be responsible for activation of the mitogen-activated protein kinase-signaling pathway and site-specific phosphorylation of tau in vivo, leading to tauopathy in NPC.

[Automated microbiology identification and antibiotic susceptibility analysis system].

This article introduces the principles, characters and application prospect of Model 2000 Automated Microbiology Identification and Antibiotic Susceptibility Analysis System.

Peroxide production and apoptosis in cultured cells carrying mtDNA mutation causing encephalomyopathy.

When cybrids with a point mutation, which locates in the tRNALeu(UUR) gene of mtDNA and causes a mitochondrial encephalomyopathy (MELAS syndrome), were exposed to a high concentration of oxygen (95%), the peroxide production markedly increased by 6 h of oxygen exposure, whereas the peroxide production was similar among the cybrids under a normal concentration of oxygen. The peroxide production by oxygen exposure was enhanced particularly in cybrids with high proportions of the mutant mtDNA and low respiratory capacities. The appearance of apoptotic cells by oxygen exposure was high in cybrids with the impaired respiratory function due to the mutation. An antioxidant NAC successfully suppressed both the peroxide production and apoptosis. These results imply that the peroxide production plays an important role in inducing apoptosis in cells carrying the mtDNA mutation causing encephalomyopathy.

Accumulation of deletions and point mutations in mitochondrial genome in degenerative diseases.

Accumulation of various mutations in the mitochondrial genome is proposed as an important contributor to aging and degenerative diseases. Extensive fragmentation of mtDNA was detected in association with increased 8-hydroxydeoxyguanosine content in the heart mitochondrial DNA (mtDNA) from a patient with premature aging and mitochondrial cardiomyopathy, who carried a mutation within the mitochondrial tRNA(Asp) gene. This result suggests that damage to mtDNA by hydroxyl radical and accumulation of deleted mtDNA can be accelerated by a specific mitochondrial genotype. Similarly, extensive fragmentation of mtDNA was also detected in cultured cells exposed to a high oxygen concentration atmosphere, implying that mtDNA is vulnerable to reactive oxygen species. To clarify the role of point mutations accumulated in mtDNA, we examined the sequence heterogeneity of mtDNA in the skeletal muscle of a MELAS patient who carried a mutation within the mitochondrial tRNA(leu)(UUR) gene. The analysis revealed that the frequency of mutant clones in the MELAS muscle was significantly higher than those in an age-matched control muscle and a control placenta. Some of these nucleotide substitutions were missense and nonsense mutations, which potentially have deleterious effects on the mitochondrial function. The frequency of nucleotide substitutions in the striatum of three patients with Parkinson's disease was also significantly higher than that in control tissues. We also observed increased protein modification by 4-hydroxy-2-nonenal, a lipid peroxidation by-product, in Parkinson's disease. These results suggests that a vicious cycle contributes to the progression of degenerative process. In this cycle, first a primary mitochondrial mutation(s) induces a mitochondrial respiratory defect, which increases the leakage of reactive oxygen species (ROS) from the respiratory chain. Then the ROS would trigger accumulation of secondary mtDNA mutations in postmitotic cells, leading to further aggravation of mitochondrial respiratory defects and increased production of ROS and lipid peroxides from mitochondria, and thus resulting in degeneration of cellular components.