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As a typical food safety industrial model strain, Bacillus subtilis has been widely used in the field of metabolic engineering due to its non-pathogenicity, strong ability of extracellular protein secretion and no obvious codon preference. In recent years, with the rapid development of molecular biology and genetic engineering technology, a variety of research strategies and tools have been used to construct B. subtilis chassis cells for efficient synthesis of biological products. This review introduces the research progress of B. subtilis from the aspects of promoter engineering, gene editing, genetic circuit, cofactor engineering and pathway enzyme assembly. Then, we also summarized the application of B. subtilis in the production of biological products. Finally, the future research directions of B. subtilis are prospected.
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Bacillus subtilis/genetics , Bacterial Proteins/genetics , Gene Editing , Metabolic Engineering , Promoter Regions, GeneticABSTRACT
Genome-scale metabolic network model (GSMM) is an extremely important guiding tool in the targeted modification of industrial microbial strains, which helps researchers to quickly obtain industrial microbes with specific traits and has attracted increasing attention. Here we reviewe the development history of GSMM and summarized the construction method of GSMM. Furthermore, the development and application of GSMM in industrial microorganisms are elaborated by using four typical industrial microorganisms (Bacillus subtilis, Escherichia coli, Corynebacterium glutamicum, and Saccharomyces cerevisiae) as examples. In addition, prospects in the development trend of GSMM are proposed.
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Corynebacterium glutamicum/genetics , Escherichia coli/genetics , Metabolic Engineering , Metabolic Networks and Pathways/geneticsABSTRACT
Chitinase has a wide industrial application prospect. For example, it can degrade shrimp shells, crab shells and other crustacean waste into high value-added chitooligosaccharides. However, the low catalytic efficiency of chitinase greatly limits the production of chitooligosaccharides. In previous study, the we expressed a chitinase Chisb with high catalytic efficiency and studied its enzymatic properties. In order to further improve the catalytic efficiency of Chisb, with R13NprB-C-SP-H as the parent, here error-prone PCR was used to construct random mutant library to conduct directed evolution of chitinase Chisb. Two mutants C43D and E336R were obtained with 96-well plate primary screening and shaker-screening, and their enzymatic properties were also studied. The optimum temperature of C43D and E336R was 55 °C, and the optimum pH of C43D was 5.0, while that of E336R was 9.0. The catalytic efficiency of C43D and E336R was 1.35 times and 1.57 times higher than that of control. The chitooligosaccharide concentration of E336R and C43D was 2.53 g/L and 2.06 g/L, improved by 2.84 times and 2.31 times compared with the control (0.89 g/L), respectively. In addition, the substrate conversion rate of mutants E336R and C43D was 84.3% and 68.7%, improved by 54.6% and 39% compared with the control (29.7%), respectively. In summary, the study indicates that random mutation introduced by error-prone PCR can effectively improve the catalytic efficiency of chitinase Chisb. The positive mutants with higher catalytic efficiency obtained in the above study and their enzymatic property analysis have important research significance and application value for the biosynthesis of chitooligosaccharides.
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Biocatalysis , Chitin , Chitinases , Hydrogen-Ion Concentration , Polymerase Chain ReactionABSTRACT
Industrial fermentation focuses on realizing the uniform of high titer, high yield, and high productivity. Multi-scale analysis and regulation, including molecule level, cell level, and bioreactor level, facilitate global optimization and dynamic balance of fermentation process, which determine high efficiency of biosynthesis, targeted directionality of bioconversion, process robustness, and well-organized system. In this review, we summariz and discuss advances in multi-scale analysis and regulation for fermentation process focusing on the following four aspects: 1) kinetic modeling of metabolic pathways, 2) characteristic of cell metabolism, 3) co-coupling fermentation and purification, and 4) bioreactor design. Integrating multi-scale analysis of fermentation process and integrating multi-scale regulation are expected as an important strategy for realizing highly efficient fermentation by industrial microorganisms.
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Bioreactors , Fermentation , Industrial Microbiology , Kinetics , Metabolic Networks and PathwaysABSTRACT
Holmes-Adie syndrome (HAS) is a clinical syndrome mainly characterized by tonic pupil and disappearance of tendon reflex. It is mostly idiopathic and can also be seen in cerebral diseases, such as trauma, infection and tumors. However, it is rarely reported to be accompanied with myasthenia gravis (MG). We report a case of MG and HAS, whose clinical manifestations were fluctuation of limb weakness, breathing difficulties, right ptosis. Her pupils were unequal: the left pupil was 3 mm, the right pupil was 2 mm, direct and indirect light reflex was slow in left pupil, and right pupil was sensitive to light reflex. The left eye pupil shrank to 2 mm after dripped pilocarpine diluent for 10 minutes, while the right pupil was still 2 mm. Chest CT examination revealed thymoma. After treatment with thymectomy, glucocorticoid, immunoglobulins and tacrolimus, her symptoms of MG were improved, but the left pupil diameter and light reflex were not changed. Combined with the patient's symptoms, physical signs and examinations, this patient was diagnosed as MG accompanied with HAS.
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Objective To compare the Poser and the McDonald diagnostic criteria for multiple sclerosis (MS).Methods Sixty-seven patients with clinical features suggestive of MS were re-evaluated by 2 neurologists utilizing both the Poser and the McDonald criteria.The Fisher' s exact test was used to statistically analyze the data from assessment by these two criteria.Results By the Poser criteria, 34 patients were classified as clinically definite MS, 24 patients as laboratory supported definite MS and 9 patients as possible MS.Under the new McDonald criteria, MS was diagnosed in 36 patients and 31 patients had possible MS.The results revealed significant differences in diagnosing MS between these 2 criteria (OR=5.549, 95% CI 2.37-13.00, P<0.01).Conclusions These 2 criteria showed remarkable differences in diagnosing MS, in particular definite MS, possibly due to greater reliance on paraclinical evidences in the Poser criteria and more stringent gnidlines of magnetic resonance imaging (MRI) in the McDonald criteria.The analysis of cerebrospinal fluid may increase the diagnostic rate of MS and specify pathologic abnormalities in MRI to some degree.
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BACKGROUND: As the second anti-Alzheimer disease drug approved by Food and Drug Administration(FDA), donepezil (Aricept) has been applied in European and American market. According to the regulation of Health Ministry of China, it needs conducting clinical trial of multiple center nationwide in order to come into Chinese market.OBJECTIVE: To evaluate the efficiency and safety of donepezil on treating mild and moderate Alzheimer disease (AD).DESIGN: Randomized, single blind and placebo control prospective study based on patients.SETTING: Neurological Department of Peking Hospital and Neurological Department of the 301 Hospital of Chinese PLA, and ect.PARTICIPANTS: Totally 188 patients with mild and moderate AD[with mini-mental state examination(MMSE) score of 10 to 24 points] from 15 big hospitals of Beijng, Shanghai and Guangzhou were conducted 12 weeks'clinical trial, among which 89 cases were of single blind and placebo control study while 99 cases were of self-controlled study. All the cases met the AD diagnostic standard of clinical neurology, linguistic dysfunction and stroke(NINCDS-ADRDA) and the 4th edition of Statistic Manual (DSM-IVR).INTERVENTIONS: Donepezil (5 rmg/tablet, ip, 5 rmg/time) or placebo with same color, shape, flavor and size with donepezil ( ip, 1 tablet/time)was taken orally for 12 consecutive weeks.MAIN OUTCOME MEASURES: MMSE, clinical dementia rating scale(CDR), activities of daily life scale(ADL), biochemical parameters, electrocardiograph(ECG) and chest x-ray were conducted once every 4 weeks before and after treatment.RESULTS: The random, single blind and placebo control study showed that the score of MMSE, CDR and ADL was greatly improved in donepezil group after 12 weeks' treatment when comparing with placebo group(P ＜ 0. 01,0.05, 0.01 ). Self-controlled study showed that the score of MMSE, CRD and ADL in donepezil group after 12 weeks' treatment increased 3.5, 0.6 and 7.1 points respectively compared with those before treatment(P ＜ 0.01,0.05, 0.01 ) . The score of MMSE was already improved in the 4th week of treatment. Among the 145 patients who took donepezil, 7 cases(4.8% )experienced side effect of mild cholinergic excitability. In the placebo group,2 of the 43 cases appeared dizziness and nausea. There was no difference between two groups( P ＞ 0.05).CONCLUSION: Donepezil can effectively treat mild and moderate AD patients and improve their cognitive functions, dementia level and daily living abilities with good tolerance and high safety.
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Objective To evaluate the roles of plasma p-tau(~ 181P)protein for clinical diagnosis of Alzheimer′s Disease (AD). Methods Subjects including 23 mild AD(19≤MMSE≤26) and 35 moderate and severe AD(MMSE0.05). Level of p-tau(~ 181P) protein was significantly higher in the moderate and severe AD group(18.3?20.3)ng/L than that in HC( P
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Objective To investigate the possible causes of the different diagnostic sensitivity of voluntary single fiber electromyography (SFEMG) and repetitive nerve stimulation(RNS) in patients with myasthenia gravis (MG). Methods The voluntary SFEMG and RNS at low rates were recorded successively from the same extensor digitorum communis (EDC) muscle on the same day in 67 patients with MG. Results The diagnostic sensitivity of SFEMG and RNS was 92.5% and 50.7%, respectively, with the former statistically significantly higher than the later. The percentage of decrement of RNS was positively correlated with 3 SFEMG parameters, i.e. the mean jitter, percentage of abnormal pairs of potential and percentage of impulse blocking. Among the 34 cases with significant decrement on RNS, 2 had no impulse blocking and the maximum decrement reached 62%, while 33 cases with normal RNS had up to 58% of impulse blocking. Conclusion The voluntary SFEMG was more sensitive than RNS in diagnosing MG even in the same muscle. The blocking phenomenon observed in voluntary SFEMG was not completely corresponding to the decrement in RNS.The possible explanations were partly because that RNS recorded the total muscle fibers response in surface of the muscle and SFEMG examined the increasing or blocking at individual motor end-plates, and partly because that the voluntary SFEMG and RNS might explored endplates belonging to different motor units.
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Objective To assess the efficacy and safety of aricept in treatment of mild to moderate Alzheimer disease(AD).Methods 188 patients with AD were followed up in a multicentre,with randomized study for 12 weeks. 89 patients had a single -blind,placebo-controlled trial and 99 a self-matched trial.Results The results of randomized single-blind,placebo-controlled trial showed that the improvements in MMSE、CDR and ADL were statistically significantly greater with aricept 5 mg/d than with placebo(P value respectively 0.05).Conclusion Aricept is effective in treating patients with mild to moderate AD. It could produce improvements in cognition and ability of daily life. Aricept was well tolerated and safe.
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Objective To assess the diagnostic value of the single-photon-emission computed tomography (SPECT) and the magnetic resonance imaging (MRI) for Alzheimer's disease (AD) and vascular dementia (VD), and to explore the correlation between cerebral atrophy and cerebral regional blood flow and the degree of cognitive impairment in AD patients. Methods The temporal and frontal lobes and hippocampus were quantitatively detected by MRI and the cerebral regional blood flow in these regions was analysed semi-quantitatively by SPECT for 36 patients with AD, 32 with VD and 30 sex-matched non-demented elderly controls (NC). Results The hippocampal atrophy of AD and VD groups detected on MRI was significantly different from that of the NC group. Left hippocampal height was (9.27?3.43)mm in AD, (11.62?5.72)mm in VD and (12.13?2.97)mm in NC(P0.05, compared to that of NC), showing that hippocampal atrophy could not be detected by MRI. However, RAR of the left hippocampus by SPECT detection was 0.61?0.07 (P
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0.05). There was no correlation between Bcl-2 and Fas positive cells and acetylcholine receptor antibody serum titre, disease grouping, age and sex in patients with or without hyperplastic thymic and thymoma.Conclusions In MG patients, the expression of Bcl-2 in hyperplastic thymus is upregulated. There is no correlation between the expression of apoptotic genes Bcl-2 and Fas and clinic relative factors.
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Objective To evaluate the expression of Sp3 gene of peripheral blood mononuclear cells (PBMC) in multiple sclerosis (MS) patients and the correlation between Sp3 gene expression and clinical manifestation Methods Two pairs of primers were used to amplify cDNAs generated from 33 MS patients and 90 controls including healthy controls, other nonimmunological neurological disease controls and the other neuroimmunological diseases than MS controls Results The deficient expression of Sp3 gene in MS patients (45%, 15/33) is statistically significantly higher than that in the other 3 control groups ( P
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Objective To evaluate the efficacy and safety in treatment of patients with mild to moderate Alzheimer’s disease (AD). Methods A total of 233 patients with mild to moderate potential AD were enrolled in a 16-week multi-center double blind clinical trial. All patients were randomized into two groups. 110 patients in galantamine group and 108 patients in donepezil group were enrolled in efficacy analysis. The scales of Alzheimer’s Disease Assessment Scale-Cognitive Subscale (ADAS-cog), Alzheimer’s Disease Cooperative Study Activities of Daily Living Scale (ADCS-ADL) and The Neuropsychiatric Inventory (NPI) were used to assess the effect at both baseline and the end of 16 weeks. Safety issues, including vital signs, lab assays and ECG examinations were measured. Results Patients in both groups were obviously improved in the total score of ADAS-cog (-5.4?6.4) in the galantamine group and (-4.0?7.3) in the donepezil group, P=0.098). 76% patients of the galantamine group had a score of ADAS-cog less than 20 at the end of 16 weeks treatment, which was higher than that of the donepezil group (58%, P=0.015). The sub-score of speech ability in ADAS-cog were improved in the galantamine group (baseline 2.8?2.9,16 weeks 1.8?2.5) compared with the donepezil group (baseline 2.8?3.0, 16 weeks 2.3?2.9, P=0.035). No significant difference of ADSC-ADL and NPI scale was found between the two groups (P=0.447 and 0.936 respectively). The sleep/night behavior was improved in the donepezil group (baseline 14%, 16 weeks 10%) compared with the galantamine group (baseline 23%, 16 weeks 22%, P=0.012). Two drug-related severe adverse events occurred during the trial, which were platelet reduction in the galantamine group and acute drug-induced hepatic injury in the donepezil group. The incidence of adverse events was 44% in the galantamine group and 47% in the donepezil group respectively. Galantamine had little influence on vital signs and lab assays. Conclusion Safe and well tolerated, galantamine improves the cognition, activities of daily living and neuropsychiatric symptoms of patients with mild to moderate AD.
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Objective To study the immune function of the subpopulation of T helper lymphocyte(Th), including Th1 and Th2 cells in myasthenic thymus and peripheral blood (PB).Methods Enzyme-linked immunospot (ELISPOT) method is adopted to detect the acetylcholine receptor (AChR)-reactive interleukin-2(IL-2)-, interleukin-4 (IL-4)-, interleukin-10 (IL-10)-, interferon-gamma(IFN-?)-secreting cell numbers in thymus and peripheral blood (PB). Results The AChR-reactive IL-2-,IL-4-,IL-10-, IFN-?-secreting cell numbers were increased in thymus as well as the AChR-reactive IL-10-, IFN-?-secreting cell numbers in PB. There is a positive correlation between thymus and PB in MG for the AChR-reactive IL-4-, IL-10-secreting cell numbers. After treatment, the AChR-reactive IL-2-,IL-4-, IFN-?-secreting cell numbers were decreased in PB, while there were 1 case and 2 cases showing a decrease and an increase in AChR-reactive IL-10-secreting cell number, respectively.Conclusions The immune dysfunction of the Th1 and Th2 subpopulation existed in the thymus and PB of MG, and the AChR-reactive IL-4-, IL-10-secreting cell numbers might be served as parameters to evaluate the degree of the immune dysfunction in the MG thymus. The role of the IL-10 in pathogenesis of MG needs further study.
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The authors give an account of the historical development of neurological intensive care and its current status in China and put forward the view that establishment of neurological intensive care units can improve the level of neurological departments in delivering emergency treatments and give impetus to the improvement of expertise in diagnosing and treating acute neurological diseases. It will also play a key role in clinically conducting encephalic resuscitation and hibernation therapy, finely monitoring changes of intracranial physiologic indexes by electro physiological methods, further clarifying organ failure resulting from cerebral and neural failure, and promoting the study of nerve grafting and regeneration. Neurological intensive care units can serve as both the teaching base for training professional staff and the research base for advancing neurological emergency medicine.
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In nine patients with epilepsy and 24 neurologically healthy controls, concentrations of IgG and albumin in cerebrospinal fluid and serum were determined,and their corresponding IgG index and intrathecal IgG synthesis rate were calculated; and cerebrospinal fluid IgG fraction bands were detected. The results revealed that immunological abnormality involving central nervous system did exist in some patients with epilepsy. These combined detecting methods provide one of the effective means for screening some epileptic patients with immunological abnormalities involving central nervous system, and for probing their aetiology and pathogenesis.