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A novel CRISPR/Cas9 system with high genomic editing efficiency and recyclable auxotrophic selective marker for multiple-step metabolic rewriting in Pichia pastoris

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Synthetic and Systems Biotechnology 2023年第3期8卷 445-451页

作者： xiang Wang Yi Li Zhehao Jin xiangjian Liu Xiang Gao Shuyuan Guo Tao Yu Center for Synthetic Biochemistry CAS Key Laboratory of Quantitative Engineering BiologyShenzhen Institute of Synthetic BiologyShenzhen Institutes of Advanced TechnologyChinese Academy of Sciences(CAS)Shenzhen518055China University of the Chinese Academy of Sciences Beijing100049China Center for Materials Synthetic Biology CAS Key Laboratory of Quantitative Engineering Biology of CASShenzhen Institute of Synthetic BiologyShenzhen Institutes of Advanced TechnologyChinese Academic of ScienceShenzhen518055China

The methylotrophic budding yeast Pichia pastoris has been utilized to the production of a variety of heterologous recombinant proteins owing to the strong inducible alcohol oxidase promoter(pAOX1).However,it is difficult to use P.pastoris as the chassis cell factory for high-valuable metabolite biosynthesis due to the low homologous recombination(HR)efficiency and the limitation of handy selective markers,especially in the condition of multistep biosynthetic pathways.Hence,we developed a novel CRISPR/Cas9 system with highly editing efficiencies and recyclable auxotrophic selective marker(HiEE-ReSM)to facilitate cell factory in P.pastoris.Firstly,we improved the HR rates of P.pastoris through knocking out the non-homologous-end-joining gene(Δku70)and overexpressing HR-related proteins(RAD52 and RAD59),resulting in higher positive rate compared to the basal strain,achieved 97%.Then,we used the uracil biosynthetic genes PpURA3 as the reverse screening marker,which can improve the recycling efficiency of marker.Meanwhile,the HR rate is still 100%in uracil auxotrophic yeast.Specially,we improved the growth rate of uracil auxotrophic yeast strains by overexpressing the uracil transporter(scFUR4)to increase the uptake of exogenous uracil from medium.Meanwhile,we explored the optimal concentration of uracil(90 mg/L)for strain growth.In the end,the HiEE-ReSM system has been applied for the inositol production(250 mg/L)derived from methanol in P.pastoris.The systems will contribute to P.pastoris as an attractive cell factory for the complex compound biosynthesis through multistep metabolic pathway engineering and will be a useful tool to improve one carbon(C1)bio-utilization.

关键词： Pichia pastoris Uracil auxotroph CRISPR/Cas9 Recyclable selective marker Homology directed repair

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Potential role of hippocampal neurogenesis in spinal cord injury induced post-trauma depression

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Neural Regeneration Research 2024年第10期19卷 2144-2156页

作者： Ying Ma Yue Qiao Xiang Gao Spinal Cord and Brain Injury Research Group Stark Neuroscience Research InstituteIndianapolisINUSA Department of Neurological Surgery Indiana University School of MedicineIndianapolisINUSA Department of Neurology Shengjing Hospital of China Medical UniversityShenyangLiaoning ProvinceChina

It has been reported both in clinic and rodent models that beyond spinal cord injury directly induced symptoms, such as paralysis, neuropathic pain, bladder/bowel dysfunction, and loss of sexual function, there are a variety of secondary complications, including memory loss, cognitive decline, depression, and Alzheimer's disease. The largescale longitudinal population-based studies indicate that post-trauma depression is highly prevalent in spinal cord injury patients. Yet, few basic studies have been conducted to address the potential molecular mechanisms. One of possible factors underlying the depression is the reduction of adult hippocampal neurogenesis which may come from less physical activity, social isolation, chronic pain, and elevated neuroinflammation after spinal cord injury. However, there is no clear consensus yet. In this review, we will first summarize the alteration of hippocampal neurogenesis post-spinal cord injury. Then, we will discuss possible mechanisms underlie this important spinal cord injury consequence. Finally, we will outline the potential therapeutic options aimed at enhancing hippocampal neurogenesis to ameliorate depression.

关键词： antidepressants chronic pain depression exercise hippocampal neurogenesis inflammation inhibition neuroinflammation physical activity deficits social isolation spinal cord injury

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Personalized pancreatic cancer therapy:from the perspective of mRNA vaccine

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军事医学研究:英文版 2023年第4期10卷 461-477页

作者： Xing Huang Gang Zhang Tian‑Yu Tang Xiang Gao Ting‑Bo Liang Zhejiang Provincial Key Laboratory of Pancreatic Disease the First Affiliated HospitalZhejiang University School of MedicineHangzhou 310009China Department of Hepatobiliary and Pancreatic Surgery the First Affiliated HospitalZhejiang University School of MedicineHangzhou 310003China Zhejiang Clinical Research Center of Hepatobiliary and Pancreatic Diseases Hangzhou 310003China The Innovation Center for the Study of Pancreatic Diseases of Zhejiang Province Hangzhou 310009China Cancer Center Zhejiang UniversityHangzhou 310058China

Pancreatic cancer is characterized by inter-tumoral and intra-tumoral heterogeneity,especially in genetic alteration and microenvironment.Conventional therapeutic strategies for pancreatic cancer usually suffer resistance,highlighting the necessity for personalized precise treatment.Cancer vaccines have become promising alternatives for pancreatic cancer treatment because of their multifaceted advantages including multiple targeting,minimal nonspecific effects,broad therapeutic window,low toxicity,and induction of persistent immunological memory.Multiple conventional vaccines based on the cells,microorganisms,exosomes,proteins,peptides,or DNA against pancreatic cancer have been developed;however,their overall efficacy remains unsatisfactory.Compared with these vaccine modalities,messager RNA(mRNA)-based vaccines offer technical and conceptional advances in personalized precise treatment,and thus represent a potentially cutting-edge option in novel therapeutic approaches for pancreatic cancer.This review summarizes the current progress on pancreatic cancer vaccines,highlights the superiority of mRNA vaccines over other conventional vaccines,and proposes the viable tactic for designing and applying personalized mRNA vaccines for the precise treatment of pancreatic cancer.

关键词： Pancreatic cancer Precise therapy Cancer vaccine mRNA vaccine Tumor antigen Immune subtype

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Prognostic role of C-reactive protein to albumin ratio in lung cancer:An updated systematic review and meta-analysis

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Chronic Diseases and Translational Medicine 2024年第1期10卷 31-39页

作者： Zhendong Lu Siyun Fu Wei Li Xiang Gao Jinghui Wang Department of Medical Oncology Beijing Tuberculosis and Thoracic Tumor Research InstituteBeijing Chest HospitalCapital Medical UniversityBeijingChina Cancer Research Center Beijing Chest HospitalBeijing Tuberculosis and Thoracic Tumor Research InstituteCapital Medical UniversityBeijingChina

Background: C-reactive protein to albumin ratio(CRP/Alb ratio,CAR)has been suggested as a potential prognostic biomarker in lung cancer.This updated systematic review and meta-analysis aimed to assess the association between CAR and lung cancer prognosis in current literature.Methods: A systematic search of databases was conducted to identify relevant studies published up to April 2023.Pooled hazard ratios(HRs)and 95%confidence intervals(CIs)were calculated to assess the association between CAR and overall survival(OS)and progression-free survival(PFS)and recurrence-free survival(RF)in lung cancer patients.Results: This meta-analysis includes 16 studies with a total of 5337 patients,indicating a significant association between higher CAR and poorer OS,PFS,and RFS in lung cancer patients,with a pooled HR of 1.78(95%CI=1.60-1.99),1.57(95%CI=1.36-1.80),and 1.97(95%CI=1.40-2.77),respectively.Conclusions: This updated meta-analysis provides evidence for the potential prognostic role of CAR in lung cancer,suggesting its utility as an effective and noninvasive biomarker for identifying high-risk patients and informing treatment decisions in a cost-effective manner.However,further large-scale studies will be necessary to establish the optimal cut-off value for CAR in lung cancer and confirm the present findings.

关键词： C-reactive protein to albumin ratio inflammation:biomarker lung cancer prognosis

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Mechanism Study on Chinese Medicine in Treatment of Nodular Goiter

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Chinese Journal of Integrative Medicine 2023年第6期29卷 566-576页

作者： WANG Chang-lin gao Ming-zhou gao xiang-ju MU xiang-yu WANG Jie-qiong gao Dong-mei QIAO Ming-qi School of Chinese Medicine Shandong University of Traditional Chinese MedicineJinan(250355)China Research and Innovation Team of Emotional Diseases and Syndromes of Shandong University of Traditional Chinese Medicine Jinan(250355)China Key Laboratory of Traditional Chinese Medicine for Classical Theory Ministry of EducationJinan(250355)China School of Pharmacy Shandong University of Traditional Chinese MedicineJinan(250355)China Youth Research and Innovation Team of Pharmacology of Liver Viscera in Emotional Disease and Syndromes Jinan(250355)China

Nodular goiter has become increasingly prevalent in recent years.Clinically,there has been a burgeoning interest in nodular goiter due to the risk of progression to thyroid cancer.This review aims to provide a comprehensive summary of the mechanisms underlying the therapeutic effect of Chinese medicine(CM)in nodular goiter.Articles were systematically retrieved from databases,including PubMed,Web of Science and China National Knowledge Infrastructure.New evidence showed that CM exhibited multi-pathway and multi-target characteristics in the treatment of nodular goiter,involving hypothalamus-pituitary-thyroid axis,oxidative stress,blood rheology,cell proliferation,apoptosis,and autophagy,especially inhibition of cell proliferation and promotion of cell apoptosis,involving multiple signal pathways and a variety of cytokines.This review provides a scientific basis for the therapeutic use of CM against nodular goiter.Nonetheless,future studies are warranted to identify more regulatory genes and pathways to provide new approaches for the treatment of nodulargoiter.

关键词： noduiar goiter Chinese medicine mechanism pharmacology review

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A novel signal feature extraction technology based on empirical wavelet transform and reverse dispersion entropy

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Defence Technology（防务技术） 2021年第5期17卷 1625-1635页

作者： Yu-xing Li Shang-bin Jiao Xiang Gao School of Automation and Information Engineering Xi’an University of TechnologyXi’an710048China School of Electrical Engineering Xi’an University of TechnologyXi’an710048China

Feature extraction is an important part of signal processing,which is significant for signal detection,classification,and recognition.The nonlinear dynamic analysis method can extract the nonlinear characteristics of signals and is widely used in different fields.Reverse dispersion entropy(RDE)proposed by us recently,as a nonlinear dynamic analysis method,has the advantages of fast computing speed and strong anti-noise ability,which is more suitable for measuring the complexity of signal than traditional permutation entropy(PE)and dispersion entropy(DE).Empirical wavelet transform(EWT),based on the theory of wavelet analysis,can decompose a complex non-stationary signal into a number of empirical wavelet functions(EWFs)with compact support set spectrum,which has better decomposition performance than empirical mode decomposition(EMD)and its improved algorithms.Considering the advantages of RDE and EWT,on the one hand,we introduce EWT into the field of underwater acoustic signal processing and fault diagnosis to improve the signal decomposition accuracy;on the other hand,we use RDE as the features of EWFs to improve the signal separability and stability.Finally,we propose a novel signal feature extraction technology based on EWT and RDE in this paper.Experimental results show that the proposed feature extraction technology can effectively extract the complexity features of actual signals.Moreover,it also has higher distinguishing ability for different types of signals than five latest feature extraction technologies.

关键词： Feature extraction Empirical mode decomposition Empirical wavelet transform Permutation entropy Reverse dispersion entropy

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Technical Perspective of Carbon Capture,Utilization,and Storage

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Engineering 2022年第7期8卷 27-32页

作者： Qingyang Lin Xiao Zhang Tao Wang Chenghang Zheng Xiang Gao State Key Laboratory of Clean Energy Utilization State Environmental Protection Engineering Center for Coal-Fired Air Pollution ControlZhejiang UniversityHangzhou 310027China

Carbon dioxide(CO_(2))is the primary greenhouse gas contributing to anthropogenic climate change which is associated with human activities.The majority of CO_(2) emissions are results of the burning of fossil fuels for energy,as well as industrial processes such as steel and cement production.Carbon capture,utilization,and storage(CCUS)is a sustainable technology promising in terms of reducing CO_(2) emissions that would otherwise contribute to climate change.From this perspective,the discussion on carbon capture focuses on chemical absorption technology,primarily due to its commercialization potential.The CO_(2) absorptive capacity and absorption rate of various chemical solvents have been summarized.The carbon utilization focuses on electrochemical conversion routes converting CO_(2) into potentially valuable chemicals which have received particular attention in recent years.The Faradaic conversion efficiencies for various CO_(2) reduction products are used to describe efficiency improvements.For carbon storage,successful deployment relies on a better understanding of fluid mechanics,geomechanics,and reactive transport,which are discussed in details.

关键词： CCUS Carbon capture Carbon utilization Carbon storage Chemical absorption Electrochemical conversion Storage mechanism

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Bidding Strategy for Coordinated Operation of Wind Power Plants and NGG-P2G Units in Electricity Market

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CSEE Journal of Power and Energy Systems 2022年第1期8卷 212-224页

作者： Xiang Gao Ka Wing Chan Shiwei Xia Xian Zhang Guibin Wang Shenzhen Polytechnic ShenzhenChina and also with the Department of Electrical Engineering The Hong Kong Polytechnic UniversityHung HomHong KongChina Department of Electrical Engineering The Hong Kong Polytechnic UniversityHung HomHong KongChina School of Electrical and Electronic Engineering North China Electric Power UniversityBeijing 102206China School of Mechanical Engineering and Automation Harbin Institute of TechnologyShenzhen 518055China College of Mechatronics and Control Engineering Shenzhen UniversityShenzhen 518060China

The synergies between electricity and other energy resources could promote energy utilization efficiency in electricity market.Within this context,this paper proposes a bi-level stochastic optimization model for the joint operation of a coordinated wind power plant(WPP)and natural gas generating(NGG)-power to gas(P2G)suppliers participating in the day-ahead(DA)market and real-time(RT)market as well as providing real-time auxiliary services.The coordinated supplier’s payoff is maximized in the upper level with consideration of the uncertainties of WPP output capacity and RT electricity price,while the social welfare of the grid is maximized in the lower level.Simulation results demonstrate the effectiveness of the proposed bidding model of the coordinated WPPs and NGG-P2G suppliers by examining its bidding behaviors and benefits with comparisons of four other bidding models.

关键词： Bi-level model bidding strategy WPP NGGP2G uncertainties

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Spatially mapping the diffusivity of proteins in live cells based on cumulative area analysis

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Science China Chemistry 2023年第11期66卷 3307-3313页

作者： Huihui gao Chu Han Limin xiang College of Chemistry and Molecular Sciences&Taikang Center for Life and Medical Sciences Wuhan UniversityWuhan 430072China

Molecular motion provides a way for biomolecules to mix and interact in living systems.Quantifying their motion is critical to the understanding of how biomolecules perform its function.However,it has been a challenged task to spatially map the fast diffusion of unbound proteins in the heterogenous intracellular environment.Here we reported a new imaging technique named cumulative area based on single-molecule diffusivity mapping(CA-SMdM).The strategy is based on the comparison of singlemolecule images between a shorter and longer exposure time.With longer exposure time,molecules will travel further,thus giving more blurred single-molecule images,hence implying its local diffusion rates.We validated our technique through measuring the fast diffusion rates(10–40μm~2/s)of fluorescent dye in glycerol-water mixture,and found the values fit well with Stokes-Einstein equation.We further showed that the spatially mapping of diffusivity in live cells is plausible through CA-SMdM,and it faithfully reported the local diffusivity heterogeneity in cytosol and nucleus.CA-SMdM provides an efficient way to mapping the local molecular motion,and therefore will have profound applications in probing the biomolecular interactions for living systems.

关键词： single molecule diffusivity cumulative area single-molecule diffusivity mapping

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Size,shape,charge and“stealthy”surface:Carrier properties affect the drug circulation time in vivo

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Asian Journal of Pharmaceutical Sciences 2021年第4期16卷 444-458页

作者： Jinwei Di Xiang Gao Yimeng Du Hui Zhang Jing gao Aiping Zheng State Key Laboratory of Toxicology and Medical Countermeasures Beijing Institute of Pharmacology and ToxicologyBeijing 100850China

The present review sets out to discuss recent developments of the effects and mechanisms of carrier properties on their circulation time.For most drugs,sufficient in vivo circulation time is the basis of high bioavailability.Drug carrier plays an irreplaceable role in helping drug avoid being quickly recognized and cleared by mononuclear phagocyte system,to give drug enough time to arrive at targeted organ and tissue to play its therapeutic effect.The physical and chemical properties of drug carriers,such as size,shape,surface charge and surface modification,would affect their in vivo circulation time,metabolic behavior and biodistribution.The final circulation time of carriers is determined by the balance between macrophage recognitions,blood vessel penetration and urine excretion.Therefore,when designing the drug delivery system,we should pay much attention to the properties of drug carriers to get enough in vivo circulation time to arrive at target site eventually.This article mainly reviews the effect of carrier size,size,surface charge and surface properties on its circulation time in vivo,and discusses the mechanism of these properties affecting circulation time.This review has reference significance for the research of long-circulation drug delivery system.

关键词： Drug carrier Circulation time Physical and chemical properties Macrophages Phagocytosis

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