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Construction of lentiviral vector carrying Rab9 gene and its expression in mouse brain

Youguo HAO, Min ZHANG, Jinzhi XU, Bitao BU, Jiajun WEI

《医学前沿（英文）》 2009年第3卷第2期页码 141-147 doi: 10.1007/s11684-009-0041-6

摘要： Rab proteins and their effectors facilitate vesicular transport by tethering donor vesicles to their respective target membranes. Rab9 mediates late endosome-to- -Golgi-network trafficking. To explore the possibility of Rab9-related gene therapy for neurodegenerative diseases, we packed Lentivirus encoding Rab9. The expressing plasmid pCDH1-MCF1-Rab9-EF1-copGFP was constructed by using molecular biological techniques. The Lentivirus encoding Rab9 cDNA was packed by Lifectamine-2000 mediated co-transfection of the plasmid pPACKH1- , pPACKH1- and pVSV- into 293T cells. DNA sequencing proved the successful construction of pCDH1-MCF1-Rab9-EF1-copGFP. After 72 hours, the expression of GFP could be detected in BV-2 cells. Western blotting revealed that the Rab9 gene expression in BALB/c mice brain was up-regulated significantly 4 weeks after injection with Lentivirus encoding Rab9, which evidenced a satisfactory increasing effect of this virus. Administration of Lenti-Rab9 to postnatal day 3 Niemann-Pick disease type C (NPC) mice reduced motor defects and prevented the weight loss associated with female NPC mice, as well as modulating the death rate of Purkinje neurons. It is concluded that the packaging of Lentivirus encoding Rab9 was successful. Lentivirus encoding Rab9 can increase the expression of Rab9 gene effectively, which might offer a novel means for the treatment of neurodegenerative diseases.

关键词： Rab9 lentivirus gene therapy gene transfer

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Optimized human factor IX expression cassettes for hepatic-directed gene therapy of hemophilia B

null

《医学前沿（英文）》 2015年第9卷第1期页码 90-99 doi: 10.1007/s11684-015-0390-2

摘要：

Gene therapy provides a potential cure for hemophilia B, and significant progress has been achieved in liver-directed gene transfer mediated by adeno-associated viral vectors. Recent clinical trials involving the use of a self-complementary adeno-associated virus serotype 8-human codon-optimized factor IX (AAV8-hFIXco) vector demonstrated encouraging efficacy with hFIX expression stabilized at 1% to 6% of normal level in patients, but safety concerns related to high vector doses are still present. Thus, further improvement of AAV vectors and hFIX expression cassette may positively contribute to the ultimate success of hemophilia B gene therapy. In this study, to obtain a higher expression level of hFIX that potentiates the coagulant capacity of recipients, human FIX expression vector was optimized by upgrading the codon adaption index and adjusting the GC content, inserting a Kozak sequence (GCCACC), and introducing a gain-of-function mutation, R338L (FIX Padua). The efficiency of the published and the presently constructed cassettes was compared through in vivo screening. In addition, the regulatory elements that control the FIX gene expression in these cassettes were screened for liver-specific effectiveness. Among all the constructed cassettes, scAAV-Pre-hFIXco-SIH-R338L, which was the construct under the control of the prothrombin enhancer and prealbumin promoter, resulted in the highest level of coagulant activity, and the expression levels of two constructed cassettes (scAAV-Chi-hFIXco-SIH-R338L and scAAV-Pre-hFIXco-SIH-R338L) were also higher than that of the published cassette (scAAV-LP1-hFIXco-SJ). In summary, our strategies led to a substantial increase in hFIX expression at the protein level or a remarkably elevated coagulant activity. Thus, these reconstructs of hFIX with AAV vector may potentially contribute to the creation of an efficacious gene therapy of hemophilia B.

关键词： factor IX hemophilia B liver-specific regulatory elements hydrodynamic gene transfer

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Bacteriophages in water pollution control: Advantages and limitations

《环境科学与工程前沿（英文）》 2021年第15卷第5期 doi: 10.1007/s11783-020-1378-y

摘要：

•Phages can be better indicators of enteric viruses than fecal indicator bacteria.

关键词： Phage Water pollution monitoring Harmful bacteria biocontrol Horizontal gene transfer Bacterial resistance

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Assessment of antibiotic resistance genes in dialysis water treatment processes

Xuan Zhu, Chengsong Ye, Yuxin Wang, Lihua Chen, Lin Feng

《环境科学与工程前沿（英文）》 2019年第13卷第3期 doi: 10.1007/s11783-019-1136-1

摘要：

• Quantitative global ARGs profile in dialysis water was investigated.

• Totally 35 ARGs were found in the dialysis treatment train.

• 29 ARGs (highest) were found in carbon filtration effluent.

• erm and mtrD-02 occurred in the final effluent.

• The effluent was associated with health risks even after RO treatment.

关键词： Dialysis water Treatment process Antibiotic resistance gene High-throughput quantitative PCR Horizontal gene transfer

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Matrix attachment regions included in a bicistronic vector enhances and stabilizes follistatin gene expressions

Xiaoming HU,Jing GUO,Chunling BAI,Zhuying WEI,Li GAO,Tingmao HU,Shorgan BOU,Guangpeng LI

《农业科学与工程前沿（英文）》 2016年第3卷第1期页码 87-96 doi: 10.15302/J-FASE-2016087

摘要： In the present study, follistatin ( ) gene expression vectors with either a bicistronic gene transfer cassette alone, or a bicistron gene cassette carrying a matrix attachment region (MAR) were constructed and transfected to bovine fetal fibroblasts. Evaluations of both the integration and expression of exogenous indicated that the pMAR-CAG-FST-IRES-AcGFP1-polyA-MAR (pMAR-FST) vector had higher capacity to form monoclonal transgenic cells than the vector without MAR, though transient transfection and integration efficiency were similar with either construct. Remarkably, protein expression in transgenic cells with the pMAR-FST vector was significantly higher than that from the bicistronic vector. Exogenous was expressed in all of the pMAR-FST transgenic mice at F , F and F . Total muscle growth in F mice was significantly greater than in wild-type mice, with larger muscles in fore and hind limbs of transgenic mice. pMAR-FST transgenic mice were also found with more evenly distributed muscle bundles and thinner spaces between sarcolemma, which suggests a correlation between transgene expression-associated muscle development and the trend of muscle growth. In conclusion, a pMAR-FST vector, which excluded the resistant genes and frame structure, enhances and stabilizes gene expressions in both transfected cells and transgenic mice.

关键词： safety of transgenic bicistron gene transfer body transgenic mice muscle development

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Invading target cells: multifunctional polymer conjugates as therapeutic nucleic acid carriers

Ulrich L?CHELT, Ernst WAGNER

《化学科学与工程前沿（英文）》 2011年第5卷第3期页码 275-286 doi: 10.1007/s11705-011-1203-z

摘要： Polymer-based conjugates are an interesting option and challenge for the design of nano-sized drug-delivery systems, as they require advanced conjugation chemistry and precise engineering. In the case of nucleic acid therapy, non-viral carriers face several biological barriers during the delivery process, namely 1) protection of the cargo from extracellular degradation, 2) avoidance of non-specific interactions with non-targeted tissues, 3) efficient entry into the target cells, 4) intracellular trafficking to the site of action and 5) cargo release. To take on these obstacles, multifunctional conjugates can act as “smart polymers” with microenvironment-sensing dynamics to facilitate the separate delivery steps. Synthesis of defined polymer architectures with precise functionalization enables structure-activity relationships to be investigated and the integration of key functions for efficient delivery. Thus bioresponsive polymer conjugates, which are equipped with molecular devices responding to the certain microenvironments within the delivery pathway (e.g. pH, redox potential, enzymes) can be assembled. This review focuses on the modular engineering and conjugation of multifunctional polymeric structures for the utilization as “tailor-made” nucleic acid carriers.

关键词： conjugate DNA gene transfer polymer RNA targeting

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人类遗传病的家系收集疾病基因定位克隆与疾病基因功能的研究

夏家辉

《中国工程科学》 2000年第2卷第11期页码 1-11

摘要：

介绍了中国医学遗传学国家重点实验室在遗传病家系收集、疾病基因定位、疾病基因克隆和疾病基因功能研究方面的研究工作。用细胞遗传学G显带技术于1975年发现了一条与鼻咽癌相关的标记染色体t（1；3）（q44；p11）；1981年将睾丸决定基因（TDF）定位于Yp11.32带；1991年以来收集遗传病家系345种共590个；1996年用显微切割、PCR、微克隆技术克隆了EXT2基因；1998年用基因家族-候选疾病基因克隆方法克隆了遗传性神经性耳聋基因GJB3；1999年用连锁分析和全基因组扫描将一种遗传性弥漫性浅表性光敏性汗孔角化症定位于12q23.2带，并在基因功能研究中发现了一个新的细胞内转运蛋白。

关键词：遗传病家系基因定位和克隆基因家族-候选疾病基因克隆基因组扫描基因功能研究

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Overexpression of netrin-1 improves neurological outcomes in mice following transient middle cerebral artery occlusion

null

《医学前沿（英文）》 2011年第5卷第1期页码 86-93 doi: 10.1007/s11684-011-0118-x

摘要：

Netrin-1 (NT-1) is one of the axon-guiding molecules that are critical for neuronal development. Because of its structural homology to the endothelial mitogens, NT-1 may have similar effects on vascular network formation. NT-1 was shown to be able to stimulate the proliferation and migration of human cerebral endothelial cells in vitro and also promote focal neovascularization in adult brain in vivo. In the present study, we reported the delivery of NT-1 using an adeno-associated virus (AAV) vector (AAV-NT-1) into mouse brain followed by transient middle cerebral artery occlusion (tMCAO). We found that AAV vectors did not elicit a detectable inflammatory response, cell loss or neuronal damage after brain transduction. The level of NT-1 was increased in the AAV-NT-1-transduced tMCAO mice compared with the control mice. Furthermore, the neurobehavioral outcomes were significantly improved in AAV-NT-1-transduced mice compared with the control animals (P<0.05) 7 days after tMCAO. Our data suggests that NT-1 plays a neuronal function recovery role in ischemic brain and that NT-1 gene transfer might present a valuable approach to treat brain ischemic disorders.

关键词： adeno-associated virus angiogenesis gene transfer ischemia middle cerebral artery occlusion netrin-1

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Molecular engineering of dendrimer nanovectors for siRNA delivery and gene silencing

Yu Cao, Xiaoxuan Liu, Ling Peng

《化学科学与工程前沿（英文）》 2017年第11卷第4期页码 663-675 doi: 10.1007/s11705-017-1623-5

摘要： Small interfering RNA (siRNA) therapeutics hold great promise to treat a variety of diseases, as long as they can be delivered safely and effectively into cells. Dendrimers are appealing vectors for siRNA delivery by virtue of their well-defined molecular architecture and multivalent cooperativity. However, the clinical translation of RNA therapeutics mediated by dendrimer delivery is hampered by the lack of dendrimers that are of high quality to meet good manufacturing practice standard. In this context, we have developed small amphiphilic dendrimers that self-assemble into supramolecular structures, which mimic high-generation dendrimers synthesized with covalent construction, yet are easy to produce in large amount and superior quality. Indeed, the concept of supramolecular dendrimers has proved to be very promising, and has opened up a new avenue for dendrimer-mediated siRNA delivery. A series of self-assembling supramolecular dendrimers have consequently been established, some of them out-performing the currently available nonviral vectors in delivering siRNA to various cell types and , including human primary cells and stem cells. This short review presents a brief introduction to RNAi therapeutics, the obstacles to their delivery and the advantages of dendrimer delivery vectors as well as our bio-inspired structurally flexible dendrimers for siRNA delivery. We then highlight our efforts in creating self-assembling amphiphilic dendrimers to construct supramolecular dendrimer nanosystems for effective siRNA delivery as well as the related structural alterations to enhance delivery efficiency. The advent of self-assembling supramolecular dendrimer nanovectors holds great promise and heralds a new era of dendrimer-mediated delivery of RNA therapeutics in biomedical applications.

关键词： gene therapy RNAi therapeutics dendrimer nanovectors gene silencing

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The antibiotic resistome: gene flow in environments, animals and human beings

null

《医学前沿（英文）》 2017年第11卷第2期页码 161-168 doi: 10.1007/s11684-017-0531-x

摘要：

The antibiotic resistance is natural in bacteria and predates the human use of antibiotics. Numerous antibiotic resistance genes (ARGs) have been discovered to confer resistance to a wide range of antibiotics. The ARGs in natural environments are highly integrated and tightly regulated in specific bacterial metabolic networks. However, the antibiotic selection pressure conferred by the use of antibiotics in both human medicine and agriculture practice leads to a significant increase of antibiotic resistance and a steady accumulation of ARGs in bacteria. In this review, we summarized, with an emphasis on an ecological point of view, the important research progress regarding the collective ARGs (antibiotic resistome) in bacterial communities of natural environments, human and animals, i.e., in the one health settings. We propose that the resistance gene flow in nature is “from the natural environments” and “to the natural environments”; human and animals, as intermediate recipients and disseminators, contribute greatly to such a resistance gene “circulation.”

关键词： antibiotic resistance resistome microbiome gene flow

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Reflections on the system of evaluation of gene-edited livestock

Ziyao FAN, Tianwen WU, Kui WU, Yulian MU, Kui LI

《农业科学与工程前沿（英文）》 2020年第7卷第2期页码 211-217 doi: 10.15302/J-FASE-2019303

摘要：

The rapid development of biotechnology has provided a greater understanding of the biological functions of major candidate genes that have important functions regarding economic traits, and new materials for livestock breeding have been obtained through gene editing (GE) and embryo manipulation with the purpose of improving quality and output and reducing the costs and risk of disease. Public concerns, particularly over safety risks and production performance, must be addressed. Evaluation is the most important component of the regulation of gene-edited livestock and is a crucial guarantee of public safety before the marketing of gene-edited animal products. Here, the system of evaluation of gene-edited livestock is discussed in terms of public safety and production performance. The search for safe and ethical applications in the GE of livestock, a case-by-case evaluation strategy, and classification and simplification are used in order to promote a more efficient, objective, comprehensive and operable evaluation system.

关键词： evaluation gene editing livestock performance safety

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Distinct gene expression pattern of mutations coordinated by target repression and promoter hypermethylation

《医学前沿（英文）》 2022年第16卷第4期页码 627-636 doi: 10.1007/s11684-020-0815-4

摘要： Runt-related transcription factor 1 (RUNX1) is an essential regulator of normal hematopoiesis. Its dysfunction, caused by either fusions or mutations, is frequently reported in acute myeloid leukemia (AML). However, RUNX1 mutations have been largely under-explored compared with RUNX1 fusions mainly due to their elusive genetic characteristics. Here, based on 1741 patients with AML, we report a unique expression pattern associated with RUNX1 mutations in AML. This expression pattern was coordinated by target repression and promoter hypermethylation. We first reanalyzed a joint AML cohort that consisted of three public cohorts and found that RUNX1 mutations were mainly distributed in the Runt domain and almost mutually exclusive with NPM1 mutations. Then, based on RNA-seq data from The Cancer Genome Atlas AML cohort, we developed a 300-gene signature that significantly distinguished the patients with RUNX1 mutations from those with other AML subtypes. Furthermore, we explored the mechanisms underlying this signature from the transcriptional and epigenetic levels. Using chromatin immunoprecipitation sequencing data, we found that RUNX1 target genes tended to be repressed in patients with RUNX1 mutations. Through the integration of DNA methylation array data, we illustrated that hypermethylation on the promoter regions of RUNX1-regulated genes also contributed to dysregulation in RUNX1-mutated AML. This study revealed the distinct gene expression pattern of RUNX1 mutations and the underlying mechanisms in AML development.

关键词： RUNX1 gene mutation acute myeloid leukemia transcriptional repression DNA methylation

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Gene delivery into isolated

Nan Zheng, Ziyuan Song, Yang Liu, Lichen Yin, Jianjun Cheng

《化学科学与工程前沿（英文）》 2017年第11卷第4期页码 521-528 doi: 10.1007/s11705-017-1612-8

摘要： The application of gene delivery materials has been mainly focused on mammalian cells while rarely extended to plant engineering. Cationic polymers and lipids have been widely utilized to efficiently deliver DNA and siRNA into mammalian cells. However, their application in plant cells is limited due to the different membrane structures and the presence of plant cell walls. In this study, we developed the cationic, -helical polypeptide that can effectively deliver DNA into both isolated protoplasts and intact leaves. The PPABLG was able to condense DNA to form nanocomplexes, and they exhibited significantly improved transfection efficiencies compared with commercial transfection reagent Lipofectamine 2000 and classical cell penetrating peptides such as poly(L-lysine), HIV-TAT, arginine9, and poly(L-arginine). This study therefore widens the utilities of helical polypeptide as a unique category of gene delivery materials, and may find their promising applications toward plant gene delivery.

关键词： α-helical polypeptide plant gene delivery protoplast intact leaves transfection

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Profiling influences of gene overexpression on heterologous resveratrol production in

Duo Liu,Bingzhi Li,Hong Liu,Xuejiao Guo,Yingjin Yuan

《化学科学与工程前沿（英文）》 2017年第11卷第1期页码 117-125 doi: 10.1007/s11705-016-1601-3

摘要： Metabolic engineering of heterologous resveratrol production in faces challenges as the precursor L-tyrosine is stringently regulated by a complex biosynthetic system. We overexpressed the main gene targets in the upstream pathways to investigate their influences on the downstream resveratrol production. Single-gene overexpression and DNA assembly-directed multigene overexpression affect the production of resveratrol as well as its precursor -coumaric acid. Finally, the collaboration of selected gene targets leads to an optimal resveratrol production of 66.14±3.74 mg·L , 2.27 times higher than the initial production in YPD medium (4% glucose). The newly discovered gene targets expressing phosphoribosylanthranilate isomerase, expressing 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase, and expressing 4-coumaryl-CoA ligase show notable positive impacts on resveratrol production in .

关键词： resveratrol aromatic amino acid DNA assembly metabolic engineering gene overexpression

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Augmentation of natural convective heat transfer by acoustic cavitation

Jun CAI, Xiulan HUAI, Shiqiang LIANG, Xunfeng LI,

《能源前沿（英文）》 2010年第4卷第3期页码 313-318 doi: 10.1007/s11708-009-0064-3

摘要： An experimental study was conducted to investigate the effects of acoustic cavitation on natural convective heat transfer from a horizontal circular tube. The experimental results indicated that heat transfer could be enhanced by acoustic cavitation and had the best effect when the head of the ultrasonic transducer was over the midpoint of the circular tube, and the distance between the head and the tube equaled 15 mm. The augmentation at low heat flux was better than that in the case of high heat flux. Based on experimental results, the correlation formula of Nusselt number for water was obtained.

关键词： heat transfer enhancement augmentation acoustic cavitation acoustic streaming convective heat transfer