Vector Insertion Research Articles

Expression of an Environmentally Friendly Enzyme, Engineered Carbonic Anhydrase, in Escherichia coli

Carbon dioxide (CO2) is one of the main causes for global warming. The most important enzyme that converts CO2 into bicarbonate and prevents CO2 emissions in the environment is carbonic anhydrase (CA). The aim of this study was to clone the heat-stable CA gene in Escherichia coli host. The CA gene coding frame of Caminibacter mediatlanticus was optimized based on the more frequently used codons by E. coli. Accordingly, nine codons were inserted in this gene fragment. After CA gene insertion in cloning vector (pGH), it was sub-cloned in pBI121 expression vector and transformed into E. coli XL1-Blue. The accuracy of gene cloning was confirmed by PCR, restriction enzyme digestion and sequencing. Cell extract was prepared from recombinant bacteria. Culture supernatants were assayed for CA activity by the color change of bromothymol blue as indicator. The time for the indicator color changes were recorded and the assays were repeated five times. The average of color change time for cell extract of recombinant, non-recombinant E. coli and control reaction were 1.5, 16, and 48 min, respectively. The amount of active enzyme was calculated to be 92.8 units/ml and the enzyme retained its activity after of incubation at 60 °C, 65 °C, and 70 °C. Due to the positive properties of CA from C. mediatlanticus, codon optimization, modification, and expression of this gene, using a simple and inexpensive method, may be used to obtain CA with enhanced properties.

Sphere-Induced Reprogramming of RPE Cells into Dual-Potential RPE Stem Cells

Background: The retinal pigment epithelium (RPE) has the potential to regenerate the entire neuroretina upon retinal injury in amphibians. In contrast, this regenerative capacity has been lost in mammals. The reprogramming of differentiated somatic cells into induced pluripotent stem cells (iPSCs) by viral transduction of exogenous stem cell factors has triggered a revolution in regenerative medicine. However, the risks of potential mutation(s) caused by random viral vector insertion in host genomes and tumor formation in recipients hamper its clinical application. One alternative is to immortalize adult stem cells with limited potential or to partially reprogram differentiated somatic cells into progenitor-like cells through non-integration protocols. Methods: Sphere-induced RPE stem cells (iRPESCs) were generated from adult mouse RPE cells. Their stem cell functionality was studied in a mouse model of retinal degeneration. The molecular mechanism underlying the sphere-induced reprogramming was investigated using microarray and loss-of-function approaches. Findings: We provide evidence that our sphere-induced reprogramming protocol can immortalize and transform mouse RPE cells into iRPESCs with dual potential to differentiate into cells that express either RPE or photoreceptor markers both in vitro and in vivo. When subretinally transplanted into mice with retinal degeneration, iRPESCs can integrate to the RPE and neuroretina, thereby delaying retinal degeneration in the model animals. Our molecular analyses indicate the Hippo signaling pathway is important in iRPESC reprogramming. Interpretation: The Hippo factor Yap1 is activated in the nuclei of cells at the borders of spheres. The factors Zeb1 and P300 downstream of the Hippo pathway are shown to bind to the promoters of the stemness genes Oct4, Klf4 and Sox2, thereby likely transactivate them to reprogram RPE cells into iRPESCs. Funding: National Natural Science Foundation of China and the National Institute of Health USA. Declaration of Interest: The authors declare no competing financial interests. Ethical Approval: All aspects of this study were conducted in accordance with the policies and guidelines set forth by the Institutional Animal Care and Use Committee and were approved by the University of Louisville, Kentucky, USA.

Two-Point One-Loop Fermionic Amplitudes in Constant Homogeneous Magnetic Field

Two-point one-loop fermionic amplitudes modifiled by a constant homogeneous magnetic fileld are studied. In addition to the amplitudes resulted after an insertion of scalar, pseudoscalar, vector and axial-vector fermionic currents, we calculate similar amplitudes with the tensor and (pseudo)scalar vertices. The crossed-field limit of these amplitudes is presented. The tensor current is a fermionic part of the Pauli Lagrangian relevant for the electromagnetic interaction of fermions through the anomalous magnetic moment and its contribution to the photon polarization operator is briefly discussed.

A Single H1 Promoter Can Drive Both Guide RNA and Endonuclease Expression in the CRISPR-Cas9 System

The RNA-guided endonuclease Cas9 (CRISPR-Cas9) genome editing system has been widely used for biomedical research and holds great potential for therapeutic applications in eukaryotes. The conventional vector-based CRISPR-Cas9 delivery system requires two different RNA polymerase promoters for expression of the guide RNA (gRNA) and Cas9 endonuclease. The large size and relative complexity of such CRISPR transgene cassettes impede their broad implementation, especially in gene therapy applications with viral vectors that have a limited packaging capacity. Here, we report the design of a single-promoter-driven CRISPR-Cas9 system that uses the dual-polymerase (Pol II and Pol III) activity of the H1 promoter. This size reduction strategy of the vector insert provides a significant titer advantage in the lentiviral vector over the regular CRISPR system.

Efficient transgene insertion in a pseudorabies virus vector by CRISPR/Cas9 and marker rescue-enforced recombination

For development of vectored vaccines against porcine pathogens the genome of the pseudorabies virus vaccine strain Bartha (PrV-Ba) was previously cloned as an infectious bacterial artificial chromosome (BAC), containing the bacterial replicon and a reporter gene cassette encoding enhanced green fluorescent protein (EGFP) at the nonessential glycoprotein G locus. To facilitate substitution of this insertion, this BAC was now modified by deletion of the adjacent promoter and initiation codon of the essential glycoprotein D (gD) gene of PrV-Ba. Furthermore, rabbit kidney (RK13) cells stably expressing Cas9 nuclease and an EGFP gene-specific guide RNA were prepared to induce site specific cleavage of the BAC DNA. After co-transfection of these cells with the modified BAC and recombination plasmids containing expression cassettes for new transgenes flanked by PrV DNA sequences including the intact 5′-end of the gD gene, >95% of the recombinants exhibited the desired gene substitutions, while no EGFP-expressing progeny virus was detectable. This approach was used for insertion and expression of the open reading frames E199L, CP204L (p30) and KP177R (p22) of African swine fever virus. The studies revealed that codon adaptation significantly enhanced expression of E199L, and that the chimeric CAG promoter increased transgene expression compared to cytomegalovirus immediate-early promoters.

Abstract 1821: Genome-wide genetic screens define the drug resistance landscape of BRAF mutant colon cancer

Abstract Background: Almost 10% of colon cancers harbor the canonical activating V600 BRAF mutation that in melanoma cancers has been shown to dramatically sensitize cells to targeted therapy and prolong survival. In the colon, such tumors are associated with worse survival. Recent clinical trials of combination therapies targeting the EGFR and MAPK pathways in these tumors have demonstrated impressive response rates. However, the clinical benefit has been limited by the rapid emergence of drug resistance. Identifying and targeting the genes complicit in this process and thus re-sensitize resistant cancer cells may be of clinical benefit. The landscape of drug resistance in cancer patients is composed of (a) loss-of-function (LoF), (b) gain-of-function (GoF) and (c) point mutation-mediated perturbations of genes, with often no single mechanism being predominant. Therefore, capturing the full breadth of resistance genes for any drug would require multiple genetic screens to be executed in parallel. Methods: We performed 3 genome-wide genetic screens in parallel in BRAF mutant colon cancer cells treated with a BRAF/MEK/EGFR inhibitor combination, to capture all of the resistance mechanisms described above. To screen for LoF events we used a genome-wide CRISPR/Cas9 sgRNA library. For GoF resistance events, a lentiviral-based insertional mutagenesis vector was used to randomly integrate the SFFV enhancer/promoter sequence throughout the genome. Finally, to saturate the genome of each cell line with point mutations we used N-Ethyl-N-nitrosourea (ENU) to randomly mutagenize every base in the genome. Each genetic screen was analysed separately to identify the relevant genes and pathways that confer drug resistance before aggregating the outputs of all three screens for a more comprehensive view of the drug resistance landscape. Results: The most recurrent and strongest hit from the CRISPR knockout screen was the inhibitor of Src family kinases, CSK. A Gaussian Kernal Convolution analysis for recurrent integrations detected in resistant clones following the insertional mutagenesis screen identified five genes as likely resistance candidates - BRAF, SOS1, MET, FRS2 and KRAS. Finally, Illumina exome sequencing of resistant clones following ENU mutagenesis revealed statistical enrichment for non-synonymous point mutations in three genes in the MAPK pathway, namely MAP2K1, NRAS and KRAS. Of note, loss of CSK was also shown to confer resistance to targeted therapies in other cancer types. Conclusion: Genome-wide genetic screens identified genes in the MAPK pathway likely to cause resistance clinically, and many of which are amenable to therapeutic targeting. Of note, the resistance as a result of loss of CSK instead activates Src signalling. It can be overcome through the addition of a Src inhibitor, and may be relevant across a range of cancer types as a resistance mechanism. Citation Format: Jonathan Brammeld, Marco Ranzani, Elizabeth A. Coker, Stacey Price, Theodoros I. Roumeliotis, Barbara Lupo, Mi Petljak, Steven P. Williams, Francesco Iorio, Francesco Sassi, Nanne Aben, Kosuke Yusa, Livio Trusolino, Lodewyk Wessels, David J. Adams, Mathew Garnett, Bissan Al-Lazikani, Jyoti S. Choudhary, Andrea Bertotti, Ultan McDermott. Genome-wide genetic screens define the drug resistance landscape of BRAF mutant colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1821.

Abstract A29: Generation of a novel transgenic mouse model with regulatable β-catenin expression to examine importance of activated β-catenin signaling to long-term maintenance of mammary tumor growth

Abstract Stabilized β-catenin expression is a well described initiator of mammary tumorigenesis in the mouse and elevated nuclear expression has been observed in human triple-negative tumor samples. However, the importance of stabilized β-catenin to continued tumor growth after initiation, and in the context of other driver mutations, has yet to be elucidated. To ascertain the importance of stabilized β-catenin after tumor initiation, we generated a novel transgenic mouse model, utilizing the tet-off system, to control expression of stabilized β-catenin. Expression was tissue restricted through insertion of a stop cassette flanked by loxP sites upstream of stabilized β-catenin and firefly luciferase, which were coexpressed to allow repeated bioluminescent imaging of experimental mice. Insertion of the expression vector was targeted to the Rosa26 locus in R1 embryonic stem cells through homologous recombination. Following aggregation and chimera generation, two founder lines were established. Pups from early litters carrying one allele of regulatable stabilized β-catenin at the Rosa26 locus, but not targeted by Cre, were smaller in size compared with wild-type littermates. They also developed perianal skin lesions around 2-3 weeks of age, with severe necrosis evident by 3-4 weeks of age, requiring the animals to be euthanized. Backcrossing to the desired FVB strain did not alter development of skin lesions, which were observed in offspring from both founder lines. While aberrant gene expression was not observed in these animals, placing breeding females on doxycycline chow through pregnancy, up until pup weaning, eliminated the condition, suggesting a role for the tet machinery in the development of the condition. Maintenance of breeding cages on doxycycline chow allowed for healthy transgenic pups to survive past 3 weeks of age. Initially, six animal cohorts were established, along with control animals, using two different mammary Cre strains, WAP-Cre and MMTV-NLST-Cre, crossed with regulatable, stabilized β-catenin (St-bcatFL) animals, as well as animals expressing mutated p53-R270H. While mammary tumors developed in animals expressing Cre-targeted regulatable St-bcatFL, the penetrance and growth kinetics were lower than observed with other mammary mouse models expressing stabilized β-catenin. As significantly fewer animals developed tumors, we were only able to test the effect of turning off stabilized β-catenin expression following tumor initiation in a small number of animals, with tumor stasis but not regression observed in animals expressing Cre-targeted St-bcatFL, but not in animals expressing both St-bcatFL and p53-R270H. The regulatable St-bcatFL mice were also crossed with animals expressing mutant PIK3CA*E545K and MMTV-NLST-Cre, to examine the importance of stabilized β-catenin to the rapid tumor growth we have observed in mice expressing stabilized β-catenin through deletion of exon 3, and PIK3CA*E545K. While all of the PIK3CA*E545K;Exon3 fl/+; Cre+ animals examined developed multiple mammary tumors by 5 months of age, the PIK3CA*E545K;St-bcatFL;Cre+ mice failed to develop tumors within the same time period. These results indicate that mammary tumor development, including penetrance and growth kinetics, differs significantly between our novel regulatable stabilized β-catenin mouse model and other more aggressive models of stabilized β-catenin. These differences will require alternative methods to determine whether stabilized β-catenin expression is required for the long-term maintenance of tumor growth. Funding for this project was provided by the Terry Fox Foundation, as well as the Canadian Breast Cancer Foundation. Citation Format: Jennifer L. Gorman, Jessica R. Adams, Emma M. Jones, Sean E. Egan, James R. Woodgett. Generation of a novel transgenic mouse model with regulatable β-catenin expression to examine importance of activated β-catenin signaling to long-term maintenance of mammary tumor growth [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A29.

Collection of homozygous mutant mouse embryonic stem cells arising from autodiploidization during haploid gene trap mutagenesis.

Haploid mouse embryonic stem cells (ESCs), in which a single hit mutation is sufficient to produce loss-of-function phenotypes, have provided a powerful tool for forward genetic screening. This strategy, however, can be hampered by undesired autodiploidization of haploid ESCs. To overcome this obstacle, we designed a new methodology that facilitates enrichment of homozygous mutant ESC clones arising from autodiploidization during haploid gene trap mutagenesis. Haploid mouse ESCs were purified by fluorescence-activated cell sorting to maintain their haploid property and then transfected with the Tol2 transposon-based biallelically polyA-trapping (BPATrap) vector that carries an invertible G418 plus puromycin double selection cassette. G418 plus puromycin double selection enriched biallelic mutant clones that had undergone autodiploidization following a single vector insertion into the haploid genome. Using this method, we successfully generated 222 homozygous mutant ESCs from 2208 clones by excluding heterozygous ESCs and ESCs with multiple vector insertions. This relatively low efficiency of generating homozygous mutant ESCs was partially overcome by cell sorting of haploid ESCs after Tol2 BPATrap transfection. These results demonstrate the feasibility of our approach to provide an efficient platform for mutagenesis of ESCs and functional analysis of the mammalian genome.

Surgical anatomy of the round window-Implications for cochlear implantation.

The round window is an important portal for the application of active hearing aids and cochlear implants. The anatomical and topographical knowledge about the round window region is a prerequisite for successful insertion for a cochlear implant electrode. To sum up current knowledge about the round window anatomy and to give advice to the cochlear implant surgeon for optimal placement of an electrode. Systematic Medline search. Search term "round window[Title]" with no date restriction. Only publications in the English Language were included. All abstracts were screened for relevance, that is a focus on surgical anatomy of the round window. The search results were supplemented with hand searching of selected reviews and reference lists from included studies. Subjective assessment. There is substantial variability in size and shape of the round window. The round window is regarded as the most reliable surgical landmark to safely locate the scala tympani. Factors affecting the optimal trajectory line for atraumatic electrode insertion are anatomy of the round window, the anatomy of the intracochlear hook region and the variable orientation and size of the cochlea's basal turn. The very close relation to the sensitive inner ear structures necessitates a thorough anatomic knowledge and careful insertion technique, especially when implanting patients with residual hearing. In order to avoid electrode migration between the scalae and to achieve protect the modiolus and the basilar membrane, it is recommended to aim for an electrode insertion vector from postero-superior to antero-inferior.

Improving the efficiency of gene insertion in a human artificial chromosome vector and its transfer in human-induced pluripotent stem cells.

A human artificial chromosome (HAC) vector has potential to overcome the problems of stable gene expression associated with plasmid, transposon, and virus-based vectors, such as insertional mutagenesis, position effect, uncontrollable copy number, unstable gene expression, and DNA size limitation. The main advantages of the HAC are its episomal nature and ability to accommodate DNA inserts of any size. However, HAC vectors have two disadvantages: low efficiency of gene insertion and lack of reports regarding the successful HAC transfer to human-induced pluripotent stem cells (iPSCs). We here provide the first report of a method for the efficient transfer of HAC to human iPSCs for obtaining reproducible experimental results. Moreover, we achieved a 10% increase in the gene insertion efficiency in the HAC vector using our new site-specific recombination systems VCre/VloxP and SCre/SloxP.

Manufacturing and Delivering Genome-Editing Proteins.

Genome-editing technologies have revolutionized the biomedical sciences by providing researchers with the ability to quickly and efficiently modify genes. While programmable nucleases can be introduced into cells using a variety of techniques, their delivery as purified proteins is an effective approach for limiting off-target effects. Here, we describe step-by-step procedures for manufacturing and delivering genome-modifying proteins-including Cas9 ribonucleoproteins (RNPs) and TALE and zinc-finger nucleases-into mammalian cells. Protocols for combining Cas9 RNP with naturally recombinogenic adeno-associated virus (AAV) donor vectors for the seamless insertion of transgenes by homology-directed genome editing are also provided.

Multi-color RGB marking enables clonality assessment of liver tumors in a murine xenograft model.

We recently introduced red-green-blue (RGB) marking for clonal cell tracking based on individual color-coding. Here, we applied RGB marking to study clonal development of liver tumors. Immortalized, non-tumorigenic human fetal hepatocytes expressing the human telomerase reverse transcriptase (FH-hTERT) were RGB-marked by simultaneous transduction with lentiviral vectors encoding mCherry, Venus, and Cerulean. Multi-color fluorescence microscopy was used to analyze growth characteristics of RGB-marked FH-hTERT in vitro and in vivo after transplantation into livers of immunodeficient mice with endogenous liver damage (uPA/SCID). After initially polyclonal engraftment we observed oligoclonal regenerative nodules derived from transplanted RGB-marked FH-hTERT. Some mice developed monochromatic invasive liver tumors; their clonal origin was confirmed both on the molecular level, based on specific lentiviral-vector insertion sites, and by serial transplantation of one tumor. Vector insertions in proximity to the proto-oncogene MCF2 and the transcription factor MITF resulted in strong upregulation of mRNA expression in the respective tumors. Notably, upregulated MCF2 and MITF expression was also observed in 21% and 33% of 24 human hepatocellular carcinomas analyzed. In conclusion, liver repopulation with RGB-marked FH-hTERT is a useful tool to study clonal progression of liver tumors caused by insertional mutagenesis in vivo and will help identifying genes involved in liver cancer.

VISPA2: a scalable pipeline for high-throughput identification and annotation of vector integration sites

BackgroundBioinformatics tools designed to identify lentiviral or retroviral vector insertion sites in the genome of host cells are used to address the safety and long-term efficacy of hematopoietic stem cell gene therapy applications and to study the clonal dynamics of hematopoietic reconstitution. The increasing number of gene therapy clinical trials combined with the increasing amount of Next Generation Sequencing data, aimed at identifying integration sites, require both highly accurate and efficient computational software able to correctly process “big data” in a reasonable computational time.ResultsHere we present VISPA2 (Vector Integration Site Parallel Analysis, version 2), the latest optimized computational pipeline for integration site identification and analysis with the following features: (1) the sequence analysis for the integration site processing is fully compliant with paired-end reads and includes a sequence quality filter before and after the alignment on the target genome; (2) an heuristic algorithm to reduce false positive integration sites at nucleotide level to reduce the impact of Polymerase Chain Reaction or trimming/alignment artifacts; (3) a classification and annotation module for integration sites; (4) a user friendly web interface as researcher front-end to perform integration site analyses without computational skills; (5) the time speedup of all steps through parallelization (Hadoop free).ConclusionsWe tested VISPA2 performances using simulated and real datasets of lentiviral vector integration sites, previously obtained from patients enrolled in a hematopoietic stem cell gene therapy clinical trial and compared the results with other preexisting tools for integration site analysis. On the computational side, VISPA2 showed a > 6-fold speedup and improved precision and recall metrics (1 and 0.97 respectively) compared to previously developed computational pipelines. These performances indicate that VISPA2 is a fast, reliable and user-friendly tool for integration site analysis, which allows gene therapy integration data to be handled in a cost and time effective fashion. Moreover, the web access of VISPA2 (http://openserver.itb.cnr.it/vispa/) ensures accessibility and ease of usage to researches of a complex analytical tool. We released the source code of VISPA2 in a public repository (https://bitbucket.org/andreacalabria/vispa2).

ExVivo Selection of Transduced Hematopoietic Stem Cells for Gene Therapy of β-Hemoglobinopathies.

Although gene transfer to hematopoietic stem cells (HSCs) has shown therapeutic efficacy in recent trials for several individuals with inherited disorders, transduction incompleteness of the HSC population remains a hurdle to yield a cure for all patients with reasonably low integrated vector numbers. In previous attempts at HSC selection, massive loss of transduced HSCs, contamination with non-transduced cells, or lack of applicability to large cell populations has rendered the procedures out of reach for human applications. Here, we fused codon-optimized puromycin N-acetyltransferase to herpes simplex virus thymidine kinase. When expressed from a ubiquitous promoter within a complex lentiviral vector comprising the βAT87Q-globin gene, viral titers and therapeutic gene expression were maintained at effective levels. Complete selection and preservation of transduced HSCs were achieved after brief exposure to puromycin in the presence of MDR1 blocking agents, suggesting the procedure’s suitability for human clinical applications while affording the additional safety of conditional suicide.

Current status of vaccines against Zika virus

The involvement of the pregnant woman during the epidemic caused by the Zika virus is one of the main reasons for the development of preventive measures. Vaccination of children or pre-fertile female may be the best approach to this epidemic infection.Different vaccine platforms are being developed based on the use of plasmid DNA including the prM/M and E structural proteins of the virus. They have shown in animal models, both mice and non-human primates, a 100% protection against exogenous infection. The main immune response is a humoral neutralising type. Some of these have already passed Phase 1 clinical trials. Chimeric vaccines are also being evaluated using the dengue virus genome, an adenoviral insertion vector and other attenuated and inactivated particle vectors. The results in animals are generally satisfactory.When validating any of these vaccines it is very important to establish their ability to respond, immunity and role of the previous antibodies against dengue or other flaviviruses. As well as the possible induction of autoimmune phenomena such as Guillain–Barre syndrome and other neurological affections.In spite of all this is progressing in a deeper knowledge not only of the virology of the Zika virus but of the development of new genetic vaccine platforms.

Situación actual de las vacunas frente al virus Zika

The involvement of the pregnant woman during the epidemic caused by the Zika virus is one of the main reasons for the development of preventive measures. Vaccination of children or pre-fertile female may be the best approach to this epidemic infection.Different vaccine platforms are being developed based on the use of plasmid DNA including the prM/M and E structural proteins of the virus. They have shown in animal models, both mice and non-human primates, a 100% protection against exogenous infection. The main immune response is a humoral neutralizing type. Some of these have already passed Phase 1 clinical trials. Chimeric vaccines are also being evaluated using the dengue virus genome, an adenoviral insertion vector and other attenuated and inactivated particle vectors. The results in animals are generally satisfactory.When validating any of these vaccines it is very important to establish their ability to respond, immunity and role of the previous antibodies against dengue or other flaviviruses. As well as the possible induction of autoimmune phenomena such as Guillain-Barre syndrome and other neurological affections.In spite of all this is progressing in a deeper knowledge not only of the virology of the Zika virus but of the development of new genetic vaccine platforms.

Defining the Hook Region Anatomy of the Guinea Pig Cochlea for Modeling of Inner Ear Surgery.

The aim of this study was to describe the hook region anatomy of the guinea pig cochlea to identify the optimal surgical approach for cochlear implantation and to determine what anatomical structures are at risk. Animal studies investigating hearing loss after cochlear implantation surgery are currently constrained by the lack of a reproducible implantation model. Guinea pig cochleae were imaged using thin-sheet laser imaging microscopy. Images were stitched, reconstructed, and segmented for analysis. Insertion vectors were determined by tracing their paths to the outer wall and converting to Cartesian coordinates. Spherical surface and multiplane views were generated to analyze outer wall and radial forces of the insertion vector. Thin-sheet laser imaging microscopy enabled quantitative, whole specimen analysis of the soft and bony tissue relationships of the complex cochlear hook region in any desired plane without loss of image quality. Round window or cochleostomy approaches in the anteroinferior plane avoided direct damage to cochlear structures. Cochleostomy approach had large interindividual variability of angular depth and outer wall forces but predictable radial force. The guinea pig hook region and lower basal turn have similar structural relationships to humans. Careful cochleostomy placement is essentially for minimizing cochlear trauma and for ensuring a straight insertion vector that successfully advances around the outer wall. Experiments with guinea pigs that control for the surgical approach are likely to provide useful insights into the aetiology and the development of therapies directed at postimplantation hearing loss.

Torque Ripple Reduction in Direct Torque Control of Five-Phase Induction Motor Using Fuzzy Controller with Optimized Voltage Vector Selection Strategy

This paper presents a torque ripple reduction method of direct torque control (DTC) using fuzzy controller with optimal selection strategy of voltage vectors in a five-phase induction motor. The conventional DTC method has some drawbacks. First, switching frequency changes according to the hysteresis bands and motor's speed. Second, the torque ripple is rapidly increased in long control period. In order to solve these problems, some/most papers have proposed torque ripple reduction methods by using the optimal duty ratio of the non-zero voltage vector. However, these methods are complicated in accordance with the parameter. If this drawback is eliminated, the torque ripple can be reduced compared with conventional method. In addition, the DTC can be simply controlled without the use of the parameter. Therefore, the proposed algorithm is changing the voltage vector insertion time by using the designed fuzzy controller. Also, the optimized voltage vector selection method is used in accordance with the torque error. Simulation and experimental results show effectiveness of the proposed control algorithm.

Simple Cloning by Prolonged Overlap Extension-PCR with Application to the Preparation of Large-Size Random Gene Mutagenesis Library in Escherichia coli.

We developed a simple method (simple cloning) for subcloning DNA fragments into any location of a targeted vector without the need of restriction enzyme, ligase, exonuclease, or recombinase in Escherichia coli. This technology can be applied to common E. coli hosts (e.g., DH5α, JM109, TOP10, BL21(DE3)). The protocol includes three steps: (1) generate DNA insert and linear vector backbone by regular high-fidelity PCR, where these two DNA fragments contain 3' and 5' overlapping termini; (2) generate DNA multimers based on these two DNA fragments by using prolonged overlap extension-PCR (POE-PCR) without primers added; and (3) transform POE-PCR product to competent Escherichia coli cells directly, yielding the desired plasmid. Simple cloning provides a new cloning method with great simplicity and flexibility. Furthermore, this new method can be modified for the preparation of a large-size mutant library for directed evolution in E. coli. Using this method, it is very easy to generate a mutant library with a size of more than 10(7) per 50 μL of the POE-PCR product within 1 day.

Role of SpdA in Cell Spreading and Phagocytosis in Dictyostelium.

Dictyostelium discoideum is a widely used model to study molecular mechanisms controlling cell adhesion, cell spreading on a surface, and phagocytosis. In this study we isolated and characterize a new mutant created by insertion of a mutagenic vector in the heretofore uncharacterized spdA gene. SpdA-ins mutant cells produce an altered, slightly shortened version of the SpdA protein. They spread more efficiently than WT cells when allowed to adhere to a glass substrate, and phagocytose particles more efficiently. On the contrary, a functional spdA knockout mutant where a large segment of the gene was deleted phagocytosed less efficiently and spread less efficiently on a substrate. These phenotypes were highly dependent on the cellular density, and were most visible at high cell densities, where secreted quorum-sensing factors inhibiting cell motility, spreading and phagocytosis are most active. These results identify the involvement of SpdA in the control of cell spreading and phagocytosis. The underlying molecular mechanisms, as well as the exact link between SpdA and cell spreading, remain to be established.