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961.
Use of historical data and real-world evidence holds great potential to improve the efficiency of clinical trials. One major challenge is to effectively borrow information from historical data while maintaining a reasonable type I error and minimal bias. We propose the elastic prior approach to address this challenge. Unlike existing approaches, this approach proactively controls the behavior of information borrowing and type I errors by incorporating a well-known concept of clinically significant difference through an elastic function, defined as a monotonic function of a congruence measure between historical data and trial data. The elastic function is constructed to satisfy a set of prespecified criteria such that the resulting prior will strongly borrow information when historical and trial data are congruent, but refrain from information borrowing when historical and trial data are incongruent. The elastic prior approach has a desirable property of being information borrowing consistent, that is, asymptotically controls type I error at the nominal value, no matter that historical data are congruent or not to the trial data. Our simulation study that evaluates the finite sample characteristic confirms that, compared to existing methods, the elastic prior has better type I error control and yields competitive or higher power. The proposed approach is applicable to binary, continuous, and survival endpoints. 相似文献
962.
Efforts to leverage clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) for targeted genomic modifications in mammalian cells are limited by low efficiencies and heterogeneous outcomes. To aid method optimization, we developed an all-in-one reporter system, including a novel superfolder orange fluorescent protein (sfOrange), to simultaneously quantify gene disruption, site-specific integration (SSI), and random integration (RI). SSI strategies that utilize different donor plasmid formats and Cas9 nuclease variants were evaluated for targeting accuracy and efficiency in Chinese hamster ovary cells. Double-cut and double-nick donor formats significantly improved targeting accuracy by 2.3–8.3-fold and 19–22-fold, respectively, compared to standard circular donors. Notably, Cas9-mediated donor linearization was associated with increased RI events, whereas donor nicking minimized RI without sacrificing SSI efficiency and avoided low-fidelity outcomes. A screen of 10 molecules that modulate the major mammalian DNA repair pathways identified two inhibitors that further enhance targeting accuracy and efficiency to achieve SSI in 25% of transfected cells without selection. The optimized methods integrated transgene expression cassettes with 96% efficiency at a single locus and with 53%–55% efficiency at two loci simultaneously in selected clones. The CRISPR-based tools and methods developed here could inform the use of CRISPR/Cas9 in mammalian cell lines, accelerate mammalian cell line engineering, and support advanced recombinant protein production applications. 相似文献
963.
POPULATION STRUCTURE OF THE BOTTLENOSE DOLPHIN (TURSIOPS TRUNCATUS) AS DETERMINED BY RESTRICTION ENDONUCLEASE ANALYSIS OF MITOCHONDRIAL DNA 总被引:1,自引:0,他引:1
Abstract: Restriction fragment length polymorphisms of mitochondrial DNA (mtDNA) were used to test for population subdivision in the bottlenose dolphin (Tursiops truncatus). Atlantic and Pacific dolphin mtDNA samples exhibited distinctly different haplotypes (approximately 2.4% sequence divergence), indicating a lack of gene exchange. Within the Atlantic Ocean, mtDNA samples from the Gulf of Mexico and the Atlantic Coast were also found to be distinct, with a sequence divergence of approximately 0.6%. The Atlantic Coast–Gulf of Mexico dichotomy is consistent with patterns of genetic variation from other marine and coastal organisms from this region, and supports the hypothesized role of bio-geographic events in promoting the divergence of these and other forms. Regional differentiation was identified along the Atlantic Coast, whereas low sequence divergences among haplotypes and consistent haplotype frequencies across populations suggested considerable gene exchange among Gulf of Mexico populations. A highly divergent haplotype found in two individuals from two localities in the Gulf of Mexico is best explained by dispersal from either a distinct offshore Gulf stock or an unsampled Atlantic Coast stock. Additional samples are required to test for the existence of a distinct offshore race and, if it exists, to identify its distribution and contribution to population structure. 相似文献
964.
965.
Citrus Tristeza Virus (CTV), usually occurs in nature as a mixture of genotypes. Six naturally infected citrus
(Citrus sinensis) trees grafted on sour orange rootstock were collected from three citrus growing governorates
in Egypt (Sharqia, Qalyubia and Garbia). In this study, RT-PCR, Single-Strand Conformation Polymorphism
(SSCP) and nucleotide sequence analysis were used for four independent CTV genomic regions (p65, p18,
p20, and p23) to detect and assess the sequence and genetic variabilities among CTV Egyptian isolates. RTPCR products (650 bp) for the CTV p23 gene obtained from the selected isolates were used for the SSCP analysis
and DNA sequencing. SSCP patterns of p23 gene for individual isolates yielded different complex haplotype patterns. Nucleotide sequence analysis of p23 region amplified from six isolates under study revealed that p23 shared
high nucleotide identity 98.7% with T36 isolate from USA, Florida. Phylogenetic analysis of p23 gene indicated a
close evolutionary relationship between all examined isolates and Qaha isolate (T36 isolate group), suggesting that
they may have originated from closely related ancestors. Nucleotide sequence analysis of the three genes located
on CTV 3′-coterminal overhang, p18, p20 and p65, amplified from isolate A3, Sharqia governorate, revealed that
the p18, p65, and p20 genes were related to the T3-KB isolate from South Africa with 99%–100% sequence
homology. Phylogenetic relationship analysis for p65, p18 and p20 ORFs clustered the current A3 isolate with
T3 genotype group. The recombination analysis identified three of six isolates from Sharqia, and Garbia as potential recombinant for p23 gene. The isolates T36 and T3 were identified as major donors for recombination events
in isolate A3. Our results concluded that p23 ORF likely to be as a hotspot region for recombination and originated through recombination event. The current study indicated that recombination is an important factor for the
origin of CTV strains in Egypt. 相似文献
966.
967.
Xuan Cuong Mai Chia-Rui Shen Chao-Lin Liu Dang Mau Trinh Minh Ly Nguyen 《Journal of phycology》2023,59(4):775-784
Species identification of Scenedesmus-like microalgae, comprising Desmodesmus, Tetradesmus, and Scenedesmus, has been challenging due to their high morphological and genetic similarity. After developing a DNA signaturing tool for Desmodesmus identification, we built a DNA signaturing database for Tetradesmus. The DNA signaturing tool contained species-specific nucleotide sequences of Tetradesmus species or strain groups with high similarity in ITS2 sequences. To construct DNA signaturing, we collected data on ITS2 sequences, aligned the sequences, organized the data by ITS2 sequence homology, and determined signature sequences according to hemi-compensatory base changes (hCBC)/CBC data from previous studies. Four Tetradesmus species and 11 strain groups had DNA signatures. The signature sequence of the genus Tetradesmus, TTA GAG GCT TAA GCA AGG ACCC, recognized 86% (157/183) of the collected Tetradesmus strains. Phylogenetic analysis of Scenedesmus-like species revealed that the Tetradesmus species were monophyletic and closely related to each other based on branch lengths. Desmodesmus was suggested to split into two subgenera due to their genetic and morphological distinction. Scenedesmus must be analyzed along with other genera of the Scenedesmaceae family to determine their genetic relationships. Importantly, DNA signaturing was integrated into a database for identifying Scenedesmus-like species through BLAST. 相似文献
968.
Molecular cytological evidence for gradual telomere synthesis at the broken chromosome ends in wheat
Hisashi Tsujimoto 《Journal of plant research》1993,106(3):239-244
Telomere formation of the normal and broken chromosomes of common wheat,Triticum aestivum, was investigated byin situ hybridization using the biotin-labeled probe of telomere repetitive sequences (pAtT4) ofArabidopsis thaliana with subsequent amplification by an antibody. After double and triple amplification, prominent signals appeared at all the
telomeric regions of the normal chromosomes.
Prominent signals also emerged at the broken ends of the telocentric and deletion chromosomes that had passed through more
than one generation since the appearance. However, broken ends that had passed through only the stages of gametogenesis, fertilization,
embryogenesis and root development did not show complete signals such as found in normal telomeres. These findings indicate
that a certain time or stage is required for synthesis of the telomeric repetitive sequences with a complete length. Nevertheless,
because the broken ends without complete telomere sequences were also healed, restoration of the normal complement of telomere
sequences is not necessary for healing of broken ends. 相似文献
969.
Yoshihiro Izumi Kanji Ono Masayuki Takamiya Kiichi Fukui 《Journal of plant research》1993,106(4):319-325
Using cultured cells of the hornwortAnthoceros punctatus, the change in the relative chloroplast DNA content in each stage of chloroplast division was investigated to clarify the
relationship between the division cycle of a chloroplast and a cell nucleus. Samples of cultured cells were stained with 4′,6-diamidino-2-phenylindole
(DAPI) and then observed with an epifluorescence microscope and a chromosome image analyzing system (CHIAS). A chloropiast
in cultured cells duplicated DNA with an increase in size. When a chloroplast began to divide, it was constricted in the middle,
taking a dumbbell shape, and then divided into two daughter chloroplasts. In cultured cells of this species, the pattern of
quantitative change of chloroplast DNA, that is, the DNA replication pattern of chloroplasts, corresponded to that of cell
nuclear DNA in mitosis. 相似文献
970.
【背景】DNA组装技术是基因组合成中的一个关键技术。探索低成本、高效率的基因组合成技术一直是合成生物学的重要研究领域。在某些细菌如变铅青链霉菌中,DNA上有磷硫酰化修饰(简称硫修饰),而在另一些细菌如天蓝色链霉菌中存在一种含有硫修饰识别结构域(sulfur-binding domain, SBD)的识别蛋白,可以特异性识别DNA上的硫修饰,这启发了我们发展出一种新的DNA组装技术。【目的】探究在DNA末端硫修饰的连接中,T4 DNA连接酶与SBD相融合蛋白和单独的T4 DNA连接酶相比,是否有更高的连接效率。【方法】根据同源重组原理,设计硫修饰引物,扩增硫修饰的DNA片段。构建T4 DNA连接酶与SBD融合蛋白的3种表达载体T4-linker-SBD(Hga)、T4-linker-SBD(Spr)和T4-linker-SBD(Mmo),表达纯化以上3种融合蛋白。比较3组浓度梯度(2.4、0.24、0.024 mg/mL) T4 DNA连接酶与融合蛋白在2.5 kb和8.0 kb DNA片段连接上的差异。【结果】DNA末端硫修饰的2.5kb和8.0kb的两端片段均能扩增,而且3种融合蛋白... 相似文献