High-efficiency and multilocus targeted integration in CHO cells using CRISPR-mediated donor nicking and DNA repair inhibitors

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.     

Study on TFF1 and PALB2 gene variants associated with gastric carcinoma risk in the Chinese Han population

ObjectiveGastric carcinoma (GC) has received extensive attention due to its complex pathogenesis. Studies have shown that the expression of Trefoil factor 1 (TFF1) and Partner and localiser of BRCA2 (PALB2) genes promotes the occurrence of GC. Therefore, we investigated whether TFF1 and PALB2 gene polymorphisms are associated with GC risk in the Chinese Han population.MethodsA total of 509 GC cases and 505 controls were recruited, and single nucleotide polymorphisms (SNPs) of TFF1 and PALB2 in these subjects were genotyped. The association between each candidate polymorphism and GC risk was assessed by calculating odds ratios (ORs) and 95% confidence intervals (CIs). The visualization of gene-gene interactions and functional enrichment analysis were then performed using Cytoscape software and the R package “cluster profile”.ResultsThe TFF1 rs2156310 polymorphism significantly reduced the predisposition to GC in people under 60 years of age (AA vs. AG - GG, OR = 0.58, 95% CI = 0.35–0.97, p = 0.036). The gender-stratified analysis found that PALB2 rs513313 was significantly associated with the risk of GC in males (CT vs. TT, OR = 1.51, 95% CI = 1.06–2.15, p = 0.022). Besides, PALB2 rs249954 significantly reduced the susceptibility to GC in females (AA vs GG, OR = 0.42, 95% CI = 0.19–0.94, p = 0.034).ConclusionOur results revealed that TFF1 and PALB2 gene polymorphisms were correlated with the genetic susceptibility to GC, providing certain data support for researchers to further study the mechanism of GC.     

2种中草药对福寿螺的杀灭效果及对肝脏影响

【目的】有效控制福寿螺产生的危害,为实现中草药的资源化利用提供依据。【方法】以甘草、陈皮为植物源灭螺剂,进行不同浓度梯度(0、0.5、1.5、3.0、4.5、6.0 g·L^-1)提取液浸泡下的灭螺活性探究。【结果】甘草组、陈皮组处理下福寿螺死亡率随时间的增长在不同浓度下毒杀效果明显。对48 h时福寿螺死亡率数据进行概率单位法回归分析,发现甘草组(LC₂₅、LC₅₀、LC₇₅)<陈皮组(LC₂₅、LC₅₀、LC₇₅),说明福寿螺对甘草更敏感。为进一步探究福寿螺死亡原因,在前期研究的基础上深入探讨甘草、陈皮提取液对福寿螺的肝脏的影响,在亚致死浓度甘草0.511 g·L^-1 (LC₂₅)、0.940 g·L^-1 (LC₅₀)、1.727 g·L^-1 (LC₇₅)和陈皮3.892 g·L^-1 (LC₂₅)、4.863 g·L^-1 (LC₅₀)、6.076 g·L^-1 (LC₇₅)处理下对福寿螺肝脏进行扫描电镜观察,结果表明,相比对照组,处理组福寿螺肝脏组织表面干瘪,失去水分,受损严重时,质地松散,表面组织脱落。【结论】2种植物提取液均可以较好地灭杀福寿螺,且严重破坏福寿螺核心组织肝脏,甘草、陈皮可作为植物源灭螺剂进一步研究开发利用。     

放散式体外冲击波穴位治疗联合中药熏洗治疗老年膝骨关节炎的疗效分析

摘要 目的：评价放散式体外冲击波（rESW）穴位治疗联合中药熏洗在老年膝骨关节炎（KOA）中的应用效果。方法：选入2021年2月~2022年8月我院收治的老年KOA患者76例,根据治疗方法不同分为对照组和观察组,各38例,两组均予以中药熏洗,观察组加用rESW穴位治疗。评价两组的治疗效果、膝关节功能、疼痛程度等指标,并进行统计比较。结果：与治疗前相比,两组治疗2周、治疗4周时的VAS评分明显下降（P＜0.05）,而观察组下降幅度更大,与对照组差异显著（P＜0.05）;两组治疗前Lysholm评分无明显差异（P＞0.05）,而观察组治疗2周、治疗4周时的Lysholm评分显著高于对照组（P＜0.05）;观察组治疗显效率和有效率均显著高于对照组（P＜0.05）;观察组治疗2周、4周时的血清IL-1β、TNF-α和MMP-13水平较对照组低（P＜0.05）。结论：rESW穴位联合中药熏洗治疗老年KOA患者疗效显著,可缓解疼痛症状,改善膝关节功能,控制炎症反应,且操作简单,值得推荐。     

Comparison of mechanistic and hybrid modeling approaches for characterization of a CHO cultivation process: Requirements,pitfalls and solution paths

Despite the advantages of mathematical bioprocess modeling, successful model implementation already starts with experimental planning and accordingly can fail at this early stage. For this study, two different modeling approaches (mechanistic and hybrid) based on a four-dimensional antibody-producing CHO fed-batch process are compared. Overall, 33 experiments are performed in the fractional factorial four-dimensional design space and separated into four different complex data partitions subsequently used for model comparison and evaluation. The mechanistic model demonstrates the advantage of prior knowledge (i.e., known equations) to get informative value relatively independently of the utilized data partition. The hybrid approach displayes a higher data dependency but simultaneously yielded a higher accuracy on all data partitions. Furthermore, our results demonstrate that independent of the chosen modeling framework, a smart selection of only four initial experiments can already yield a very good representation of a full design space independent of the chosen modeling structure. Academic and industry researchers are recommended to pay more attention to experimental planning to maximize the process understanding obtained from mathematical modeling.     

中国菌物分类学和多样性研究的历史与现状概况

我国菌物分类学研究始于20世纪初,经过百余年的不断探索和发展,取得了丰硕的成果,并逐渐走进世界前列。本研究通过对世界菌物名称信息库Fungal Names进行数据统计,对发现自中国的菌物新物种和中国学者发表菌物新分类单元等数据开展分析,从中揭示中国菌物分类学的历史和发展趋势。过去,一共有2 214位中国学者参与发表了15 626个菌物新分类单元,包括 3个新纲、27个新目及亚目、117个新科及亚科、769个新属及亚属、11 100个新种、322个新种下单元和3 288个新组合。在全球已知的菌物物种中,自中国发现的新物种有10 233种,隶属于 3界13门44纲174目572科2 379属,占全球已知物种多样性的6.84%,居世界第二位。地理分布上,我国西南地区(云南、四川、贵州、西藏)和低纬度的热带、亚热带地区(中国台湾、广东)发现的新物种最多。根据每年发现的新分类单元数量趋势和命名作者的构成,可将中国菌物分类学的发展历史分为五个阶段：外人在华采菌及研究(1750s-1929)、中国菌物分类学起步(1930-1949)、新中国菌物分类学早期发展(1950-1977)、全国性菌物标本采集与研究(1978-2010)、走进世界前列(2011至今)。本研究对每个发展时期的分类学概况和重要历史事件进行了总结和回顾,通过上述综述性研究,有助于系统地了解中国菌物分类学不同阶段的发展趋势和研究概况,为学科当下和未来的发展提供参考。     

中医正骨手法改善桡骨远端伸直型骨折患者腕关节功能的临床研究及疗效的影响因素分析

摘要 目的：观察中医正骨手法在桡骨远端伸直型骨折中的临床应用价值,并分析疗效的影响因素。方法：选择四川省骨科医院2020年1月~2022年1月期间收治的桡骨远端伸直型骨折患者152例,按照治疗方式的不同将患者分为对照组（给予石膏固定）和研究组（应用中医正骨手法治疗）,例数分别为77例和75例。对比两组优良率、临床指标、腕关节活动度和X线相关影像学指标。同时采用多因素Logistic回归分析影响研究组腕关节功能疗效的相关因素。结果：治疗后,研究组的优良率明显高于对照组（P<0.05）。研究组手背消肿时间、疼痛缓解时间、骨折愈合时间均短于对照组（P<0.05）。两组治疗后12周掌屈、背伸、桡偏、尺偏、旋前、旋后活动度均扩大,且研究组均大于对照组（P<0.05）。两组治疗后12周掌倾角、尺偏角、桡骨高度均增加,且研究组均大于对照组（P<0.05）。单因素分析显示,研究组腕关节功能优良率与年龄、性别、骨质疏松、功能锻炼、掌倾角、尺偏角、桡骨高度、骨折端稳定性、受伤能量、利手情况有关（P<0.05）,而与体质量指数、就诊时间、基础疾病、骨折类型、固定时间无关（P>0.05）。多因素Logistic回归分析显示：性别为女、骨质疏松、无功能锻炼、掌倾角偏小、桡骨高度偏短、骨折端不稳定、受伤能量为高能量、利手是影响腕关节功能优良率的危险因素（P<0.05）。结论：中医正骨手法可有效改善桡骨远端伸直型骨折患者腕关节功能,减少骨折愈合及疼痛缓解时间。此外,患者腕关节功能的优良率还受到性别、骨质疏松、功能锻炼、掌倾角、桡骨高度、骨折端稳定性、受伤能量、利手情况的影响,值得引起临床重视。     

A genome-wide perspective on the diversity and selection signatures in indigenous goats using 53 K single nucleotide polymorphism array

Tibetan goats, Taihang goats, Jining grey goats, and Meigu goats are the representative indigenous goats in China, found in Qinghai-Tibet Plateau, Western pastoral area, Northern and Southern agricultural regions. Very few studies have conducted a comprehensive analysis of the genomic diversity and selection of these breeds. We genotyped 96 unrelated individuals, using goat 53 K Illumina BeadChip array, of the following goat breeds: Tibetan (TG), Taihang (THG), Jining grey (JGG), and Meigu (MGG). A total of 45 951 single nucleotide polymorphisms were filtered to estimate the genetic diversity and selection signatures. All breeds had a high proportion (over 95%) of polymorphic loci. The observed and excepted heterozygosity ranged from 0.338 (MGG) to 0.402 (JGG) and 0.339 (MGG) to 0.395 (JGG), respectively. Clustering analysis displayed a genetically distinct lineage for each breed, and their Fst were greater than 0.25, indicating that they had a higher genetic differentiation between groups. Furthermore, effective population size reduced in all four populations, indicating a loss of genetic diversity. In addition, runs of homozygosity were mainly distributed in 5–10 Mb. Lastly, we identified signature genes, which were closely related to high-altitude adaptation (ADIRF) and prolificity (CNTROB, SMC3, and PTEN). This study provides a valuable resource for future studies on genome-wide perspectives on the diversity and selection signatures of Chinese indigenous goats.     

安息香科植物地理分布研究

安息香科植物共１２属,约１５０种,分布于亚洲和美洲的热带亚热带地区及欧洲南部的温暖地区。亚洲是现代安息香科植物的主要分布区,有１１个属,集中分布于中国南部和西南部附近。中国南岭山地及其附近汇集了８属２０多种,包括中国特有的陀螺果属、秤锤树属和长果安息香属以及东亚北美间断分布的银钟花属。这些呈星散分布的残余成分是从当地起源的。中国西南地区的安息香科植物仅云南一地就达８属３０余种,其中不少为局部分化形成的狭域分布特有种。这里是安息香科植物的分化中心,茉莉果属和歧序野茉莉属可能即是由此发端的。后者和安息香属经中南半岛一直分布到热带亚洲地区,并在马来西亚地区形成一次生分化中心。欧洲安息香科植物可能来自亚洲,并于第三纪得到发展。安息香科植物在欧洲的衰落,可能是遭到第四纪冰川严重破坏的结果。北美安息香科植物和欧亚大陆的有着共同起源,主要分布于东南部,并通过中美向南美发展。南美安息香科植物达５０余种,集中分布于亚马逊河流域。这里是安息香科植物的另一次生分化中心。特产巴西的花弄蝶属即是从当地分化出来的。     

Glutamine repeats and inherited neurodegenerative diseases: molecular aspects

Several dominantly inherited, late onset, neurodegenerative diseases are due to expansion of CAG repeats, leading to expansion of glutamine repeats in the affected proteins. These proteins are of very different sizes and, with one exception, show no sequence homology to known proteins or to each other; their functions are unknown. In some, the glutamine repeat starts near the N-terminus, in another near the middle and in another near the C-terminus, but regardless of these differences, no disease has been observed in individuals with fewer than 37 repeats, and absence of disease has never been found in those with more than 41 repeats. Protein constructs with more than 41 repeats are toxic to E. coli and to CHO cells in culture, and they elicit ataxia in transgenic mice. These observations argue in favour of a distinct change of structure associated with elongation beyond 37–41 glutamine repeats. The review describes experiments designed to find out what these structures might be and how they could influence the properties of the proteins of which they form part. Poly- -glutamines form pleated sheets of β-strands held together by hydrogen bonds between their amides. Incorporation of glutamine repeats into a small protein of known structure made it associate irreversibly into oligomers. That association took place during the folding of the protein molecules and led to their becoming firmly interlocked by either strand- or domain-swapping. Thermodynamic considerations suggest that elongation of glutamine repeats beyond a certain length may lead to a phase change from random coils to hydrogen-bonded hairpins. Possible mechanisms of expansion of CAG repeats are discussed in the light of looped DNA model structures.