Centrosome reorientation in regenerating endothelial monolayers requires bFGF

Monolayers of endothelial cells respond to physical denudation with a characteristic sequence of lamellipodia extrusion, cell migration, and cell proliferation. Basic fibroblast growth factor (bFGF) has been implicated as a necessary component of this process: addition of exogenous bFGF enhances monolayer regeneration both in vitro and in vivo, and monolayer regeneration can be inhibited in vitro by treatment with neutralizing antibodies raised against bFGF. Centrosome reorientation from a random location to one preferentially situated between the nucleus and the denudation edge has been postulated as a mechanism essential for cell polarization and subsequent migration. This present study examined the effects of a polyclonal antibody to bFGF and suramin on monolayer regeneration, actin microfilament staining, and centrosome orientation at the wound edge of partially denuded bovine large vessel endothelial monolayers. Treatment with anti-bFGF or suramin abolished monolayer repair in these cultures. Cells at the denudation edge showed altered actin staining patterns and reduced lamellipodia extrusion, and there was complete inhibition of centrosome reorientation in treated cultures. Monolayer repair and centrosome reorientation could be restored by addition of exogenous bFGF in antibody but not suramin treated cultures. Recent evidence suggests that preferential centrosome location in migrating cells may be a consequence of lamellipodia protrusion and cell spreading, rather than an indication of cell polarization. However, these results indicate that agents which interfere with bFGF availability prevent endothelial monolayer regeneration via mechanisms involving cell spreading and/or centrosome reorientation.     

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.     

泛素蛋白磷酸化修饰的功能与解析

泛素化是存在于真核生物中一种重要的翻译后修饰过程,参与调控包括蛋白质降解在内的多种生命活动。实现这一调控过程需要将一个由76个氨基酸组成的泛素蛋白共价连接到底物蛋白上。同时,泛素本身也存在多种翻译后修饰,包括泛素化、磷酸化、乙酰化等,进一步丰富了泛素的修饰类型,决定了底物蛋白不同的命运。近年来,伴随着第65位丝氨酸磷酸化泛素蛋白参与调控线粒体自噬这一突破性进展,泛素蛋白其余磷酸化位点的功能研究也获得越来越多的关注。本文根据目前已有的国内外研究和报道,总结了泛素蛋白已知的磷酸化修饰位点,梳理了泛素蛋白第12位和66位苏氨酸、第57位和65位丝氨酸等位点的磷酸化修饰对其生物物理特性带来的改变,并对相应修饰位点所涉及的生物学功能调控进行了综述。     

Current status and prospects of basic research and clinical application of mesenchymal stem cells in acute respiratory distress syndrome

Acute respiratory distress syndrome (ARDS) is a common and clinically devastating disease that causes respiratory failure. Morbidity and mortality of patients in intensive care units are stubbornly high, and various complications severely affect the quality of life of survivors. The pathophysiology of ARDS includes increased alveolar–capillary membrane permeability, an influx of protein-rich pulmonary edema fluid, and surfactant dysfunction leading to severe hypoxemia. At present, the main treatment for ARDS is mechanical treatment combined with diuretics to reduce pulmonary edema, which primarily improves symptoms, but the prognosis of patients with ARDS is still very poor. Mesenchymal stem cells (MSCs) are stromal cells that possess the capacity to self-renew and also exhibit multilineage differentiation. MSCs can be isolated from a variety of tissues, such as the umbilical cord, endometrial polyps, menstrual blood, bone marrow, and adipose tissues. Studies have confirmed the critical healing and immunomodulatory properties of MSCs in the treatment of a variety of diseases. Recently, the potential of stem cells in treating ARDS has been explored via basic research and clinical trials. The efficacy of MSCs has been shown in a variety of in vivo models of ARDS, reducing bacterial pneumonia and ischemia-reperfusion injury while promoting the repair of ventilator-induced lung injury. This article reviews the current basic research findings and clinical applications of MSCs in the treatment of ARDS in order to emphasize the clinical prospects of MSCs.     

Spontaneous allelic variant in deafness–blindness gene Ush1g resulting in an expanded phenotype

Relationships between novel phenotypic behaviors and specific genetic alterations are often discovered using target-specific, directed mutagenesis or phenotypic selection following chemical mutagenesis. An alternative approach is to exploit deficiencies in DNA repair pathways that maintain genetic integrity in response to spontaneously induced damage. Mice deficient in the DNA glycosylase NEIL1 show elevated spontaneous mutations, which arise from translesion DNA synthesis past oxidatively induced base damage. Several litters of Neil1 knockout mice included animals that were distinguished by their backwards-walking behavior in open-field environments, while maintaining frantic forward movements in their home cage environment. Other phenotypic manifestations included swim test failures, head tilting and circling. Mapping of the mutation that conferred these behaviors showed the introduction of a stop codon at amino acid 4 of the Ush1g gene. Ush1g^bw/bw null mice displayed auditory and vestibular defects that are commonly seen with mutations affecting inner-ear hair-cell function, including a complete lack of auditory brainstem responses and vestibular-evoked potentials. As in other Usher syndrome type I mutant mouse lines, hair cell phenotypes included disorganized and split hair bundles, as well as altered distribution of proteins for stereocilia that localize to the tips of row 1 or row 2. Disruption to the bundle and kinocilium displacement suggested that USH1G is essential for forming the hair cell's kinocilial links. Consistent with other Usher type 1 models, Ush1g^bw/bw mice had no substantial retinal degeneration compared with Ush1g^bw/+ controls. In contrast to previously described Ush1g alleles, this new allele provides the first knockout model for this gene.     

植入了骨修复材料的小型猪腰椎椎体不脱钙病理组织切片制备方法

摘要 目的：建立植入了骨修复材料小型猪腰椎椎体骨组织标本的不脱钙病理组织切片制备方法。方法：将含骨修复材料的腰椎椎体骨组织标本进行分割暴露组织切面,梯度浓度乙醇脱水后经Technovit 7200 VLC光聚树脂浸润,经黄蓝光共同辐照进行光聚合包埋,借助硬组织病理切磨系统制备含骨修复材料不脱钙病理组织切片。结果：结果显示通过上述方法制备的病理组织切片,经苏木精-伊红（HE）染色及甲苯胺蓝染色后光学显微镜下观察能较好地显示骨的各种组织细胞结构,可清晰的观察到骨小梁的走向及连接情况。结论：研究建立了含骨修复材料骨组织标本病理组织切片制备方法,实现了含骨修复材料不脱钙骨组织病理切片的制备,经病理染色后实现了带植入物的组织学观察,为生物材料及医疗器械动物试验研究提供了新的病理检测手段及组织学评价途径。     

比较CSPO和ISR治疗超低位直肠癌的保肛效果

摘要 目的：比较适形切除保肛术（CSPO）和经内外括约肌间切除术（ISR）治疗超低位直肠癌的保肛效果。方法：选择2020年6月至2022年3月选择在本院诊治的超低位直肠癌患者63例作为研究对象,根据随机分配原则把患者分为ISR组31例与CSPO组32例,ISR组予经内外括约肌间切除术治疗,CSPO组予适形切除保肛术治疗,记录与随访患者的近期与远期预后。结果：所有患者都顺利完成保肛,两组的术中出血量、淋巴结清扫数目对比无差异（P>0.05）,CSPO组的手术时间、术后肠道功能恢复时间、术后住院时间明显少于ISR组（P<0.05）。CSPO组的术后7 d的切口感染、泌尿系感染、吻合口狭窄、吻合口瘘等并发症发生率为6.3 %,明显低于ISR组的29.0 %（P<0.05）。CSPO组术后1 d、3 d、7 d的血清降钙素原（PCT）、超敏C-反应蛋白（hs-CRP）含量都明显少于ISR组（P<0.05）。ISR组术后3个月的满意度为80.6 %,明显低于CSPO组100.0 %（P<0.05）。结论：相对经内外括约肌间切除术,适形切除保肛术治疗超低位直肠癌能有效抑制血清PCT与hs-CRP的表达,具有很好的保肛效果,能减少并发症的发生,还可促进患者康复,从而持续提高患者的随访生存率。     

Human Interindividual Variability in Cancer Risks—Technical and Management Challenges

Existing risk assessment procedures for carcinogens are intended to be “conservative” in the uncertainty dimension—giving estimates that are expected to be higher than true risks for typical people. However, these procedures do not consider the likely variability in susceptibility among individual people. This paper updates previous estimates of the likely extent of this variability for metabolically activated, genetically-acting carcinogens based on recent information on human interindividual variability in metabolic activation, detoxification, and DNA repair. The resulting expected skewness of cancer risk distributions is estimated using Monte Carlo simulations of both variability and uncertainty.

Some risk management implications are:

1. When evaluating the fairness of a particular risk distribution, managers need to gain familiarity with a three-dimensional characterization—X level of risk, for the Yth percentile individual (addressing variability) with Z degree of confidence (addressing uncertainty).

2. To the extent that variability distributions are skewed (e.g., with a long tail extending to high values) population mean risks will tend to exceed risks for median individuals. Together with the skewness in uncertainty distributions, this implies that “expected value” estimates of aggregate population risks—the estimates of interest for cost benefit analyses—are likely to be closer to traditional upper confidence limit risk estimates than has often been assumed in the past.

The binding motifs for Ac transposase are absolutely required for excision of Ds1 in maize

A reverse genetic system for studying excision of the transposable elementDs1 in maize plants has been established previously. In this system, theDs1 element, as part of the genome of maize streak virus (MSV), is introduced into maize plants via agroinfection. In the presence of theAc element, excision ofDs1 from the MSV genome results in the appearance of viral symptoms on the maize plants. Here, we used this system to study DNA sequences requiredin cis for excision ofDs1. TheDs1 element contains theAc transposase binding motif AAACGG in only one of its subterminal regions (defined here as the 5′ subterminal region). We showed that mutation of these motifs abolished completely the excision capacity ofDs1. This is the first direct demonstration that the transposase binding motifs are essential for excision. Mutagenesis with oligonucleotide insertions in the other (3′) subterminal region resulted in elements with either a reduced or an increased excision efficiency, indicating that this subterminal region also has an important function.