RS‐1 enhances CRISPR‐mediated targeted knock‐in in bovine embryos

Targeted knock‐in (KI) can be achieved in embryos by clustered regularly interspaced short palindromic repeats (CRISPR)‐assisted homology directed repair (HDR). However, HDR efficiency is constrained by the competition of nonhomologous end joining. The objective of this study was to explore whether CRISPR‐assisted targeted KI rates can be improved in bovine embryos by exposure to the HDR enhancer RS‐1. In vitro produced zygotes were injected with CRISPR components (300 ng/µl Cas9 messenger RNA and 100 ng/µl single guide RNA against a noncoding region) and a single‐stranded DNA (ssDNA) repair template (100 ng/µl). ssDNA template contained a 6 bp XbaI site insert, allowing targeted KI detection by restriction analysis, flanked by 50 bp homology arms. Following microinjection, zygotes were exposed to 0, 3.75, or 7.5 µM RS‐1 for 24 hr. No differences were noted between groups in terms of development or genome edition rates. However, targeted KI rates were doubled in the group exposed to 7.5 µM RS‐1 compared to the others (52.8% vs. 25% and 23.1%, for 7.5, 0, and 3.75 µM, respectively). In conclusion, transient exposure to 7.5 µM RS‐1 enhances targeted KI rates resulting in approximately half of the embryos containing the intended mutation, hence allowing direct KI generation in embryos.     

Association of age with class VI composite restoration

Dental caries is the major oral health problem in most of the countries, affecting 60-90% of school children and a vast majority of adults. Therefore, it is of interest to evaluate the association of age with Class VI defects restored with composite restorations. We used 102 cases with data regarding Class VI composite restorations in a datasheet of 86,000 records at Saveetha Dental College, India for this study. Data shows that Class VI restorations were commonly seen in upper anterior teeth in the age group of 51 and above. The cavities prepared to receive Class VI restoration followed a conservative design of caries removal and used direct restoration techniques for reconstruction of the lost tooth structure.     

超声引导下QLB复合气管插管全麻对于老年患者TEP腹股沟疝无张力修补术影响因素分析

摘要 目的：探讨与分析超声引导下腰方肌阻滞(quadratus lumborum block,QLB)复合气管插管全麻对于老年患者腹腔镜下全腹膜外（totally extraperitoneal prosthetic,TEP）腹股沟疝无张力修补术的影响,以促进该方法的临床使用。方法：2014年9月到2020年6月选择在本院诊治的腹股沟疝老年患者180例,根据随机数字表法分为QLB组与对照组各90例。所有患者都给予腹腔镜下全腹膜外腹股沟疝无张力修补术,对照组给予气管插管全麻,QLB组在对照组麻醉的基础上给予超声引导下QLB,记录两组镇痛与麻醉效果。结果：两组的术中出血量、手术时间等对比差异无统计学意义(P>0.05),QLB组的术后住院时间、术后胃肠功能恢复时间、术后下床活动时间显著短于对照组(P<0.05)。与术后12 h对比,两组术后24 h与36 h的疼痛VAS评分均降低(P<0.05),且QLB组术后12 h、24 h与36 h的疼痛VAS评分都显著低于对照组(P<0.05)。QLB组术后7 d的血肿、呼吸抑制、脏器损伤、腹股沟区包块等并发症发生率为8.9 %,显著低于对照组的21.1 %(P<0.05)。QLB组的瑞芬太尼用量、术后48 h内有效按压自控静脉镇痛泵次数、自控静脉镇痛泵累计用量都显著少于对照组(P<0.05)。结论：超声引导下QLB复合气管插管全麻在老年患者腹腔镜下全腹膜外腹股沟疝无张力修补术中的应用能提高镇痛与麻醉效果,减少术后并发症的发生,有利于促进患者康复。     

Oligonucleotide/oligosaccharide-binding fold proteins: a growing family of genome guardians

The maintenance of genomic stability relies on the coordinated action of a number of cellular processes, including activation of the DNA-damage checkpoint, DNA replication, DNA repair, and telomere homeostasis. Many proteins involved in these cellular processes use different types of functional modules to regulate and execute their functions. Recent studies have revealed that many DNA-damage checkpoint and DNA repair proteins in human cells possess the oligonucleotide/oligosaccharide-binding (OB) fold domains, which are known to bind single-stranded DNA in both prokaryotes and eukaryotes. Furthermore, during the DNA damage response, the OB folds of the human checkpoint and DNA repair proteins play critical roles in DNA binding, protein complex assembly, and regulating protein–protein interactions. These findings suggest that the OB fold is an evolutionarily conserved functional module that is widely used by genome guardians. In this review, we will highlight the functions of several well-characterized or newly discovered eukaryotic OB-fold proteins in the DNA damage response.     

SLX4: multitasking to maintain genome stability

The SLX4/FANCP tumor suppressor has emerged as a key player in the maintenance of genome stability, making pivotal contributions to the repair of interstrand cross-links, homologous recombination, and in response to replication stress genome-wide as well as at specific loci such as common fragile sites and telomeres. SLX4 does so in part by acting as a scaffold that controls and coordinates the XPF–ERCC1, MUS81–EME1, and SLX1 structure-specific endonucleases in different DNA repair and recombination mechanisms. It also interacts with other important DNA repair and cell cycle control factors including MSH2, PLK1, TRF2, and TOPBP1 as well as with ubiquitin and SUMO. This review aims at providing an up-to-date and comprehensive view on the key functions that SLX4 fulfills to maintain genome stability as well as to highlight and discuss areas of uncertainty and emerging concepts.     

Base Excision Repair and its Role in Maintaining Genome Stability

For all living organisms, genome stability is important, but is also under constant threat because various environmental and endogenous damaging agents can modify the structural properties of DNA bases. As a defense, organisms have developed different DNA repair pathways. Base excision repair (BER) is the predominant pathway for coping with a broad range of small lesions resulting from oxidation, alkylation, and deamination, which modify individual bases without large effect on the double helix structure. As, in mammalian cells, this damage is estimated to account daily for 10⁴ events per cell, the need for BER pathways is unquestionable. The damage-specific removal is carried out by a considerable group of enzymes, designated as DNA glycosylases. Each DNA glycosylase has its unique specificity and many of them are ubiquitous in microorganisms, mammals, and plants. Here, we review the importance of the BER pathway and we focus on the different roles of DNA glycosylases in various organisms.     

Interleukin-1 beta and tumor necrosis factor alpha inhibit migration activity of chondrogenic progenitor cells from non-fibrillated osteoarthritic cartilage

Introduction

The repair capability of traumatized articular cartilage is highly limited so that joint injuries often lead to osteoarthritis. Migratory chondrogenic progenitor cells (CPC) might represent a target cell population for in situ regeneration. This study aims to clarify, whether 1) CPC are present in regions of macroscopically intact cartilage from human osteoarthritic joints, 2) CPC migration is stimulated by single growth factors and the cocktail of factors released from traumatized cartilage and 3) CPC migration is influenced by cytokines present in traumatized joints.

Methods

We characterized the cells growing out from macroscopically intact human osteoarthritic cartilage using a panel of positive and negative surface markers and analyzed their differentiation capacity. The migratory response to platelet-derived growth factor (PDGF)-BB, insulin-like growth factor 1 (IGF-1), supernatants obtained from in vitro traumatized cartilage and interleukin-1 beta (IL-1β) as well as tumor necrosis factor alpha (TNF-α) were tested with a modified Boyden chamber assay. The influence of IL-1β and TNF-α was additionally examined by scratch assays and outgrowth experiments.

Results

A comparison of 25 quadruplicate marker combinations in CPC and bone-marrow derived mesenchymal stromal cells showed a similar expression profile. CPC cultures had the potential for adipogenic, osteogenic and chondrogenic differentiation. PDGF-BB and IGF-1, such as the supernatant from traumatized cartilage, induced a significant site-directed migratory response. IL-1β and TNF-α significantly reduced basal cell migration and abrogated the stimulative effect of the growth factors and the trauma supernatant. Both cytokines also inhibited cell migration in the scratch assay and primary outgrowth of CPC from cartilage tissue. In contrast, the cytokine IL-6, which is present in trauma supernatant, did not affect growth factor induced migration of CPC.

Conclusion

These results indicate that traumatized cartilage releases chemoattractive factors for CPC but IL-1β and TNF-α inhibit their migratory activity which might contribute to the low regenerative potential of cartilage in vivo.     

The XRCC3 Thr241Met polymorphism and breast cancer risk: a case–control study in a Thai population

The X-ray repair cross-complementing group 3 gene (XRCC3) belongs to a family of genes responsible for repairing DNA double-strand breaks caused by normal metabolic processes and exposure to ionizing radiation. Polymorphisms in DNA repair genes may alter an individual's capacity to repair damaged DNA and may lead to genetic instability and contribute to malignant transformation. We examined the role of a polymorphism in the XRCC3 gene (rs861529; codon 241: threonine to methionine change) in determining breast cancer risk in Thai women. The study population consisted of 507 breast cancer cases and 425 healthy women. The polymorphism was analysed by fluorescence-based melting curve analysis. The XRCC3 241Met allele was found to be uncommon in the Thai population (frequency 0.07 among cases and 0.05 among controls). Odds ratios (OR) adjusted for age, body mass index, age at menarche, family history of breast cancer, menopausal status, reproduction parameters, use of contraceptives, tobacco smoking, involuntary tobacco smoking, alcohol drinking, and education were calculated for the entire population as well as for pre- and postmenopausal women. There was a significant association between 241Met carrier status and breast cancer risk (OR 1.58, 95% confidence interval (CI) 1.02–2.44). Among postmenopausal women, a slightly higher OR (1.82, 95% CI 0.95–3.51) was found than among premenopausal women (OR 1.48, 95% CI 0.82–2.69). Our findings suggest that the XRCC3 Thr241Met polymorphism is likely to play a modifying role in the individual susceptibility to breast cancer among Thai women as already shown for women of European ancestry.     

Bulky DNA adduct levels in normal-appearing colon mucosa,and the prevalence of colorectal adenomas

Abstract

Purpose: Examine the association between bulky DNA adduct levels in colon mucosa and colorectal adenoma prevalence, and explore the correlation between adduct levels in leukocytes and colon tissue.

Methods: Bulky DNA adduct levels were measured using ³²P-postlabelling in biopsies of normal-appearing colon tissue and blood donated by 202 patients. Multivariable logistic regression was used to examine associations between DNA adducts, and interactions of DNA adduct-DNA repair polymorphisms, with the prevalence of colorectal adenomas. Correlation between blood and tissue levels of DNA adducts was evaluated using Spearman’s correlation coefficient.

Results: An interaction between bulky DNA adduct levels and XPA rs1800975 on prevalence of colorectal adenoma was observed. Among individuals with lower DNA repair activity, increased DNA adduct levels were associated with increased colorectal adenoma prevalence (OR?=?1.41 per SD increase, 95%CI: 0.92–2.18). Conversely, among individuals with normal DNA activity, an inverse association was observed (OR?=?0.60 per SD increase, 95%CI: 0.34–1.07). Blood and colon DNA adduct levels were inversely correlated (ρ?=??0.20).

Conclusions: Among genetically susceptible individuals, higher bulky DNA adducts in the colon was associated with the prevalence of colorectal adenomas. The inverse correlation between blood and colon tissue measures demonstrates the importance of quantifying biomarkers in target tissues.     

Epithelial cell adhesion molecule (EpCAM) is associated with prostate cancer metastasis and chemo/radioresistance via the PI3K/Akt/mTOR signaling pathway

Prostate cancer (CaP) is the second leading malignancy in men. The role of epithelial cell adhesion molecule (EpCAM), also known as CD326, in CaP progression and therapeutic resistance is still uncertain. Here, we aimed to investigate the roles of EpCAM in CaP metastasis and chemo/radioresistance. Expression of EpCAM in CaP cell lines and human CaP tissues was assessed using immunofluorescence and immunohistochemistry, respectively. EpCAM was knocked down (KD) in PC-3, DU145 and LNCaP-C4-2B cells using small interfering RNA (siRNA), and KD results were confirmed by confocal microscope, Western blotting and quantitative real time polymerase chain reaction (qRT-PCR). Cell growth was evaluated by proliferation and colony formation assays. The invasive potential was assessed using a matrigel chamber assay. Tumorigenesis potential was measured by a sphere formation assay. Chemo-/radiosensitivity were measured using a colony formation assay. Over-expression of EpCAM was found in primary CaP tissues and lymph node metastases including cancer cells and surrounding stromal cells. KD of EpCAM suppressed CaP proliferation and invasive ability, reduced sphere formation, enhanced chemo-/radiosensitivity, and down-regulated E-cadherin, p-Akt, p-mTOR, p-4EBP1 and p-S6K expression in CaP cells. Our findings suggest that EpCAM plays an important role in CaP proliferation, invasion, metastasis and chemo-/radioresistance associated with the activation of the PI3K/Akt/mTOR signaling pathway and is a novel therapeutic target to sensitize CaP cells to chemo-/radiotherapy.