Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR):A critical overview on the most promising applications of molecular scissors in oral medicine

The scientific community is continuously working to translate the novel biomedical techniques into effective medical treatments.CRISPR-Cas9 system(Clustered Regularly Interspaced Short Palindromic Repeats-9),commonly known as the“molecular scissor”,represents a recently developed biotechnology able to improve the quality and the efficacy of traditional treatments,related to several human diseases,such as chronic diseases,neurodegenerative pathologies and,interestingly,oral diseases.Of course,dental medicine has notably increased the use of biotechnologies to ensure modern and conservative approaches:in this landscape,the use of CRISPR-Cas9 system may speed and personalize the traditional therapies,ensuring a good predictability of clinical results.The aim of this critical overview is to provide evidence on CRISPR efficacy,taking into specific account its applications in oral medicine.

Comb-Based Filter Design Using Sharpening Technique and Certain Palindromic Polynomial

This paper presents a novel approach to improve aliasing rejection in comb-based decimation filters. The method is established on certain palindromic polynomials with all zeros on the unit circle and the sharpening technique. As a result, aliasing rejection and the passband characteristic are improved. The method is illustrated with various examples and compared with the methods from the literature. .

Polynomials with Palindromic and Unimodal Coefficients

Let f（q） = arq^r ＋…＋asqs, with ar ≠ 0 and as ≠ 0, be a real polynomial. It is a palindromic polynomial of darga n if r＋s= n and ar＋i =as-i for all i. Polynomials of darga n form a linear subspace Pn（q） of R（q）n＋l of dimension [n/2] ＋ 1. We give transition matrices between two bases {q^j（1 ＋ q ＋… q^n-2j）}, {q^j（1 ＋ q）^n-2j } and the standard basis {q^j（1 ＋ q^n-2j）} of Pn （q）. We present some characterizations and sufficient conditions for palindromic polynomials that can be expressed in terms of these two bases with nonnegative coefficients. We also point out the link between such polynomials and rank-generating functions of posets.

Overexpression of proteasome 26S subunit non-ATPase 6 protein and its clinicopathological significance in intrahepatic cholangiocarcinoma

BACKGROUND Currently,intrahepatic cholangiocarcinoma(ICC)poses a continuing,significant health challenge,but the relationship has yet to be established between ICC and the proteasome 26S subunit non-ATPase 6(PSMD6).AIM To investigate the protein expression and clinicopathological significance of PSMD6 in ICC.METHODS The potential impact of the PSMD6 gene on the growth of ICC cell lines was analyzed using clustered regularly interspaced short palindromic repeat knockout screening technology.Forty-two paired specimens of ICC and adjacent noncancerous tissues were collected.PSMD6 protein expression was determined by immunohistochemistry.Receiver operating characteristic curve analysis was performed to validate PSMD6 expression level,and its association with ICC patients’various clinicopathological characteristics was investigated.RESULTS The PSMD6 gene was found to be essential for the growth of ICC cell lines.PSMD6 protein was significantly overexpressed in ICC tissues(P<0.001),but showed no significant association with patient age,gender,pathological grade,or tumor-node-metastasis stage(P>0.05).CONCLUSION PSMD6 can promote the growth of ICC cells,thus playing a pro-oncogenic role.

The Applications of CRISPR Screening in Aging Research

The advancement of Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)gene editing technology has revolutionized the comprehension of human genome,propelling molecular and cellular biology research into unexplored realms and accelerating progress in life sciences and medicine.CRISPR-based gene screening,recognized for its efficiency and practicality,is widely utilized across diverse biological fields.Aging is a multifaceted process governed by a myriad of genetic and epigenetic factors.Unraveling the genes regulating aging holds promise for understanding this intricate phenomenon and devising strategies for its assessment and intervention.This review provides a comprehensive overview of the progress in CRISPR screening and its applications in aging research,while also offering insights into future directions.CRISPR-based genetic-manipulation tools are positioned as indispensable instruments for mitigating aging and managing age-related diseases.

转录因子HNF1A、HNF4A和FOXA2调节肝细胞蛋白质N-糖基化

Hepatocyte nuclear factor 1 alpha(HNF1A),hepatocyte nuclear factor 4 alpha(HNF4A),and forkhead box protein A2(FOXA2)are key transcription factors that regulate a complex gene network in the liver,cre-ating a regulatory transcriptional loop.The Encode and ChIP-Atlas databases identify the recognition sites of these transcription factors in many glycosyltransferase genes.Our in silico analysis of HNF1A,HNF4A.and FOXA2 binding to the ten candidate glyco-genes studied in this work confirms a significant enrich-ment of these transcription factors specifically in the liver.Our previous studies identified HNF1A as a master regulator of fucosylation,glycan branching,and galactosylation of plasma glycoproteins.Here,we aimed to functionally validate the role of the three transcription factors on downstream glyco-gene transcriptional expression and the possible effect on glycan phenotype.We used the state-of-the-art clus-tered regularly interspaced short palindromic repeats/dead Cas9(CRISPR/dCas9)molecular tool for the downregulation of the HNF1A,HNF4A,and FOXA2 genes in HepG2 cells-a human liver cancer cell line.The results show that the downregulation of all three genes individually and in pairs affects the transcrip-tional activity of many glyco-genes,although downregulation of glyco-genes was not always followed by an unambiguous change in the corresponding glycan structures.The effect is better seen as an overall change in the total HepG2 N-glycome,primarily due to the extension of biantennary glycans.We propose an alternative way to evaluate the N-glycome composition via estimating the overall complexity of the glycome by quantifying the number of monomers in each glycan structure.We also propose a model showing feedback loops with the mutual activation of HNF1A-FOXA2 and HNF4A-FOXA2 affecting glyco-genes and protein glycosylation in HepG2 cells.

Is 26S proteasome non-ATPase regulatory subunit 6 a potential molecular target for intrahepatic cholangiocarcinoma?

In this editorial we comment on the article by Tang et al published in the recent issue of World Journal of Hepatology.Drug therapy of intrahepatic cholangiocarcinoma(iCCA)poses an enormous challenge since only a small proportion of patients demonstrate beneficial responses to therapeutic agents.Thus,there has been a sustained search for novel molecular targets for iCCA.The study by Tang et al evaluated the role of 26S proteasome non-ATPase regulatory subunit 6(PSMD6),a 19S regulatory subunit of the proteasome,in human iCCA cells and specimens.The authors employed clustered regularly interspaced short palindromic repeat(CRISPR)knockout screening technology integrated with the computational CERES algorithm,and analyzed the human protein atlas(THPA)database and tissue microarrays.The results show that PSMD6 is a gene essential for the proliferation of 17 iCCA cell lines,and PSMD6 protein was overexpressed in iCCA tissues without a significant correlation with the clinicopathological parameters.The authors conclude that PSMD6 may play a promoting role in iCCA.The major limitations and defects of this study are the lack of detailed information of CRISPR knockout screening,in vivo experiments,and a discussion of plausible mechanistic cues,which,therefore,dampen the significance of the results.Further studies are required to verify PSMD6 as a molecular target for developing novel therapeutics for iCCA.In addition,the editorial article summarizes the latest advances in molecular targeted drugs and recently emerging immunotherapy in the clinical management of iCCA,development of proteasome inhibitors for cancer therapy,and advantages of CRISPR screening technology,computational methods,and THPA database as experimental tools for fighting cancer.We hope that these comments may provide some clues for those engaged in the field of basic and clinical research into iCCA.

Decoding the genetic landscape of autism:A comprehensive review

BACKGROUND Autism spectrum disorder(ASD)is a complex neurodevelopmental condition characterized by heterogeneous symptoms and genetic underpinnings.Recent advancements in genetic and epigenetic research have provided insights into the intricate mechanisms contributing to ASD,influencing both diagnosis and therapeutic strategies.AIM To explore the genetic architecture of ASD,elucidate mechanistic insights into genetic mutations,and examine gene-environment interactions.METHODS A comprehensive systematic review was conducted,integrating findings from studies on genetic variations,epigenetic mechanisms(such as DNA methylation and histone modifications),and emerging technologies[including Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)-Cas9 and single-cell RNA sequencing].Relevant articles were identified through systematic searches of databases such as PubMed and Google Scholar.RESULTS Genetic studies have identified numerous risk genes and mutations associated with ASD,yet many cases remain unexplained by known factors,suggesting undiscovered genetic components.Mechanistic insights into how these genetic mutations impact neural development and brain connectivity are still evolving.Epigenetic modifications,particularly DNA methylation and non-coding RNAs,also play significant roles in ASD pathogenesis.Emerging technologies like CRISPR-Cas9 and advanced bioinformatics are advancing our understanding by enabling precise genetic editing and analysis of complex genomic data.CONCLUSION Continued research into the genetic and epigenetic underpinnings of ASD is crucial for developing personalized and effective treatments.Collaborative efforts integrating multidisciplinary expertise and international collaborations are essential to address the complexity of ASD and translate genetic discoveries into clinical practice.Addressing unresolved questions and ethical considerations surrounding genetic research will pave the way for improved diagnostic tools and targeted therapies,ultimately enhancing outcomes for individuals affected by ASD.

Dissemination of carbapenem-non-susceptible Acinetobacter baumannii isolates collected from educational hospitals in Qazvin province of Iran

Background:Acinetobacter baumannii(A.baumannii)is known as an opportunistic pathogen related to health-care-associated infection that has a high antibiotic resistance potential,notably against carbapenems that are widely used to combat A.baumannii infections.This study aimed to detect oxacillin-hydrolyzing(OXA)carbapenemases and metallo-β-lactamases(MBL)among carbapenem-resistant A.baumannii isolated strains and to determine their clonal relationship by repetitive extragenic palindromic PCR(rep-PCR).Methods:In the present study,a total of 211 non-repetitive isolates of A.baumannii were collected from Qazvin educational hospitals(2016–2017).The disk diffusion method was used to investigate the antibiotic susceptibility of studied strains,followed by the detection of MBL and OXA-type genes using polymerase chain reaction(PCR)and sequencing methods.The rep-PCR method assessed the clonal relationship of carbapenem-non-susceptible A.baumannii isolates.Result:The obtained results showed that 87.2%and 86.7%of isolates were non-susceptible to imipenem and meropenem.The blaOXA-24(93.5%)was the most frequent gene,followed by the blaOXA-23(4.34%),blaIMP-1(1.63%),and blaVIM-1(0.54%).Meanwhile,blaOXA-58 and blaOXA-143 genes were not found.81.5%and 66.1%of isolates contained ISAba1 upstream of the blaOXA-23 and blaOXA-58 genes,respectively.Rep-PCR results revealed the carbapenem non-susceptible isolates belonged to three distinct clones:A 171(81%),B 34(16.1%),and C 6(2.8%).Conclusions:The results indicated a high prevalence of carbapenem-non-susceptible A.baumannii,with the emergence of the blaOXA-24 gene as the most common gene and the notable prevalence of MBL genes.These results revealed the need for appropriate therapeutic and infection control strategies and monitoring susceptibility patterns for controlling A.baumannii infections.

嗜热链球菌中CRISPR序列的检测与同源性分析

为探明内蒙古的8株嗜热链球菌的CRISPR,用PCR方法扩增了其CRISPR序列,并采用生物信息学方法分析和预测了重复序列(direct repeat,DR)的同源性及二级结构。结果表明:除S4只含有一个CRISPR结构外,其余7株嗜热链球菌均检测出3条CRISPR序列,远高于CRISPR database公布细菌中具有该结构的比例(46.4%),且分别具特异性的重复序列(DR1～DR3);CRISPR最长达2853bp,最短仅101bp。对DR的二级结构预测发现,DR1～DR3均能形成回文结构,但茎环大小会有差异;在同源性比对中发现,除多数同属或同种外,供试DR还与个别远缘菌种具有高同源性,这种现象证明了供试菌株的CRISPR序列同样存在的水平基因转移,并可能存在其他的进化进程。

Lychrel Numbers in Base 10: A Probabilistic Approach

For decades, Lychrel numbers have been studied on many bases. Their existence has been proven in base 2, 11 or 17. This paper presents a probabilistic proof of the existence of Lychrel number in base 10 and provides some properties which enable a mathematical extraction of new Lychrel numbers from existing ones. This probabilistic approach has the advantage of being extendable to other bases. The results show that palindromes can also be Lychrel numbers.

Genome engineering using the CRISPR/Cas system

Recently, an epoch-making genome engineering technology using clustered regularly at interspaced short palindromic repeats(CRISPR) and CRISPR associated(Cas) nucleases, was developed. Previous technologies for genome manipulation require the time-consuming design and construction of genome-engineered nucleases for each target and have, therefore, not been widely used in mouse research where standard techniques based on homologous recombination are commonly used. The CRISPR/Cas system only requires the design of sequences complementary to a target locus, making this technology fast and straightforward. In addition, CRISPR/Cas can be used to generate mice carrying mutations in multiple genes in a single step, an achievement not possible using other methods. Here, we review the uses of this technology in genetic analysis and manipulation, including achievements made possible to date and the prospects for future therapeutic applications.

乳酸菌CRISPR-Cas系统同源性分析

CRISPR-Cas系统为乳酸菌抵抗噬菌体等外源基因元件提供获得性免疫,利用生物信息学方法分析了CRISPR序列及cas基因在乳酸菌种间的同源性关系。利用生物信息学方法预测NCBI中已测序的部分乳酸菌所含CRISPRCas系统,并对重复序列和Cas蛋白序列进行系统发生学分析。结果显示,嗜热链球菌、德氏乳杆菌和瑞士乳杆菌标准菌株均含不同数目的 CRISPR-Cas系统,各类型的重复序列高度保守,Cas蛋白序列同源性较高。CRISPR-Cas系统的同源性与其亚型密切相关,而与菌种亲缘关系有较大差异,证明了CRISPR-Cas系统能够通过基因水平转移传播这一结论。

CRISPR家族新成员:CRISPR-Cpf1

近年来,基因组编辑技术得到了飞速发展,该技术正在基础生物学研究、医学、生物技术等多个领域引起一场新的变革.Cpf1,作为CRISPR系统的新成员,极大地扩展了基因编辑靶位点的选择范围,同时其介导的多基因编辑具有明显的优势.另外,较短的crRNA序列也使Cpf1更容易产业化.本文将从Cpf1的结构和编辑特点、应用进展、目前面临的问题及展望等方面进行介绍和总结.

Gene editing for corneal disease management

Gene editing has recently emerged as a promising technology to engineer genetic modifications precisely in the genome to achieve long-term relief from corneal disorders.Recent advances in the molecular biology leading to the development of clustered regularly interspaced short palindromic repeats(CRISPRs) and CRISPR-associated systems,zinc finger nucleases and transcription activator like effector nucleases have ushered in a new era for high throughput in vitro and in vivo genome engineering.Genome editing can be successfully used to decipher complex molecular mechanisms underlying disease pathophysiology,develop innovative next generation gene therapy,stem cell-based regenerative therapy,and personalized medicine for corneal and other ocular diseases.In this review we describe latest developments in the field of genome editing,current challenges,and future prospects for the development of personalized genebased medicine for corneal diseases.The gene editing approach is expected to revolutionize current diagnostic and treatment practices for curing blindness.

Palindrome H新型肝素涂层导管在血液透析患者中的应用

目的:比较Palindrome H新型肝素涂层导管和Palindrome非肝素涂层导管在血液透析患者中透析充分性、导管功能及并发症的异同。方法:选取2014年1月至2016年6月,需维持性血液透析,而无法建立内瘘的患者,采用前瞻开放对照的方式,分为2组,分为Palindrome H肝素涂层带隧道带涤纶套导管组和Palindrome带隧道带涤纶套导管组,分别置管,对比两组尿素清除率(Kt/V)、尿素下降率(URR)、导管相关感染、导管功能不良率等指标。结果:共纳入患者50例,其中Palindrome H肝素涂层导管组有26例,使用Palindrome导管组有24例。两组患者年龄差异无统计学意义(62±19.72 vs.67±13.99,P=0.284)。Palindrome H肝素涂层导管组患者Kt/V优于Palindrome导管组(P=0.038),Palindrome H肝素涂层导管组患者导管功能不良率明显低于Palindrome导管组(P=0.022)。结论:Palindrome H肝素涂层带隧道带涤纶套的导管在透析充分性、导管功能不良率等方面均优于非肝素涂层Palindrome导管,在带隧道带涤纶套导管的临床应用中,Palindrome H肝素涂层导管比Palindrome导管更具优势。

新型长期血液透析导管Palindrome的临床应用

目的比较新型长期透析导管Palindrome同以往长期透析导管Permcath及动静脉内瘘为血管通路的血液透析患者在透析充分性、导管功能及并发症方面的异同。方法以使用Permcath导管临床资料完整43例为Permcath组,Palindrome导管49例为Palindrome组,同期动静脉内瘘56例为AVF组。观察各组尿素清除率(Kt/V)、尿素下降率(URR),导管最大血流量,各组导管相关感染、血栓发生及导管1年通畅率等。结果选择长期透析导管占同期透析通路比例为49.2%,首次直接选择长期透析导管比例为17.4%。使用长期透析导管患者病因中,继发性肾损害(代谢性疾病、高血压、缺血性肾病等)占73.1%,明显高于AVF组的51.5%(t=13.064,P=0.000,)。Palindrome组在Kt/V、URR、1年通畅率上明显优于Permcath组(P分别=0.02,0.007,0.021),与AVF组无显著差异。Palindrome组通路相关感染发生率明显高于AVF组(ρ~2=4.5946,P=0.032),血栓发生率为30.6%,明显低于Permcath组的48.8%(ρ~2=7.8125,P=0.005),高于AVF组的5.4%(ρ~2=5.9019,P=0.015)。结论 Palindrome导管在最大血流量,透析充分性、1年通畅率等方面优于Permcath导管,与AVF患者相当,但仍有较高的感染率和血栓发生率。长期透析导管只能作为维持性透析患者血管通路的较好补充形式,不作为首选。

68例Palindrome导管的临床应用观察

目的探讨palindrome导管在长期深静脉导管留置术中的应用效果。方法登记自2009年5月至2011年5月在第二军医大学附属长征医院肾内科接受长期深静脉导管留置术的患者,选取其中采用palindrome导管与相同手术时间的非palindrome导管病例在进行比较。2组感染及低流量发生率用次/1000导管日表示,比较用χ2检验,平均带管时间比较用t检验。结果总手术234例次(229人),Palindrome导管组68例次,带管时间(381.08±44.61)d,发生感染2例次,平均感染率为0.08次/1000导管日,发生低流量23例次,平均发生率为0.5/1000导管日,拔管6例;普通导管组166例次,带管时间(273.11±25.41)d,感染19例次,平均感染率0.42次/1000导管日,发生低流量213例次,平均发生率为4.7次/1000导管日,拔管21例。2组在感染、低流量发生率及平均带管时间上存在统计学差异,Palindrome组优于普通导管组。6例Palindrome导管再循环为0,反接时未发生流量不佳。结论 Palindrome导管是临床深静脉长期留置导管术很好的选择,值得对其进行更大样本及更长时间的进一步临床观察。

Palindrome H新型肝素涂层导管与Permcath导管临床应用效果比较

目的比较Palindrome H肝素涂层带隧道带涤纶套导管和Permcath带隧道带涤纶套导管在血液透析充分性、导管功能及并发症的异同。方法选取维持性血液透析使用带隧道带涤纶套导管的患者47例,最终以使用Permcath导管临床资料完整21例为Permcath组,使用Palindrome H肝素涂层导管26例为Palindrome H肝素涂层组,对比两组尿素清除率(Kt/V)、尿素下降率(URR)、导管相关感染率、导管功能不良率等指标。结果两组患者年龄差异无统计学意义[(62±19.72)岁vs(64±12.22)岁,P=0.728]。Palindrome H肝素涂层导管组患者Kt/V、URR均优于Permcath组(P=0.000),Palindrome H肝素涂层导管组患者导管功能不良率明显低于Permcath组(P=0.000),Palindrome H肝素涂层导管组患者导管相关感染率低于Permcath组(P=0.048)。结论 Palindrome H肝素涂层带隧道带涤纶套的导管在透析充分性、导管功能不良率、导管相关感染率等方面均优于非肝素涂层Permcath导管,在带隧道带涤纶套导管的应用中,Palindrome H肝素涂层导管比Permcath导管更有优势。

Palindrome导管在老年维持性血液透析患者中的临床应用

目的:比较新型Palindrome导管与Permcath导管在老年维持性血液透析患者中的临床应用效果。方法:纳入2013年5月至2015年5月我院采用Palindrome导管的老年维持性血液透析患者33例(试验组)与同期采用Permcath导管的老年维持性血液透析患者29例(对照组),观察比较两组尿素清除率(Kt/V)、尿素下降率(URR),导管相关感染、功能不良发生率及导通畅率。结果 :试验组的Kt/V、URR及导管通畅率显著高于对照组,平均感染率及功能不良发生率显著低于对照组,差异有统计学意义(P<0.05)。结论:对于动静脉内瘘建立困难的老年维持性透析患者,使用Palindrome导管在透析充分性、导管感染率、通畅率等方面优于Permcath导管,是临床较好的选择。