Construction of Marker-Free GFP Transgenic Tobacco by Cre/lox Site-Specific Recombination System

Marker-free GFP transgenic tobacco plants were constructed based on Cre/lox site-specific recombination system. A GFP gene was introduced into the tobacco genome using the Bar gene as a linked selectable marker flanked by recombination sites in a directed orientation. The Bar gene expression box was subsequently excised from the plant genome by a strategy of Cre gene retransformation. After removal of the Cre-NPT Ⅱ locus by genetic segregation through self-cross, plants that incorporated only the GFP transgene were obtained. Transgenic tobacco plants mediated by Agrobacterium tumefaciens were obtained, which resisted herbicide Basta and GFP expressed well, then the Cre gene was subsequently introduced into 5 plants of them, respectively, by retransformation. The leaf disks from Cre transgenic plants were used to test the resistance to Basta on the medium with 8 mg L-1 of PPT. The results showed that few discs were able to regenerate normally, and the excision at 76-100% efficiency depended on individual retransformation events. Evidence for a precise recombination event was confirmed by cloning the nucleotides sequence surrounding the lox sites of the Basta sensitive plants. The result indicated that the excision event in the recombination sites was precise and conservative, without loss or alteration of any submarginal nucleotides of the recombination sites. Bar gene excised plants were selfpollinated to allow segregation of the GFP gene from the Cre-NPT Ⅱ locus. The progenies from self-pollinated plants were scored for Kan senstivity, then the segregation of GFP gene from Cre-NPT Ⅱ locus in the Kan senstive plants were confirmed by PCR analysis subsequently. Hence, constructing marker-free transgenic tobacco plants by Cre/lox sitespecific recombination system was reliable, and the strategy presented here should be applicable to other plants for the construction of marker-free transgenic plants as well.

A novel color image encryption algorithm based on genetic recombination and the four-dimensional memristive hyperchaotic system

Recently, many image encryption algorithms based on chaos have been proposed. Most of the previous algorithms encrypt components R, G, and B of color images independently and neglect the high correlation between them. In the paper, a novel color image encryption algorithm is introduced. The 24 bit planes of components R, G, and B of the color plain image are obtained and recombined into 4 compound bit planes, and this can make the three components affect each other. A four-dimensional(4D) memristive hyperchaotic system generates the pseudorandom key streams and its initial values come from the SHA 256 hash value of the color plain image. The compound bit planes and key streams are confused according to the principles of genetic recombination, then confusion and diffusion as a union are applied to the bit planes,and the color cipher image is obtained. Experimental results and security analyses demonstrate that the proposed algorithm is secure and effective so that it may be adopted for secure communication.

Establishment and application of both FLP and Cre site-specific recombination systems at the same position in the genome

Both FRT-FRT and LoxP-LoxP sites that are the target sepuences of site-specific recombinases have been constructed in a vector, called C4LFY, using the recombinant DNA technigue. C4LFY also contains P elements, 2 exons and 1 intron of Drosophila yellow gene, yellow promoter and enhancers, and flanking DNA. Since C4LFY made use of two pairs of FRT and LoxP sites, this vector included two site-specific recombination systems. C4LFY was then integrated into Drosophila genome by P-element-mediated germ line transformation. in the presence of the FLP or Cre recombinase, either FLP/FRT or Cre/LoxP recombination reaction was successfully created at the same position in the genome. Using this system, the molecular basis of yellow gene expression and regulation during development have been investigated. Results indicate that the tissue-specific expression of yellow gene is directly regulated by transcriptional enhancers. in addition, the 5’ and 3’ genomic sequences flanking the yellow gene have been

Co-transformation to tobacco of Cre/lox site-specific recombination system and its precise recombination

For the temporally and spatially regulated expression of the barnase gene in plant, two kinds of plasmids with ere gene and its directly repeat recognition sites lox from bacteriophage P1 were constructed and co-transformed into tobacco by agrobacterium mediated procedure. The transgenic plants were conformed by PCR analysis. The blocking fragment between the two lox directly repeat sites was excised by Cre protein in the transgenic plant genome. Cloning and seguencing the DNA fragment from the co-transformed plant DNA showed that the precise DNA excision occurred in transgenic tobacco genome directed by Cre//ox site-specif ic recombination .

Impact of Sequence Non-Identities on Recombination within the <i>pil</i>System of <i>Neisseria gonorrhoeae</i>

Neisseria gonorrhoeae engages in extensive intra-cellular gene conversion between the PilE-expression locus (pilE) and the transcriptionally-silent pil gene copies (pilS). In silico analyses were applied to investigate the extent of sequence heterogeneity between the various pilS gene copies. Analysis of synonymous and non-synonymous substitutions between the different pilS genes indicated that relatively few amino acid changes would occur due to nucleotide polymorphisms towards the 5’ end of the pilS genes whereas more frequent amino acid substitutions would be incorporated within the “hypervariable” region. The lack of non-synonymous substitutions at the 5’ end of the genes was found to be under selective pressure as indicated by a positive DT score utilizing the Tajima test. The presence or absence of mismatch repair appeared to only impact recombination when non-identical DNAs recombined via the DNA transformation route, where small pil sequence heterogeneities were sufficient to terminate recombination tracts, with these sequence constraints being relieved in cells carrying a mutS mutation. Therefore, the data indicate that the effect of sequence heterogeneity on recombination within the pil system appears to depend upon the context with which the non-identical DNAs recombine.

Clinical and molecular significance of homologous recombination deficiency positive non-small cell lung cancer in Chinese population:An integrated genomic and transcriptional analysis

Objective:The clinical significance of homologous recombination deficiency(HRD)in breast cancer,ovarian cancer,and prostate cancer has been established,but the value of HRD in non-small cell lung cancer(NSCLC)has not been fully investigated.This study aimed to systematically analyze the HRD status of untreated NSCLC and its relationship with patient prognosis to further guide clinical care.Methods:A total of 355 treatment-naïve NSCLC patients were retrospectively enrolled.HRD status was assessed using the AmoyDx Genomic Scar Score(GSS),with a score of≥50 considered HRD-positive.Genomic,transcriptomic,tumor microenvironmental characteristics and prognosis between HRD-positive and HRDnegative patients were analyzed.Results:Of the patients,25.1%(89/355)were HRD-positive.Compared to HRD-negative patients,HRDpositive patients had more somatic pathogenic homologous recombination repair(HRR)mutations,higher tumor mutation burden(TMB)(P<0.001),and fewer driver gene mutations(P<0.001).Furthermore,HRD-positive NSCLC had more amplifications in PI3K pathway and cell cycle genes,MET and MYC in epidermal growth factor receptor(EGFR)/anaplastic lymphoma kinase(ALK)mutant NSCLC,and more PIK3CA and AURKA in EGFR/ALK wild-type NSCLC.HRD-positive NSCLC displayed higher tumor proliferation and immunosuppression activity.HRD-negative NSCLC showed activated signatures of major histocompatibility complex(MHC)-II,interferon(IFN)-γand effector memory CD8+T cells.HRD-positive patients had a worse prognosis and shorter progressionfree survival(PFS)to targeted therapy(first-and third-generation EGFR-TKIs)(P=0.042).Additionally,HRDpositive,EGFR/ALK wild-type patients showed a numerically lower response to platinum-free immunotherapy regimens.Conclusions:Unique genomic and transcriptional characteristics were found in HRD-positive NSCLC.Poor prognosis and poor response to EGFR-TKIs and immunotherapy were observed in HRD-positive NSCLC.This study highlights potential actionable alterations in HRD-positive NSCLC,suggesting possible combinational therapeutic strategies for these patients.

Mechanochemical strategy assisted morphology recombination of COFs for promoted kinetics and LiPS transformation in Li-S batteries

A covalent organic frameworks(COFs)material with regular pores and stable structure can be used as the host of lithium-sulfur batteries to improve battery kinetics and polysulfides conversion.Herein,we designed and synthesized two kinds of rod-liked bulk COFs by adjusting different pore sizes(COF-BTD and COF-TFB),unfortunately,the active sites masking and sluggish kinetics have not met our expectations.Generally,the available layered COFs prepared from mechanochemical can expose abundant active sites and favorable kinetics than bulk COFs.Thus,simple mechanical ball milling is applied to activate the above COFs(M-COFs group).It is worth noting that layered R-COF-BTD is directly synthesized from rod-liked precursors by simple morphological reconstruction.A series of characterization methods are used to systematically explore the advantages of the group of M-COFs@S electrodes in the cycling process,including the effects of specific morphology on the kinetics and transformation of polysulfides.Our research provides a feasible plan for the development and selection of the host material of lithium-sulfur batteries.

Physico−mathematical model of the voltage−current characteristics of light-emitting diodes with quantum wells based on the Sah−Noyce−Shockley recombination mechanism

Herein,a physical and mathematical model of the voltage−current characteristics of a p−n heterostructure with quantum wells(QWs)is prepared using the Sah−Noyce−Shockley(SNS)recombination mechanism to show the SNS recombination rate of the correction function of the distribution of QWs in the space charge region of diode configuration.A comparison of the model voltage−current characteristics(VCCs)with the experimental ones reveals their adequacy.The technological parameters of the structure of the VCC model are determined experimentally using a nondestructive capacitive approach for determining the impurity distribution profile in the active region of the diode structure with a profile depth resolution of up to 10Å.The correction function in the expression of the recombination rate shows the possibility of determining the derivative of the VCCs of structures with QWs with a nonideality factor of up to 4.

Initial measurement of ultrafast charge exchange recombination spectroscopy on the EAST tokamak

Ultrafast charge exchange recombination spectroscopy(UF-CXRS)has been developed on the EAST tokamak(Yingying Li et al 2019 Fusion Eng.Des.146522)to measure fast evolutions of ion temperature and toroidal velocity.Here,we report the preliminary diagnostic measurements after relative sensitivity calibration.The measurement results show a much higher temporal resolution compared with conventional CXRS,benefiting from the usage of a prismcoupled,high-dispersion volume-phase holographic transmission grating and a high quantum efficiency,high-gain detector array.Utilizing the UF-CXRS diagnostic,the fast evolutions of the ion temperature and rotation velocity during a set of high-frequency small-amplitude edgelocalized modes(ELMs)are obtained on the EAST tokamak,which are then compared with the case of large-amplitude ELMs.

Low testing rates and high BRCA prevalence: Poly (ADP-ribose) polymerase inhibitor use in Middle East BRCA/homologous recombination deficiency-positive cancer patients

BACKGROUND Poly(ADP-ribose)polymerase inhibitors(PARPis)are approved as first-line therapies for breast cancer gene(BRCA)-positive,human epidermal growth factor receptor 2-negative locally advanced or metastatic breast cancer.They are also effective for new and recurrent ovarian cancers that are BRCA-or homologous recombination deficiency(HRD)-positive.However,data on these mutations and PARPi use in the Middle East are limited.AIM To assess BRCA/HRD prevalence and PARPi use in patients in the Middle East with breast/ovarian cancer.METHODS This was a single-center retrospective study of 57 of 472 breast cancer patients tested for BRCA mutations,and 25 of 65 ovarian cancer patients tested for HRD.These adult patients participated in at least four visits to the oncology service at our center between August 2021 and May 2023.Data were summarized using descriptive statistics and compared using counts and percentages.Response to treatment was assessed using Response Evaluation Criteria in Solid Tumors criteria.RESULTS Among the 472 breast cancer patients,12.1%underwent BRCA testing,and 38.5%of 65 ovarian cancer patients received HRD testing.Pathogenic mutations were found in 25.6%of the tested patients:26.3%breast cancers had germline BRCA(gBRCA)mutations and 24.0%ovarian cancers showed HRD.Notably,40.0%of gBRCA-positive breast cancers and 66.0%of HRD-positive ovarian cancers were Middle Eastern and Asian patients,respectively.PARPi treatment was used in 5(33.3%)gBRCA-positive breast cancer patients as first-line therapy(n=1;7-months progression-free),for maintenance(n=2;>15-months progression-free),or at later stages due to compliance issues(n=2).Four patients(66.6%)with HRD-positive ovarian cancer received PARPi and all remained progression-free.CONCLUSION Lower testing rates but higher BRCA mutations in breast cancer were found.Ethnicity reflected United Arab Emirates demographics,with breast cancer in Middle Eastern and ovarian cancer in Asian patients.

Establishing the homologous recombination score threshold in metastatic prostate cancer patients to predict the efficacy of PARP inhibitors

Background:The homologous recombination deficiency(HRD)score serves as a promising biomarker to iden-tify patients who are eligible for treatment with PARP inhibitors(PARPi).Previous studies have suggested a 3-biomarker Genomic Instability Score(GIS)threshold of≥42 as a valid biomarker to predict response to PARPi in patients with ovarian cancer and breast cancer.However,the GIS threshold for prostate cancer(PCa)is still lacking.Here,we conducted an exploratory analysis to investigate an appropriate HRD score threshold and to evaluate its ability to predict response to PARPi in PCa patients.Methods:A total of 181 patients with metastatic castration-resistant PCa were included in this study.Tumor tissue specimens were collected for targeted next-generation sequencing for homologous recombination repair(HRR)genes and copy number variation(CNV)analysis.The HRD score was calculated based on over 50,000 single-nucleotide polymorphisms(SNP)distributed across the human genome,incorporating three SNP-based as-says:loss of heterozygosity,telomeric allelic imbalance,and large-scale state transition.The HRD score threshold was set at the last 5th percentile of the HRD scores in our cohort of known HRR-deficient tumors.The relation-ship between the HRD score and the efficacy in 16 patients of our cohort who received PARPi treatment were retrospectively analyzed.Results:Genomic testing was succeeded in 162 patients.In our cohort,61 patients(37.7%)had HRR mutations(HRRm).BRCA mutations occurred in 15 patients(9.3%).The median HRD score was 4(ranged from 0 to 57)in the total cohort,which is much lower than that in breast and ovarian cancers.Patients who harbored HRRm and BRCA or TP53 mutations had higher HRD scores.CNV occured more frequently in patients with HRRm.The last 5th percentile of HRD scores was 43 in the HRR-mutant cohort and consequently HRD high was defined as HRD scores≥43.In the 16 patients who received PARPi in our cohort,4 patients with a high HRD score achieved an objective response rate(ORR)of 100%while 12 patients with a low HRD score achieved an ORR of 8.3%.Progression-free survival(PFS)in HRD high patients was longer compared to HRD low patients,regardless of HRRm.Conclusions:A HRD score threshold of 43 was established and preliminarily validated to predict the efficacy of PARPi in this study.Future studies are needed to further verify this threshold.

Features of Recombination Radiation of GaAs Type Semiconductors with the Participation of Fine Acceptor Levels in a Magnetic Field

Using the method of Picus and Beer invariants, general expressions are obtained for the total intensity I and the degree of circular polarization Рcirc.of the luminescence of GaAs-type semiconductors with the participation of shallow acceptor levels in a longitudinal magnetic field H. Special cases are analyzed depending on the value and direction of the magnetic field strength, as well as on the constants of the g-factor of the acceptor g1,g2and the conduction band electron ge. In the case of a strong magnetic field H// [100], [111], [110], a numerical calculation of the angular dependence of the quantities I and Рcirc.was performed for some critical values of g2/g1, at which Рcirc.exhibits a sharp anisotropy in the range from −100% to +100%, and the intensity of the crystal radiation along the magnetic field tends to a minimum value.

Crystallization Modulation and Holistic Passivation Enables Efficient Two‑Terminal Perovskite/CuIn(Ga)Se_(2)Tandem Solar Cells

Two-terminal(2-T)perovskite(PVK)/CuIn(Ga)Se_(2)(CIGS)tandem solar cells(TSCs)have been considered as an ideal tandem cell because of their best bandgap matching regarding to Shockley–Queisser(S–Q)limits.However,the nature of the irregular rough morphology of commercial CIGS prevents people from improving tandem device performances.In this paper,D-homoserine lactone hydrochloride is proven to improve coverage of PVK materials on irregular rough CIGS surfaces and also passivate bulk defects by modulating the growth of PVK crystals.In addition,the minority carriers near the PVK/C60 interface and the incompletely passivated trap states caused interface recombination.A surface reconstruction with 2-thiopheneethylammonium iodide and N,N-dimethylformamide assisted passivates the defect sites located at the surface and grain boundaries.Meanwhile,LiF is used to create this field effect,repelling hole carriers away from the PVK and C60 interface and thus reducing recombination.As a result,a 2-T PVK/CIGS tandem yielded a power conversion efficiency of 24.6%(0.16 cm^(2)),one of the highest results for 2-T PVK/CIGS TSCs to our knowledge.This validation underscores the potential of our methodology in achieving superior performance in PVK/CIGS tandem solar cells.

Recombinant chitinase-3-like protein 1 alleviates learning and memory impairments via M2 microglia polarization in postoperative cognitive dysfunction mice

Postoperative cognitive dysfunction is a seve re complication of the central nervous system that occurs after anesthesia and surgery,and has received attention for its high incidence and effect on the quality of life of patients.To date,there are no viable treatment options for postoperative cognitive dysfunction.The identification of postoperative cognitive dysfunction hub genes could provide new research directions and therapeutic targets for future research.To identify the signaling mechanisms contributing to postoperative cognitive dysfunction,we first conducted Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses of the Gene Expression Omnibus GSE95426 dataset,which consists of mRNAs and long non-coding RNAs differentially expressed in mouse hippocampus3 days after tibial fracture.The dataset was enriched in genes associated with the biological process"regulation of immune cells,"of which Chill was identified as a hub gene.Therefore,we investigated the contribution of chitinase-3-like protein 1 protein expression changes to postoperative cognitive dysfunction in the mouse model of tibial fractu re surgery.Mice were intraperitoneally injected with vehicle or recombinant chitinase-3-like protein 124 hours post-surgery,and the injection groups were compared with untreated control mice for learning and memory capacities using the Y-maze and fear conditioning tests.In addition,protein expression levels of proinflammatory factors(interleukin-1βand inducible nitric oxide synthase),M2-type macrophage markers(CD206 and arginase-1),and cognition-related proteins(brain-derived neurotropic factor and phosphorylated NMDA receptor subunit NR2B)were measured in hippocampus by western blotting.Treatment with recombinant chitinase-3-like protein 1 prevented surgery-induced cognitive impairment,downregulated interleukin-1βand nducible nitric oxide synthase expression,and upregulated CD206,arginase-1,pNR2B,and brain-derived neurotropic factor expression compared with vehicle treatment.Intraperitoneal administration of the specific ERK inhibitor PD98059 diminished the effects of recombinant chitinase-3-like protein 1.Collectively,our findings suggest that recombinant chitinase-3-like protein 1 ameliorates surgery-induced cognitive decline by attenuating neuroinflammation via M2 microglial polarization in the hippocampus.Therefore,recombinant chitinase-3-like protein1 may have therapeutic potential fo r postoperative cognitive dysfunction.

秩序·扰动·RECOMBINATION

这个事实我们知道了很久:一种生物,若遇到人为的物理、化学、生物方法引导,或者遗传因子得到重组,就可能在合适的新环境中发生多元突变。世界美术的古典风格、中世纪风格、文艺复兴风格、巴洛克风格,艺术设计的现代理念、后现代理念,手工艺的传统方式、"莫里斯方式"、"柳宗悦方式"……我们看到了,一部历史,就是传统秩序,经演进中的社会经济、政治、哲学、文化、生活方式等新生因子的扰动催化,最后产生复合变异的结果。

Homologous recombination in DNA repair and DNA damage tolerance

Homologous recombination （HR） comprises a series of interrelated pathways that function in the repair of DNA double-stranded breaks （DSBs） and interstrand crosslinks （ICLs）. In addition, recombination provides critical support for DNA replication in the recovery of stalled or broken replication forks, contributing to tolerance of DNA damage. A central core of proteins, most critically the RecA homolog Rad51, catalyzes the key reactions that typify HR： homology search and DNA strand invasion. The diverse functions of recombination are reflected in the need for context-specific factors that perform supplemental functions in conjunction with the core proteins. The inability to properly repair complex DNA damage and resolve DNA replication stress leads to genomic instability and contributes to cancer etiology. Mutations in the BRCA2 recombination gene cause predisposition to breast and ovarian cancer as well as Fanconi anemia, a cancer predisposition syndrome characterized by a defect in the repair of DNA interstrand crosslinks. The cellular functions of recombination are also germane to DNA-based treatment modalities of cancer, which target replicating cells by the direct or indirect induction of DNA lesions that are substrates for recombination pathways. This review focuses on mechanistic aspects of HR relating to DSB and ICL repair as well as replication fork support.

Support vector machine for prediction of meiotic recombination hotspots and coldspots in Saccharomyces cerevisiae

A novel method for predicting hotspots and coldspots using support vector machine （SVM） based on statistical learning theory is developed. This method is applied to published 303 hot and 48 cold open reading frames （ORFs） in Saccharomyces cerevisiae. The sequence features of general dinucleotide abundance and dinucleotide abundance based on codon usage are extracted, and then the data sets are classified with different parameters and kernel functions combined with the method of two-fold cross validation. The result indicates that 87.47% accuracy can be reached when classifying hot and cold ORF sequences with the kernel of radial basis function combined with dinucleotide abundance based on codon usage.

The multi-factor recombination and processes superimposition model for hydrocarbon accumulation:application to the Silurian in the Tarim Basin

The multi-factor recombination and processes superimposition model for hydrocarbon accumulation is put forward in view of the hydrocarbon geological characteristics of multiple episodes of structural evolution, multiple sets of source-reservoir-seal assemblage, multiple cycles of hydrocarbon accumulation and multiple episodes of readjustment and reconstruction in the complex superimposed basins in China. It is a system including theories and methods that can help to predict favorable exploration regions. According to this model, the basic discipline for hydrocarbon generation, evolution and distribution in the superimposed basins can be summarized in multi-factor recombination, processes superimposition, multiple stages of oil filling and latest stage preservation. With the Silurian of the Tarim basin as an example, based on the reconstruction of the evolution history of the four factors （paleo-anticline, source rock, regional cap rock and kinematic equilibrium belt） controlling hydrocarbon accumulation, this model was adopted to predict favorable hydrocarbon accumulation areas and favorable exploration regions following structural destruction in three stages of oil filling, to provide guidance for further exploration ofoil and gas in the Silurian of the Tarim basin.

Vleiotic recombination and male infertility： from basic science to clinical reality？

Infertility is a common problem that affects approximately 15% of the population. Although many advances have been made in the treatment of infertility, the molecular and genetic causes of male infertility remain largely elusive. This review will present a summary of our current knowledge on the genetic origin of male infertility and the key events of male meiosis. It focuses on chromosome synapsis and meiotic recombination and the problems that arise when errors in these processes occur, specifically meiotic arrest and chromosome aneuploidy, the leading cause of pregnancy loss in humans. In addition, meiosis-specific candidate genes will be discussed, including a discussion on why we have been largely unsuccessful at identifying disease-causing mutations in infertile men. Finally clinical applications of sperm aneuploidy screening will be touched upon along with future prospective clinical tests to better characterize male infertility in a move towards personalized medicine.

Strategies for production of active eukaryotic proteins in bacterial expression system

Bacteria have long been the favorite expression system for recombinant protein production.However,the flaw of the system is that insoluble and inactive proteins are co-produced due to codon bias,protein folding,phosphorylation,glycosylation,mRNA stability and promoter strength.Factors are cited and the methods to convert to soluble and active proteins are described,for example a tiglit control of Escherichia coli milieu,refolding from inclusion body and through fusion technology.