Functional Cloning Research Articles

Application of Next-Generation Sequencing Analysis in the Directed Evolution for Creating Antibody Mimic

Molecular evolution with a variant library is used to obtain a protein with the binding affinity for a target molecule. In the study on antibody mimics, a scaffold protein is selected from structural database, and the selected protein is functionalized by means of the molecular evolution. In a conventional molecular evolution, sub-libraries including functional variants with higher probability than initial library is generated by selection method, and functional variants are identified after the sub-libraries are monoclonalized; whereas, next-generation sequencing (NGS) is recently applied for identifying positive clones in the sub-library. In this study, we tried to monitor how functional clones behaved in the sub-libraries in the directed evolution of antibody mimics. A M13 phage displayed library, containing approximately 1.0 x 109 variants was constructed by randomizing 11 amino acids in two loops of a scaffold protein selected from Protein Data Bank. After four rounds selection, nearly 400 clones were randomly monoclnalized from third and fourth round sub-libraries and a variant with specific binding affinity (∼3µM) was identified. NGS analysis was performed to observe the distributions of variant frequencies at each round sub-library. As a result, although the distribution has little change after the elution process at first round, subsequent phage infection and bacterial amplification processes changed the distribution of the sub-library and the obtained functional variant was ranked within tenth at all the rounds. These results indicate that the enrichment of functional variants depends on the phage infection and bacterial amplification processes at the first round.

Maize kernel development.

Maize (Zea mays) is a leading cereal crop in the world. The maize kernel is the storage organ and the harvest portion of this crop and is closely related to its yield and quality. The development of maize kernel is initiated by the double fertilization event, leading to the formation of a diploid embryo and a triploid endosperm. The embryo and endosperm are then undergone independent developmental programs, resulting in a mature maize kernel which is comprised of a persistent endosperm, a large embryo, and a maternal pericarp. Due to the well-characterized morphogenesis and powerful genetics, maize kernel has long been an excellent model for the study of cereal kernel development. In recent years, with the release of the maize reference genome and the development of new genomic technologies, there has been an explosive expansion of new knowledge for maize kernel development. In this review, we overviewed recent progress in the study of maize kernel development, with an emphasis on genetic mapping of kernel traits, transcriptome analysis during kernel development, functional gene cloning of kernel mutants, and genetic engineering of kernel traits.

Synthetic protein switches: Combinatorial linker engineering with iFLinkC.

Linker engineering constitutes a critical, yet frequently underestimated aspect in the construction of synthetic protein switches and sensors. Notably, systematic strategies to engineer linkers by predictive means remain largely elusive to date. This is primarily due to our insufficient understanding how the biophysical properties that underlie linker functions mediate the conformational transitions in artificially engineered protein switches and sensors. The construction of synthetic protein switches and sensors therefore heavily relies on experimental trial-and-error. Yet, methods for effectively generating linker diversity at the genetic level are scarce. Addressing this technical shortcoming, iterative functional linker cloning (iFLinkC) enables the combinatorial assembly of linker elements with functional domains from sequence verified repositories that are developed and stored in-house. The assembly process is highly scalable and given its recursive nature generates linker diversity in a combinatorial and exponential fashion based on a limited number of linker elements.

Abstract PR04: Understanding tumor clonal evolution by single-cell transcriptomic analysis in liver cancer

Abstract Tumor evolution is a key feature intrinsic to tumor biology and contributes to intratumor heterogeneity, escape of immune surveillance, treatment failure, and patients’ prognosis. The evolutionary process of tumor is driven by selecting favorable phenotypes in terms of their fitness and survival in a tumor ecosystem. While genomic alterations provide rich materials for tumor evolution, only a few can induce a recognizable phenotypic change with even fewer for a fitness advantage. Thus, transcriptomics, a major molecular feature reflecting functional activities, will be informative in modeling tumor heterogeneity and crucial in understanding tumor evolution. Here, we aim to study tumor clonal evolution by single-cell transcriptomic profiling of hepatocellular carcinoma and intrahepatic cholangiocarcinoma from 37 patients participating the immune checkpoint inhibition trials. By analyzing core biopsies before or after treatment, we determined the single-cell atlas of liver tumors and confidently separated malignant cells and non-malignant cells by inferring chromosomal copy number variations. We developed a consensus clustering model based on machine learning algorithms and statistical methods to identify functional clones from malignant cells within each tumor. We further determined the clonal relationship by constructing the phylogenetic tree of the clones from all tumors. The clonal relationship within each tumor was independently assessed by manifold based single-cell trajectory and RNA-velocity based cell lineage. The analyses revealed a tumor branching evolutionary architecture of the clones. Noticeably, tumor branching evolution was associated with patient outcomes, which was also validated by using bulk transcriptomic data from 765 liver tumors. We found tumors in the poor prognosis branch were enriched in the pathways of hypoxia, epithelial-mesenchymal transition and angiogenesis. Remarkably, the functional role of the clones within a tumor varied, indicating a cooperative tumor cell community. We found a polarization of immune landscape associated with tumor branching evolution driven by tumor cell-specific cytokines. Our results offer insight into the collective behavior of tumor cell communities in liver cancer as well as potential drivers for tumor evolution in response to immunotherapy. Citation Format: Lichun Ma, Limin Wang, Ching-Wen Chang, Sophia Franck, Dana Dominguez, Marshonna Forgues, Julian Candia, Maria O. Hernandez, Michael Kelly, Yongmei Zhao, Bao Tran, Jonathan M. Hernandez, Jeremy L. Davis, David E. Kleiner, Bradford J. Wood, Tim F. Greten, Xin Wei Wang. Understanding tumor clonal evolution by single-cell transcriptomic analysis in liver cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PR04.

Neighborhoods and isolated points in spaces of functional clones on sets

In a previous paper, on a collection FA of functional clones on a set A, we introduced a natural metric d turning it into a topological (metric) space $$ {\mathfrak{F}}_A=\left\langle {F}_A;d\right\rangle . $$ In this paper, we describe the structure of neighborhoods of clones in spaces $$ {\mathfrak{F}}_A $$ and establish a number of consequences of this result.

Functional expression cloning of molecules inducing CD34 expression in bone marrow-derived stromal myofibroblasts

We have previously shown a fraction of stromal fibroblasts/myofibroblasts (Fibs) from leukemic bone marrow cells expresses leukemia-specific transcripts along with hematopoietic and Fib-related markers. Normal bone marrow-derived Fibs (nFibs) do not express CD34 or CD45; however, nFibs may express hematopoietic markers with some specific stimulations. CD34 expression was detected in nFib cultures following the addition of a culture supernatant of blood mononuclear cells stimulated with phytohemagglutinin (PHA)-P. To identify the molecules responsible for inducing CD34 expression in nFibs, cDNA clones were isolated using functional expression cloning with a library constructed from PHA-P-stimulated human blood mononuclear cells. Positive clones inducing CD34 transcription in nFibs were selected. We confirmed that an isolated positive cDNA clone encoded human interleukin (IL)-1 beta (β). CD34 expression was observed in the nFib cultures with recombinant human (rh) IL-1β protein. And CD34 transcription was suppressed when a rhIL-1β neutralizing antibody was added to the IL-1β-stimulated nFib cultures. nFibs expressed gp130 and IL-6 receptors, and CD45 expression was detected in nFibs cultured with rhIL-1β and rhIL-6. Chronic myelogenous leukemia (CML) cells reportedly respond well to IL-1β. When CML-derived Fibs were cultured with rhIL-1β and rhIL-6, CD45-positive cells increased in number. Cell fate may be influenced by an external specific stimulation without gene introduction.

A Rare Mutation in an Infant-Derived HIV-1 Envelope Glycoprotein Alters Interprotomer Stability and Susceptibility to Broadly Neutralizing Antibodies Targeting the Trimer Apex.

The envelope glycoprotein (Env) of human immunodeficiency virus type 1 (HIV-1) is the sole target of broadly neutralizing antibodies (bnAbs). Several mechanisms, such as the acquisition of mutations, variability of the loop length, and alterations in the glycan pattern, are employed by the virus to shield neutralizing epitopes on Env to sustain survival and infectivity within the host. The identification of mutations that lead to viral evasion of the host immune response is essential for the optimization and engineering of Env-based trimeric immunogens. Here, we report a rare leucine-to-phenylalanine escape mutation (L184F) at the base of hypervariable loop 2 (population frequency of 0.0045%) in a 9-month-old perinatally HIV-1-infected infant broad neutralizer. The L184F mutation altered the trimer conformation by modulating intramolecular interactions stabilizing the trimer apex and led to viral escape from autologous plasma bnAbs and known N160 glycan-targeted bnAbs. The L184F amino acid change led to the acquisition of a relatively open trimeric conformation, often associated with tier 1 HIV-1 isolates and increased susceptibility to neutralization by polyclonal plasma antibodies of weak neutralizers. While there was no impact of the L184F mutation on free virus transmission, a reduction in cell-to-cell transmission was observed. In conclusion, we report a naturally selected viral mutation, L184F, that influenced a change in the conformation of the Env trimer apex as a mechanism of escape from contemporaneous plasma V2 apex-targeted nAbs. Further studies should be undertaken to define viral mutations acquired during natural infection, to escape selection pressure exerted by bnAbs, to inform vaccine design and bnAb-based therapeutic strategies.IMPORTANCE The design of HIV-1 envelope-based immunogens capable of eliciting broadly neutralizing antibodies (bnAbs) is currently under active research. Some of the most potent bnAbs target the quaternary epitope at the V2 apex of the HIV-1 Env trimer. By studying naturally circulating viruses from a perinatally HIV-1-infected infant with plasma neutralizing antibodies targeted to the V2 apex, we identified a rare leucine-to-phenylalanine substitution, in two out of six functional viral clones, that destabilized the trimer apex. This single-amino-acid alteration impaired the interprotomeric interactions that stabilize the trimer apex, resulting in an open trimer conformation and escape from broadly neutralizing autologous plasma antibodies and known V2 apex-directed bnAbs, thereby favoring viral evasion of the early bnAb response of the infected host. Defining the mechanisms by which naturally occurring viral mutations influence the sensitivity of HIV-1 to bnAbs will provide information for the development of vaccines and bnAbs as anti-HIV-1 reagents.

FCCA: Hybrid Code Representation for Functional Clone Detection Using Attention Networks

Code cloning, which reuses a fragment of source code via copy-and-paste with or without modifications, is a common way for code reuse and software prototyping. However, the duplicated code fragments often affect software quality, resulting in high maintenance cost. The existing clone detectors using shallow textual or syntactical features to identify code similarity are still ineffective in accurately finding sophisticated functional code clones in real-world code bases. This article proposes functional code clone detector using attention (FCCA), a deep-learning-based code clone detection approach on top of a hybrid code representation by preserving multiple code features, including unstructured (code in the form of sequential tokens) and structured (code in the form of abstract syntax trees and control-flow graphs) information. Multiple code features are fused into a hybrid representation, which is equipped with an attention mechanism that pays attention to important code parts and features that contribute to the final detection accuracy. We have implemented and evaluated FCCA using 275 777 real-world code clone pairs written in Java. The experimental results show that FCCA outperforms several state-of-the-art approaches for detecting functional code clones in terms of accuracy, recall, and F1 score.

Neighborhoods and Isolated Points in Spaces of Functional Clones on Sets

Neighborhoods and Isolated Points in Spaces of Functional Clones on Sets

Development, Characterization, and Application of Two Reporter-Expressing Recombinant Zika Viruses.

Zika virus (ZIKV), a mosquito-borne transplacentally transmissible flavivirus, is an enveloped virus with an ~10.8 kb plus-strand RNA genome that can cause neurological disease. To facilitate the identification of potential antivirals, we developed two reporter-expressing ZIKVs, each capable of expressing an enhanced green fluorescent protein or an improved luminescent NanoLuc luciferase. First, a full-length functional ZIKV cDNA clone was engineered as a bacterial artificial chromosome, with each reporter gene under the cap-independent translational control of a cardiovirus-derived internal ribosome entry site inserted downstream of the single open reading frame of the viral genome. Two reporter-expressing ZIKVs were then generated by transfection of ZIKV-susceptible BHK-21 cells with infectious RNAs derived by in vitro run-off transcription from the respective cDNAs. As compared to the parental virus, the two reporter-expressing ZIKVs grew to lower titers with slower growth kinetics and formed smaller foci; however, they displayed a genome-wide viral protein expression profile identical to that of the parental virus, except for two previously unrecognized larger forms of the C and NS1 proteins. We then used the NanoLuc-expressing ZIKV to assess the in vitro antiviral activity of three inhibitors (T-705, NITD-008, and ribavirin). Altogether, our reporter-expressing ZIKVs represent an excellent molecular tool for the discovery of novel antivirals.

A novel proteomic mass spectrometry-based approach to reveal functionally heterogeneous tumor clones in breast cancer metastases and identify clone-specific drug targets.

e13063 Background: Breast cancer mortality is expected to rise by almost 30% by 2030 worldwide, mainly due to the occurrence of distant metastases. The development of drugs specifically targeted at tumor drivers has not yet curbed resistance to treatment, which prevents metastases curability. There is a need for new molecular approaches to tackle metastases complex biology, particularly tumor heterogeneity, a main determinant of resistance. The aim of this study was to use a proteomic mass spectrometry-based approach to reveal functionally heterogeneous’ tumor subpopulations in breast cancer metastases, and identify clone specific drug targets. Methods: Metastasis biopsies (n = 21) were collected retrospectively from patients with advanced breast cancer treated at Oscar Lambret Cancer Center (Lille, France). Tumor heterogeneity was analyzed directly on FFPE tissue sections using MALDI mass spectrometry imaging (MSI) on a RapifleX Tissuetyper. Unsupervised spatial segmentation was performed to reveal tumor subpopulations with distinct proteomic profiles within each metastasis. The full proteomic characterization of these tumor clones was further performed with spatially resolved proteomic mass spectrometry. Results: MSI revealed that breast cancer metastases contained 2 to 5 functionally distinct tumor clones (proteomic clones). Although the clone profiles within a metastasis were correlated, unsupervised hierarchical clustering showed a clear distinction between them and specific proteomic signatures. Enrichment analysis showed that differentially expressed proteins were involved in a variety of biological processes or pathways including regulation of histone acetylation, extracellular matrix degradation, DNA repair, NOTCH pathway, estrogen-responsive target genes or exocytosis. The evolution of the proteomic clones profile during disease progression was also determined by comparison of paired biopsies. To identify the candidate treatments best fitted to metastasis heterogeneity, the specific proteomic signatures of the clones were matched against a druggable genome database. It was possible to unveil candidate drug targets personalized to each metastasis functional clone. Conclusions: MALDI mass spectrometry imaging combined with spatially resolved proteomics has the potential to tackle breast cancer metastases heterogeneity, and identify candidate drug targets specific to functional clones to personalize treatments.

Holant Clones and the Approximability of Conservative Holant Problems

We construct a theory of holant clones to capture the notion of expressibility in the holant framework. Their role is analogous to the role played by functional clones in the study of weighted counting Constraint Satisfaction Problems. We explore the landscape of conservative holant clones and determine the situations in which a set F of functions is “universal in the conservative case,” which means that all functions are contained in the holant clone generated by F together with all unary functions. When F is not universal in the conservative case, we give concise generating sets for the clone. We demonstrate the usefulness of the holant clone theory by using it to give a complete complexity-theory classification for the problem of approximating the solution to conservative holant problems. We show that approximation is intractable exactly when F is universal in the conservative case.

Abstract A69: Identification of HLA-A0201 restricted epitope of cancer/testis antigen (CTA) Hormad1 and generation of antigen-specific T-cell receptor-engineered T cells (TCR-T) for treatment of solid tumor malignancies

Abstract Purpose: Hormad1 is a novel cancer/testis antigen (CTA) that is highly expressed in several solid tumor types such as lung cancer, esophageal carcinoma, as well as head and neck cancer. High expression of Hormad1 is correlated with elevated mutational burden and reduced survival in lung adenocarcinoma, indicating that Hormad1 could be a potential target for immunotherapy. The aims of this study are to identify HLA-A0201-restricted epitope of Hormad1 and generate antigen-specific T cells and T-cell receptor (TCR)-engineered T cells (TCR-T) for adoptive cell therapy (ACT) to treat patients with Hormad1-expressing solid tumors. Methods: Candidate HLA-A0201 restricted epitopes of Hormad1 were identified by reverse-immunology and MHC class I stabilization assay. Mature HLA-A0201+ dendritic cells (mDC) were pulsed with the candidate epitope peptides and used to stimulate autologous CD25 depleted T cells. Tetramer guided sorting was used to enrich for Hormad1-specific T cells from which CTL cell lines and clones were generated. The alpha and beta TCR chains were sequenced from HORMAD1-specific, tumor-reactive CTL and cloned into a retroviral vector construct to introduce the TCR to allogeneic PBMC to generate functional TCR-T cells. Results: From 6 candidate peptides, peptide 4, which showed highest HLA-A2 binding characters, was chosen for T-cell generation. After stimulation with peptide pulsed mDC, functional CTL cell line and clones were generated from two healthy donor PBMC. Both the CTL cell line and clone selectively recognize tumor targets expressing HLA-A0201 and Hormad1. Specificity of the CTL for the HORMAD1 epitope presented by HLA-A0201 was confirmed using lines coexpressing HLA-A0201 and/or HORMAD1. Cold target inhibition assays further confirmed that the Hormad1 peptide 4 was naturally processed and presented by tumor targets. After transduction with the recombinant retroviral vector expressing the alpha and beta TCR chains of the functional CTL cell line, the resultant TCR-T cells displayed high affinity, specific tumor target killing at E:T of 1: 1.25. Moreover, these TCR-T cells specifically enhanced CD137, CD69, IFN-γ, and TNF-α expression in response to HLA-A0201-positive and Hormad1-positive tumor target cells. Conclusion: The HLA-A0201 restricted Hormad1 epitope identified in this study is a novel candidate peptide for antigen-specific vaccine development and endogenous T-cell therapy. A Hormad1-specific TCR-encoding vector was also created that conferred tumor reactivity and antigen specificity among TCR-T cells, offering a high-value product for use in adoptive T-cell therapy for patients with Hormad1-expressing solid tumor malignancies. Citation Format: Ke Pan, Yulun Chiu, Farah Hasan, Natalia Miroballi, Angelique Whitehurst, Cassian Yee. Identification of HLA-A0201 restricted epitope of cancer/testis antigen (CTA) Hormad1 and generation of antigen-specific T-cell receptor-engineered T cells (TCR-T) for treatment of solid tumor malignancies [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A69.

Molecular mapping and identification of quantitative trait loci for domestication traits in the field cress (Lepidium campestre L.) genome

Lepidium campestre (L.) or field cress is a multifaceted oilseed plant, which is not yet domesticated. Moreover, the molecular and genetic mechanisms underlying the domestication traits of field cress remain largely elusive. The overarching goal of this study is to identify quantitative trait loci (QTL) that are fundamental for domestication of field cress. Mapping and dissecting quantitative trait variation may provide important insights into genomic trajectories underlying field cress domestication. We used 7624 single nucleotide polymorphism (SNP) markers for QTL mapping in 428 F2 interspecific hybrid individuals, while field phenotyping was conducted in F2:3 segregating families. We applied multiple QTL mapping algorithms to detect and estimate the QTL effects for seven important domestication traits of field cress. Verification of pod shattering across sites revealed that the non-shattering lines declined drastically whereas the shattering lines increased sharply, possibly due to inbreeding followed by selection events. In total, 1461 of the 7624 SNP loci were mapped to eight linkage groups (LGs), spanning 571.9 cM map length. We identified 27 QTL across all LGs of field cress genome, which captured medium to high heritability, implying that genomics-assisted selection could deliver domesticated lines in field cress breeding. The use of high throughput genotyping can accelerate the process of domestication in novel crop species. This is the first QTL mapping analysis in the field cress genome that may lay a foundational framework for positional or functional QTL cloning, introgression as well as genomics-assisted breeding in field cress domestication.

Identification of the novel tigecycline resistance gene tet(X6) and its variants in Myroides, Acinetobacter and Proteus of food animal origin

To report a novel tigecycline resistance gene, tet(X6), and its variants in four bacterial species isolated from chickens and pigs in China. WGS was conducted to identify the suspected resistance genes in the tigecycline-resistant Myroides phaeus 18QD1AZ29W. Functional cloning, homology modelling and molecular docking were performed to compare the function with other Tet(X) variants. Retrospective screening for tet(X6) was conducted for 80 isolates in our WGS data collection, and all genomic environments of tet(X6)-positive isolates were analysed. The tigecycline-resistant M. phaeus 18QD1AZ29W isolated from a pig farm in Shandong in 2018 was positive for tet(X2) and a novel tet(X) gene, designated tet(X6). Tet(X6) could increase the MICs of all tested tetracyclines/glycylcyclines for Escherichia coli only 2- to 4-fold, which was possibly due to a lower tetracycline binding capacity of Tet(X6) compared with that of other Tet(X) variants. Retrospective screening showed that seven other isolates (7/80, 8.8%), comprising four Proteus spp. and three Acinetobacter spp. from chickens and pigs in Shandong and Guangdong, were positive for three different variants of tet(X6). The analysis of the genomic environment revealed that two tet(X6)-positive isolates from M. phaeus and Proteus cibarius, respectively, contained ISCR2, which may play a role in tet(X6) transmission. This study identified a novel type of tigecycline resistance gene, tet(X6), in Myroides, Acinetobacter and Proteus from chickens and swine. Tet(X6) conferred lower tetracycline/glycylcycline MICs than other Tet(X) variants, and ISCR2 may play a role in the transmission of tet(X6).

IFLinkC: an iterative functional linker cloning strategy for the combinatorial assembly and recombination of linker peptides with functional domains.

Recent years have witnessed increasing efforts to engineer artificial biological functions through recombination of modular-organized toolboxes of protein scaffolds and parts. A critical, yet frequently neglected aspect concerns the identity of peptide linkers or spacers connecting individual domains which remain poorly understood and challenging to assemble. Addressing these limitations, iFlinkC comprises a highly scalable DNA assembly process that facilitates the combinatorial recombination of functional domains with linkers of varying length and flexibility, thereby overcoming challenges with high GC-content and the repeat nature of linker elements. The capacity of iFLinkC is demonstrated in the construction of synthetic protease switches featuring PDZ-FN3-based affinity clamps and single-chain FKBP12-FRB receptors as allosteric inputs. Library screening experiments demonstrate that linker space is highly plastic as the induction of allosterically regulated protease switches can vary from >150-fold switch-ON to >13-fold switch-OFF solely depending on the identity of the connecting linkers and relative orientation of functional domains. In addition, Pro-rich linkers yield the most potent switches contradicting the conventional use of flexible Gly-Ser linkers. Given the ease and efficiency how functional domains can be readily recombined with any type of linker, iFLinkC is anticipated to be widely applicable to the assembly of any type of fusion protein.

Increased O-GlcNAcylation of c-Myc Promotes Pre-B Cell Proliferation

O-linked β-N-acetylglucosamine (O-GlcNAc) modification regulates the activity of hundreds of nucleocytoplasmic proteins involved in a wide variety of cellular processes, such as gene expression, signaling, and cell growth; however, the mechanism underlying the regulation of B cell development and function by O-GlcNAcylation remains largely unknown. Here, we demonstrate that changes in cellular O-GlcNAc levels significantly affected the growth of pre-B cells, which rapidly proliferate to allow expansion of functional clones that express successfully rearranged heavy chains at the pro-B stage during early B cell development. In our study, the overall O-GlcNAc levels in these proliferative pre-B cells, which are linked to the glucose uptake rate, were highly induced when compared with those in pro-B cells. Thus, pharmacologically, genetically, or nutritionally, inhibition of O-GlcNAcylation in pre-B cells markedly downregulated c-Myc expression, resulting in cell cycle arrest via blockade of cyclin expression. Importantly, the population of B cells after the pro-B cell stage in mouse bone marrow was severely impaired by the administration of an O-GlcNAc inhibitor. These results strongly suggest that O-GlcNAcylation-dependent expression of c-Myc represents a new regulatory component of pre-B cell proliferation, as well as a potential therapeutic target for the treatment of pre-B cell-derived leukemia.

A Novel Method of Clone Detection by Neural Networks

Code clone is that type of engine that helps to find duplicate code patterns find within the whole code. Programmers usually adopt code reusability task from previous few years, so that time consumption can be reduces. Code reusability can be done via replication or by just copy-paste. Code reusability leads to not writing code from scratch, just copy paste the useful part of the code. In finding of duplicated code fragment or text, plagiarism detection also work pretty well but it is not applicable to the large system in finding functional clone and also it is more time consuming even at small scale which make the detection method inappropriate. In this paper, we proposed a pattern similarity conditions on the basis of textual similarity for finding the code or text clones in the large content on the basis of SVM, Neural Network using Java coding, Neural Network and Sim Cad. This approach detects code or text clones from original one. The resultant simulation is taken place in the MATLAB environment, and it has shown that it is providing better results. The proposed algorithm performance is measured using parameters i.e. FRR, FAR and Accuracy.

Novel Method of Clone Detection by Neural Networks

Code clone is that type of engine that helps to find duplicate code patterns find within the whole code. Programmers usually adopt code reusability task from previous few years, so that time consumption can be reduces. Code reusability can be done via replication or by just copy-paste. Code reusability leads to not writing code from scratch, just copy paste the useful part of the code. In finding of duplicated code fragment or text, plagiarism detection also work pretty well but it is not applicable to the large system in finding functional clone and also it is more time consuming even at small scale which make the detection method inappropriate. In this paper, we proposed a pattern similarity conditions on the basis of textual similarity for finding the code or text clones in the large content on the basis of SVM, Neural Network using Java coding, Neural Network and Sim Cad. This approach detects code or text clones from original one. The resultant simulation is taken place in the MATLAB environment, and it has shown that it is providing better results. The proposed algorithm performance is measured using parameters i.e. FRR, FAR and Accuracy.

A recombination bin-map identified a major QTL for resistance to Tomato Spotted Wilt Virus in peanut (Arachis hypogaea)

Tomato spotted wilt virus (TSWV) is a devastating disease to peanut growers in the South-eastern region of the United States. Newly released peanut cultivars in recent years are crucial as they have some levels of resistance to TSWV. One mapping population of recombinant inbred line (RIL) used in this study was derived from peanut lines of SunOleic 97R and NC94022. A whole genome re-sequencing approach was used to sequence these two parents and 140 RILs. A recombination bin-based genetic map was constructed, with 5,816 bins and 20 linkage groups covering a total length of 2004 cM. Using this map, we identified three QTLs which were colocalized on chromosome A01. One QTL had the largest effect of 36.51% to the phenotypic variation and encompassed 89.5 Kb genomic region. This genome region had a cluster of genes, which code for chitinases, strictosidine synthase-like, and NBS-LRR proteins. SNPs linked to this QTL were used to develop Kompetitive allele specific PCR (KASP) markers, and the validated KASP markers showed expected segregation of alleles coming from resistant and susceptible parents within the population. Therefore, this bin-map and QTL associated with TSWV resistance made it possible for functional gene mapping, map-based cloning, and marker-assisted breeding. This study identified the highest number of SNP makers and demonstrated recombination bin-based map for QTL identification in peanut. The chitinase gene clusters and NBS-LRR disease resistance genes in this region suggest the possible involvement in peanut resistance to TSWV.