Functional Cloning Research Articles

Evolutionary-Related High- and Low-Virulent Classical Swine Fever Virus Isolates Reveal Viral Determinants of Virulence.

Classical swine fever (CSF) has been eradicated from Western and Central Europe but remains endemic in parts of Central and South America, Asia, and the Caribbean. CSF virus (CSFV) has been endemic in Cuba since 1993, most likely following an escape of the highly virulent Margarita/1958 strain. In recent years, chronic and persistent infections with low-virulent CSFV have been observed. Amino acid substitutions located in immunodominant epitopes of the envelope glycoprotein E2 of the attenuated isolates were attributed to positive selection due to suboptimal vaccination and control. To obtain a complete picture of the mutations involved in attenuation, we applied forward and reverse genetics using the evolutionary-related low-virulent CSFV/Pinar del Rio (CSF1058)/2010 (PdR) and highly virulent Margarita/1958 isolates. Sequence comparison of the two viruses recovered from experimental infections in pigs revealed 40 amino acid differences. Interestingly, the amino acid substitutions clustered in E2 and the NS5A and NS5B proteins. A long poly-uridine sequence was identified previously in the 3' untranslated region (UTR) of PdR. We constructed functional cDNA clones of the PdR and Margarita strains and generated eight recombinant viruses by introducing single or multiple gene fragments from Margarita into the PdR backbone. All chimeric viruses had comparable replication characteristics in porcine monocyte-derived macrophages. Recombinant PdR viruses carrying either E2 or NS5A/NS5B of Margarita, with 36 or 5 uridines in the 3'UTR, remained low virulent in 3-month-old pigs. The combination of these elements recovered the high-virulent Margarita phenotype. These results show that CSFV evolution towards attenuated variants in the field involved mutations in both structural and non-structural proteins and the UTRs, which act synergistically to determine virulence.

Functional Screening of cDNA Expression Library for Novel Ctenophore Photoproteins.

The functional screening of cDNA libraries (or functional cloning) enables isolation of cDNA genes encoding novel proteins with unknown amino acid sequences. This approach is the only way to identify a protein sequence in the event of shortage of biological material for obtaining pure target protein in amounts sufficient to determine its primary structure, since sensitive functional test for a target protein is only required to successfully perform functional cloning. Commonly, bioluminescent proteins from representatives belonging to different taxa significantly differ in sequences due to independent origin of bioluminescent systems during evolution. Nonetheless, these proteins are frequently similar in functions and can use even the same substrate of bioluminescence reaction, allowing the use of the same functional test for screening. The cDNA genes encoding unknown light-emitting proteins can be identified during functional screening with high sensitivity, which is provided by modern light recording equipment making possible the detection of a very small amount of a target protein. Here, we present the protocols for isolation of full-size cDNA genes for the novel bioluminescent protein family of light-sensitive Ca2+-regulated photoproteins in the absence of any sequence information by functional screening of plasmid cDNA expression library. The protocols describe all the steps from gathering animals to isolation of individual E. coli colonies carrying full-size cDNA genes using photoprotein berovin from ctenophore Beroe abyssicola as an illustrative example.

Unveiling miRNA30b’s Role in Suppressing ADAM12 to Combat Triple‐Negative Breast Cancer

Background: Triple‐negative breast cancer, a subtype of breast cancer, is characterized by a poor prognosis. Recent studies have shown that miRNA30b acts as an oncogene and is vital for the proliferation of malignancies across various systems. This study aimed to elucidate the impact of miRNA30b on the proliferation, migration, and invasion capabilities of breast cancer cells in vitro.Methods: Triple‐negative breast cancer cell lines MDA‐MB‐231 were transiently transfected with miRNA30b inhibitor, mimic, or the negative control by Lipofectamine 2000. Successful transfection was confirmed by quantitative real‐time polymerase chain reaction (qRT‐PCR). Functional assays, including CCK8, clone formation, scratch, and transwell assays, were conducted to evaluate the proliferation, invasion, and migration ability of MDA‐MB‐231 cells in each group. The target protein of miRNA30b was determined using an online prediction data website, and the dual‐luciferase assay confirmed whether there was a binding site between miRNA30b and ADAM12. The effect was further verified by Western blot analysis.Results: MDA‐MB‐231 cells were transfected with miRNA30b inhibitor, mimic, and negative control. miRNA30b expression was downregulated in the cells. Relative to the negative control group, miRNA30b expression significantly increased in the mimic group and decreased in the miRNA30b inhibitor group, with the differences being statistically significant. The miRNA30b mimic group exhibited a significant increase in miRNA30b expression and a corresponding promotion of cell proliferation, colony formation, and migration. Conversely, the miRNA30b inhibitor group displayed significantly reduced miRNA30b expression and suppressed cell proliferation, colony formation, and migration abilities compared to the negative control cells. Bioinformatics software predicted ADAM12 as a potential target of miRNA30b. Dual‐luciferase assays confirmed the presence of a binding site between miRNA30b and ADAM12. Western blot analysis revealed that overexpression of miRNA30b downregulated ADAM12 expression in MDA‐MB‐231 cells.Conclusions: miRNA30b could promote proliferation, migration, and invasion of TNBC cell lines MDA‐MB‐231. The effect of miRNA30b on triple‐negative breast cancer would be achieved partly at least through inhibiting the expression of ADAM12.

Instagram Clone Using Python

Social Media gives the common man a platform to voice with complete freedom of expression, be it for supporting a cause or for addressing a national or international issue of any sensitivity. Business prospects and job opportunities gear up as social media is a stage with global audience to showcase our talents. Social media started off as a trend among youth but today it has emerged as a medium that influences even Presidential elections. Instagram a is a social media app that has grown immensely over the years, and it continues to broaden its horizons. The photo-sharing app has gone a long way from once being a favorite for vacationers sharing their holiday pictures to now appealing to all kinds of influencers, advertisers, and brands. The early version of Instagram was a html prototype for photo-sharing. But as time progressed, a bundle of languages were used to create this awesome clutter free app. Through this project, we’ll discover a way to build a basic functional clone of Instagram, relying on Kotlin and Firebase.A growing number of people are interacting on the web to express and share their views

QTL analysis of traits related to seed size and shape in sesame (Sesamum indicum L.).

Seed size and shape are important traits that determine seed yield in sesame. Understanding the genetic basis of seed size and shape is essential for improving the yield of sesame. In this study, F2 and BC1 populations were developed by crossing the Yuzhi 4 and Bengal small-seed (BS) lines for detecting the quantitative trait loci (QTLs) of traits related to seed size and shape. A total of 52 QTLs, including 13 in F2 and 39 in BC1 populations, for seed length (SL), seed width (SW), and length to width ratio (L/W) were identified, explaining phenotypic variations from 3.68 to 21.64%. Of these QTLs, nine stable major QTLs were identified in the two populations. Notably, three major QTLs qSL-LG3-2, qSW-LG3-2, and qSW-LG3-F2 that accounted for 4.94-16.34% of the phenotypic variations were co-localized in a 2.08 Mb interval on chromosome 1 (chr1) with 279 candidate genes. Three stable major QTLs qSL-LG6-2, qLW-LG6, and qLW-LG6-F2 that explained 8.14-33.74% of the phenotypic variations were co-localized in a 3.27 Mb region on chr9 with 398 candidate genes. In addition, the stable major QTL qSL-LG5 was co-localized with minor QTLs qLW-LG5-3 and qSW-LG5 to a 1.82 Mb region on chr3 with 195 candidate genes. Gene annotation, orthologous gene analysis, and sequence analysis indicated that three genes are likely involved in sesame seed development. These results obtained herein provide valuable in-formation for functional gene cloning and improving the seed yield of sesame.

Semantic-Enriched Code Knowledge Graph to Reveal Unknowns in Smart Contract Code Reuse

Programmers who work with smart contract development often encounter challenges in reusing code from repositories. This is due to the presence of two unknowns that can lead to non-functional and functional failures. These unknowns are implicit collaborations between functions and subtle differences among similar functions. Current code mining methods can extract syntax and semantic knowledge (known knowledge), but they cannot uncover these unknowns due to a significant gap between the known and the unknown. To address this issue, we formulate knowledge acquisition as a knowledge deduction task and propose an analytic flow that uses the function clone as a bridge to gradually deduce the known knowledge into the problem-solving knowledge that can reveal the unknowns. This flow comprises five methods: clone detection, co-occurrence probability calculation, function usage frequency accumulation, description propagation, and control flow graph annotation. This provides a systematic and coherent approach to knowledge deduction. We then structure all of the knowledge into a semantic-enriched code Knowledge Graph (KG) and integrate this KG into two software engineering tasks: code recommendation and crowd-scaled coding practice checking. As a proof of concept, we apply our approach to 5,140 smart contract files available on Etherscan.io and confirm high accuracy of our KG construction steps. In our experiments, our code KG effectively improved code recommendation accuracy by 6% to 45%, increased diversity by 61% to 102%, and enhanced NDCG by 1% to 21%. Furthermore, compared to traditional analysis tools and the debugging-with-the-crowd method, our KG improved time efficiency by 30 to 380 seconds, vulnerability determination accuracy by 20% to 33%, and vulnerability fixing accuracy by 24% to 40% for novice developers who identified and fixed vulnerable smart contract functions.

Recent Advances in Metagenomic Approaches, Applications, and Challenge.

Advances in metagenomics analysis with the advent of next-generation sequencing have extended our knowledge of microbial communities as compared to conventional techniques providing advanced approach to identify novel and uncultivable microorganisms based on their genetic information derived from a particular environment. Shotgun metagenomics involves investigating the DNA of the entire community without the requirement of PCR amplification. It provides access to study all genes present in the sample. On the other hand, amplicon sequencing targets taxonomically important marker genes, the analysis of which is restricted to previously known DNA sequences. While sequence-based metagenomics is used to analyze DNA sequences directly from the environment without the requirement of library construction and with limited identification of novel genes and products that can be complemented by functional genomics, function-based metagenomics requires fragmentation and cloning of extracted metagenome DNA in a suitable host with subsequent functional screening and sequencing clone for detection of a novel gene. Although advances were made in metagenomics, different challenges arise. This review provides insight into advances in the metagenomic approaches combined with next-generation sequencing, their recent applications highlighting the emerging ones, such as in astrobiology, forensic sciences, and SARS-CoV-2 infection diagnosis, and the challenges associated. This review further discusses the different types of metagenomics and outlines advancements in bioinformatics tools and their significance in the analysis of metagenomic datasets.

Automatic continuity, unique Polish topologies, and Zariski topologies on monoids and clones

In this paper we explore the extent to which the algebraic structure of a monoid M M determines the topologies on M M that are compatible with its multiplication. Specifically we study the notions of automatic continuity; minimal Hausdorff or T 1 T_1 topologies; Polish semigroup topologies; and we formulate a notion of the Zariski topology for monoids and inverse monoids. If M M is a topological monoid such that every homomorphism from M M to a second countable topological monoid N N is continuous, then we say that M M has automatic continuity. We show that many well-known, and extensively studied, monoids have automatic continuity with respect to a natural semigroup topology, namely: the full transformation monoid N N \mathbb {N} ^\mathbb {N} ; the full binary relation monoid B N B_{\mathbb {N}} ; the partial transformation monoid P N P_{\mathbb {N}} ; the symmetric inverse monoid I N I_{\mathbb {N}} ; the monoid Inj ⁡ ( N ) \operatorname {Inj}(\mathbb {N}) consisting of the injective transformations of N \mathbb {N} ; and the monoid C ( 2 N ) C(2^{\mathbb {N}}) of continuous functions on the Cantor set 2 N 2^{\mathbb {N}} . The monoid N N \mathbb {N} ^\mathbb {N} can be equipped with the product topology, where the natural numbers N \mathbb {N} have the discrete topology; this topology is referred to as the pointwise topology. We show that the pointwise topology on N N \mathbb {N} ^\mathbb {N} , and its analogue on P N P_{\mathbb {N}} , is the unique Polish semigroup topology on these monoids. The compact-open topology is the unique Polish semigroup topology on C ( 2 N ) C(2 ^\mathbb {N}) , and on the monoid C ( [ 0 , 1 ] N ) C([0, 1] ^\mathbb {N}) of continuous functions on the Hilbert cube [ 0 , 1 ] N [0, 1] ^\mathbb {N} . The symmetric inverse monoid I N I_{\mathbb {N}} has at least 3 Polish semigroup topologies, but a unique Polish inverse semigroup topology. The full binary relation monoid B N B_{\mathbb {N}} has no Polish semigroup topologies, nor do the partition monoids. At the other extreme, Inj ⁡ ( N ) \operatorname {Inj}(\mathbb {N}) and the monoid Surj ⁡ ( N ) \operatorname {Surj}(\mathbb {N}) of all surjective transformations of N \mathbb {N} each have infinitely many distinct Polish semigroup topologies. We prove that the Zariski topologies on N N \mathbb {N} ^\mathbb {N} , P N P_{\mathbb {N}} , and Inj ⁡ ( N ) \operatorname {Inj}(\mathbb {N}) coincide with the pointwise topology; and we characterise the Zariski topology on B N B_{\mathbb {N}} . Along the way we provide many additional results relating to the Markov topology, the small index property for monoids, and topological embeddings of semigroups in N N \mathbb {N}^{\mathbb {N}} and inverse monoids in I N I_{\mathbb {N}} . Finally, the techniques developed in this paper to prove the results about monoids are applied to function clones. In particular, we show that: the full function clone has a unique Polish topology; the Horn clone, the polymorphism clones of the Cantor set and the countably infinite atomless Boolean algebra all have automatic continuity with respect to second countable function clone topologies.

Pilot Study to Detect the Repertoires of T-Cell Receptor Gamma Chains in Patients with Juvenile Schizophrenia

Disfunction of immune system and neuroinflammation may play a role in pathogenesis of schizophrenia. The role of adaptive immune system has to be elucidated. We present a pilot study to test the methodology of profiling of immune repertoires of the TCR gamma chain by deep sequencing using several patients with juvenile schizophrenia and unaffected control subjects. The clonotype profiles were revealed and their diversity and presumable differences in structure of CDR3 TRG region of functional clones in juvenile schizophrenia and controls were estimated. This approach is perspective for further comprehensive study of changes in adaptive immune system in representative cohorts of patients with different forms of schizophrenia.

Java Code Clone Detection by Exploiting Semantic and Syntax Information From Intermediate Code-Based Graph

Code clone detection plays a critical role in the field of software engineering. To achieve this goal, developers are required to have rich development experience for finding the &#x201C;functional&#x201D; clone code. However, this is unfriendly to novice developers. Although many approaches were proposed to automatically detect code clones, the results are not satisfactory. A major reason is that it is difficult to extract syntax and semantic information from the source code. To resolve this problem, in this article, we develop a novel graph representation approach based on intermediate code to detect the functional code clones. This graph representation is built based on intermediate code compiled from the source code. By using it, we can easily utilize graph embedding techniques to extract syntactic and semantic features from abstract syntax tree, control flow graph, and DFG generated from intermediate code. After that, we use the Softmax classifier to detect functional code clone pairs. We evaluate the performance of the proposed graph representation approach based on intermediate code for the code clone detection task on the BigCloneBench dataset. In order to improve performance, the embedded representation of intermediate code is initialized based on pretrained vectors learned from the collected LLVM IR dataset in advance. The experimental results show that our proposed intermediate code-based graph approach performs better than existing functional code clone detection approaches. Especially for the type-4 code clone detection, our approach outperforms the baseline approaches by an average of 33.49&#x0025; in the term of <i>F</i>1 score.

Characterization of two novel colistin resistance gene mcr-1 variants originated from Moraxella spp.

This study aimed to characterize two novel mcr-1 variants, mcr-1.35 and mcr-1.36, which originated from Moraxella spp. that were isolated from diseased pigs in China. The Moraxella spp. carrying novel mcr-1 variants were subjected to whole-genome sequencing (WGS) and phylogenetic analysis based on the 16S rRNA gene. The mcr-1 variants mcr-1.35 and mcr-1.36 were characterized using phylogenetic analysis, a comparison of genetic environments, and protein structure prediction. The WGS indicated that two novel mcr-1 variants were located in the chromosomes of three Moraxella spp. with a genetic environment of mcr-1-pap2. In addition to the novel colistin resistance genes mcr-1.35 and mcr-1.36, the three Moraxella spp. contained other antimicrobial resistance genes, including aac(3)-IId, tet(O), sul2, floR, and blaROB-3. A functional cloning assay indicated that either the mcr-1.35 or mcr-1.36 gene could confer resistance to colistin in Escherichia coli DH5α and JM109. The nucleotide sequences of mcr-1.35 and mcr-1.36 presented 95.33 and 95.33% identities, respectively, to mcr-1.1. The phylogenetic analysis showed that mcr-1.35 and mcr-1.36 were derived from Moraxella spp. that belonged to subclades that were different from those of the mcr-1 variants (mcr-1.1 to mcr-1.34 except mcr-1.10) originating from Enterobacteriaceae. The deduced amino acid sequences of MCR-1.35 (MCR-1.36) showed 96.67% (96.49%), 82.59% (82.04%), 84.07% (83.52%), 55.52% (55.17%), 59.75% (59.57%), and 61.88% (61.69%) identity to MCR-1.10, MCR-2.2, MCR-6.1, MCR-LIN, MCR-OSL, and MCR-POR, respectively, that originated from Moraxella sp. Notably, protein structure alignment showed only a few changes in amino acid residues between MCR-1.1 and MCR-1.35, as well as between MCR-1.1 and MCR-1.36. In conclusion, this study identified Moraxella spp. carrying two novel mcr-1 variants, mcr-1.35 and mcr-1.36, conferring resistance to colistin, which were isolated from pig farms in China. In addition, mcr-like variants were observed to be located in the chromosomes of some species of Moraxella isolated from pig samples.

GENOME-WIDE IDENTIFICATION AND CHARACTERIZATION OF PLANT-SPECIFIC DOF TRANSCRIPTION FACTOR GENE FAMILY IN CASHEW (ANACARDIUM OCCIDENTALE)

DNA binding with one-finger (Dof) protein belongs to the plant-specific transcription factors (PSTFs) gene family. These transcription factors have a variety of roles in many biological processes in plants. However, there is limited research on their role in Cashew. A total of 67 Dof genes were found in the cashew genome and were classified into 11 subgroups (A, B1, B2, C1, C2, D, E1, E2, F1, F2, and F3) by comparing them with Dof genes from Arabidopsis and lettuce. Cashew Dof genes were present in 9 of these subgroups, except for A and F1. This article provides a detailed discussion of the gene structures, chromosome positions, phylogeny, subcellular localization, cis-regulatory analysis, protein motifs, and evolutionary patterns of Dof genes in cashew. The only type of duplication found in cashew was segmental duplication, which mainly contributes to the large Dof gene family. The analysis of cis-regulatory elements (CREs) revealed the presence of light, ethylene, seed, circadian, meristem, and auxin-sensitive elements, which are particularly sensitive to these factors. The article also includes a comparative analysis of the evolutionary or phylogenetic relationships between Dof genes from lettuce, cashew, and Arabidopsis. This study provides a comprehensive understanding of the Dof gene family in cashew and can serve as a guide for functional analysis and cloning of its gene family members.

A high throughput bispecific antibody discovery pipeline

Bispecific antibodies (BsAbs) represent an emerging class of immunotherapy, but inefficiency in the current discovery has limited their broad clinical availability. Here we report a high throughput, agnostic, single-cell-based functional screening pipeline, comprising molecular and cell engineering for efficient generation of BsAb library cells, followed by functional interrogation at the single-cell level to identify and sort positive clones and downstream sequence identification and functionality characterization. Using a CD19xCD3 bispecific T cell engager (BiTE) as a model, we demonstrate that our single-cell platform possesses a high throughput screening efficiency of up to one and a half million variant library cells per run and can isolate rare functional clones at a low abundance of 0.008%. Using a complex CD19xCD3 BiTE-expressing cell library with approximately 22,300 unique variants comprising combinatorially varied scFvs, connecting linkers and VL/VH orientations, we have identified 98 unique clones, including extremely rare ones (~ 0.001% abundance). We also discovered BiTEs that exhibit novel properties and insights to design variable preferences for functionality. We expect our single-cell platform to not only increase the discovery efficiency of new immunotherapeutics, but also enable identifying generalizable design principles based on an in-depth understanding of the inter-relationships between sequence, structure, and function.

An integrated mammalian library approach for optimization and enhanced microfluidics-assisted antibody hit discovery

Recent years have seen the development of a variety of mammalian library approaches for display and secretion mode. Advantages include library approaches for engineering, preservation of precious immune repertoires and their repeated interrogation, as well as screening in final therapeutic format and host. Mammalian display approaches for antibody optimization exploit these advantages, necessitating the generation of large libraries but in turn enabling early screening for both manufacturability and target specificity. For suitable libraries, high antibody integration rates and resulting monoclonality need to be balanced – we present a solution for sufficient transmutability and acceptable monoclonality by applying an optimized ratio of coding to non-coding lentivirus. The recent advent of microfluidic-assisted hit discovery represents a perfect match to mammalian libraries in secretion mode, as the lower throughput fits well with the facile generation of libraries comprising a few million functional clones. In the presented work, Chinese Hamster Ovary cells were engineered to both express the target of interest and secrete antibodies in relevant formats, and specific clones were strongly enriched by high throughput screening for autocrine cellular binding. The powerful combination of mammalian secretion libraries and microfluidics-assisted hit discovery could reduce attrition rates and increase the probability to identify the best possible therapeutic antibody hits faster.

Stability of Boolean Function Classes with Respect to Clones of Linear Functions

We consider classes of Boolean functions stable under compositions both from the right and from the left with clones. Motivated by the question how many properties of Boolean functions can be defined by means of linear equations, we focus on stability under compositions with the clone of linear idempotent functions. It follows from a result by Sparks that there are countably many such linearly definable classes of Boolean functions. In this paper, we refine this result by completely describing these classes. This work is tightly related with the theory of function minors, stable classes, clonoids, and hereditary classes, topics that have been widely investigated in recent years by several authors including Maurice Pouzet and his coauthors.

D-amino acid auxotrophic Escherichia coli strain for invivo functional cloning of novel d-amino acid synthetic enzyme.

Various d-amino acids have been found in a wide range of organisms, including mammals. Although the physiological functions of various d-amino acids have been reported or suggested, the molecular basis of these biological functions has been elucidated in only a few cases. The identification of a d-amino acid biosynthetic enzyme is a critical step in understanding the mechanism of the physiological functions of d-amino acids. While invivo functional screening can be a powerful tool for identifying novel metabolic enzymes, none of the existing organisms exhibit growth dependent on d-amino acid other than d-Ala and d-Glu. Here, we report the first organism that exhibits non-canonical d-amino acid auxotrophy. We found that an Escherichia coli strain lacking the major d-Ala and d-Glu biosynthetic enzymes, alr, dadX, and murI, and expressing the mutated d-amino acid transaminase (DAAT) gene from Bacillus sp. YM-1 (MB3000/mdaat+ ) grew well when supplemented with certain d-amino acid. A multicopy suppression study with plasmids encoding one of the 51 PLP-dependent enzymes of E.coli showed that MB3000/mdaat+ could detect weak and moonlighting racemase activity, such from cystathionine β-lyase (MetC) and a negative regulator of MalT activity/cystathionine β-lyase (MalY)-these exhibit only a few tenths to a few thousandths of the racemization activity of canonical amino acid racemases. We believe that this unique platform will contribute to further research in this field by identifying novel d-amino acid-metabolizing enzymes.

Reaumuria soongorica-plant model to understand drought adaptive mechanisms of xerophyte and their potentials in improving stress tolerance in plants

Reaumuria soongorica plays critical role in withstanding wind-induced soil erosion and combating desertification due to its unique drought adaptive networks involved in physiological, morphological, biochemical, and molecular biological levels. This review was conceived to summarize the most updated information on drought adaptive mechanisms of R. soongorica to formulate valuable strategies for non-xerophytes crop species to be drought tolerant. Research indicates that R. soongorica can be drought resistant via posing a high root to shoot ratio, having salt secreting gland, subsidized stomata, hard leaf texture, pallet leaf shape, high seeds viability, actively working antioxidative enzymes, secondary metabolites, phytohormones, and differentially expressed drought resistant genes. These characteristics interlink at morpho-physiological, biochemical, metobolic, molecular, and genetic level in R. soongorica to adapt the extreme abitotic stress conditions in desert regions as a plant model in xerophyte. The potentials of using the genetic elements in R. soongorica to produce drought tolerant crop species for yield production on growing on marginal lands could be vital for maintaining future food security. However, functional gene cloning and their transformation in crop species should be conducted as pre-requisite. Key words: Abiotic stress, Crop improvement, Drought adaptive mechanisms, Reaumuria soongorica, Xerophytes

Disruption to the FOXO-PRDM1 axis resulting from deletions of chromosome 6 in acute lymphoblastic leukaemia

A common problem in the study of human malignancy is the elucidation of cancer driver mechanisms associated with recurrent deletion of regions containing multiple genes. Taking B-cell acute lymphoblastic leukaemia (B-ALL) and large deletions of 6q [del(6q)] as a model, we integrated analysis of functional cDNA clone tracking assays with patient genomic and transcriptomic data, to identify the transcription factors FOXO3 and PRDM1 as candidate tumour suppressor genes (TSG). Analysis of cell cycle and transcriptomic changes following overexpression of FOXO3 or PRDM1 indicated that they co-operate to promote cell cycle exit at the pre-B cell stage. FOXO1 abnormalities are absent in B-ALL, but like FOXO3, FOXO1 expression suppressed growth of TCF3::PBX1 and ETV6::RUNX1 B-ALL in-vitro. While both FOXOs induced PRDM1 and other genes contributing to late pre-B cell development, FOXO1 alone induced the key transcription factor, IRF4, and chemokine, CXCR4. CRISPR-Cas9 screening identified FOXO3 as a TSG, while FOXO1 emerged as essential for B-ALL growth. We relate this FOXO3-specific leukaemia-protective role to suppression of glycolysis based on integrated analysis of CRISPR-data and gene sets induced or suppressed by FOXO1 and FOXO3. Pan-FOXO agonist Selinexor induced the glycolysis inhibitor TXNIP and suppressed B-ALL growth at low dose (ID50 < 50 nM).

Cloning and Analysis of T-DNA Targeted Gene of Colletotrichum gloeosporioides Pathogenicity-Defective Strain Cs16

In preliminary stage, the nucleotide sequence of Cs16 Colletotrichum gloeosporioides associated gene obtained from database of (NCBI), analyzed with Clustalx 1.83 in order to assess the deduced amino acid sequences. The full-length sequences of Cs16 gene is including with specific Open Reading Frame (ORF) having 3’ and 5’ UTR was obtained. In order to assess the relationship of C. gloeosporioides associated CS16 gene with other members, a combined rooted neighbor-joining (NJ) tree was generated through MEGA 4.5 by following 1000 bootstrap replicates. The resulted alignment predicts that Cs16 gene showed entirely diverse sequences from other species and has no any conserved domain sequences between them. In order to scrutinize the full length nucleotide sequences, the gene functional cloning of Cs16 gene has been carried out by PCR amplification. To examine the functional analysis of Cs16 gene in C. gloeosporioides and its regulatory mechanisms, the over expressing vector (pGapneoR26) was transformed into Agrobacterium strain LBA4404 that resulted successful detection of GFP-Cs16 expression into C. gloeosporioides transformants and it was also substantially detected by fluorescence microscopy. The T-DNA intentional mutants of C. gloeosporioides mycelia illustrated phonotypic different intensification, colour, colony growth rate than wild type isolated C. gloeosporioides STIJ6 strain.

Honing-in antigen-specific cells during antibody discovery: a user-friendly process to mine a deeper repertoire

Immunization based antibody discovery is plagued by the paucity of antigen-specific B cells. Identifying these cells is akin to finding needle in a haystack. Current and emerging technologies while effective, are limited in terms of capturing the antigen-specific repertoire. We report on the bulk purification of antigen-specific B-cells and the benefits it offers to various antibody discovery platforms. Using five different antigens, we show hit rates of 51–88%, compared to about 5% with conventional methods. We also show that this purification is highly efficient with loss of only about 2% antigen specific cells. Furthermore, we compared clones in which cognate chains are preserved with those from display libraries in which chains either from total B cells (TBC) or antigen-specific B cells (AgSC) underwent combinatorial pairing. We found that cognate chain paired clones and combinatorial clones from AgSC library had higher frequency of functional clones and showed greater diversity in sequence and paratope compared to clones from the TBC library. This antigen-specific B-cell selection technique exemplifies a process improvement with reduced cycle time and cost, by removing undesired clones prior to screening and increasing the chance of capturing desirable and rare functional clones in the repertoire.