Candidate Clones Research Articles

Enzymatic oxidation of polyethylene by Galleria mellonella intestinal cytochrome P450s

Polyethylene is widely used but highly resistant to biodegradation, owing to its composition of only a hydrocarbon backbone. For biodegradation to occur, oxidation within the polymer needs to be initiated. Galleria mellonella was the first insect discovered to autonomously oxidize polyethylene without the aid of gut microbes. However, the specific enzyme remains unidentified. Here, we identified for the first time two polyethylene oxidation enzyme candidates of cytochrome P450 (CYP), 6B2-GP04 and CYP6B2-13G08, from the G. mellonella midgut. Both candidate clones oxidized polyethylene efficiently, generating short-chain aliphatic compounds, with CYP6B2-GP04 exhibiting higher activity than CYP6B2-13G08 in yeast and insect cells. In silico structural modeling approaches revealed that the CYP6B2-GP04 Phe118 was essential for interacting with hydrocarbons, which was further validated by mutating it to glycine. Furthermore, directed enzyme evolution led to the identification of an enzyme variant with significantly increased oxidation efficiency. Our findings offer promising enzyme-based solutions for polyethylene biodegradation, potentially mitigating polyethylene-driven plastic pollution.

Construction and validation of a novel multi-epitope in silico vaccine design against the paramyosin protein of Opisthorchis viverrini using immunoinformatics analyses

Liver fluke infection caused by Opisthorchis viverrini (O. viverrini) remains a significant but neglected health threat across Southeastern Asia. The early infective anabolic growth stage of O. viverrini expresses and exposes proteins integral for the growth and maturation of immature worms to the adult catabolic stage. Among these proteins, paramyosin emerged as a distinct immunogenic protein during opisthorchiasis. The functional region of the paramyosin protein known as myosin tail was selected to design a multi-epitope vaccine (MEV) to elicit T and B cell immune responses in susceptible human hosts utilizing various immunoinformatics and in silico vaccinology tools. The vaccine candidate had several B- and T-cell epitopes that stimulate both humoral and cellular immune responses. Moreover, in silico structural, docking, and dynamic analyses showed that the construct interacted with target immune receptors effectively, which may result in sufficient immunological stimulation. Analysis of simulated coverage efficacy also supports vaccine application in the field. Cloning and expression of the vaccine candidate were determined to be viable based on physicochemical and in silico assessments. These results reveal that the vaccine candidate developed herein is stable and potentially useful in addressing opisthorchiasis. The promising result of this study establishes a strong platform for initiating laboratory and efficacy trials for the vaccine candidate.

Variation in survival, growth and stem straightness in Eucalyptus urophylla provenances in the Southern Highlands region of Tanzania

Lack of quality planting materials has significantly limited productivity and quality of commercial forestry plantations in Tanzania. A provenance/progeny trial of Eucalyptus urophylla was established to test performance in order to select the best planting materials for future plantation activities. A complete randomised block design was used whereby 30 families from 8 provenances within 6 replications were tested. At 13 years tree survival, growth (diameter at breast height and height) and stem straightness were assessed. The results indicate that there were highly significant differences (p < 0.05) among provenances and families in survival, height, breast height diameter (DBH) and stem straightness. The overall survival ranged from 79.0% to 96.2%; height ranged between 24.9 and 32.5 m; and DBH ranged from 18.7 to 24.5 cm. Overall, results from this study show that provenances Alassannaru, Elun kirpas, Nesunhuhun and Nakana ulam have better growth performance and stem form than others. These results provide valuable information on the growth performance of different provenances and families of E. urophylla. The provenances and families selected with best performance can be used in developing improved seeds for plantation establishment and/or selection of plus trees for vegetative propagation as candidate clones for a clonal testing programme in Tanzania.

Prioritisation of code clones using a genetic algorithm

Context:Code clones are prevalent, and due to their diverse impact on projects’ quality they require a proper management strategy. Objectives:Develop GA-based Refactoring-Aware Detection (RAD) approach for prioritisation of code clones. Method:A genetic algorithm (GA) that balances estimated gain and cost/risk of refactoring to select the optimal clone candidate to refactor. Results:GA converges on a solution, with diverse variance. The value of fitness function is higher for multi-objective approaches, but they also exhibit higher variance. Conclusion:GA can be effectively applied for clone prioritising.

Screening Non-neutralizing Anti-idiotype Antibodies Against a Drug Candidate for Total Pharmacokinetic and Target Engagement Assay.

Non-neutralizing anti-idiotype antibodies against a therapeutic monoclonal antibody (mAb) play a crucial role in the creation of total pharmacokinetic (PK) assays and total target engagement (TE) assays during both pre-clinical and clinical development. The development of these anti-idiotype antibodies is challenging. In this study, we utilized a hybridoma platform to produce a variety of anti-idiotype antibodies against GSK2857914, a humanized IgG1 anti-BCMA monoclonal antibody. The candidate clones were evaluated using surface plasmon resonance (SPR) and bio-layer interferometry (BLI) for binding affinity, binding profiling, matrix interference, and antibody pairing determination. We discovered that three anti-idiotype antibodies did not prevent BCMA from binding to GSK2857914. All three candidates demonstrated high binding affinities. One of the three exhibited minimal matrix inference and could pair with the other two candidates. Additionally, one of the three clones was biotinylated as a capture reagent for the total PK assay, and another was labeled with ruthenium as a detection reagent for both the total PK assay and total TE assay. The assay results clearly show that these reagents are genuine non-neutralizing anti-idiotypic antibodies and are suitable for total PK and TE assay development. Based on this and similar studies, we conclude that the hybridoma platform has a high success rate for generating non-neutralizing anti-idiotype antibodies. Our methodology for developing and characterizing non-neutralizing anti-idiotype antibodies to therapeutic antibodies can be generally applied to any antibody-based drug candidate's total PK and total TE assay development.

Clonal Preselection in Grape (Vitis vinifera L.) Varieties of Ekşi Kara and Gök Üzüm

Ekşi Kara (functional female flowers) and Gök Üzüm (hermaphroditic flowers) are the two most important autochthonous varieties of middle Anatolia. This clone selection study started with mass-selection in producer vineyards consisting of approximately 5000 vines by The International Organization of Vine and Wine (OIV) clonal selection procedure. Two-years genetic and sanitation were examined visually in population and 220 clone candidates were ampelography and fertilization biology and bud fertility determined for Ekşi Kara variety. The clone candidates were ranked at the level of sums, with weighted grading of three-year yield, growth, and quality records. Sanitation analyses of the superior clones were made. 17 clones in the Ekşi Kara grape variety were selected according to their superior scores in genetic selection and sanitation analyses. Eleven clones were selected by mass selection from Gök Üzüm carried out in a single location, and 2 clones were selected with genetic selection scores and health tests. Grapevine fleck virus (GfKV) was the most common ( 29%) in the samples tested, Grapevine leafroll-associated virus 1+3 (GLRaV-1 + 3,  26%), Grapevine virus A (GVA, 12%), Grapevine leafroll associated virus-2 (GLRaV-2,  3%), Arabis mosaic virus (ArMV) / Grapevine fanleaf virus (GFLV) ( 1%) are fallowed with indicated percentage. Although virus and bacterial infections are common in the vineyards, enough healthy clones were selected. 17 Ekşi Kara and 2 Gök Üzüm clones selected as pollinators were grafted onto the 110R rootstock for clone comparison in homogeneous conditions, and a "Clone Comparison Vineyard" was established in Selçuk University.

Serum Amyloid A1 (SAA1) Secreted By the Stromal Microenvironment Drives Malignant Clonal Proliferation in Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML)

Drivers of malignant hematopoietic stem cell clonal proliferation from age-related clonal hematopoiesis (ARCH) to Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML) are not well understood. However, a pro-inflammatory and immune tolerant aging bone marrow (BM) microenvironment is thought to contribute to disease development. Serum Amyloid A1 (SAA1) has been identified as a novel pro-inflammatory oncoprotein the levels of which progressively increase with MDS and AML progression. SAA1 also selectively promotes growth of patient-derived MDS and AML cells independent of cytogenetic or mutational profile. We aimed to characterize the mechanism by which SAA1 drives oncogenesis within the microenvironment. Herein we further stratified the disease marking properties of SAA1 by clarifying the association of its BM levels with disease progression in MDS and AML. SAA1 levels were significantly elevated in BM aspirates from MDS and AML patients as compared to those from healthy age-matched subjects (p=0.0002 and p=0.0052, respectively). The effects of SAA1 on mutated hematopoietic cells were assessed in a model of age-related clonal hematopoiesis (ARCH). Utilizing a CRISPR-Cas9 system, ASXL1-mutant CD34+ hematopoietic stem cells (HSCs) were generated; the replating capacity of both mutated and healthy CD34+ cells were assessed in the presence or absence of SAA1. Notably, SAA1 increased the replating capability assessed by an increase in the number of colonies formed on serial replating of ASXL1-mutant CD34+ cells, but not those of SAA1-treated healthy CD34+ or vehicle treated ASXL1-mutated CD34+ cells, suggesting that the presence of SAA1 favors stemness of mutated HSCs, and implicating SAA1 as a driver of malignant clonality. To investigate the mechanism of action of SAA1 BM mononuclear cells from AML patients or healthy age-matched subjects were treated with SAA1 or vehicle overnight and single-cell RNA sequencing analysis was performed. Seurat and cell cluster annotation analysis showed that AML samples treated with SAA1 had increased numbers of hematopoietic stem and progenitor cells (HSPCs) and plasmacytoid dendritic cells as compared to matched untreated AML samples. Further, Enrichr analysis showed that pathways upregulated by SAA1 in AML samples included pro-inflammatory molecules, anti-apoptotic, and antiviral defense mechanisms (p&amp;lt;0.0001). Shared genes upregulated in these pathways in HSPCs include interferon-induced signals including JAK2, STAT1, and OAS3, and genes implicated in resistance to chemotherapy, including CD44 and CD36, as well as the anti-phagocytosis signal CD47. Lastly, we initiated the development of a blocking monoclonal antibody against SAA1 as a means of inhibiting its potent oncogenic effects on malignant HSCs and examining its potential therapeutic activity. Utilizing a RAW 264.7 NFκB-luciferase reporter cell line, we screened 51 clones for their ability to selectively block SAA1-mediated NFκB activation. 7 optimal candidates were identified which demonstrated dose-dependent reduction in NFκB activity upon SAA1 challenge. Importantly, these candidate clones were specific for SAA1 and did not affect NFκB activation mediated by lipopolysaccharide (LPS)-mediated activation. We observed up to an 85% reduction of SAA1-mediated activity using supernatants from further subcloning of previously selected candidates. Moreover, cell proliferation of a human AML cell line (OCI-AML3) stimulated by SAA1 was markedly reduced in the presence of candidate antibodies, suggesting anti-proliferative activity. Our findings show that SAA1 levels correlates with disease severity in MDS and AML progression, selectively promotes stemness and proliferative potential of ASXL1-mutant cells, and demonstrates unique immune-related mechanisms selecting for malignant clonality. Further development and purification of a novel inhibitory antibody against SAA1 for future in vivo and ex vivo testing is underway as a promising novel target to reduce SAA1-mediated, niche-driven effects with broad implications in the treatment of hematological malignancies.

Rooting screening in Eucalyptus dunnii clones: inverting the order in a clonal selection process

One of the main goals of a tree breeding program is to increase forest productivity, adaptation and profitability of forest plantations. Eucalyptus dunnii is a main species used for pulp production in Uruguay. Montes del Plata (MdP) is one of the largest forest companies in Uruguay and has been focused on the development, testing and deployment of E. dunnii clones to establish commercial plantations in Uruguay. MdP clonal strategy starts with the generation, testing and selection of families (Open-pollination and|or Control-pollination) with a breeding population with a wide genetic diversity in order to have a sustainable clonal program. Seeds from the best families are used for clonal development in a two-phase strategy that takes place in the company research nursery. On the first phase, all potential clones are screened for rooting and only the ones that root more than 60% go to the next phase. An average of 4,000 E. dunnii clones is evaluated every year, and 200 clones overpass 60% rooting threshold. This represents 5% of screened clones. The second phase is to produce plants in the nursey for the establishment of clonal trials in field, and, based on the performance, to select the candidate clones for deployment and breeding. Although the strategy and the breeding program are relatively young, promising results are seen. In the last 6 years, 1,225 E. dunnii clones developed by this rooting strategy were successfully established in clonal field trials. For species of Eucalyptus that have an undesirable trait of difficulty-to-root (recalcitrance), the screening of genotypes by rooting allows to establish in field testing clones with efficient clonal propagation. This way, the measurement and wood sampling in field trials is funnelled to clones that, if selected, could be scaled to clonal plantations.

Eucalyptus dunnii clonal strategy implementation in Montes del Plata

One of the main goals of a tree breeding program is to increase forest productivity, adaptation and profitability of forest plantations. Eucalyptus dunnii is a main species used for pulp production in Uruguay. Montes del Plata (MdP) is one of the largest forest companies in Uruguay and has been focused on the development, testing and deployment of E. dunnii clones to establish company plantations in Uruguay. On this presentation, the MdP ongoing clonal strategy for E. dunnii is presented. MdP clonal strategy starts with the generation, testing and selection of families in progeny tests (Open-pollination and Control-pollination) with a breeding population with a wide genetic diversity in order to have a sustainable clonal program. Seeds from the best families are used for clonal development in a two-phase strategy. In the first phase all potential clones are screened for rooting, in the company research nursery, and only the ones that root more than 60% go to the next phase, which is to produce plants for the establishment of clonal trials in field and, based on the performance, to select the candidate clones for deployment. On average, 4,000 clones|year are evaluated in the first phase, from which 200 go to clonal field trials every year. The strategy also includes a protocol for other traits assessment, like tree form, health and wood properties; the selection trait being tons of cellulose per tree (ADt). Wood properties are evaluated at early age using a company near infrared model (NIR). Then, values of predicted wood properties are projected to rotation age. This presentation will be complemented with extra information related to the different strategy phases in several posters. The MdP clonal strategy is a rolling front strategy, where clones from the best families are tested and selected for deployment and breeding to have continuous gain over time for E. dunnii.

Human Single Domain Antibody-Based CAR-T Cells Targeting BAFF-R Demonstrate Promising Preclinical Activity in B Cell Malignancies

Background: B-cell activating factor-receptor (BAFF-R) is a potential B-cell specific target, highly expressed in B cell malignancies and regulates B cell proliferation and survival. Single-chain antibody-based CAR-T cells targeting BAFF-R (PMB101) has demonstrated antitumor effects against human B cell malignancies and can overcome CD19 antigen loss (Qin et al. Sci Transl Med. 2019;11(511)). However, CARs built on antigen-specific single chain antibody variable fragment (scFv) may have some immunological risks. Recently, single variable domain of heavy chain antibodies are becoming an alternative to scFv to construct CARs. To improve the function of BAFF-R CAR-T cells, we designed CARs with only a fully human heavy-chain variable domain. Methods: A fully human antibody phage display library (IMARS, IASO Biotherapeutics) was used to select anti-BAFF-R clones using optimal protein/cell alternate panning. The enzyme-linked immunosorbent assay (ELISA) and flow cytometry were applied to evaluate the specificity of clones. The screened clones were grafted into a second-generation CAR with CD8α hinge, 4-1BB costimulatory and CD3ζ intracellular signaling domains to generate CAR-T cells. PMB101 and CAR-T cells constructed with VAY736 were used as positive controls. Non-transduced T cells from the same donor were used as a negative control. Results: Four candidate clones including clone 1, 5, 77 and 80 were finally screened by in vitro functional evaluation, among which clone 5, 77 and 80 were variable domain heavy chain antibodies. The expression levels of the degranulation marker CD107a on CAR-T cells constructed with the four candidate clones were significantly upregulated after coculture with BAFF-R positive tumor cells, while BAFF-R negative tumor cells could not effectively activate CAR-T cells (data not shown). The four clones exhibited similar cytotoxicity in CAR-T cells compared with the PMB101 and VAY736 CAR-T cells using the luciferase-based cytotoxicity assay (data not shown). Xenografts were established in NPG mice following intravenous injection of 1 x 10 6 jeko-1 cells expressing the firefly luciferase gene on day - 5, and a single dose of 4 x 10 6 CAR T cells were infused IV on day 0. Compared to the other groups, no progressive tumor growth was discovered in mice treated with clone 5 CAR-T cells until day 49. Clone 5 CAR-T cells showed excellent in vivo antitumor activity to PMB101 and conferred long-term survival (data not shown). Conclusion: We developed a novel BAFF-R CAR-T cell product structured on single-domain antibody. This product demonstrated promising preclinical activity, and it may provide an alternative choice for patients with B cell malignancies.

T cell receptor-engineered T cells derived from target human leukocyte antigen-DPB1-specific T cell can be a potential tool for therapy against leukemia relapse following allogeneic hematopoietic cell transplantation.

Human leukocyte antigen (HLA)-DPB1 antigens are mismatched in approximately 70% of allogeneic hematopoietic stem cell transplantations (allo-HSCT) from HLA 10/10 matched unrelated donors. HLA-DP-mismatched transplantation was shown to be associated with an increase in acute graft-versus-host disease (GVHD) and a decreased risk of leukemia relapse due to the graft-versus-leukemia (GVL) effect. Immunotherapy targeting mismatched HLA-DP is considered reasonable to treat leukemia following allo-HCT if performed under non-inflammatory conditions. Therefore, we isolated CD4+ T cell clones that recognize mismatched HLA-DPB1 from healthy volunteer donors and generated T cell receptor (TCR)-gene-modified T cells for future clinical applications. Detailed analysis of TCR-T cells expressing TCR from candidate clone #17 demonstrated specificity to myeloid and monocytic leukemia cell lines that even expressed low levels of targeted HLA-DP. However, they did not react to non-hematopoietic cell lines with a substantial level of targeted HLA-DP expression, suggesting that the TCR recognized antigenic peptide is only present in some hematopoietic cells. This study demonstrated that induction of T cells specific for HLA-DP, consisting of hematopoietic cell lineage-derived peptide and redirection of T cells with cloned TCR cDNA by gene transfer, is feasible when using careful specificity analysis.

A high-resolution genotype–phenotype map identifies the TaSPL17 controlling grain number and size in wheat

BackgroundLarge-scale genotype–phenotype association studies of crop germplasm are important for identifying alleles associated with favorable traits. The limited number of single-nucleotide polymorphisms (SNPs) in most wheat genome-wide association studies (GWASs) restricts their power to detect marker-trait associations. Additionally, only a few genes regulating grain number per spikelet have been reported due to sensitivity of this trait to variable environments.ResultsWe perform a large-scale GWAS using approximately 40 million filtered SNPs for 27 spike morphology traits. We detect 132,086 significant marker-trait associations and the associated SNP markers are located within 590 associated peaks. We detect additional and stronger peaks by dividing spike morphology into sub-traits relative to GWAS results of spike morphology traits. We propose that the genetic dissection of spike morphology is a powerful strategy to detect signals for grain yield traits in wheat. The GWAS results reveal that TaSPL17 positively controls grain size and number by regulating spikelet and floret meristem development, which in turn leads to enhanced grain yield per plant. The haplotypes at TaSPL17 indicate geographical differentiation, domestication effects, and breeding selection.ConclusionOur study provides valuable resources for genetic improvement of spike morphology and a fast-forward genetic solution for candidate gene detection and cloning in wheat.

BLINCAR: a reusable bioluminescent and Cas9-based genetic toolset for repeatedly modifying wild-type Scheffersomyces stipitis.

Scheffersomyces stipitis is a yeast that robustly ferments the 5-carbon sugar xylose, making the yeast a valuable candidate for lignocellulosic ethanol fermentation. However, the non-canonical codon usage of S. stipitis is an obstacle for implementing molecular tools that were developed for other yeast species, thereby limiting the molecular toolset available for S. stipitis. Here, we developed a series of molecular tools for S. stipitis including BLINCAR, a Bio-Luminescent Indicator that is Nullified by Cas9-Actuated Recombination, which can be used repeatedly to add different exogenous DNA payloads to the wild-type S. stipitis genome or used repeatedly to remove multiple native S. stipitis genes from the wild-type genome. Through the use of BLINCAR tools, one first produces antibiotic-resistant, bioluminescent colonies of S. stipitis whose bioluminescence highlights those clones that have been genetically modified; then second, once candidate clones have been confirmed, one uses a transient Cas9-producing plasmid to nullify the antibiotic resistance and bioluminescent markers from the prior introduction, thereby producing non-bioluminescent colonies that highlight those clones which have been re-sensitized to the antibiotic and are therefore susceptible to another round of BLINCAR implementation. IMPORTANCE Cellulose and hemicellulose that comprise a large portion of sawdust, leaves, and grass can be valuable sources of fermentable sugars for ethanol production. However, some of the sugars liberated from hemicellulose (like xylose) are not easily fermented using conventional glucose-fermenting yeast like Saccharomyces cerevisiae, so engineering robust xylose-fermenting yeast that is not inhibited by other components liberated from cellulose/hemicellulose will be important for maximizing yield and making lignocellulosic ethanol fermentation cost efficient. The yeast Scheffersomyces stipitis is one such yeast that can ferment xylose; however, it possesses several barriers to genetic manipulation. It is difficult to transform, has only a few antibiotic resistance markers, and uses an alternative genetic code from most other organisms. We developed a genetic toolset for S. stipitis that lowers these barriers and allows a user to deliver and/or delete multiple genetic elements to/from the wild-type genome, thereby expanding S. stipitis's potential.

Endoplasmic Reticulum Stress and Cellular Homeostasis in Genetically Engineered Porcine Donors for Xenotransplantation.

Genetically engineered pigs with multiple gene deletions and insertions are predicted to extend porcine to human xenograft survival. Several genes have been successfully knocked out and inserted, yet more have failed to produce viable animals for unexplained reasons. The effects of gene editing on cellular homeostasis may be the cause of reduced embryo fitness, failed pregnancies, or poor piglet viability. The elements of cellular dysfunction such as endoplasmic reticulum stress and oxidative stress induced by gene editing may additively affect the quality of genetically engineered cells to be used for cloning. Evaluating the impact of each gene edit on cellular fitness for cloning will allow researchers to maintain the cellular homeostasis of engineered cells that were validated as candidates for cloning and the production of porcine organ donors.

Abstract 2914: iPSC-derived CAR-NK cell therapy: nominating clinical candidate clones through integrated multi-functional analysis

Abstract CNTY-101 is an induced Pluripotent Stem Cell (iPSC) derived Chimeric Antigen Receptor (CAR) NK cell clinical candidate for the treatment of B-cell malignancies. It incorporates six gene edits. A CAR targeting CD19 mediates tumor cell engagement and killing, the homeostatic cytokine IL-15 improves functional persistence, and EGFR acts as a safety switch, providing an antibody target for elimination of the engineered cells, if ever necessary. Further edits are designed to reduce graft rejection due to alloreactivity, including disruption of beta-2-microglobulin (B2M) to prevent major histocompatibility complex class I (MHC-I) surface expression, disruption of class II major histocompatibility complex transactivator (CIITA), and expression of HLA-E fused with B2M. Our data indicate that iPSC lines show significant functional variability dependent on the original somatic cell and donor used for reprogramming. Additional functional variability is induced when the iPSC are genetically edited and differentiated to NK cells. This complexity necessitates careful methodology for nominating clinical candidate clones with desirable functional attributes and presents an opportunity for future refinement. Here, we evaluate 28 clinical candidate clones for CNTY-101, integrating in-process data from genetic engineering, differentiation, and a battery of functional assays into a unified analysis framework. We find that the donor and the number of integrated transgene alleles (ex. monoallelic vs biallelic) have a large impact on clone function. In silico correction for donor and allele number reveals that transgene surface expression correlates with functions including short-term killing, serial-killing, and persistence. We show that IL-15 transgene expression, serial-killing, and persistence correlate along one functional axis, while CAR expression, short-term killing, and cytokine secretion correlate on a second functional axis. In addition, we identify biomarkers measured during the differentiation process that are correlated with functional performance, such as the level of CD94 surface expression and tumor killing. Finally, we discuss the application of functional multi-omics to candidate clone nomination for Century Therapeutics’ pipeline of future products. Citation Format: Matthew S. Hall, Ohad Manor, Liam Campion, Chiamin Bullaughey, Barry A. Morse, Buddha Gurung, Luis Borges. iPSC-derived CAR-NK cell therapy: nominating clinical candidate clones through integrated multi-functional analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2914.

Cloning, Expression Analysis, and Functional Characterization of Candidate Oxalate Transporter Genes of HbOT1 and HbOT2 from Rubber Tree (Hevea brasiliensis)

Secretion of oxalic acid from roots is an important aluminum detoxification mechanism for many plants such as Hevea brasiliensis (rubber tree). However, the underlying molecular mechanism and oxalate transporter genes in plants have not yet been reported. In this study, the oxalate transporter candidate genes HbOT1 and HbOT2 from the rubber tree were cloned and preliminarily identified. It was found that HbOT1 had a full length of 1163 bp with CDS size of 792 bp, encoding 263 amino acids, and HbOT2 had a full length of 1647 bp with a CDS region length of 840 bp, encoding 279 amino acid residues. HbOT1 and HbOT2 were both stable hydrophobic proteins with transmembrane structure and SNARE_assoc domains, possibly belonging to the SNARE_assoc subfamily proteins of the SNARE superfamily. qRT-PCR assays revealed that HbOT1 and HbOT2 were constitutively expressed in different tissues, with HbOT1 highly expressed in roots, stems, barks, and latex, while HbOT2 was highly expressed in latex. In addition, the expressions of HbOT1 and HbOT2 were up-regulated in response to aluminum stress, and they were inducible by metals, such as copper and manganese. Heterologous expression of HbOT1 and HbOT2 in the yeast mutant AD12345678 enhanced the tolerance to oxalic acid and high concentration aluminum stress, which was closely correlated with the secretion of oxalic acid. This study is the first report on oxalate transporter genes in plants, which provides a theoretical reference for the study on the molecular mechanism of oxalic acid secretion to relieve aluminum toxicity and on aluminum-tolerance genetic engineering breeding.

Identification of QTL and candidate genes associated with biomass yield and Feed Quality in response to water deficit in alfalfa (Medicago sativa L.) using linkage mapping and RNA-Seq

Biomass yield and Feed Quality are the most important traits in alfalfa (Medicago sativa L.), which directly affect its economic value. Drought stress is one of the main limiting factors affecting alfalfa production worldwide. However, the genetic and especially the molecular mechanisms for drought tolerance in alfalfa are poorly understood. In this study, linkage mapping was performed in an F1 population by combining 12 phenotypic data (biomass yield, plant height, and 10 Feed Quality-related traits). A total of 48 significant QTLs were identified on the high-density genetic linkage maps that were constructed in our previous study. Among them, nine main QTLs, which explained more than 10% phenotypic variance, were detected for biomass yield (one), plant height (one), CP (two), ASH (one), P (two), K(one), and Mg (one). A total of 31 candidate genes were identified in the nine main QTL intervals based on the RNA-seq analysis under the drought condition. Blast-P was further performed to screen candidate genes controlling drought tolerance, and 22 functional protein candidates were finally identified. The results of the present study will be useful for improving drought tolerance of alfalfa varieties by marker-assisted selection (MAS), and provide promising candidates for further gene cloning and mechanism study.

Production of an anti-TNFα antibody in murine myeloma cells by perfusion culture.

Infliximab is a mouse/human chimeric IgG1 monoclonal antibody which recognizes the proinflammatory cytokine, tumor necrosis factor α (TNFα), and inhibits receptor interactions, thereby decreasing inflammation and autoimmune response in patients. This monoclonal antibody has been successfully used to treat rheumatoid arthritis, ankylosing spondylitis, and psoriatic arthritis. However, the high treatment cost limits patient access to this biotherapy. One alternative to this problem is the use of biosimilars. In this work, we describe the stable expression and physicochemical characterization of an anti-TNFα antibody. While infliximab is produced in recombinant murine SP2/0 cells, our anti-TNFα IgG antibody was expressed in recombinant murine NS0 myeloma cells. The best anti-TNFα antibody-expressing clone was selected from three clone candidates based on the stability of IgG expression levels, specific productivity as well as TNFα-binding activity compared to commercial infliximab. Our results indicate that the selected cell clone, culture medium, and fermentation mode allowed for the production of an anti-TNFα antibody with similar characteristics to the reference commercially available product. An optimization of the selected culture medium by metabolomics may increase the volumetric productivity of the process to satisfy the demand for this product. Further experiments should be performed to evaluate the biological properties of this anti-TNFα antibody. KEY POINTS: • An anti-TNFα antibody was produced in NS0 cells using perfusion culture. • A proprietary chemically defined culture medium was used to replace commercially available protein-free medium. • The purified anti-TNFα antibody was comparable to the reference marketed product.

Genetic Association between Blast Resistance and Yield Traits in Rice Detected Using a High-Density Bin Map

Avoiding linkage drag of the resistance genes will facilitate the use of gene resources for rice breeding. This study was conducted to confirm the avoidance of linkage drag due to Pi26 and Pi25 blast resistance genes, and to analyze the association of Pi26, Pi25, Pib and Pita with quantitative trait loci (QTL) for yield traits. A recombinant inbred line population was derived from an indica rice cross Dan 71/Zhonghui 161. A linkage map consisting of 1219 bin markers, 22 simple sequence repeats and five gene markers was constructed. A total of 75 QTL were identified, including 2 for leaf blast resistance and 73 for eight yield traits. The two QTL for blast resistance were closely linked and located in the Pi26 and Pi25 regions, explaining 69.06 and 12.73% of the phenotypic variance, respectively. In a region covering Pi26 and Pi25, QTL were detected for grain yield and its key components. The alleles for enhancing blast resistance and grain yield were all from Dan 71. Not only was the linkage drag due to Pi26 and Pi25 avoided, but the results also indicate that these resistance genes may be used for simultaneously enhancing blast resistance and grain yield in rice. In the Pib and Pita regions, QTL was not detected for blast resistance, but was for yield traits. In each region, the allele for improving trait performance was derived from the parent carrying the resistance allele. In addition, four QTL clusters for grain weight and size, qGL4/qGW4.1, qGL11.2/qRLW11, qTGW11/qGW11 and qGL12/qGW12/qRLW12, were shown to be promising candidates for map-based cloning.

Fine mapping of powdery mildew resistance gene MlWE74 derived from wild emmer wheat (Triticum turgidum ssp. dicoccoides) in an NBS-LRR gene cluster.

Powdery mildew resistance gene MlWE74, originated from wild emmer wheat accession G-748-M, was mapped in an NBS-LRR gene cluster of chromosome 2BS. Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a globally devastating disease. Wild emmer wheat (Triticum turgidum var. dicoccoides) is a valuable genetic resource for improving disease resistance in common wheat. A powdery mildew resistance gene was transferred to hexaploid wheat line WE74 from wild emmer accession G-748-M. Genetic analysis revealed that the powdery mildew resistance in WE74 is controlled by a single dominant gene, herein temporarily designated MlWE74. Bulked segregant analysis (BSA) and molecular mapping delimited MlWE74 to the terminal region of chromosome 2BS flanking by markers WGGBD412 and WGGBH346 within a genetic interval of 0.25cM and corresponding to 799.9kb genomic region in the Zavitan reference sequence. Sequence annotation revealed two phosphoglycerate mutase-like genes, an alpha/beta-hydrolases gene, and five NBS-LRR disease resistance genes that could serve as candidates for map-based cloning of MlWE74. The geographical location analysis indicated that MlWE74 is mainly distributed in Rosh Pinna and Amirim regions, in the northern part of Israel, where environmental conditions are favorable to the occurrence of powdery mildew. Moreover, the co-segregated marker WGGBD425 is helpful in marker-assisted transfer of MlWE74 into elite cultivars.