Recombinant Lines Research Articles

Splicing by Overlap Extension PCR for the Production of Fusion Proteins.

Fusion proteins are valuable molecules to meet different demands related to the development of biopharmaceuticals and bioprocesses. In human therapy, they are used to improve the half-life of biologics by modifying the biophysical properties of the proteins. In biotechnology, the design of fusion proteins can standardize the establishment of production clones and the purification process. Analytical detection capabilities of the fusion partner and binding to affinity ligands play a crucial role. For the generation of the recombinant cell line, the maturation of the protein and the secretion are also crucial factors, which can be significantly influenced by the fusion partner and candetermine the final yield of the bioprocess. Here we present a protocol to recombine the human extracellular domain of CD19 with the human serum albumin domain 2 resulting in a fusion protein CD19-AD2 including a flexible linker sequence in the interface between the C-terminus of CD19 and the N-terminus of HSA-D2 and a terminal His12-tag. The two fragments are independently amplified with primers allowing to genetically connect the two fragments in the next step by overlap extension PCR. By this strategy, the linker sequence as well as the albumin fragment can be chosen individually to be flexible in the fine-tuning of the final protein. The amplified product is then cloned into a mammalian expression vector suitable to generate a recombinant transient or stable cell culture. This workflow can be applied to any protein sequence by adapting the specific primer sequences.

Wild introgression as an effective tool for aiding the expansion and adaptation of cultivated safflower

AbstractSafflower is a multipurpose crop grown in different regions, mainly for its high oil quality. Crop wild relatives serve as a valuable reservoir of genes that have been depleted due to evolutionary bottlenecks, which are poorly applied in safflower. During the last decade, we developed three populations from hybridization of safflower with its wild relatives and selected the superior lines to develop new varieties. From each of three different interspecific populations (TP: Carthamus tinctorius × Carthamus palaestinus, PO: C. palaestinus × Carthamus oxyacantha, and TO: C. tinctorius × C. oxyacantha), 10 genotypes were selected (a total of 30 lines) in the “F8” generation and were evaluated along with their parents (T, P, and O) and one control cultivar (Golsfid) at the field during 2019–2022 to assess genetic variation, estimate genetic parameters, and evaluate the stability. Considerable genetic variability for oil, seed yield, and other agronomic traits between and within the interspecific populations suggests the high potential of these new recombinant lines for introducing beneficial alleles. Our results indicated that recombinant inbred lines derived from the hybridization of TP were superior in terms of seed yield, oil content, and stability parameters. The use of stability indices of Wricke, Lin and Binns, Eberhart and Russell, and HMRPGVi, along with the biplot analysis, allowed the identification of preferable and stable safflower genotypes. Moderately high values of heritability were found for yield‐related traits. New recombinant lines can be introduced to the safflower gene pool to improve the genetic base of this valuable oil seed crop.

Exploring Thinopyrum spp. Group 7 Chromosome Introgressions to Improve Durum Wheat Performance under Intense Daytime and Night-Time Heat Stress at Anthesis.

Durum wheat (DW) is one of the major crops grown in the Mediterranean area, a climate-vulnerable region where the increase in day/night (d/n) temperature is severely threatening DW yield stability. In order to improve DW heat tolerance, the introgression of chromosomal segments derived from the wild gene pool is a promising strategy. Here, four DW-Thinopyrum spp. near-isogenic recombinant lines (NIRLs) were assessed for their physiological response and productive performance after intense heat stress (IH, 37/27 °C d/n) had been applied for 3 days at anthesis. The NIRLs included two primary types (R5, R112), carriers (+) of a differently sized Th. ponticum 7el1L segment on the DW 7AL arm, and two corresponding secondary types (R69-9/R5, R69-9/R112), possessing a Th. elongatum 7EL segment distally inserted into the 7el1L ones. Their response to the IH stress was compared to that of corresponding non-carrier sib lines (-) and the heat-tolerant cv. Margherita. Overall, the R112+, R69-9/R5+ and R69-9/R112+ NIRLs exhibited a tolerant behaviour towards the applied stress, standing out for the maintenance of leaf relative water content but also for the accumulation of proline and soluble sugars in the flag leaf and the preservation of photosynthetic efficiency. As a result, all the above three NIRLs (R112+ > R69-9/R5+ > R69-9/R112+) displayed good yield stability under the IH, also in comparison with cv. Margherita. R112+ particularly relied on the strength of spike fertility/grain number traits, while R69-9/R5+ benefited from efficient compensation by the grain weight increase. This work largely confirmed and further substantiated the value of exploiting the wild germplasm of Thinopyrum species as a useful source for the improvement of DW tolerance to even extreme abiotic stress conditions, such as the severe heat treatment throughout day- and night-time applied here.

Atp1a2 and Kcnj9 are candidate genes underlying sensitivity to oxycodone-induced locomotor activation and withdrawal-induced anxiety-like behaviors in C57BL/6 substrains.

Opioid use disorder is heritable, yet its genetic etiology is largely unknown. C57BL/6J and C57BL/6NJ mouse substrains exhibit phenotypic diversity in the context of limited genetic diversity which together can facilitate genetic discovery. Here, we found C57BL/6NJ mice were less sensitive to oxycodone (OXY)-induced locomotor activation versus C57BL/6J mice in a conditioned place preference paradigm. Narrow-sense heritability was estimated at 0.22-0.31, implicating suitability for genetic analysis. Quantitative trait locus (QTL) mapping in an F2 cross identified a chromosome 1 QTL explaining 7-12% of the variance in OXY locomotion and anxiety-like withdrawal in the elevated plus maze. A second QTL for EPM withdrawal behavior on chromosome 5 near Gabra2 (alpha-2 subunit of GABA-A receptor) explained 9% of the variance. To narrow the chromosome 1 locus, we generated recombinant lines spanning 163-181 Mb, captured the QTL for OXY locomotor traits and withdrawal, and fine-mapped a 2.45-Mb region (170.16-172.61 Mb). Transcriptome analysis identified five, localized striatal cis-eQTL transcripts and two were confirmed at the protein level (KCNJ9, ATP1A2). Kcnj9 codes for a potassium channel (GIRK3) that is a major effector of mu opioid receptor signaling. Atp1a2 codes for a subunit of a Na+/K+ ATPase enzyme that regulates neuronal excitability and shows functional adaptations following chronic opioid administration. To summarize, we identified two candidate genes underlying the physiological and behavioral properties of opioids, with direct preclinical relevance to investigators employing these widely used substrains and clinical relevance to human genetic studies of opioid use disorder.

Exotic alleles from the wild Hordeum spontaneum contribute to drought tolerance and grain quality in modern Hordeum vulgare

Conventional breeding relies on genetic variability in crop germplasm and wild species. This study evaluated 137 barley Recombinant Chromosome Substitution Lines (RCSLs), originating from crossing a drought and salinity-tolerant wild Hordeum spontaneum with the high-quality malting H. vulgare cv. Harrington. The objectives of this work were to i) improve understanding of the genetic diversity and introgression levels in RCSLs using a comprehensive set of high-density single nucleotide polymorphism (SNP) markers; ii) investigate the phenotypic variability of physiological, agronomic, and malting quality traits in RCSLs under rainfed and fully irrigated conditions; iii) unravel the relationship between these traits and the resilience of crops in drought-prone regions; and iv) identify specific SNPs associated with both drought tolerance indices and malting quality traits. Variability in physiological, agronomic, and malting quality traits was examined under rainfed and irrigated conditions, aiming to identify specific SNPs associated with both drought tolerance indices and malting quality traits. Additionally, a subset of three recombinant lines and the parent cv. Harrington was tested in growth chambers under well-watered and water-limited conditions. Agronomic traits were influenced by genotype-by-environment interaction, with notable drought tolerance lines. The grain yield was significantly correlated with yield-related traits and carbon isotope discrimination in grains. Drought stress impacted agronomic and physiological traits, with a strong reduction in leaf gas exchange and chlorophyll and protein content. The H. spontaneum genome introgression into H. vulgare cv. Harrington varied across 137 RCSLs, averaging 13.0 %. Associations between traits and genetic markers were found, particularly for the stress tolerance index in chromosome 1H and for grain quality traits (alfa-amylase, wort color, and protein) in chromosomes 1H, 4H, and 5H. These findings uncovered valuable barley RCSLs that demonstrate drought tolerance, offering promising prospects for breeding programs targeted at improving stress resilience.

Effect of ABCB1 most frequent polymorphisms on the accumulation of bictegravir in recombinant HEK293 cell lines

Bictegravir, a key second-generation integrase strand transfer inhibitor in the treatment of HIV, is subject to active efflux transport mediated by ABCB1 (P-glycoprotein). Several coding variants of ABCB1 have been described and associated with variable effects on substrate drugs pharmacokinetics. Here, we investigated the effect of the four most common coding ABCB1 single nucleotide polymorphisms (i.e., c.1199G > A, c.1236C > T, c.2677G > T and c.3435C > T) on the intracellular accumulation of bictegravir. Using a previously validated HEK293 recombinant cell line model, we found decreased bictegravir intracellular concentrations in cell lines overexpressing ABCB1 as compared to control cell lines, in line with the known role of ABCB1 in bictegravir transport. However, we were unable to demonstrate any significant difference in bictegravir intracellular accumulation when comparing HEK293 cells overexpressing the wild type (1236C-2677G-3435C, 1199G) or the variant (1236C-2677G-3435T, 1236T-2677T-3435T or 1199A) proteins. These findings suggest that the ABCB1 c.1199G > A and c.1236C > T-c.2677G > T-c.3435C > T variants have no or at least limited impact on the active transport of bictegravir by ABCB1.

In vitro assessment of the risk of ABCB1-mediated drug–drug interaction between rivaroxaban and tacrolimus in human embryonic kidney 293 recombinant cell lines

BackgroundIn lung transplant patients, direct oral anticoagulants are often taken in combination with immunosuppressive drugs such as tacrolimus. Since tacrolimus is a substrate and inhibitor of the efflux protein ABCB1, also transporting direct oral anticoagulants, a possible drug–drug interaction mediated by competition for this transporter needs to be investigated. ObjectivesTo determine the in vitro effect of tacrolimus on ABCB1-mediated rivaroxaban transport in order to support clinician practice. MethodsRecombinant cell line models, based on human embryonic kidney 293 cells, were generated by a stable transfection process to overexpress ABCB1 or not (control cells). The impact of tacrolimus on ABCB1-mediated rivaroxaban transport was assessed by accumulation experiments. ResultsABCB1 expression decreased the cellular accumulation of rivaroxaban and tacrolimus at their respective clinically relevant concentrations when compared with control cells. This confirms the involvement of ABCB1 in the active transport of tacrolimus and rivaroxaban. However, tacrolimus had no significant influence on rivaroxaban disposition at those clinically relevant concentrations. ConclusionOur study does not provide evidence for a possible interaction between tacrolimus and rivaroxaban when used together in practice.

An electroporation cytometry system for long-term, live cell cycle analysis.

Electric fields are used in biology to address a broad range of questions and through a variety of techniques, including electroporation, gene electrotransfer (GET), electrostimulation (ES), and electrochemotherapy. Each of these modalities requires specific conditions and has drastically different target outcomes on the cell. ES has demonstrated that non-pore forming electric fields alter cell cycle progression. However, pore forming electric fields such as with GET have not been as widely explored despite major clinical advancements. Additionally, the real-time visual analysis of electrical field effects on mammalian cell culture is currently lacking among most commercial systems. To facilitate investigations into these research areas, an electroporation cytometry system was developed including a custom chamber compatible with live cell imaging and exponential decay pulse generator for live cell analysis. The functionality of the system was demonstrated using a recombinant cell line using U-2 OS cells and FUCCI(CA)5 cell cycle reporter. The exposure of the cells to a 180 V pulse in both unsynchronized and synchronized populations revealed an effect on the cell cycle.

High-density linkage to physical mapping in a unique Tall × Dwarf coconut (Cocos nucifera L.) outbred F2 uncovers a major QTL for flowering time colocalized with the FLOWERING LOCUS T (FT).

The coconut tree crop (Cocos nucifera L.) provides vital resources for millions of people worldwide. Coconut germplasm is largely classified into 'Tall' (Typica) and 'Dwarf' (Nana) types. While Tall coconuts are outcrossing, stress tolerant, and late flowering, Dwarf coconuts are inbred and flower early with a high rate of bunch emission. Precocity determines the earlier production of a plantation and facilitates management and harvest. A unique outbred F2 population was used, generated by intercrossing F1 hybrids between Brazilian Green Dwarf from Jiqui (BGDJ) and West African Tall (WAT) cultivars. Single-nucleotide polymorphism (SNP) markers fixed for alternative alleles in the two varieties, segregating in an F2 configuration, were used to build a high-density linkage map with ~3,000 SNPs, anchored to the existing chromosome-level genome assemblies, and a quantitative trait locus (QTL) mapping analysis was carried out. The linkage map established the chromosome numbering correspondence between the two reference genome versions and the relationship between recombination rate, physical distance, and gene density in the coconut genomes. Leveraging the strong segregation for precocity inherited from the Dwarf cultivar in the F2, a major effect QTL with incomplete dominance was mapped for flowering time. FLOWERING LOCUS T (FT) gene homologs of coconut previously described as putatively involved in flowering time by alternative splice variant analysis were colocalized within a ~200-kb window of the major effect QTL [logarithm of the odds (LOD) = 11.86]. Our work provides strong phenotype-based evidence for the role of the FT locus as the putative underlying functional variant for the flowering time difference between Dwarf and Tall coconuts. Major effect QTLs were also detected for developmental traits of the palm, plausibly suggesting pleiotropism of the FT locus for other precocity traits. Haplotypes of the two SNPs flanking the flowering time QTL inherited from the Dwarf parent BGDJ caused a reduction in the time to flower of approximately 400 days. These SNPs could be used for high-throughput marker-assisted selection of early-flowering and higher-productivity recombinant lines, providing innovative genetic material to the coconut industry.

Coping with salinity stress: segmental group 7 chromosome introgressions from halophytic Thinopyrum species greatly enhance tolerance of recipient durum wheat.

Increased soil salinization, tightly related to global warming and drought and exacerbated by intensified irrigation supply, implies highly detrimental effects on staple food crops such as wheat. The situation is particularly alarming for durum wheat (DW), better adapted to arid/semi-arid environments yet more sensitive to salt stress than bread wheat (BW). To enhance DW salinity tolerance, we resorted to chromosomally engineered materials with introgressions from allied halophytic Thinopyrum species. "Primary" recombinant lines (RLs), having portions of their 7AL arms distally replaced by 7el1L Th. ponticum segments, and "secondary" RLs, harboring Th. elongatum 7EL insertions "nested" into 7el1L segments, in addition to near-isogenic lines lacking any alien segment (CLs), cv. Om Rabia (OR) as salt tolerant control, and BW introgression lines with either most of 7el1 or the complete 7E chromosome substitution as additional CLs, were subjected to moderate (100 mM) and intense (200 mM) salt (NaCl) stress at early growth stages. The applied stress altered cell cycle progression, determining a general increase of cells in G1 and a reduction in S phase. Assessment of morpho-physiological and biochemical traits overall showed that the presence of Thinopyrum spp. segments was associated with considerably increased salinity tolerance versus its absence. For relative water content, Na+ accumulation and K+ retention in roots and leaves, oxidative stress indicators (malondialdehyde and hydrogen peroxide) and antioxidant enzyme activities, the observed differences between stressed and unstressed RLs versus CLs was of similar magnitude in "primary" and "secondary" types, suggesting that tolerance factors might reside in defined 7el1L shared portion(s). Nonetheless, the incremental contribution of 7EL segments emerged in various instances, greatly mitigating the effects of salt stress on root and leaf growth and on the quantity of photosynthetic pigments, boosting accumulation of compatible solutes and minimizing the decrease of a powerful antioxidant like ascorbate. The seemingly synergistic effect of 7el1L + 7EL segments/genes made "secondary" RLs able to often exceed cv. OR and equal or better perform than BW lines. Thus, transfer of a suite of genes from halophytic germplasm by use of fine chromosome engineering strategies may well be the way forward to enhance salinity tolerance of glycophytes, even the sensitive DW.

Context-dependent genomic locus effects on antibody production in recombinant Chinese hamster ovary cells generated through random integration

High-yield production of therapeutic protein using Chinese hamster ovary (CHO) cells requires stable cell line development (CLD). CLD typically uses random integration of transgenes; however, this results in clonal variation and subsequent laborious clone screening. Therefore, site-specific integration of a protein expression cassette into a desired chromosomal locus showing high transcriptional activity and stability, referred to as a hot spot, is emerging. Although positional effects are important for therapeutic protein expression, the sequence-specific mechanisms by which hotspots work are not well understood. In this study, we performed whole-genome sequencing (WGS) to locate randomly inserted vectors in the genome of recombinant CHO cells expressing high levels of monoclonal antibodies (mAbs) and experimentally validated these locations and vector compositions. The integration site was characterized by active histone marks and potential enhancer activities, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) mediated indel mutations in the region upstream of the integration site led to a significant reduction in specific antibody productivity by up to 30%. Notably, the integration site and its core region did not function equivalently outside the native genomic context, showing a minimal effect on the increase in exogenous protein expression in the host cell line. We also observed a superior production capacity of the mAb expressing cell line compared to that of the host cell line. Collectively, this study demonstrates that developing recombinant CHO cell lines to produce therapeutic proteins at high levels requires a balance of factors including transgene configuration, genomic locus landscape, and host cell properties.

Using single antigen specificity magnetic beads for the isolation of specific antibodies against HLA antigens.

The presence of multiple donor-specific antibodies (DSAs) targeting HLA antigens poses a challenge to transplantation. Various techniques, including the use of recombinant cell lines and crossmatch cells have been developed to isolate DSAs. To simplify the extraction of HLA-specific DSAs from complex sera, we introduced magnetic beads with single HLA specificity (MagSort). Sera were treated with MagSort, allowing HLA-specific antibodies to bind to the beads, and these specific antibodies were subsequently eluted. MagSort beads, coated with 59 different HLA variants, underwent testing through 1329 adsorption/elution processes, demonstrating their effectiveness and specificity in adsorbing and eluting HLA-specific antibodies. The MagSort method proves comparable to the cell method, showing similar isolated antibody binding patterns. The isolated antibody binding patterns from MagSort reveal both known eplets and unknown patterns, suggesting its utility for eplet discovery. Additionally, MagSort proved effective in extracting signals for flow cytometry cross-matching, offering a means to assess the binding capability of isolated antibodies against specific donor cells.

Abstract 1563: Anoikis associated metabolic modulation by UCA1 in CRC

Abstract Colorectal Cancer (CRC) is the second most lethal cancer in estimated deaths for 2023 according to the American Cancer Society. This is partially due to the low survival rate once CRC is diagnosed at a distant location. This warrants early diagnostic markers and an understanding of the mechanisms underlying the metastatic process, anoikis resistance being one of them. Our laboratory analysis of the TCGA database comprising 519 human CRC tissues shows lncRNA UCA1 overexpressed in tumors compared to normal, and higher expression of lncRNA UCA1 is responsible for the progression, metastasis, and poor survival of CRC patients. In our anchorage-independent growth model (anoikis resistant), using isogenic (oncogenic and metastatic) cell lines, we observed a very high expression of lncRNA UCA1 along with GluT1 with increased cell survival. Cancer cells, however, appear to rapidly increase their glucose uptake and directly ferment it into lactate, even in the presence of oxygen and functional mitochondria. UCA1 has been found to play an essential role in the development of metastasis and dysregulation of glycolysis in various cancers. To verify if lncRNA UCA1 modulates anoikis resistance and glucose metabolism, we generated SW480+UCA1 overexpression (OE) and SW620+shUCA1 knockdown (KD) stable cell lines using lentiviral transduction. The OE GFP-expressing cell lines were further sorted, and grown in puromycin. The KD cell lines were selected with puromycin for the generation of stable cell lines. The stable cell lines were characterized through different phenotypic assays. UCA1 OE cell lines have a higher glucose uptake and the UCA1 KD cell lines have a lower glucose uptake. The lactate production is high in the KD and low in OE cell lines. The OE and KD cell lines along with the control cell lines were subjected to an anoikis model, and glucose and lactate analysis were performed accordingly. RT-PCR and western blot analysis were performed for the target genes responsible for cell survival, stemness, and glucose metabolism. We are analyzing the Extracellular Acidification Rate (ECAR), Oxidative Consumption Rate (OCR), ATP production rate the rate and potential max rate of glycolysis, and the mitochondrial stress of the recombinant cell lines using the “Agilent Seahorse XF”. The role of lncRNA UCA1 in anoikis resistance leading to metastasis and modulating glucose metabolism will provide new insights into the pathophysiology of CRC metastasis. This information will be beneficial for developing more unique strategies for the treatment of this metastatic disease. Citation Format: Sophia Leslie, Kyle Doxtater, Samantha Lopez, Bilal Hafeez, Tamer Oraby, Timothy Loy, Manish K. Tripathi. Anoikis associated metabolic modulation by UCA1 in CRC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1563.

Principal Component Analysis of Yield and Its Attributing Traits in Recombinant Inbred Lines of Rice Under Submerged Condition (Oryza sativa L.)

Rice, Oryza sativa L., is the world's most important staple crop, feeding more than half of the world's population. The phenotype of plant is the result of interaction of many factors and final yield is the sum of total effect of several component factors. Therefore evaluation of genetic variability forms the basis for any crop improvement programme, the success of which depends on sufficient genetic variability among genotypes so as to permit effective selection. Hence Evaluation of Principle Component Analysis (PCA)of recombinant inbred lines (RILs) was done at phenotypic level under submerged conditions to reduce a large series of data into smaller number of components by looking for groups that have very strong inter-correlation in a set of variables and each component explained per cent variation to the total variability. The RIL population was derived from an inter-specific cross between BPT5204 and HPR14 parents. A study was conducted using 1256 Recombinant Inbreed Lines submerged condition in the two seasons at College of Agriculture V.C. Farm, Mandya with nine agro-morphological traits and a principle component analysis was carried out. Out of nine principle components, four exhibited Eigenvalue more than one governing 77.74% variance and 69.86% variance in the summer and kharif seasons respectively. The highest positive Eigenvalue was observed for total number of tillers, productive tillers, non-productive tillers and fallowed by single plant yield in PC1 in the summer and kharif season respectively. The highest positive Eigenvalue was observed for five panicle weight, single panicle length, single plant yield and plant height in PC2 of summer and kharif season respectively. Indicating their pronounced effect on the overall variation in the Recombinant Inbreed Lines of Rice.

Metabolism-dependent mutagenicity of two structurally similar tobacco-specific nitrosamines (N-nitrosonornicotine and N-nitrosoanabasine) in human cells, partially different CYPs being activating enzymes

N-nitrosonornicotine (NNN) and N-nitrosoanabasine (NAB) are both tobacco-specific nitrosamines bearing two heterocyclic amino groups, NAB bearing an extra -CH2- group (conferring a hexa- rather than penta-membered cycle) but with significantly decreased carcinogenicity. However, their activating enzymes and related mutagenicity remain unclear. In this study, the chemical-CYP interaction was analyzed by molecular docking, thus the binding energies and conformations of NNN for human CYP2A6, 2A13, 2B6, 2E1 and 3A4 appeared appropriate as a substrate, so did NAB for human CYP1B1, 2A6, 2A13 and 2E1. The micronucleus test in human hepatoma (HepG2) cells with each compound (62.5–1000 μM) exposing for 48 h (two-cell cycle) was negative, however, pretreatment with bisphenol AF (0.1–100 nM, CYPs inducer) and ethanol (0.2% v:v, CYP2E1 inducer) potentiated micronucleus formation by both compounds, while CITCO (1 μM, CYP2B6 inducer) selectively potentiated that by NNN. In C3A cells (endogenous CYPs enhanced over HepG2) both compounds induced micronucleus, which was abolished by 1-aminobenzotriazole (60 μM, CYPs inhibitor) while unaffected by 8-methoxypsoralen (1 μM, CYP2A inhibitor). Consistently, NNN and NAB induced micronucleus in V79-derived recombinant cell lines expressing human CYP2B6/2E1 and CYP1B1/2E1, respectively, while negative in those expressing other CYPs. By immunofluorescent assay both compounds selectively induced centromere-free micronucleus in C3A cells. In PIG-A assays in HepG2 cells NNN and NAB were weakly positive and simply negative, respectively; however, in C3A cells both compounds significantly induced gene mutations, NNN being slight more potent. Conclusively, both NNN and NAB are mutagenic and clastogenic, depending on metabolic activation by partially different CYP enzymes.

Flax lines of hybrid origin homozygous for genes of chlorophyll coloration and other morphological traits in the VIR flax genetic collection

Due to climate change and the industrialization of agriculture, the role of genetic collections is increasing. The paper characterizes 41 flax lines with mutations regarding chlorophyll coloration from the VIR genetic collection: 5 parental lines created on the basis of the VIR global flax collection, and 36 recombinant ones.Among the 36 created recombinant lines, homozygous for two or more genes of morphological traits, there are those with chlorophyll deficiency and differences in anthocyanin color, stem shape and ciliation of the septa of the boll. Anthocyanin coloration and other morphological features were controlled by one or two of the 22 genes identified by us. Four of the recombinant lines were also homozygous for two or three independent chlorophyll coloration genes.It was established that the genes of chlorophyll coloration and 22 genes controlling other morphological features act independently.The ygp1 and ygp2 genes do not have a significant effect on most economically valuable traits, except for early flowering, and can be used for labeling varieties.The genes s1, YSED1, ysed2 and rs1, which determine the yellow seed color, are necessary for the creation of flax varieties for food purposes, which makes the 11 lines based on these genes in demand for breeding purposes.In flax, the molecular genetic function of none of the chlorophyll coloration genes is known, so the created genetic collection will be in demand to solve this problem.

MicroRNA-502-3p regulates GABAergic synapse function in hippocampal neurons.

JOURNAL/nrgr/04.03/01300535-202412000-00026/figure1/v/2024-04-08T165401Z/r/image-tiff Gamma-aminobutyric acid (GABA)ergic neurons, the most abundant inhibitory neurons in the human brain, have been found to be reduced in many neurological disorders, including Alzheimer's disease and Alzheimer's disease-related dementia. Our previous study identified the upregulation of microRNA-502-3p (miR-502-3p) and downregulation of GABA type A receptor subunit α-1 in Alzheimer's disease synapses. This study investigated a new molecular relationship between miR-502-3p and GABAergic synapse function. In vitro studies were performed using the mouse hippocampal neuronal cell line HT22 and miR-502-3p agomiRs and antagomiRs. In silico analysis identified multiple binding sites of miR-502-3p at GABA type A receptor subunit α-1 mRNA. Luciferase assay confirmed that miR-502-3p targets the GABA type A receptor subunit α-1 gene and suppresses the luciferase activity. Furthermore, quantitative reverse transcription-polymerase chain reaction, miRNA in situ hybridization, immunoblotting, and immunostaining analysis confirmed that overexpression of miR-502-3p reduced the GABA type A receptor subunit α-1 level, while suppression of miR-502-3p increased the level of GABA type A receptor subunit α-1 protein. Notably, as a result of the overexpression of miR-502-3p, cell viability was found to be reduced, and the population of necrotic cells was found to be increased. The whole cell patch-clamp analysis of human-GABA receptor A-α1/β3/γ2L human embryonic kidney (HEK) recombinant cell line also showed that overexpression of miR-502-3p reduced the GABA current and overall GABA function, suggesting a negative correlation between miR-502-3p levels and GABAergic synapse function. Additionally, the levels of proteins associated with Alzheimer's disease were high with miR-502-3p overexpression and reduced with miR-502-3p suppression. The present study provides insight into the molecular mechanism of regulation of GABAergic synapses by miR-502-3p. We propose that micro-RNA, in particular miR-502-3p, could be a potential therapeutic target to modulate GABAergic synapse function in neurological disorders, including Alzheimer's disease and Alzheimer's disease-related dementia.

Assessment of Genetic Variability for Seed Yield and Related Traits in Some Mutant and Non-mutant Lines of Indian Mustard (Brassica juncea L.)

A study was conducted to evaluate the genetic variability for seed yield and related traits in 29 genotypes of Indian mustard during Rabi 2019-20 and Rabi 2020-21 at Experimental Farm, Department of Plant Breeding and Genetics, AAU, Jorhat. The genotypes comprised of 21 M4-5 mutant lines developed by gamma irradiation of the parent varieties TM-2 and PM-27, 5 newly developed F6-7 lines, and 3 check varieties (TM-2, PM-27 and NRCHB-101). Pooled analysis of variances over the years revealed significant variation for most of the characters observed. The characters seed yield per plant, number of primary branches per plant and number of secondary branches per plant exhibited moderate genetic variation. Moderate heritability coupled with high genetic advance was observed for number of secondary branches per plant whereas, number of primary branches per plant showed high heritability with moderate genetic advance. The genotypes JMM-TM2-17, JMM-TM2-15, JMM-TM2-34, JMM-TM2-10, JMM-PM27-1, JM13-6 and the variety NRCHB-101 were found promising for most of the yield attributing characters. The mutant line JMM-TM2-28 and recombinant line JM13-4 may be exploited for the development of early-maturing mustard varieties.

Hernandulcin Production in Elicited Hairy Roots of Phyla scaberrima: Toward Sustainable Production of a Non-Caloric Sweetener with Nutraceutical Properties.

Herein we report on the generation of hairy root lines of P. scaberrima able to produce hernandulcin (HE), a non-caloric sweetener with nutraceutical properties. From ten different lines analyzed, three synthesized up to 100 mg ⋅ L-1 HE under the batch culture conditions standardized in this investigation. Adding elicitors (salicylic acid, chitin, Glucanex, polyethylene glycol) and biosynthetic precursors (farnesol and (+)-epi-alpha-bisabolol) significantly altered HE accumulation. Chitin and Glucanex enhanced HE production from 130 to 160 mg ⋅ L-1 , whereas farnesol and (+)-epi-alpha-bisabolol from 165 to 200 mg ⋅ L-1 without dependence on biomass accumulation. Improved batch cultures containing liquid Murashige & Skoog medium (MS; pH 7), added with 4 % sucrose, 0.5 mg ⋅ L-1 naphthaleneacetic acid, 100 mg ⋅ L-1 Glucanex, 150 mg ⋅ L-1 chitin, 250 mg ⋅ L-1 farnesol, and 150 mg ⋅ L-1 (+)-epi-alpha-bisabolol at 25 °C (12 h light/12 h darkness), triggered HE accumulation to 250 mg ⋅ L-1 in 25 days. The efficiency of each recombinant line is discussed.

Strain-specific requirements of susceptibility to rhesus enteric calicivirus infection.

Recently, we identified the coxsackie and adenovirus receptor (CAR) as the entry receptor for rhesus enteric calicivirus (ReCV) isolate FT285 and demonstrated that co-expression of the CAR and the type B histo-blood group antigen (HBGA) is required to convert the resistant CHO cell line susceptible to infection. To address whether the CAR is also the functional entry receptor for other ReCV isolates and the requirement for specific HBGAs or other glycans, here we used a panel of recombinant CHO cell lines expressing the CAR and the type A, B, or H HBGAs alone or in combination. Infection studies with three diverse ReCV strains, the prototype GI.1 Tulane virus (TV), GI.2 ReCV-FT285, and GI.3 ReCV-FT7, identified that cell surface expression of the CAR is an absolute requirement for all three strains to promote susceptibility to infection, while the requirement for HBGAs varies among the strains. In addition to the CAR, ReCV-FT285 and TV require type A or B HBGAs for infection. In the absence of HBGAs, TV, but not Re-CV FT285, can also utilize sialic acids, while ReCV-FT7 infection is HBGA-independent and relies on CAR and sialic acid expression. In summary, we demonstrated strain-specific diversity of susceptibility requirements for ReCV infections and that CAR, type A and B HBGA, and sialic acid expression control susceptibility to infection with the three ReCV isolates studied. Our study also indicates that the correlation between in vitro HBGA binding and HBGAs required for infection is relatively high, but not absolute. This has direct implications for human noroviruses.IMPORTANCEHuman noroviruses (HuNoVs) are important enteric pathogens. The lack of a robust HuNoV cell culture system is a bottleneck for HuNoV cell culture-based studies. Often, cell culture-adapted caliciviruses that rapidly replicate in conventional cell lines and recapitulate biological features of HuNoVs are utilized as surrogates. Particularly, rhesus enteric caliciviruses (ReCVs) display remarkable similarities, including the primate host, clinical manifestation of gastroenteritis, genetic/antigenic diversity, and reliance on histo-blood group antigens (HBGAs) for attachment. While the HuNoV entry receptor(s) is unknown, the coxsackie and adenovirus receptor (CAR) has recently been identified as the ReCV entry receptor. Here, we identified the CAR, the type A and B HBGAs, and sialic acids as critical cell surface molecules controlling susceptibility to ReCV infections. The CAR is required for all ReCV isolates studied. However, the requirement for the different carbohydrate molecules varies among different ReCV strains. Our findings have direct implications for HuNoVs.