Hemoglobin is an oxygen-dependent glutathione buffer adapting the intracellular reduced glutathione levels to oxygen availability

Fast changes in environmental oxygen availability translate into shifts in mitochondrial free radical production. An increase in intraerythrocytic reduced glutathione (GSH) during deoxygenation would support the detoxification of exogenous oxidants released into the circulation from hypoxic peripheral tissues. Although reported, the mechanism behind this acute oxygen-dependent regulation of GSH in red blood cells remains unknown. This study explores the role of hemoglobin (Hb) in the oxygen-dependent modulation of GSH levels in red blood cells. We have demonstrated that a decrease in Hb O2 saturation to 50% or less observed in healthy humans while at high altitude, or in red blood cell suspensions results in rising of the intraerythrocytic GSH level that is proportional to the reduction in Hb O2 saturation. This effect was not caused by the stimulation of GSH de novo synthesis or its release during deglutathionylation of Hb's cysteines. Using isothermal titration calorimetry and in silico modeling, we observed the non-covalent binding of four molecules of GSH to oxy-Hb and the release of two of them upon deoxygenation. Localization of the GSH binding sites within the Hb molecule was identified. Oxygen-dependent binding of GSH to oxy-Hb and its release upon deoxygenation occurred reciprocally to the binding and release of 2,3-bisphosphoglycerate. Furthermore, noncovalent binding of GSH to Hb moderately increased Hb oxygen affinity. Taken together, our findings have identified an adaptive mechanism by which red blood cells may provide an advanced antioxidant defense to respond to oxidative challenges immediately upon deoxygenation

Genome of Linum usitatissimum convar. crepitans expands the view on the section Linum

Sequencing whole plant genomes provides a solid foundation for applied and basic studies. Genome sequences of agricultural plants attract special attention, as they reveal information on the regulation of beneficial plant traits. Flax is a valuable crop cultivated for oil and fiber. Genome sequences of its representatives are rich sources of genetic information for the improvement of cultivated forms of the plant. In our work, we sequenced the first genome of flax with the dehiscence of capsules—Linum usitatissimum convar. сrepitans (Boenn.) Dumort—on the Oxford Nanopore Technologies (ONT) and Illumina platforms. We obtained 23 Gb of raw ONT data and 89 M of 150 + 150 paired-end Illumina reads and tested different tools for genome assembly and polishing. The genome assembly produced according to the Canu—Racon ×2—medaka—POLCA scheme had optimal contiguity and completeness: assembly length—412.6 Mb, N50—5.2 Mb, L50—28, and complete BUSCO—94.6% (64.0% duplicated, eudicots_odb10). The obtained high-quality genome assembly of L. usitatissimum convar. crepitans provides opportunities for further studies of evolution, domestication, and genome regulation in the section Linum

Donor age and red cell age contribute to the variance in lorrca indices in healthy donors for next generation ektacytometry: a pilot study

The ability of red blood cells (RBCs) to transport gases, their lifespan as well as their rheological properties invariably depend on the deformability, hydration, and membrane stability of these cells, which can be measured by Laser optical rotational red cell analyser (Lorrca® Maxsis, RR Mechatronics). The osmoscan mode of Lorrca is currently used in diagnosis of rare anemias in clinical laboratories. However, a broad range of normal values for healthy subjects reduces the sensitivity of this method for diagnosis of mild disease phenotype. In this pilot study, we explored the impact of age and gender of 45 healthy donors, as well as RBC age on the Lorrca indices. Whereas gender did not affect the Lorrca indices in our study, the age donors had a profound effect on the O_hyper parameter. To study the impact of RBC age on the osmoscan parameters, we have isolated low (L)-, medium (M)-, or high (H)- density fractions enriched with young, mature, and senescent RBCs, respectively, and evaluated the influence of RBC age-related properties, such as density, morphology, and redox state, on the osmoscan indices. As before, O_hyper was the most sensitive parameter, dropping markedly with an increase in RBC density and age. Senescence was associated with a decrease in deformability (EI_max) and tolerability to low and high osmolatites (Area). L-fraction was enriched with reticulocytes and cells with high projected area and EMA staining, but also contained a small number of cells small in projected area and most likely, terminally senescent. L-fraction was on average slightly less deformable than mature cells. The cells from the L-fraction produced more oxidants and NO than all other fractions. However, RBCs from the L-fraction contained maximal levels of reduced thiols compared to other fractions. Our study suggests that reference values for O_hyper should be age-stratified, and, most probably, corrected for the average RBC age. Further multi-center study is required to validate these suggestions before implementing them into clinical practice

ITS and 16S rDNA metagenomic dataset of different soils from flax fields

Flax (Linum usitatissimum L.), one of the important and versatile crops, is used for the production of oil and fiber. To obtain high and stable yields of flax products, L. usitatissimum varieties should be cultivated under optimal conditions, including the composition of the soil microbiome. We evaluated the diversity of microorganisms in soils under conditions unfavorable for flax cultivation (suboptimal acidity or herbicide treatment) or infected with causative agents of harmful flax diseases (Septoria linicola, Colletotrichum lini, Melampsora lini, or Fusarium oxysporum f. sp. lini). For this purpose, twenty-two sod-podzolic soil samples were collected from flax fields and their metagenomes were analyzed using the regions of 16S ribosomal RNA gene (16S rDNA) and internal transcribed spacers (ITS) of the ribosomal RNA genes, which are used in phylogenetic studies of bacteria and fungi. Amplicons were sequenced on the Illumina MiSeq platform (reads of 300 + 300 bp). On average, we obtained 8,400 reads for ITS and 43,300 reads for 16S rDNA per sample. For identification of microorganisms in the soil samples, the Illumina reads were processed using DADA2. The raw data are deposited in the Sequence Read Archive under the BioProject accession number PRJNA956957. Tables listing the microorganisms identified in the soil samples are available in this article. The obtained dataset can be used to analyze the fungal and bacterial composition of flax field soils and their relationship to environmental conditions, including suboptimal soil acidity and infection with fungal pathogens. In addition, it can help to understand the influence of herbicide treatment on the microbial diversity of flax fields. Another useful application of our data is the ability to assess the suitability of the soil microbiome for flax cultivation

Hemoglobin is an oxygen-dependent glutathione buffer adapting the intracellular reduced glutathione levels to oxygen availability

Fast changes in environmental oxygen availability translate into shifts in mitochondrial free radical production. An increase in intraerythrocytic reduced glutathione (GSH) during deoxygenation would support the detoxification of exogenous oxidants released into the circulation from hypoxic peripheral tissues. Although reported, the mechanism behind this acute oxygen-dependent regulation of GSH in red blood cells remains unknown.This study explores the role of hemoglobin (Hb) in the oxygen-dependent modulation of GSH levels in red blood cells. We have demonstrated that a decrease in Hb O2 saturation to 50% or less observed in healthy humans while at high altitude, or in red blood cell suspensions results in rising of the intraerythrocytic GSH level that is proportional to the reduction in Hb O2 saturation. This effect was not caused by the stimulation of GSH de novo synthesis or its release during deglutathionylation of Hb's cysteines. Using isothermal titration calorimetry and in silico modeling, we observed the non-covalent binding of four molecules of GSH to oxy-Hb and the release of two of them upon deoxygenation. Localization of the GSH binding sites within the Hb molecule was identified. Oxygen-dependent binding of GSH to oxy-Hb and its release upon deoxygenation occurred reciprocally to the binding and release of 2,3-bisphosphoglycerate. Furthermore, noncovalent binding of GSH to Hb moderately increased Hb oxygen affinity. Taken together, our findings have identified an adaptive mechanism by which red blood cells may provide an advanced antioxidant defense to respond to oxidative challenges immediately upon deoxygenation

Isolating Linum usitatissimum L. Nuclear DNA Enabled Assembling High-Quality Genome

High-quality genome sequences help to elucidate the genetic basis of numerous biological processes and track species evolution. For flax (Linum usitatissimum L.)—a multifunctional crop, high-quality assemblies from Oxford Nanopore Technologies (ONT) data were unavailable, largely due to the difficulty of isolating pure high-molecular-weight DNA. This article proposes a scheme for gaining a contiguous L. usitatissimum assembly using Nanopore data. We developed a protocol for flax nuclei isolation with subsequent DNA extraction, which allows obtaining about 5 μg of pure high-molecular-weight DNA from 0.5 g of leaves. Such an amount of material can be collected even from a single plant and yields more than 30 Gb of ONT data in two MinION runs. We performed a comparative analysis of different genome assemblers and polishers on the gained data and obtained the final 447.1-Mb assembly of L. usitatissimum line 3896 genome using the Canu—Racon (two iterations)—Medaka combination. The genome comprised 1695 contigs and had an N50 of 6.2 Mb and a completeness of 93.8% of BUSCOs from eudicots_odb10. Our study highlights the impact of the chosen genome construction strategy on the resulting assembly parameters and its eligibility for future genomic studies

Development and Complex Application of Methods for the Identification of Mutations in the <i>FAD3A</i> and <i>FAD3B</i> Genes Resulting in the Reduced Content of Linolenic Acid in Flax Oil

Flax is grown worldwide for seed and fiber production. Linseed varieties differ in their oil composition and are used in pharmaceutical, food, feed, and industrial production. The field of application primarily depends on the content of linolenic (LIN) and linoleic (LIO) fatty acids. Inactivating mutations in the FAD3A and FAD3B genes lead to a decrease in the LIN content and an increase in the LIO content. For the identification of the three most common low-LIN mutations in flax varieties (G-to-A in exon 1 of FAD3A substituting tryptophan with a stop codon, C-to-T in exon 5 of FAD3A leading to arginine to a stop codon substitution, and C-to-T in exon 2 of FAD3B resulting in histidine to tyrosine substitution), three approaches were proposed: (1) targeted deep sequencing, (2) high resolution melting (HRM) analysis, (3) cleaved amplified polymorphic sequences (CAPS) markers. They were tested on more than a thousand flax samples of various types and showed promising results. The proposed approaches can be used in marker-assisted selection to choose parent pairs for crosses, separate heterogeneous varieties into biotypes, and select genotypes with desired homozygous alleles of the FAD3A and FAD3B genes at the early stages of breeding for the effective development of varieties with a particular LIN and LIO content, as well as in basic studies of the molecular mechanisms of fatty acid synthesis in flax seeds to select genotypes adequate to the tasks