Immature Erythrocytes Research Articles

Hi-C analysis of genomic contacts revealed karyotype abnormalities in chicken HD3 cell line

BackgroundKaryotype abnormalities are frequent in immortalized continuous cell lines either transformed or derived from primary tumors. Chromosomal rearrangements can cause dramatic changes in gene expression and affect cellular phenotype and behavior during in vitro culture. Structural variations of chromosomes in many continuous mammalian cell lines are well documented, but chromosome aberrations in cell lines from other vertebrate models often remain understudied. The chicken LSCC-HD3 cell line (HD3), generated from erythroid precursors, was used as an avian model for erythroid differentiation and lineage-specific gene expression. However, karyotype abnormalities in the HD3 cell line were not assessed. In the present study, we applied high-throughput chromosome conformation capture to analyze 3D genome organization and to detect chromosome rearrangements in the HD3 cell line.ResultsWe obtained Hi-C maps of genomic interactions for the HD3 cell line and compared A/B compartments and topologically associating domains between HD3 and several other cell types. By analysis of contact patterns in the Hi-C maps of HD3 cells, we identified more than 25 interchromosomal translocations of regions ≥ 200 kb on both micro- and macrochromosomes. We classified most of the observed translocations as unbalanced, leading to the formation of heteromorphic chromosomes. In many cases of microchromosome rearrangements, an entire microchromosome together with other macro- and microchromosomes participated in the emergence of a derivative chromosome, resembling “chromosomal fusions'' between acrocentric microchromosomes. Intrachromosomal inversions, deletions and duplications were also detected in HD3 cells. Several of the identified simple and complex chromosomal rearrangements, such as between GGA2 and GGA1qter; GGA5, GGA4p and GGA7p; GGA4q, GGA6 and GGA19; and duplication of the sex chromosome GGAW, were confirmed by FISH.ConclusionsIn the erythroid progenitor HD3 cell line, in contrast to mature and immature erythrocytes, the genome is organized into distinct topologically associating domains. The HD3 cell line has a severely rearranged karyotype with most of the chromosomes engaged in translocations and can be used in studies of genome structure–function relationships. Hi-C proved to be a reliable tool for simultaneous assessment of the spatial genome organization and chromosomal aberrations in karyotypes of birds with a large number of microchromosomes.

Safety Evaluation and Anti-Inflammatory Efficacy of Lacticaseibacillus paracasei PS23

Lacticaseibacillus paracasei strain PS23 (PS23) exhibits some probiotic properties. In this study, a genomic analysis of PS23 revealed no genes related to virulence or antibiotic resistance. Moreover, ornithine decarboxylase activity was not detected in vitro. In addition, PS23 was sensitive to the tested antibiotics. Genotoxicity tests for PS23 including the Ames test and chromosomal aberrations in vitro using Chinese hamster ovary cells and micronuclei in immature erythrocytes of ICR mice were all negative. Moreover, following a 28-day study involving repeated oral dose toxicity tests (40, 400, and 4000 mg/kg equal 1.28 × 1010, 1.28 × 1011, and 1.28 × 1012 CFU/kg body weight, respectively) using an ICR mouse model, no adverse effects were observed from any doses. In addition, supplementation with live or heat-killed PS23 ameliorates DSS-induced colonic inflammation in mice. Our findings suggest that PS23 is safe and has anti-inflammatory effects and may therefore have therapeutic implications.

Assessment of Micro-R/Macro-R values and other hemogram parameters for the diagnosis of early neonatal sepsis

Aims: Neonatal sepsis is a systemic condition that results in hemodynamic and clinical findings. We aimed to determine the role of hemogram parameters in the diagnosis of early neonatal sepsis.
 Methodology: This retrospective study was conducted with 126 neonates hospitalized in Kastamonu Training and Research Hospital Neonatal Intensive Care Unit between March 2018 to March 2022. There were 65 term neonates diagnosed with early neonatal sepsis in the patient group. In the control group, there were 61 term neonates who underwent hemogram within the first 72 hours (postnatal period) due to physiological jaundice and did not receive any antibiotic treatment. The assessment was made by comparing the hemogram parameters of these groups.
 Results: The Macro-R value, white blood cell count, absolute neutrophil count, monocyte count, immature erythrocyte count and percentage, immature granulocyte count and percentage and neutrophil-lymphocyte ratio were found to be significantly higher in the patient group than in the control group (p<0.05). Macro R, white blood cell count, absolute neutrophil count, immature erythrocyte count and percentage, immature granulocyte count and neutrophil/lymphocyte ratio had high sensitivity and specificity for the diagnosis of early neonatal sepsis.
 Conclusion: We think that these simple and easily accessible parameters have potential value in the diagnosis of ENS if they are used together with the clinical symptoms of ENS.

Loss of DHX15 Function Impairs Hematopoiesis in Vav-Dhx15-KO Mice and Cause DNA Damage-Induced Cell Cycle Arrest and Apoptosis in Leukemia Cell Lines

DHX15, a member of the DEAD-box RNA helicase family, acts as splicing factor in pre-messenger RNA splicing, pre-ribosomal RNA, and RNA metabolism. Our previous studies showed that DHX15 overexpression is associated with poor prognosis in acute myeloid leukemia (AML), and DHX15 regulates cell apoptosis via the NF-kB signaling pathway(Pan et al., 2017). However, its molecular function remains obscure in the blood system. First, with the Cre-loxp knockout system, Dhx15fl/fl male mice were crossed with vav-icre female mice (specifically expressed in blood system), we found that Dhx15fl/fl; vav-icre homozygous knockout mice (vav-Dhx15-KO) are lethal by E17.5-E18.5. Then, to explore the inner difference in blood system, the hematopoietic stem cells (HSC) were analyzed by flow cytometry in fetal liver (FL) (E13.5, E15.5). Compared with Dhx15fl/fl (wild-type, WT) mice, the lineage-negative cells (Lin- cells; Lin+ Cocktail, CD5, CD45R (B220), CD11b, Anti-Gr-1 (Ly-6G/C), 7-4 and Ter-119) significantly increased in vav-Dhx15-KO FL cells (P<0.0001) and the short-term HSC(ST-HSC) proportion compensatively increased in LSK (Lin-c-Kit+Sca1+cells) population (P<0.01), while the proportion of LK(Lin-c-Kit+Sca1-cells) (P<0.0001 in E13.5, P<0.001 in E15.5), LSK(Lin-c-Kit+Sca1+cells)(P<0.1 in E15.5), and multipotent progenitor (MPP) (P<0.0001 in E15.5) all witnessed decrease in the Lin-/LSK population respectively. Meanwhile, vav-Dhx15-KO mice exhibited impaired multi-lineage hematopoiesis especially in erythroid, where the precursors and immature erythrocytes increased, mature erythrocytes decreased (P<0.001 in E13.5). Next, in vivo competitive repopulation assay were performed to further examine any potential cell-intrinsic defect of HSC function caused by Dhx15-inactivation. Thus, 1,000,000 E13.5/E15.5 total FL donor cells (CD45.2, vav-Dhx15-KO or WT) together with 1,000,000 CD45.1 unfractionated BM competitor cells were intra-venous transplanted into lethally irradiated (9Gy) B6.SJL (CD45.1) recipient mice. Recipient mice were sacrificed 8-weeks after transplantation, flow cytometry was conducted to analysis the ratio of CD45.2 in mice, the results showed that vav-Dhx15-KO FL was severely deficient compared to WT FL(0.1±0.04%vs 62.2±2.8% in BM, 0.2±0.05% vs 64.5±6.1% in SP, 0.1±0.05% vs 45.1±5.5% in PB). These results indicate that Dhx15 is crucial in mice blood system, loss of its function may block cell differentiation, leading to the reduction of mature cells. At the same time, to further explore the molecular mechanism of DHX15 in blood system, MOLM-13 and NB4 leukemia cell lines were selected for shRNA-KD (knockdown) experiments due to the lethality of vav-Dhx15-KO mice. We found that Dhx15-KD significantly impaired the proliferation ability of these cell lines in vitro and in vivo, by experiments of survival competition, clone formation, subcutaneous and intra-venous tumorigenesis. Besides, our data demonstrate DHX15-KD-mediated G0/G1 cell cycle arrest and increase in early-late apoptotic cells by Hoechest 33342-EdU cell cycle and DAPI-Annexin V cell apoptosis assays. Mechanistically, we found that UV_RESPONSE signaling pathway which is mainly related to DNA damage was up-regulated by RNAseq and bioinformatics analysis (DHX15-KD cell line vs WT cell line). Correspondingly, the DNA damage indicators γH2AX and 53BP1 were also increased in DHX15-KD cell lines detected by WB and immunofluorescence experiments, suggesting that the deletion of DHX15 could lead to DNA damage-mediated cell cycle arrest and apoptosis in leukemia cell lines. In summary, these results show that DHX15 deficiency leads to DNA damage-induced cell cycle arrest and apoptosis which further impairs the proliferative capacity of leukemia cell lines and lost function of Dhx15 impairs multi-lineage hematopoiesis in mouse embryos, which may lead to its perinatal death.

The assessment of cytokine-dependent hematopoietic cell linker and interleukin-3 levels in children with beta-thalassemia major

Beta-thalassemia is caused by a lack of or failure to synthesize beta globin chains in hemoglobin resulting in an excess of alpha chains. Cytokine-dependent hematopoietic cell linker (CLNK) is an adapter protein which is involved in the regulation of immunoreceptor signaling. It was found to be associated with a tyrosine-phosphorylated polypeptide (p92) in response to immunoreceptor stimulation. In thalassemia, oxidative stress causes tyrosine phosphorylation of the cytoplasmic domain of band 3. Therefore, we aimed to see how serum CLNK and interleukin-3 correlated with serum ferritin and annual transfusion index in children with beta-thalassemia major (b-TM). This case-control study included 100 non-splenectomized, transfusion-dependent b-TM pediatric patients receiving oral deferasirox and 100 healthy controls. The study was approved by the Institutional Review Board (IRB) of the Menoufia Faculty of Medicine, the approval number is 19/4/2021.PEDI. All procedures were carried out in accordance with relevant guidelines and regulations. In both groups, serum ferritin, interleukin-3, hemoglobin and CLNK levels were measured. They were found to be significantly higher in the b-TM patients than in the controls (p 0.001). There was a negative correlation between serum CLNK and hemoglobin (r = –0.483, p &lt; 0.001), and a positive correlation between serum CLNK and ferritin levels (r = 0.855, p &lt; 0.001). There was a positive correlation between serum CLNK, ferritin, and annual transfusion index. Increased serum CLNK in transfusion-dependent b-TM patients was associated with elevated serum ferritin concentrations and high annual transfusion index. This could be explained by reciprocal effects between immune signaling system and immature erythrocytes which release signaling molecules, such as CLNK, in the blood.

The Phosphodiesterase Inhibitor Tadalafil Promotes Splenic Retention of Plasmodium falciparum Gametocytes in Humanized Mice.

The persistence of erythrocytes infected with Plasmodium falciparum gametocytes in the bloodstream is closely related to the modulation of their mechanical properties. New drugs that increase the stiffness of infected erythrocytes may thus represent a novel approach to block malaria parasite transmission. The phosphodiesterase inhibitor tadalafil has been shown to impair the ability of infected erythrocytes to circulate in an in vitro model for splenic retention. Here, we used a humanized mouse model to address in vivo the effect of tadalafil on the circulation kinetics of mature gametocyte-infected erythrocytes. We show that stiff immature gametocyte-infected erythrocytes are retained in the spleen of humanized mice at rates comparable to that of the in vitro model. Accordingly, tadalafil-induced stiffening of mature gametocyte-infected erythrocytes impairs their circulation in the bloodstream and triggers their retention by the spleen. These in vivo results validate that tadalafil is a novel drug lead potentially capable of blocking malaria parasite transmission by targeting GIE mechanical properties.

Genotoxicity and 28-day repeated dose oral toxicity study of garlic essential oil in mice

Background and aimGarlic essential oil (GEO) isolated from Garlic (Allium sativum L.) exerts biological activities in disease prevention, particularly in metabolic and liver diseases, and is used for a dietary therapy for centuries. However, due to the side effects associated with the excessive consumption of GEO, there is a need to evaluate the safety of the GEO. Experimental procedureAmes test using five Salmonella typhimurium strains (TA98, TA100, TA102, TA1535, and TA1537) and Chinese hamster ovary (CHO–K1) cells with or without metabolic activation (S9 system), and mammalian erythrocyte micronucleus test were used to assess the genotoxicity and clastogenic effects of GEO. A repeated dose of GEO (15, 25, and 50 mg/kg body weight, p.o.) were administrated to ICR mice for 28 days to ascertain the subacute toxicity of GEO. Results and conclusionsThe results of the Ames test with or without S9 system indicated that GEO did not induce mutagenicity nor have clastogenic effects in CHO–K1 cells with or without S9 activation. Furthermore, GEO did not affect the ratio of immature to total erythrocytes or the number of micronuclei in immature erythrocytes of ICR mice after 24 and 48 h. In a 28-day oral toxicity assessment, GEO (15, 25, and 50 mg/kg body weight, p.o.)-fed ICR mice exhibited normal behaviors, mortality, body weight, daily intake, hematology, clinical biochemistry, and organ weight. GEO shows no genotoxicity, and the no-observed-adverse-effect level (NOAEL) for GEO is considered to be greater than 50 mg/kg bw/day orally for 28 days in mice.

Mutagenicity and safety pharmacology of a standardized antidiabetic polyherbal formulation

Synacinn is a standardized polyherbal extract formulated for the treatment of diabetes mellitus and its complications. This study aims to assess the mutagenicity potential of Synacinn by Ames assay and in vivo bone marrow micronucleus (MN) test on Sprague Dawley rat. Human ether-a-go-go-related gene (hERG) assay and Functional Observation Battery (FOB) were done for the safety pharmacology tests. In the Ames assay, Dose Range Finding (DRF) study and mutagenicity assays (+/− S9) were carried out. For the MN test, a preliminary and definitive study were conducted. In-life observations and number of immature and mature erythrocytes in the bone marrow cells were recorded. The hERG assay was conducted to determine the inhibitory effect on hERG potassium channel current expressed in human embryonic kidney cells (HEK293). FOB tests were performed orally (250, 750, and 2000 mg/kg) on Sprague Dawley rats. Synacinn is non-mutagenic against all tested strains of Salmonella typhimurium and did not induce any clastogenicity in the rat bone marrow. Synacinn also did not produce any significant inhibition (p ≤ 0.05) on hERG potassium current. Synacinn did not cause any neurobehavioural changes in rats up to 2000 mg/kg. Thus, no mutagenicity, cardiotoxicity and neurotoxicity effects of Synacinn were observed in this study.

To Evaluate Diagnostic Efficacy of Nucleated Red Blood Cells (NRBCs) in Neonatal Sepsis

BACKGROUND &#x0D; Early diagnosis of sepsis in neonates is cardinal. The isolation of the organism from the body fluids is time-consuming and does not reliably exclude the infection even if it is negative. Nucleated red blood cells (NRBCs) are immature erythrocytes that are elevated in association with foetal distress, hypoxia, and inflammation. The purpose of the study was to evaluate the diagnostic efficacy of nucleated red blood cells (NRBCs) in the detection of neonatal sepsis.&#x0D; METHODS &#x0D; This was a prospective study which was conducted in the section of haematology, Department of Pathology of Shri Guru Ram Rai Institute of Health and Medical Sciences, Dehradun, Uttarakhand. A total of 92 term neonates admitted to NICU with clinical features and risk factors of sepsis from October 2020 to March 2021 were included in the study. All study neonates were classified into proven sepsis, probable clinical sepsis and no sepsis groups. The NRBC count and haematological parameters were obtained by a fully automated analyzer SYSMEX XN1000 and corroborated with a peripheral blood smear. The data obtained were analyzed using one-way ANOVA with Fisher’s LSD multiple comparison tests or t-test using GraphPad Prism 7 software. A P-value of 0.001 was taken to be statistically significant.&#x0D; RESULTS&#x0D; Among the 92 cases studied, 23 were culture-positive sepsis, 31 were of probable clinical sepsis or were culture-negative and the rest 38 cases had no sepsis. In the present study, the difference in NRBC count between the sepsis and no sepsis group was statistically significant (P-value &lt; 0.001). The sensitivity, specificity, positive predictive value and negative predictive value of NRBC count for sepsis were found to be 77. 78 %, 60.52 %, 73.68 % and 65.71 % respectively. The average NRBC count in the mortality group was higher than in live neonates.&#x0D; CONCLUSIONS &#x0D; The NRBC count showed comparable or higher sensitivity than haematological parameters. Higher NRBC counts in the mortality group correlated with adverse neonatal outcomes, hence carrying a prognostic value.

Weaning differentially affects the maturation of piglet peripheral blood and jejunal Peyer\u2019s patches

The study aimed to assess how the post-weaning condition changes piglet peripheral blood (PB) and jejunal Peyer’s patches (JPPs) as compared to the suckling period, and how these changes are associated with intestinal microbiota evolution. Sixteen pigs were slaughtered and sampled for PB, JPPs and jejunal content (JC) at weaning (26 days) or at 12 days fed on a pre-starter diet. The PB and JPP transcriptomes were analysed using mRNA-seq. The Gene Set Enrichment Analysis was used to demonstrate enriched gene clusters, depending on sampling time. Jejunal microbiota was profiled using 16S rRNA gene sequencing. Post-weaning JPPs were enriched for processes related to the activation of IFN-γ and major histocompatibility complex (MHC) class I antigen processing which clustered with the reduced abundance of the Weisella genus and Faecalibacterium prausnitzii in JC. The post-weaning microbiome differed from that seen in just-weaned pigs. For just-weaned PB, the enrichment of genes related to hemoglobin and the iron metabolism indicated the greater presence of reticulocytes and immature erythrocytes. The JPP genes involved in the I MHC and IFN-γ activations were markers of the post-weaning phase. Several genes attributable to reticulocyte and erythrocyte maturation could be interesting for testing the iron nutrition of piglets.

NONPIGMENTED VERSUS PIGMENTED: HEALTH VARIABLES AND GENETICS OF ALBINO FLORIDA GREEN TURTLE (CHELONIA MYDAS) HATCHLINGS COMPARED WITH NORMALLY PIGMENTED HATCHLINGS FROM THE SAME CLUTCH.

At the time of hatchling emergence from a nest laid on Juno Beach, Florida, US, by a normally pigmented green turtle (Chelonia mydas), 23 albino hatchlings and 75 normally pigmented hatchlings were observed. This condition is rarely seen in sea turtles, and little is known about blood analytes and genetics of albino wildlife to date. Therefore, the objective of our study was to assess and compare morphometric measurements (mass, minimum straight carapace length, body condition index), carapacial scute anomalies, a suite of hematologic and plasma biochemical analytes, and two glucose analysis methodologies (glucometer and dry chemistry analysis) in albino (n=20) versus normally pigmented (n=24) hatchlings from this nest. Genetic analyses were completed to identify paternal contributions of hatchlings and to test Mendelian inheritance assumptions. Although morphometric measurements, scute anomalies, and leukocyte morphology were similar between albino and normally pigmented hatchlings, several differences were observed in blood analyte data: immature erythrocytes, packed cell volume, heterophil:lymphocyte ratio, and glucose concentrations (by both methodologies) were significantly higher, whereas absolute immature heterophils, absolute lymphocytes, number of erythrocyte micronuclei, sodium, and chloride were significantly lower in albino hatchlings compared with normally pigmented hatchlings. Considerations for these differences include a stress response from sampling (e.g., timing of procedures or possibly from photosensitivity or reduced visual acuity in albinos) and different osmoregulation, which may reflect physiologic variations or stress. There was a small positive bias (0.10 mmol/L) with glucose by glucometer, similar to reports in other sea turtle species and confirming its suitability for use in hatchlings. All albino hatchlings analyzed (n=10) were from the same father, but the normally pigmented hatchlings (n=24) were from two other fathers. These findings provide insight into the physiology and genetics of albinism in sea turtles.

Immature Erythroid Precursors and/or Erythrocyte Dysplasia in Peripheral Blood.

With the progress of technology in automated hematology analyzers, in the vast majority of cases, nucleated red corpuscles (NRC) can be automatically identified by most types of automated hematology analyzers, thus correcting the leukocyte count and avoiding pseudoleukocytosis by the analyzers themselves. The objective of the study was to explore pseudoleukocytosis due to immature erythroid precursors and/or erythrocyte dysplasia in the peripheral blood resulting from different rare situations. Four rare cases showing pseudoleukocytosis due to immature erythroid precursors and/or erythrocyte dysplasia in the peripheral blood were analyzed and the effects on complete blood count (CBC) performed on a Sysmex XN-2000 analyzer and microscopic morphological features of the peripheral blood were investigated. These cases were selected for their vital value in describing all pseudoleukocytosis due to immature erythroid pre-cursors and/or erythrocyte dysplasia in the peripheral blood. The causes of immature erythroid precursors and/or erythrocyte dysplasia in the peripheral blood were analyzed. In all these cases, proportions of NRC and leukocyte counts were affected to varying degrees by the presence of numerous immature erythroid precursors and/or obvious erythrocyte dysplasia in the peripheral blood. All cases associated with an alarm concerning NRC presentation, and abnormal scattergrams of WDF and WNR, thus leading to pseudoleukocytosis. Laboratory artifacts led by NRC may be an indicator towards the occurrence of numerous immature erythroid precursors and/or obvious erythrocyte dysplasia in the peripheral blood, and active extramedullary hematopoiesis, breakdown of the bone marrow barrier, and the stress response of acute bleeding and severe multiple infection.

HDAC1 is required for GATA-1 transcription activity, global chromatin occupancy and hematopoiesis.

The activity of hematopoietic factor GATA-1 is modulated through p300/CBP-mediated acetylation and FOG-1 mediated indirect interaction with HDAC1/2 containing NuRD complex. Although GATA-1 acetylation is implicated in GATA-1 activation, the role of deacetylation is not studied. Here, we found that the FOG-1/NuRD does not deacetylate GATA-1. However, HDAC1/2 can directly bind and deacetylate GATA-1. Two arginine residues within the GATA-1 linker region mediates direct interaction with HDAC1. The arginine to alanine mutation (2RA) blocks GATA-1 deacetylation and fails to induce erythroid differentiation. Gene expression profiling and ChIP-seq analysis further demonstrate the importance of GATA-1 deacetylation for gene activation and chromatin recruitment. GATA-12RA knock-in (KI) mice suffer mild anemia and thrombocytopenia with accumulation of immature erythrocytes and megakaryocytes in bone marrow and spleen. Single cell RNA-seq analysis of Lin− cKit+ (LK) cells further reveal a profound change in cell subpopulations and signature gene expression patterns in HSC, myeloid progenitors, and erythroid/megakaryocyte clusters in KI mice. Thus, GATA-1 deacetylation and its interaction with HDAC1 modulates GATA-1 chromatin binding and transcriptional activity that control erythroid/megakaryocyte commitment and differentiation.

Cell composition of blood for Salvelinus malma (Salmonidae) from the Raduga River (Kamchatka)

Hematopoiesis is a mechanism of multifunctional adaptation in vertebrates, including fish. Adaptive capabilities of populations are studied for a species of genus Salvelinus with high ecological plasticity. Char Salvelinus malma is represented in Kamchatka by the anadromous, river, brook, and several lake forms. Peripheral blood cells are investigated for this species juveniles and adults from the Raduga River in the lower Kamchatka River basin. The fish sampled in August 2013 were divided into two groups: sexually mature individuals (10 specimens) and juveniles (10 specimens). The body length (AC) of mature chars (mean ± standard deviation, range in brackets) was 338.90 ± 12.64 (304.0–380.0) mm, the body weight — 437.40 ± 57.74 (235.0–735.0) g, their sex ratio was close to 1 : 1. The length of juvenile chars was 150.50 ± 9.14 (104.0–195.0) mm, the weight — 33.90 ± 6.19 (10.1–68.8) g. Blood was sampled from the caudal vessel of alive fish immediately after catching, the blood smears were dried and fixed with May-Grunwald dye and then stained with azure-eosin. Composition of leukocytes and erythrocytes was determined on the stained blood smears. The blood cells were classified according to Ivanova (1983) and Zhiteneva et al. (1989). Statistical significance of the blood cells composition differences between fish groups was assessed using the Mann-Whitney U-test, after checking for normal distribution of characteristics (Kolmogorov-Smirnov test). The erythroid row of S. malma was represented by proliferating (erythroblasts, basophilic and polychromatophilic normoblasts), maturing (oxyphilic normoblasts) and mature erythrocytes, the leukocyte row — by blast cells, agranulocytes and granulocytes. The juveniles were characterized by intense proliferative processes (great number of immature erythrocytes, large and medium lymphocytes, presence of blast cells) and appearance of poikilocytes; the sexually mature chars were distinguished by greater number of granulocytes, mainly due to stab and segmented neutrophils (p &lt; 0.01). Parasitological analysis showed different infestation of the juvenile and adult chars by pathogenic helminths (Anisakis simplex, Eubothrium salvelini, Neoechinorhynchus salmonis) that influenced on their blood. Broad adaptive capabilities of S. malma hematopoiesis provided by wide morphological and functional diversity of blood cells are concluded that maintains homeostasis of the species. Blood indices for char from the Raduga River can be used to form reference intervals for monitoring physiological state of this species.

Identification of the causative gene of a transparent phenotype of juvenile red sea bream Pagrus major.

Deformities in cultured fish species may be genetic, and identifying causative genes is essential to expand production and maintain farmed animal welfare. We previously reported a genetic deformity in juvenile red sea bream, designated a transparent phenotype. To identify its causative gene, we conducted genome-wide linkage analysis and identified two single nucleotide polymorphisms (SNP) located on LG23 directly linked to the transparent phenotype. The scaffold on which the two SNPs were located contained two candidate genes, duox and duoxa, which are related to thyroid hormone synthesis. Four missense mutations were found in duox and one in duoxa, with that in duoxa showing perfect association with the transparent phenotype. The mutation of duoxa was suggested to affect the transmembrane structure and thyroid-related traits, including an enlarged thyroid gland and immature erythrocytes, and lower thyroxine (T4) concentrations were observed in the transparent phenotype. The transparent phenotype was rescued by T4 immersion. Loss-of-function of duoxa by CRISPR-Cas9 induced the transparent phenotype in zebrafish. Evidence suggests that the transparent phenotype of juvenile red sea bream is caused by the missense mutation of duoxa and that this mutation disrupts thyroid hormone synthesis. The newly identified missense mutation will contribute to effective selective breeding of red sea bream to purge the causative gene of the undesirable phenotype and improve seed production of red sea bream as well as provide basic information of the mechanisms of thyroid hormones and its related diseases in fish and humans.

Do blood parasites increase immature erythrocytes and mitosis in amphibians?

Introduction: In amphibians, blood may act as a hematopoietic tissue. However, the knowledge concerning hematological features is scarce, there is not much information that allows an analysis about the possible explanations of this physiological feature. Objective: This study aimed to evaluate the relationship between immature red blood cells (RBCs) mitosis and the presence of blood parasites in amphibians. Methods: We sampled 116 amphibians (31 species) in six Colombian localities. Blood was taken by cardiac puncture or maxillary vein puncture. Smears were prepared, fixed, and Giemsa stained for microscopical analysis. The variables analyzed were the percentage of immature RBCs, mitotic cells in peripheral blood, and blood parasite infection. Data were analyzed using Wilcoxon's rank test and exact Fisher statistical tests. Results: Sixty-two individuals showed mitosis in peripheral blood, and these mitotic RBCs shared morphological features with immature RBCs. Overall, parasite prevalence was 30.1 %, distributed as follows: Trypanosoma (24.1 %), Hepatozoon-like (6 %), Dactylosoma (4.3 %), Karyolysus-like (0.9 %), and Filarioidea (2.6 %). A positive association between the percentage of immature RBCs and the presence of mitotic RBCs was found, and also between the blood parasite infection and the percentage of immature RBCs. Conclusions: In this study, we found that the presence of blood parasites, immature RBCs, and RBCs mitosis are frequent events in amphibians' peripheral blood, and our analysis suggests an association between those features. Thus, the release of immature RBCs and the mitosis of those cells in peripheral blood may be a physiological response to blood parasite infection. Further studies characterizing hematology in amphibians and wildlife, in general, are desirable.

Cytokine dependent hematopoietic cell linker (CLNK) is highly elevated in blood transfusion dependent beta-thalassemia major patients.

Transfusion-dependent β-thalassemia (TDT) is a severe form of thalassemia caused by mutations in the β-globin gene, resulting in partial or complete deficiency of β-globin chains. This deficiency results in oxidative stress, dyserythropoiesis, and chronic anemia. Cytokine-dependent hematopoietic cell linker (CLNK) belongs to adaptor proteins that have the capacity to interact with multiple signalling proteins and function in the organisation of the molecular components required for signal transduction. This is the first study which measured serum CLNK in TDT patients and examines the correlation between CLNK and iron overload biomarkers. Sixty children with TDT and 30 normal children (aged 3-12 years old) participated in the present study. The patients were on blood transfusion as a part of their treatment regimen. Serum C-reactive protein was negative in all samples. The results showed significantly higher (P<0.001) serum CLNK levels in TDT patients as compared with controls. The TDT diagnosis explained 19.4% of the variance in CLNK levels. The increased levels of CLNK were significantly associated with indicants of iron overload, namely increased ferritin levels. Increased CLNK levels in TDT may be explained by reciprocal effects between immune signalling and immature erythrocytes, which release soluble receptors and signalling molecules, including CLNK, in the blood.

Quantitative and qualitative changes in blood cells associated with COVID-19

Aim. To establish the relationship of hematological disorders with the pathogenesis, course and outcomes of COVID-19.&#x0D; Methods. We examined 235 hospitalized patients with moderate and severe forms of acute COVID-19 receiving anticoagulants and immunosuppressive drugs. We studied the full blood cell counts and morphology along with the platelet function by flow cytometry in comparison with the clinical features and synthesis of inflammatory markers. To assess platelet contractility, blood clot contraction (retraction) kinetics was used in combination with scanning electron microscopy of platelets and blood clots.&#x0D; Results. Hemolytic anemia, neutrophilia and lymphopenia were associated with immature erythrocytes and leukocytes, indicating activation of hematopoiesis. Contraction of blood clots in COVID-19 was impaired, especially in severe and lethal cases, as well as in the presence of comorbidities, including myeloproliferative and coronary heart diseases and acute cerebrovascular disease. In male patients, the changes in clot contraction were more pronounced. Suppression of clot contraction correlated directly with anemia and coagulopathy, including a high D-dimer level, which confirms the pathogenetic significance of blood clot contraction in COVID-19. A decrease in platelet contractility was due to moderate thrombocytopenia in combination with chronic platelet activation and secondary platelet dysfunction. The structure and cellular composition of blood clots depended on the extent of contraction; clots with impaired contraction were porous, had a low content of deformed polyhedral erythrocytes (polyhedrocytes) and an even distribution of fibrin.&#x0D; Conclusion. Blood cells undergoing both quantitative and qualitative changes are involved in the pathogenesis of COVID-19; the suppressed platelet-driven contraction of intravital blood clots may be a part of the prothrombotic mechanisms.

High expression of BCL6 inhibits the differentiation and development of hematopoietic stem cells and affects the growth and development of chickens

BackgroundB-cell CLL/lymphoma 6 (BCL6) is a transcriptional master regulator that represses more than 1200 potential target genes. Our previous study showed that a decline in blood production in runting and stunting syndrome (RSS) affected sex-linked dwarf (SLD) chickens compared to SLD chickens. However, the association between BCL6 gene and hematopoietic function remains unknown in chickens.MethodsIn this study, we used RSS affected SLD (RSS-SLD) chickens, SLD chickens and normal chickens as research object and overexpression of BCL6 in hematopoietic stem cells (HSCs), to investigate the effect of the BCL6 on differentiation and development of HSCs.ResultsThe results showed that comparison of RSS-SLD chickens with SLD chickens, the BCL6 was highly expressed in RSS-SLD chickens bone marrow. The bone marrow of RSS-SLD chickens was exhausted and red bone marrow was largely replaced by yellow bone marrow, bone density was reduced, and the levels of immature erythrocytes in peripheral blood were increased. At the same time, the hematopoietic function of HSCs decreased in RSS-SLD chickens, which was manifested by a decrease in the hematopoietic growth factors (HGFs) EPO, SCF, TPO, and IL-3, as well as hemoglobin α1 and hemoglobin β expression. Moreover, mitochondrial function in the HSCs of RSS-SLD chickens was damaged, including an increase in ROS production, decrease in ATP concentration, and decrease in mitochondrial membrane potential (ΔΨm). The same results were also observed in SLD chickens compared with normal chickens; however, the symptoms were more serious in RSS-SLD chickens. Additionally, after overexpression of the BCL6 in primary HSCs, the secretion of HGFs (EPO, SCF, TPO and IL-3) was inhibited and the expression of hemoglobin α1 and hemoglobin β was decreased. However, cell proliferation was accelerated, apoptosis was inhibited, and the HSCs entered a cancerous state. The function of mitochondria was also abnormal, ROS production was decreased, and ATP concentration and ΔΨm were increased, which was related to the inhibition of apoptosis of stem cells.ConclusionsTaken together, we conclude that the high expression of BCL6 inhibits the differentiation and development of HSCs by affecting mitochondrial function, resulting in impaired growth and development of chickens. Moreover, the abnormal expression of BCL6 might be a cause of the clinical manifestations of chicken comb, pale skin, stunted growth and development, and the tendency to appear RSS in SLD chickens.

Oral deoxynivalenol toxicity in Harlan Sprague Dawley (Hsd:Sprague Dawley® SD®) rat dams and their offspring

There is widespread human exposure to deoxynivalenol (DON), a fungal mycotoxin found globally in many grain-based foods and animal feed. Acute exposures to high levels of DON are associated with gastrointestinal effects and emesis in humans and some animals, but the effects of low-dose exposures throughout the lifetime, a more likely exposure scenario in humans, are understudied. Therefore, this study was designed to identify doses of DON that could be used to evaluate long-term toxicity following perinatal exposure. Time-mated Harlan Sprague Dawley (Hsd:Sprague Dawley® SD®) rats were administered 0, 0.03, 0.1, 0.3, 1, or 3 mg/kg/day of DON once daily via gavage starting on gestational day 6 through postnatal day (PND) 27. F1 animals were administered the same dose as their respective dams via gavage starting on PND 12 until PND 27. Animals were euthanized on PND 28. DON had no effect on maternal body weight or feed consumption at any dose. Findings were limited to the 3 mg/kg/day group: F0 females had smaller live litter sizes than controls and F1 pups had lower body weight (4–13%) compared to controls. By PND 28, F1 body weight, after adjustments for litter effects, was 10–13% lower than controls. Blood samples obtained on PND 28 showed no increases in frequencies of micronucleated immature erythrocytes in either F0 or F1 animals. In summary, doses of DON up to 3 mg/kg/day did not affect maternal survival or body weight. Doses of 3 mg/kg/day resulted in slight toxicity manifested as decreased body weight in the offspring. The no-observed effect level was 1 mg/kg/day.