MSTN) was knocked out in rabbits at two novel sites in exon3, and the function of these mutations was determined in subsequent generations. The prominent double muscle phenotype with hyperplasia or hypertrophy of muscle fiber was observed in the MSTN-KO rabbits, and a similar phenotype was confirmed in the F1 generation. Moreover, the average weight of 80-day-old MSTN-KO rabbits  was higher than that of wild-type rabbits , and also the bodyweight of MSTN-KO rabbits  was significantly higher (P < 0.001) at the age of 180 days than wild-type (WT) rabbits . In MSTN-KO rabbits, fourteen rabbit pups from the F1 generation and thirteen from the F2 generation stably inherited the induced MSTN gene mutations. Totally, 194 pups were produced in the F1 generation of which 49 were MSTN-KO rabbits, while 47 pups were produced in the F2 generation of which 20 were edited rabbits, and the ratio of edited to wild-type rabbits in the F2 generation was approximately 1:1. Thus, we successfully generated a heritable double muscle buttocks rabbits via myostatin mutation with CRISPR/Cas9 system, which could be valuable in rabbit's meat production and also a useful animal model to study the development of muscles among livestock species and improve their important economic traits as well as the human muscle development-related diseases.Rabbits have been domesticated for meat, wool, and fur production, and have also been cherished as a companion, artistic inspiration, and an experimental model to study many human diseases. In the present study, the muscle mass negative regulator gene myostatin ( Leporidae order Lagomorpha and domesticated about 1,400 years ago  is a member of the transforming growth factor (TGF-β) superfamily which is considered to be a negative regulator through inhibiting muscle development and regeneration  and a significant increase in muscle mass  and named as g1, g2, g3, g4, g5, g6, g7, and g8  for sequencing to analyze the editing effect of the gene fragment. Optimal sgRNA was selected for the following experiment.Primary rabbit fibroblasts were cultured in 10% (v/v) FBS, 35 mm glutamine, and 1 × DMEM (Gibco). The plasmid was transfected into fibroblasts through electroporation (Gene Pulser Xcell & BIO-RAD) at a pulse of 225 volts for 10 ms. Briefly, 4 μg of pCMV-T7-NLS-hSpCas9-NLS (hSpCas9) DNA plasmid and 2 μg of gRNA plasmid were used to transfect 5 × 10n = 3) in estrus were mated, the female rabbits were anesthetized 16–20 h after mating, and the fertilized eggs were collected from the oviduct with a cell culture medium consisting of M199 , 3% bovine serum , 5 mM HEPES , and 5 mM NaHCO3 . Then, the zygotes were microinjected with Cas9 protein and sgRNA and the blastocysts were cultured in vitro. The mutation of the target region was analyzed by PCR and T7EI digestion assay.Rabbits (n = 30). The pregnancy phenomenon was checked 10 days after the transplantation and the pups were born after 30 days.The zygote cytoplasm was injected with a 10-pl CRISPR/Cas9 mixture containing 200 ng/μl Cas9 protein , 20 ng/μl sgRNA6, and 20 ng/μl sgRNA8, and the zygotes were transferred back to the oviduct of the recipient rabbit  and CRISPR/Cas9-induced mutations were studied by PCR and T7E1 assay. PCR was performed using primers flanking the target site and the product was sequenced  and digested with T7EI  to characterize the mutation.http://crispor.tefor.net/) and the top 4 potential off-target sites of each sgRNA were selected and amplified by PCR with specific primers , the tissues were ground in liquid nitrogen and 2.5 μl/ml protease inhibitor was added and kept in ice for 30 min. Protein concentration was determined using the Bradford method (Bio-Rad), and 35 μg of protein sample was subjected to a 5–12% separation by SDS-polyacrylamide gel. Anti-MSTN polyclonal antibody (Abcam) and goat anti-rabbit IgG were coupled to horseradish peroxidase  antibodies for protein detection, while β-actin antibody (Santa Cruz) was used as an internal control.MSTN-KO and 5 WT, were fed at the same conditions and weaned on the 30th day. Rabbits were kept in separate cages and their body weight was recorded after every 10 days for 200 days. At the age of 6 months, the rabbits were anesthetized with pentobarbital sodium (1 mL/kg) to take gluteal muscle mass tissue. The gluteus maximus tissues of the F0 generation MSTN-KO and WT rabbits were fixed with 4% paraformaldehyde at 4°C. Different concentrations of dehydrated sucrose  were also used and frozen at −25°C for histological examination. The tissue slices of 5 μm thickness were first stained with hematoxylin and eosin (H&E) and then examined by a fluorescence inverted microscope . The integral optical density (IOD) analysis of histological sections was performed by using Image-Pro Plus 6.0 software.A total of 10 rabbits, including 5 F0 The F0 generation edited rabbits were bred with wild type to obtain the F1 generation rabbits with the edited genotype. There were 3 male parents of edited rabbits and 22 wild-type female rabbits. All the F1 generation-produced edited rabbits were well developed and had significant differences in body weight from the wild type and were reserved for seed use. After sexual maturity, two breeding methods were adopted for breeding with the same genotype, and the edited male rabbits were bred with wild-type female rabbits to produce the F2 generation. Among them, there were 4 male-edited rabbits, 5 female-edited rabbits, and 9 wild-type female rabbits of the F1 generation. Genotype identification was carried out and two genotypes insertion type and deletion type were selected. The F2 generation edited rabbits were crossbred with the same genotype and also with the wild type. The weight recording method is consistent with the F0 generation edited rabbit recording method. Among them, the breeding and farrowing information of the F1 generation male rabbits were recorded.T-test) and the p < 0.05 was considered statistically significant  which were cloned into the pMD18T vector as shown in The exon 1 and 3 of the The rabbit primary fibroblasts were obtained from newborns  and the MSTN gene. A total of 16 zygotes were used for injection purposes, of which 10 developed to the blastocyst stage through in vitro maturation . But after growing to the age of 3rd month, the five MSTN-KO rabbits' weight began to appear slightly higher than that of the control group . Furthermore, the bodyweight of MSTN-KO rabbits  was significantly (P < 0.01) more at 160 days as compared to the control WT rabbit . Additionally, the weight gain data from two selected genotypes (insertion and deletion type) of the F1 generation rabbit are shown in P < 0.05, n = 5), respectively. The gluteus of the MSTN-KO rabbits was also bigger than the WT rabbits, and histological analysis showed that in MSTN-KO rabbits the density and diameter of the myofiber were significantly large  has been reported in MSTN-deficient animals  superfamily ligand that can bind with different TGF-beta receptors and ultimately recruit or activate the SMAD family transcription factors, which regulate the gene expression  and body weight in the first 60 days compared with the WT group. This might be because of the IGF-1 which is an important positive regulator of muscle cell proliferation and differentiation in the skeletal muscle . Written informed consent was obtained from the owners for the participation of their animals in this study.SR, KC, and QL: conceptualization and resources. YZ, YLZ, and LW: data curation. YLZ, YZ, and ZL: methodology. HR, ZL, and DS: software. QL and KC: supervision. YZ and YLZ: writing—original draft preparation. YLZ, YZ, HR, ZL, DS, SR, QL, and KC: writing—review and editing. All authors have read and agreed to the published version of the manuscript.The present study was granted and supported by the National Natural Science Fund (Grant Nos. 31760648 and 31860638) and Guangxi Natural Science Foundation (Grant No. AB16380042).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


KCNN1 (encoding the KCa2.1 channel) that we also verified in samples from >200 patients. Yet, we found that the channel is not functional and does not modulate Ewing sarcoma cell behavior. We could explain this lack of functional impact by the surprising absence of any KCa2.1-carried K+ current in Ewing sarcoma cells. However, we show in a proof-of-principle study that the essential lack of a K+ conductance can be exploited by applying hypoosmotic stress and effectively and selectively killing the Ewing sarcoma cells.The main oncogene in Ewing sarcoma directly drives a high expression of a previously unknown variant 2+ homeostasis, which is severely disturbed by osteolytic processes in EwS. Therefore, we aimed to investigate how ion transport proteins involved in Ca2+ homeostasis affect EwS pathophysiology. We characterized the expression of 22 candidate genes of Ca2+-permeable or Ca2+-regulated ion channels in three EwS cell lines and found the Ca2+-activated K+ channel KCa2.1 (KCNN1) to be exceptionally highly expressed. We revealed that KCNN1 expression is directly regulated by the disease-driving oncoprotein EWSR1-FL1. Due to its consistent overexpression in EwS, KCNN1 mRNA could be a prognostic marker in EwS. In a large cohort of EwS patients, however, KCNN1 mRNA quantity does not correlate with clinical parameters. Several functional studies including patch clamp electrophysiology revealed no evidence for KCa2.1 function in EwS cells. Thus, elevated KCNN1 expression is not translated to KCa2.1 channel activity in EwS cells. However, we found that the low K+ conductance of EwS cells renders them susceptible to hypoosmotic solutions. The absence of a relevant K+ conductance in EwS thereby provides an opportunity for hypoosmotic therapy that can be exploited during tumor surgery.Ewing sarcoma (EwS) is a rare and highly malignant bone tumor occurring mainly in childhood and adolescence. Physiologically, the bone is a central hub for Ca Ewing sarcoma (EwS) is the second most common bone malignancy in children and adolescents, driven by a disease-defining translocation between chromosomes 11 and 22 q24;q12) . Standar;q12 . St2+, derived from calcium phosphate in the inorganic bone matrix. Consequently, it is expected that tumor cells have to adapt to an increased extracellular Ca2+ ([Ca2+]e) concentration. Moreover, as Ca2+ as well as inflammatory and cellular degradation products are osmotically active, cells have likely to cope with a hyperosmotic milieu. Whether the particularly Ca2+-rich microenvironment is relevant to EWS tumorigenesis and progression, how EwS cells regulate their intracellular Ca2+ concentration ([Ca2+]i) and cell volume and how EwS cells cope with osmotic stress is unknown.Bone is a central hub for body mineral and pH homeostasis . As EwS 2+ and volume homeostasis are tightly regulated. Spatially and temporally coded miniscule changes in [Ca2+]i and volume can induce and fine-tune various elements of cell physiology including cell migration, viability and proliferation. Cancer cells can differentially regulate ion channels involved in these procedures as means for developing (chemo-)therapy and apoptosis resistance and enhancing their invasiveness and metastatic potential i was calibrated using Ca2+-free Ringer solution supplemented with the Ca2+ ionophore 1 µM ionomycin , then 5 mM Ca2+-containing Ringer solution + 1 µM ionomycin. Images were acquired every 20 s using dual excitation with wavelengths of 340 nm (F340) and 380 nm (F380), corresponding to the excitation maxima of the Ca2+-bound and Ca2+-free form of the fluorophore, respectively, and emission was detected at 520 nm. F340/F380 ratio was derived from these values in each cell over time and [Ca2+]i was calculated as described by Grynkiewicz et al. e by expressing Ca2+-regulated ion channels (2+-modulated ion channels are involved directly or indirectly in store-operated entry (SOCE). Intracellular Ca2+ homeostasis as well as the relevant Ca2+ regulatory ion channels in EwS have not yet been studied. We conducted intracellular Ca2+ measurements in cells from two established EWS cell lines . Moreover, EwS cells contain functional Ca2+ stores and utilize SOCE triggered by thapsigargin (TG) stimulation . Osteolytic growth of EwS is coupled with marked local Cachannels A. Multipll lines B–D, wher2+ signaling in EwS cells, we inspected the expression of [Ca2+]i regulating ion channel genes in a published EwS RNAseq surfaceome database . We. WeKCNN1Ca2.1 is involved in EwS cell migration in a more complex system, we created spheroids from SK-ES-1 cells, embedded them into a complex collagen-based extracellular matrix similar to the bone . . Ca2.1 cse (RVD) C. Moreovol siRNA C, or upool siRNA  at any tSAOS = 33, nTC-71 = 26, nSK-ES-1 = 42, pSAOS-TC—71 < 0.0001, pSAOS-SK-ES-1 = 0.0001). Notably, in a 220 mOsm solution, the TC-71 cells do not exhibit any RVD at all, which is highly uncommon in eukaryotic cells . To evaluate whether the poor RVD of the TC-71 is a general feature of EwS cells, the RVD-assays were repeated with a milder hypotonic shock (220 mOsm) and with an additional EwS cell line (SK-ES-1) as well as with an osteosarcoma cell line (SAOS) B. Comparp < 0.0001). The IC50 value of osmolarity-dependent survival of TC-71 was 65.7 mOsm. In sharp contrast, SAOS cells were still viable even after 15 min in distilled water . To verify whether this poor volume regulation of the EwS cells results in a vulnerability for hypotonic stress, EwS and osteosarcoma cells were exposed to different hypotonic solutions for 15 min, and cell survival was subsequently measured with a sulforhodamine B (SRB)-assay E. After r, TC-71  versus Sr, TC-71  . In contrast, two of three osteosarcoma cell lines were osmoresistant .To test whether this effect is common in EwS cells compared to osteosarcoma cells, we repeated the assay for three additional EwS cell lines  and two additional osteosarcoma cell lines (HOS and ZK-58) at 55 mOsm, where the survival difference between TC-71 and SAOS cells was the highest F. Indeed2+ homeostasis of an osteolytic tumor, EwS, and specifically to outline the “Ca2+ toolkit” necessary for EwS cells to cope with increased environmental [Ca2+]. Our work sheds first light on the expression of ion channel genes that may coordinate Ca2+ homeostasis in EwS. Among them, we found the expression of genes that encode channels permeable to Ca2+, such as SOCE-mediating channels, Piezo 1 and 2, as well as voltage-gated Ca2+ channels  and TRP channels . We also found prominent gene expression in the case of K+ channels that are activated by a rise in the intracellular Ca2+ concentration, specifically in KCNN1 encoding KCa2.1. To understand why KCNN1 expression is prominent, we assessed the epigenetic landscape of the locus. Previously, it has been described that the promoters of multiple voltage-gated K+ channels’ genes are differentially methylated in EwS when compared with nonmalignant adult tissues  upon tumor-induced osteolysis. From these genes, we followed up on the prominent expression of KCNN1, a channel that is normally only expressed in excitable cells. We elucidated in detail that the oncoprotein EWSR1-FLI1 directly activates an enhancer in the KCNN1 locus by directly binding to GGAA microsatellites. The activation of the enhancer leads to the transcription of a novel transcript variant EWSKCNN1. In a large EwS patient cohort, we found that KCNN1 mRNA does not correlate with any of the investigated clinical parameters. In hindsight, this is not surprising, as we found that KCa2.1, the channel encoded by KCNN1, elicits no K+ currents in EwS cells and has no measurable implication in numerous aspects of EwS cell physiology. Lastly, we found that the generally low K+ conductance of EwS cells leads to an ineffective volume homeostasis, which we could then exploit by inducing EwS cell death by hypoosmotic stress. In summary, numerous Ca


So far, the cytogenomic and molecular features and clinical correlation of 21q22 amplification in AML have not been well-characterized.RUNX1 probe. Two of these patients presented with therapy-related AML (t-AML) secondary to chemotherapy, while the third had de novo AML. There was one case each of FAB M0, M1 and M4. Morphologic evidence of dysplasia was identified in both t-AML cases. Phenotypic abnormalities of the myeloblasts were frequently observed. Extra copies of 21q22 were present on chromosome 21 and at least one other chromosome in two cases. Two showed a highly complex karyotype. Microarray analysis of 21q22 amplification in one case demonstrated alternating levels of high copy number gain split within the RUNX1 locus at 21q22. The same patient also had mutated TP53. Two patients died at 1.5 and 11 months post-treatment, while the third elected palliative care and died within 2 weeks.Here, we describe a case series of three AML patients with amplified 21q22 identified by fluorescence in situ hybridization using a TP53 aberrations, and poor outcomes. Furthermore, we demonstrate that 21q22 amplification is not always intrachromosomally localized to chromosome 21 and could be a result of structural aberrations involving 21q22 and other chromosomes.Our results provide further evidence that 21q22 amplification in AML is associated with complex karyotypes,  Acute myeloid leukemia (AML) is a genetically heterogeneous disease including several distinct subtypes based on cytogenetic and molecular characteristics , 2. ChroMYC and KMT2A(MLL). Amplification of KMT2A (located at 11q23) in AML has been reported in patients with older age, complex karyotypes, TP53 aberrations, and inferior outcomes /45,sl,-4,-12,+r,+mar[15]/46,sdl1,+8[2] in three clones, each having monosomy 5 and 7 and an hsr on chromosome 21  and one RUNX1 signal each on chromosome 14 and a marker chromosome of unknown origin  and mild dysmegakaryopoiesis. By immunohistochemistry, the blasts/blast equivalents were positive for CD34, CD117 and negative for CD61. Flow cytometry data were not available. The findings were diagnostic for t-AML by WHO classification and FAB subtype M4.Patient 3’s initial bone marrow biopsy showed 67% blasts without morphologic evidence of myeloid differentiation. By flow cytometry, the blasts expressed CD34, HLA-DR, CD117, increased CD33, CD11b, TdT and partial dim CD79a without expression of MPO. The case was diagnosed as AML, NOS by WHO classification or FAB subtype M0.RUNX1 that includes non-coding exons 1–2 and coding exons 3–6, (NM_001754.4) while the rest of the RUNX1 gene (exons 7–9) is within the low-copy-number gain segment .Chromosomal microarray was performed on DNA extracted from bone marrow of patient 1 and the analysis was focused on chromosome 21. The results showed alternating levels of gain at 21q21.3q22.3 , with high-copy-number (~ 3 to 6 copies) at distal 21q22.12q22.3  compared to more proximal 21q22.12 segment   with follicular lymphoma, and Patient 2  with Hodgkin lymphoma; both developed therapy-related AML (t-AML). Patient 3  had no history of malignancy and was diagnosed with AML when she was 25 weeks pregnant. Patient 1 was treated with venetoclax/azacitidine and died 1.5 months after diagnosis. Patient 3 deferred treatment until after delivery and then received “7 + 3” induction chemotherapy (idarubicin/cytarabine). She then went on to receive an allogeneic stem cell transplantation in the setting of persistent measurable residual disease and died of disease relapse 11 months after diagnosis. Patient 2 elected palliative care and received no treatment for AML; he died 2 weeks after diagnosis . A similar observation was reported by Nguyen and colleagues who described an AML patient with 21q22 amplification covering a portion of the 5′ region of RUNX1 and extending to the 21q terminus, albeit with different breakpoints than the ones observed in our patient [ERG by FISH, Weber and colleagues reported the ERG locus as the commonly amplified in 21q22 region while the RUNX1 locus was amplified in only about half of the cases [RUNX1 expression is not upregulated in AML with amplified RUNX1 identified by RUNX1 FISH [RUNX1 FISH does not include the entire RUNX1 locus [ERG locus is within the high-copy-number gain region. The RUNX1 FISH approach is likely to lead to an underestimation of the incidence of 21q22 amplification in AML because it would not identify those cases that do not involve the RUNX1 locus. Xie et. al reported a prevalence of 0.1% using RUNX1 FISH to ascertain 21q22 amplification in their AML cohort and using the same approach, we found a frequency of 0.2% in our AML cohort, while Weber et al. described a higher prevalence of 0.7% using ERG FISH [There is limited understanding of cytogenomic features of 21q22 amplification in AML. In this study, the high-resolution microarray study of Patient 1 showed high-copy-number gain (~ 3 to 6 copies) of the 21q22 segment  covering the 5′ portion of the  patient . In theihe cases . This isX1 locus  and the ERG FISH , 14.ERG encodes a proto-oncogenic transcription factor expressed in hematopoietic stem cells including those present in AML and myelodysplastic syndrome (MDS) [ERG overexpression has been shown to be an adverse biomarker for cytogenetically normal AML [Erg overexpression in mice induced the development of an erythro-megakaryocytic leukemia [ERG overexpression is correlated with ERG high-copy-number gain, indicating that amplification may result in upregulation of ERG expression [RUNX1 truncating and frameshift mutations have been reported in myeloid neoplasms, supporting its role as a tumor suppressor [RUNX1 mutations and ERG amplification was observed in 25% of AML cases with 21q22 amplification [RUNX1 mutation cooperates with ERG amplification to promote AML leukemogenesis remains unclear at the present time.me (MDS)  ERG overrmal AML , 20. Morleukemia . Weber epression . RUNX1 tppressor . Co-exisfication . HoweverRUNX1 FISH probe signals were dispersed in interphase nuclei and metaphase chromosomes rather than as a clustering pattern seen in hsr(21). Jain and colleagues described AML in a 6-year-old child who had an hsr(21) with additional 21q22 material inserted into chromosome 2q31 [In this study, two patients, Patients 1 and 3, had an hsr on an abnormal chromosome 21. These results are in agreement with the observations by others who described hsr(21) as a common form of 21q22 amplification in AML , 12, 14.ome 2q31 . Similarome 2q31 , 11. TheTP53 is common in AML with 21q22 amplification and accordingly, Patient 1 in our study also harbored dual TP53 mutations [TP53 deletion detected by FISH in an AML patient with 21q22 amplification [TP53 plays a role in maintaining genomic stability and has been linked to chromothripsis [Previous mutational analysis revealed that mutant utations , 14. Ngufication . TP53 plthripsis , therefoTP53 aberrations and complex karyotypes are known adverse risk features for AML [TP53 aberrations.So far, there is limited information on clinical characteristics and outcomes in AML with 21q22 amplification , 10, 14. for AML , 25 and ERG gene due to the lack of materials. Third, molecular studies were only done on one patient, and lastly, high resolution genomic analysis of 21q22 amplification using chromosomal microarray was performed on one case, again due to the lack of materials. Future large international collaborations are necessary to further characterize the genomic features and the prognostic significance of 21q22 amplification in AML.This case series study has several limitations. First, this is a small cohort of three patients, rendering it difficult to adequately assess whether 21q22 amplification is a poor prognostic factor independent from other known adverse risk factors in AML, including complex karyotype and chromosome 7 abnormalities present in 2 of 3 our patients. Second, we were not able to perform gene expression studies on the RUNX1 locus is not a primary target of 21q22 amplification in AML.In summary, we report the cytogenomic features, histopathologic findings, and clinical outcomes in three adult AML patients with 21q22 amplification. Our results show that AML with 21q22 amplification is associated with a complex karyotype, poor prognosis, and occurs in both de novo AML and t-AML. 21q22 amplification may result from either a cluster on a single chromosome in the form of hsr(21) or multiple 21q22 rearrangements scattered across different chromosomes. Findings from this study support that the


N-ethylmaleimide modified hemoglobin (NEMHb); and equine myoglobin (Mb). These molecules efficiently sequester CO from hemoglobin in erythrocytes in vitro. A kinetic model was developed to predict the CO binding efficacy for hemoproteins, based on their measured in vitro oxygen and CO binding affinities, suggesting that the therapeutic efficacy of hemoproteins for CO poisoning relates to a high M value, which is the binding affinity for CO relative to oxygen . In a lethal CO poisoning mouse model, StHb, NEMHb, and Mb improved survival by 100%, 100%, and 60%, respectively, compared with saline controls and were well tolerated in 48-hour toxicology assessments. In conclusion, both StHb and NEMHb have high CO binding affinities and M values, and they scavenge CO efficiently in vitro and in vivo, highlighting their therapeutic potential for point-of-care antidotal therapy of CO poisoning.I.v. administration of a high-affinity carbon monoxide–binding (CO-binding) molecule, recombinant neuroglobin, can improve survival in CO poisoning mouse models. The current study aims to discover how biochemical variables of the scavenger determine the CO removal from the RBCs by evaluating 3 readily available hemoproteins, 2,3-diphosphoglycerate stripped human hemoglobin (StHb);  In the case of hemoglobin, CO binding has 2 main detrimental effects: (a) CO binds tightly to the heme iron, decreasing the number of oxygen-binding sites available and thereby decreasing oxygen binding capacity, and (b) the CO-bound hemoglobin (carboxyhemoglobin [COHb]) allosterically stabilizes the high-affinity R-state tetramer, increasing the affinity for hemoglobin-bound oxygen and, therefore, decreasing oxygen release in the tissues. At high CO concentrations, CO also binds to the heme a3 group of cytochrome c oxidase, arresting electron transfer and aerobic metabolism (Carbon monoxide (CO) is the most common non–drug-related human poisoning, accounting for tens of thousands of emergency department visits every year in the United States alone . CO exhitabolism , 2. The tabolism , 5. Up ttabolism , 7. Survtabolism . There rc oxidase activity compared with CO-poisoned mice treated with only saline . This protein, which exhibits a CO affinity 500-fold higher than that of hemoglobin , rapidlyy saline . While ty saline , 12.KA,CO and KA,O2) on efficacy must be determined. Ngb-H64Q-CCC shows high affinity for both CO and oxygen, but it shows a higher overall selectivity for CO. This selectivity parameter is represented numerically by the M value, or the ratio of ligand binding affinities, KA,CO/KA,O2. It is logical to consider that high CO affinity will be advantageous for a CO scavenger, but selectivity for CO over oxygen must also be considered under aerobic conditions in the bloodstream. The exact relationship between in vitro ligand affinity and selectivity and the CO-scavenging efficacy in vivo is not well understood. Thus, a primary goal of this study is to establish benchmark criteria for CO binding affinity and selectivity that confer CO-scavenging efficacy in vivo.To extend our knowledge about the role of CO-scavenging hemoproteins in antidotal pharmacology, the impact of ligand binding parameters  to form NEM-modified hemoglobin (NEMHb). This chemical modification also results in allosteric stabilization of R-state hemoglobin and enhanced ligand binding affinity compared with StHb  were determined for StHb and NEMHb by directly monitoring CO rebinding following laser flash photolysis (https://doi.org/10.1172/jci.insight.153296DS1), and CO dissociation rate constants  were measured by the nitric oxide (NO) displacement method  were determined by CO displacement  were calculated indirectly from M value and other measured kinetic parameters. Kinetic parameters and binding rate constants are summarized in 6 M–1s–1) compared with StHb (5.1 ± 0.2 × 106 M–1s–1). The dissociation rate constants were similar between NEMHb  and StHb , with NEMHb exhibiting a slightly larger dissociation rate constant  and StHb  were similar. Measured M value for StHb (242 ± 4) was greater than the measured M value for NEMHb (231 ± 3), as shown in kon,O2 calculated for NEMHb (3.1 ± 0.3 × 107 M–1s–1) and StHb (2.4 ± 0.3 × 107 M–1s–1) were similar. These data show that NEMHb and StHb have very similar affinities for CO and are both similar to the reported CO affinity for R-state hemoglobin  with a concomitant rise in fraction of CO-bound scavenger to total extracellular scavenger  under both anaerobic and aerobic conditions  compared with StHb (M = 242) and NEMHb (M = 231)  .RBC reached 96.9% ± 0.2 % at the end of the 4.5-minute CO inhalation period, resulting in 0% survival and a median survival time of 12.0 minutes in mice treated with NS  and StHb (absolute change in COHbRBC% = 17.1% ± 0.9%), and the magnitudes of these changes were significantly greater than that of the NS control  and StHb (COsc = 65.9% ± 4.9%), determined from spectral deconvolutions of blood plasma, were observed  nor NEMHb   , A and BRBC%, including CO elimination via lungs, redistribution of CO from blood to tissues, and sequestration by exogenous hemoprotein scavengers. Fraction of CO scavenged by hemoproteins to total initial CO was defined as CO sequestration (COseq), which was not affected by CO elimination via lungs and redistribution to tissues. It was also used for comparison of CO-binding efficacy and was estimated by blood concentration of RBC-encapsulated hemoglobin, initial COHbRBC percentage, hematocrit, and correction for blood dilution by infusions  kept decreasing after infusion in the NS group, while a recovery of PO2 was observed with StHb treatment , heart rate (HR), and survival for each treatment group. As shown previously , 10, allDose-response studies for StHb and NEMHb were evaluated using the same severe–CO-poisoning model as for the hemoprotein comparison study. Mice were administered doses between 0 and 150 μmol/kg body weight for StHb or NEMHb  in a NS vehicle, and outcomes were compared with NS infusion alone. A volume of 10 mL/kg body weight was infused at an initial hemoprotein concentration ranging from 1 to 15 mM to achieve target dose levels. At least 5 mice were tested at each dosing level.RBC was observed in StHb and was found to be significantly correlated with dose , and hemoprotein concentration in blood was measured at the end of the 2-minute infusion period. There was no significant difference of hemoprotein scavenger concentrations in blood between the 2 moderate models. Measured hemoprotein blood concentrations immediately after infusion were 0.8 ± 0.6 mM in model A and 0.8 ± 0.1 mM in model B for Mb, and they were 0.8 ± 0.1 mM in model A and 0.9 ± 0.1 mM in model B for StHb . Estimated fraction for COseq were significantly smaller for Mb  compared with StHb . Interestingly, estimates of COseq for Mb and StHb do not vary as a function of initial COHbRBC levels in the moderate–CO-poisoning models. RBC% was higher for StHb  but not significantly different for Mb  compared with NS .StHb exhibited significantly greater efficacy than Mb . In bothI.v. infusion of NEMHb and StHb at a therapeutic dose was well tolerated in a separate cohort of nonpoisoned, healthy animals. For safety studies, mice were infused with exogenous hemoprotein at a dose of 100 μmol/kg body weight via tail vein catheter and monitored for 48 hours after infusion. Outcomes for mice treated with NEMHb and StHb were compared with those of animals infused with NS, and there were 5 mice for each treatment group and the NS control group. All mice survived 48 hours after i.v. infusion, and no differences in behavior were observed between NEMHb and StHb treatment groups and the NS control group. No significant changes in biomarkers for liver function   , CO trankon and koff are the binding and dissociation rate constants of the indicated ligand (oxygen or CO), the scavenger (X), and RBC hemoglobin (R). Additionally, irreversible loss of CO from the system (kCOloss), either through diffusion at the liquid-air interface in vitro or through tissue distribution and exhalation in the circulatory system in vivo, is included in the model by Equation 5:Where kon) and ligand dissociation (koff) for hemoglobin and extracellular CO scavengers were determined from in vitro experiments and used to compute ligand affinity values , and root mean squared error (RMSE). Although there is no published prediction of CO scavenging efficacy for direct comparison, the predictive performance is considered accurate with R2 of 0.80–0.99, a slope of 0.72–0.97, and a Y-intercept and RMSE less than half SD of the measured data. The intercept for COseq in the hemoprotein study was higher than other data sets, and this difference could possibly be related to lower blood concentration of Mb at the same molar dose.To determine the important kinetic parameters for design of an ideal in vivo CO scavenger, we modeled a series of theoretical scavengers. Intuitively, a scavenger with high selectivity for CO over oxygen , will effectively sequester CO under aerobic conditions. This point is illustrated when comparing the in vivo scavenging kinetics of StHb and Mb, simulated using experimentally determined rate constants . StHb hakoff,O2 , no changes in rate were observed  and the M value of Mb readjusted to 242 (dotted orange lines) by adjusting their respective koff,O2  does notolecules . We baseO and O2  did not observed , likely affinity  yielded In the present work, the CO scavenging efficacy of 3 hemoproteins — StHb, NEMHb, and Mb — was investigated as treatments for CO poisoning. While our previous study demonstrated that a hemoprotein with very high CO affinity and selectivity (Ngb-H64Q-CCC) functions as an effective antidote against CO poisoning , here, aRBC% = 52.6%) and Mb (absolute change in COHbRBC% = 28.7%). Importantly, in vitro total CO scavenging under aerobic conditions accurately reflected in vivo outcomes: CO scavenging efficacy and survival were significantly lower for Mb compared with StHb and NEMHb in vivo in our severe–CO-poisoning mouse model  resulted in the highest survival (100% for StHb and NEMHb) in our severe model . These sseq, CO elimination from the cardiopulmonary circulation, and oxygen delivery. Under similar conditions in the severe–CO-poisoning mouse model, heme sites in Ngb-H64Q-CCC are almost fully saturated (99.9% ± 0.1%) by CO  by CO . Sites n%) by CO , and pre%) by CO . Thus, SRBC level before infusion  reducing CO exposure time and (b) increasing the latency period between CO exposure and infusion. Estimated values for COseq for NEMHb and StHb do not vary significantly as a function of initial COHb levels in this moderate–CO-poisoning model. Furthermore, COseq values observed in moderate CO models are comparable with those observed at the same dose of NEMHb and StHb in the severe model, where the initial COHbRBC is ~97%  and resuspension. The RBCs were then lysed under hypotonic conditions by resuspension in excess deionized water. Cellular debris was removed by centrifugation at 23,000g for 30 minutes at 4°C. The hemoglobin-containing supernatant was passed through a desalting column  equilibrated with NS to remove molecules smaller than 6 kDa. The hemoglobin solution was further purified and concentrated by differential ultracentrifugation . The isolation and purification processes were conducted at 4°C and completed within 1 week. To assess holoprotein concentration and species fractionation, UV-Visible spectra of freshly prepared hemoglobin samples were deconvoluted in terms standard reference spectra for ferric (Fe3+), deoxyferrous (Fe2+), and oxyferrous (Fe2+-O2) hemoglobin. Concentration of hemoproteins reported in this paper refers to heme concentration. UV-Vis spectra were recorded using a Cary 50 spectrophotometer (Varian). Most preparations yielded > 99% Fe2+-O2 hemoglobin, and any protein batches with more than 1% metHb were discarded. Finally, StHb was filtered using a GD/X 0.2 μm PES filter (Whatman) and stored in aliquots at –80°C.Expired, leukoreduced human donor packed RBC units were used as a source of hemoglobin. The isolation and purification process was carried out as previously reported . Briefly2+-O2 hemoglobin were stored for further use.NEM  was incubated with hemoglobin, isolated, and purified as described above in a 3:1 ratio of NEM/heme  with gentle rocking (Labnet ProBlot Rocker 25 XLD) for 1 hour at room temperature. Excess NEM was removed by a gravity-flow chromatography column  preequilibrated with NS. Then the NEMHb was concentrated by differential ultracentrifugation , aliquoted, and stored at –80°C. As above, holoprotein concentration and species fractionation were assessed using UV-Vis spectral deconvolution, and only preparations that yielded > 99% FeM values were measured as previously described  by the fO2 and PCO were the partial pressure of oxygen and CO in the equilibrium gas phase, respectively. In brief, 2 mL of oxy-StHb or oxy-NEMHb in PBS (pH 7.4) was added to a 4.9 mL quarts cuvette capped with septum. Final concentration of StHb or NEMHb was around 20 μM. CO gas with known concentration in air was injected to the headspace of the cuvette. Equilibrium between gaseous and aqueous phase was reached by gently shaking on a plate rotator at 25°C for 90 minutes until the ratio of COHb/oxyHb was not changed. The absorbance spectra of the Hb solution was measured by spectrophotometer (Cary 100) at 25°C. [COHb], [oxyHb], and [metHb] concentrations were determined by deconvolution.Where COHb and oxyHb were the fraction of COHb and oxyhemoglobin, Pkon,CO was determined by laser flash photolysis for StHb and NEMHb as previous described . The absorption at 419 nm were fit to single exponential decay for the values of koff,CO.The koff,O2 was measured in a stopped-flow spectrophotometer using CO replacement for oxygenate StHb and NEMHb in PBS at 25°C. Oxygenated protein 5 or 20 μM was mixed with CO-saturated PBS (CO concentration between 0.8 and 1 mM) in a stopped-flow spectrophotometer. The absorption at 421 nm was fit to single exponential decay for the koff,O2.The g for 2 minutes at 4°C) and resuspension to further remove CO in the buffer. The CO saturated RBCs were resuspended in PBS with dithionite for anaerobic experiments or without dithionite for aerobic experiments. The concentration was adjusted to 0.4 mM in heme, measured by UV-Vis spectroscopy.The CO transfer from RBC-encapsulated COHb to cell-free hemoproteins was monitored as previously reported . Mouse bg for 10 seconds at room temperature. The supernatant and the RBC pellet were separated and stored on ice. When all the samples were collected, absorbance spectroscopy was used to measure the saved supernatant and the RBC pellet heme ligand binding state . The supernatants were measured after diluting with PBS with 10 mM dithionite. The RBC pellets were measured after lysis with 0.5% NP40 in PBS with 10 mM dithionite.For anaerobic experiments, a 1 cm quartz cuvette sealed by a cap with septum was filled with nitrogen to maintain an oxygen-free environment. CO-saturated RBCs were equilibrated to the set temperature in the cuvette and were gently stirred. CO transfer was initiated by mixing the hemoprotein solution  with the CO-saturated RBCs in the cuvette with 10 mM dithionite. The ratio of free hemoprotein to hemoglobin encapsulated within the RBCs was 1:1 (by heme concentration). A 100 μL sample was taken out from the cuvette at predefined time points. The sample was then centrifuged at 2,000For aerobic experiments, the procedures were the same as the anaerobic experiments, except that no dithionite was used in the making and washing of CO-saturated RBCs and reaction mixtures, and the cuvette was not capped or purged with nitrogen; the reaction system was open to air, and oxygen partial pressure in the reaction mixture was estimated to be 160 mmHg.UV-Vis spectroscopy was used to determine the transfer of CO between RBC-encapsulated COHb and extracellular hemoproteins. Concentration and species fractionation were assessed using UV-Vis spectral deconvolution with the CO saturated RBCs lysed by hypotonic 20 mM sodium dithionite solution. Multiple replicate measurements were performed to ensure the accuracy. Equimolar amounts of fully reduced Mb, NEMHb, or StHb were mixed with mouse RBCs saturated by CO.Adult male C57BL/6J mice  were used for all animal experiments. Mouse blood was collected and used in the same day for the in vitro CO-RBC experiments.In vivo therapeutic effects of hemoproteins in moderate and severe CO poisoning were investigated, adapting previously described mouse models . Prior tOne severe–CO-poisoning model and 2 moderate–CO-poisoning models were tested in this project. For the severe model , the CO For in-life studies, mice were anesthetized with 2%–3% isoflurane. An incision was made on the neck. A tracheal tube was introduced via a tracheotomy for mechanical ventilation with a 20 gauge cannula. Catheters (0.025” × 0.012”) were inserted into the right carotid artery and jugular vein. The incision was closed with 6-0 sutures. The anesthetized mice were then moved into a fume hood to protect the operators from CO poisoning. Volume-controlled mechanical ventilation  was applied with a small animal ventilator . Arterial blood pressure was continuously recorded  via the arterial catheter. The MAP and pulse rate were calculated by Labchart software (ADInstrument).Arterial blood samples were taken through the arterial catheters at preset time points to determine COHb levels and plasma protein levels. A total of 15 μL of blood was taken each time with a Hamilton syringe (Hamilton Company). Blood was centrifuged for 1 minute at 10,000g at room temperature to separate the plasma from cells. Then, the plasma and cell pellet were frozen on dry ice and store in –80°C for later analysis.Venous injection was controlled by an injection pump with the volume of 10 mL/kg body weight and the injection time of 2 minutes for both test articles  and control (NS or PBS). NS was used as control group for the severe–CO-poisoning model, including the hemoprotein comparison study and the dose-response study of StHb and NEMHb. PBS was used in the moderate–CO-poisoning models. Hemoproteins were administered in the oxygenated ferrous form in the treatment groups with a concentration of 10 mM heme, to achieve a dose of 100 μmol/kg body weight, except for the dose-response study of StHb and NEMHb.g for 1 minute to separate the RBCs pellet and plasma. Samples were then immediately frozen and stored at –80°C. To determine the fraction of CO-bound hemoglobin, each frozen blood cell pellet was thawed and lysed by dilution into a hypotonic 20 mM sodium dithionite solution to prevent adventitious oxygen binding and facilitate spectral deconvolution. To determine the fraction of CO-bound extracellular scavenger, frozen plasma was thawed and diluted into 20 mM sodium dithionite solution to facilitate spectral deconvolution. Absorption spectra of lysed RBC and diluted plasma samples were recorded from 450–700 nm using a Cay 50 spectrophotometer (Varian). Spectral deconvolution was employed to determine the concentrations and relative fractions of hemoprotein species for RBC-derived hemoglobin and extracellular hemoproteins using standard references for Fe3+, Fe2+, Fe2+-O2, and CO-bound species, along with a linear scattering correction (Mouse blood samples (15 μL) withdrawn in the CO-poisoning study were immediately centrifuged at 10,000seq is as follows.Blood concentration of RBC-encapsulated hemoglobin and plasma concentration of exogenous hemoprotein were measured directly with RBC lysates or plasma, respectively, and calculated by dilution factors. Blood concentration of exogenous hemoprotein were then calculated from measured plasma concentration and plasma volume percent of the whole blood, which was 100% minus hematocrit . Considering the correction for blood dilution by infusion, estimation of COsc,end] is the plasma concentration of CO-bound hemoprotein scavenger at the end of infusion. Hematocritend is the hematocrit at the end of infusion,  and  is the blood concentration of total RBC-encapsulated hemoglobin at the start and end of infusion, and COHbRBC,start is the RBC-encapsulated COHb percent at the start of infusion.Where [Blood CO-bound scavenger] and [plasma CO-bound scavenger] are the blood concentrations and plasma concentration of CO-bound scavenger at the end of infusion, respectively. [plasma CORBC% (after infusion) = COHbRBC % (start infusion) – COHbRBC% (after infusion)Absolute change in COHbCO transfer from RBC-encapsulated COHb to extracellular hemoproteins was simplified to a single- compartment system containing RBCs and extracellular hemoproteins. Reactions were described in Equations 1–5.Equations 1–5 were used to generate a kinetic model using the COPASI software, version 4.29 . Total vkon) and ligand dissociation (koff) for RBC-encapsulated COHb and exogenous CO scavengers were determined from in vitro experiments and used to compute ligand affinity values  and serum chemistry measurement (Marshfield Labs).Healthy C57BL/6J mice were anesthetized with inhaled isoflurane and body temperature maintained at 37°C. A catheter was implanted into the lateral tail vein, through which the Fet test was used for comparison of 2 groups and normal distribution. One-way or 2-way ANOVA was used for comparison of more than 2 groups. Kaplan-Meier survival curves were compared with log-rank (Mantel-Cox) test. Bonferroni corrected threshold for P value was adopted for each individual comparison in the survival curves. MAP data were compared by 2-way repeated-measures ANOVA. Agreement between the prediction and observation was analyzed by Spearman correlation. * shows p value of multiple comparison of Tukey test . A p value less than 0.05 was considered significant.Data are shown in the form of mean ± SEM and analyzed with GraphPad Prism software version 8.4.2. Unpaired 2-tailed All animal studies were performed in accordance with protocols approved by the IACUC at the University of Pittsburgh and NIH Guide for Care and Use of Laboratory Animals.MTG conceived and supervised the project. QX designed and performed all in vitro experiments and some in vivo experiments. JJR designed and performed some in vivo experiments and assisted with critical review of the manuscript. XC performed most in vivo experiments. LW performed some in vivo experiments. AWD assisted with the in vitro experiments and development of the kinetic model. MRD assisted with the mathematical simulation. ST isolated, purified, and made the StHb and NEMHb. KB assisted with the in vivo experiments. XNH assisted with some in vitro experiments. QT assisted with some in vitro experiments. CFM assisted with experimental design. LG assisted with in vivo experiments. EA, TCJ, and KBU conducted laser photolysis and NO displacement experiments and analyzed data. DBKS designed and supervised laser photolysis and NO displacement experiments. JT conducted mathematical simulation and critical review of the manuscript.