Currently, high doses of cytarabine arabinoside (Ara-C)-based combined chemotherapy are commonly used in acute myeloid leukemia (AML) therapy, but severe adverse effects and poor suppression effects in leukemia cells limit the clinical therapeutic efficiency of Ara-C-based chemotherapy due to a lack of targeting selectivity. To improve the therapeutic effect of Ara-C in AML, here, since we confirmed that transferrin receptor 1 (TFRC) expression in AML cells was constant, we generated Ara-C@HFn by encapsulating free Ara-C into self-assembled heavy ferritin chain  nanocages.The analysis of clinically relevant data suggested that the high expression levels of TFRC from AML cells would not decrease significantly after treatment with Ara-C. Ara-C@HFn can be efficiently internalized by leukemia cells, showing stronger cytotoxic effects in vitro and reducing the burden of leukemia in AML mice more effectively in vivo than free Ara-C. Ara-C@HFn treatment showed no acute toxicity in visceral organs of mice. Moreover, the analysis of clinically relevant data also suggested that there are several drugs (such as tamibarotene and ABT199) that would not cause significant expression down-regulation of TFRC in AML cells (after treatment).The above results suggested that TFRC can be used as a constant and effective target for drug targeting delivery of AML cells. Thus Ara-C@HFn treatment can become a safe and efficient strategy for AML therapy by specifically delivering Ara-C to AML cells. Besides, the HFn nanocages are promising for improving antineoplastic effect of other AML-related therapy drugs that do not cause downregulated expression of TFRC in AML cells.The online version contains supplementary material available at 10.1186/s12951-023-01881-8. As a highly heterogeneous hematological malignancy, acute myeloid leukemia (AML) is characterized by immature myeloid cell proliferation and bone marrow failure . AML accTo improve the uptake efficiency of Ara-C, as well as reduce drug-related complications, targeted delivery is a particularly attractive method that can improve the local effective anticancer drug concentration at target cells . The pree.g., pH = 7.4) [As the TFRC ligand , ferritiH = 7.4) –25. In gAlthough it is known that TFRC has a high expression level in cancerous cells in AML patients, whether the expression of TFRC in AML cells decreases after treatment with Ara-C has not been reported; the downregulated expression of TFRC will interfere the long-term effectiveness of targeted therapy based on TFRC. In this context, this study first analyzed some sequencing data of related patients’ sample from the Gene Expression Omnibus (GEO) database and concluded that the expression of TFRC in AML cells would not significantly decrease after Ara-C treatment. These findings further encouraged us to develop an HFn nanocarrier loaded with Ara-C (Ara-C@HFn) in the ferritin cavity. The nanocarrier can be specifically recognized by AML cells and can rapidly release Ara-C into leukemia cells following intravenous injection, which has the potential to enhance the therapeutic effect and reduce the side effects to normal body cells induced by Ara-C Scheme .Scheme 12. HL-60 cells transduced with luciferase were used in some experiments as indicated.HL-60 (1101HUM-PUMC000037) and K562 (1101HUM-PUMC000039) cells were sourced from the National Infrastructure of Cell Line Resource. No mycoplasma contamination was detected in the above cell lines. The cells were maintained in RPMI-1640 media  supplemented with 10% fetal bovine serum , 100 U/mL penicillin, and 10 mg/mL streptomycin  at 37 °C with 5% COPrkdcscidIL2rgtm1/Bcgen) severe combined immune deficiency (SCID) mice that were female and 5–6 weeks old were purchased from Beijing Biocytogen Co., Ltd. Specific pathogen‑free (SPF) female BALB/c mice (6‑8 weeks old) were obtained from Vital River Laboratory . Mice were maintained in SPF conditions with a 12 h/12 h light/dark cycle, and the mice received food and water ad libitum. This study was performed in strict accordance with the Regulations for the Care and Use of Laboratory Animals and Guideline for Ethical Review of Animals . All animal experiments in this study were reviewed and approved by the Animal Ethics Committee of the Institute of Process Engineering (IPEAECA2021081).B-NDG  using a C18 column  with a linear gradient of acetonitrile containing 0.1% TFA from 0 to 100% in 10 min at a flow rate of 1.0 mL/min. Initially, the peak area of the Ara-C standard with various concentrations was detected by HPLC, and the standard curve was fitted accordingly. The Ara-C concentration in Ara-C@HFn was determined by HPLC, and the levels of Ara-C encapsulated by HFn were measured according to the concentration of HFn (approximately 56 Ara-C molecules were encapsulated by one HFn nanocage).The blank HFn nanocage, disassembled HFn nanocage (pH = 2.0), and Ara-C@HFn were prepared for transmission electron microscopy (TEM) by directly dropping these aqueous solutions on 400 mesh ultrathin carbon film (Beijing Zhongjingkeyi). These samples were then negatively stained with uranyl acetate and observed by TEM .The size distributions of HFn and Ara-C@HFn were determined by dynamic light scattering  in a Nano series. Samples were tested in triplicate, and the average peak size (nm) was recorded (n = 3).The molecular weight of the protein subunit from Ara-C@HFn, HFn using the same amount of protein as that in Ara-C@HFn, and Ara-C using the same amount of Ara-C as that in Ara-C@HFn was determined by SDS–polyacrylamide gel electrophoresis (SDS‒PAGE) using a standard operation procedure (n = 3).Confocal laser scanning microscopy (Nikon) was used to observe the internalization of Ara-C@HFn. Before observation, Cy5-labeled Ara-C@HFn was prepared. Briefly, Cy5NHS ESTER (Meilunstar) was dissolved in water and incubated with Ara-C@HFn at a molar ratio of more than 20:1. The mixed solution was stirred at 4 °C overnight, and the free dye was removed by ultrafiltration. Then, HL-60 or K562 cells were incubated with Cy5-labeled Ara-C@HFn for 24 h at 37 ℃ (in RPMI-1640 media) and fixed in 4% cold formaldehyde for approximately 15 min. Subsequently, the cell membrane was stained with rhodamine phalloidin (Invitrogen) for 30 min, and the nucleus was stained with Hoechst 33342 (Solarbio) for 20 min. Finally, the prepared samples were observed with a confocal laser scanning microscope (Nikon) by NIS Elements AR 5.20 (n = 3). Note that for lysosomal staining, it is not necessary to fix the cells before staining. After incubating the lyso-Tracker Green (Beyotime) and Hoechst 33342 (Solarbio) with cells for 20 min at 37 ℃, then the prepared samples could be observed with a confocal laser scanning microscope.5 cells/well were treated with PBS buffer, various concentrations of free Ara-C or various concentrations of Ara-C@HFn with the equivalent amount of Ara-C for 48 h. After incubating for 2 h, the absorbance value at 450 nm in each well was measured by the Infinite M200 microplate spectrophotometer , and cell viability was assessed by using the CCK-8 assay .The cell viability assay was similar to that used in our previously published paper . BrieflyAn antibody blocking assay was performed according to the published procedures , 19 to cTo assay for the release of Ara-C, Ara-C@HFn samples  were placed in Eppendorf centrifuge tube and incubated in either PBS buffer (pH 7.4) or hydrochloric acid buffer (pH 5.0) at 37 °C in the dark. After removing the Ara-C@HFn by using ultrafiltration membrane columns , the released free Ara-C from Ara-C@HFn at different incubation times was determined by HPLC with a C18 column.6 cells/well and incubated with 0.6 μM Ara-C or Ara-C@HFn with the equivalent amount of Ara-C for 48 h. The cell cycle was assessed using a DNA Content Quantitation Assay (Solarbio) through the flow cytometry system .HL-60 cells were cultured in 12-well plates at a density of 1 × 106 AML cells intravenously the following day. At 15 days post HL-60 cell injection, the mice were injected with Cy5-labeled Ara-C@HFn, and 2 h later, the peripheral blood, spleen and hindlimbs of the AML-bearing mice were collected to evaluate targeting capacity. Then, the spleen and hindlimbs were used to prepare single-cell suspensions, red blood cells (RBCs) were removed from the peripheral blood and single-cell suspensions by using RBC lysis buffer (Solarbio). Subsequently, these samples were incubated with EV450-conjugated anti-human CD45 (anti-hCD45) antibody (eBioscience) for 30 min, and the proportion of hCD45+ cells (the marker of HL-60 cells) in Cy5+ (the label of Ara-C@HFn) cells was analyzed by flow cytometry (n = 3).The AML-bearing mouse model was established as described in a previously published paper . BrieflyThe AML mouse models were established with Luc-HL-60 cells (HL-60 cells that stably expressed luciferase) as described above. All mice were randomly divided into three groups (n = 4): control , free Ara-C and Ara-C@HFn. Four days later, the mice received six-round treatments of PBS, Ara-C (8 mg/kg) or Ara-C@HFn  (once every 2 days). Then, leukemia progression was monitored by an In vivo Imaging System (IVIS) optical imaging system (PerkinElmer) once every 5 days. AML-bearing mice were injected with D-luciferin potassium  intraperitoneally, and then the bioluminescence signals from AML-bearing mice were determined by Living Image 4.5.2. All mice were euthanized at 20 days after the HL-60 cell transplantation, following which the peripheral blood, backbone, hindlimbs were isolated. The leukemia burden in interested tissues was evaluated by measuring the bioluminescence levels. Subsequently, a small amount of peripheral blood was used for white blood cell (WBC) counting. In addition, most of the peripheral blood and the spine were used to prepare single-cell suspensions, which were stained with EV450-conjugated anti-human CD45 antibody and/or APC-conjugated anti-human TFRC antibody (Biolegend) for subsequent flow cytometry analysis.The Balb/C mice were treated with PBS, free Ara-C or Ara-C@HFn . Forty-eight hours later, peripheral blood and the major organs, such as the heart, liver, spleen, lung, and kidney, were collected for acute toxicity assessment. Peripheral blood was used for routine blood tests, and the major organs were used for histopathological analysis (n = 3).The histopathological analyses were performed according to standard operating procedures as described in a previously published paper . Brieflyhttp://www.ncbi.nlm.nih.gov/projects/geo/) over the past two years . To facilitate further analysis, the inclusion criteria of the data analyzed in this study mainly included the following: the library preparation strategy was for RNA-seq (including single-cell RNA sequencing data); the sequencing data were directly obtained from human or transgenic mice that were treated with transplanted human volunteer cells; and if the transgenic mice were treated with drugs, the drugs were used in the clinic or in clinical trials.This study further analyzed AML-related transcriptome sequencing data uploaded to the GEO database  data was performed by R software (v4.1.0), and the statistical analysis of other data was performed by SAS software . Among them, for statistical analysis of two groups of data, when the data conformed to a normal distribution and exhibited homogeneity of variance, the t test was used; otherwise, the Wilcoxon rank sum test was used. For statistical analysis of three or more groups of data, when the data conformed to a normal distribution and exhibited homogeneity of variance, one-way ANOVA was used; otherwise, the Kruskal‑Wallis test was performed, and the Student‑Newman‑Keuls test was used for multiple comparisons of data. In addition, these statistical data were plotted using GraphPad Prism (v8.4.3) except for scRNA-seq data, principal component analysis (PCA) and correlation analysis, which were plotted using R software. Note that each experiment in this study has no less than 2 technical replicates.Previously, based on the expression of TFRC in a large number of AML patient cells assessed by flow cytometry, we concluded that TFRC has a high expression level on AML cells . HoweverConsidering the important role of Ara-C in AML treatment and the important advantages of HFn in targeted drug delivery (such as good biosafety and ease of expression and purification), the above analysis results encouraged us to further analyze whether TFRC expression was downregulated in AML cells post Ara-C treatment based on the GEO database. If Ara-C leads to significant downregulation of the expression of the HFn receptor TFRC, HFn-based targeted drug delivery will be difficult to achieve, and the effect of HFn-based targeted drug delivery will be greatly reduced. Although the available effective data are only a little, it is exciting that our analysis results suggested that the expression level of TFRC in AML cells from AML patients was upregulated after Ara-C treatment in vitro  of Ara-C@HFn was approximately 50% lower than that of free Ara-C  cell line, K562. Flow cytometry indicated that TFRC exhibited a high expression level on the surface of CML cells  were leukemia cells (hCD45+ cells)   in the two groups of mice were significantly different, which again suggested that Ara-C@HFn induced a blockade of AML progression.We also analyzed the counts of WBCs in the peripheral blood because elevated WBC counts are a common indication of AML. The results showed that the counts of WBCs in the peripheral blood in mice treated with Ara-C@HFn were significantly lower than those in the Ctrl group and free Ara-C group Fig. a. In addls) Fig. b, c. Incice Fig. d, e. To In general, the above results indicate that Ara-C@HFn has a better AML blockade effect than free Ara-C.Although many previous studies have reported that HFn has good biosafety in vivo , 30, 31,The above results confirmed our hypothesis based on the analysis of data obtained from the GEO database; that is, the expression of TFRC in AML cells treated with Ara-C was not significantly decreased, which enables the effective inhibition of the development of AML cells by Ara-C@HFn. This result also motivated us to analyze the prospect of applying HFn nanocages by loading drugs for AML treatment based on the relevant data from the GEO database. Initially, we analyzed the clinical data and RNA sequencing data obtained from 81 AML patients GSE165656) and found that there was no significant correlation (positive or negative correlation) between the sex or age of diagnosed AML patients and the TFRC expression level in AML cells  in all relevant clinical scenarios. The results of this study suggest that using existing open-source public databases, such as GEO and The Cancer Genome Atlas (TCGA), to summarize the relevant data can also provide useful guidance for research designs, which will greatly accelerate the process of future drug development. We believe that this research method of mining and sorting data from public databases as used in this study is expected to accelerate the process through which we develop antitumor drugs.In conclusion, after determining that the expression of TFRC in AML cells from AML patients did not significantly decrease after treatment with Ara-C, we generated Ara-C@HFn, which can be used to deliver Ara-C to AML cells specifically by recognizing TFRC that is expressed on AML cells at a high level. Ara-C@HFn had significantly higher suppression efficiency in AML cells than free Ara-C in vitro or in vivo, and effectively reduced the leukemia burden in AML mice. Therefore, Ara-C@HFn is very promising for clinical application in the future, as it has good biocompatibility and an excellent antineoplastic effect in AML cells, as observed in this study. Furthermore, by analyzing the sequencing data obtained from clinical samples from the GEO database, we identified a variety of related drugs that did not cause significant downregulation of TFRC expression in AML cells, as well as 2 individual drugs and 1 combination drug that caused significant downregulation of TFRC expression in AML cells, which provided important reference data for our further efforts to develop a targeted drug delivery system based on HFn.Additional file 1:Fig. S1 Characterization of TFRC gene expression in cells obtained from different sources. a The expression levels of TFRC in bone marrow endothelial progenitor cells isolated from AML patients or healthy donors . *p<0.05, t test. b The expression levels of TFRC in bone marrow immune cells isolated from AML patients or healthy donors . *p<0.05, t test. c TPM changes in TFRC gene expression in AML cells based on RNA-seq data after treatment with Ara-C . *p<0.05, t test. d FPKM changes in TFRC gene expression in AML cells based on RNA-seq data after treatment with Ara-C . *p<0.05, Student‑Newman‑Keuls test. TPM, transcripts per million; FPKM, fragment counts per kilobase of transcript sequence per million base pairs sequenced. Fig. S2 Assessments of intracellular internalization and cytotoxicity of Ara-C@HFn on HL-60 cells blocking with anti-TFRC antibodies. a, b Representative confocal laser scanning microscope images of Ara-C@HFn (Cy5 labeled) endocytosed by normal HL-60 a and anti-TFRC antibody blocked HL-60 b cells . c Relative viability analysis of normal HL-60 and anti-TFRC antibody blocked HL-60 cells after treatment with different concentrations of Ara-C@HFn for 48 h (n=4). d The kinetics of Ara-C release from Ara-C@HFn in vitro at pH 5.0 and pH 7.0 at 37 ℃ (n=3); e Percentage analysis of the G0/G1, S and G2/M phases of the cell cycle in HL-60 cells treated with free Ara-C or Ara-C@HFn (n=3). f Ratio analysis of the percentage of S phase and G0/G1 phase of the cell cycle in HL-60 cells after treatment with free Ara-C or Ara-C@HFn (n=3). *p<0.05, Student‑Newman‑Keuls test. Fig. S3 Assessments of Ara-C@HFn targeting ability in leukemia cells in vivo and expression characteristics of TFRC in AML cells after Ara-C treatment. a Flow cytometry analysis of Cy5-Ara-C@HFn binding to cells extracted from the peripheral blood, hindlimbs or spleen of leukemia-bearing mice (n=3). b Percentage of hCD45+ cells from HFn+ cells extracted from the peripheral blood, hindlimbs or spleen of leukemia-bearing mice (n=3). c TFRC expression in AML cells (hCD45+ cells) in peripheral blood obtained from mice in the three treatment groups (n=4). Fig. S4 Biosafety evaluation of Ara-C@HFn. a–c WBC, PLT and RBC counts in peripheral blood obtained from mice treated with PBS, free Ara-C or Ara-C@HFn (n=3). *p<0.05; **p<0.01; Student‑Newman‑Keuls test. d The concentration of HGB in peripheral blood obtained from mice treated with PBS, free Ara-C or Ara-C@HFn (n=3). e Histopathological analysis of tissue sections from mice treated with PBS, free Ara-C or Ara-C@HFn (n=3). Fig. S5 Characteristics of TFRC expression in AML patient-derived leukemia cells in various clinical scenarios. a TPM expression of TFRC in AML cells with different expression levels of GPR56 or CD34 based on RNA-seq data . b–d The TFRC levels in AML cells from newly diagnosed and relapsed AML patients b, GSE156008, n=9 in the newly diagnosed group and n=15 in the relapsed group; c, GSE199451, n=11; d, GSE153348, n=6). e, f The TFRC levels in AML cells from the CR group and non-CR group (e), GSE103424, n=18; f GSE164894, n=15 in the CR group and n=9 in the non-CR group). (g) The TFRC levels in AML cells obtained from progressor and non-progressor groups among AML patients administered combined venetoclax and azacitidine treatment . h TPM expression of TFRC in AML cells of non-responder, partial responder and responder groups among AML patients treated with fedratinib . TPM transcripts per million, CR complete remission, non-CR noncomplete remission.


Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and mammals. During apoptosis, the exposure of phosphatidylserine to the outer surface of a cell is driven by the membrane scramblases and flippases, the activities of which are regulated by caspases. Cells undergoing necrosis, a kind of cell death frequently associated with cellular injuries and morphologically distinct from apoptosis, were initially believed to allow passive exposure of phosphatidylserine through membrane rupture. Later studies revealed that necrotic cells actively expose phosphatidylserine before any rupture occurs. A recent study in C. elegans further reported that the calcium ion (Ca2+) plays an essential role in promoting the exposure of phosphatidylserine on the surfaces of necrotic cells. These findings indicate that necrotic and apoptotic cells, which die through different molecular mechanisms, use common and unique mechanisms for promoting the exposure of the same “eat me” signal. This article will review the mechanisms regulating the exposure of phosphatidylserine on the surfaces of necrotic and apoptotic cells and highlight their similarities and differences.Phosphatidylserine (PS) is a lipid component of the plasma membrane. It is asymmetrically distributed to the inner leaflet in live cells. In cells undergoing apoptosis, phosphatidylserine is exposed to the outer surfaces. The exposed phosphatidylserine acts as an evolutionarily conserved “eat-me” signal that attracts neighboring engulfing cells in metazoan organisms, including the nematode  These p (TAT-1) , whereas (TAT-1) . Both TAn tested . The cleof CED-8 . Whetherating it , castingC. elegans necrotic neurons expose PS while maintaining the integrity of the plasma membrane , allowing Ca2+ to enter the cytoplasm through this channel  mutant embryos, it was observed that the cytoplasmic Ca2+ level increases in the touch neurons before cell swelling, a feature of necrosis, prior to PS exposure on necrotic cell surfaces 2+ and the levels of PS exposure. The endoplasmic reticulum (ER) is a major intracellular Ca2+ storage pool  mutants , the Ca2+ reuptake pump on the ER membrane  The Ca2+ leaked into touch neurons through the mutant DEG/ENaC channel in the mec-4(d), unc-8(d), or deg-1(d) mutant strains is necessary but not sufficient to induce PS exposure or necrosis; instead, the Ca2+-induced Ca2+ release from the ER, which further elevates the cytoplasmic Ca2+ level, is necessary for the induction of PS exposure and necrosis, and 3) the induction of PS exposure is independent of the induction of necrosis  mutations, results in the excitotoxic necrosis of neurons (mec-4(d) mutations, this mutation induces necrosis and PS exposure in a manner independent of the contribution of the ER Ca2+ pool  dominant mutation causes this channel to remain open constitutively (deg-3(d), a dominant mutation in trp-4, which encodes a transient receptor potential (TRP) channel, another Ca2+ channel (trp-4(d)-induced necrosis is dependent on the ER Ca2+ release (trp-4(d)-induced PS exposure is not  and trp-4(d), suggesting that the Ca2+-induced PS exposure is dependent on ANOH-1 (anoh-1(−); mec-4(d) double mutant larvae, necrotic touch neurons linger much longer before being engulfed, presumably as a result of the reduction of PS exposure activity  to “maintain” the exposed PS on the surface of necrotic cells. 2+-ionophore is quickly internalized when the ionophore is removed. Yet, in ATP11A−/−ATP11C−/- double deletion cells, PS remains on the outer leaflet persistently. These observations indicate that cytoplasmic Ca2+ regulates flippase activities. Together, all the above information indicates that in necrotic cells, the increase of the cytoplasmic Ca2+ level might activate scramblase(s) and inactivate flippase(s) simultaneously to achieve continuous PS exposure. The mechanisms of how cytoplasmic Ca2+ induces PS exposure on necrotic cells need much further investigation.In addition to the scramblase activity, Ca2+ influx and PS exposure occur when cells undergo necroptosis, PS exposure does not require either Ca2+ influx or caspase activity  in living cells, or that CED-7 can be activated by multiple upstream signals. Nevertheless, both common and differential mechanisms are utilized to regulate PS exposure on dying cell surfaces. Interestingly, necroptotic cells and certain types of cells induced to undergo necrosis by ATP seem to apply specific Ca2+-independent mechanisms for PS exposure. Necrosis can be induced by various kinds of stimuli. The detailed mechanisms of PS exposure, which might be stimuli-specific and cell type-specific, await further investigation. Further dissecting the PS exposure mechanisms would help us understand the cell clearance mechanisms, especially in the disease context, and may serve as a critical step for future therapeutic intervention.Here we summarize a few features behind the molecular mechanisms that regulate PS exposure on cell surfaces. First, cells die of apoptosis and necrosis, two different death mechanisms, expose the same “eat me” signal–PS and likely attract the same phagocytic receptor(s). For example, in


Encephalitozoon cuniculi is a microsporidial parasite that primarily infects domestic rabbits (Oryctolagus cuniculus). It is the causative agent of encephalitozoonosis, a disease with an internationally recognized seroprevalence among rabbits. This study determines the presence, clinical manifestation, and serological status of encephalitozoonosis in pet rabbits in Slovenia using various diagnostic procedures. From 2017 to 2021, 224 pet rabbit sera were collected and tested for encephalitozoonosis with the indirect immunofluorescence assay. Immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies against E. cuniculi were confirmed in 160 (65.6%) cases. Most seropositive rabbits suffered from neurological clinical signs or signs of gastrointestinal disorders such as recurrent hypomotilities, chronic weight loss, cachexia, or anorexia, and fewer showed clinical signs related to the urinary system or phacoclastic uveitis. A quarter of the positively tested rabbits presented without clinical signs. Hematological and biochemical blood analysis confirmed that seropositive animals had elevated globulin and deviated albumin levels in comparison to the normal reference values of non-infected animals. Furthermore, rabbits with neurological clinical signs showed statistically significant higher levels of globulins and total protein. Sixty-eight whole-body radiographs and thirty-two abdominal ultrasound reports were analyzed, looking for changes in the shape or size of the urinary bladder, presence of urinary sludge or uroliths, and any abnormalities related to the kidneys . The results suggest that neurological defects in the urinary bladder caused by E. cuniculi lead to a distended urinary bladder and consequently dysuria, incontinence, urine scalding, and sludgy urine. Encephalitozoon cuniculi (E. cuniculi) is a single-celled intracellular pathogen related to fungi and classified in the phylum Microsporidia [E. cuniculi is now also considered an opportunistic pathogen in immunocompromised people  causing multiple syndromes, such as encephalitis, keratoconjunctivitis, nephritis, hepatitis, myositis, peritonitis, sinusitis, and pneumonia [Oryctolagus cuniculus), which are considered the main host [E. cuniculi infection is ubiquitous in rabbits [sporidia . It is tsporidia . E. cunineumonia . Additioain host ,5,6. Theain host ,7,8. The rabbits ,10.E. cuniculi [In rabbits, the central nervous system, kidneys, and eyes are usually predilection sites of infection with cuniculi . Howevercuniculi ,12. Evencuniculi ,14,15. E. cuniculi is mediated mainly through cell-mediated responses involving CD8+T and CD4+ cells, and to a lesser degree through humoral immunity. There is typically an increase in pathogen-specific immunoglobulin M (IgM) from day 0 to day 35 post-exposure and an increase in immunoglobulin G (IgG) at 2 or 3 weeks after exposure [E. cuniculi indicates past exposure to the parasite, without any indication of the timing of infection. However, high levels of IgM can be observed in very early infection (less than 30 days after infection), reactivated infection or reinfection. High levels of both IgG and IgM suggest an acute recent infection, while high levels of IgG with non-existing levels of IgM suggest past exposure to E. cuniculi [E. cuniculi to evade the immune response of the host [The protective immunity following infection with cuniculi ,16. RabbE. cuniculi infection, even though the presence of antibodies does not confirm the organism as a causative agent of disease. Positive results can indicate chronic E. cuniculi infection [Whereas clinically manifested neurological signs can be easily recognized, the in vivo diagnosis of encephalitozoonosis is often difficult because of the high percentage of subclinically infected animals . In livenfection .E. cuniculi infection in the pet rabbit population in Slovenia is unknown. In this article, the results of the data analysis of (suspected and diagnosed) E. cuniculi infections of domestic rabbits in Slovenia between 2017 and 2021 are presented. The main purpose was to obtain an overview of the clinical presentation and serological status of E. cuniculi infection in Slovenia. Furthermore, the results of various diagnostic procedures (particularly indirect immunofluorescence assay (IFA), blood analysis, radiographic imaging, or ultrasound examination) were used, and their effectiveness was rated for further information. The information gathered should aid clinicians through easier and better evaluation of clinical signs (in possible combination with other diagnostic procedures) to determine whether a rabbit could have encephalitozoonosis.To date, the status and prevalence of E. cuniculi infection with the IFA. If needed, other diagnostic methods to assess their general health condition were also performed, such as blood analysis, radiography, and abdominal ultrasound.The data from 224 pet rabbits were included in a retrospective study of encephalitozoonosis in Slovenia. All rabbits were companion animals, living in the same household as their owners. The majority of the rabbits were mixed breeds, weighed approximately 2 kilograms, and were between 5 months and 10.5 years old, with a mean age of 4.98 years. The animals were presented to the University Clinic of Birds, Small Animals, and Reptiles  between January 2017 and July 2021. The animals were either brought to the clinic due to illness or for preventive clinical examination and testing for encephalitozoonosis. All the admitted rabbits were tested for n = 160), meaning they had specific immunoglobulin M (IgM) or immunoglobulin G (IgG) antibodies against E. cuniculi, were used for further statistical analysis. The positive population was our main study subject of interest. Animals that tested negative (n = 64) were not included in further statistical analyses or division into clinical signs groups because encephalitozoonosis was not considered the main cause of their clinical condition.Information about different types of clinical signs, laboratory results from hematological and biochemical assays, ultrasound reports and radiographic image analyses of animals that tested positive (n = 160) were divided into five groups. Group 1  was specified as the preventive health check group. Animals in this group showed no obvious clinical signs but had elevated IgM and/or IgG. Group 2  consisted of animals with neurological clinical signs including vestibular disease , tremors, or weakness of hind legs. In Group 3 (urinary system signs), signs of urinary tract disorders were seen, including polyuria, polydipsia, dehydration, anorexia, weight loss, sludgy urine, urine scalding, and difficulty urinating. Group 4 (eye disorder signs) consisted of rabbits with signs of phacoclastic uveitis . Group 5  included rabbits with recurrent gastrointestinal hypomotilities, cachexia, anorexia, and chronic weight loss that could have been caused by a chronic E. cuniculi infection.All studied rabbits that were presented to the clinic underwent a prompt clinical examination. Depending on their clinical status, seropositive animals . Anti-rabbit IgM and IgG antibodies, conjugated with Fluorescein-Iso-Tio-Cyanate (FITC), were added . Each serum was tested and titrated from 1:40 to 1:1280. Conjugates were added according to manufacturers’ instructions. With an intravenous catheter, blood was collected from the marginal ear vein of all 224 rabbits. BD Microtainer tubes containing Clot Activator / SST™ Gel were used for collecting blood . After centrifugation at 2000 rpm for 10 min, sera were collected and tested for the presence of specific antibodies against Blood samples from seropositive rabbits that needed additional diagnostics to assess their general health were tested at the laboratory of the Small Animal Clinic of the Veterinary Faculty, University of Ljubljana, for a hematological and biochemical analysis. For both the hematological and biochemical assays, the individual blood samples were collected in BD Microtainer blood collection tubes containing lithium heparin as an anticoagulant . Since the proceeding of diagnostic procedures on the animals depended on the owner’s permission and their financial limitations, unfortunately not all seropositive rabbits had their blood samples taken and sent to the laboratory for hematological and biochemical assays. Consequently, the number of animals for which hematological and biochemical data were available varied.E. cuniculi-positive rabbits were analyzed using an automated laser-based ADVIA 120 hematology analyzer with a species-specific setting for rabbits in the multi-species software developed by the manufacturer of the analyzer . Factory software settings were used without adjustments or modifications. The analyzer utilizes the principle of automated cytochemistry coupled with flow cytometry. The hematological parameters included white blood cell (WBC) count, red blood cell (RBC) count, platelet count (PLT), procalcitonin (PCT), number of neutrophils (NEUT), lymphocytes (LYMPH), monocytes (MONO), eosinophils (EOS), basophils (BASO), large unstained cells (LUC), hematocrit (HCT), and hemoglobin (HGB). The distribution of hematological assays between the groups of clinical signs was as follows: 17 hematological analyses from clinically healthy animals, 42 hematological analyses from rabbits with neurological clinical signs, 9 hematological analyses from rabbits with urinary system signs, 28 hematological analyses from rabbits with gastrointestinal signs and zero hematological analysis from rabbits with eye disorder signs.For hematological analyses, the blood samples of 96 E. cuniculi-positive rabbits underwent a biochemical analysis with the portable VetScan® VS2 Chemistry Analyzer . VetScan Comprehensive Diagnostic Profile Reagent or (mainly) VetScan Equine Profile Plus reagent rotors  were used. For the statistical analyses, we focused on the following parameters: albumins (ALB), globulins (GLOB), alkaline phosphatase (ALP), alanine aminotransferase (ALT), amylase (AMY), aspartate aminotransferase (AST), blood urea nitrogen (BUN), calcium (Ca), sodium (Na), creatine kinase (CK), creatinine (CRE), gamma-glutamyl transferase (GGT), glucose (GLU), potassium (K), inorganic phosphorus (PHOS), total protein (TP), and total bilirubin (TBIL). The distribution of biochemical assays between the groups of clinical signs was as follows: 19 biochemical analyses from clinically healthy animals, 49 biochemical analyses from rabbits with neurological clinical signs, 10 biochemical analyses from rabbits with urinary system signs, 34 biochemical analyses from rabbits with gastrointestinal signs and only 1 biochemical analysis from rabbits with eye disorder signs.Individual blood samples of 113 n = 68) and ultrasound (n = 32). Distribution of abdominal ultrasound reports between the groups of clinical signs was as follows: 6 reports from clinically healthy animals, 14 reports from rabbits with neurological clinical signs, 4 reports from rabbits with urinary system signs, 8 reports from rabbits with gastrointestinal signs and none reports from rabbits with eye disorder signs. Distribution of radiographic images among the clinical signs groups was as follows: 8 radiographs from clinically healthy rabbits, 27 radiographs from rabbits with neurological clinical signs, 7 radiographs from animals with urinary system signs, 24 radiographs from rabbits with gastrointestinal signs and only 2 radiographic images from rabbits with eye disorder signs. For abdominal ultrasound, the Logiq S7 Expert/Pro  machine was used. Abdominal examination was performed with the 11L linear ultrasound probe. For the radiographic analysis and measurements of urinary bladder length (mm), Vet-Exam plus 7.3.0 Imaging Software  was used.If additional diagnostic procedures were necessary, individual seropositive rabbits underwent other diagnostic tests, such as abdominal radiography  were non-clinical for any type of encephalitozoonosis-linked signs and were classified into Group 1 . The other 120 seropositive rabbits (75.0%) showed various kinds of clinical signs, upon which they were divided into four further groups. Animals with central vestibular disease including clinical signs such as torticollis with head-tilt, nystagmus, imbalance, rolling or leaning to one side, or weakness of the hind legs represented 62 (38.8%) positive-tested rabbits and were classified into Group 2 . Rabbits showing clinical signs associated with urinary tract disorders represented 7.5% (n = 12) of positive-tested animals and were assigned to Group 3 (urinary system signs). Usually, clinical presentation of polyuria/polydipsia, dehydration, anorexia, weight loss, sludgy urine, urine scald, and difficulty urinating were observed. Signs of phacoclastic uveitis was observed in three seropositive rabbits (1.9%) and was therefore the least represented sign of encephalitozoonosis. These rabbits were categorized in Group 4 (eye disorder signs). Finally, clinical signs of gastrointestinal disorders  were observed in 43 seropositive rabbits (26.9%), which were categorized into Group 5 .Out of all 224 rabbit cases included in the study, the owners of 169 animals (75.4%) made a veterinary appointment because they had noticed signs of illness in their animals. The other 55 rabbits (24.6%) were tested for encephalitozoonosis at the owners’ request and were classified as overall healthy animals. With IFA, 160 (71.4%) rabbits tested positive for the presence of either IgM and/or IgG antibodies against In the table below , a displE. cuniculi, 65 female and 95 male rabbits were positive. We examined the relationship between age and infection with E. cuniculi. The relationship was tested using the χ² test. The results show that the association between age and infection with E. cuniculi is not statistically significant (χ2 (1) = 0.42, p = 0.518). We also tested the relationship between infection with E. cuniculi and the sex of the animal. Similarly, the results were not statistically significant (χ2 (1) = 0.38, p = 0.537).Out of all IFA-positive animals tested for the presence of E. cuniculi exposure. Out of 224 rabbits, 64 animals tested negative, meaning that both IgM- and IgG-specific antibodies against E. cuniculi were absent . Among all the animals that tested positive (n = 160), the results of their serological status are the following. Rabbits with both IgM and IgG antibodies  comprised 74.4% of all animals that tested positive. Thirty-six  animals only had specific IgG antibodies elevated , whereas a smaller number of rabbits (n = 5) tested positive for only IgM antibodies , which is 3.1% of the animals that tested positive. In all five groups, the number of rabbits with the combination of elevated IgM and IgG specific antibodies against E. cuniculi was the highest, whereas the presence of only IgM antibodies was the lowest or even absent in some groups.With the IFA, we were able to gather information regarding previous Information about the serological status of rabbits in individual groups is presented in p < 0.05). Furthermore, cases with neurological signs (Group 2) had higher medians of globulins and total protein compared to other groups. Due to missing values or insufficient sample size, we did not include the following parameters: ALT, AST, ALP, AMY, CK, PHOS, and GGT.Out of the seropositive group, blood samples from 96 and 113 rabbits were available for hematological and biochemical analyses, respectively, to investigate possible dissimilarities between the rabbits in the five different groups. The differences in hematological parameters between groups were tested using the Kruskal–Wallis test. The results show that the differences in the distribution of hematological parameters were not statistically significant between groups. The same test was also performed for the biochemical blood parameters between the five groups. These results show that the differences in the distribution of globulins (GLOB) and total protein (TP) between groups were statistically significant  by James W. Carpenter [In addition, we tested whether the median values of the hematological or biochemical parameters for rabbits infected with arpenter  and the n = 27) or urinary system disorders (n = 7) were examined especially closely for any changes in the size of the urinary bladder, the presence of urinary sludge or uroliths, or any abnormalities related to the kidneys .Abdominal radiographs of 68 rabbits with confirmed encephalitozoonosis were available during our data research. Radiographic images in the latero–lateral and ventro–dorsal view were used. Images of rabbits with neurological clinical signs . In addition, all five animals with urinary sludge simultaneously had a largely distended urinary bladder. The measured lengths of the urinary bladder of rabbits with neurological or urinary system signs are listed in Of all the rabbits, no animal had any obvious alterations of the kidneys seen on the radiographs. In addition, no nephroliths or uroliths were discovered. Four rabbits (14.8%) with neurological clinical signs had a moderate to large amount of urinary sludge present. Twelve rabbits (44.4%) with neurological clinical signs had a lightly to moderately enlarged/distended urinary bladder. In rabbits with urinary system disorder, the presence of urinary sludge was stronger , the measurements from Banzato et al. were used [The abdominal ultrasound examination reports of 32 ere used . DependiE. cuniculi IgM and/or IgG serological-positive rabbits (n = 160), there was a strong presence of both neurological clinical signs (38.8%) and, as now registered as nonspecific disease presentations, gastrointestinal disorders (27.5%), such as recurrent hypomotilities, chronic weight loss, cachexia, or anorexia. In the setting of an active E. cuniculi infection, focal granulomatous infiltrates can be histologically observed in various organs and tissues, also in the intestines [n = 12) and central nervous system (n = 62)) can cause nausea, apathy, and anorexia, and consequently development of gut hypomotility [E. cuniculi-seropositive rabbits that present to the clinic with repetitive gastrointestinal hypomotility with no other identified problems respond well to treatment with fenbendazole  and metamizole . After the treatment, the frequency of hypomotilities successfully decreases.This study examines encephalitozoonosis among Slovenian rabbits, showing that its clinical presentation is quite variable. In testines . The effmotility . Furthermotility ,21. We bE. cuniculi-related disorders, such as stomatology problems, other bacterial, viral, or parasitic pathogens , improper diet, and so on [Toxoplasma gondii infection that would show some similar neurological clinical signs as E. cuniculi infection. Any changes in the rabbits’ diet, consummation of toxins or other stressful events in the animals’ life can also lead to gastrointestinal problems. After gathering information about these topics, some differential diagnosis mentioned above can be eliminated. With a careful clinical examination, the veterinarian can once again exclude an ear infection as the causative for neurological signs, stomatology problems or fur shedding that could lead to hypomotilities, and any  discharges that could be present in, for instance, an infection with Pasteurella multocida. With the microscopic examination of feces, Eimeria spp. can easily be ruled out as a causative of gastrointestinal disorders, apathy, dehydration, or lethargy. After the conversation with the owner and clinical examination of the rabbit, the clinician should have gathered enough information about the animals’ clinical status to see whether there is still a chance for an E. cuniculi infection. Further diagnostic methods, such as ultrasound examination of the abdomen to eliminate any (tumorous) growths on the organs and any obvious pathology or inflammation of the urinary bladder, should be used. Radiographic images of the body in the latero–lateral and ventro–dorsal view are advised to be taken to exclude any pathological findings on the spine or other bone structures that could cause neurological clinical signs or chronic pain and stress related to it. Radiographic images of the rabbits’ skull could give us an insight into possible stomatology problems, or any tympanic bullae pathology, which can cause vestibular signs  [E. cuniculi some other disguised health condition is ongoing in the animals’ body. Certainly, it is important to rule out other causes that could lead to gastrointestinal or other nd so on ,22. A clad tilt) . All of E. cuniculi infection (25.0%) with none of the clinical signs mentioned in this study. These animals were mainly brought to the clinic for testing by owners, who are aware of encephalitozoonosis and its risks. This might show that there is a rising awareness of E. cuniculi among owners of pet rabbits, which is of great importance because it is a potential zoonosis [Signs of phacoclastic uveitis (1.9%) and especially urinary tract problems (7.5%) were not as strongly represented clinical signs among our tested rabbit population in comparison to the number of rabbits suffering from other clinical signs in the study. Similar to our findings, in other studies the eye disorder signs and urinary system signs were also less represented in the rabbit study population than, for example, neurological clinical signs ,24,25. Tzoonosis . The infE. cuniculi infection, which is in agreement with studies performed on domestic rabbits [This study confirmed that the rabbit’s age and sex had no significant effect on  rabbits ,11,27.E. cuniculi are an indicator of past exposure to E. cuniculi or resolved infection, the specific IgM antibodies reflect an acute, reactivated infection or reinfection [E. cuniculi antibodies present. The detection of IgM antibodies in combination with clinical signs of encephalitozoonosis requires administration of the proper antimicrosporidial therapy [E. cuniculi antibody titers and the severity of the clinical signs in positive rabbits.Simultaneous testing of both IgM and IgG offers an indication of the animal’s infection status. Whereas IgG antibodies against nfection ,22. The nfection . In addi therapy . Therefo therapy . For furE. cuniculi--positive animals to the reference values of healthy rabbits. The results showed that rabbits with neurological signs (Group 2) had statistically significant higher levels of globulins and total protein in comparison to other clinical signs/groups. Similar, rabbits with E. cuniculi infection showed elevated globulin and deviated albumin levels in comparison to normal upper and lower reference values of non-infected animals. These results were not unexpected since some previous studies [E. cuniculi infections may appear clinically acute, they are typically a chronic inflammatory disease process; hence a rise in gamma globulins is expected in active cases [E. cuniculi serum samples and referral values of healthy animals or between the same parameters in different groups. Even though it was expected that Group 3 animals (urinary system signs) would have higher parameters connected to urinary tract problems, such as creatinine (CREA), urea (BUN), phosphorus (P), sodium (Na), potassium (K), and so on, their levels surprisingly exhibited non-significant changes when compared to the same parameters in other groups or values from healthy rabbits. The reason for these results probably lies in the small number of animals with renal clinical signs in the study. On the other hand, previous studies show that elevations in serum/plasma creatinine can occasionally be noted in rabbits with encephalitozoonosis, but these findings are not reliable indicators of E. cuniculi infections [Hematological and biochemical analysis was carried out to compare different parameters between the five groups of animals and to compare the parameters of  studies ,29 showe studies . In addive cases . In our fections ,14.n = 32) and radiographic images (n = 68), we mainly focused on finding any potential changes in the size of the urinary bladder or the presence of urinary sludge, uroliths, or abnormalities related to the kidneys . Radiographs in fact did not show any obvious alterations of the kidneys or the presence of nephroliths or uroliths. Some interesting results were found regarding active encephalitozoonosis serological status and changes to the urinary bladder. To date, there have been no studies investigating the direct clinical impact of E. cuniculi on the urinary bladder size or distension in rabbits. Namely, it is suspected that encephalitozoonosis is linked to formation of sludge, incontinence, and dysuria among other kidney-related pathologies [E. cuniculi-associated neurologic effects could disturb the normal urinary bladder function/contractions and consequently distend the urinary bladder wall because urine is not passed normally. Unfortunately, no reference values of a normal and healthy rabbit urinary bladder size or volume to compare with our measurements were found in the literature. Consequently, we used the calculated urinary bladder mean size from the group of clinically healthy and E. cuniculi-negative rabbits included in our study. The calculated mean length of 11 urinary bladders was 32.2 mm. The mean weight of the animals was 2.3 kg. Urinary bladders that were larger than the calculated control group mean size were perceived as enlarged/distended, especially if the rabbit was suffering from dysuria, incontinence, urine scalding, and sludge urine. Even though our study population of E. cuniculi-positive rabbits with largely distended urinary bladders and clinical signs associated with this was quite small, serologically all the animals had an active form of encephalitozoonosis. Certainly, other neurological and urinary tract diseases can also lead to incontinence, difficulty urinating, and consequently urinary retention in the urinary bladder. A veterinarian must always consider this possibility and decide for a suitable diagnostic method based on the rabbits’ clinical signs, medical history, and the owners’ anamnesis to rule out other causes for the observed clinical presentation. Further studies about a possible link between encephalitozoonosis and its effects on the urinary system should be undertaken to determine whether our theory about bladder enlargement could be of any significance. A pathological investigation of deceased seropositive rabbits that suffered from urinary system signs to obtain additional information about the urinary bladder status would be useful as well.With the ultrasound examination . One rabbit had nephroliths in the left kidney and a slightly enlarged right kidney. Its serological status showed active encephalitozoonosis . Finally, two rabbits had some renal pathology present  but did not have elevated IgM antibodies . The number of available ultrasound reports and the study population are both unfortunately too small to draw any conclusions between the correlation with active ongoing encephalitozoonosis and the pathologies mentioned above. Further research should be dedicated to this topic. Regarding the correlation between the size of kidneys and encephalitozoonosis, some authors have reported that chronic interstitial nephritis, often associated with subclinical infections caused by ney size ,32. In oE. cuniculi infection. For now, in Slovenia, the most common and reliable method is still the use of an indirect immunofluorescence assay. In the future, a more detailed observation plan and anamnesis during clinical examination could be developed, as well as a more advanced overview of some ultrasonographic or radiographic features. Abroad, the use of polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and even Western blot is increasing [Some parameters and results of the diagnostic methods used in the study  may help the veterinary clinician to raise the suspicion of encephalitozoonosis. Still, simultaneous serological testing is needed to confirm creasing . The menE. cuniculi is present in pet rabbits in Slovenia. The infected rabbits mostly suffer from neurological clinical signs and urinary tract disorders, but a high percentage of gastrointestinal disorders were also seen. More studies regarding rabbits with this clinical manifestation and pertaining serological status should be performed in the future. Some rabbits with active encephalitozoonosis that suffered from urinary tract disorders and neurological clinical signs had an enlarged urinary bladder, as seen on radiographs. We believe that there may be a positive correlation between the neurological defects caused by E. cuniculi and a distended urinary bladder that clinically manifests as dysuria, incontinence, urine scalding, and sludgy urine. A large number of positive rabbits had serologically elevated titers of IgM antibodies, which indicates an active infection, and therefore preventive serological testing for E. cuniculi in pet rabbits with or without clinical signs should be performed regularly. For clinicians, the criteria used to suspect encephalitozoonosis would be clarified as a rabbit showing neurological clinical signs, urinary system signs or gastrointestinal signs discussed in this study, with simultaneously elevated IgM and IgG antibody levels. Based on zoonotic potential for owners, knowledge about the serological status of pet rabbits is also of great importance. In conclusion, the findings of this present survey, the first performed in Slovenia, show that


Secondary acute myeloid leukemia (S-AML) patients generally have a poor prognosis, but the chromosomal aberrations of S-AML have been rarely reported. We aimed to explore the chromosomal aberrations and clinical significance in patients with S-AML.i.e., at S-AML diagnosis).The clinical characteristics and karyotypes of 26 patients with S-AML were retrospectively analyzed. The overall survival (OS) was measured from the time of the patients’ transition to AML (P < 0.05).The study included 26 S-AML patients , with a median age of 63 years . They transformed from various hematologic malignancies or solid tumors; most of them were secondary to myelodysplastic syndrome (MDS). About 62% of the S-AML patients showed chromosomal aberrations. The serum lactate dehydrogenase (LDH) level in S-AML patients with abnormal karyotype was higher than those with normal karyotype. Apart from the differences in treatment regimens, S-AML patients with chromosomal aberrations had shorter OS (S-AML patients with abnormal karyotype have higher LDH levels and shorter OS than normal karyotype patients, and the OS of hypodiploidy was much shorter than hyperdiploid. Secondary acute myeloid leukemia (S-AML) refers to AML developing either after a prior hematologic disorder, usually myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPN), or MDS/MPN . CompareSome predictors of transformation have been identified, including mutations in genes involved in growth-signaling pathways  and mutations commonly seen in AML  . Recent Some major clinical features are poor prognostic factors for AML. For instance, high WBC and/or LDH levels were identified as significant predictive features for OS . Here, wA total of 26 S-AML participants diagnosed or treated in the Second Affiliated Hospital of Anhui Medical University from January 2009 to January 2020 were collected. All the newly diagnosed S-AML participants met the 2008 or 2016 WHO criteria . In the i.e., at S-AML diagnosis). All cytogenetic analyses were carried out in a standardized fashion at the Chromosome Laboratory, Department of Hematology, The Second Affiliated Hospital of Anhui Medical University. Bone marrow specimens were prepared by the short-term culture method and the G-banding method. Twenty (20) metaphase spreads were examined per patient, if available. The International System for Human Cytogenetic Nomenclature (ISCN) was used for karyotyping  counts, white blood cell (WBC) counts, platelet counts (PLT), lymphocyte counts (LYM), mononuclear cell counts (MO), neutrophil counts (NEUT), hemoglobin (Hb), hypersensitive c-reactive protein (Hs-CRP) and lactate dehydrogenase (LDH), using the fully automated hematology analyzer Sysmex XE-2100  and the fully automated biochemical analyzer AU5831 .Participants were followed till death, loss to follow-up, or the end of the study follow-up period on July 20, 2020. OS was calculated from the time of S-AML diagnosis to the date of death or last follow-up. Medical record retrieval and telephone follow-up were performed during the study period.t-test was used to test the differences between the two groups for quantitative and normally distributed variables; the Mann–Whitney U test was used for non-parametric variables. Kaplan–Meier survival curves were used to estimate OS. Statistical analyses were performed with the IBM SPSS 25.0 software. Results were considered significant at p < 0.05.The student’s Half (n =13/26) of these 26 S-AML participants were men. In addition to unclassified AML , M2 was the most common FAB subtype in 42.3% (n =11/26) of the participants, followed by M4 , M3 , M5 , M7 . As for the diagnosis prior to AML, 57.7% (n =15/26) of the participants were secondary to MDS ), 3.8% (n =1/26) of the participants were secondary to myelodysplastic-myeloproliferative neoplasms (MDS/MPN), 11.5% (n =3/36) of the participants were secondary to chronic myeloid leukemia (CML), and 7.7% (n =2/26) of the participants were secondary to chronic myeloid monocytic leukemia (CMML), primary myelofibrosis (PMF) and rectal cancer respectively. 3.8% (n =1/26) of the participants were secondary to gastric diffuse large B cell lymphoma. The basic characteristics of 26 S-AML participants was shown in The treatment of AML is complex, and multiple subentities of AML often require different therapies. In our study, many participants were treated with decitabine in combination with other regimens. Other optional regimens such as azacytidine, IA/IAG regimen, and intrathecal injection have also been used to treat participants, depending on the patient’s condition. The detailed therapeutic regimen of 26 S-AML participants is shown in In this study, a total of 16 participants had chromosome abnormalities, 50% (n =8/16) of the participants had chromosome 5 abnormalities, 43.8% (n =7/16) of the participants had chromosome 7-8 and 11-13 abnormalities, 37.5% (n =6/16) of the participants had chromosome 9 and 22 abnormalities. Chromosome 15 and 21 abnormalities were found in 31.3% (n =5/16) of the participants. The frequencies of the other types of chromosomes were relatively low. The detail of each chromosome abnormality was shown in P < 0.05). The scatter diagram for the LDH levels between the 2 groups is shown, which only contains 23 participants  (P = 0.038) (P > 0.05). However, the OS of chromosomes 5, 7, 9, 11, 12 and 22 was statistically different from that of the normal chromosome (P < 0.05).Except for participants who were lost to follow-up, there were two deaths in the normal karyotype group and 12 deaths in the abnormal group. The median OS of normal and abnormal karyotypes was 212 days and 162 days, respectively. In addition, the abnormal karyotype included seven hyperdiploid and five hypodiploid participants, and the median OS was 211 days and 62 days, respectively. The Kaplan–Meier (KM) survival curve results showed that the OS of S-AML participants with abnormal karyotypes was shorter than those with normal karyotypes (= 0.038) . Also, c= 0.038) . KM survS-AML is a heterogeneous disease; its incidence increases with age, but therapy remains a challenge . MyelodyChromosomal aberrations are associated with progression to S-AML and deserved further study. The purpose of this study was to analyze the chromosomal aberrations of S-AML participants and further explore the factors connected with the survival and prognosis of S-AML in combination with relevant laboratory examinations. Our results indicated that most S-AML participants had abnormal karyotypes, including autosomal and sex chromosome aberrations. Abnormal changes in autosomal karyotypes were more common in S-AML participants and were closely related to survival and prognosis. Studies have demonstrated an increased incidence of abnormalities on chromosomes 5 and 7 in patients with S-AML . In our Most AML patients with chromosome number abnormalities may manifest with an increase of 1–2 chromosomes (47–48 chromosomes), known as low hyperdiploid, or rare high hyperdiploidy (49–65 chromosomes), both of which are associated with poor outcome in AML ; Sandah. HolmfelIn addition to other factors affecting OS, such as various treatment regimens, our research found that karyotypes were closely related to S-AML participants’ survival; participants with abnormal karyotypes demonstrated inferior OS compared with those with normal karyotype. What is more, S-AML participants with hypodiploidy showed worse outcomes than those with hyperdiploidy.LDH not only plays a vital role in the early diagnosis and prognosis of many solid tumors but also plays a crucial role in evaluating the severity of leukemia patients . LDH pose.g., CAG , retinoic acid) results in a higher OS rate than decitabine alone (It is of great significance to choose the appropriate chemotherapy regimen to manage patients with AML effectively. In clinical practice, an individualized treatment regimen is often tailored to the patient’s tolerance and other specific conditions. In our study, many participants were treated with decitabine in combination with other regimens. Decitabine is a demethylation agent that is effective and safe in older patients with AML; its combination with other regimens (ne alone . It is tThere are some limitations to our study. Firstly, the abnormality of sex chromosomes may relate to the survival and prognosis of S-AML, but no definite conclusion could be drawn because of the small number of sex chromosome aberrations in our study. A small number of patients did not get 20 metaphase spread, and there may be chromosomal abnormalities that have not been found, leading to a certain bias in the results. Apart from this, the accurate information of all participants could not be obtained through telephone follow-up in this study, which may interfere with the experimental results. Additionally, this study is a single-center retrospective study; the number of included cases was relatively small, so further study expanding the sample size is needed to validate our results. Moreover, with the heterogeneity of the individualized treatment among AML participants, the treatment regimens could constitute an important source of limitation, which may have influenced the results.In conclusion, our research highlights chromosomes and LDH contributions to the poor prognosis of S-AML participants. Also, the abnormality of sex chromosomes may be associated with the survival and prognosis of S-AML participants. Understanding the multifactorial contributions will lead to more precise risk classification and treatment strategies. More factors related to the survival and prognosis of S-AML need to be explored, which may contribute to monitoring the progression of the disease, early diagnosis, and improved treatment.10.7717/peerj.15333/supp-1Data S1Click here for additional data file.


A key aspect of the DMD clinical phenotype is dilated cardiomyopathy, affecting virtually all patients by the end of the second decade of life. Furthermore, despite respiratory complications still being the leading cause of death, with advancements in medical care in recent years, cardiac involvement has become an increasing cause of mortality. Over the years, extensive research has been conducted using different DMD animal models, including the mdx mouse. While these models present certain important similarities to human DMD patients, they also have some differences which pose a challenge to researchers. The development of somatic cell reprograming technology has enabled generation of human induced pluripotent stem cells (hiPSCs) which can be differentiated into different cell types. This technology provides a potentially endless pool of human cells for research. Furthermore, hiPSCs can be generated from patients, thus providing patient-specific cells and enabling research tailored to different mutations. DMD cardiac involvement has been shown in animal models to include changes in gene expression of different proteins, abnormal cellular Ca2+ handling, and other aberrations. To gain a better understanding of the disease mechanisms, it is imperative to validate these findings in human cells. Furthermore, with the recent advancements in gene-editing technology, hiPSCs provide a valuable platform for research and development of new therapies including the possibility of regenerative medicine. In this article, we review the DMD cardiac-related research performed so far using human hiPSCs-derived cardiomyocytes (hiPSC-CMs) carrying DMD mutations.Duchenne muscular dystrophy (DMD) is an X-linked progressive muscle degenerative disease caused by mutations in the  These results demonstrate that impaired Ca2+ handling is involved in DMD pathophysiology at the tissue level.Chang and colleagues  devised 2+] and increased transient peak, but overall decreased amplitude compared to healthy cells. DMD cells also exhibited slower rates of rise and decay of Ca2+ transients. These results emphasize once more abnormal Ca2+ handling as a key pathophysiological feature of DMD. Consistent with our previous report [Bremner and coauthors  measureds report , DMD EHT2+ handling machinery and adaptation to changes in substrate stiffness. Analysis of Ca2+ transients revealed that DMD cardiomyocytes exhibited an initial smaller Ca2+ transient amplitude and a smaller increase in the transient amplitude with maturation compared to healthy cardiomyocytes. Additionally, post-rest contraction potentiation was lower in DMD compared to healthy cardiomyocytes, indicating lower SR calcium storage and release. Ca2+/calmodulin-dependent protein kinase II (CaMKII) levels were higher in DMD compared to healthy cardiomyocytes; consequently, the RyR2 CaMKII phosphorylation site S2814, was more phosphorylated. This increased phosphorylation may have accounted for the SR Ca2+ leak, resulting in the reduced Ca2+ transient amplitude in DMD cardiomyocytes. Compared to healthy cells, DMD hiPSC-CMs exhibited reduced Ca2+ transient amplitude on both soft and stiff substrates. Furthermore, while healthy cardiomyocytes displayed an accelerated decay of Ca2+ transient amplitude when cultured on a stiffer substrate, DMD cells did not differ in their kinetics between the substrates, indicating failure to adapt normally to differences in tissue stiffness. These findings demonstrate the abnormal Ca2+ handling of DMD cardiomyocytes and specifically the inability to adapt their Ca2+ homeostasis in response to changes in ECM stiffness, which may be a key factor of the DMD cardiac pathophysiology.Pioner et al.  investigATP channel opener and an antioxidant [2O2-induced stress related LDH release and cell death to levels similar to healthy cells, through a mechanism involving the NO–cGMP pathway and KATP channel opening. Nicorandil also decreased ROS levels and maintained mitochondrial membrane integrity following oxidative stress, an effect mediated via increase of SOD2 expression which converts superoxide to hydrogen peroxide. Lastly, nicorandil was also shown to decrease cytosolic hydrogen peroxide production in DMD hiPSC-CMs. Although further research is required, these findings provide evidence that nicorandil may have a potential to protect against stress-induced cardiac involvement in DMD.Emerging evidence from recent years suggests that metabolic and mitochondrial dysfunction play prominent roles in DMD pathophysiology ,136,137.ioxidant ,140,141 mdx mice, rendering it a potential cardiomyopathy-sensitive target in DMD. Interestingly, in DMD hiPSC-CMs, miR-339-5p-containing exosomes were found to be involved in stress-related mitochondrial-dependent cell death. Specifically, miR-339-5p, known to directly bind to MDM2, a p53 regulator leading to its downregulation [Gartz and colleagues  investiggulation , was alsmdx mice [Ca,L density, although, similarly to our findings, they observed arrhythmogenic firing patterns including DADs and oscillatory prepotentials (OPPs). Another finding in line with ours was APD prolongation in DMD cells. Cell death, measured by flow cytometric analyses using annexin V and 7-amino-actinomycin D (7AAD), revealed that DMD cardiomyocytes underwent accelerated cell death compared to healthy cells. Furthermore, DMD cells had a higher ROS content compared to healthy cardiomyocytes. Treatment with N-acetyl-L-cysteine (NAC), ataluren (PTC124) and idebenone increased DMD cell survival and reduced ROS levels. By means of JC-1, a fluorescent voltage-sensitive dye with membrane-permeant fluorescent lipophilic cationic properties, Lin and coworkers measured mitochondrial membrane potential. Similar to Lin et al. [Duelen and coauthors  investigmdx mice ,106, DMDn et al. , DMD hiPn et al. ,156,157,Our group investigated various metabolic and bioenergetic aberrations in DMD cardiomyocytes . We firsde novo mutations with no familial background [Current treatments for DMD can at best slow the disease course and alleviate some symptoms ,159,160.ckground ,163, predystrophin gene. Zatti and coauthors [dystrophin into DMD hiPSC-CMs; these authors found that differentiated cardiomyocytes expressed all dystrophin isoforms and displayed corrected protein localization, in addition to normal Ca2+ transients. Choi and colleagues [dystrophin gene expression profiles upon HAC delivery into DMD cardiomyocytes. Dick and coauthors [dystrophin expression in DMD cardiomyocytes. Additionally, Howard et al. [Different methods can be used to attempt genetic repair . Early eoauthors  successflleagues  also repoauthors  chose mid et al.  found thd et al. . In addid et al. ,171.Another approach to correct DMD mutations is by editing the mutated gene directly. Gene editing requires a break in the DNA sequence at a specific position, followed by repair, which includes replacement of excised DNA with a new sequence. Cellular DNA repair mechanisms include two options ,173,174.Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) endonuclease comprises an anti-bacteriophage bacterial system . CRISPR Zinc finger nucleases (ZFNs) are a group of enzymes capable of cleaving specific sites in DNA. ZFNs consist of DNA-binding zinc finger arrays and a FokI-based endonuclease . DespiteXanthomonas bacteria which are capable of plant gene expression alteration [Transcription activator-like effector nucleases (TALENs) are based on proteins produced by teration ,183. Likteration ,185.To achieve safeness and effectiveness, the process of introducing an exogenous endonuclease alongside a DNA template must be highly precise with minimal off-target effects. To date, three main systems are in use for preforming DNA DSBs and editing:Off-target effects are a major concern when considering the clinical application of gene editing. These include possible disruption of oncogenes, tumor suppression genes and DNA repair genes ,186,187.dystrophin provided sufficient membrane stability. Furthermore, miR-31 levels, which were reportedly high in DMD cells [scid IL2Rgamma (NSG) mdx mice, and dystrophin and β-dystroglycan were found only in the corrected and healthy cardiomyocytes. In conclusion, ORF restoration reinstated key functional cellular aspects of dystrophin, and this approach was proven as an important therapeutic strategy.To develop an approach which potentially benefits a larger target population, Young et al.  utilizedMD cells ,194, werZhang and colleagues  applied 2+ transients were analyzed in hiPSC-CMs. Indeed, release and reuptake parameters, including time to peak, Ca2+ decay rate, and transient duration, were higher in the uncorrected DMD hiPSC-CMs compared to WT and isogenic cells. Interestingly, while corrected cells with exons 3–9 deleted displayed normalized Ca2+ transient properties, corrected hiPSC-CMs carrying a deletion of exons 6–9 or 7–11 displayed only partial improvement of the Ca2+ transient parameters. These results indicate that, among the three approaches of ORF restoration, the one leading to a deletion of exons 3–9 yields the most functioning protein. Additional functional assessment was performed by means of engineered heart muscle (EHM). Consistent with the Ca2+ transient parameters results, enhanced contractile performance was observed in all corrected cell lines, with the most prominent effect in hiPSC-CMs carrying the deletion of exons 3–9. Overall, these results suggest that, of the three approaches, the correction leading to a deletion of exons 3–9 was the most effective in restoring functionality, implying the importance of ABS-3 over ABS-1 and ABS-2 of the ABD-1 domain. This also demonstrates the importance of functional testing, as they serve as a measure to evaluate the different approaches, specifically because there is no conclusive explanation as to why the correction resulting in the three ABS regions being intact (and a deletion of exons 7–11) was less effective than the approach leaving out ABS-1 and ABS-2.Kyrychenko and coauthors  comparedLong et al.  identifiC to T or G to A, and subsequently restore the ORF in DMD hiPSCs lacking exon 51 [dystrophin, thus restoring the ORF in 99.9% of dystrophin transcripts, as demonstrated by high-throughput sequencing (HTS). Corrected hiPSC-CMs released normal levels of creatine kinase (CK). Additionally, expression of miR-31 was suppressed in the corrected hiPSC-CMs, and β-dystroglycan levels were increased to levels comparable with healthy hiPSC-CMs. Importantly, contrary to CRISPR/Cas9 approaches which involve DSBs, targeted AID-mediated mutagenesis (TAM) does not require NHEJ or HDR; therefore, it is significantly less prone to indels, with higher correction efficiency [Yuan and coauthors  utilized exon 51 ,202. Theficiency . These b2+ handling in DMD hiPSC-CMs. They used phosphorodiamidate morpholino oligomers (PMOs) to skip exon 45 and restore the ORF in cells carrying a deletion of dystrophin exons 46–55. By means of Fluo-4 they measured Ca2+ transient parameters following treatment and detected increased amplitude, as well as a decrease in the rates of rise and decay, all improvements resembling healthy cells. Additionally, the number of fluorescent-detected irregular pattern events suggestive of arrhythmogenicity decreased following exon 45 skipping. Taken together, these results demonstrate the effectiveness of the exon-skipping strategy to improve functional abnormalities in DMD cardiomyocytes.Sato and colleagues  investigGT to GC sequence and restoring the ORF in DMD cells carrying an exon 51 deletion. The prime-editing strategy was used to introduce a swap of the TT to GT sequence in exon 52, resulting in ORF restoration. RT-PCR, sequencing, WB analysis, and immunocytochemistry confirmed correction and the resulting truncated protein. Next, the researchers tested the effect of correction on cardiomyocytes treated with isoproterenol. Indeed, corrected DMD cardiomyocytes displayed a reduction in arrhythmogenic Ca2+ transients compared to DMD cardiomyocytes, similar to healthy cardiomyocytes. Overall, these results demonstrate the feasibility of both genetic correction approaches in alleviating DMD cardiac pathophysiology.Chemello et al.  investigStreptococcus pyogenes Cas9 (SpCas9), which requires a dedicated adeno-associated virus (AAV) vector aside from the sgRNA vector, they used a Cas9 ortholog from Staphylococcus aureus (SaCas9), which is small enough to be packed together with sgRNA into a single AAV vector. This strategy was then utilized to deliver SaCas9-mediated single-cut gene editing to correct an exon 48–50 DMD deletion, avoiding possible unwanted genomic modifications due to the “double-cut” strategy with dual sgRNAs [2+ transient parameters in hiPSC-CMs, such as time to peak and rate of decay, were comparable to healthy cells, contrary to their abnormal elevation prior to correction. Additionally, genotoxicity analysis demonstrated no significant off-target editing, suggesting that this method is safe and effective at restoring the ORF and achieving functional restoration in DMD hiPSC-CMs.Zhang and coauthors  devised l sgRNAs ,208. TheDMD exon 44: one using a single-nucleotide insertion to achieve reframing and the other by skipping exon 45 to restore the ORF. The correction of DMD hiPSC-CMs restored cellular area, volume and sarcomere length back to normal. DMD cardiomyocytes showed Ca2+ handling abnormalities including increased release and reuptake times, in addition to impaired localization of RYR2s, all of which were corrected with the truncated dystrophin lines. To test the effects of the corrections on contractility, the researchers used a polydimethylsiloxane matrix as a substrate for seeding cardiomyocytes, allowing auxotonic contraction. By means of video detection they demonstrated in DMD cells decreased systolic force and prolonged relaxation time, which were corrected in the truncated DMD lines. Furthermore, they found that ORF restoration reversed changes in gene expression, including two hallmark DCM transcripts, NPPA and NPPB [Atmanli and colleagues  investigand NPPB , which wand NPPB . These rmdx mouse [This article reviewed the DMD cardiomyopathy-related research conducted in recent years in hiPSCs-CMs, as summarized in dx mouse ,28,212. dx mouse ,51,55,56


Alterations in free Ca2+ concentration, known as Ca2+ signals,2+ signalling has been implicated in various human disorders such as cancer, immunodeficiency, myocardial hypertrophy, and neurodegenerative diseases.2+ signals possess the capacity to convey crucial information through three‐dimensional variations in amplitude, temporal and spatial distribution  have gained widespread popularity for tracking intracellular Ca2+ signals or Ca2+‐modulated cell activities.2+‐sensing moiety (sensor) and a fluorescence reporting module (reporter). Upon binding or dissociation of Ca2+, the sensor often undergoes conformational changes that modify the spectral properties and/or brightness of the reporter,2+ signals by quantifying changes in fluorescence intensities (F)  Figure .2+ signal. Through several generations of evolution over the past two decades, GECIs have shown significantly enhanced sensitivity and diversified kinetics.2+ signals seen in excitable cells including neurons and cardiomyocytes.2+ signal amplitudes, have progressed at a comparatively slower pace since the inception of the GCaMP6 series. The dynamics of GECIs are commonly gauged by the dynamic range (DR) or the signal‐to‐baseline ratio (SBR). DR represents the responsive capacity of GECI, which is calculated from its fluorescence intensity at Ca2+‐saturated (maxF) or Ca2+‐free states (minF) and expressed as (maxF—minF)/minF.F—F0, or ΔF) over the basal fluorescence intensity (F0). Among the green GECIs utilizing circularly permuted enhanced green fluorescent protein (cpEGFP) as the reporter , has led to the creation of a new series of ultra‐sensitive mNG‐based calcium indicators, designated as NEMO Figure .10 NEM2+ signal amplitudes. Indeed, NEMO sensors could resolve Ca2+ oscillations  responses (Figure 2+ transients in neurons (Figure As aforementioned, a larger DR or SBR facilitates better resolution Cas Figure  and stors Figure  in non‐es Figure , with mu2+ transients with enhanced sensitivity. These NEMO sensors have undergone rigorous validation in diverse settings, encompassing non‐excitable cell lines, dissociated cultured rat neurons, acute mouse brain slices, in vivo two‐photon laser imaging (Figure 2+ signals within cancer cells under hypoxia conditions. By optimizing the Ca2+‐binding affinities and incorporating appropriate targeting sequences, modified NEMO indicators can be tailored to discern Ca2+ signals within acidic organelles like the endosomal compartments, as well as synaptic vesicles, secretory vesicles, and the trans‐Golgi apparatus. Looking ahead, subcellular NEMO indicators may also hold the promise of effectively resolving localized Ca2+ signals at membrane contact sites formed between the endoplasmic reticulum and mitochondria, or the plasma membrane.The extra‐large DR of NEMO indicators allows real‐time quantification of subtle Cag Figure , fibre r2+ signal reporting. Moreover, they exhibit substantially improved photostability, enabling them to withstand considerably stronger illumination, along with enhanced resistance to pH fluctuations. Collectively, these desirable characteristics position NEMO sensors as the preferred choice for monitoring Ca2+ dynamics within mammalian cells, tissues, living animals, as well as in plants.In summary, in contrast to most existing GECIs, which often trade their DR for heightened sensitivity and faster kinetics, NEMO variants manage to maintain rapid responsiveness while preserving superior dynamic ranges for accurate CaThe authors declare no conflicts of interest.


Diarrhea in weaned rabbits is one of the most common problems in early weaned rabbits, which can lead to slow growth, decreased immunity, and even death. Against the background of a total ban on the use of antibiotics in feed, the development and utilization of antibiotic substitutes have become top priorities. Mixed organic acids (MOAs), as a residue-free green additive, have the ability to enhance immunity and intestinal digestion, regulate pH, and improve intestinal microbiota. Although there are more studies on MOAs, the current domestic and international studies on them mainly focus on pigs and chickens, and the effects on rabbits are less reported. Therefore, this experiment used weaned rabbits as the experimental object to explore the effects of the dietary addition of MOAs on the growth, immunity, and intestinal function of rabbits, to provide a theoretical basis for the application of MOAs in the production of meat rabbits. The results showed that MOAs could improve the growth performance, immunity, and antioxidant ability and maintain the intestinal barrier function of weaned rabbits, which has a promising application prospect in meat rabbit breeding.p < 0.05). The serum IL-6 and liver MDA levels of Ira rabbits in the SAL group, MOA1 group, and MOA2 group were lower than those in the CON group (p < 0.05). In addition, sIgA levels in the jejunal mucosa of Ira rabbits in the SAL group and MOA1 group were increased compared with those in the CON group (p < 0.05). Compared with the CON group, the gene expression of IL-6 was decreased (p < 0.05) in the jejunal mucosa of Ira rabbits in the SAL, MOA1, and MOA2 groups, while the gene expression of IL-1β tended to decrease (p = 0.077) and the IL-10 content tended to increase (p = 0.062). Moreover, the gene expression of ZO-1 in the jejunal mucosa of Ira rabbits was elevated in the MOA1 group compared with the CON group (p < 0.05). In conclusion, dietary supplementation with MOAs can improve growth performance, enhance immune function and antioxidant capacity, and maintain the intestinal barrier in weaned Ira rabbits.The aim of this study was to investigate the effects of mixed organic acids (MOAs) on growth performance, immunity, antioxidants, intestinal digestion, and barrier function in Ira rabbits. A total of 192 weaned male Ira rabbits at 35 days of age were randomly assigned to four groups with six replicates of eight rabbits each. The rabbits in the control group (CON) were fed a basal diet, and the antibiotic group (SAL) was fed a basal diet supplemented with 60 mg/kg salinomycin. The test groups were fed a basal diet supplemented with 1000 mg/kg and 2000 mg/kg MOAs . The experiment lasted for 55 days. The results showed that the ADG of Ira rabbits in the SAL group and MOA1 group was higher than that in the CON group ( With the expanding demand for food variety, the consumption of meat rabbits in recent years has increased greatly. In addition to its food value, rabbit fur can also be used to make leather garments, which shows the promising future of the rabbit farming industry. However, diarrhea is a common problem in the process of meat rabbit breeding, causing significant losses to the rabbit breeding industry. Diarrhea in rabbits is affected by a variety of factors, such as feeding density, environmental temperature and humidity, feed formulation, and weaning . Among t+, so their pH-lowering effect is also worse than that of small molecules of organic acids [Organic acid, a common additive, is commonly used in piglet feed, poultry feed, silage, and other fields because of its good flavor and strong antibacterial effect . Accordiic acids . The secic acids . Some scic acids . It has ic acids . Currentic acids . Many stic acids  found thic acids  and promic acids . Propionic acids . In viewE. coli vaccine; at 30 days of age, 2 mL of inactivated bacillus and bordetella vaccine; and at 40 days of age, 2 mL of inactivated rabbit distemper vaccine. All of the above vaccines were administered by subcutaneous injection. The health and mental conditions of the rabbits were observed every day. All rabbits had ad libitum access to feed and water. The indoor temperature was controlled at 24 °C.A total of 192 male rabbits with similar body weight and weaned at the age of 35 ± 2 days were randomly divided into 4 treatments with 6 replicates of 8 rabbits each. The four treatments were as follows: (1) control group , (2) antibiotic group , (3) MOA1 group , and (4) MOA2 group . The basal diet was formulated according to the dietary requirements of China’s agricultural industry standards (NY/T 4049-2021). The diet  was pelleted, and its composition and nutritional levels are shown in Rabbits from each replication were weighed at 90 days old, and the total feed consumption of each replicate was recorded. The diarrhea of the rabbits was visually checked every day, and records were made. The ADFI, ADG, F/G, and diarrhea rates were calculated.g for 15 min at 4 °C. The collected serum samples were stored at −20 °C prior to being tested. After blood collection, rabbits were euthanized, and liver tissue samples were collected and stored at −80 °C for testing antioxidant-related indicators. Tissue was collected from the mid-jejunum (approximately 3 cm), and the segment was then opened longitudinally and gently rinsed with 4 °C phosphate-buffered saline (PBS). A sample of the jejunal mucosa was gently scraped with a sterile slide. The scraped intestinal mucosa was stored in liquid nitrogen and then quickly frozen at −80 °C for further analysis.On day 90, one rabbit that was close to the average weight of each replicate was selected. Blood was collected from the jugular vein and left at room temperature for 2 h, and serum was separated by centrifugation at 1000× The concentrations of interleukin-6 , interleukin-10 , interleukin-1β , and tumor necrosis factor-α  in serum were measured using ELISA kits purchased from Enzymatic Biotechnology Co., Ltd.  according to the manufacturer’s instructions. Serum samples were diluted 5-fold during the assay. Concentrations of the standards in the kit were 10, 20, 40, 80, and 160 pg/mL. More detailed steps can be found in our previous reports . The absg for 10 min, the supernatant was collected, and the 10% homogenate was then diluted to 1% with saline. As Kesik et al. [Liver tissue was accurately weighed to approximately 0.1 g, and a 10% tissue homogenate was prepared by adding 9 times the volume of saline at a ratio of liver weight (g): volume (mL) = 1:9 and mechanically homogenized  in an ice-water bath. The resulting homogenate was centrifuged at 1000× k et al.  reportedg for 10 min, and the supernatant was collected and then assayed for mucosal lipase and trypsin activities as per the instructions. For determination of α-amylase activity, a 0.1 g sample of mucosa was weighed, 1 mL of distilled water was added, and the homogenate was ground. The homogenate was poured into a centrifuge tube, and then the extraction solution was added and left to extract for 15 min at room temperature, with shaking every 5 min to allow full extraction. After extraction, the mixture was centrifuged at 1000× g for 10 min at room temperature, the supernatant was collected, distilled water was added to 10 mL, and the mixture was shaken well to obtain the amylase reserve solution. The above amylase stock solution (1 mL) was pipetted, and 4 mL of distilled water was added and shaken well to make an amylase dilution solution. The amylase stock solution and dilution solution were used for the determination of amylase activity. The absorbance was detected by the iMark™ Microplate Absorbance Reader . The above results were corrected for total protein determination using the BCA protein quantification kit (ZJ101) purchased from Epizyme Biomedical Technology Co., Ltd. .Referring to Marchioro et al. , with sog for 10 min to collect the supernatant. Total protein was determined using a BCA protein quantification kit (ZJ101) purchased from Epizyme Biomedical Technology Co., Ltd.  for correction of subsequent results. The jejunal mucosa immune factor concentrations of IL-6 (ml027844), IL-10 (ml027828), IL-1β (ml027836), and secretory immunoglobulin A  were measured using ELISA kits purchased from Enzymatic Biotechnology Co., Ltd. . The absorbance was detected by the iMark™ Microplate Absorbance Reader .The jejunal mucosa samples were homogenized using ice-cold saline (mucosa weight (g): saline volume (mL) = 1:9), and then the samples were centrifuged at 1000× ® qPCR Master Mix , and the amplification conditions were as follows: initial denaturation at 94 °C for 30 s, followed by 40 cycles of denaturation at 94 °C for 5 s, annealing at 56 °C for 15 s, and extension at 72 °C for 10 s. GADPH was used as a reference gene, and the relative mRNA expression level was calculated using the 2−∆∆CT method.The method to determine gene expression was generally the same as we previously reported . Total Rp < 0.05 was considered statistically significant, and 0.05 ≤ p ≤0.10 was taken to indicate a statistical tendency.Statistical analysis was performed using SPSS, version 26.0 . The Shapiro–Wilk test was used to check the normality of all data, and Levene’s test was used to check the homogeneity of variance. A one-way analysis of variance (ANOVA) was used for data analysis, and Tukey’s multiple range tests were used for multiple comparisons. The results are presented as the mean ± standard deviation. p = 0.051). However, there was no significant difference in ADFI, F/G, or diarrhea rate of Ira rabbits among groups (p > 0.05).As shown in p < 0.05) in Ira rabbits in the SAL, MOA1, and MOA2 groups compared to the CON group (Serum IL-6 levels were reduced (ON group .p < 0.05). In addition, the content of CAT in the livers of Ira rabbits in the MOA2 group was also higher than that in the CON group (p < 0.05).As shown in p > 0.05).As can be seen from p < 0.05), and the IL-10 content in the SAL, MOA1, and MOA2 groups showed an increasing trend (p = 0.062). In addition, the gene expression of IL-6 in the jejunal mucosa of Ira rabbits in the SAL, MOA1, and MOA2 groups was lower than that in the CON group (p < 0.05), and there was a trend of decreasing gene expression of IL-10 (p = 0.077) (As shown in = 0.077) .ZO-1 was significantly increased in the jejunal mucosa of Ira rabbits in the MOA1 group compared with the CON group (p < 0.05). However, there was no significant change in the gene expression of Occuldin and Claudin-3 (p > 0.05).As shown in Clarias gariepinus had no significant effect on its body weight gain, feed conversion, and survival rate. This may be because, compared to terrestrial animals such as pigs and chickens, aquatic animals are characterized by short and simple digestive organs, poor activity of digestive enzymes, and short retention time of food in the intestinal tract [Organic acids have been extensively studied in livestock, poultry, and aquatic animals. The results of their studies on animal growth performance vary. In an experiment by Kinza Saleem et al. , organical tract . Organic2O2, thereby reducing the damage caused by organic hydroperoxides in the body. In order to inhibit the accumulation of oxygen radicals, SOD catalyzes the conversion of superoxide anion to hydrogen peroxide, which inhibits superoxide anion from causing cellular damage. A large amount of hydrogen peroxide can damage tissue cells, and CAT catalyzes hydrogen peroxide into water and oxygen, which reduces the damage to the organism. T-AOC indicates the total antioxidant capacity of the organism [2O2 concentration, thereby alleviating oxidative stress in broilers [Antioxidant capacity can reflect the health status of the organism, and the oxidative and antioxidant systems in the organism are jointly involved in regulating the growth and elimination of free radicals . Oxidatiorganism , and theorganism . In the organism , which cbroilers . Therefobroilers .The intestinal tract is the main place for digestion and absorption of nutrients in rabbits and an important gateway to resist the invasion of foreign pathogenic microorganisms . TherefoIntestinal digestive enzyme activity is an important indicator of intestinal digestive capacity . Lipase,The mortality rate of rabbits caused by diarrhea is extremely high. Since the banning of antibiotics in feed, the mortality rate of diarrhea caused by bacterial enteritis in rabbits accounts for the main factor, so the important guarantee to improve the survival rate of rabbits is to strengthen the prevention of bacterial enteritis in rabbits. Bacterial enteritis is due to microorganisms or pathogens invading the intestine, stimulating immune cells, and leading to the production of a large number of inflammatory cytokines and mediators. Inflammatory molecules will destroy the tight junctions, exacerbating the damage to the intestinal mucosal barrier function . TherefoThe intestinal mucosal immune barrier is mainly composed of intestinal-associated lymphocyte tissues and diffuse immune cells . When anThe intestinal mechanical barrier is composed of intestinal mucosal epithelial cells and intercellular tight junctions . InterceIt can be seen that MOAs maintain both immune and mechanical barriers in rabbits, which lays a theoretical foundation for their use in rabbits against bacterial enteritis in breeding production. The protective effect of MOAs on the barrier is closely related to their bacteriostatic ability. Studies have shown that MOAs can lower the pH of the gastrointestinal tract, which not only helps to stimulate the metabolic activity of beneficial bacteria such as lactobacilli, accelerating their growth and reproduction but also inhibits the growth and reproduction of bacteria with neutral or alkaline internal environments (most of them are pathogenic bacteria) ,56. HoweIn conclusion, dietary supplementation with MOA reduced serum IL-6 levels and MOA content in the liver and enhanced immunity and antioxidant capacity. In addition, MOA could increase the secretion of sIgA, inhibit the gene expression of IL-6, and promote the gene expression of ZO-1 in the jejunal mucosa, thus maintaining the integrity of the intestinal immune system and mechanical barriers in rabbits. The antioxidant, immune, and intestinal barrier-maintaining effects of MOAs collectively improved the growth performance of Ira rabbits.