RUNX1 was mainly reclassified as AML-MR . Different inclusion criteria of AML-CEBPA and AML-MR (i.a. exclusion of TP53 mutated cases according to ICC) were associated with differences in overall survival. In conclusion, both classifications focus on more genetics-based definitions with similar basic concepts and a large degree of agreement. The remaining non-comparability  needs additional studies to definitely answer open questions on disease categorization in an unbiased way.In parallel to the 5th edition of the World Health Organization Classification of Haematolymphoid Tumours (WHO 2022), an alternative International Consensus Classification (ICC) has been proposed. To evaluate the impact of the new classifications on AML diagnoses and ELN-based risk classification, we analyzed 717 MDS and 734 AML non-therapy-related patients diagnosed according to the revised 4th WHO edition (WHO 2017) by whole genome and transcriptome sequencing. In both new classifications, the purely morphologically defined AML entities decreased from 13% to 5%. Myelodysplasia-related (MR) AML increased from 22% to 28% (WHO 2022) and 26% (ICC). Other genetically-defined AML remained the largest group, and the abandoned AML- KMT2A and MECOM rearrangements (-r), inclusion of NUP98 and other DGAs and abandoning the previous category of AML with RUNX1 mutation. The definition of AML with myelodysplasia-related changes (AML-MRC) has been modified – mainly abandoning the morphologic criteria of dysplasia and newly incorporating molecular genetics  as well as single mutations of any kind that are located in the bZIP region of the gene. ICC also includes KMT2A- and MECOM-r with other partner genes than MLLT3 or GATA2 but specifically defines the partner genes. With respect to AML-MR, ICC separates this entity into AML with MR gene mutations (here including RUNX1) and with MR cytogenetic abnormalities, with mutations having the higher hierarchy. In contrast to WHO 2022, ICC does not consider a documented MDS or MDS/MPN history for diagnosing AML-MR but uses it as a diagnostic qualifier.In parallel to the WHO, an international expert panel proposed an independent classification named ICC for International Consensus Classification . In contMECOM-r and MR-defining gene mutations are added to the adverse risk-defining genetic abnormalities in ELN 2022. The FLT3-ITD allelic ratio used in ELN 2017 has been abolished in ELN 2022 risk classification mainly affecting NPM1 mutated patients. Within the favorable CEBPA mutated risk group, ELN 2017 considers biallelic CEBPA mutations, but ELN 2022 only includes bZIP in-frame mutated CEBPA in line with ICC.These changes described by ICC regarding AML classification are also mainly reflected within ELN 2022 recommendations for the diagnosis and management of AML in adults, guidelines for risk prediction in AML . Here, dThe aim of our study was to evaluate the impact of the new WHO classification on AML patients and to quantify differences in disease categorization compared to the previous WHO classification as well as the ICC.n = 717; AML: n = 734) with material available to perform whole genome and transcriptome sequencing sent to the MLL Munich Leukemia Laboratory between 09/2005 and 01/2020. Demographic, pathologic, and clinical characteristics of the cohort are described in Table For this analysis we selected 1,451 non-therapy-related MDS or AML samples  was used. Analyses for overall survival (OS) were performed according to Kaplan-Meier and compared using two-sided log rank tests. The OS was calculated as time from diagnosis to death or last follow-up. To assess the correlation between categories with real outcomes we used the Harrell’s concordance index (c-index ). All reDEK::NUP214 fusion did not change at all. Major additional contributors were AML with KMT2A-r (n = 45) now including 19 cases (42%) with a different partner gene but MLLT3  now including 33 cases (48%) not comprising GATA2  and one case to AML with another defined genetic aberration (KAT6A::CREBBP). In contrast, the now abandoned entity AML with mutated RUNX1 was mainly reclassified as AML-MR  based on a large overlap with ASXL1 and splicing factor mutations  and 5 cases as AML with mutated CEBPA .According to WHO 2022, 746 patients were diagnosed as AML and 705 patients as MDS Fig. . Within RUNX1). The largest contributor to this increase were mutations in the defining genes solely leading to this classification in 44% (92/208) of cases  and three were defined by differentiation as the morphologic dysplasia had been the only MRC-defining criterion according to WHO 2017  AML-MRC to 28% (208/746) AML-MR in WHO 2022 .Overall, reclassification from MDS according to WHO 2017 to AML according to WHO 2022 was a rare event affecting < 1% of cases of the total cohort . Within TP53 mutated AML we observed a high fraction of cases with complex karyotype and biallelic TP53 mutations but only a weak overlap with MR mutations , AML-NOS (n = 52) and AML-RUNX1 (n = 46). As in ICC – in contrast to WHO 2022 – RUNX1 mutations were qualifying mutations for AML-MR, 46 of the previous 48 (96%) AML-RUNX1 cases were classified as AML-MR while two had other MECOM-r. In line, AML-NOS was reduced from 99 to 34 cases (5%) not further characterized with respect to morphology . AML with MR gene mutations mainly composed former AML-MRC (p < 0.001). Across the different classifications, major changes regarding OS were observed for AML-CEBPA and AML-MR both entities present in all classification systems but with different inclusion criteria. CEBPA mutated AML showed a median OS of 5.0 and 4.1 years based on WHO 2017 and WHO 2022, respectively, while it was not reached according to ICC. Notably, based on WHO 2017 this entity comprised cases with biallelic CEBPA mutations (n = 56), WHO 2022 additionally considered single CEBPA mutations located in bZIP region  and ICC only included in-frame bZIP mutations (n = 47). Concerning myelodysplasia-related AML, AML-MRC based on WHO 2017 had the shortest median OS with 0.4 years compared to AML-MR according to WHO 2022 with a median OS of 0.5 years and AML-MR according to ICC with a median OS of 1.0 years. The later was mediated by excluding TP53 mutated cases which showed the worst OS (0.1 years) within all AML entities. Within TP53 mutated cases we observed a trend towards effects of TP53 mutation status  on OS  revealed marked differences between AML entities classified according to WHO 2017, WHO 2022 and ICC. For all classifications prognostic significance was confirmed .A direct comparison of the ICC with the WHO 2022 highlighted several differences Fig. . OverallKMT2A- and MECOM-rearranged cases formed two entities in WHO 2022 but were split into four different entities following ICC separating other partner genes than MLLT3 or GATA2. However, there was no significant difference in OS between KMT2A::MLLT3 and other KMT2A-r cases  of AML-MR leading to a classification as AML-TP53 according to ICC. Notably, AML-TP53 showed shorter OS compared to AML-MR according to WHO 2022 , with MR gene mutations , with MR cytogenetic abnormalities  and MDS/AML, NOS .The largest difference between WHO 2022 and ICC was mediated by the introduction of the new category MDS/AML including mainly MDS-IB2, but also MDS and AML cases . The general concept of a more genetics based definition of AML entities is consistent between the new classification systems substantially reducing purely morphologically defined cases. Despite this and many other concordant definitions and entity criteria between ICC and WHO 2022, there are several key differences that could influence patients’ diagnoses and risk stratification as well as therapeutic decisions and clinical trials in AML.CEBPA mutated AML. While WHO 2022 includes biallelic CEBPA mutations (independent of the gene region) and single mutations located in the bZIP region, ICC only accepts in-frame bZIP CEBPA mutations . The inclusion of monoallelic bZIP mutations is supported by several recent studies [CEBPA double-mutant patients (defined as a second mutation in addition to a bZIP mutation) [CEBPA mutated entities. Thus, regarding outcome of CEBPA mutated AML patients, the prerequisite of an (in-frame) bZIP mutation seems reasonable.One point is the discrepant definition of  studies –11. The  studies . Wakita  studies . TarlockKMT2A-r and MECOM-r cases from MLLT3- and GATA2- rearranged cases and accepting only specific partner genes according to ICC did not show differences regarding OS neither within rearranged cases nor compared to WHO 2022. Our findings regarding MECOM-r cases are in line with a previous study of 120 patients demonstrating no difference in outcome between cases with GATA2::MECOM as compared to MECOM-r with other partners [KMT2A-r AML, the prognostic impact in ELN guidelines [KMT2A::MLLT3 indicating intermediate risk and other KMT2A-r indicating adverse risk. However, we could not validate this discrimination in our cohort. This could be due to the small sample size of KMT2A-r cases (n = 45). Notably, in line with our results, another study analyzing 172 KMT2A-r AML showed that KMT2A-r was associated with adverse outcomes regardless of translocation subtype [MECOM-r or KMT2A-r cases within the same category.In contrast, discriminating partners . Regardiidelines , 7 is st subtype . TogetheRUNX1 mutations). In our cohort 79% (38/48) of previous AML-RUNX1 harbored either ASXL1 or splicing mutations in line with previous reports showing the association of RUNX1 mutations with other genetic features [TP53 mutated patients according to ICC. The different MR entity criteria and the introduction of the new ICC entity AML-TP53 affects not only the composition but also the outcome of AML-MR. The newly defined AML-MR entities potentially impact patient care as CPX-351 (Vyxeos®) was approved amongst others for the treatment of adults with newly diagnosed AML-MRC as defined by WHO 2017 [TP53 mutations had poorer responses to CPX-351 [Another major difference is the definition of AML-MR. In contrast to WHO 2017, WHO 2022 includes defining mutations as entity criteria for AML-MR and does not consider morphologic criteria anymore (same as in the ICC). In line with WHO 2022, ICC also incorporates MR gene mutations  entity.In line with our data, several studies reported a worse prognosis of patients –23. Howeerations . We obse cohorts . WHO autLastly, one main difference between WHO 2022 and ICC with respect to diagnosing myeloid malignancies is the recognition of the MDS/AML overlap category as a novel disease entity in ICC but not in WHO 2022, affecting 9% of patients within our cohort. As described by Zeidan et al. introducing this novel MDS/AML entity may enable more flexibility for trial enrollment  and clinical practice, and may also better reflect the natural history of high-risk MDS . In contIn parallel to the changes in disease classification, adjustments have also been made for risk classification in AML as the new ELN recommendations follow criteria proposed by ICC. This has the potential to substantially complicate diagnosis and treatment of AML patients when applying different in- and exclusion criteria for diagnosis according to WHO 2022 and risk stratification according to ELN 2022. When we compared ELN 2017 and ELN 2022, considerable difference in outcome was only observed for patients falling into the favorable risk group. The later was also shown by Lachowiez et al. analyzing patients of the multi-center Beat AML cohort . HoweverOur cohort is derived from a single diagnostic laboratory with multiple referring centers. Strengths of this study design include a homogeneous diagnostic workup according to gold standard methods and a powerful genetic analysis with WGS and WTS. Limitations of our study include the heterogeneity in treatment and follow-up assessment in different referral centers. The inclusion of cases diagnosed as early as 2005 provides a long follow-up but reduces the fraction of patients treated with novel therapies. Referral patterns and an enrichment of rare genetic subtypes in our WGS/WTS cohort potentially lead to an overrepresentation of de novo AML in our study compared to other cohorts, and cases diagnosed as therapy-related myeloid neoplasm according to WHO 2017 have not been investigated here.Overall, we observed a large degree of agreement between the WHO 2022 and the ICC classification with 86% (643/750) AML cases being assigned to corresponding subgroups. The remaining differences are either due to biological aspects/subgroups included in one classification but not the other or by small differences in the inclusion criteria of corresponding subgroups. The first affects a relevant number of patients and should be clarified based on available data to reach a broad consensus. Here, the WHO provides an ideal format to incorporate additional  entities given that sufficient data were provided. The later affects a relatively small number of patients and should not hamper a common classification. The parallel usage of two different classifications for AML confuses the diagnostic language for physicians, patients and legal authorities. Here it has to be noted that a unified global standard for cancer classification, in which the WHO 2022 is included, is of utmost value for the field. A commonly accepted classification is essential for comparability of diagnostic data in- and outside of clinical studies. We should therefore make all efforts to avoid a Babylonian confusion of languages in the diagnoses of AML and argue for a common diagnostic classification as a foundation to further build upon the tower of knowledge in hematology.Supplemental Material


Acute myeloid leukaemia (AML) is characterized by impaired myeloid differentiation resulting in an accumulation of immature blasts in the bone marrow and peripheral blood. Although AML can occur at any age, the incidence peaks at age 65. The pathobiology of AML also varies with age with associated differences in incidence, as well as the frequency of cytogenetic change and somatic mutations. In addition, 5-year survival rates in paediatrics are 60–75% but fall to 5–15% in older AML patients. This systematic review aimed to determine whether the altered genes in AML affect the same molecular pathways, indifferent of patient age, and, therefore, whether patients could benefit from the repurposing drugs or the use of the same immunotherapeutic strategies across age boundaries to prevent relapse. Using a PICO framework and PRISMA-P checklist, relevant publications were identified using five literature databases and assessed against an inclusion criteria, leaving 36 articles, and 71 targets for therapy, for further analysis. QUADAS-2 was used to determine the risk of bias and perform a quality control step. We then priority-ranked the list of cancer antigens based on predefined and pre-weighted objective criteria as part of an analytical hierarchy process used for dealing with complex decisions. This organized the antigens according to their potential to act as targets for the immunotherapy of AML, a treatment that offers an opportunity to remove residual leukaemia cells at first remission and improve survival rates. It was found that 80% of the top 20 antigens identified in paediatric AML were also within the 20 highest scoring immunotherapy targets in adult AML. To analyse the relationships between the targets and their link to different molecular pathways, PANTHER and STRING analyses were performed on the 20 highest scoring immunotherapy targets for both adult and paediatric AML. There were many similarities in the PANTHER and STRING results, including the most prominent pathways being angiogenesis and inflammation mediated by chemokine and cytokine signalling pathways. The coincidence of targets suggests that the repurposing of immunotherapy drugs across age boundaries could benefit AML patients, especially when used in combination with conventional therapies. However, due to cost implications, we would recommend that efforts are focused on ways to target the highest scoring antigens, such as WT1, NRAS, IDH1 and TP53, although in the future other candidates may prove successful. Acute myeloid leukaemia (AML) is an aggressive cancer that occurs due to the clonal expansion of immature white blood cells, known as blasts . The accThe frequency, pathophysiology, treatment approaches, and survival rates vary between adult and paediatric AML, with age being a major factor in the frequency of AML diagnosis. In patients below 65 years of age, there are ~1.3 cases per 100,000 compared to ~12.2 cases per 100,000 in those above 65 years of age , inactivRecently there has been a shift towards antigen-targeting immunotherapy in the treatment of cancers, including AML, to trigger anti-leukaemic responses . In 2007We wanted to determine whether the molecular pathways affected in AML were the same in adults and paediatrics, and whether the same immunotherapy targets could be used to treat AML across age groups. AML is diagnosed by molecular findings as described in the 2016 revision of the World Health Organisation (WHO) classification of myeloid neoplasms and acute leukaemia  which weStandard systematic review methods were used and reported according to the PRISMA guidelines , includiThe study inclusion criteria were composed of both study and report characteristics . The PICTwo independent reviewers (V.M. and H.G.) performed the search of the literature and screening process. Any disagreements were then settled by a third independent reviewer (B.G.) as and when required. MeSH terms and Boolean operators were used to search five databases   from incThe studies found within each database were exported into a single page of a Microsoft Excel Office 2019 sheet, combined, and duplicates were removed . The remReview articles were only removed once the cited articles in all selected manuscripts had been screened against the inclusion/exclusion criteria, as detailed above. This “backward snowballing” step helps ensure that relevant literature is successfully found as part of a systematic review .A study specific version of QUADAS-2  was applFrom the final list of articles after quality assurance and risk of bias analysis, the potential targets of interest were separated into two data collection files, one for the antigens found in adult AML tissues and one for the antigens found in paediatric AML tissues . The NCBPANTHER was used to illustrate which pathways are affected by events within the genome through predictions of gene interactions/functions. The 20 potential targets with the highest prioritization scores for both adult and paediatric AML underwent PANTHER analysis independently to produce a bar chart of the affected pathways.Homo sapiens from a wide range of sources for both diseases. For both networks, the confidence levels of interactions were set to 0.7 to increase the accuracy of the network without excluding predicted connections. The database has in-built quality control and provides enrichment analysis to ensure the accuracy and relevance of the networks produced [STRING was also utilized to analyse the 20 highest scoring targets in both adult and paediatric AML separately. This created networks of known and predicted protein–protein interactions within produced . The ideproduced .The search strategy used identified a total number of 8577 articles . Of thesA total of 90.7% of the genes identified as altered in paediatric patients were also identified in the literature describing altered gene expression in adults with AML . HoweverWe identified and assessed 63 targets and found that they ranged in score from 0.85 (WT1) down to the lowest scoring target at 0.17 (VEGFR-2) . A totalTo determine which pathways were predominantly affected within adult and paediatric AML cells, the NCBI numbers of the top 20 targets, as identified by the modified Cheever model in , were inInputting the top 20 targets into the Search Tool for the Review of Interacting Genes/Protein (STRING) for paediatric and adult AML patients showed their inter-relationships . STRING ® includes results from PubMed. It is possible that relevant studies may have been missed if they were not written entirely in English or if the title or abstract did not make it clear that the outcome of the article was focussed on antigen expression in AML. However, the use of “reverse snowballing”, whereby citations in the selected articles were also subjected to the systematic review process, was used to mitigate this risk [The databases selected meant that a large number of articles were systematically reviewed. This is because the Cochrane Library includes articles from the CINAHL database and MEDLINEhis risk .The databases used for the literature search have built in quality assessment. For example, Scopus openly advertises “a rigorous evaluation and selection process to ensure it meets the high-quality title selection criteria required for acceptance” and that journals “must demonstrate their ability to maintain quality status each year” with a “multi-step re-evaluation process” . AdditioThe criteria used excluded animal models. Traditionally animal models, predominantly rodent, have been utilized to investigate the underlying mechanisms of biological processes in a multiorgan setting, and can avoid some of the ethical and practical concerns that are associated with human studies. However, there are several drawbacks to the use of animal models within cancer research, which has called for a shift in how these models are used. The average success rate within clinical trials of the translation from animal models and patients is stated to be less than 8% . This ilSignificantly fewer articles described the testing of paediatric patient samples, probably reflecting their relative scarcity compared with adult AML, and this may have contributed to the lower number of targets identified in younger AML patients. In addition, the reduced number of articles describing genetic change in paediatric AML may reflect historical concerns with the use of samples from paediatric patients in clinical trials due to the age of consent being 18 years old. However, it was recently stated that treatment intended for paediatrics should only be tested on that age group due to pathophysiological differences between adults and children . In addiThere were a vast number of similarities seen between adult and paediatric AML with 90.7% of antigens in paediatric samples also being described in adult AML. In fact, only four targets identified in paediatric AML were not shared with adult AML . The large number of similarities observed between adult and paediatric AML supported the possibility of treatment repurposing, such as targeting BIRC5 (survivin) that had high expression levels in both adult and paediatric AML ,67. BIRCThe Initial extraction of data identified a large amount of targets; therefore, we chose the Cheever et al.  model ofLooking at both the amount of the literature that focussed on it and its top position after prioritization, WT1 was the most promising immunotherapeutic target for AML patients. This was the same outcome found by Cheever et al.  in a stuCD13 could prove a viable target due to the high scores it achieved in key categories, such as immunogenicity  and the PANTHER was used to determine which pathways were affected by the 20 highest scoring targets in both adult and paediatric AML patients independently. In addition to paediatric AML sharing 96% of their immunotherapeutic targets with adults, both adult and paediatric AML have the most targets involved in angiogenesis and inflammation mediated by the cytokine and chemokine pathway(s). Angiogenesis is the generation of new blood vessels, which are key for cancer survival through the fresh supply of oxygen and nutrients to cancer cells . UsuallyInflammation mediated by the cytokine and chemokine pathway was involved in both adult and paediatric AML development and had an equal number of targets involved in angiogenesis. Cytokines that are secreted within the bone marrow microenvironment are crucial in modulating cell survival, proliferation, differentiation, and the immune response . The exaThe adult AML STRING network was created using the 20 highest scoring targets after Cheever prioritization to ensure the network only included the most relevant targets. TP53 was the target that displayed the most connections within the adult network. It has been predicted that ~10% of AML patients have mutations in TP53, which are associated with poor prognosis . TP53 muIn paediatric patients, STRING indicated that the target with the most connections was PTPN11. PTPN11 also had a lot of evidence types within the STRING network which supports its potential success as a target despite not being frequently represented within this literature review. PTPN11 was found to be mutated in both adult and paediatric patients with AML and, therefore, could provide a unified approach. Mutations within PTPN11 were found in ~12% of adult AML patients and only 4% of paediatric AML patients ,96. ReceIn concurrence with PANTHER, STRING networks showed vast similarities between adult and paediatric patients when considering the top 20 antigenic targets in each age group. In adults, BMI1, CA9, PRAME, and TERT only featured in adults with AML, while CD44, CD244, CLEC12A, and IL-3RA only featured in paediatrics. One of the most connected genes within the top 20 for paediatric AML was JAK2, which, as previously mentioned, is part of the cytokine-mediated inflammatory pathway. JAK2 has been previously utilized as an immunotherapeutic target for breast cancer; however, there is little evidence of its successful targeting in AML so far, although it could provide a novel therapeutic approach ,99. ThisFrom the information presented thus far, there are a number of potential targets identified for the therapy of AML; however, due to the high costs of developing treatments and the length of time that the approval of a treatment takes, it may be beneficial to focus on a few prioritised targets for treatment development over the next few years. In order to decide which targets should be selected, there are additional factors to consider including broad applicability across subtypes and age-boundaries. FLT3 mutations have been found in ~25% of paediatric patients and ~30% of adult patients , with soAnother key factor to consider when selecting which of the targets should be pursued are the toxicity issues caused by a lack of specificity. There are ways to mitigate this type of toxicity either through a combinational CAR T cell approach  or the dAML is a rare but progressive form of blood cancer, which continues to have poor prognosis in both adult and paediatric patients. Low survival rates indicate a clear need for the development of new treatment approaches that will universally improve prognosis. The most promising approach are immunotherapeutic methods that target specific antigens that have a key role in the development and survival of AML cells. This could create an approach with higher efficacy and fewer side effects than high-dose chemotherapy alone. Adult and paediatric AML are currently treated as different conditions due to differences in underlying pathophysiology; however, immunotherapy offers one way to cross age boundaries by targeting common antigens. This review has identified antigens that could or do offer an opportunity to develop a unified treatment approach. Throughout all analyses in this review, there were significant overlaps between the antigens expressed in adult and paediatric AML, including within the most promising targets. A few examples of the overlapping targets that stood out at multiple stages were WT1, TP53, BIRC5, and JAK2, with some of these already being effectively targeted within clinical trials using a variety of delivery systems. However, there are a lot more, such as CD13 and PTPN11, that have been identified as potentially viable novel targets for a future unified approach. With the correct target selection and delivery method it may be possible to mitigate a lot of the side effects associated with the immunotherapeutic treatment of AML, such as toxicity caused by the lack of specificity. It is likely that there are ways to develop a unified approach of immunotherapy that could treat both adult and paediatric patients simultaneously and benefit from the cost savings that drug repurposing offers.


Lactiplantibacillus plantarum supernatant (LP-1S) on PEDV and its mechanism. We used IPEC-J2 cells as a model to assess the inhibitory effect of LP-1S on PEDV and to further investigate the relationship between LP-1S, Ca2+, and PEDV. The results showed that a divalent cation chelating agent (EGTA) and calcium channel inhibitors (Bepridil hydrochloride and BAPTA-acetoxymethylate) could inhibit PEDV proliferation while effectively reducing the intracellular Ca2+ concentration. Furthermore, LP-1S could reduce PEDV-induced loss of calcium channel proteins (TRPV6 and PMCA1b), alleviate intracellular Ca2+ accumulation caused by PEDV infection, and promote the balance of intra- and extracellular Ca2+ concentrations, thereby inhibiting PEDV proliferation. In summary, we found that LP-1S has potential therapeutic value against PEDV, which is realized by modulating Ca2+. This provides a potential new drug to treat PEDV infection.Porcine epidemic diarrhea virus (PEDV) mainly invades the small intestine and promotes an inflammatory response, eventually leading to severe diarrhea, vomiting, dehydration, and even death of piglets, which seriously threatens the economic development of pig farming. In recent years, researchers have found that probiotics can improve the intestinal microenvironment and reduce diarrhea. At the same time, certain probiotics have been shown to have antiviral effects; however, their mechanisms are different. Herein, we aimed to investigate the inhibitory effect of  The vir2+ acts as an important second messenger in intracellular signal transduction processes. Ca2+ is essential for the transmembrane transmission of biological signals, the regulation of glandular secretion, and the maintenance of acid-base homeostasis in the body ; (ii) binding to proteins with high Ca2+ affinity to form calcium-binding protein-D9k (CaBPD9k), which in turn moves from the BBM to the basolateral membrane (BLM); and (iii) extracellular excretion via the plasma membrane Ca2+-ATPase (PMCA1b/Ca2+ pump) and Na+/Ca2+ exchanger (NCX1). Among them, TRPV6 and PMCA1b, as calcium channel proteins, have received much research attention because they are the major Ca2+ transport proteins in the intestinal epithelium and have important roles in maintaining intra- and extracellular homeostasis and controlling ions entering and leaving the cell  Crothe cell .Lactiplantibacillus plantarum (Lp) significantly reduced the cytopathic effects of respiratory syncytial virus (RSV), influenza B virus, and HCoV-229E coronavirus . Its main metabolites were found to include acids (33.96%), amino acids (and their derivatives) (32.08%), and polysaccharides (15.1%) through the pristine analysis using gas chromatography–mass spectrometry (GC–MS) (Lactiplantibacillus plantarum supernatant (LP-1S) on PEDV and its mechanism.In recent years, research has shown that a variety of probiotics can maintain microbial homeostasis in the intestinal environment and prevent diarrhea . Antivironavirus . Lp is a (GC–MS)  LP, as a2.2.1.2 cell incubator. PEDV-LJX strain and PEDV N mouse-derived monoclonal antibodies were kindly donated by GuangLiang Liu, Researcher, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Lactiplantibacillus plantarum LP-1 was obtained by pre-laboratory isolation. TRPV6 polyclonal rabbit antibodies , horseradish peroxidase (HRP)-goat anti-mouse IgG (H + L)  and β-actin goat anti-rabbit antibody  were purchased from Proteintech. PMCA1b Polyclonal Rabbit Antibodies  were purchased from Abcam . Fura-2-acetoxymethyl ester (Fura-2 AM) and CaCl2 were purchased from Beyotime Biotechnology Co. . EGTA, Fluo3-AM, and BAPTA-AM were purchased from Solarbio . Bepridil hydrochloride (BP) was purchased from MCE . Ca2+ standard solution  was purchased from National Research Center for Reference Materials, Beijing, China.African green monkey kidney cells  and porcine small intestinal epithelial cells  were purchased from Suer Biotech  and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) medium with 10% fetal bovine serum, at 37°C, in a 5% CO2.2.Lactiplantibacillus plantarum LP-1 was inoculated onto MRS solid medium and incubated at 37°C. After 24 h, single colonies were picked, inoculated in MRS liquid medium, incubated at 37°C with shaking 180 rpm for 12 h until they reached a measured OD595 value between 0.8 ~ 0.9, and centrifuged at 3,000 × g for 10 min. The supernatant was retained and filtered using a 0.44 μm–0.22 μm microporous filter membrane gradient. The filtered supernatant (LP-S1) was stored at −20°C for a short time.2.3.6 cells per well. A positive control group (PEDV-infected group) and a negative control were set, and the adsorption culture was replaced with DMEM maintenance solution after 90 min. When the cytopathic effect (CPE) of the PEDV-infected group reached 70%, the maintenance solution was discarded, the wells were washed three times with 0.01 M phosphate-buffered saline (PBS), 5 mg/mL MTT reagent was added to each well, the plate was incubated at 37°C for 4 h to allow the formation of formazan crystals, and the OD575 values were then measured. Cell viability was calculated, and the IC50 of LP-S1 was calculated: lgIC50 = Xm-I (P- (3-Pm-Pn)/4) .Firstly, LP-1S was diluted with DMEM basal medium to 0, 1/2, 1/4, 1/8, 1/16, 1/32, and 1/64 times the original concentration. Different dilutions of LP-1S were added to each well of a microtiter plate, containing 3.6 × 102.4.50 of each PEDV group was measured according to the Reed-Muench method , which were blocked in 5% skim milk for 1.5 h, followed by incubation with the corresponding primary antibodies overnight at 4°C. Next day, the membranes were incubated with the corresponding secondary antibodies for 90 min at 37°C. The immunoreactive protein bands were visualized using the FX5 imaging system , and the grayscale values were analyzed.2.6.2.6.1.Total RNA was extracted from cells and reverse transcribed to cDNA using RNAiso plus  and 5 × PrimeScript RT Master Mix .2.6.2.TRPV6, PMCA1b, and ACTB (β-actin genes) were downloaded from GenBank. Primers were designed against the sequences using Primer Premier 5.0 software . The primer sequences are shown in −ΔΔCt method was used to analyze the expression of the corresponding mRNAs (The full sequences of ng mRNAs .2.6.3.PEDV M gene.The PEDV-M primer sequences were downloaded from GenBank . The PED2.6.4.The shRNA sequence was designed and synthesized by Wuhan Jinkairui Biotechnology Co.  and then2.7.6 cells per well) and incubated for 60 min at 37°C with 5% CO2. The wells were washed using 0.1 M HEPES  buffered saline three times, added with HBSS containing 1% fetal bovine serum, and incubated for 30 min. Changes in intracellular calcium ion content were observed using fluorescence microscopy.Fluo3-AM was diluted to a 2 mM Fluo3-AM stock solution using dimethyl sulfoxide and stored at −20°C for later use. The preserved Fluo3-AM stock solution was diluted with 0.1 M Hanks’ Balanced Salt Solution , added to cells in a microtiter plate (3.6 × 102.8.3 + 2 mg/mL K+). Then, the standard concentration was measured and the standard curve was plotted. Finally, the collected cell supernatant and intracellular fluid were diluted 5,000 times with medium solution and then assayed on the flame atomic absorption spectrophotometer. The absorbance and concentration values of each sample were recorded. Changes in extracellular calcium ion content were then analyzed with reference to the standard curve.0.25 mL, 0.5 mL, 1 mL, 1.5 mL, and 2 mL of a Ca standard solution (10 μg/mL) were added to a 50 mL volumetric flask and added with 50 mL with medium solution  using the statistical software GraphPad Prism 8.0  and IBM SPSS Statistics 23 to determine statistical differences between multiple groups. A p-value > 0.05 (ns) indicates a non-significant difference, a p-value < 0.05 indicates a significant difference (*), a p-value < 0.01 expresses a highly significant difference (**), and a p-value < 0.001 expresses a very significant difference (***).All results were subjected toa 3.3.1.2+ concentrations is unknown. We used the calcium ion fluorescence probe Fluo-3 AM to detect the intracellular Ca2+ concentration under PEDV infection, which showed that the intracellular Ca2+ fluorescence intensity after 2 h, 8 h, 24 h, and 48 h of PEDV infection was extremely significantly higher than that in the negative control group, under fluorescence microscopy  compared with that in the negative control group, in which the calcium ion concentration in the extracellular fluid was significantly lower in the 2 h, 8 h, and 24 h infection groups compared with that in the negative control group  and the CaTT assay . IPEC-J2TT assay ; the PEDTT assay . Thus, BTT assay ; the PEDTT assay .3.5.2+ transmembrane transport mainly involves membrane transporter proteins, carrier proteins, and channel proteins. To further investigate the role played by calcium channel proteins during PEDV infection, we selected two typical Ca2+ transport channels, TRPV6 and PMCA1b, as the subjects of our study. Firstly, we constructed vectors for RNA interference of TRPV6 and PMCA1b (shTRPV6/shPMCA1b) separately to determine the association between TRPV6/PMCA1b and PEDV. The results showed that both interfering vectors were stably expressed in IPEC-J2 cells and significantly reduced the protein levels of TRPV6 and PMCA1b  at 12 h , calcium channel inhibitors (BP and BAPTA-AM), and calcium channel proteins (TRPV6 and PMCA1b). Pretreatment with 2 mM EGTA significantly inhibited PEDV M expression, and PEDV expression M was significantly increased when the cells were supplemented with Ca2+, suggesting that EGTA effectively reduced the inward flow of extracellular free Ca2+ by chelating it, thereby inhibiting PEDV proliferation. In contrast, CaCl2 supplementation induced an increase in intracellular Ca2+ flow, leading to an increase in PEDV expression , among which PMCA1b was more sensitive to PEDV infection than TRPV6. LP-1S might precisely enhance the regulation of Ca2+ by PMCA1b and TRPV6 to reduce the intracellular Ca2+ concentration and maintain an intracellular low calcium state, thus exerting an anti-PEDV effect  can be found in the article/supplementary material.Ethical approval was not required for the studies on animals in accordance with the local legislation and institutional requirements because only commercially available established cell lines were used.ZK, SZ, and GL: conceptualization. ZK: methodology. ZN: software. ZK, SZ, and ZN: validation. ZSu and XL: formal analysis. XH: investigation. ZSo: resources, supervision, project administration, and funding acquisition. YZ: data curation. SZ: writing—original draft preparation. ZN: writing—review and editing. SX, JZ, and HZ: visualization. All authors contributed to the article and approved the submitted version.


V3.2‐RyR axis for extracellular Ca2+ influx to trigger Ca2+ sparks in vascular smooth muscle cells (VSMCs). We hypothesize that the administration of senolytics, which can selectively clear senescent cells, could preserve the caveolar CaV3.2‐RyR axis in aging VSMCs. In this study, 10‐month‐old mice were administered the senolytics cocktail consisting of dasatinib (5 mg/kg) and quercetin (50 mg/kg) or vehicle bi‐weekly for 4 months. Using VSMCs from mouse mesenteric arteries, we found that Ca2+ sparks were diminished after caveolae disruption by methyl‐β‐cyclodextrin (10 mM) in cells from D + Q treated but not vehicle‐treated 14‐month‐old mice. D + Q treatment promoted the expression of CaV3.2 in 14‐month‐old mesenteric arteries. Structural analysis using electron tomography and immunofluorescence staining revealed the remodeling of caveolae and co‐localization of CaV3.2‐Cav‐1 in D + Q treatment aged mesenteric arteries. In keeping with theoretical observations, Cav3.2 channel inhibition by Ni2+ (50 μM) suppressed Ca2+ in VSMCs from the D + Q group, with no effect observed in vehicle‐treated arteries. Our study provides evidence that age‐related caveolar CaV3.2‐RyR axis malfunction can be alleviated by pharmaceutical intervention targeting cellular senescence. Our findings support the potential of senolytics for ameliorating age‐associated cardiovascular disease.Aging is a major risk factor for cardiovascular diseases. Our previous studies demonstrate that aging impairs the caveolar T‐type Ca V3.2 channels and caveolae remodeling, resulting in an improved CaV3.2‐RyR axis and subsequent increase in Ca2+ spark generation. Utilizing this senescent cell clearance approach has the potential to be a promising therapeutic method in enhancing vascular function among elderly individuals.The use of a senolytic cocktail, consisting of dasatinib and quercetin, could potentially improve the functionality of aging arteries by targeting and eliminating senescent cells. This treatment may lead to enhancements in the expression of Ca BKcalarge‐conductance Ca2+‐sensitive K+Cav‐1caveolin‐1DdasatinibGSEAgene set enrichment analysisQquercetinSASPsenescence‐associated secretory phenotypeSRsarcoplasmic reticulumSTOCsspontaneous transient outward K+ currentsVSMCsvascular smooth muscle cells12+ release events (known as Ca2+ sparks) has been identified to attenuate arterial tone and limit excessive vasoconstriction. T‐type CaV3.2 channels, which are localized in caveolae, mediate Ca2+ influx and stimulates the cytosolic domain of ryanodine receptors (RyRs) to induce Ca2+ release from the sarcoplasmic reticulum (SR) in the form of Ca2+ sparks, and thus opens numerous large‐conductance Ca2+‐sensitive K+ (BKCa) channels causing spontaneous transient outward K+ currents (STOCs). As a result, Ca2+ spark–BKCa channel coupling induces VSMCs hyperpolarization and the attenuation of arterial constriction . Furthermore, there was no difference in the myogenic tone between aged mesenteric arteries from CaV3.2 Ca2+ channel deficient (CaV3.2−/−) and wild‐type mice, despite an enhanced tone in young CaV3.2−/− mice compared to controls  and endothelial function, which may potentially lead to cardiovascular disease .There is strong experimental and clinical evidence suggesting that targeting cellular senescence could delay the aging process and alleviate age‐related diseases , a cholesterol‐depleting drug known to disturb caveolae and inhibit a significant fraction of Ca2+ sparks in VSMCs  in aged and D + Q‐treated VSMCs. STOCs were measured in the presence of Ni2+ or not. The holding potential was set to −40 mV, a physiological membrane potential that should drive T‐type Ca2+ channel‐mediated Ca2+ sparks, enabling the activation of BKCa channels. We found that Ni2+ blocked STOCs in cells from D + Q‐treated mice but not in cells from aged mice ‐, middle aged (vehicle‐treated) (14 months)‐, D + Q‐treated aged (14 months)‐male mice were used. Mice were maintained at the breeding facility of the Animal Center of Huazhong University of Science and Technology Union Shenzhen Hospital in individually ventilated cages under standardized conditions that included a 12‐h dark–light cycle and free access to standard chow, and drinking water. All mice were deeply anaesthetized by inhalation of isoflurane until cessation of breathing, then killed by cervical dislocation and the mesentery arteries removed. Experiments were performed on the same day with arteries from litter‐matched old versus D + Q mice. All animal protocols were approved by the local animal care committee of Huazhong University of Science and Technology Union Shenzhen Hospital. There are no ethical concerns.5.2Mice were administered a senolytic cocktail containing 5 mg/kg dasatinib  and 50 mg/kg quercetin  as described previously  physiological salt solution (PSS) of the following composition (mM): 119 NaCl, 4.7 KCl, 1.2 KH2PO4, 25 NaHCO3, 1.2 MgSO4, 1.6 CaCl2, and 11.1 glucose. The arteries were cleaned, cut into pieces, and placed into a Ca2+‐free Hank‘s solution (mM): 55 NaCl, 80 sodium glutamate, 5.6 KCl, 2 MgCl2, 1 mg/mL bovine serum albumin , 10 glucose, and 10 HEPES (pH 7.4 with NaOH) containing 0.5 mg/mL papain (Sigma) and 1.0 mg/mL DTT for 37 min at 37°C. The segments were then placed in Hank‘s solution containing 1 mg/mL collagenase  and 0.1 mM CaCl2 for 17 min at 37°C. Following several washes in Ca2+‐free Hank‘s solution (containing 1 mg/mL BSA), single cells were dispersed from artery segments by gentle triturating. Cells were then stored in the same solution at 4°C.Arterial VSMCs from mesenteric arteries were isolated as previously described  and pluronic acid  for 60 min at room temperature in Ca2+‐free Hanks' solution. After loading, cells were washed with bath solution for 10 min at room temperature. Isolated cells and intact arterial segments were imaged in a bath solution containing (mM): 134 NaCl, 6 KCl, 1 MgCl2, 2 CaCl2, 10 glucose and 10 HEPES . Images were recorded using confocal microscope . Images were obtained by illumination with an argon laser at 488 nm, and recording all emitted light above 515 nm. Ca2+ spark analyses were performed line‐scan using ImageJ software. The entire area of each image was analyzed to detect Ca2+ sparks. Ca2+ sparks were defined as local fractional fluorescence increase (F/F0) above the noise level of 1.5. The frequency was calculated as the number of detected sparks divided by the total scan time.Ca5.5V3.2‐rabbit , anti‐rabbit‐caveolin‐1 , and anti‐rabbit‐β‐actin . The relative amount of each protein was quantified via the peak areas detected in the chemiluminescence electropherogram generated by the Compass for SW software (ProteinSimple), following the default settings. A standard curve based on the serial dilutions of the input was used to estimate the absolute amount of protein in each sample. Finally, the recovery of input for each identified interacting protein was calculated through normalization to the percentage recovery of CTCF. All antibodies were approved of only if they detected the correct bands upon WES/JESS analyses.The samples were analyzed with a Simple Western assay using the WES™ system , anti‐caveolin‐1‐mouse , DAPI .Mice were anesthetized with 2% ketamine/10% rompun, perfused by 30 mL PBS and 50 mL 4% PFA  and after wards vessels were dissected, and tissue pieces were further fixed for 4 h in 4% PFA, transferred to 15% sucrose  for 4 h and incubated in 30% sucrose overnight. After embedding in TissueTek (Sakura), the tissue is frozen at −80°C and 8 μm sections were obtained in a Leica cryostat at −30°C. For immunostainings, the cryostat sections were incubated with blocking buffer (1% donkey serum/1% TritonX100/PBS), the first antibody was applied overnight at 4°C, and after washing with PBS/1% Tween, the secondary antibody and DAPI were applied for 2 h. Afterward the sections were embedded in ImmoMount (ThermoScientific #9990402). The stained sections were analyzed with confocal microscope , and images were analyzed by ImageJ. Antibodies: anti‐Ca5.82 and 0.05 EGTA (pH 7.2). The external bath solution contained (in mM): 134 NaCl, 6 KCl, 1 MgCl2, 2 CaCl2, 10 glucose and 10 HEPES (pH 7.4); holding potential was −60 mV. Whole cell currents were recorded using an Axopatch 200B amplifier (Axon Instruments/Molecular Devices) or an EPC 7 amplifier (List) at room temperature. Data were digitized at 5 kHz, using a Digidata 1440A digitizer  and pCLAMP software versions 10.1 and 10.2. STOC analysis was performed off‐line using IGOR Pro (WaveMetrics) and Microsoft Excel software. A STOC was identified as a signal with at least three times the BKCa single channel current amplitude.Potassium currents were measured in the whole‐cell perforated‐patch mode of the patch‐clamp technique. Patch pipettes  were filled with a solution containing (in mM): 110 potassium aspartate, 30 KCl, 10 NaCl, 1 MgCl5.9n = 4/group) were used to isolate RNA with the Qiagen miRNeasy® Mini Kit . RNA concentration was determined by utilizing a Nanodrop 2000 (Thermo Fisher) and RNA integrity was quantified using an Agilent Bioanalyzer 2100 . High‐quality RNA (RIN >8.0) of 500 nanograms was then sent to Novogene for sequencing. RNAseq analyses were carried out using Partek® Flow® software, v10.0. The default QA/QC tool was employed for pre‐alignment quality control. The splice‐aware program STAR (v2.7.8a) was used for aligning sequencing reads to the mouse genome (GRCm39). Gene counts were quantified using Partek E/M against transcriptome release 103 with a minimum expression cutoff of 10 counts to filter out low expression genes. Differential gene expression was examined by using DESeq2 (v3.5) with FDR <0.05.Mesenteric arteries from vehicle‐ and D + Q‐treated mice . All salts and other drugs were obtained from Sigma‐Aldrich or Merck. In cases where DMSO was used as a solvent, the maximal DMSO concentration after application did not exceed 0.5%.5.11t test or one‐way analysis of variance (ANOVA) or Mann–Whitney U test. p < 0.05 were considered statistically significant; “n” represents the number of cells.Data are presented as mean ± SEM. Statistically significant differences in mean values were determined by Student's unpaired All authors were responsible for interpretation of the data, contributed to the drafting and revised the manuscript critically for important intellectual content. All authors have approved the final version of the manuscript and agree to be accountable for all aspects of the work. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.The authors have no conflict of interest to declare.Figure S1.Click here for additional data file.


In this work, we induced the generation of A1-like reactive astrocytes after the co-treatment of U251 human astroglioma cells with a cocktail of the cytokines TNF-α, IL1-α and C1q. These A1-like astrocytes show increased production of APP and Aβ peptides compared to untreated U251 cells. Additionally, A1-like astrocytes show a (75 ± 10)% decrease in the Ca2+ stored in the endoplasmic reticulum (ER), (85 ± 10)% attenuation of Ca2+ entry after complete Ca2+ depletion of the ER, and three-fold upregulation of plasma membrane calcium pump expression, with respect to non-treated Control astrocytes. These altered intracellular Ca2+ dynamics allow A1-like astrocytes to efficiently counterbalance the enhanced release of Ca2+ from the ER, preventing a rise in the resting cytosolic Ca2+ concentration.Highly neurotoxic A1-reactive astrocytes have been associated with several human neurodegenerative diseases. Complement protein C3 expression is strongly upregulated in A1 astrocytes, and this protein has been shown to be a specific biomarker of these astrocytes. Several cytokines released in neurodegenerative diseases have been shown to upregulate the production of amyloid β protein precursor (APP) and neurotoxic amyloid β (Aβ) peptides in reactive astrocytes. Also, aberrant Ca Neuroinflammatory cytokines secreted by activated microglia in the brain can induce the generation of A1-reactive astrocytes that are highly neurotoxic ,3. Also,In addition, it has been shown that reactive astrocytes induced via 2-chloroethanol poisoning can stimulate microglia polarization and microglia activation , leading2+ signals have been proposed as a hallmark of astrocyte functional remodeling in Alzheimer’s disease (AD) mouse models  piperazine-N′-[2-ethanesulfonic acid] (HEPES)/KOH, at pH 7.4, with 0.32 M sucrose, 0.5 mM MgSOd method .Proteins from 20 µg homogenates were separated using 10% or 7.5–20% gradient polyacrylamide sodium dodecyl sulfate (SDS) gels  and tran® SF . Total protein was visualized via Ponceau S protein staining. After blocking and washing, the membrane was incubated with the anti-Aβ antibody  overnight at 4 °C. The membrane was incubated with peroxidase-conjugated anti-mouse secondary antibody and developed using the Enhanced Chemiluminescence (ECL) substrate.Immunodetection of total Aβ peptides was carried out via dot blot. Homogenates (12 µg) from U251 cells, untreated (C) or treated (A1-like astrocytes) with TNF-α (30 ng/mL), IL-1α (3 ng/mL) and C1q (400 ng/mL), were spotted onto a nitrocellulose membrane, sealed on a Bio-Dot2+ concentration ([Ca2+]i) was measured in Control U251 cells and in A1-like astrocytes with the fluorescent probes Fluo3 AM and Fura2 AM, as previously described i using the Fluo3 probe, 5 mM MnCl2 and 5 μg/mL of the nonfluorescent Ca2+ ionophore 4-Bromo-A23187 were added to Control U251 cells and to A1-like astrocytes, and the fluorescence microscopy images were recorded in kinetic mode to obtain the fluorescence intensity of Fluo3 saturated with Mn2+ [F(Mn-Fluo3)], which has been reported to be 20% of the value of fluorescence intensity upon saturating Ca2+ (Fmax) i was measured using the formula: [Ca2+]i = Kd i was 390 nM, reported in the Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies, Tenth Edition (2005).For the determination of [Ca+ (Fmax) . The flu.01·Fmax . [Ca2+]i2+]i using the Fura2 probe, [Ca2+]i was calculated using the following equation: [Ca2+]i = Kd × [(R − Rmin)/(Rmax − R)], where R is the measured fluorescence ratio (340/380), and Rmax and Rmin are the ratio values (340/380) for Ca2+-bound and Ca2+ -free Fura2-loaded cells. Rmax and Rmin were experimentally determined from steady-state fluorescence ratio (340/380) measurements after sequential addition to the culture medium of Fura2-loaded cells of (i) nonfluorescent Ca2+ ionophore 4-Bromo-A23187 (5 μg/mL) or ionomycin (5 μg/mL), and (ii) 10 mM EGTA, respectively. To obtain the values of [Ca2+]I, we used the reported dissociation constant (Kd) of Fura2/Ca2+ of 224 nM [For the determination of [Caf 224 nM .2+ stores was triggered by adding 2 µM of the sarcoendoplasmic calcium pump blocker Tg plus 1 mM EGTA in Ca2+ -free MLocke’s K5 buffer [2 to the Tg-containing medium. Data acquisition and analysis were carried out using HCImage software after the selection of the cells using the ROI tool of this software. The response of SOCE was determined in Fluo3-loaded Control U251 cells and in A1-like astrocytes. The depletion of the Ca5 buffer . Then, St-test, and the results are expressed as the mean ± standard error of the mean (SEM). A significant difference was defined as p < 0.05. All results were obtained at least in triplicate, with the number of cells (n) indicated for each experimental condition.Statistical analysis was carried out using the Student’s


FLT3-ITD and TP53 mutant AML cell lines and a variety of patient-derived AML cells. Results: PU-H71 and combination treatments with MCL1 inhibitor S63845 or BCL2 inhibitor venetoclax induced cell cycle arrest and apoptosis in susceptible AML cell lines and primary AML. The majority of the primary AML samples were responsive to PU-H71 in combination with BH3 mimetics. Elevated susceptibility to PU-H71 and S63845 was associated with FLT3 mutated AML with CD34 < 20%. Elevated susceptibility to PU-H71 and venetoclax was associated with primary AML with CD117 > 80% and CD11b < 45%. The combination of HSP90 inhibitor PU-H71 and MCL1 inhibitor S63845 may be a candidate treatment for FLT3-mutated AML with moderate CD34 positivity while the combination of HSP90 inhibitor PU-H71 and BCL2 inhibitor venetoclax may be more effective in the treatment of primitive AML with high CD117 and low CD11b positivity.Targeting the molecular chaperone HSP90 and the anti-apoptotic proteins MCL1 and BCL2 may be a promising novel approach in the treatment of acute myeloid leukemia (AML). The HSP90 inhibitor PU-H71, MCL1 inhibitor S63845, and BCL2 inhibitor venetoclax were assessed as single agents and in combination for their ability to induce apoptosis and cell death in leukemic cells. AML cells represented all major morphologic and molecular subtypes including  Heat-shock protein 90 (HSP90) is involved in the folding and maturation of a wide range of client proteins, including diverse kinases and transcription factors . Over thFLT3) growth factor receptor gene. FLT3-induced signaling pathways are highly active in AML cells, leading to elevated protein translation and cell proliferation as well as reduced apoptosis. In hematological malignancies including AML, tumor cells prevent apoptosis by the elevated expression of anti-apoptotic proteins of the B-cell lymphoma 2 (BCL2) family. Elevated levels of BCL2 protein in acute myeloid leukemia cells are associated with poor responses to chemotherapy [BCL2 mRNA is upregulated in 84% of AML patients at diagnosis and 95% at relapse [In acute myeloid leukemia (AML), a hyperactive signalosome drives addiction to oncogenic HSP90 species, thus rendering the cells vulnerable to HSP90-directed therapy . PU-H71  relapse . The mye relapse . Hematol relapse ,13. This relapse . S63845  relapse .FLT3-ITD and FLT3 wild type, NPM1 mutant and wild type, and TP53 mutant and wild type AML cell lines as well as a variety of patient-derived AML cells.Here, we assessed the HSP90 inhibitor PU-H71, the MCL1 inhibitor S63845, and the BCL2 inhibitor venetoclax as single agents and in combination for their ability to induce apoptosis and cell death in leukemic cells in vitro. AML cells represented all major morphologic and molecular subtypes including the ASXL1, ASXL2, ATRX, BCOR, BCORL1, BRAF, CALR, CBL, CDKN2A, CEBPA, CREBBP, CSF3R, CSNK1A1, CTCF, CTNNA1, CUX1, DDX41, DNMT3A, EP300, ETV6, EZH2, FBXW7, FLT3, GATA1, GATA2, GNAS, HRAS, IDH1, IDH2, IKZF1, JAK2, KDM5A, KDM6A, KIT, KMT2D, KMT2C, KRAS, MLL, MPL, MYC, MYD88, NF1, NPM1, NRAS, PHF6, PPM1D, PTEN, PTPN11, RAD21, RB1, RUNX1, SETBP1, SF3B1, SH2B3, SMC1A, SMC3, SRSF2, STAG2, SUZ12, TET2, TP53, U2AF1, WT1, ZBTB7A, and ZRSR2. Flow-cytometry immunophenotyping was conducted according to international consensus with CD markers in the initial evaluation of myeloid leukemia including CD7, CD11b, CD13, CD14, CD15, CD16, CD33, CD34, CD35, CD36, CD45, CD56, CD64, CD71, CD105, CD117, and HLA-DR.Mononuclear cells of AML patients diagnosed and treated at the University Hospital, Bern, Switzerland, between 2015 and 2022 were included in this study. Informed consent was obtained according to the Declaration of Helsinki and the studies were approved by the decisions of the Ethics Committee of the Canton of Bern, Switzerland. Peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMCs) were collected at the time of diagnosis before the initiation of treatment. The AML cells were analyzed at the central hematology laboratory of the University Hospital Bern according to state-of-the-art techniques . ConventFLT3wt, DNMT3A R882C, NPM1mut, TP53wt), MOLM-13 , FLT3-ITD, TP53wt), MOLM-16 , ML-2 , FLT3wt, TP53wt), SKM-1 , and PL-21  cells were supplied by the Leibniz Institute DSMZ, German Collection of Microorganisms and Cell Cultures. AML cells were grown in RPMI 1640 media  supplemented with 20% fetal bovine serum  in a standard cell culture incubator at 37 °C with 5% CO2.OCI-AML3 . Cell viability was determined after 20 h of treatment using the MTT-based cell proliferation kit I . This time point was selected because the cellular responses were effectual for the calculation of combination indexes after 20 h of treatment with two compounds in leukemic cells. For AML cell lines, four independent assays  with four measurements  per dosage were performed. For hematological patient samples, two independent assays with three technical replicates per dosage were performed. The concentration of the drug resulting in 50% inhibition of cell viability (IC50) was calculated using three-parameter logistic curve fitting. For the calculation of combination indexes, three dosages of PU-H71 and two dosages of the other compounds were applied alone and in combination. Combination indexes were calculated on Compusyn software . Data are depicted as XY graphs, column plots, or scatter plots with mean values and SD. Statistical analysis was performed on GraphPad Prism  in grouped analysis and the significance was calculated by t-test for column graphs or Mann–Whitney test for scatter plots.The HSP90 inhibitor PU-H71 and the MCL1 inhibitor S63845 were purchased at MedChem-Express . A stock solution of Venetoclax was prepared by dissolving a tablet in DMSO (VenclextaCDKN1A), Hs01034249_m1 (TP53) and Hs02758991_g1 (GAPDH). Measurements for CDKN1A and TP53 were normalized with GAPDH values (ddCt relative quantitation). Assays were performed in three or more independent experiments. Statistical analysis was performed on GraphPad Prism 9 software using unpaired t-tests. Data are depicted in column bar graphs with SD values.RNA was extracted from AML cells and quantified using qPCR. The RNA extraction kit was supplied by Macherey-Nagel, Düren, Germany. Reverse transcription was performed with MMLV-RT . Real-time PCR was performed on the Quant-studio 7 Flex PCR Instrument using FAST Start Universal probe master mix  and gene-specific probes : Hs00355782_m1  with reagents supplied by ChemoMetec. To determine the induction of apoptotis, cells were stained with AnnexinV-CF488A conjugate  in AnnexinV buffer and Hoechst 33,342 (10 μg/mL) for 15 min at 37 °C followed by several washes. Propidium iodide was added shortly before imaging. According to Annexin V and PI staining intensity, cells were classified as vital , early apoptotic , late apoptotic , or necrotic . For cell cycle analysis, cells were incubated in a lysis buffer with DAPI (10 μg/mL) for 5 min at 37 °C before imaging on the NC-3000 cell analyzer. According to DAPI staining, cell intensities were classified as subG1 (<2N), G0/G1 (2N), S phase (2–4N), or G2 phase (4N). Statistical analysis was performed using an unpaired t-test on GraphPad Prism 9 software. Data are depicted as column bar graphs with SD values.Protein extraction was conducted according to the standard protocol. In short, cell pellets were lysed in RIPA buffer containing protease and kinase inhibitors. The protein concentration of the total lysate was calculated by a Bradford assay. Concentrations of specific proteins, namely FLT3, MCL1, and BCL2, were determined with double-antibody sandwich ELISA assays with a detection range of 0.312–20 ng/mL and intra-assay CV < 10% . Two independent assays with three technical replicates were performed per sample. Statistical analysis was performed using unpaired t-tests on GraphPad Prism 9 software. Data are depicted as column bar graphs with SD values.FLT3-ITD and FLT3 wild type, NPM1 mutant and NPM1 wild type as well as TP53 wild type, mutant, and hemizygous cells  or bone marrow (BM) and to mononuclear cells isolated from the peripheral blood of healthy donors. A total of 27 primary AML and 5 healthy donor cells (HD) were subjected to single compound and combination treatments . CytotoxFLT3-ITD positive AML. Intermediate and minor responses to PU-H71 monotherapy were detected in ten AML samples each. Fourteen primary AML samples had substantial responses to S53845 monotherapy, with nine FLT3 mutated AML. Eight AML samples presented intermediate responses to S63845 monotherapy, with three NRAS and one BRAF-mutated AML. Seventeen primary AML samples had substantial responses to venetoclax monotherapy, with nine FLT3 mutated AML. Ten AML and five healthy donor cells had minor responses to venetoclax.The patient samples were sorted into response groups, namely substantial response (SR), intermediate response (IR), and minor  response (NR); compared to healthy donor cells; and treated with 100 nM PU-H71 and 100 nM S63845 C, 100 nMTP53 mutated samples (A1 and A2) were in response categories C2 and C3. The four samples with different PTPN11 mutations  fell into four response categories, Y62D:C1, E69K:C2, F285I: C3, and A72V:C4. The six samples with RAS/RAF mutations  fell into four response categories, one C1, two C2, two C3, and one C4. The eight samples with different TET2 mutations fell into four response categories: one C1, three C2, one C3, and three C4. The only sample with an AML-ETO (A21) mutation was in response category C2.According to the magnitude of in vitro response to combination treatment, the primary AML cells were grouped in four categories. C1: Elevated susceptibility to both combination treatments (17 of 27). C2: Substantial response to PU-H71 and S63845 but not to PU-H71 and venetoclax (6 of 27). C3: Substantial response to PU-H71 and venetoclax but not to PU-H71 and S63845 (2 of 27). C4: Minor response to both combination treatments (2 of 27). The two FLT3 status appeared to be the main biomarker of response to PU-H71 and S63845 treatment with elevated susceptibility of the FLT3 mutated samples .  samples A–E. The  samples F–J. The  samples A–E. The 17 > 80% F–J. The 17 > 80% K–O.In summary, elevated susceptibility to PU-H71 and S63845 was present in FLT3 mutated AML with CD34 < 20% and to PU-H71 and venetoclax in primary AML with CD117 > 80% and CD11b < 45%. A possible scenario of intracellular signaling in AML cells affected by chemical inhibition of HSP90, BCL2, and MCL1 is presented in HSP90 inhibitors may destabilize oncoproteins associated with the cell cycle, angiogenesis, RAS-MAPK activity, histone modification, kinases, and growth factors. However, due to toxicity and limited efficacy, no HSP90 inhibitor has been approved for clinical use as a single agent. Combining HSP90 inhibitors with other anticancer therapies might be a more advisable strategy. Here, we assessed the HSP90 inhibitor PU-H71, the MCL1 inhibitor S63845, and the BCL2-inhibitor venetoclax as single agents and in combination for their ability to induce apoptosis and cell death in leukemic cells in vitro.PU-H71 and combination treatments with MCL1 inhibitor S63845 or BCL2 inhibitor venetoclax induced cell cycle arrest and apoptosis in susceptible AML cell lines and primary AML samples. Drug induced changes in cellular protein levels were detected in AML cell lines treated with all three compounds. PU-H71 treatment led to reduced protein levels of FLT3, BCL2, and MCL1. Similar effects have been described where HSP90 inhibitor treatment destabilized oncoproteins and caused the degradation of cellular FLT3, BCL2, and MCL1 proteins ,21. S638FLT3-mutated primary AML samples and the FLT3-ITD-positive cell line MOLM-13. FLT3-ITD is a constitutively active growth factor receptor signaling via PI3K-AKT, RAS-MEK-ERK, and STAT5, leading to cell growth and proliferation via p53 inhibition and MCL1 induction. Treatment with the HSP90 inhibitor PU-H71 may lead to degradation of client proteins including AKT, FLT3-ITD, STAT5, and BCL2 [The majority of the tested AML patient samples were susceptible to PU-H71 in combination with S63845, with few exceptions. Biomarkers of response were deduced from mutation profiles and target protein levels. In this study, FLT3 status appeared to be the main biomarker of response to HSP90-inhibitor PU-H71 and MCL1 inhibitor S63845 with elevated susceptibility of and BCL2 ,21. In aand BCL2 , thus reand BCL2  and descand BCL2 .The marker CD34 was negatively associated with the response to PU-H71 and S63845 treatment with elevated susceptibility of primary AML samples with CD34 < 20%. CD34 is a cell surface selectin-binding glycoprotein with pro- or anti-adhesive function depending on its context and expressed glyco-form . LeukemiIn the combination treatment with PU-H71 and BCL2 inhibitor venetoclax, two different markers were associated with responses: CD117 (c-KIT) and CD11b (MAC-1). CD117 was positively associated with a response with elevated susceptibility of primary AML samples with CD117 > 80%. CD-117 is a tyrosine kinase receptor expressed on the surface of hematopoietic stem cells (HSC), multipotent progenitors (MPP), and common myeloid progenitors (CMP) as well as on the majority of AML leukemic cells. Compared to CD34, which is expressed in leukemic cells of various origins, CD117 is a specific marker for leukemia of myeloid origin. Notably, both CD117 and FLT3-ITD can induce the same downstream signaling components including PI3K-AKT, RAS-MEK-ERK, and STAT5 . The marTP53 and PTPN11 (SHP2) may also be associated with the treatment response. The tumor suppressor protein p53 and the cellular p53 inhibitor MDM2 are HSP90 clients. The conformations of both wild-type and mutant TP53 isoforms may be modified by HSP90 binding [PTPN11 gene may also be an HSP90 client. HSP90 inhibition may have opposing effects on wild-type and mutant SHP2, with downregulation of wild-type SHP2 protein and upregulation of mutant SHP2 with varied effects depending on the specific amino acid change present in the particular SHP2 mutant protein. The vast majority of cases of de novo AML carry wild-type TP53 and PTPN11 alleles. Only 8% of primary AML cases carry mutated TP53 or mutated PTPN11 genes with single or multiple mutations with varied allele frequencies [TP53 and PTPN11 mutations in primary AML and to confirm the response markers CD34, CD117, and CD11b, preclinical studies in larger cohorts are required.Other genetic markers including  binding . HSP90 i binding . Moreove binding . The SHPquencies ,39,40,41FLT3-mutated AML with CD34 < 20% and to PU-H71 and venetoclax in primary AML with CD117 > 80% and CD11b < 45%. The combination of PU-H71 and BH3-mimetics may be effective in the treatment of AML with differential target specificity for MCL1 and BCL2 inhibitors. The combination of HSP90 inhibitor PU-H71 and MCL1 inhibitor S63845 may be a candidate treatment for FLT3-mutated AML with moderate CD34 positivity. The combination of HSP90 inhibitor PU-H71 and BCL2 inhibitor venetoclax may be a candidate treatment for primitive AML with high CD117 and low CD11b positivity.In this preclinical study, we assessed the HSP90 inhibitor PU-H71 in combination with the MCL1 inhibitor S63845 or the BCL2 inhibitor venetoclax in the treatment of acute myeloid leukemia and investigated the associated biomarkers of response. Our data suggest that both treatment combinations can effectively induce cell cycle arrest, apoptosis, and cell death in AML cells. Elevated susceptibility to PU-H71 and S63845 was present in


Herein, we describe the impact of a multifaceted stewardship initiative on the sequence of antibiotic administration across three hospitals.Appropriate antibiotic sequence  across three hospitals: a major university hospital and two community hospitals. Our initiative consisted of 1) a health-system wide adult beta-lactam order panel combining load and maintenance doses for select beta-lactams  2) nursing administration instructions facilitating the appropriate sequential order of antibiotic administration (i.e. beta-lactam before vancomycin)  and 3) system-wide education to physicians, pharmacists, and nurses. The rate of beta-lactam first administration was compared between groups. Beta-lactam first rates were further stratified by hospital and antibiotic administration locations. Chi-square tests were used to compare rates between groups.We performed a pre-post analysis describing the impact of a multifaceted stewardship initiative from March 1, 2021 – May 22, 2022 (pre) and May 31, 2022 – Feb 12, 2023 (post) on the sequence of antibiotic administrations in adults Beta-Lactam Order Panel, Piperacillin-tazobactamBeta-Lactam First: Nursing Administration Instructions, Cefepime361 patients were included for analysis: 224 pre- and 137 post-intervention. Piperacillin-tazobactam was the most common beta-lactam among studied patients . Beta-lactam first rates were already high at baseline, then higher post-intervention compared to pre-intervention  (Table 2).A multifaceted initiative to promote beta-lactam before vancomycin administration increased adherence to this preferred practice in our health system, though baseline rates were high. Clinical decision support paired with system-wide education involving key stakeholders can help standardize appropriate antibiotic sequence. Future study could evaluate impact on a larger scale, on patient outcomes, and evaluate sustainability of beta-lactam first practice.Rebekah W. Moehring, MD, MPH, FIDSA, FSHEA, UpToDate, Inc.: Author Royalties


Growth traits are important parameters in husbandry. Especially for meat-type animals, understanding the genetic background and identifying naturally occurring polymorphisms can enhance production. Also, meat quality is a very important factor for food processing and consumers. In this study, we analyse IGFBP genes for possible polymorphisms that may influence growth traits and/or meat quality traits in rabbits. We found one polymorphism that influences the pH value, one of the most important indicators of meat quality, and therefore, this polymorphism may be used in the selection process.IGFBP1, IGFBP2, IGFBP4, IGFBP5, and IGFBP6 for possible polymorphisms. Based on a bioinformatics analysis in an association study on 466 animals of different breeds , Termond White (TW), Popielno White (PW), and Flemish Giant (FG)), we analyse the influence of five polymorphisms within the IGFBP genes. Statistically significant differences were found among the carcass and meat quality traits but not for all of the analysed rabbit breeds. The most promising polymorphism was g.158093018A>T within the IGFBP5 gene. The values of pH24 of m.longissimus lumborum (m.l.l.) and biceps femoris muscles (m.b.f.) were higher for the AT genotypes compared to the AA genotypes for the TW and NZWxFG crossbreeds. Also, for pH24, we found differences in ing.41594308T>C for NZWxFG, where the TT genotype values were higher than the TC values. We found differences in L*24 on m.l.l. for g.41592248A>C for NZWxFG. For m.b.f., significant differences were found in b*45 for g.3431insAC in the FG population and a*45 for g.41592248A>C and g.158093018A>T in the TW population. The shear force statistically differed for g.158093018A>T in TW rabbits and g.41592248A>C for NZWxFG. We conclude that this polymorphism may be promising for better quality rabbit meat and may be implemented in selection processes.Rabbits are important livestock animals, popular for their nutritional value. Nowadays, the molecular background of traits influencing the quality of meat and meat products is in high demand. Therefore, in the current study, we analyse the sequences of  Growth is an important aspect of animal production. Growth traits are considered moderately to highly heritable. It must be said that growth is a non-linear process, and growth patterns can differ among animals, ages, and breeds ,2. As reFor rabbits, a GWAS was successfully used to identify the genetic background of the long-hair trait of Angora rabbits  and alsoA highly conserved signalling pathway involving insulin-like growth factor 1 (IGF1) plays a major role in regulating skeletal muscle growth. The activity of this pathway may be regulated by many factors and pathways at different steps. One of these factors includes IGF binding proteins (IGFBPs) with IGFB5, as it can block IGF1 by inhibiting its binding to IGF1 receptors . The binIGFBP genes for possible polymorphisms associated with the growth, slaughter, carcass, and meat quality traits in different rabbit breeds.Therefore, the current study analyses The authors confirm that the experiment complied with the European Union’s Directive on Animal Experimentation (Directive 2010/63/EU) and ARRIVE guidelines. All experimental protocols were approved by the 2nd Local Institutional Animal Care and Use Committee (IACUC) in Krakow, agreement No.267/2018, and the Institutional Animal Care Review Board of the Faculty of Animal Sciences, the University of Agriculture in Krakow permissions, 29/2016, 37/2016, and 2/2018. All procedures were performed in accordance with the relevant guidelines and regulations.The experiment was conducted under standardised conditions at the Experimental Station in the Department of Genetics, Animal Breeding and Ethology, University of Agriculture in Krakow. In the present study, we analysed data from 466 animals : 130 Termond White (TW) rabbits (from 20 bucks); 40 Flemish Giant (Belgian Giant Grey) rabbits (FG) (from 8 bucks); 71 Popielno White (PW) rabbits (from 10 bucks); and 225 crossbred rabbits of the F2 generation of New Zealand White × Flemish Giant (NZWxFG) (18 bucks). The crossbred population was a part of the experiment conducted at the Department of Genetics, Animal Breeding and Ethology for possibly increasing the slaughter weight of NZW rabbits by mating them with a large breed (FG). The crossbred population was derived from 48 litters, and subpopulations of the animals with the highest, medium, and lowest slaughter weights were chosen for the experiment. The purebred animals  were under the control of the National Centre for Animal Breeding. The animals were kept in a heated hall with a water supply (nipple drinkers), lighting (14L/10D), and exhaust ventilation. From weaning (on the 35th day of life) until the 84th day of life, the animals were kept in wire metal cages intended for the commercial rearing of rabbits (with two rabbits per cage). Water and feed were available ad libitum; their pelleted commercial diet contained 15% crude protein, 16.1% crude fibre, and 3.5% crude fat.The rabbits were weaned in their fifth week of life and slaughtered in week 12 (BW12). The standardised methodology for slaughter was used as follows: After 24 h of fasting, the slaughter body weight (SW) was recorded, and the animals were subsequently slaughtered. The rabbits were stunned, immediately bled, pelted, and eviscerated. After slaughter, the hot carcass weight (HCW) was recorded, and after 24 h of storage at 4 °C, the chilled carcass weight (CCW) was recorded. The dressing-out percentages (%) were calculated—both the dressing-out percentage hot (DPH) = [HCW/SW]*100 and the dressing-out percentage chilled (DPC) = [CCW/SW]*100. Each carcass was divided between the seventh and the eighth thoracic vertebrae and between the sixth and seventh lumbar vertebrae, and the dissected meat, bone, and fat weights from each part were recorded. All measurements used Łucznik KS-205 electronic scales .m.longissimus lumborum (loin—m.l.l.) and m.biceps femoris (hind leg—m.b.f.).The colour of the meat was determined using Minolta CR-400 chroma meters  calibrated with a white plate supplied by the manufacturer and programmed to use a D65 standard illuminant, a 10° observer with an illuminating/viewing aperture size of 11 mm, and a blooming time of 45 min. The average value of three readings was recorded for each sample and expressed as the CIE lightness (L*), redness (a*), and yellowness (b*). The pH values in the meat were determined using the Consort C561 pHmeter  with a temperature probe with temperature compensation . The colouring and pH were recorded 45 min after slaughter and 24 h after chilling on m. longissimus lumborum were cut from the right half of the loin. The samples were vacuum-packed in foil used for food storage, frozen for 72 h at −18 °C, thawed at room temperature, and boiled in a water bath at 80 °C for 40 min. The shear force and texture parameters were measured using the TA.XTplus Texture Analyser . The shear force was measured from three cylindrical samples (15 mm in diameter and 15 mm in height) using a Warner–Bratzler attachment and a triangular notch in the blade. Meat samples were cut perpendicular to the direction of the muscle fibres. The blade speed during the test was 2 mm/s. The texture  was analysed using the attached cylinder, 50 mm in diameter. The three samples were subjected to a double pressing test, applying a force of 10 g to 70% of their height. The cylinder speed was 5 mm/s, and the interval between presses was 5 s.Cylindrical samples from the The DNA was collected during slaughter into tubes containing EDTA. The DNA was extracted using a WIZARD Genomic Kit  from 300 µL of blood collected after slaughter into tubes containing EDTA.https://bioinfo.ut.ee/primer3/) (accessed on 13 May 2020) using the sequences of rabbit IGFBP1 (ENSOCUG00000012831), IGFBP2 (ENSOCUG00000008561), IGFBP4 (ENSOCUG00000002828), IGFBP5 (ENSOCUG00000025513), and IGFBP6 (ENSOCUG00000001412). All chromatograms were visually inspected in FinchTV v1.4.0 . The online sequence alignment BLAST tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi) (accessed on 20 October 2020) was used to confirm the obtained sequences. The quality of the sequences was inspected using the CodonCode Aligner  (accessed on 20 October 2020). The SNPs were identified by aligning the reference sequence of OryCun 2.0 and aligned sequencing readings in MEGAX [The primers for sequencing were designed with Primer3 software (v. 0.4.0)  according to protocol and the MIC qPCR cycler   and PCR- restriction fragment length polymorphism (RFLP). The primers for both methods are listed in stralia) . PCR-RFLp < 0.05) was divided by the number of tests. Thus, the Bonferroni-corrected significance level of 0.05/16 = 0.0031 was applied.Associations between the SNPs and quantitative traits within each breed were investigated using the analysis of variance using the general linear model (GLM) procedure of SAS 9.4  and the The estimation of the likelihood of SNP’s impact on proteins was analysed using PANTHER 16.0 software , where phttps://www.ebi.ac.uk/ena/data/view/PRJEB42486) (accessed on 20 January 2021), using the PCR-RFLP/PCR-HRM method, we analysed 5 SNPs. Those SNPs were located in coding regions (non-synonymous mutations), 5′ UTRs, and close to coding regions. Therefore, we hypothesised that those SNPs could have an impact on the protein function  and influence some growth, slaughter, and meat traits. In the IGFBP1 gene sequence, we analysed mutations within intron 2_3—g.4988G>A and the insertion of AC within 3′ UTR. For the IGFBP4 gene, a missense mutation in exon 3 g.41592248A>C resulted in an I182L substitution (Pdel = 0.57 with a preservation time of 456′ was probably damaging), and g.41594308T>C on the last position of 5′ UTR. For the IGFBP5 gene, a substitution g.158093018G>T in 5′ UTR was analysed. The analysed polymorphism frequencies are presented in Out of the 37 identified SNPs (IGFBP1). IGFBP4), IGFBP4), and IGFBP5). The statistical analysis did not show significant differences between the genders, so in the tables, those results are excluded. Moreover, the interaction between gender and genotypes was not significant. The association results with the growth and slaughter traits are presented in IGFBP4, the AA genotypes had significantly higher a* values after 45 min on m.b.f. compared to the AC for the TW rabbits. For the NZWxFG rabbits, the L* value on m.l.l. after 24 h and the shear force for the AA genotypes showed significantly higher values than the AC genotypes. The most promising results were obtained for g.158093018A>T within IGFBP5. For the carcass traits, the pH24 values measured on m.l.l. and m.b.f. showed significantly higher values for the AT genotypes compared to the AA genotypes for the TW and NZWxFG rabbits. Also, for the TW rabbits, a *45 value measured on m.b.f. showed a significantly higher value for AT compared to AA. We did not find significant differences in the a* and b* values after 24 h of chilling on both muscles. The shear force of the TW rabbits was lower for the AT genotypes compared to AA for g.158093018A>T. Meanwhile, for the NZWxFG rabbits, the AC genotypes had a significantly lower shear force value than AC for g.41592248A>C. We did not find differences in the texture profile analysis values. The statistical analysis did not show significant differences between the genders, so those results are excluded from the tables. Moreover, the interaction between gender and genotypes was not significant.IGFBP5 on a meat quality parameter—pH24—as the main point of discussion. Meat quality is an important parameter that modern husbandry pays attention to. The quality of meat depends on many traits, which are affected by different genes and, therefore, different metabolic pathways. As reviewed by Blasco et al. [FGD4 and DNM1L genes. Sanchez et al. [Our results highlight the influence of the g.158093018A>T polymorphism within o et al. ,11,12,25As presented in the tables, many traits that impact the quality of carcasses were associated with the analysed SNPs; however, these results are limited to one or two breeds. In the present study, we analysed phenotypically different breeds: a common broiler breed—Termond White (TW), and a crossbreed of another broiler breed—New Zealand White and Flemish Giant (NZWxFG). Also, we included in the analysis a native breed (Popielno White rabbits—PW), a Polish medium breed with good maternal and growth traits , and theMeat quality is a composite concept and is difficult to measure simply . From maIGFBP5 mRNA. In particular, these authors found under-expression in the Duroc breed. Acidity affects meat processing suitability and its culinary uses. Defects such as pale, soft, exudative (PSE) and dark, firm, and dry (DFD) lead to a lower culinary value of meat and decreased consumer acceptance. In this context, the halothane gene can be mentioned as a good example in pigs [The pH of rabbit meat is well described in the literature. As there are many rabbit breeds, different breeding systems that affect the pH value and differences between muscles were observed. Additionally, pH was reported to have a reliable estimate of heritability , and the in pigs . The sla in pigs  analysedIGFBP5 and pH values. For pigs, on the SSC15 region, SNPs between 114.8 and 121.4 MB affect, among other factors, the level of residual glycogen and glucose, pH36, drip loss, and the colour parameters L*, a*, and b* were also identified [PRKAG3 (at 120.86 MB), close to the IGFBP5 gene (at 118.87 MB). In rabbit chromosome 7 (OCU7), where the IGFBP5 is mapped (at 158.1 MB), the PRKAG3 gene is also located (at 160 MB), which may suggest that this region is important for meat traits in rabbits. As mentioned above, most traits are polygenic, which can also be seen in the pH, as reports show the influence of different polymorphisms within genes on the pH. Wang et al. [POU1F1 influence the pH of m. biceps femoris. Also, Wang et al. [CAST (g.16443397 T>G) influence the pH of both muscles.There are no reports about the associations of SNPs within rabbits between the entified ,38. Thisg et al.  showed tg et al.  showed tCAST gene that the GT (2.46 ± 0.25) genotype was significantly higher compared to GG (1.81 ± 0.23) and TT (1.07 ± 0.19) in Hyla, Champagne, and Tianfu black rabbits. Our results for each analysed breed were lower for the b* value on m.b.f. compared to those reported by other authors [Colour is considered one of the most important characteristics of meat quality that most influence consumer choice . Meat co authors ,23,33. T authors ,30. From authors  and, mos authors . As Maj  authors  reported authors , Chodova authors . For the authors , but the authors  reportedRabbit meat texture may differ among breeds, thermal treatments, the composition of muscle fibres, and the transport and handling before slaughter ,47,48,49IGFBP family for possible SNPs and their association with growth and meat traits. We found that g.158093018A>T in Termond White rabbits and Flemish Giant and New Zealand White crossbreed rabbit populations influenced the pH after 24 h on m. longissimus lumborum and m. biceps femoris. Also, other associations were found, like shear force and colour parameters, but these findings were breed-specific. As in rabbit husbandry, a number of different breeds that are used in different countries and companies may be mainly limited to molecular approaches for selection. However, these findings may be interesting, as the pH value is one of the most important indicators of meat quality. There is a lack of reports about meat conditions for rabbits, like what exists for pigs and poultry (PSE). We hypothesised that our results might be interesting in future research to help with a better understanding of unacceptable meat conditions in rabbits. Based on our results, we hypothesise that primitive breed populations (like PW and FG) do not carry this mutation, and this SNP may occur in meat-type breeds , which may encourage additional analyses regarding the influence of breeds on meat quality. As additional analyses must be performed on other meat and local rabbit breeds, this SNP may be used in the future as a marker in MAS.Here, we have analysed the sequences of rabbit genes from the