The non-homologous end joining pathway is vital for repairing DNA double-strand breaks (DSB), with DNA-dependent protein kinase (DNA-PK) playing a critical role. Altered DNA damage response (DDR) in chronic (CML) and acute myeloid leukemia (AML) offers potential therapeutic opportunities. We studied the therapeutic potential of AZD-7648 (DNA-PK inhibitor) in CML and AML cell lines. This study used two CML (K-562 and LAMA-84) and five AML  cell lines. DDR gene mutations were obtained from the COSMIC database. The copy number and methylation profile were evaluated using MS-MLPA and DDR genes, and telomere length using qPCR. p53 protein expression was assessed using Western Blot, chromosomal damage through cytokinesis-block micronucleus assay, and γH2AX levels and DSB repair kinetics using flow cytometry. Cell density and viability were analyzed using trypan blue assay after treatment with AZD-7648 in concentrations ranging from 10 to 200 µM. Cell death, cell cycle distribution, and cell proliferation rate were assessed using flow cytometry. The cells displayed different DNA baseline damage, DDR gene expressions, mutations, genetic/epigenetic changes, and p53 expression. Only HEL cells displayed inefficient DSB repair. The LAMA-84, HEL, and KG-1 cells were the most sensitive to AZD-7648, whereas HL-60 and K-562 showed a lower effect on density and viability. Besides the reduction in cell proliferation, AZD-7648 induced apoptosis, cell cycle arrest, and DNA damage. In conclusion, these results suggest that AZD-7648 holds promise as a potential therapy for myeloid leukemias, however, with variations in drug sensitivity among tested cell lines, thus supporting further investigation to identify the specific factors influencing sensitivity to this DNA-PK inhibitor.  DNA douaneously . One of aneously ,5. The Dectively ,6.BCR-ABL1, which encodes an oncoprotein with abnormally high tyrosine kinase activity [Genomic stability is crucial for faithful genome replication, therefore, cells have developed a complex DNA damage response (DDR) network to protect genome integrity, and repair damaged DNA . Dysfuncactivity . AML is activity ,15. Someactivity ,16,17.50 value of 0.6 nM in biochemical assays and a >100-fold selectivity against many closely related kinases, such as PI3Ks [Genomic instability associated with cancer may provide targetable vulnerabilities for cancer therapy , and givas PI3Ks ,19,21. Cas PI3Ks . Considering the doubt regarding the therapeutic potential of AZD-7648 in the treatment of myeloid leukemias, studies in this area are necessary to elucidate the relevance of this approach. Therefore, our study aimed to assess the therapeutic potential of AZD-7648 in monotherapy for myeloid leukemia treatment using in vitro models of CML and AML. We hypothesized that AZD-7648 may have promise as a new therapeutic strategy for these myeloid leukemias in monotherapy. The results from our study may help shed some light on the efficacy of this DNA-PK inhibitor and may help clarify the relevance of this therapeutic line for these diseases.The mutational status of genes involved in the DDR of CML and AML cell lines included in this study was retrieved from the COSMIC platform . CML celConsidering the functions associated with the mutated genes, particularly those linked with DNA repair pathways, distinct patterns are evident among the cell lines. The NER pathway was the most affected repair mechanism in the K-562, HEL, and KG-1 cell lines and the MMR pathway in THP-1 cells. LAMA-84 presented more alterations in the Fanconi anemia pathway, NB-4 in the HRR pathway, and HL-60 with the same number of mutations in the BER and HRR pathways. Further information involving the mutations can be found in the MGMT), were assessed in CML and AML cell lines using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA). As shown in MLH1, MSH2, and PMS2 genes appeared to be the most altered, as each presented genetic abnormalities in five different cell lines. The MSH6 gene was only altered on one CML cell line (LAMA-84). The HEL cell line presented only a genetic abnormality, specifically an amplification within the MSH2 gene. Similarly, both K-562 (CML) and THP-1 (AML) cell lines just carried amplifications, affecting three genes each. On the other hand, the KG-1 cell line carried only deletions in the MLH1 and MSH2 genes. The HL-60 and NB-4 cell lines carried the highest number of genetic alterations, each with five genes affected. The MGMT gene exhibited the highest degree of methylation  were evaluated via qPCR. As illustrated in RAD51, significantly surpassing HL-60, K-562, NB-4, and THP-1 (p < 0.01), and KG-1 (p < 0.001), which was the cell line with the lowest expression levels. Both the LAMA-84 and HL-60 cell lines presented the highest expression levels of CHEK2, significantly above those of HEL , K-562, KG-1, NB-4, and THP-1 (p < 0.001). Among these cell lines, NB-4 had the lowest expression levels. The PRKDC gene’s lowest expression levels were observed in HL-60 cells, significantly lower than in the KG-1 cell line (p < 0.05). KG-1, followed by LAMA-84 cells, exhibited the highest expression levels for this gene. Although without statistical significance, the NB-4 cell line displayed the highest TP53 expression levels, followed by HEL. Among the remaining cell lines, except HL-60 cells that do not express the gene TP53, noticeably lower expression levels were observed. Lastly, regarding the PARP1 and XRCC6 genes, the expression levels were mostly similar across the cell lines. However, for both genes, particularly for the PARP1 gene, LAMA-84 cells appeared to have the highest expression levels, while NB-4 had the lowest.The baseline expression levels of the genes involved in NHEJ and alternative repair pathways , K-562 (p < 0.01), and THP-1 (p < 0.05).We began by characterizing the frequency of DSB in the CML and AML cell lines, as well as in IMC . Baseline levels of phosphorylated-H2AX (γH2AX), a biomarker of DSB, were quantified through flow cytometry . The HELp < 0.001), as well as KG-1 (p < 0.01).The chromosomal damage was assessed using the cytokinesis-block micronucleus cytome assay (CBMN), wherein binucleated (BN) viable cells were categorized into cells lacking chromosomal damage biomarkers (CDBs), cells with micronuclei (MNi), cells with nuclear buds (NBUDs), and cells with nucleoplasmic bridges (NPBs). Firstly, total chromosomal damage was quantified as a percentage of cells with CDBs. As detailed in p < 0.01). Within this cell line, the MNi frequency was 1.6-fold and 8.4-fold higher than the NBUDs and NPBs, respectively. The KG-1 cell line also presented MNi as the dominant chromosomal damage. Here, MNi were 1.8-fold and 2.2-fold more frequent than the NBUDs and NPBs, respectively. NPBs were the prevalent damage type in the LAMA-84 cell line, with the percentage of this damage being 2.0-fold and 1.9-fold higher than in MNi and NBUDs, respectively. When compared to IMC cells, the levels of NPBs were significantly higher, not only in the LAMA-84 (p < 0.001) but also in the K-562 (p < 0.01), HEL, KG-1, and NB-4 (p < 0.05) cell lines. Lastly, NBUDs were the predominant type of damage in the remaining cell lines, specifically HEL, HL-60, K-562, NB-4, and THP-1. In these cases, the percentage of NBUDs was 2.1-fold, 1.7-fold, 2.6-fold, 1.6-fold, and 1.9-fold higher than that of MNi, respectively. When comparing NPBs and NBUDs, the latter percentage was 2.4-fold, 3.5-fold, 1.9-fold, 2.3-fold, and 4.7-fold higher in the HEL, HL-60, K-562, NB-4 and THP-1 cells, respectively. Among these cell lines, THP-1 showed the highest NBUDs percentage, significantly surpassing that of the IMC cells (p < 0.05).Additionally, the percentage of BN cells displaying each type of damage  was calculated . The CMLp < 0.01).The telomere length was assessed using qPCR and normalized to the telomere length of IMC cells . The cel2O2. As shown in 2O2 in the K-562 , KG-1 , and THP-1  cells, compared to the baseline (0 hour time point). Then, this increase was followed by a significant decrease in γH2AX levels at the 24 h time point in the K-562  and THP-1  cell lines, reaching the baseline levels of damage. Though this decrease was not statistically significant in KG-1 cells (1.9-fold), no statistically significant differences were found between the baseline DSB levels and the levels 24 h after H2O2 exposure in any of these three cell lines. In the HL-60, LAMA-84, and NB-4 cells, while not statistically significant, a peak 1 h after the genotoxic stimuli was observed. This peak resulted from respective 1.8-fold, 1.4-fold, and 1.2-fold increases in γH2AX levels compared to the baseline. A decrease in DSB levels between the 1 and 24 h time points was observed , showing a recovery of the original γH2AX levels. Consequently, no statistical differences were observed between these cell lines’ 0 and 24 h time points. The HEL cell line showed a significant 1.9-fold increase (p < 0.05) in γH2AX levels 1 h after exposure to H2O2, followed by a 1.2-fold decrease at 24 h. When comparing the damage quantified at 24 h with that at the baseline levels, a significant 1.2-fold (p < 0.05) difference remains evident.DSB repair kinetics were established by assessing γH2AX levels through flow cytometry at different time points following exposure to a genotoxic stimulus with H50) was not reached in the HL-60, K-562, NB-4, and THP-1 cells at any time point (IC50 > 200 µM). However, different behaviors were observed among these cell lines, and in HL-60 and K-562, time seemed to have limited influence on the effect of the tested doses on cell viability. In contrast, the NB-4 and THP-1 cells presented a more pronounced decrease in cell viability over time, particularly at 72 h. After 24 h of incubation, HEL cells required the lowest concentration of AZD-7648, showing an IC50 ranging between 150 µM and 200 µM  failed to achieve a 50% decrease in cell viability. However, following 48 and 72 h of incubation required the lowest concentrations for these time points, resulting in IC50 values of 92.6 µM and 81.6 µM, respectively. Like this cell line, in KG-1 cells, a 50% decrease in cell viability was not reached at the tested concentrations at 24 h. At 48 h, a decrease in viability of approximately 50% was obtained with the 200 µM dose, yet the mathematical IC50 was only reached at 72 h, with a value of 159.9 µM.The effects of AZD-7648 on cell density and viability are represented in d 200 µM b. After p < 0.001; KG-1: 7.9-fold, p < 0.001) and late apoptotic/necrotic cells  was observed in both cell lines. This increase was linked to a decrease in the percentage of viable cells (p < 0.001). In the LAMA-84 cell line, treatment with 100 µM of AZD-7648 led to an increase in the percentage of early apoptotic (2.8-fold) and late apoptotic/necrotic  cells, compared to untreated cells. The treatment with 200 µM of the inhibitor significantly increased the early and late apoptotic LAMA-84 cells . The effect of the highest tested dose was evaluated in the cell lines where the IC50 was not reached . Mainly, the K-562 and THP-1 cells treated with 200 µM of the inhibitor showed a decrease in viable cells, accompanied by a significant increase of 2.6-fold (p < 0.001) and 1.7-fold (p < 0.05) in the early apoptotic and 2.1-fold (p < 0.01) and 2.4-fold (p < 0.01) in the late apoptotic/necrotic cells, respectively, compared to the control. Despite the absence of statistical difference, a 2-fold increase in the late apoptotic/necrotic population was noticeable in the HL-60 and NB-4 cells following inhibitor treatment  . No statreatment a. In agreement with these results, cells treated with AZD 7648 showed morphological aspects typical of apoptosis, like blebbing, nuclear fragmentation, and cellular contraction b. After 50 was reached , further supporting apoptosis as the main cell death mechanism due to a significant increase in cleaved PARP and activated caspase-3-positive cells (p < 0.001) was observed after treatment with 50 µM and 150 µM of AZD-7648, respectively, compared to untreated cells. Similarly, in the LAMA-84 cell line, the inhibitor increased the percentage of cells with cleaved PARP . In the KG-1 cell line, after 48 h of treatment, similar results were observed, with a 2.6-fold, and 7.8-fold (p < 0.001) increase in the cleaved PARP levels after treatment with 100 µM and 200 µM, respectively. Likewise, an increase in activated caspase-3-positive cells was observed after 24 h of treatment  compared to the control. The KG-1 cell line presented a similar trend after 48 h of treatment with 100 µM and 200 µM of AZD-7648, increasing the percentage of activated caspase-3 cells by 4.9-fold and 13.3-fold (p < 0.001), correspondingly. Afterwards, we quantified the levels of cleaved PARP and activated caspase-3 in the cell lines where the ICve cells a. Followp < 0.01) for 24 h. The tested doses in LAMA-84, 100 µM, and 200 µM increased γH2AX-positive cells by 1.9-fold and 4.4-fold (p < 0.001), respectively. For the KG-1 cell line, treatment with 100 µM and 200 µM of AZD-7648 for 48 h led to similar increases in γH2AX-positive cells .As represented in 0/G1 phase, leading to an increased percentage of cells in this cell cycle phase compared to untreated cells. The treatment with the IC25 dose induced a significant increase in the proportion of cells in the G0/G1 phase in HEL , KG-1 , and LAMA-84  cells. A similar trend in cell cycle distribution was observed for these cell lines after exposure to higher doses . The HL-60, NB-4 and THP-1 cell lines exhibited a significant increase of 2.2-fold (p < 0.001), 1.9-fold (p < 0.001), and 1.3-fold (p < 0.001), correspondingly, in G0/G1 cells after the treatment with 200 µM of AZD-7648. On the contrary, in K-562 cells, the cell cycle distribution remained similar between the control and treated cells. Additionally, the presence of an apoptotic peak (Sub-G1), corresponding to the DNA fragmentation typical of apoptotic cells, was observed across all tested concentrations of AZD-7648, except in HL-60, particularly in the HEL cells treated with 150 µM (p < 0.01) and KG-1 cells treated with 200 µM (p < 0.01), thus reinforcing the induction of apoptosis as demonstrated in previous studies  hours of treatment with AZD-7648 . This DN studies .p < 0.001; LAMA-84: 6.7-fold, p < 0.01). In the KG-1 cell line, treatment with 100 µM and 200 µM of the inhibitor for 48 h resulted in significant decreases of 8-fold (p < 0.05) and 9-fold (p < 0.01) in cell proliferation, respectively.The incorporation of BrdU, a marker for cell proliferation, was assessed in the LAMA-84, HEL, and KG-1 cell lines after treatment with different concentrations of AZD-7648 for 24 h, or 48 h in the case of KG-1 . CompareSignificant progress has been made in the CML and AML treatment options. However, CML patients develop resistance or are intolerant to tyrosine kinase inhibitors  and AML Of the tested cell lines, the cellular density and viability decrease induced by AZD-7648 was more evident in the LAMA-84, HEL, and KG-1 cell lines. A decrease in cell viability after treatment with a DNA-PK inhibitor was also observed in previous studies . As prevBRCA1, RAD51D, PAXIP1, and RBBP8), three of which are only mutated in this cell line. Considering that the HR repair pathway is responsible for the accurate repair of DSBs [BCR-ABL1. This fusion gene has been linked to resistance to apoptosis and the down-regulation of DNA-PKcs, leading to deficient DNA repair [The NB-4 cell line also presented high γH2AX levels. This cell line has the highest number of mutated genes related to HR repair  repair pathways, was associated with high basal levels of MNi . Among tg DNA-PK ,7, as weg DNA-PK ,39. The MLH3 in the LAMA-84 cell line. Elevated methylation levels were mainly detected in the direct reversal damage repair gene (MGMT). The copy number amplification potentially triggers the increased expression of corresponding MMR proteins. This increase could result in a more effective MMR pathway, increasing the cell’s capacity to repair DNA mismatches and errors caused by DDR inhibitors. Furthermore, MMR and HR pathways are connected, and studies showed that MMR proteins, such as MSH2 and MSH3, are involved in HR repair choice [The HL-60 and K-562 cell lines, closely followed by the NB-4 and THP-1 cells, demonstrated resistance to AZD-7648. As is known, the same type of DNA damage can be repaired by different repair pathways, thus showing the redundancy in DNA repair ,41,42. Tr choice ,44. ThisA previous study proved that a population of p53-deficient cells was not sensitive to DNA-PK inhibition . This woTP53 gene were detected in all cell lines; however, the nature and severity of these mutations varied depending on the specific cell line. In the case of the HL-60 cell line, the extensive deletion of the TP53 gene was detected [TP53 expression. In the KG-1 cell line, the TP53 mutation originated from a substitution in a non-coding region that functions as a splice donor site. In the HEL and NB-4 cells, a missense mutation led to the replacement of one amino acid, and the SIFT (Sorting Intolerant From Tolerant) score predicted that the mutations are likely to have a harmful or damaging effect on the protein’s function. For the K-562 and THP-1 cell lines, the TP53 mutation resulted in a frameshift due to an insertion or deletion, respectively. Notably, these mutations in the above-mentioned five cell lines are located within the p53 DNA binding domain. Lastly, in LAMA-84 cells, a nonsense mutation is found in the p53 tetramerization domain, which could account for the observed lower molecular weight of p53 in this cell line. All these mutations are considered oncogenic  or likely oncogenic .Genetic alterations in the detected  and corrAs previously shown by Fok et al., the treatment with AZD-7648 induces apoptosis . We also0/G1 phase. Cell cycle arrest in this phase allows cells to repair damaged DNA or follow the apoptotic pathway [0/G1 and reduction in BrdU incorporation after treatment with a DNA-PK inhibitor were also previously observed by Hafsi et al. [Besides the cytotoxic effect observed after treatment with AZD-7648, this inhibitor also presented a cytostatic effect. This effect was observed as an increase in the percentage of cells in the Gi et al. . The cyti et al. ,50. In a50 values of less than 3 nM and 14 nM, respectively [50 values of 21 nM and 13 nM, respectively [50 of 0.6 nM), AZD-7648 also presents a high selectivity against closely related kinases  [While previous works explored the potential of DNA-PK inhibition as a therapeutic pathway in AML, limited effects were observed when treating the cell lines only with this inhibitor ,52,53. Iectively ,54. CC-1ectively . In the , PI3Kδ) ,21. ThisAlthough this study gives information about the therapeutic potential of AZD-7648 in myeloid leukemias, it is essential to identify some limitations that could provide direction for future research. Firstly, this study used in vitro cell culture models, which do not fully represent the complexity of leukemia in a living organism and the diversity of genetic and molecular alterations found in patients. Therefore, future research could use primary cell line cultures and in vivo studies to better assess the drug’s effectiveness and safety. Furthermore, in this study, a human lymphocyte cell line was used as the model for normal cells, instead of a myeloid cell line. This choice was due to the difficulties associated with establishing and maintaining normal myeloid cell lines, making the lymphocyte cell line a better alternative. Secondly, at the tested concentrations, AZD-7648, in addition to inhibiting DNA-PK, may also present off-target effects on other kinases involved in DSB repair, such as ATM and ATR. Hence, in future studies, it would be valuable to investigate the status and activity of these proteins to clarify the mechanism of action of this drug at the tested concentrations. Lastly, the differences in drug sensitivity observed among the tested cell lines highlight the heterogeneity of leukemia cells and the specific factors influencing these differences should be studied.6 cells/mL and maintained at 37 °C in a humidified atmosphere containing 5% CO2. The cell lines were negative for Mycoplasma and were authenticated using short tandem repeat analysis.We used two CML in blast crisis cell lines (K-562 and LAMA-84) and five AML cell lines: HEL (human erythroleukemia), HL-60 (acute myeloid leukemia), KG-1 (human erythroleukemia), NB-4 (acute promyelocytic leukemia), and THP-1 (acute monocytic leukemia). K-562, HEL, HL-60, and KG-1 were purchased from the ATCC, and LAMA-84, NB-4, and THP-1 were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ). Additionally, an immortalized normal human lymphocyte cell line (IMC) was used and obtained from the Cytogenetics and Genomics Laboratory, Faculty of Medicine, University of Coimbra. All the cells were cultured in Roswell Park Memorial Institute 1640 medium  supplemented with 10% or 20% (IMC) of heat-inactivated fetal bovine serum , 2 mM of L-glutamine, 100 U/mL of penicillin, and 100 μg/mL of streptomycin . Cells were grown at initial densities of 0.3 (HL-60), 0.4 (HEL and NB-4), or 0.5  × 10AZD-7648 (AZD—DNA-PK inhibitor) was purchased from MedChemExpress and was prepared in dimethyl sulfoxide (DMSO). Cell lines were incubated in the absence or presence of increasing doses of AZD-7648, ranging from 10 to 200 µM, for different periods depending on the assay. The stocks were prepared in the medium on the day of use to avoid DMSO cytotoxicity and ensure consistency between conditions.https://cancer.sanger.ac.uk/cell_lines accessed on 10 August 2023. The genes were grouped according to human DNA repair genes (mdanderson.org accessed on 10 August 2023). The oncogenic classification and the mutations coding impact were obtained from two databases, the Franklin by Genoox  and the Varsome (https://varsome.com/ accessed on 10 August 2023). Lastly, the SIFT and PolyPhen scores and predictions were gathered from the Variant Effect Predictor (https://useast.ensembl.org/Homo_sapiens/Tools/VEP accessed on 10 August 2023). The DDR pathways mutations data were retrieved from the COSMIC platform, available at MS-MLPA (ME011) probe panel was applied as described in the manufacturer’s protocol . This panel determines the methylation status of selected GCGC sites in the promoter regions of human repair genes. It also detects deletions or duplications in the human 3′ region of these genes. Succinctly, 100 ng of each DNA sample was used, and the amplification products were detected and measured using capillary electrophoresis on an ABI-3500 genetic analyzer . Subsequently, the results were evaluated using Coffalyser software version v.220513.1739 . Each MS-MLPA assay also included three control DNA samples obtained from healthy individuals and negative control (no template control). Methylation analysis involved the identification of distinct methylation ranges: demethylation (0–9%), hemi-methylation (10–50%), and methylation (51–100%). For copy number analysis, a value below 0.8 was categorized as deletion (copy number loss), while a value surpassing 1.2 was categorized as duplication (copy number gain).CHEK2, PARP1, PRKDC, RAD51, TP53, and XRCC6) was evaluated by qPCR  in a QuantStudio™ 3 System . Hypoxanthine phosphoribosyltransferase (HPRT) was used as the endogenous control gene. Relative gene expression was calculated using the 2−ΔΔCt formula, and the results represent the fold change mean ± SEM of three independent experiments. The total RNA of the seven cell lines was extracted using TripleXtractor  and converted to cDNA using Xpert cDNA Synthesis Kit (GRiSP), according to the manufacturer’s protocol. Expression of six DDR genes . Afterwards, cells were stained with 1 µL of anti-γH2AX antibody  for 20 min, in the dark, at room temperature. Lastly, cells were washed, resuspended in PBS, and analyzed in a FACS Calibur flow cytometer (Becton Dickinson). Through CellQuest software v.3.3 (Becton Dickinson), 50,000 cells were acquired, and data were analyzed using Paint-a-Gate software v.3.0 (Becton Dickinson). The results were expressed in mean fluorescence intensity  and represented mean ± SEM of three independent experiments.The γH2AX expression levels were evaluated through flow cytometry. Briefly, 0.5 × 10Chromosomal damage biomarkers were evaluated using the cytokinesis-block micronucleus cytome assay. Cell lines were cultured at their optimal density and incubated for 24 h with 4.5 µg/mL or 3 µg/mL (NB-4) of cytochalasin-B . Cells were then treated and analyzed as described by Costa et al. . Chromos−ΔΔCt formula. The results represent the fold change mean ± SEM of three independent experiments.The genomic DNA used was isolated using the salting out method. According to the manufacturer’s instructions, the telomere length of the tested cell lines was assessed using Relative Human Telomere Length Quantification qPCR Assay Kit . Briefly, two primer sets were used: the telomere primer that recognizes and amplifies telomere sequences and the single copy reference primer that serves as a reference for data normalization. Equal amounts of DNA (5 ng/µL) were used for each reaction. qPCR reactions were performed in a QuantStudio™ 3 System . The telomere length of the IMC cell line was used as a reference for calculating telomere length. Relative telomere length was calculated using the 22O2   in a humidified atmosphere containing 5% CO2. The repair capacity of double-strand breaks was evaluated by assessing its kinetics through the expression levels of γH2AX. Following the genotoxic exposure, the cell suspensions were harvested, centrifuged for 5 min at 42× g, and cultured in a new medium. Then, at 1 h and 24 h post-exposure to H2O2, 0.5 × 106 cells were collected and subjected to a 5 min PBS wash at 300× g. The levels of γH2AX were quantified using the intracellular staining protocol outlined in The cell lines were cultured at their optimal density and were exposed, for 30 min, to HThe trypan blue exclusion assay assessed cell density and viability for 72 h. Briefly, after every 24 h of incubation, an aliquot of cells was harvested and stained with an equal volume of trypan blue (Sigma-Aldrich), loaded in a hemocytometer, and counted resorting to the Countess™ II Automated Cell Counter (ThermoFisher Scientific). The instrument automatically calculates cell density and viability based on the number of viable cells per mL or the percentage of viable cells, respectively. The results were expressed as mean ± SEM of five independent experiments.6 cells were collected and washed with PBS by centrifugation at 1000× g for 5 min. Afterwards, cells were resuspended in 100 μL of AV binding buffer and incubated with 5 μL of AV-APC  and 1 μL of PI (Biolegend) for 15 min in the dark at room temperature. Cells were then diluted in 300 µL of AV binding buffer, and the analysis was run on a FACSCalibur (Becton Dickinson) flow cytometer. Through CellQuest software v.3.3 (Becton Dickinson), 50,000 events were acquired, and data were analyzed using Paint-a-Gate (Becton Dickinson). Results were expressed as a percentage of viable cells (AV−/PI−), early apoptotic (AV+/PI−), late apoptotic/necrotic (AV+/PI+), and necrotic cells (AV−/PI+) and represent the mean ± SEM of five independent experiments. Cell death was assessed using flow cytometry, through annexin V (AV) and propidium iodide (PI) double staining, and by morphological analysis using optic microscopy. Succinctly, after 24 h or 48 h (KG-1) of incubation in the absence or presence of 50 µM, 100 µM, 150 µM, or 200 µM of AZD-7648, 0.5 × 106 cells from the different conditions were collected and seeded in glass slides. The smears were subjected to staining and analysis following the procedures defined by Lapa et al. [For morphological analysis, 1 × 10a et al. .6 cells were collected and washed with PBS by centrifugation for 5 min at 1000× g. The cells were fixed by incubation at 4 °C for 30 min with 200 μL of 70% ethanol solution added during vortex agitation. Afterwards, cells were washed with PBS, resuspended in 300 µL of propidium iodide/RNase, and analyzed on a FACSCalibur flow cytometer (Becton Dickinson). Cell cycle distribution was analyzed using ModFitLT Software v. 2.0 . Results were expressed as the percentage of cells in the different cell cycle phases  according to propidium iodide fluorescence intensity and represent the mean ± SEM of five independent experiments. When present, apoptotic cells were identified as a sub-G1 population.Cell cycle analysis was performed using flow cytometry using a propidium iodide/RNase cell cycle analysis kit (Immunostep). Cells were grown in the absence or presence of 50 µM, 100 µM, 150 µM or 200 µM of AZD-7648 for 24 h or 48 h (KG-1). Then, 1 × 1050 was reached, particularly LAMA-84, HEL, and KG-1. After 24 h or 48 h (KG-1) of treatment with different concentrations of AZD-7648, 1 × 106 cells were washed, fixated, and permeabilized using BD Citofix Perm solution. Three hours before the end of this incubation, the cells were labeled with BrdU at 37 °C. Cells were stained with anti-cleaved PARP (PE), anti-activated caspase 3 (FITC), anti-H2AX (Alexa Fluor™ 647), and anti-BrdU (PerCP-Cy5.5). These markers were analyzed using flow cytometry in a FACSCalibur cytometer (Becton Dickinson). Results represent the mean ± SEM of five independent experiments and are expressed as the percentage of positive cells for each molecular marker.The Apoptosis, DNA damage, and Cell Proliferation Kit  and anti-activated caspase 3 (BD Pharmingen) were used to analyze molecular markers of apoptosis, DNA damage, and cell proliferation, according to the manufacturer’s instructions. This method was exclusively performed in cell lines where the IC50 was calculated to the data concerning the exposure of cells at 24, 48, and 72 h, and the determination was performed by a non-linear curve fit dose-response. The normality of the samples was assessed using the Shapiro–Wilk test. Statistical significance was determined using a range of methods. These included unpaired t-tests (with or without Welch’s correction), one sample t test, Wilcoxon signed-rank test, the Mann–Whitney test, and ordinary one-way ANOVA. Following the ANOVA, either Tukey’s or Dunnett’s multiple comparisons tests were used. Moreover, Welch and Brown–Forsythe ANOVA, followed by Dunnett’s T3 multiple comparisons test, and Kruskal–Wallis test, followed by Dunn’s multiple comparisons test were also used. One-way ANOVA for repeated measures followed by Tukey’s multiple comparisons test or the Friedman test followed by Dunn’s multiple comparisons test was used to evaluate the effects of H2O2 through time. In this study, a significance level of p < 0.05 was considered statistically significant. All values were expressed as the mean ± SEM of the number of independent experiments (indicated in the figure legends).Statistical analysis was performed in GraphPad Prism 9 software . The ICIn conclusion, these results using CML and AML models support the further investigation of the potential of AZD-7648 as a new therapy for myeloid leukemias. The tested cell lines presented different drug sensibilities, showing that the value of this therapy may depend, for example, on the patient’s genetic background, the expression of specific genes, and DDR efficiency and damage at the baseline level. Therefore, subsequent studies are necessary to determine with more precision what may confer sensitivity to this inhibitor. However, this work opens up the possibility of testing new combination schemes with other agents using lower doses that could synergistically induce a cytostatic and cytotoxic effect and overcome the resistance to AZD-7648. Lastly, studies that would assess the therapeutic potential of AZD-7648 in combination with conventional therapy may also help to elucidate this inhibitor’s promise further, considering that previous studies already showed the advantage of combining this drug with other DDR inhibitors, chemotherapy, or radiation ,53,59,60


At present, the influence of the internal metallic endoskeleton of Spacer on the biomechanical strength of Spacer remains unclear. The aim of this study was to analyze the mechanical stability of a novel Spacer applying a annular skeleton that mimics the structure of trabecular bone using finite element methods.The femur models of three healthy individuals and skeletonless Spacer, K-Spacer, and AD-Spacer were assembled to create 15 3D models. Finite element analysis was performed in an Ansys Bench2022R1. Biomechanical parameters such as stress and strain of the Spacer, internal skeleton and femur were evaluated under three loads, which were applied with the maximum force borne by the hip joint (2100 N), standing on one leg (700 N), and standing on two legs (350 N). The mechanical properties of the new hip Spacer were evaluated.The stresses on the medial and lateral surfaces of the AD-Spacer and K-Spacer were smaller than the stresses in the state without skeletal support. The maximum stresses on the medial and lateral surfaces of the AD-Spacer were smaller than those of the inserted K-Spacer, and the difference gradually increased with the increase of force intensity. When the skeleton diameter was increased from 3 to 4 mm, the stresses in the medial and lateral sides of the AD-Spacer and K-Spacer necks decreased. The stress of both skeletons was concentrated at the neck, but the stress of the annular skeleton was evenly distributed on the medial and lateral sides of the skeleton. The mean stress in the proximal femur was higher in femurs with K-Spacer than in femurs with AD-Spacer.AD-Spacer has lower stress and higher load-bearing capacity than K-Spacer, and the advantages of AD-Spacer are more obvious under the maximum load state of hip joint. Total hip arthroplasty (THA) is the ideal treatment for advanced hip disease, achieving remarkable results in relieving pain, correcting deformity, and improving joint mobility. Periprosthetic joint infection (PJI) is a catastrophic complication after prosthetic joint replacement, with an incidence of 0.4–1.44% . PJI meaThe use of Spacer containing antibiotics maintains hip stability, lower extremity length and patient mobility while the infection is eradicated –10 SpaceToday, studies on the mechanical strength of the Spacer are imperfect and controversial. Schollner et al.  performeFor this purpose, our team has developed a two-layer Spacer Antibiotic Delayed Release System (AD-Spacer) with an applied ring skeleton. Inside it can be placed antibiotic-loaded calcium sulfate. The long-term and massive release of antibiotics from calcium sulfate for the treatment of periarticular prosthetic infections has been demonstrated . And an Three healthy participants between the ages of 20 and 60 years without any history of hip trauma, hormone use, or chronic alcohol use were used in this study Table . 3D modeThe surface errors such as spikes, intersections etc. of the femur models were corrected using Geomagic Studio 10 software (Raindrop Inc. USA). After these corrections, the 3D smooth solid model was developed and imported into SolidWorks program  in STEP format.The smoothed femurs were imported into SolidWorks software  in STEP format. The head and neck of the femoral solid model were cut off 2 mm above the base of the femoral neck and perpendicular to the femoral neck.  , 24. . With the increase of force intensity, the difference gradually increased. Fig. A Under aAs the diameter of the skeleton increased, the stress on the medial and lateral sides of the Spacer neck also decreased.K-Spacer internal skeleton increased from 3 to 4 mm. The medial wall stress of Spacer decreases by 1.18%, 4.43% and 3.94%, respectively, under the load of 2100 N, 700 N and 350 N. The lateral wall stress decreases by 0.44%, 3.05% and 9.98% under the load of 2100 N, 700 N and 350 N, respectively.  Fig. B.From the cloud diagram, the stresses of the AD-Space, K-Spacer and skeletonless Spacer were all concentrated in the neck, but the stress distribution of the AD-Spacer was more uniform Fig. .The maximum stress of the internal metal skeleton is proportional to the magnitude of the force applied, and the maximum stress of the annular skeleton is smaller than that of the Kirschner wire monolayer skeleton. Figure  CThe strComparing the Von Mises stresses of the two endoskeletons, under the same load, the stress carried by the 3 mm endoskeletons was less than that carried by the 4 mm endoskeletons. (P < 0.05) the hip joint is subject to multiple muscles and ligaments pulling in the normal state, this experiment simplified force model was used for finite element analysis; (2) this experiment will femur assumed as Homogeneous, isotropic material. This is different from the actual bone material properties of non-homogeneous, anisotropic different (3) The hip joint is subjected to cyclic loading in daily life, and only a few of its several static loads mechanical strengths under static loads. Regarding the new Hip Spacer of mechanical strength of the related issues. and the incidence of complications remain to be further investigated through various experiments and clinical analysis of large samples.It was shown that the AD-Spacer with simulated bone trabecular structure had higher stress compared to the conventional K-Spacer, while the AD-Spacer with applied annular skeleton had better weight-bearing capacity. The AD-Spacer has less chance of fracture than the K-Spacer and the skeletonless Spacer. It is our future research direction to Further optimization of Spacer structure and apply the high strength material into the skeleton of AD-Spacer to achieve the normal function of patient Spacer implantation.


In vitro analysis revealed that inhibition of PVT1 impeded the proliferation, migration, and colony formation of ESCA cells, increased the expressions of p-RIP1 and p-MLKL and promoted necroptosis. By contrast, PVT1 overexpression resulted in a decrease in necroptotic cell death events, thus promoting tumor progression. Collectively, the established 6-NRLs panel was a promising biomarker for the prognostic prediction of ESCA. Moreover, our current findings provided potential targets for individualized therapy for ESCA patients.Necroptosis is a newly identified programmed cell death associated with the biological process of various cancers, including esophageal carcinoma (ESCA). Meanwhile, the dysregulation of long non-coding RNAs (lncRNAs) is greatly implicated in ESCA progression and necroptosis regulation. However, the lncRNAs involved in regulating necroptosis in ESCA are still unclear. In this study, we aim to explore the expression profile of necroptosis-related lncRNAs (NRLs), and evaluate their roles in ESCA prognosis and treatment. In the present study, 198 differentially expressed NRLs were identified between the ESCA and adjacent normal tissues through screening the data extracted from the Cancer Genome Atlas (TCGA) database. And, a prognostic panel consisting of 6 NRLs was constructed using the LASSO algorithm and multivariate Cox regression analysis. The ESCA patients with high risks had a markedly reduced survival time and higher mortality prevalence. Moreover, C-index of 6 NRLs-panel was superior to 48 published prognostic models based on lncRNAs or mRNAs for ESCA. There were significant differences between the high-risk and low-risk groups in tumor-related pathways, genetic mutations, and drug sensitivity responses.  As one of the most common gastrointestinal tumors worldwide, esophageal carcinoma (ESCA) is often associated with delayed diagnosis and inferior outcomes , 2. At pAs a newly identified programmed cell death, necroptosis is regulated by TNF-α and RIPK1/RIPK3-dependent phosphorylation of MLKL , 9, and Long non-coding RNAs (lncRNAs) are a group of special non-coding RNAs with transcript lengths over 200 nucleotides , which aLncRNA plasmacytoma variant translocation 1(PVT1) is located on chromosome 8 of the c-Myc gene and is abnormally expressed in multiple malignancies, including nasopharyngeal carcinoma , colorecIn the present work, we thoroughly explored the expression pattern of NRLs in ESCA and established a prognostic panel based on six NRLs by least absolute shrinkage and selection operator (LASSO) and multi-Cox regression. Moreover, we assessed its predictive value using principal component analysis (PCA), time-dependent receiver operating characteristic (ROC), concordance index (C-index), nomogram, tumor mutational burden (TMB) analysis, and chemotherapy response analysis of ESCA patients. Besides, we further performed the gene set enrichment analysis (GSEA) to explore its underlying mechanisms. Finally, the expression of one NRL, PVT1, in ESCA cells was detected, suggesting its role in necroptosis. Collectively, these data provided valuable insights into the progression and prognosis of ESCA.https://portal.gdc.cancer.gov/), and their corresponding transcriptomic FPKM data, somatic mutations, and clinical information were also collected. Patients with missing information on survival time or those with survival time < 1 month were excluded from the analysis. In addition, 159 necroptosis-related genes (NRGs) were acquired from the Kyoto Encyclopedia of Genes and Genomes   .https://metascape.org/gp/index.html#/) was adopted to conduct the functional enrichment analysis and protein-protein interaction (PPI) network of DE-NRGs. Besides, NRG mutations were also assessed in ESCA using the cBioPortal website .The DE-NRGs between ESCA and adjacent normal tissues were identified using the “limma” package. Subsequently, Metascape  were identified using Pearson correlation analysis  based on their expression. Subsequently, DE-NRLs were screened using the “limma” package.The optimal panel of prognostic NRLs was determined using the LASSO-Cox algorithm, and a corresponding prognostic panel was constructed. In the meantime, we equally divided all patients into training and testing groups, the risk score of each patient with ESCA was calculated using the formula as follows:Coef (i) represents the coefficient, and x(i) represents the standardized level of each NRL. According to the median risk score of each patient, all patients were categorized into low-risk and high-risk groups. Subsequently, the predictive capacity of the 6-NRLs prognostic panel was assessed for various clinicopathological characteristics by the log-rank method. Besides, a nomogram was generated, and the 1-, 3- and 5-year recurrence rates of ESCA patients were predicted based on such a nomogram.We retrieved the published prognostic signatures of ESCA constructed based on lncRNAs or mRNAs since 2017 using multiple databases. Then, “timeROC” and “survcomp” packages were applied for model comparison to evaluate the predictive ability of each model.The potential enrichment pathways among different risk groups were identified using the GSEA with the “clusterProfiler” package. Moreover, “maftools”, “survival,” and “survminer” were applied to reveal the difference and survival of the TMB between the above-mentioned two groups of patients.The “pRRophetic”, “ggpub”, and “ggplot2” packages were used to identify the potential chemotherapeutic drugs that might be applied for ESCA therapy. Moreover, the Wilcoxon signed-rank test was adopted to determine the half-maximal inhibitory concentration (IC50) of common compounds between high-risk and low-risk groups.2. TSZ   and necrostatin-1  were used as previously described [The human esophageal epithelial cells (HEECs) and human esophageal cell lines, including ECA109, KYSE150, and KYSE510, were maintained in RPMI-1640 medium  supplemented with 10% fetal bovine serum  and penicillin-streptomycin cocktail  at 37° C in a humidified atmosphere containing 5% COescribed .−ΔΔCt method. Total RNA was isolated from the cells using TRIzol Reagent . Subsequently, purified RNA was reversely transcribed to cDNA using HiScript III RT SuperMix . RT-qPCR was performed using ChamQ Universal SYBR qPCR Master Mix . The PVT1 plasmid was adopted as a positive control, while the negative control contained all components except for cDNA. Each experiment was carried out three times. GAPDH was employed as the housekeeping gene. The relative expression of PVT1 was determined using the 2^For PVT1 overexpression, the full-length sequence of PVT1 was synthesized and cloned into the pcDNA3.1(+) vector . The shRNAs targeting PVT1 were synthesized by Sangon Biotech Co., Ltd. , and To conduct migration assays, uncoated transwell inserts with 8 μm pores were inserted into 24-well plates. Next, we added 700 μl of RPMI-1640 medium with 20% FBS to each lower chamber, and a total of 5×104 infected ECA-109, KYSE-150, or KYSE-510 cells were resuspended in 200 μl serum-free medium in each upper chamber. The remaining cells in the upper chamber were wiped clean with a cotton swab after 24 hours of incubation (48 hours for KYSE-510 cells) at 37° C, 5%CO2. The bottom membrane with invaded cells was fixed with 4% paraformaldehyde and dyed with 0.1% crystal violet  for 30 min, respectively. The migrated cells were counted in five random fields at 200× magnification using ImageJ software. The results were expressed as the mean number of migrated cells per field.For colony formation assay, 1000 infected ECA-109, KYSE-150, or KYSE-510 cells were inoculated into the six-well plates and incubated for 12 days at 37° C, 5% CO2. When clones were visible to the naked eye, the cells were washed with phosphate-buffered saline (PBS) twice and fixed with 4% paraformaldehyde for 20 min. When clonal colonies were visible (≥ 50 cells/colony), the cells were washed twice with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 20 minutes, followed by staining with 0.1% crystal violet solution for 30 min. After being air-dried, the colonies were photographed and counted.To evaluate the proliferative capacity of infected cells, a cell counting kit-8 (CCK-8) experiment was conducted. Specifically, infected ECA-109, KYSE-150, or KYSE-510 cells were inoculated into 96-well plates at a density of 3000 cells per well for proliferation for 0, 1, 2, 3, 4, and 5 days, then 10ul CCK-8 reagent  was added to each well and incubated for 2 h at 37° C. for 2 hours. The absorbance at 450 nm was measured using a microplate reader .Briefly, ESCA cells under different treatments were rinsed with cold PBS two times and then lysed using RIPA buffer  containing protease inhibitors  and phosphatase inhibitors . Moreover, the contents of soluble proteins were determined by the BCA protein detection kit . Subsequently, Western blotting analysis was carried out as previously described  using anhttps://portal.gdc.cancer.gov/) and KEGG website (https://www.kegg.jp/). Other details are available from the corresponding author upon a reasonable request.The data supporting the conclusions of this study could be acquired from TCGA database , Nutlin.3a (Rebemadlin), BIRB.0796 (Doramapimod), and PD.0332991 , in the low-risk group, were higher compared with the high-risk group , while tIn vitro experiments revealed that depletion of PVT1 significantly inhibited the proliferation and migration of ECA-109 and KYSE-150 cells compared with the controls  based on LASSO and multi-Cox regression. Simultaneously, a nomogram, internal testing cohort, PCA, and TMB analysis were adopted to assess the validity of the prognostic panel. Besides, we further investigated the predictive capability of this prognostic model in various clinicopathological features and multiple prognostic signatures. The results showed that regardless of age, sex, grade, and other clinicopathologic indicators, the prognosis model could effectively divide ESCA patients into low- and high-risk groups. On the other hand, this 6-NRLs panel has more predictive power than other prognostic models. Overall, our results showed that the 6-NRLs prognostic panel had a good performance in predicting the prognosis of ESCA patients.As one of the most common malignant tumors worldwide, ESCA originates from the esophageal mucosa epithelium. Some studies have confirmed that the dysregulation of lncRNAs is extensively involved in the biological processes of ESCA, including proliferation, apoptosis, metastasis, angiogenesis, and treatment resistance , 40, 41.To further investigate the underlying molecular mechanisms of the signature, the GSEA was carried out between different risk groups. The signaling pathways related to metabolisms, such as the TCA cycle, ribosome, glycosaminoglycan degradation, mTOR signaling pathway, and primary bile acid biosynthesis, were mainly enriched in the high-risk group. Conversely, TGF, notch, pentose phosphate, and Hedgehog pathways were enriched in the low-risk group. It has been found that the inhibition of mitochondrial oxidative phosphorylation mediated via clomipramine helps restrain ESCC progression . The mutIn vitro analysis revealed that overexpression of PVT1 facilitated the proliferation, migration, and colony formation in ESCA cells, and depletion of PVT1 effectively alleviated this phenotype. Interestingly, inhibiting PVT1 increased the expressions of p-RIP1 and p-MLKL and enhanced necroptosis, suggesting that PVT1 was a potential NRLs in ESCA.In addition, the functional phenotype of PVT1 in ESCA cell lines was explored through experimental studies. We first verified the expression level of PVT1 from the TCGA database and cell lines. in vitro and in vivo experiments.However, our research has some limitations. First, our results were mainly based on the TCGA dataset. Therefore, other large-scale case series data and clinical samples for external verification are required to further evaluate the applicability of the signature. Second, we only verified that PVT1 was a possible oncogenic lncRNA, while the specific mechanism of necroptosis regulating ESCA is still unclear, and the remaining NRGs and NRLs and more detailed molecular mechanisms need to be further proved via In this study, we constructed a promising prognostic panel consisting of six NRLs, which was not only an independent predictor but also a potential therapeutic target. Furthermore, we also found that PVT1 was a potential regulator of necroptosis, and it participated in the development of ESCA. Collectively, our current findings provided valuable insights into the tumor progression and clinical outcomes of ESCA patients.Supplementary FiguresSupplementary Table 1Supplementary Tables 2 and 3Supplementary Table 4Supplementary Table 5Supplementary Tables 6 and 7


First, the hemodynamic response of SSPN−/− mice was tested and was similar to SSPN+/+ (wild-type) mice after isoproterenol injection. Using the in situ Langendorff perfusion method, SSPN−/− hearts were subjected to IR injury and found to have increased infarct size and arrhythmia susceptibility compared to SSPN+/+. Ca2+ handling was assessed in single cardiomyocytes and diastolic Ca2+ levels were increased after acute β-AR stimulation in SSPN+/+ but not SSPN−/−. It was also found that SSPN−/− cardiomyocytes had reduced Ca2+ SR content compared to SSPN+/+ but similar SR Ca2+ release. Next, we used qRT-PCR to examine gene expression of Ca2+ handling proteins after acute IR injury. SSPN−/− hearts showed a significant decrease in L-type Ca2+ channels and a significant increase in Ca2+ release channel (RyR2) expression. Interestingly, under oxidizing conditions reminiscent of IR, SSPN−/− cardiomyocytes, had increased H2O2-induced reactive oxygen species production compared to SSPN+/+. Examination of oxidative stress proteins indicated that NADPH oxidase 4 and oxidized CAMKII were increased in SSPN−/− hearts after acute IR injury. These results suggest that increased arrhythmia susceptibility in SSPN−/− hearts post-IR injury may arise from alterations in Ca2+ handling and a reduced capacity to regulate oxidative stress pathways.The protein sarcospan (SSPN) is an integral member of the dystrophin-glycoprotein complex (DGC) and has been shown to be important in the heart during the development and the response to acute stress. In this study, we investigated the role of SSPN in the cardiac response to acute ischemia-reperfusion (IR) injury in SSPN-deficient (SSPN DYS1 gene leading to dystrophin loss and the mdx mouse model was developed to study the disease [Pathogenic variants in the dystrophin-glycoprotein complex (DGC) lead to cardiac and skeletal muscle disorders . The DGC disease ,3. Loss  disease ,5,6,7. SSSPN as a candidate gene for atrial fibrillation (AF) [−/− mice resulted in a blunted cardiac functional response compared to wild type mice [2+ handling. Overall, this study is aimed at understanding the normal functions of SSPN in the developing and adult heart that protect the heart from injury and arrhythmias [The protein SSPN has been shown to be important in maintaining cardiac and skeletal muscle function and integrity ,9. Unlikion (AF) . This stion (AF) ,12,13. Sype mice . Since βhythmias ,16.−/− myocardium, we utilized several arrhythmia-inducing stressors, including β-adrenergic stimulation and ischemia-reperfusion (IR) injury. We also compared the impact of SSPN loss to that of its closely associated proteins. One protein that SSPN makes significant associations with is β1D integrin [To uncover arrhythmogenic triggers for the SSPNintegrin ,17,18 anintegrin . Integriintegrin . Integriintegrin . The losintegrin . Since S2+ handling and response to IR injury. To investigate a role for SSPN in arrhythmia and acute IR injury, we examined ex vivo Langendorff perfused SSPN−/− hearts to assess the intrinsic mechanisms governing electrical activity and conduction independent of autonomic nervous regulation.This is the first study examining the role of SSPN in cardiomyocyte Ca−/− mice compared to age-matched SSPN+/+ controls. In +/+ or SSPN−/− mice. After the administration of the isoproterenol bolus, both SSPN+/+ and SSPN−/− mice exhibited a similar increase in heart rate, recorded as beats per minute (bpm), increasing from baseline values of ~450 bpm to ~550 bpm for SSPN+/+ and ~600 bpm for SSPN−/− as shown in +/+ or SSPN−/− mice before and after the isoproterenol treatment indicate a similar decrease in HRV during stress testing. Next, we assessed the in vivo cardiac contractility in mice using echocardiography under 2% isoflurane with 100% O2. The SSPN−/− mice displayed similar left ventricular fractional shortening  and isovolumic relaxation time  compared to SSPN+/+ mice  injury and the development of arrhythmias, we subjected ex vivo perfused SSPN+/+ and SSPN−/− hearts to IR injury, according to the schematic shown in +/+ and SSPN−/− perfused hearts. Post-IR arrhythmia risk was assessed using an ECG during perfusion and the number of premature ventricular contractions (PVCs) per minute after IR injury were found to be significantly increased in SSPN−/− compared to SSPN+/+ hearts.To determine whether the SSPN−/− myocardium after acute IR injury cardiomyocytes were isolated from SSPN+/+ and SSPN−/− hearts to assess perturbations in cellular Ca2+ handling. Cellular Ca2+ handling was reported in Fura-2 AM-loaded cardiomyocytes after 10 s of electrical stimulation under baseline conditions and after acute exposure to 1 μM isoproterenol (Iso). In +/+ and SSPN−/− cardiomyocytes exhibited similar Fratio, at baseline indicating similar levels of diastolic Ca2+.However, after acute Iso exposure SSPN+/+ cardiomyocytes exhibited a significantly increased Fratio. This suggested that there was an increase in diastolic Ca2+ in SSPN+/+ cardiomyocytes; however, it remained unchanged in SSPN−/− cardiomyocytes. In 2+ transient is shown and reports the peak amplitude of the Ca2+ transient under resting (-Iso) and stimulated conditions (+Iso). Under resting conditions, the Ca2+ T height was significantly lower in SSPN−/− compared to SSPN+/+ cardiomyocytes. Overall, there were no differences in the peak amplitude between SSPN+/+ and SSPN−/− cardiomyocytes upon acute Iso exposure.To investigate the underlying factors contributing to the heightened risk of arrhythmias in the SSPN2+ transient height to decay is reported in 2+ T decay. A delay in Ca2+ T decay suggests action potential prolongation, which were significantly longer under stimulated conditions (+Iso), while there was also a trend toward delayed Ca2+ T decay under resting conditions. This provides important information about the balance between inward ICa and outward Ik currents; therefore, the prolonged Ca2+ transients seen in SSPN−/− may increase the risk of early afterdepolarizations, leading to PVCs under stress conditions. In 2+ T height was measured after caffeine-induced SR Ca2+ release to estimate the SR Ca2+ content. The Fratio increased after Iso treatment, indicating higher diastolic Ca2+ (greater Ca2+ SR release) for both SSPN+/+ and SSPN−/− cardiomyocytes. However, SSPN−/− cardiomyocytes had significantly lower SR Ca2+ T height compared to SSPN+/+ after Iso treatment. After caffeine administration, Iso treatment SR Ca2+ content was significantly higher for both SSPN+/+ and SSPN−/− cardiomyocytes indicated by Fratio. To better understand the functional SR Ca2+ release, the Ca2+ T height was normalized to caffeine induced SR Ca2+ T height, +/+ and SSPN−/− under both conditions tested.The length of time it takes the peak Ca2+ release and cardiac rhythm in response to IR injury. The expression of β-adrenergic receptor 1 (β-AR1) appeared unchanged between the groups after acute IR injury as shown in −/− and SSPN+/+ hearts. Whereas expression of the plasma membrane Ca2+ handling protein L-type Ca2+ channel (LTCC) was increased in SSPN+/+ hearts upon IR injury with a concomitant decrease in in SSPN−/− hearts as indicated in +/Ca2+ exchanger (NCX) and RyR2 expression were similar; however, RyR2 expression was significantly increased while NCX expression trended higher in SSPN−/− hearts as shown in +/+ hearts as shown in 2+ uptake through SERCA2.To assess acute changes in gene expression, qRT-PCR was utilized to assess the rapid regulatory responses of key genes responsible for modulating Ca−/− hearts exhibited larger infarct size in response to acute IR injury, we assessed whether increased reactive oxygen species (ROS) production under oxidative stress could be a contributing factor to heightened myocardial damage in SSPN−/− hearts. To assess this, in +/+ and SSPN−/− cardiomyocytes were loaded with fluorogenic dye DCFDA/H2DCFDA to obtain a quantitative assessment of ROS produced in response to cellular oxidants mimicked by H2O2 exposure. In the left panel of +/+ and SSPN−/− cardiomyocytes exposed to oxidizing conditions for 30 min. In +/+ and SSPN−/− cardiomyocytes due to the increased oxygen species. It is evident from these measurements that SSPN−/− cardiomyocytes produced greater amounts of oxidant-induced excessive oxygen species ROS compared to SSPN+/+.Since perfused SSPN+/+ and SSPN−/− perfused hearts were assessed for alterations in protein expression to better understand immediate alterations in protein stress responsive pathways. In −/− hearts undergo a rapid antioxidant response since NOX4 protein levels increased upon acute IR injury. Furthermore, excessive ROS in the SSPN−/− cytosol after IR injury increased oxidized CAMKII/total CAMKII as shown in 2+ release and uptake channels (RyR2 or SERCA2) [After acute IR injury the SSPN SERCA2) .−/− hearts responsive to an acute β-adrenergic stimulus? (2) Does SSPN-deficiency render the myocardium more susceptible to acute IR injury? (3) Does acute IR injury reveal arrhythmogenesis in SSPN−/− hearts?As a tetraspanin-like protein, SSPN participates in stabilizing protein-protein interactions between important sarcolemma adhesion complexes, including the DGC, UGC and integrin complexes ,23,24. T+/+ and SSPN−/− hearts. SSPN−/− hearts were responsive to acute β-adrenergic challenges with similar increases in heart rate (bpm) and decreases in heart rate variability as the SSPN+/+ hearts  to assess the hemodynamic response of SSPN+ hearts . As SSPNR injury , global R injury , and carR injury . Similar−/− hearts were found to have increased incidence of PVCs, and, therefore, an increased risk of arrhythmias compared to SSPN+/+ hearts. After acute IR injury, the SSPN−/− myocardium exhibited larger infarcted areas and more widespread damage. This suggests that SSPN affords global protection to cells throughout the heart. The increased vulnerability of the SSPN−/− myocardium to acute IR can be attributed to a variety of factors, including reduced localization of dystrophin at the sarcolemma [In rcolemma , alteredrcolemma  and the 2+ handling and redox proteins. Studies in the mdx mouse model are highly informative regarding the impact of complete dystrophin deficiency, destabilization of the DGC and perturbations in Ca2+ handling. Studies in mdx cardiomyocytes show that they have reduced resting sarcomere lengths and increased passive tension, which contributes to poor passive compliance [mdx cardiomyocyte experiments by Viola et al. suggest that the absence of dystrophin disrupts the normal cytoskeletal architecture, affecting LTCC activation and superoxide generation [Perturbations to key pathways can increase cell death in response to acute IR injury; therefore, we examined alterations in key Campliance . Examinimpliance . Additioneration . Whereasneration  and cardneration  muscle s−/− cardiomyocytes may diminish the “shock absorbing” capacity of the sarcolemma by weakening the connection between the ECM and intracellular actin and/or perturb its scaffolding function [2+ handling and redox homeostasis. Relatedly, the absence of dystrophin in DMD leads to mis-localization and abnormal expression/activity of Ca2+ handling and oxidative stress responsive proteins [−/− myocardium does not exhibit overt signs of membrane instability—the increased vulnerability of the SSPN−/− myocardium to IR injury could be attributed to aberrant cardiomyocyte Ca2+ entry or a reduction in submembrane scaffolding orchestrated by dystrophin and/or SSPN.The reduction of dystrophin in SSPNfunction ,34. Thisproteins ,36,37. S2+ handling in SSPN−/− cardiomyocytes to better understand their cellular phenotype compared to the dystrophic cellular phenotype. Our results indicate that mechanisms leading to cytosolic Ca2+ overload in mdx cardiomyocytes may not be substantially activated in SSPN−/− cardiomyocytes. To assess this, we examined intracellular Ca2+ handling in SSPN−/− cardiomyocytes after exposure to acute isoproterenol regimen. We found that in contrast to chronic in vivo β-adrenergic stimulation of SSPN-deficient hearts, isolated SSPN−/− cardiomyocytes remained responsive to acute isoproterenol exposure [In this study, we examine alterations in Caexposure , see Fig2+ entry and efflux across the cell membrane to achieve a given average systolic and diastolic Ca2+ level. Stress induction regimens using acute β-adrenergic stimulation, provide information on the responsive balance of stress and adaptations of excitation-contraction coupling (ECC) [−/− cardiomyocyte Ca2+ handling. Our Ca2+ handling data indicated that SSPN−/− cardiomyocytes maintained responsiveness to acute isoproterenol exposure. Overall, isoproterenol exposure activates PKA through β-adrenergic receptors and increases the amount of SR Ca2+ release through increased Ca2+ current (ICa) and SR Ca2+ contents [2+ height estimates the SR Ca2+ content of cardiomyocytes since caffeine triggers Ca2+ release by reducing the RyR2 activation threshold for luminal but not cytosolic Ca2+ activation [−/− cardiomyocytes had alterations in these two parameters (diastolic Ca2+ and SR Ca2+ content) revealing reduced Ca2+ SR content under two conditions: β-adrenergic and caffeine stimulation. After β-adrenergic stimulation, both the diastolic Ca2+ levels and SR Ca2+ contents were lower, which may be explained by the reduced SR Ca2+ release accompanied by the lower SR Ca2+ content.To maintain normal function, cardiomyocytes undergo infinite adaptations to Cang (ECC)  to uncovcontents . Furthercontents . SR Ca2+tivation . SSPN−/−2+ handling gene expression occur upon acute IR injury [2+ handling gene expression and protein levels. The initial response of healthy cardiac myocytes to electrical stimulation is an increase in intracellular Ca2+. This initially occurs as a small influx of Ca2+ through LTCC that triggers a larger Ca2+ release from the SR through RyR2 [2+ handling proteins essential to ECC we anticipated affected proteins may exhibit transient gene expression changes in response to acute IR injury. The most striking rapid alteration in gene expression in SSPN−/− hearts after acute IR injury was a reduction in LTCC and an increase in RyR2 expression see 2+ homeostasis. Our initial findings suggest that tight transcriptional regulatory mechanisms may compensate for cellular perturbations that exist within SSPN−/− cardiomyocytes.It has been shown that transient alterations in CaR injury . These cR injury . Therefough RyR2 . If loss−/− hearts exhibited widespread damage after acute IR injury, we were interested in whether the antioxidative capacity of the SSPN−/− myocardium was compromised. To assess this, we exposed SSPN−/− cardiomyocytes to oxidizing conditions and measured the production of ROS in response to H2O2, see 2O2-induced ROS production in SSPN−/− cardiomyocytes compared to SSPN+/+ may be a contributing factor to the increased susceptibility of the SSPN−/− myocardium to IR injury. Ca2+ overload also leads to increased ROS in the heart; however, the SSPN−/− cardiomyocyte Ca2+ handling data presented here does not suggest major perturbations in Ca2+ regulation. Therefore, it is more likely that SSPN deficiency has a greater impact on the antioxidant mechanisms within the heart.The scaffold role of dystrophin was demonstrated and the loss of SSPN may potentially influence these processes by altering the activity/sarcolemma localization of proteins regulating redox homeostasis. Excessive ROS production that outpaces the antioxidant capacity of the heart contributes to myocardial damage in ischemic heart disease and heart failure . Since S−/− cardiomyocytes produce higher levels of ROS under oxidative conditions, we examined the protein expression of key oxidative stress markers in SSPN−/− hearts after acute IR injury. This data is shown in 2- or H2O2 and are the major source of ROS. The suppression of either NOX2 or NOX4 has been demonstrated to reduce ROS and protect against IR injury [−/− hearts after IR injury. The previous studies of DMD-associated cardiomyopathy found increased induction of NOX4 in the diseased hearts associated with increased fibrosis and functional decline [2+ regulation in cardiomyocytes and activate CAMKII via oxidation-dependent mechanisms. Remodeling of the cardiac action potential by higher baseline oxidative stress-induced cellular memory may lead to pathological processes, increasing the susceptibility to cardiac arrhythmia.To address the finding that SSPNR injury . Our stu decline . In resp2+ release. These alterations in SSPN−/− hearts can contribute to increased number of premature ventricular contractions and altered propagation of signals throughout the heart leading to an increased risk of arrhythmias.Our findings suggest that SSPN deficiency increases myocardial risk upon IR injury by enhancing pathways that increase oxidative stress. This can cause increased damage to heart tissue under adverse conditions and affect systolic Ca+/+ and SSPN−/−) and their age range was between 4 and 8 months. Differences between the groups were assessed using one-way ANOVA; whereas, the incidence of arrhythmia was compared using the non-parametric Mann-Whitney test.All animal protocols used in this study conformed to the Guiding Principles in the Care and Use of Animals of the American Physiological Society and were approved by The University Animal Care and Use Committee  at Florida State University. The sspntm1Kcam (SSPN-deficient) mice were purchased from The Jackson Laboratory and developed by the Campbell laboratory  these mi2 for 15 min [Echocardiography was performed under 2% of Isoflurane with 100% Or 15 min . The echTotal RNA was isolated from heart tissue using Trizol reagent . For cDNA synthesis the qPCRBIO cDNA Synthesis Kit (PCR Biosystems) was used and each reaction contained 0.4 μg total RNA. The expression of mRNA was analyzed by Real-time quantitative PCR using 2X qPCRBIOSyGreen Mix (PCR BIOsystems) in QuantStudio 3 (Applied Biosystems). β-actin was used to normalize the mRNA. The relative expression was determined using the ΔΔCt method.The sequences of the primers utilized are as follows:β-AR1-F: 5′-CTC ATC GTG GTG GGT AAC GTG-3′ and R: 5′-ACA CAC AGC ACA TCT ACC GAA-3′;LTCC-F: 5′-ATG AAA ACA CGA GGA TGT ACG TT-3′ and R: 5′-ACT GAC GGT AGA GAT GGT TGC-3′;NCX1-F: 5′-AAA GAG TGC AGT TTC TCC CTT G-3′ and R: 5′-TGA AGC CAC CTT TCA ATC CTC-3′;RyR2-F: 5′-ACG GCG ACC ATC CAC AAA G-3′ and R: 5′-AAA GTC TGT TGC CAA ATC CTT CT-3′;SERCA2-F: 5′-GAG AAC GCT CAC ACA CAA AGA CC-3′ and R: 5′-CAA TTC GTT GGA GCC CCA T-3′;PLN-F: 5′-AAA GTG CAA TAC CTC ACT CGC-3′ and R: 5′-GGC ATT TCA ATA GTG GAG GCT C-3′; andβ-actin-F: 5′-GGG TGT ATT CCC CTC CAT CG-3′ and R: 5′-CCA GTT GTT GGT AAC AAT GCC ATG-3′.n = 6 per group. One point was removed from the NCX1 data for SSPN−/− controls as it was identified as an outlier by Prism Graphpad using the ROUT (Q = 1%) method to facilitate plotting but did not alter the results, with nonsignificant trends between the groups. The SSPN−/− IR injury NCX1 consists of n = 6; however, one point is obscured by a similar value.The number of samples used was 2 for 10 min while electrocardiography (ECG) traces were recorded for a period of 5 min while at rest and then after stress induction  for 5 min. The ECG recording and analysis were performed using Powerlab and Lab chart .The mice were anesthetized under 2% Isoflurane with 100% O3, 1.2 NaH2P04, 1 MgCl2, 1.5 CaCl2 and 10 glucose as previously described [2/5% CO2) to achieve a pH of 7.4. The heart was allowed to equilibrate for 10 min prior to initiation of the ischemia-reperfusion (IR) protocol. The baseline ECG measurements were collected for ten minutes prior to exposing the heart to ischemic conditions (no flow) for 30 min and the ECG readings were recorded during continuous reperfusion for an additional 60 min. After completion of the experiment, the hearts were used for protein, mRNA and histological (infarct size) analyses.Mice were euthanized under isoflurane anesthesia and the heart was removed after performing a thoracotomy. The aorta was then cannulated in the Langendorff mode, which involved retrograde perfusion of the aorta at 37 °C with a perfusion buffer containing (mM) 139 NaCl, 4 KCl, 14 NaHCOescribed ,48. The 2-cooled 2-methylbutane [2. For each heart, three adjacent sections were used to calculate the infarct volume, and the number of pixels in the infarct zones were counted to obtain the infarct size in mm2 for each heart.Histological analysis of the heart tissues was performed to measure infarct size. To this end the heart tissues were embedded horizontally in OCT and flash-frozen in liquid Nylbutane . Next, tylbutane . The sel2, 10 glucose, and 10 HEPES (pH 7.4) at room temperature. Intracellular Ca2+ regulation was measured using the previously described protocol [2+ content was then quantified by the application of caffeine . The Ca2+ fluorescence ratios (Fratio) were recorded and normalized with an average vehicle group. The data were analyzed using the commercially available data analysis software IonWizardTM 6.6, IonOptix, Milton, MA, USA.Myocytes were isolated using a modified collagenase/protease method ,52,53, aHeart samples were lysed at 4 °C using a urea-based extraction buffer . After qIsolated cardiomyocytes were loaded with DCFDA/H2DCFDA (3 μM) for 15 min. After they were washed three times with dye. The fluorescent signals were imaged using fluorescence microscopy at baseline for up to 60 min. The fluorescent intensity of the captured images was measured using ImageJ 1.53k.


Background and Objectives: Milk is healthy and includes several vital nutrients and microbiomes. Probiotics in milk and their derivatives modulate the immune system, fight inflammation, and protect against numerous diseases. The present study aimed to isolate novel bacterial species with probiotic potential for neuroinflammation. Materials and Methods: Six milk samples were collected from lactating dairy cows. Bacterial isolates were obtained using standard methods and were evaluated based on probiotic characteristics such as the catalase test, hemolysis, acid/bile tolerance, cell adhesion, and hydrophobicity, as well as in vitro screening. Results: Nine morphologically diverse bacterial isolates were found in six different types of cow’s milk. Among the isolates, PO3 displayed probiotic characteristics. PO3 was a Gram-positive rod cell that grew in an acidic (pH-2) salty medium containing bile salt and salinity (8% NaCl). PO3 also exhibited substantial hydrophobicity and cell adhesion. The sequencing comparison of the 16S rRNA genes revealed that PO3 was Lactococcus raffinolactis with a similarity score of 99.3%. Furthermore, PO3 was assessed for its neuroanti-inflammatory activity on human oligodendrocyte (HOG) cell lines using four different neuroimmune markers: signal transducer and activator of transcription (STAT-3), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), and GLAC in HOG cell lines induced by MOG. Unlike the rest of the evaluated neuroimmune markers, STAT-3 levels were elevated in the MOG-treated HOG cell lines compared to the untreated ones. The expression level of STAT-3 was attenuated in both PO3-MOG-treated and only PO3-treated cell lines. On the contrary, in PO3-treated cell lines, MBP, GFAP, and GLAC were significantly expressed at higher levels when compared with the MOG-treated cell lines. Conclusions: The findings reported in this article are to be used as a foundation for further in vivo research in order to pave the way for the possible use of probiotics in the treatment of neuroinflammatory diseases, including multiple sclerosis. Milk and its products are habits for beneficial bacteria known as probiotics, which have a key role in immunomodulatory, anti-inflammatory, and antimicrobial action against several diseases. They have been part of human food and culture for a long time due to their use in medicine, food manufacturing, and preservation . Their e per day ,3. Certa per day . The comproducts ,5,6. Thelococcus ,9,10,11.lococcus . Hence, Lactococci, which is helpful in developing various cheese products [Lactococcus (38.6%) and Streptococcus quantity increased significantly after 24 h of refrigeration [Lactic acid bacteria are widely used in dairy industries to ferment food products through acidification or low pH to preserve them. Moreover, they contribute proteolytic, lipolytic, and organoleptic characteristics to the fermented foodstuffs, such as flavor and texture ,14. Geneproducts ,17. One geration .Gut microbiota plays a key role in regulating the interactions among the central nervous system, endocrine glands, and inflammatory markers ,20. TheyLactococcus raffinolactis and its other members belong to the family Streptococcaceae, and they are used in different industries for meat preservation, starters, and flavoring purposes [Lactobacillus and Lactococcus. However, few studies correlate the probiotic effect on neurological inflammation. Therefore, this study aims to focus on the probiotic potential of Lactococcus raffinolactis and its role in the modulation of neuroinflammation. It has recently been reported that Lactococcus lactis displayed a significant positive impact in an LPS-induced depression-like model in mice [purposes . There a in mice , allevia in mice , attenua in mice , and pre in mice . DespiteFor the bacterial analysis, 6 samples of lactating dairy cows (one-week lactating milk (Colostrum) and 25th-week lactating milk) were collected from January to March 2023 in the mid-winter season . A total of 400–500 mL of milk was collected in sterilized containers. The samples were labeled and stored at 4 °C until use.Isolation of beneficial bacteria from lactating cow milk was performed using the serial dilution method. A total of 1 mL of cow milk was added to 24 mL of de Man, Rogosa, and Sharpe (MRS) broth, and diluted milk (0.2 mL) was spread and plated on an MRS agar plate for 48 h. The single colonies were selected and stored in 30% glycerol stock with 10% skim milk with consecutive −70 °C storage conditions .2O2 slide drop assay. The hemolytic activity of active isolates was examined on Colombia blood agar  [The Gram-positive isolates from cow milk were screened for catalase activity using the H, India) .w/v) NaCl and at pH 3.0 according to the method described earlier by [Colony morphology, cell shape, Gram staining, catalase activity, and hemolytic activity were investigated after 48 h on MRS agar plates at 30 °C. The isolates were checked for physiological traits, including growth in 8% (rlier by . Based ow/v)) and taurodeoxycholic acid (0.5% (w/v)). Plates were incubated anaerobically at 37 °C for 72 h. Zone forming colonies or white colonies indicated signs of BSH activity. The baseline was MRS agar that had not been changed.The activity of BSH was determined following the method mentioned earlier by , with slThe ability of bacterial cells to adhere to the mucus environment was achieved by using octane solvent in an in vitro environment . The assay was performed according to a previously described method .The hydrophobicity index (HBI) was calculated as follows:−1. The MRS-cholesterol-PEG 600 mixture was incubated at 37 °C for 24 h with each 1% (v/v) inoculum. The turbidity of MRS broth was measured by CFU methods. The assimilation of cholesterol was detected by centrifuging the broth at 4000 rpm for 10 min, and the supernatant was collected for absorbance analysis using a microplate spectrum reader (570 nm). The concentrations of cholesterol were compared to the absorbance of the standard curve, with cholesterol concentrations ranging from 0 to 500 µg/mL in MRS (R2 = 0.987). The efficiencies of beneficial bacteria to cholesterol assimilation in MRS were determined as follows:The ability of beneficial bacteria to assimilate cholesterol was assessed in MRS broth supplemented with cholesterol-polyethylene glycol (PEG) 600  at a final concentration of 100 µg mLg for 10 min at 4 °C and resuspended in sterile physiology saline (0.88%) to adjust the cell culture (OD600 = 1 and 109 CFU/mL).Preparation of bacterial cell suspensions was performed as described by Pithva et al.  to evaluThe simulated gastric acid and intestinal bile fluids were prepared as described previously . The aciThe percentage of bacterial survival was calculated as follows:v/v) fetal bovine serum (FBS) and 100× antimycotic solution under humidified and 5% CO2 conditions.Human oligodendrocyte (HOG) cell lines  were routinely grown at 37 °C in Dulbecco’s Modified Eagle’s Medium (DMEM) with serum supplemented with 10% (5 cells) were cultivated for 72 h in cell culture inserts (Boyden chamber). After 72 h, the solution containing 5 mM sodium butyrate was replaced, and cells were cultured for 96 h to promote differentiation. HOG cells were aspirated with sterile PBS and bacterial cell suspension and were resuspended in antibiotics-free DMEM. After 90 min in a 37 °C 5% CO2 incubator, each cell suspension was plated on wells. Unbound bacterial cells were removed by sterile PBS aspiration and were subsequently lysed with 0.05% Triton X-100 solution. Spot plate counts on MRS agar were performed after 48 h of incubation at 37 °C to determine the total number of attached bacteria. The following equation was used to determine the adhering abilities of the chosen isolate:Human oligodendrocyte cells were used to study the adherence characteristics of PO3 . HOG cells (1.5 × 104 cells/well) were inoculated in 48-well plates for 24 h. The serum-free DMEM (1% FBS) was stored overnight before treatment. The starved cells were pre-incubated with 1 µg/mL MOG for 4 h, and the active isolate PO3 was inoculated and co-cultured for 24 h. The supernatant was examined for NO production, which was determined using an NO detection kit (Griess reagent kit) in accordance with the manufacturer’s protocol.HOG cell lines induced with MOG at 1 µg/mL  in accordance with the manufacturing guidelines. The amounts of cytokines on the plates were measured using an automated ELISA plate reader at 450 nm . The standard was used for each cytokine marker with Rhttp://pga.mgh.harvard.edu/primerbank (accessed on 15 March 2023).Total RNA was extracted from MOG-induced HOG cell lines in accordance with the instructions provided by the manufacturer . The cDNA synthesis kit was used to generate complementary DNA (cDNA), and the process was carried out in compliance with the guidelines provided by the manufacturer. The qPCR primers that are shown in −ΔΔCt [In order to determine the optimal annealing temperature for each primer set, gradient PCR was used. The quantity of mRNA produced by the genes under investigation was measured using the detection instrument ABI step one plus  and was supplied with SYBR Green master mix manufactured by Amplicon Bio in Denmark. In order to ensure accuracy, all reactions were carried out twice. The relative amount of gene expression was calculated using the formula RQ = 2−ΔΔCt , which wThe isolates of 16S rRNA gene sequences were amplified using PCR following the method mentioned earlier by the authors of . PCR prop < 0.05, Duncan’s Multiple Range Test (DMRT) was used as the appropriate statistical tool. Welch’s t-test, with a significance threshold of p < 0.05, was used to evaluate the immunomodulatory effects of the treatment.All the tests were carried out three times to ensure accurate data, and the findings are presented as the mean along with the standard deviation (SD). The results of tests on acid and bile tolerance and adhesion capacity were analyzed using ANOVA and the SPSS 22.0 program. When comparing mean values at a significance level of 2O2 was added. PN3 and PN4 exhibited hemolytic activities, whereas the remaining isolates did not.Nine morphologically distinct bacterial isolates were obtained from six cow’s milk samples. There were four  and five isolates  obtained from the 1st and 25th week of lactating cows, respectively . As can Four isolates, PN1, PN2, PO2, and PO3, were selected for further screening using bile salt hydrolase (BSH) activity, and the results are presented in The capacity of probiotics to attach to epithelia is assessed in vitro by measuring the cells’ hydrophobicity at the surface for certain solvents (octane). This determines whether or not the probiotics are able to attach to the epithelia. It was discovered that the isolate has a significant degree of hydrophobicity . The resp < 0.01). Unlike IL-6, a comparative evaluation of IFN-γ levels in MOG-treated cells was compared to the negative control ones. The presence of PO3 significantly reduced the increased levels of NO seen in MOG-treated cells (The findings for the pro-inflammatory cytokines (IL-6 and IFN-γ) are shown in ed cells A.The impact of PO3 on the induction of neuroimmune markers  in HOG cell lines by MOG (1 µg) was studied and the results are presented in Lactococcus raffinolactis at a similarity level 99.3%. The submitted 16S rDNA sequences received the accession number for PO3 (OQ914364). The neighbor-joining method revealed the active isolate was Lactococcus raffinolactis, with the 0.001 bootstrap closer to Lactococcus raffinolactis NR044359  indicated that AggLr is a member of the SFCBAF protein family [Disturbance of the gut–brain axis, a bidirectional relationship between the microbiota in the digestive tract and the brain, has been linked to neuroinflammatory response and neurological disorders ,30. Cow eactions . It has n family .Limosilactobacillus walteri as a novel probiotic bacterial strain [PO3 was unable to generate the catalase that is required to produce oxygen and water by splitting hydrogen peroxide. This finding also implies that PO3 can live in environments with extremely low concentrations of oxygen. These findings were comparable to those that were published by Saroha et al. (2023) , who desl strain . ScreeniLactococcus lactis has very high hydrophobicity, as measured by microbial adherence to xylene, with a value of 89.3% [Hydrophobicity at the surface of cells is measured in vitro to evaluate the ability of probiotics to attach to epithelia. The ability of the probiotics to bind to the epithelia depends on this. According to the findings, PO3 exhibited the maximum adhesion towards xylene (3%). Selecting probiotic bacteria for use as therapeutic agents requires consideration of their ability to attach to the gut. Colonization of the human gut by probiotics is believed to be needed for them to perform their therapeutic effects, such as the avoidance of enteropathogenic bacteria . The coaof 89.3% ,52.Lactobacillus sp. strain in the earlier investigation [A crucial biological barrier that a probiotic strain has to overcome to reach its target is the acid that is found in the stomach, as well as the bile salts that are found in the intestine. In environments with a low pH, metabolic activity may be restricted, and the growth and survival of probiotics may be hindered. Therefore, the ability of probiotic strains to tolerate acid, gastric juice, and bile is critical to their capacity for survival and proliferation in the gastrointestinal system. In the research that we conducted, we found that PO3 was resistant to bile salt, acidic pH, and stimulated gastric juice. These findings are comparable to those obtained with the probiotic tigation ,54.Lactococcus lactis inhibits experimental autoimmune encephalomyelitis by preventing cell migration into the spinal cord [Bacillus amyloliquifaciens-supplemented camel milk suppress the neuroinflammation of autoimmune encephalomyelitis by regulating inflammatory markers [The modulatory impact of PO3 on the expression of myelin basic protein (MBP) neuroimmune markers , glial fibrillary acidic protein (GFAP), and GLAC) was studied in HOG cell lines induced by MOG. Unlike the rest of the evaluated neuroimmune markers, STAT-3 levels were elevated in the MOG-treated HOG cell lines compared to the untreated ones. The expression level of STAT-3 was attenuated in both PO3-MOG treated cell lines and those only treated with PO3. In contrast, in PO3-treated cell lines, MBP, GFAP, and GLAC were significantly expressed at higher levels when compared with the MOG-treated cell lines. It has been reported that Hsp65-producing nal cord . Further markers , as well markers . STAT-3 H. pylori infection by stimulating phagocytoses, natural killer (NK) cells, DCs, and antibody and anti-inflammatory cytokines [Lactococcus sp. PO3 abrogated STAT-3 and activated the neuroprotective markers to improve cell communication and the functionality of the blood–brain barrier. In support of that, L. rhamnosus and L. acidophilus have been found to block STAT-3 and NF-B activation in dendritic cells via upregulation of certain anti-inflammatory markers (IL-10 and Foxp3+ Treg) [The activation of STAT-3 signaling may be triggered by a variety of stimuli and is crucial for the functions of many distinct types of cells . By maniytokines . STAT-3 3+ Treg) .Lactococcus raffinolactis through 99.3% 16S rRNA gene sequence similarity. PO3 displayed substantial modulatory impacts on the immune markers in the MOG-induced cell lines. The results presented herein are considered a base for further in vivo investigation in order to open the door for potential application of the probiotic to combat neuroinflammation disorders.The six cow’s milk samples yielded nine morphologically distinct bacterial isolates. PO3, from 25th-week lactating cow milk, was a probiotic. PO3 was Gram-positive, and rod cells were developed in highly acidic medium (pH 3), with bile salt, and in a saline environment (8% NaCl). PO3 was hydrophobic and cell-adhesive. The isolate altered cell line proinflammatory markers. PO3 was shown to be