TSC1 was identified as a direct miR-130b target in luciferase-3’UTR assays, and shRNA-mediated knockdown of TSC1 replicated miR-130b effects. Loss-of-function and gain-of-function studies showed that miR-130b levels regulate cell morphology and motility. Following miR-130b suppression, LMS cells adopted a rounded morphology, amoeboid mode of cell movement and enhanced invasive capacity that was Rho/ROCK dependent. Conversely, miR-130b-overexpressing LMS cells exhibited Rho-independent invasion, accompanied by down-regulation of Rho-pathway effectors. In mesenchymal stem cells, both miR-130b overexpression and TSC1 silencing independently impaired SM differentiation in vitro. Together, the data reveal miR-130b as a pro-oncogenic miRNA in LMS and support a miR-130b-TSC1 regulatory network that enhances tumor progression via inhibition of SM differentiation.Leiomyosarcoma (LMS) is an aggressive, often poorly differentiated cancer of the smooth muscle (SM) lineage for which the molecular drivers of transformation and progression are poorly understood. In microRNA (miRNA) profiling studies, miR-130b was previously found to be upregulated in LMS vs. normal SM, and down-regulated during the differentiation of mesenchymal stem cells (MSCs) into SM, suggesting a role in LMS tumor progression. In the present study, the effects of miR-130b on human LMS tumorigenesis were investigated. Stable miR-130b overexpression enhanced invasion of LMS cells  Leiomyosarcoma (LMS) is an aggressive malignancy of smooth muscle (SM) origin for which the molecular basis of transformation and mechanisms of progression are mostly unknown. Given the diversity of anatomical locations of SM tissues throughout the body, LMS can arise in various regions; however, the most common sites are the smooth muscle lining of the uterus  and the retroperitoneal cavity. Although tumors from different locations are histologically similar, they often have distinct clinical courses and response to chemotherapy. While studies have shown that LMS of cutaneous origin have a more favorable outcome , LMS of Molecular features of soft tissue LMS have been investigated using cytogenetic and genomic approaches to reveal multiple gene alterations and complex karyotypes . ComparaMicroRNAs are small, non-coding RNAs that exert important roles in a variety of biological processes by negative regulation of their mRNA targets . MiRNAs TSC1 and blockade of SM differentiation.In an effort to better understand LMS pathogenesis, we previously performed profiling studies to identify candidate miRNAs involved in cellular transformation and SM differentiation . Among tHuman tissue specimens for microarray analysis  were colhttp://medicine.tamhsc.edu/irm/msc-distribution.html) and cultured as previously described [Human BM-MSCs were obtained from the Texas University Institute of Regenerative Medicine MSC Distribution Program  as follows: MIRH empty (#CD511B-1), MIRH-SCR (#PMIRH000PA-1), MIRH-miR130b (#PMIRH130bPA-1), ZIP-SCR (#MZIP000-PA-1), ZIP-miR130b (#MZIP130b-PA-1). For shRNA-mediated knockdown studies, expression plasmids were obtained from Dharmacon  encoding scrambled, GIPZ-SCR (#RHS4346), or shTSC1 sequences (#RHS4430-99147968). Expression plasmids were co-transfected into 293T cells with the second-generation packaging plasmids pSPAX-2 (Addgene ID# 12260) and pMD2.G (Addgene ID# 12259) using Lipofectamine 2000 transfection reagent (Invitrogen), and viral supernatants were harvested 48 h later and used directly for cell transduction. For infection, cell monolayers were transduced with viral supernatants 1-3x in the presence of polybrene (8 μg/ml).TSC1 inhibition, non-targeting control siRNA  or siTSC1 (#L-003028-00-0005) were transfected (100 nM) under the same conditions. For gene expression profiling studies, SK-LMS1 and SK-UT1 were transfected with control or miR-130b-3p mimics (50 nM) and cells harvested for RNA preparation 36 h later.To investigate miR-130b on SM myogenesis, hMSCs were transiently transfected with control  or miR-130b-3p (#C-300660-05) oligonucleotide miRNA mimics (50 nM) using Lipofectamine, 24 h prior to induction of differentiation. For Total RNA was extracted from cell cultures using the miRNeasy Mini Kit . For determination of miRNA levels, qRT-PCR was performed on 500 ng total RNA using Taqman reverse transcription reagents and miRNA assay  using the small RNA, RNU44 for normalization. For assay of mRNA levels, qRT-PCR was performed using the Superscript III reverse transcription kit (Invitrogen) and the FastStart SYBR Green MasterMix  with gene-specific PCR primers designed from human sequences .B2M genomic DNA levels using gene-specific primers . Genomic amplification was analyzed by qPCR as above, and values were normalized to  primers .P < 0.05, alpha level) and fold-change thresholding (>33% reproducible change) using the volcano plot feature. Lists of modulated genes were overlapped using the Venn diagram feature to identify transcripts altered in both cell lines, and further filtered by in silico miRNA target prediction (www.targetscan.com).Gene expression profiling of two LMS cell lines (SK-LMS1 and SK-UT1) transfected with control or miR-130b oligonucleotide mimics was obtained with the Affymetrix Genechip system . Cells were harvested 36 h post transfection and 100 ng of total RNA were used to prepare cRNA following the Affymetrix 3’IVT Express Kit labeling protocol. Standardized array processing procedures including hybridization, fluidics processing and scanning of the Affymetrix HG-U133 Plus 2.0 arrays were performed according to the manufacturer’s instructions. GeneSpring GX11 software  was used to normalize the raw data (Affymetrix CEL files) using the Robust Multichip Average algorithm (RMA) , to filtCells were harvested for protein extraction and immunoblotting as described previously . PrimaryFor phalloidin staining, as-miR-130b or control transduced cells were seeded on cover slips overnight, fixed with 4% formaldehyde, permeabilized with 0.1% triton, and incubated with Phalloidin , followed by DAPI counterstaining. Cultures were then mounted with ProLong Gold mounting reagent (Invitrogen), imaged using a Leica SP5 confocal microscope  and analyzed using image J software 3 cells plated into 6-well glass bottom dishes and videomicroscopy images were taken every 7 minutes for 2 h. Cell positions  were tracked manually and marked by the centroid of the nucleus in each frame. Cell migration speed was determined using ImageJ software. All analyses were performed in a blinded fashion.Wound healing ‘scratch’ assays were performed by seeding cells at 80% confluence and scratching the surface of the well through the center using a 1 ml pipette tip after 24 h. Images of the ‘wound gap’ were taken under phase-contrast and fluorescence microscopy over a 24 h time period and measured using Image J software, to determine the extent of closure. For time-lapse videography, 5 x 104 cells in IMDM (serum-free) and allowed to invade for 16–20 h. Following removal of non-invasive cells, the bottom of the insert membrane was fixed with 0.1% glutaraldehyde, stained with crystal violet and analyzed by phase contrast microscopy to determine the number of cells per field. For ROCK inhibition, LMS1 cells were pre-incubated for 3 h with either vehicle or Y27632  prior to seeding and maintained in the presence of the inhibitor for the experiment duration.Transwell invasion assays were performed using 8 μm pore inserts coated with 100 μg/ml fibronectin (Sigma) for 1 h, followed by blocking of insert membrane with 2.5% BSA for 30 minutes. For matrigel coated inserts, a 1:40 dilution of matrigel  was incubated on the membrane for 30 minutes and then washed with serum-free media before seeding cells. Inserts were seeded at 2 x 106 cells were combined 1:1 with matrigel and injected subcutaneously into the flanks of 6-weeks old immunocompromised mice . Once tumors were palpable, tumor length and width were measured every other day and volume was calculated using the following formula: a2*b/2, where a is the width and b is the length. After 7 weeks, primary tumors were excised, weighed and processed for paraffin histology according to standard protocols. Cell proliferation was assessed by immunostaining for Ki67  and counterstaining with hematoxylin. Lung metastasis was assessed by fluorescence microscopy of whole lung tissue recovered at endpoint, for quantification of GFP+ foci. Primary tumors were graded according to WHO classification for soft tissues [All animal experiments were performed in accordance with the NYU IACUC approved protocols (#061108–03 and #100108–01). To determine the tumor growth and metastatic properties of LMS cell lines, 5 x 10 tissues .TSC1) were purchased from Switchgear Genomics . For generation of a TSC1-3’UTR reporter construct, genomic DNA containing a portion of the TSC1-3’UTR sequence was PCR-amplified using the primers TSC1-3’UTR-Fw (XhoI): 5’ CCCTCGAGGGGTTTGGGCAGAGCGAAGACC−3’; and TSC1-3’UTR-Rv (NotI): 5’-TTGCGGCCGCAAGCCACTCATTGAGGAAGAGC-3’, subcloned into the pCR-II vector using TOPO-TA cloning kit (ThermoFisher) for sequencing, and then cloned into a pLS-Empty Vector (Switchgear genomics) by XhoI/NotI ligation. For generation of luciferase constructs containing individual miR-130b target sites and target site mutants, double stranded DNA fragments (~80 bp), corresponding to regions of TSC1 3’UTR genomic DNA, were synthesized using Genscript Gene Synthesis Services  and cloned downstream of luciferase into the pLS-Empty Vector by Nhe1/XhoI ligation. The partial 3’UTR sequences are shown in Human-3’UTR luciferase reporter constructs  (P = 0.003) in tumor samples  MM and LMS clinical specimens  . Validat samples . Amplifipecimens . Gene amin vitro oncogenic assays were performed using the LMS cell line, SK-LMS-1 (hereafter LMS1) stably transduced with a dual expression lentiviral vector encoding miR-130b and GFP (P = 0.016)  .in vivo, cells were xenotransplanted in immunocompromised mice. At endpoint (7 weeks), miR-130b overexpressing tumors exhibited significant increases in volume (P = 0.006) (P = 0.012) (top panels). While controls displayed a fascicular pattern of cells with cigar shaped nuclei, common histological features of LMS, miR-130b overexpressing tumors presented a storiform appearance with pleomorphic nuclei, characteristic of pleomorphic undifferentiated sarcomas. Grading of tumors revealed a higher score in the miR-130b group (2.0 ± 0.5) compared to controls (1.4 ± 0.3) (P = 0.037) (P = 0.0036) (bottom panels) consistent with increased tumor growth.To investigate whether miR-130b enhances LMS tumorigenesis = 0.006)  and weig= 0.012)  relative= 0.037) . ImmunohEx vivo fluorescent imaging revealed a marked increase in GFP positive lung foci in mice with miR-130b overexpressing tumors compared to controls (P = 0.029) (right panel). Altogether, the data demonstrates that miR-130b promotes an aggressive pleomorphic tumor phenotype in LMS1 cells, by accelerating tumor growth and enhancing metastatic potential.Metastatic burden at endpoint was also assessed in resected lungs of xenotransplanted mice by monitoring GFP expression. controls . FluoresP < 0.05) (www.targetscan.org). Functional annotation of those potential targets revealed a set of genes involved in GTPase signaling, cell projection and leading edge, suggesting a role for miR-130b in the regulation of cell motility  following transient transfection with miR-130b or miRNA mimic controls (SCR). Expression profiling of RNA extracted from LMS cells 36 h post-miRNA transfection identified 224 transcripts significantly down-regulated in both LMS1 and UT1  . Among tmotility . This waARFGEF1, RAB5A, and TSC1 were found to be targets, as evidenced by significant repression of luciferase activity following transfection with miR-130b (P < 0.05)  . Among t samples  Fig 3C)ARFGEF1, 1 levels  followinTSC1, target site prediction of the 3’UTR was performed using TargetScan and miRDB databases, which identified putative non-conserved sites at seed locations 2688 (S1) and 3948 (S2), and conserved sites at 4453 (S3) and 4879 (S4)  . Truncat879 (S4) . To iden879 (S4) . Three s879 (S4) . To dete879 (S4) . MiR-130879 (S4) . To dete879 (S4) . In 293T879 (S4) , indicat879 (S4) . In UT-1879 (S4) , suggestTSC1 was directly repressed by miR-130b, shRNA-mediated TSC1 knockdown was performed in LMS1 cells to determine if the pro-tumorigenic effects of miR-130b were replicated. Cells were stably transduced with dual expression lentiviral vectors encoding either a short hairpin RNA against TSC1 (shTSC1) or a non-target control, and GFP. Transduction of shTSC1 resulted in a ~50% reduction of TSC1 mRNA levels compared to the non-target control vector (TSC1 knockdown had no effects on cell proliferation or colony formation (P = 0.011)   , consist= 0.011) , S1E Figcontrol) .TSC1 knockdown also enhances LMS tumor growth in vivo, shTSC1- and control vector-transduced LMS1 cells were xenotransplanted into immunocompromised mice. Similar to miR-130b overexpressing cells, shTSC1 cells exhibited greater tumor volume (P = 0.002) and tumor weight at endpoint (P = 0.001), compared to controls    supportiright panels). This morphological change was accompanied by an altered rate and mode of migration. In scratch assays, as-miR-130b cells closed the wound gap more rapidly (P < 0.001) (right panel), and did not exhibit leading edge protrusions compared to controls (left panel). Time-lapse microscopy further confirmed increased migration rates and non-lamellipodia driven movement (P < 0.05)  and GFP . MiR-130 < 0.05)  Movies.P = 0.0038)  . Since m 0.0038) , and Rhoin vitro [SMMHC mRNA levels, indicating impaired SMC maturation  in hMSCs also resulted in impaired SMC maturation, indicated by a partial reduction of SMMHC mRNA levels in differentiating cultures compared to non-target siRNA controls (SCR)  . These dntiation . AccordiMS cells . Thus, iin vivo tumor growth, cell invasion and metastatic potential, and inhibits SM differentiation. These findings are consistent with miRNA expression profiles that indicate miR-130b upregulation of in LMS relative to normal SM, and miR-130b downregulation during SM differentiation of MSCs [TSC1, which was also found to be differentially expressed in both LMS and SM differentiation, and a target of miR-130b. These observations indicate that miR-130b targets effectors of both, cellular transformation and differentiation, and may critically contribute to the undifferentiated and aggressive nature of LMS  [TSC1 expression in LMS cells by directly binding to conserved sites at +4453 (S3) and +4879 (S4) of its 3’-UTR. Moreover, TSC1 was significantly downregulated in LMS patient samples, and shRNA-mediated TSC1 knockdown replicated the effects of miR-130b in cell invasion, in vivo tumor growth and metastasis. These observations further support TSC1 as a LMS tumor suppressor, and are consistent with AKT-mTOR pathway activation as a major oncogenic driver of LMS [SM22a-Pten knockout mice [Our data support a role for miR-130b in promoting the aggressiveness of LMS, in part by repressing n (mTOR) . Luciferout mice .in vivo, this process was Rho/ROCK-independent; characteristic of a mesenchymal mode of movement that uses elongated protrusions [TSC1, the endocytosis regulator RAB5A, and the ADP-ribosylation factor guanine exchange factor, ARFGEF1 , as miR-130b targets which can act as regulators of Rho activation and signaling [ARHGAP1, which was not found to be regulated by miR-130b in LMS cells Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 TableTSC1 3’UTR constructs for luciferase reporter assays. Putative miR-130b binding sites are indicated in bold. Mutated sequence is shown in red. Sequence locations are indicated relative to the beginning of the 3’UTR.DNA sequences of truncated (XLSX)Click here for additional data file.S1 Figlower panels). (E) Colony forming capacity of control and miR-130b-transduced LMS1 cells, 9 days after plating. Images (lower panels) show crystal violet-stained colonies at endpoint. (F) Representative images of miR-130b- and control-transduced cells cultured as sarcospheres in methyl cellulose. At day 7, cells were isolated from a subset of each group and single cell suspensions were reseeded in methyl cellulose to assess for self-renewal. Data represent mean ± SD of triplicate measurements; experiments were performed in 2 stably transduced LMS1 cell lines. Scale bars: 100 μM.(A) Relative miR-130b expression levels, measured by RT-qPCR, in LMS cells (LMS1) stably transduced with a lentiviral construct containing miR-130b sequence (miR-130b) relative to control cells transduced with a non-targeting miRNA (Cntrl). (B) Growth curves of miR-130b- or control-transduced LMS1 in monolayer cultures in media with serum (S) (solid lines) and under serum-free conditions (SF) (hashed lines). Proliferation was assessed by crystal violet staining and measuring absorbance at 595 nm. (C) Cell survival under attachment-free conditions. Transduced cells were plated on ultra-low attachment plates, forming cell clusters which were dissociated and counted every 24 h for 3 days. Representative bright field images for each group are shown under graph. (D) Scratch assay of control and miR-130b transduced LMS1 showing percentage of wound closure (relative to t = 0) over 24 h. Representative bright field images showing extent of wound closures over time are shown under graph ((TIF)Click here for additional data file.S2 FigP < 0.05, vs Ctrl HI. Values are normalized to the mock controls for each luciferase construct. Data represent mean ± SD, n = 4. Note that miR-130b inhibition did not significantly increase luciferase activity of the wild type S4 construct or either of the mutant S3 and S4 constructs.Values represent luciferase units in UT-1 cells of wild type or mutant S3 and S4 constructs following mock (no inhibitor), control (ctrl HI) or miR-130b (miR-130b HI) hairpin inhibitor cotransfection. *(TIF)Click here for additional data file.S3 Figtop panels) and fluorescence images (bottom panels) indicating transduction efficiency of LMS1 cells transduced with dual expression lentiviral vectors encoding shTSC1or non-target shRNA (Cntrl), and GFP. (B) Relative TSC1 expression levels in LMS1 cells in control and knockdown groups, measured by RT-qPCR. (C) Growth curves of shTSC1 and control-transduced LMS1 cells cultured in standard growth media (10% serum). (D) Colony forming capacity of control and shTSC1-transduced LMS1 cells, 9 days after plating. Images (lower panels) show crystal violet-stained colonies at endpoint. (E) Scratch assay of control and shTSC1 transduced LMS1 showing percentage of wound closure (relative to t = 0) over 24 h. Representative bright field images showing extent of wound closures over time are shown. Data represent mean ± SD of triplicate measurements; experiments were performed in 2 stably transduced LMS1 cell lines. Scale bars: 100 μM. Statistical analyses were performed by two-tailed students t-test; * P < 0.05, ** P < 0.005.(A) Representative bright field ((TIF)Click here for additional data file.S4 Figtop panels) and fluorescence images (bottom panels) indicating transduction efficiency of LMS1 cells transduced with dual expression lentiviral vectors encoding the antisense sequence of miR-130b (as-miR-130b) or non-target RNA control sequence (Cntrl), and GFP. Scale bar: 100 μM. (B) Colony forming capacity of control and as-miR-130b-transduced LMS1 cells, 9 days after plating. Images (lower panels) show crystal violet-stained colonies at endpoint. (C) Growth curves of as-miR-130b and control cells grown in standard growth media (10% serum). Data represent mean ± SD of triplicate measurements; experiments were performed in 2 stably transduced LMS1 cell lines.(A) Representative bright field ((TIF)Click here for additional data file.S5 FigDICER1, measured by RT-qPCR, in UT1 and LMS1 cells transfected with miR-130b or control (SCR) oligonucleotide mimics. (B) Western blot analysis of DICER1 protein expression in miR-130b and control mimic transfected cells. (C) Luciferase-3’UTR reporter assay for validation of DICER1 as a direct target of miR-130b. Values represent relative luciferase units (RLU) in 293T cells transfected with a luciferase expression vector containing the 3’UTR of DICER1 without mimics (-), scrambled control (SCR), a non-targeting mimic (NT), or miR-130b mimic. Values are normalized to the mock (-) control group (= 1). Data represent mean ± SD, n = 3–5. * P < 0.05 vs all control groups.(A) Relative expression levels of (TIF)Click here for additional data file.S1 MovieA representative control cell is shown exhibiting an elongated morphology and directional lamellopodia driven movement.(MOV)Click here for additional data file.S2 MovieA representative as-miR-130b modified cell is shown, exhibiting a rounded ameboid morphology and non-lamellopodia driven movement.(MOV)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.


Poloxamer-188 (P188) is a nonionic triblock linear copolymer that can be used as a pharmaceutical excipient because of its amphiphilic nature. This study investigated whether P188 can act as an adjuvant to improve the immunogenicity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor binding domain (RBD) subunit vaccine. BALB/c mice were vaccinated twice with the RBD antigen alone or in combination with P188 or MF59 . The resulting humoral and cellular immunity were assessed. Results showed that P188 helped elicit higher neutralizing activity than MF59 after vaccination. P188 induced significant humoral immune response, along with type 1 T helper (Th1) and type 2 T helper (Th2) cellular immune response when compared with MF59 due to repressing p38MAPK phosphorylation. Furthermore, P188 did not result in adverse effects such as fibrosis of liver or kidney after vaccination. In conclusion, P188 is a novel adjuvant that may be used for safe and effective immune enhancement of the SARS-CoV-2 RBD antigen. COVID-19 is a novel coronavirus SARS-CoV-2) that causes severe respiratory syndromes and was first identified in 2019  that cau. SARS-CoAfter the widespread use of the novel vaccines, technical problems started to surface, such as instability of the mRNA vaccines  and rarePoloxamer-188 (P188) is a safe, nonionic triblock copolymer composed of a central hydrophobic chain of poly(propylene oxide) sandwiched between two hydrophilic chains of poly(ethylene oxide), giving it an amphiphilic nature useful as an emulsifier ,20. P188Given the multiple useful characteristics of P188, we aimed to evaluate whether it may serve as an effective adjuvant for the SARS-CoV-2 RBD subunit vaccine. Mice were vaccinated twice with the RBD antigen alone or in combination with P188 or MF59. Antibody and cellular immune responses were examined.2 euthanasia for pathological analysis and splenocyte isolation. For vaccine formulation per dose, 1 or 5 μg of SARS-CoV-2 RBD (S1 + S2 fragments) was combined with 50 μL of P188  or 50 μL of MF59 . Then, 6-week-old male BALB/c mice were intramuscularly immunized twice 3 weeks apart. After vaccination, ELISA was performed to examine the levels of glutamate pyruvate transaminase (GPT), creatinine (Cr), blood urea nitrogen (BUN), neutralizing antibody titer, and plasma cytokines on Day 49. The mice were then sacrificed by CO50.Human ACE (hACE) gene-transfected HEK293 cells  were seeded and incubated overnight in 96-well plates. Sera from vaccinated mice were diluted by MEM containing 1% FBS, and then mixed with an equal volume of SARS-CoV-2 pseudoviruses  expressing full-length wild-type Wuhan-Hu-1 strain SARS-CoV-2 spike protein. After incubation at 37 °C for 1 h, the sera-virus mixture was then added to the hACE-transfected HEK293 cells. After 1 h incubation at 37 °C, the virus-containing medium was then replaced with fresh medium and the cells were incubated for 72 h. Relative luciferase units (RLU) from lysed hACE-transfected HEK293 cells were measured by Tecan i-control . The valence of neutralizing antibody is presented as the reciprocal of ID2. To detect S-protein immunoprecipitated by hACE, hACE of cell lysate was first immunoprecipitated by hACE monoclonal antibody  with Protein G plus/Protein A agarose beads . Thereafter, the precipitates were blotted with monoclonal anti-S-protein antibody . To establish a human ACE2 (hACE2)-overexpressing cell line, human ACE2 (NM_021804) cDNA was inserted into the Sgfl/Mlul site of the pCMV6-GFP expression vector plasmid . The resulting pCMV6-ACE2-GFP was then transfected into HepG2 cells (ATCC HB-8065) using Lipofectamine 2000 . Cells were cultured in Opti-MEM  at 37 °C for 5 h and then placed in freshly changed culture medium. The diluted sera from vaccinated mice were mixed with SARS-CoV-2 pseudovirus and incubated at 37 °C for 1 h before adding to hACE2-transfected HepG2 cells in 48-well culture plate for a 24-h incubation period in DMEM  at 37 °C, 5% COAfter secondary vaccination, levels of plasma Interferon-γ (IFN-γ), Interleukin 2 (IL-2), Interleukin 4 (IL-4), and Interleukin 10 (IL-10) were determined by enzyme-linked immunosorbent assay (ELISA) purchased from MyBioSsource . Isolation of primary splenocytes from spleens of vaccinated mice was conducted using the Spleen Dissociation Kit . Splenocytes were adherently cultured in 96-well plates containing DMEM  supplemented with 10% fetal bovine serum , 2-mercaptoethanol , 0.01% penicillin G , gentamycin , 0.01% HEPES , and L-arginine . Splenocytes were stimulated with RBD or RBD plus P188 (or MF59) for the detection of IFN-γ, IL-2, IL-4, and IL-10 mRNA and protein expression. Human peripheral blood mononuclear Cells (PBMCs) from Kaohsiung Medical University , were cultured in 96-well plates containing RPMI-1640 medium  and P188  with RBD (5 μg) stimulation for 24 h. IFN-γ was measured by quantitative reverse transcription PCR (RT-qPCR) with qPCR primer: F:5′-GAGTGTGGAGACCATCAAGGAAG-3′ and R:5′-TGCTTTGCGTTGGACATTCAAG TC-3′, and apoptosis was detected by ApoStrand™ ELISA Apoptosis Detection Kit  according to manufacturer’s protocol. Total RNA from splenocytes cell lysate was extracted with Trizol  and converted to cDNA with the Super Script III cDNA Synthesis Kit . IFN-γ, IL-2, IL-4, and IL-10 cDNA were amplified using the SYBR Green I qPCR master mix kit  and qPCR primers. Respective forward and reverse primers  were as follows: qPCR primer included IFN-γ: F:5′-CAGCAACAGCA AGGCGAAAAAGG-3′ and R:5′-TTTCCGCTTCCTGAG GCTGGAT-3′; IL-2: F:5′-GCGGCATGTTCTGGATTTGACTC-3′ and R:5′-CCACCACAGT TGCTGACTCATC-3′; IL-4: F:5′-ATCATCGGCATTTTGAACGAGGTC-3′ and R:5′-ACCTTGGAAGCCCTACA GACGA-3′, IL-10: F:5′-CGGGAAGACAATAACTGCACCC-3′ and R:5′-CGGTTAGC AGTATGTTGTCCAG C-3′. Data analysis was performed by Foldchange = 2 (∆Ct treatment-∆Ct control).Proteins of primary splenocytes were extracted with the M-PER Mammalian Protein Extraction Reagent  and were then separated with SDS-PAGE. The separated proteins on SDS-PAGE were transferred onto a PVDF membrane . Then, the PVDF membrane was blocked with Tris-buffered saline with 0.2% Tween 20 (TBS-T) containing 5% skim milk at 4 °C overnight. For protein detection, the PVDF membrane was incubated with diluted primary antibodies. After washing with TBST, the PVDF membrane was incubated with a 1:10,000 dilution of horseradish peroxidase-conjugated secondary antibody in TBS-T containing 5% skim milk. Western blots were detected using the ECL Detection Kit . In this experiment, anti-p-p38, p38, IFN-γ, IL-2, IL-4, and IL-10 antibodies  were used. t test with the Bonferroni test. The GraphPad Prism software  was used for statistical analysis. Data are presented as mean ± SEM and significant difference between groups were calculated using one-way ANOVA and paired To confirm dose-responsive immune response and safety of P188, human PBMC were cultured by P188  and stimulated RBD (5 μg) for 24 h. RT-qPCR showed that P188  significantly enhanced TNF-γ A. HoweveTo evaluate the adjuvant effect, mice were vaccinated twice with the RBD antigen (1 or 5 μg) alone or in combination with P188 or MF59. Neutralization assay using pseudoviruses showed that P188 increased the titer of neutralizing antibody in groups of 1 μg RBD immunization when compared to the MF59-adjuvanted group . When 5 To verify the binding specificity of the antibodies elicited by the vaccines, sera of vaccinated mice were evaluated for the ability to block the interaction between recombinant S protein and hACE expressed on cells. Co-immunoprecipitation analysis showed that antibodies elicited by adjuvanted RBD vaccines successfully disrupted S protein binding to hACE  while anAfter immunization, mice splenocytes were isolated and evaluated for cytokine expression. Compared to the MF59-adjuvanted group, splenocytes from the P188-adjuvanted group synthesized higher levels of T-helper 1-type cytokines, IFN-γ A and IL-To determine whether p38MAPK may play a role in P188-induced cytokine expression, Western blot analysis of p38 and the cytokines was performed. Results showed that P188 significantly repressed p38MAPK phosphorylation and enhanced IFN-γ and IL-2 and T-helper type 2 cytokines, IL-10, or IL-4 in primary splenocytes of mice . For verAfter vaccination, biochemical analysis of plasma was performed, and pathological characters of liver and kidney sections were investigated. On day 49 post vaccination, there was no mortality, abnormal body weight, and biochemical changes  between plasma samples of PBS; RBD-vaccinated mice (RBD); MF59-vaccinated mice (RBD + MF59); and P188-vaccinated mice (RBD + P188). Ideally, the efficiency of controlling SARS-CoV-2 spread is reliant on vaccination with the RBD subunit antigen combined with adaptive adjuvant to stimulate innate and efficient adaptive immunities. This study showed that injection of SARS-CoV-2 RBD vaccine adjuvanted with P188 was useful to enhance neutralizing antibody against S protein-expressing the pseudovirus. P188 induced Th1 and Th2 immune responses and higher levels of neutralizing antibody than MF59 without adverse effects during in vivo studies. To improve the immune efficacy of SARS-CoV-2 RBD vaccines, P188 first was used for adjuvant to effectively enhanced production of anti-SARS-CoV-2 antibody for a more sustained period. In terms of the immunization schedule, we saw that immunization significantly induced anti-SARS-CoV-2 RBD antibody titer after two-dose vaccination and successfully neutralized recombinant S protein of SARS-CoV-2 in vitro. Specifically, P188 induced higher antibody titers than MF59 under the same concentration of RBD antigen vaccination in vivo study. After vaccination, we still detect higher titers of neutralizing antibody in plasma of vaccinated mice with P188-adjuvanted RBD than MF59-adjuvanted RBD during the third week. In addition, vaccination with poloxmer188-adjuvanted RBD did not significantly alter GPT, BUN, and Cr in serum, and fibrosis of liver and kidney. These studies suggest that P188 is a safe, efficient, and novel adjuvant for the SARS-CoV-2 RBD vaccine. For considering clinical immune responsive pathway, the adjuvant of SARS-CoV-2 RBD vaccine must be suitable for inducing sufficient humoral and cellular immune response against SARS-CoV-2 infection. A current study showed that P188 enhanced higher Th1 cytokines (IFN-γ and IL-2) and Th2 cytokines (IL-10 and IL-4) in plasma compared with MF59 after vaccination. These results pointed out that P188-induced immunization is dominant Th1 and Th2 immune response to be adjuvant of SARS-CoV-2 RBD vaccine.50 5700 mg/Kg). In clinical trials of healthy people, P188 of intravenous injection  causes mild and frequent events, including back pain, leg pain, headache, and nausea. Clinical trial of patients with sickle cell disease showed P188 of intravenous therapy (30 mg/kg/h) for 72 h could induce mild and frequent events such as injection site pain, nausea, headache, vomiting, abdominal pain, or constipation [P188 exhibits extremely low toxicity (oral LDtipation . In a cltipation . In addiRecent study confirms that P188 can keep good chemical stability in solution under environment stress such as temperature and pH alteration . MoreoveOur study showed that P188 can induce better Th1 and Th2 immune responses than MF59. Co-administration of P188 with SARS-CoV-2 RBD allowed a significant boost in neutralizing antibody titer without adverse effect. This use of P188 as adjuvant is an attractive strategy for the development of commercial vaccines against COVID-19.


Dear Editor,1 Perivascular stem cells (PSCs), known as mural cells covering the vasculature, are essential for blood vessel formation and postulated as progenitors of mesenchymal stem cells (MSCs).3 We previously established umbilical cord artery-derived PSCs (UCA-PSCs) and Wharton’s jelly-derived MSCs (WJ-MSCs) and UCA-PSCs display optimal angiogenic capacity in vitro.4 Therefore, we explored the angiogenesis and pro-angiogenesis mechanisms of UCA-PSCs and provided them as an efficient treatment strategy for ovarian failure.Chemotherapy-induced ovarian failure significantly diminishes ovarian blood flow, ovarian size, and follicular development. Angiogenesis plays a vital role in repairing ovarian damage.5In this study, we isolated and cultured UCA-PSCs and WJ-MSCs, which morphologically resembled fibroblasts, and differentiated into adipocytes, osteoblasts, or neural-like cells after induction in vitro  were used for sequencing  mouse model  levels to normal controls  but similar to that in the normal control group (26.33 ± 1.36)  mouse model  Fig. .In conclusion, our study found that UCA-PSCs showed greater angiogenic and proangiogenic potential than WJ-MSCs in vivo and ex vivo, which was related to cell-to-cell communication through the CD146/AKT/FHL1/Jagged1 signaling pathway and IL6 paracrine activity Fig. . MoreoveSupplementary Figure1Supplementary Figure2Supplementary Figure3Supplementary Figure4Supplementary Figure5Supplementary Figure6Supplementary Figure7Supplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4Supplementary video1-UCA-PSCsSupplementary video2-WJ-MSCsSupplementary MaterialsRaw data


A novel population of age-associated Granzyme K (GZMK)-expressing CD8 T cells was found in mice and humans. These cells enhance local tissue senescence and inflammation and are distinct from central memory and conventional Granzyme B- or Interferon γ-producing effector memory CD8 T cells. It is unknown whether these cells drive chronic disease; thus, we induced atherosclerosis in young (3-mo) and aged (18-mo) wild-type mice via the PCSK9-AAV model and used scRNAseq to demonstrate that this GZMK-CD8 T cell population homes to atherosclerotic lesions exclusively in aged mice. Neutralizing CD8 T cells demonstrates that GZMK-CD8 cells drive age-enhance atherosclerosis. Finally, we transferred GZMK-CD8 cells from different aged donors into young CD8-/- hosts and demonstrate that GZMK-CD8 cells from aged but not young donors drive atherosclerosis. In conclusion, we identified a novel role for this recently described population of aging-specific GZMK-expressing CD8+ T cells as a critical driver of chronic disease.


The ongoing monkeypox outbreak, a WHO-declared PHEIC, requires fast and broad rollout of MVA-BN vaccinations among risk groups. An initial single-dose strategy, followed by a delayed second dose beyond the approved 4-week interval, could help managing tight vaccine resources.Bavarian Nordic has run a study in vaccinia-naïve participants, randomized to receive either 1 MVA-BN vaccination (N = 181) or 2 MVA-BN vaccinations 4 weeks apart (N = 183). Of note, this study also evaluated participants with prior smallpox vaccination, who received 1 MVA-BN boost (N = 200). Subsets of approximately 75 participants of the vaccinia-naïve subjects having received either 1 or 2 doses of the vaccine were administered an MVA-BN boost 2 years later. Neutralizing antibodies (nAb) were measured using Vaccinia PRNT.The 1xMVA-BN and 2xMVA-BN groups responded as expected to the primary regimen, with modest increases in nAb GMTs at Week 2  that further increased at Week 4 (7.2 and 7.5). Two weeks after the second primary vaccination (at Week 6) (2xMVA-BN) nAb GMTs peaked (45.6) before stabilizing at Week 8 (34.0). In both the 1x and 2xMVA-BN groups, the nAb levels were near baseline 2 years after primary vaccination. After the MVA-BN boost at 2 years, both groups exhibited rapid anamnestic responses with nAb GMTs that peaked 2 weeks post-boost (80.7 and 125.3), at higher levels than those observed at any timepoint following primary vaccination, remaining elevated at 6 months post-boost (25.6 and 49.3). These anamnestic responses support the presence of durable immunological memory in both groups. No safety concerns were identified and the most common adverse event following a 2-year MVA-BN boost was injection site erythema in 82.2% of participants.A single dose of MVA-BN elicits a durable memory immune response for at least 2 years. Similarly, high nAb responses following a boost dose among subjects in the initial 1xMVA-BN or 2xMVA-BN groups indicate that the approved 4-week interval of the two-dose regimen could optionally be extended up to at least 2 years.NCT00316524 and NCT00686582Heinz Weidenthaler, MD, Bavarian Nordic GmbH: Employee and received warrants Florian Lienert, PhD, Bavarian Nordic GmbH: employee Employee and received warrants Daniela Reichhardt, PhD, Bavarian Nordic A/S: employee Employee and received warrants Darja Schmidt, Dr, Bavarian Nordic GmbH: Employee and received warrants Günter Silbernagl, MSc, Bavarian Nordic: Employed|Bavarian Nordic GmbH: employee Employee and received warrants Paul Chaplin, PhD, Bavarian Nordic A/S: employee (CEO)|Bavarian Nordic A/S: Stocks/Bonds.


NPM1 mutations are the most common genetic alteration in acute myeloid leukemia (AML), detected in about 30–35% of adult AML and more than 50% of AML with normal karyotype. Because of its peculiar molecular and clinico-pathological features, including aberrant cytoplasmic dislocation of the NPM1 mutant and wild-type proteins, lack of involvement in driving clonal hematopoiesis, mutual exclusion with recurrent cytogenetic abnormalities, association with unique gene expression and micro-RNA profiles and high stability at relapse, NPM1-mutated AML is regarded as a distinct genetic entity in the World Health Organization (WHO) classification of hematopoietic malignancies. Starting from the structure and functions of NPM1, we provide an overview of the potential targeted therapies against NPM1-mutated AML and discuss strategies aimed at interfering with the oligomerization (compound NSC348884) and the abnormal traffic of NPM1  as well as at inducing selective NPM1-mutant protein degradation -epigallocatechin-3-gallate, imidazoquinoxaline derivatives) and at targeting the integrity of nucleolar structure (actinomycin D). We also discuss the current therapeutic results obtained in NPM1-mutated AML with the BCL-2 inhibitor venetoclax and the preliminary clinical results using menin inhibitors targeting HOX/MEIS1 expression. Finally, we review various immunotherapeutic approaches in NPM1-mutated AML, including immune check-point inhibitors, CAR and TCR T-cell-based therapies against neoantigens created by the NPM1 mutations.Nucleophosmin 1 (NPM1) is a nucleus-cytoplasmic shuttling protein which is predominantly located in the nucleolus and exerts multiple functions, including regulation of centrosome duplication, ribosome biogenesis and export, histone assembly, maintenance of genomic stability and response to nucleolar stress.  Nucleophosmin 1 (NPM1) is a ubiquitous nucleus-cytoplasmic shuttling protein .NPM1 gene are the most frequent genetic lesion in acute myeloid leukemia (AML), being detectable in about one-third of adult AML and 50–60% of AML with normal karyotype [DNMT3A and TET2 [NPM1-mutated AML include the mutual exclusion with recurrent cytogenetic abnormalities, the association with specific gene expression [NPM1 mutations at relapse [NPM1-mutated AML is the aberrant cytoplasmic dislocation of the NPM1 mutant and NPM1 wild-type proteins (through heterodimerization) [NPM1-mutated AML still remain largely unknown [NPM1-mutated AML can be diagnosed irrespective of the percentage of blasts, based on previous observations that cases classified as MDS or MDS/MPN with NPM1 mutations quickly progressed to AML [NPM1-mutated AML is recognized as a distinct entity, within the category of AML with recurrent genetic abnormalities of the WHO classification [NPM1-mutated AML are summarized in Table Mutations of aryotype , 4. Thes relapse . Anotherization) , 9. Moreization)  which ard to AML . Becausefication . The maiNPM1-mutated AML in young adults is based upon induction chemotherapy (±FLT3 inhibitors) and consolidation cycles with high/intermediate dose of cytarabine (ARA-C) ± allogeneic hematopoietic stem cell transplantation (HSCT) in first complete remission (CR), depending on the status of the FLT3 gene and measurable residual disease-MRD [NPM1-mutated AML, about 50% of patients still died of progressive disease. Thus, there is a need for new therapeutic opportunities. Whole genomic approaches have unraveled the molecular heterogeneity of AML [The standard therapy of ease-MRD , 13. Howy of AML , 15 and y of AML , 17. Becy of AML . In the y of AML , 20. ConNPM1-mutated AML.Here, we provide an overview of the current status and future perspectives of targeted therapies in Small molecules can be potentially designed to bind to any portion of a given protein to modulate its function . The mulThe N-term domain contains two nuclear export signals (NES) that promote the shuttling of NPM1 from the nucleus to the cytoplasm through the interaction with the nuclear exporter XPO1  . The N-tNPM1-mutated OCI-AML3 cells than in NPM1 wild-type HL-60 and OCI-AML2 cells [NPM1 mutations target the C-terminal domain whilst NSC348884 targets the N-terminal domain.Therefore, disruption of NPM1 oligomers may promote unfolding of the protein and alter NPM1 structure and functions. Targeting NPM1 oligomerization can be achieved using NSC348884, a water-insoluble compound that interacts with the high hydrophobic NPM1 dimerization surface . NSC3488L2 cells  The N-term region is critical for the interaction of NPM1 with its partners through positively charged surface. These molecular interactions are critical to build-up the structure of the nucleolus , 29. In NPM1-mutated AML cells [Many NPM1 interactors that are thought to be involved in leukemogenesis have been claimed to be dislocated in the cytoplasm by the NPM1 mutant , althougML cells . Thus, sNPM1 mutations [The NPM1 C-terminus consists of a three-helix bundle  stabilizutations , 9.NPM1-mutated than wild-type AML cells [NPM1-mutated AML [The C-term of NPM1 mutated protein is unfolded and therefore difficult to target , 25. TheML cells , probablML cells . Moreoveated AML .NPM1-mutated AML cells may be particularly vulnerable to stress because it is NPM1 depleted due to both haploinsufficiency and cytoplasmic delocalization [NPM1-mutated AML [NPM1-mutated cells [NPM1-mutated AML [NPM1-mutated AML.NPM1 acts as a nucleolar stress sensor . The nuclization . Therefoated AML  ed cells , 41 and ated AML , 40. Othated AML  should bNPM1-mutated AML may be even more complex. In fact, NPM1 mutants can impede the formation of acute promyelocytic leukemia (APL) nuclear bodies (NBs) [NPM1-mutated AML cells are characterized by defective mitochondrial function [NPM1-mutated AML cells. Interestingly, actinomycin D appears to potentiate the activity of venetoclax on mitochondrial function and apoptosis [However, the mechanism of action of actinomycin D in es (NBs) , which ies (NBs) . Thus, Nfunction . Actinompoptosis .NPM1 mutations [NPM1-mutated AML cell lines OCI-AML3 with deguelin, a rotenoid isolated from several plant species [p53 [NPM1-mutated AML xenografts [NPM1-mutation into cells normally bearing wild-type NPM1 sensitized them to EAPB0503, leading to their growth arrest [Targeting the levels of mutant NPM1 represents another therapeutic opportunity  and rednografts , 50. MorNPM1-mutated AML. In this regard, selective degradation of mutant NPM1 through degron-tag, invariably induced differentiation, and growth arrest of NPM1-mutated cell lines [Mutant NPM1 can be also targeted through proteolysis targeting chimera (PROTAC) which allows ubiquitination and proteasome-mediated degradation of the target protein  ll lines , confirmThe multilayered nucleolar structure  is dictated by the “liquid-liquid” phase separation involving nucleolar proteins, primarily native NPM1 and fibrillarin that segregate in the granular component and dense fibrillar component, respectively . NPM1 isNPM1-mutated AML [NPM1-mutated AML patients in early-phase clinical trials [NPM1-mutated AML cells in vitro and in vivo (unpublished data). Interestingly, eltanexor synergizes with a BCL-2 inhibitor by increasing apoptosis in primary AML cells [Altered nuclear-cytoplasmic shuttling is a peculiar characteristic of ated AML . Thus, tated AML  but its ated AML . More reated AML   or low-dose cytarabine (LDAC), shows anti-leukemic activity in 60-70% of AML patients . By downOX genes , remainsOX genes .Fig. 2OtNPM1-mutated AML particularly benefits from venetoclax-based regimens, both at first diagnosis [FLT3 status [NPM1-mutated AML patients achieved CR + CRi [NPM1-mutated AML exceeded 80%, with an estimated 2-year OS of 70% [NPM1-mutated AML patients [NPM1-mutated AML patients, when venetoclax was combined with 5 + 2 (cytarabine + idarubicin) or FLAG + IDA   [iagnosis , 71 (ind3 status ) and in 3 status –75  , 80.NPM1-mutated AML are lower, ranging between 46% and 66.8%. In general, venetoclax plus 5-azacitidine was associated with better results than venetoclax plus decitabine or LDAC. Therapy was usually administered continuously until progression. However, about half of AML patients carrying NPM1 and/or IDH2 mutations and/or achieving a molecular CR after at least 12 months of a venetoclax-based regimen, experienced a long treatment-free remission, after therapy cessation [NPM1-mutated AML patients with persistent or relapsed/progressed MRD [NPM1-mutated MRD-positive patients, as bridge to allotransplant , decreasing Bcl-2 and Bcl-xL levels [NPM1-mutated AML patients [Unfortunately, most patients treated with these regimens develop resistance over time and eventually relapse. To circumvent this problem, new combinations are currently tested, including venetoclax plus XPO1  or meninL levels , and sigL levels . ATO canpatients .Menin (MEN1) gene is located on chromosome 11q13 and encodes for a protein which regulates tissue-specific gene expression [MEN1 mutations cause the multiple endocrine neoplasia type-1 syndrome [Men1 gene leads to reduced expression of Hoxc6 and Hoxc8 genes during embryogenesis [MEIS1 contributes to the gene signature typical of NPM1-mutated AML [NPM1-mutated AML overlap with those of normal hematopoietic stem/progenitor cells, pointing to common mechanisms regulating HOX expression and to persistent expression rather than upregulation of HOX genes in NPM1-mutated AML [The pression . In humasyndrome . In miceogenesis . Menin iogenesis . High exated AML , 5. Inteated AML .More recently, we proved that HOX expression is directly dependent upon the aberrant cytoplasmic dislocation of NPM1 mutants . Indeed,NPM1-mutated AML cells on epigenetic machinery for HOX regulation [NPM1-mutated AML cells, causing a dose-dependent reduction in cell proliferation, a significant downregulation of HOXA/B clusters and MEIS1 gene expression, a marked differentiation of leukemic cells, a reduction of AML engraftment and a prolonged survival in mice PDX models [MEIS1 and PBX3), the effect on expression of HOXA and HOXB genes being not relevant [NPM1-mutated AML cells, independently from the coexistence of other mutations in patients’ samples, and in reducing blast infiltration of organs and expression of MEIS1 and FLT3 in PDX NPM1-mutated mouse models [Dependence of gulation  providesgulation , 94 . SNDX-5613  was very active in PDX mouse models of NPM1-mutated AML, including animals remaining in CR 1 year after cessation of therapy [NPM1-mutated or KTM2A-rearranged AML is being evaluated in the AUGMENT-101 phase I/II trial (NCT04065399). Preliminary results of this study were released by Syndax in April 2021. By March 2021, the trial had enrolled in the phase 1 cohort, 43 patients (median age 54 years) who had received a median of 3 previous lines of treatment. The most common side effects (>5%) included QT prolongation (14%), differentiation syndrome (5%) and anemia (5%). The overall response rate in the 7 patients with NPM1-mutated AML was 29% (2/7). In keeping with the proposed mechanism of menin inhibitors, RNA-Seq analysis of the bone marrow samples from responding patients exhibited downregulation of MEIS and HOXA9 genes and upregulation of the differentiation antigens CD11b, CD14 and CD13. The phase 2 part of the trial is ongoing. JNJ-75276617 is a potent inhibitor of the binding between menin and KTM2A. Its safety and activity are being tested in NCT04811560 that enrolls AML patients harboring NPM1 mutations or KMT2A rearrangements. However, no data have been released so far. The safety and efficacy of DS-1594b menin inhibitor will be evaluated as single drug or in combination with azacytidine and venetoclax regimens in a phase 1/2 clinical trial (NCT04752163). To maximize the depth and durability of clinical response, the Biomea Fusion, Inc. has recently developed BMF-219, an orally bioavailable, potent and selective irreversible covalent menin inhibitor. Clinical trial with this compound is ongoing (NCT05153330).Menin inhibitors, including KO-539, SNDX-5613, JNJ-75276617 and DS-1594b, are currently evaluated in clinical trials , 101. KO therapy , 102. ThHOXA9/MEIS1 signature (as in NPM1-mutated AML) than in the whole patient population [HOXA and MEIS genes [NPM1-mutated AML (NCT05020665).In a phase 1b/2 trial (NCT02343939), entospletinib, a selective oral inhibitor of the spleen tyrosine kinase (SYK) which is constitutively activated in AML promoting survival and proliferation , when copulation  IS genes . Based oNPM1-mutated AML cells [Ideally, any target antigen for AML immunotherapy should be expressed at high levels in the whole leukemic population, including leukemic stem cells, and to be absent or low expressed in normal hematopoietic cells and other tissues. Leukemia-associated antigens  are usually strongly expressed in AML cells  butNPM1), are specifically expressed in malignant clones and therefore represent ideal targets. In particular, the NPM1 mutant neoantigen can be considered an ideal AML target for a number of reasons. First, NPM1 mutations are common driver, gate-keeper events [NPM1 mutated proteins are detectable in chemoresistant leukemic stem cells [NPM1 mutations have been identified, the 4 bp frameshift insertion occurring in NPM1 mutant A is responsible for almost 80-85% of all mutations and more rare NPM1 mutations lead to the same amino acidic changes at NPM1 C-terminus. Fourth, the newly acquired amino acid C-terminus sequence of NPM1 mutant proteins is highly immunogenic in animals, eliciting specific antibodies. Fifth, the aberrant localization of the NPM1 mutant proteins in the cytoplasm of leukemic cells may favor their processing by the Human Leukocyte Antigen (HLA) MHC class I degradation pathway leading to HLA presentation and anti-cancer immune response. Indeed, using in silico analysis, we predicted that several peptides could bind to specific HLA class I molecules [NPM1-mutated AML patients [NPM1-mutated AML [NPM1-mutated AML sensitivity to T-cell immunity has been observed not only in the autologous but also in allogeneic setting. Although NPM1-mutated AML patients without FLT3-ITD has a good prognosis, those who underwent allogeneic HSCT showed a particularly long-term disease control [NPM1-mutated clone to achieve cure of AML is exemplified by the clinical observation of patients with NPM1-DNMT3A double-mutated AML after cessation of therapy. These cases, when achieve long-term molecular (MRD-negative) remission, are likely to be cured, even though the persistence of detectable copies of the DNMT3A mutant  may expose them to a low risk of developing a second AML [Conversely, leukemia-specific antigens deriving from altered proteins encoded by leukemogenic mutations  [NPM1-mutated/FLT3-ITD genotype predicted inferior survival [NPM1 and FLT3 preferentially associated with low T-cell cytolytic activity and a reduced expression of HLA-II  [CIITA methylation may limit antigen presentation by primary NPM1mutIDH1mut AML blasts through downregulation of MHC-II, thereby inducing immune evasion [NPM1-mutated AML is also contributed by VISTA (V-domain Ig suppressor of T-cell activation) and ULBP1 (NKG2 ligand) immunoregulatory circuitries that are both significantly upregulated in NPM1-mutated AML patients [AML is a poorly immunogenic and highly immune-suppressive hematological malignancy. High expression of PD-L1 has been found in + CD38−) . High PDsurvival . Moreoves CIITA) . Interes evasion . Immune patients  Fig. 4)NPM1-mutapatients .NPM1 mutations [NPM1-mutated AML, combining anti-PD-1 and anti NPM1-mutation specific immunotherapy (see below). Moreover, targeted immune gene expression and multiplexed digital spatial profiling showed distinct AML immune microenvironments [immune-infiltrated microenvironment that was characterized by severe immune suppression , higher dependence from IFN-γ driven adaptive immune responses, high T-cell infiltration and expression of major histocompatibility complex, closely clustered with the adverse-risk genetic AML categories  [NPM1-mutated cases (with or without FLT3-ITD) more frequently showed an immune-depleted microenvironment [NPM1-mutated AML harboring concomitant clonal hematopoiesis driven mutations  showed an enriched tumor inflammation signature score, predicting a clinical benefit from anti-PD-1 treatment [NPM1mut myeloproliferative phenotype in vivo [More recently, the anti-PD-1 antibody, nivolumab, was found to increase leukemia-associated antigen-stimulated cytotoxic T cells and cytotoxicity against stem cell-like cells, especially those carrying utations . These fronments . The immted AML) . Conversironment . Finallyreatment . This fi in vivo .NPM1-mutated AML may be a potential candidate for immune check-point inhibition  has been assessed also in association with high-dose cytarabine in 37 R/R AML patients  [Although the above findings suggest that ion Fig. , the few T cells . The clitations) . The ovetations) .NPM1-mutated AML [As previously mentioned, venetoclax is very active in ated AML . This efated AML  and incrated AML . The NCTNPM1-mutated AML. Immune targeting can be distinguished into: (1) HLA-dependent therapies relying on the presentation of NPM1 neoantigen [CAR T cells or T-cell receptor (TCR) gene therapy could be promising approaches against oantigen ; or (2) NPM1 mutation types. These peptides are able to efficiently bind to at least most common HLA types  which are often detected in the Caucasian population [NPM1-mutated AML cells carrying NPM1-mutant/HLA-A2 complex but not against NPM1 wild-type/HLA-A2+ AML cells or HLA-A2 negative tumor cells [NPM1-mutated AML in absence of toxicity [Searching for HLA class I ligandome of primary AMLs, multiple ΔNPM1-derived immunogenic peptides, have been identified, including AIQDLCLAV, AIQDLCVAV, CLAVEEVSL, LAVEEVSLR, AVEEVSLRK 9-mer, and CLAVEEVSLRK 11-mer, representative of the more common pulation , 140–142toxicity .NPM1-mutated AML cells and healthy tissue. Thus, aiming to improve selectivity for leukemic cells while minimizing toxicity towards normal cells, a dual targeting model was exploited through Cytokine Induced Killer (CIK) cells co-expressing a first-generation low affinity anti-CD123 and an anti-CD33 as costimulatory receptor without activation signaling domains. This trans-signaling strategy could allow: i) low toxicity profile against CD123+ endothelial cells and HSPC, due to a reduced cell activation given by the suboptimal first-generation CAR signal; ii) no or low myelotoxicity against CD33+ HSPC cells, due to absence of CIK cell activation upon the sole costimulatory signal engagement; and iii) full CAR-CIK activation only against double expressing CD123+/CD33+ leukemic cells [CD123 and CD33 are strongly expressed both in ic cells  Fig. 5)NPM1-mutaNPM1-mutated OCI-AML3 human cells [NPM1-mutated TCR gene-engineered T-cell therapy.Specific T cells for HLA-A*02:01-binding CLAVEEVSL have been searched in healthy individuals using peptide-HLA tetramers. Tetramer-positive CD8+ T cells were isolated and their activity towards primary AMLs investigated. The TCR was then isolated from a clone with high anti-leukemic reactivity and its capability to specifically recognize and lyse HLA-A*02:01-positive ΔNPM1 AML demonstrated after retroviral transduction of CD8+ and CD4+ T cells . Moreovean cells  NPM1-mutated AML with persistent NPM1 transcripts or in molecular relapse.DLI using T cells derived from healthy donors and specifically directed against the NPM1-mutated neoantigen with the aim to elicit graft-versus-leukemia may be a therapeutic option in patients experiencing molecular relapse following allogeneic HSCT. Eliciting endogenous immune responses through vaccination with NPM1 neoantigens is unlikely to be effective in patients with high-burden newly diagnosed or relapsed AML but could be of benefit, possibly in combination with immune check-point inhibitors, to treat pre-emptively NPM1-mutated AML are still poorly understood. Endogenous tagging of wild-type and mutated NPM1 proteins followed by mass spectrometry may unravel their interactions with other partners and functions in the cytoplasm. How NPM1 mutants deregulate the HOX program remains also to be better defined. Moreover, all current experimental data are based upon the analysis of OCI-AML3 and IMS-M2 human AML cell lines that both carry the most frequent NPM1 mutation . Whether similar results can be extended also to the rarer NPM1 variants remains to be determined. Clarifying these issues may lead to the development of new targeted therapeutic strategies.Molecular mechanisms underlying NPM1-mutated AML patients relapsing after venetoclax-based regimens represent a medical need. The mechanisms of resistance to venetoclax and its use in combination with other drugs to prevent relapse should be better investigated. Menin inhibitors are emerging as the most promising agents for targeted therapy of NPM1-mutated AML. The ongoing trials will tell us which is the real impact of these compounds in NPM1-mutated AML and suggest which are the best combinations to maximize the clinical benefit. Menin inhibitors, alone or in combination with venetoclax or other agents, could be incorporated in the treatment algorithm, as that shown in Fig. NPM1-related MRD, possibly as bridge to allo-HSCT, in eligible patients. Moreover, menin and XPO1 inhibitors have the potential to be used alone or in combination , at initial diagnosis, especially in patients who are older or unfit for intensive chemotherapy.Unfit NPM1-mutated AML using synthetic lethality approaches are under way [Identifying novel drugs for nder way . High-thnder way  allows tnder way , 149. Se


Caenorhabditis elegans as a model system to study presenilin function, we found that loss of C. elegans presenilin orthologue SEL-12 results in elevated mitochondrial and cytosolic calcium levels. Here, we provide evidence that elevated neuronal mitochondrial generated reactive oxygen species (ROS) and subsequent neurodegeneration in sel-12 mutants are a consequence of the increase of mitochondrial calcium levels and not cytosolic calcium levels. We also identify mTORC1 signaling as a critical factor in sustaining high ROS in sel-12 mutants in part through its repression of the ROS scavenging system SKN-1/Nrf. Our study reveals that SEL-12/presenilin loss disrupts neuronal ROS homeostasis by increasing mitochondrial ROS generation and elevating mTORC1 signaling, which exacerbates this imbalance by suppressing SKN-1/Nrf antioxidant activity.Mitochondrial dysfunction and oxidative stress are major contributors to the pathophysiology of neurodegenerative diseases, including Alzheimer’s disease (AD). However, the mechanisms driving mitochondrial dysfunction and oxidative stress are unclear. Familial AD (fAD) is an early onset form of AD caused primarily by mutations in the presenilin-encoding genes. Previously, using  Oxidative stress has emerged as a key driver of many neurodegenerative disorders, including Parkinson’s disease, amyotrophic lateral sclerosis, multiple sclerosis, and Alzheimer’s disease (AD) . The agiPSEN1 and PSEN2. The presenilins are conserved transmembrane proteins located primarily on the endoplasmic reticulum (ER) and function as the catalytic subunit of the gamma-secretase complex, which cleaves single pass transmembrane proteins , dvIs19 [(pAF15)gst-4p::GFP::NLS] III, goeIs22 [mec-4p::SL1::GCaMP3.35::SL2::mKate2::unc-54 3’UTR + unc-119(+)], jsIs609 [mec-4p::MLS::GFP], uthIs248 [aak-2p::aak-2(genomic aa1-321)::GFP::unc-54 3′UTR + myo-2p::tdTOMATO], zcIs4[hsp-4p::GFP], zcIs9[hsp-60p::GFP], zcIs13[hsp-6p::GFP], zdIs5 [mec-4p::GFP + lin-15(+)] I, takEx641[mec-7p::mito-GCaMP6f::SL2::mCherry], and zhsEx17 [mec-4p::MLS::ROGFP]. Genotypes were determined by PCR and DNA sequencing. sel-12(ty11) mutants were used as the canonical sel-12 loss of function mutant unless otherwise indicated.The following strains were used in the study: N2 was the wild type, skn-1 or sca-1 double stranded RNAi acquired from the Ahringer library ) were immobilized in 100 mM levamisole and mounted on 2% agarose pads on glass slides, then imaged using a 63× oil objective on a Zeiss Axio Observer microscope equipped with an Andor Clara CCD camera. Metamorph software was used to compile images. Samples were sequentially excited with a 405 nm light and 488 nm light with GFP emission detection. Exposure times were kept consistent between samples. The ratio of 405 nm to 488 nm fluorescence intensity was quantified using Fiji software. gst-4p::GFP (dvIs19) transgenic animals were used as a transcriptional reporter of the SKN-1 target GST-4. hsp-6p::GFP (zcIs13) and hsp-60p::GFP (zcIs9) transgenic animals were used as reporters for the UPRmt, and hsp-4p::GFP (zcIs4) animals were used as a reporter for UPRER. For all animals, day 1 adults were imaged using a 10x objective lens on a Zeiss Axio Observer microscope. Exposure times were kept consistent between samples.Animals expressing redox sensor roGFP1 targeted to the TRNs . The fluorescence intensity of mito-GCaMP6 or GCaMP3.35 was normalized to mCherry or mKate2 fluorescent intensity, respectively, as an expression control, and this ratio was quantified using ImageJ .Mitochondrial calcium was measured in the TRNs in animals expressing Day 1 adult worms were washed twice in PBS. Half the worm pellet was resuspended in RIPA buffers with protease inhibitors , lysed via sonication, and then used to determine protein concentration with a BCA assay . The other half of the sample was lysed via sonication in 2x Laemmli sample buffer  containing 5% beta-mercaptoethanol. From this lysate, 20 ug of each sample was loaded and separated with a 10% tris-glycine gel (BioRad). The separated proteins were transferred to a 0.2 μm nitrocellulose membrane (Invitrogen), then incubated in primary antibodies (phospho-Drosophila p70 S6 Kinase (Thr398), 1:500, Cell Signaling #9209, and beta-actin, 1:1000, MP Biomedicals #8691002) in TBS overnight. The membrane was incubated for 1 h in secondary antibodies (IRDye 800CW Goat anti-rabbit (LI-COR), 1:20,000 and IRDye 680RD Goat anti-mouse, 1:20,000 (LI-COR)). LiCor Odyssey CLx infrared imaging system was used to image the blot and the Odyssey Image Studio software  was used to quantify band intensity.p value of less than 0.05 is considered significant. Graph Pad Prism software  was used for all analyses.Statistical difference comparing three or more treatment groups was determine using a one-way analysis of variance and a Kruskal–Wallis test used for multiple comparisons of nonparametric data. Non-parametric tests were utilized because all analyses had at least one sample that did not show a normal distribution. Analysis of paraquat data was conducted using a two-way analysis of variance and a Bonferroni correction post hoc analysis. For the mitochondrial and ALM neuronal morphology analyses, a chi-square test was used to determine statistical difference between genotypes. A C. elegans, sel-12, also increase cytosolic calcium signaling . Both the egl-19(n2368gf) and unc-2(zf35gf) mutations have been shown to increase the activation state of the EGL-19 VGCC and UNC-2 VGCC, respectively [egl-19(n2368gf) gain-of-function mutation in egl-19 increasing the activity of the EGL-19 VGCC, we found a ~4-fold increase in cytosolic calcium in egl-19(n2368gf) mutants compared to wild type animals (unc-2(zf35gf) mutants  and unc-2(zf35gf) mutants. Using a mitochondrial calcium biosensor (mito-GCaMP6f::SL2::mCherry) expressed in the TRNs [sel-12 mutants, both egl-19(n2368gf) and unc-2(zf35gf) mutants were indistinguishable from wild type animals  or unc-2(zf35gf) mutants (sel-12 mutants it appears discontinuous disorganized and disorganized (egl-19(n2368gf) or unc-2(zf35gf) mutants did not show defects in mitochondrial morphology  mutants disrupts redox homeostasis. To accomplish this, we analyzed animals expressing roGFP1, a redox sensitive GFP, targeted to the mitochondria in TRNs [C. elegans [sel-12 null mutants had a significant increase in neuronal oxidation, which is rescued by the application of mitoTEMPO, a mitochondrial targeted superoxide scavenger (egl-19(n2368gf) mutants had neuronal oxidation levels indistinguishable from wild type animals (sel-12 mutants and not in egl-19(n2368gf) mutants is the cause of the elevated oxidative stress and neuronal degeneration observed in sel-12 mutants. To test this notion, we introduced a null mutation in the mitochondrial calcium uniporter, encoded by mcu-1, which we and others have shown reduces mitochondrial calcium uptake in wild type and sel-12 mutants  that is required for aspartyl protease activity [sel-12 null mutants, we did not observe an increase in neuronal mitochondrial oxidation in the sel-12 mutants carrying the D226A mutation  [mt is a phylogenetically conserved adaptive response that functions to maintain mitochondrial proteostasis during mitochondrial dysfunction. Since sel-12 mutants show disrupted mitochondrial redox homeostasis, we investigated whether sel-12 mutants have activated UPRmt. To accomplish this, we utilized two UPRmt reporters, hsp-6p::GFP and hsp-60p::GFP [mt reporters demonstrated robust activity  . The UPR60p::GFP ,40. As p60p::GFP ,42, bothactivity A–C. Surp animals A-C. ThesER) in sel-12 mutants. Similar to UPRmt, UPRER is a phylogenetically conserved adaptive pathway that maintains ER proteostasis during ER dysfunction. Using the UPRER reporter, hsp-4p::GFP [ER reporter in sel-12 mutants compared to wild type animals (sca-1(RNAi), which knocks down the expression of the sarco-endoplasmic reticulum calcium ATPase and induces a robust UPRER response  pathway [sel-12 mutants, including impairments to neuronal and mitochondrial morphology, protein homeostasis, neurodegeneration, and behavior, yet did not reduce mitochondrial calcium levels [sel-12 mutants [sel-12 background by crossing in a null mutation in raga-1, which encodes RagA, a GTPase whose activation is required for full mTORC1 activity. We also crossed in a null mutation of rsks-1, which encodes ribosomal protein S6 kinase and as an effector of the mTORC1 pathway promotes protein translation. When we compared the oxidation state of roGFP1 in the TRNs between sel-12 and raga-1; sel-12 mutants, we found that raga-1; sel-12 animals showed a significant reduction in oxidized roGFP1, indicating that mTORC1 inhibition reduces neuronal oxidative stress , a sensor of cellular energy and a major negative regulator of mTORC1 signaling [aak-2(ca); sel-12 mutants had a significant increase in survival rate compared to sel-12 mutants when exposed to paraquat ; sel-12 animals. To this end, we inhibited SKN-1 activity using RNA interference (RNAi) and found that skn-1 RNAi prevented the increased survival rate in raga-1; sel-12 and aak-2(ca); sel-12 animals following paraquat exposure  [sel-12 mutants  . Surpris mutants A–C, gst- animals D,E, indi mutants A and impsel-12 animals, we asked if sel-12 mutants can activate SKN-1 in response to mitochondrial oxidative stress. To induce mitochondrial oxidative stress, we treated sel-12 mutants with paraquat to induce mitochondrial superoxide production and examined gst-4p::GFP expression as a readout for SKN-1 activity. While treatment of wild type animals with 0.01 mM paraquat was able to activate the gst-4p::GFP SKN-1 reporter, this concentration only showed baseline gst-4p::GFP reporter activity in sel-12 mutants  into the sel-12 mutant background [sel-12 null mutants show defects in soft touch response at day 1 of adulthood ([skn-1(lax120); sel-12, or skn-1(lax188); sel-12 animals showed a significant increase in soft touch response compared to sel-12 mutants alone, indicating that increasing skn-1 activity improves the health of the TRNs in sel-12 mutants (skn-1(lax120); sel-12 and skn-1(lax188); sel-12 animals showed an increased survival rate following paraquat exposure , we found that inhibition of protein translation via a SK6/rsks-1 loss-of-function mutation was not sufficient to reduce ROS levels in sel-12 mutants . Initial presenilin data provided support for the amyloid cascade hypothesis, which postulates that AD is caused by the pathological build-up of amyloid beta peptides forming plaques in the brain. However, efforts to treat AD by reducing amyloid plaque load have been clinical failures, with immunotherapies targeting amyloid beta showing no effect on disease progression ,80. Rece mutants ,37. It i elegans ,84, furtegl-19 and unc-2. However, unlike egl-19(gf) mutants, we did not observe elevated cytosolic calcium in unc-2(gf) mutants, despite a previous report showing that this mutation increases calcium currents and causes hyperactive animal behavior [unc-2 encodes a subunit of the CaV2.1 VGCC, which acts at the presynaptic terminals to promote synaptic transmission [sel-12 mutants. In agreement with this notion, it was previously shown that reducing ER calcium release in sel-12 mutants was able to restore normal mitochondrial and neuronal function in sel-12 mutants [We determined that elevated cytosolic calcium is not sufficient to promote ROS levels and neurodegeneration. We manipulated cytosolic calcium levels through gain-of-function mutations in two voltage-gated calcium channels: behavior . Additiosmission . It migh mutants ,14.sel-12 mutants is dependent on ER calcium release [In congruence, using multiple model systems, the loss of presenilin function has been found to increase ER calcium release into the cytoplasm ,10,11,12 release . However release . fAD mut release ,87. HoweC. elegans indicates that small increases in superoxide generation may promote longevity by activating the mitochondrial stress response, which upregulates the SKN-1-mediated stress response [sel-12 mutants have reduced, not increased longevity [sel-12 mutants triggers any mitochondrial stress response (There is an interesting and complex relationship between ROS and longevity. Evidence in response ,89,90. Hongevity . Notablyresponse A–C, nor response D,E. Thissel-12 mutants [Altogether, our study highlights the critical role of mitochondrial calcium in disrupting redox homeostasis and promoting neurodegeneration in animals lacking SEL-12/presenilin function. Additionally, we identified mTORC1 signaling as a mediator of increased mitochondrial ROS, and discovered that its effects on ROS levels are, in part, due to the inhibition of SKN-1/Nrf function. However, the elevation of mTORC1 signaling alone is not sufficient to cause neurodegeneration . It is i mutants , suggest


Correction: BMC Pharmacol Toxicol 23, 81 (2022)https://doi.org/10.1186/s40360-022-00607-0During the publication process of the original article  there waFigure 1 Incorrect figure 3 as originally publishedFigure 2 correct version of figure 3


Caenorhabditis elegans to investigate whether MA-5 improves the Duchenne muscular dystrophy (DMD) model. Firstly, we confirmed the efficient penetration of MA-5 in the mitochondria of C. elegans. MA-5 also alleviated symptoms such as movement decline, muscular tone, mitochondrial fragmentation and Ca2+ accumulation of the DMD model. To assess the effect of MA-5 on mitochondria perturbation, we employed a low concentration of rotenone with or without MA-5. MA-5 significantly suppressed rotenone-induced mitochondria reactive oxygen species (ROS) increase, mitochondrial network fragmentation and nuclear destruction in body wall muscles as well as endogenous ATP levels decline. In addition, MA-5 suppressed rotenone-induced degeneration of dopaminergic cephalic (CEP) neurons seen in the Parkinson’s disease (PD) model. Furthermore, the application of MA-5 reduced mitochondrial swelling due to the immt-1 null mutation. These results indicate that MA-5 has broad mitochondrial homing and MINOS stabilizing activity in metazoans and may be a therapeutic agent for these by ameliorating mitochondrial dysfunction in DMD and PD.Mitochonic Acid 5 (MA-5) enhances mitochondrial ATP production, restores fibroblasts from mitochondrial disease patients and extends the lifespan of the disease model “Mitomouse”. Additionally, MA-5 interacts with mitofilin and modulates the mitochondrial inner membrane organizing system (MINOS) in mammalian cultured cells. Here, we used the nematode  We have developed a mitochondrial homing drug, Mitochonic Acid 5 -2-1H-indol-3-yl)-4-oxobutanoic acid), which was synthesized as a derivative of the plant hormone, indole-3-acetic acid (IAA) -yl-4-oxo,7.The most severe and common muscular dystrophy, Duchenne muscular dystrophy (DMD), is a serious progressive muscle disease caused by mutations in the DMD (dystrophin-encoding) gene. In the meantime, Parkinson’s disease (PD) is the second most prevalent neurodegenerative disease in the world. There is increasing evidence that mitochondrial dysfunction leads to progressive deterioration in patients with DMD or PD ,9. The uCaenorhabditis elegans (C. elegans) provides the advantage of conducting aging and disease model studies due to the homology it shares with human genome cyto was around 10 s for each cycle  mutants, a severely fragmented mitochondrial network shown in the BWM cells of young adults, was improved by prednisone and NaGYY treatments, and restored to motility [2+ in the mitochondria ([Ca2+]mito), due to the dys-1 defect, also improved with MA-5 treatment  were treated with a low concentration of 2 μM rotenone, an inhibitor of ETC complex I. The fluorescent signals of the mitochondria-specific ROS generation with MitoTracker Red CMXRos significantly increased after exposure to rotenone for 6 h . In contccIs4251 transgene, mtGFP and nuclear targeted GFP (nucGFP), with 2 μM rotenone from synchronous D1 adulthood for 48 h significantly suppressed body growth associated with increased muscle mass  mito is associated with the excessive opening of the mitochondrial permeability transition pore (mPTP), loss of mitochondrial membrane potential and mitochondrial depolarization [2+]cyto is observed in the C. elegans. dys-1 mutants, which in turn activates various matrix metalloproteinase-mediated extracellular matrix degradation [C. elegans. tissues  [2+]cyto accumulates longer in the dys-1 (eg33) mutants immobilized with microspheres    supplementation (GY4137 and AP39) acts similar to prednisone and improves neuromuscular health using the C. elegans DMD model. They also find a decline in total sulfide and H2S-producing enzymes in dystrophin/utrophin knockout mice, suggesting the deficit with H2S may contribute to DMD pathology. On the other hand, the loss of Ca2+ homeostasis in C. elegans DMD model does not appear to be corrected by either prednisone or H2S supplementation cyto levels in vitro and ex vivo, reversed the mitochondrial swelling, increased OCR and reduced ROS production in isolated mitochondria of mdx mice. Taking together, controlling Ca2+ homeostasis in muscular mitochondria and cytoplasm by MA-5 and CND1163 is effective in treating and alleviating muscular dystrophy.Intriguingly, we found that the mitochondrial homing drug MA-5 significantly improved elevated  V, ATU2301: goeIs3 [myo-3p::SL1::GCamP3.35::SL2::unc54 3’UTR + unc-119(+)] V, acels1[myo-3p::mitochondrial LAR-GECO+myo-2p::RFP] II, ATU2305: dys-1(eg33) goeIs3 , aceIs1[Pmyo3::mitochondrial LAR-GECO + Pmyo2::RFP] [ccIs4251 [(pSAK2) myo-3p::GFP::LacZ::NLS + (pSAK4)myo-3p::mitochondrialGFP+dpy-20(+)] I, acels1 II, ATU3305::dys-1(eg33) ccIs4251 [Pmyo3::nucGFP-LacZ + Pmyo-3::mitochondrial GFP], aceIs1 [Pmyo-3::mitochondrial LAR-GECO+ Pmyo2::RFP], SD1347: ccIs4251 [(pSAK2) myo-3p::GFP::LacZ::NLS+ (pSAK4) myo-3p::mitochondrial GFP + dpy-20(+)] I and ATU3307: ccIs4251 I, acels1 II, immt-1 (tm1730) X.The standard procedures for at 20 °C . MA-5 , which stains mitochondrial with blue fluorescence at final concentration 25 μM was performed with 1 μM BODIPY-MA-5 mito) was calculated using the following equation [2+ and the LAR-GECO probe [ccIs4251 transgene in a constant area.Mitochondrial Caur group . The Ca2equation .[Ca2+]mCO probe  and R indat-1p::GFP (TG2435) were used in this experiment. Approximately 12 worms were analyzed for each condition. Images were obtained using confocal microscopy and ImageJ software was used to calculate the number of beads in all four cephalic (CEP) neurons [Age-synchronized two-day-old adult worms with  neurons .® Red CMXRos  and 25 μM AIE™ Mitochondria Blue  for 3 h. Nematodes were then transferred to fresh NGM agar plates and incubated overnight. Images were obtained using confocal microscopy and fluorescence intensity was analyzed using FV10-ASW Viewer software .Wild-type (N2) one-day-old adult nematodes were incubated with rotenone for 6 h in the presence or absence of MA-5. Subsequently, worms were treated with 0.5 µM MitoTrackerWild-type (N2) worms on two-day-old adults were collected in 100 µM M9 buffer for further ATP assays. An ATP determination kit  was used to measure endogenous ATP levels, as previously reported for rotenone treatment .immt-1 mutant (ATU3307) on a four-day-old adult was used in this experiment. Approximately 50 muscle cell images were taken from 10 worms for each treatment. Images of mitochondrial morphology were observed using confocal microscopy and analyzed using ImageJ software.The synchronized p value of < 0.05 was considered significant. Different letters indicate significant differences between the groups.The one-way ANOVA with post hoc Tukey’s HSD and Dunn’s test were used for comparisons between groups as appropriate (R or Origin software). All data points including outliers were used for means and statistical significance. A


Potential signaling networks in different cell types induced by apigenin were identified, suggesting that the molecular mechanisms of apigenin in cardiometabolic diseases vary with cell types. Additionally, the mechanisms of apigenin-induced biological response in different cardiometabolic diseases were analyzed, including obesity, diabetes, hypertension and cardiovascular diseases. This review provides novel insights into the potential role of apigenin in cardiometabolic diseases.Apigenin is a flavonoid with antioxidant, anti-inflammatory, and anti-apoptotic activity. In this study, the potential effects of apigenin on cardiometabolic diseases were investigated  Apium belonging to family Apiaceae  is named after the genus Apiaceae . It is wApiaceae , and is Apiaceae   that are predictive of cardiovascular disease and Type 2 diabetes (T2DM) . RecentlObesity is attributed to chronic energy imbalance, including excessive energy intake and limited energy expenditure , 12. AntIn vitro and in vivo studies confirmed that apigenin upregulates the expression of anorexigenic neuropeptides pro-opiomelanocortin (POMC) and cocaine- and amphetamine-related transcript (CART), resulting in inhibition of food intake. N-29-2 and SH-SY5Y cells transfected with pPOMC-Luc and pCART-Luc vectors were treated with 0.2–5 μM apigenin for 6 h, resulting in upregulation of pPOMC-Luc and pCART-Luc activity. In an in vivo study, 6-week-old C57BL/6J mice were injected (every 24 h) intraperitoneally with 1 or 10 mg/kg of apigenin in a short-term intervention. Male C57BL/6J mice fed with a high-fat diet (HFD) or a standard laboratory chow diet received 0.05% apigenin for 30 days to demonstrate that apigenin reduces food intake and visceral fat over a long-term period -CD36-PPARγ (peroxisome proliferator-activated receptor-gamma) axis  to downregulate adipogenesis via AMPK/PPARγ axis in 3T3-L1 cells treated with 10 μM apigenin for 1 h or 4 days ((1) ma) axis  and AMPKma) axis . One stufat mass . Apigenifat mass , followefat mass . PPARγ ir 4 days  and in Hr 4 days . AMPK acr 4 days , 22 and r 4 days .Repression of enzyme activity. Guo et al. reported that 0.6 mM apigenin directly inhibits pancreatic lipase activity in vitro ((2) in vitro . Pancreain vitro .Activation of lipolysis-related genes. Apigenin regulates lipolysis via activation of lipolysis-related genes. In a recent study, 3-week-old HFD mice  treated with 0.04% apigenin for 12 weeks showed upregulation of lipolysis-related genes in white adipose tissues (WAT), such as FOXO1 (Forkhead Box O1) and SIRT1 (Sirtuin 1) ((3) rtuin 1) .Induction of fatty acid oxidation. Dietary apigenin induces phosphorylation of AMPK and 1-aminocyclopropane-1-carboxylic acid (ACC) in brown adipose tissues (BAT) to utilize free fatty acids synthesized from white adipose tissues (WAT) ((4) es (WAT) .via following mechanisms:Liver, in addition to adipose tissues, is essential for lipid metabolism. Abnormal lipid metabolism in the liver induced by obesity may cause hepatic steatosis. Apigenin also improves lipid metabolism in the liver to alleviate hepatic steatosis Stimulation of PPARγ signaling. Apigenin modulates PPARγ expression in hepatic lipid metabolism via Nrf2-PPARγ axis. In Hep1-6 cells, apigenin activates nuclear factor erythroid 2-related factor 2 (Nrf2) via translocation into the nucleus to upregulate downstream antioxidant enzymes and downregulate lipid synthesis ((1) ynthesis . Activatynthesis .Regulation of SREBP family. Apigenin treatment inhibits lipid homeostasis by the sterol regulatory element-binding protein (SREBP) family. Apigenin significantly decrease lipid accumulation, total intracellular cholesterol (TC), and intracellular triglyceride (TG) levels via the AMPK-SREBP-1/2 (sterol regulatory element-binding protein-1/2) axis in HepG2 cells. Apigenin-induced activation of AMPK downregulates the levels of SREBP-1 and SREBP-2 to reduce the synthesis of cholesterol, fatty acids, and triglycerides in the liver. The inhibition of 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR), which is the downstream target gene of SREBP-1 and FAS, the downstream target gene of SREBP-2 also regulates fatty acid and cholesterol synthesis ((2) ynthesis .Activation of genes related to fatty acid oxidation and cholesterol homeostasis. Other genes related to fatty acid oxidation and cholesterol homeostasis in the liver, such as short/branched-chain acyl-CoA dehydrogenase (ASADSB), enoyl-CoA-hydratase and 3-hydroxyacyl-CoA dehydrogenase (EHHADH), Niemann-Pick type C 2 (NPC2) ((3) 2 (NPC2) , HMG-CoA2 (NPC2)  have bee2 (NPC2) .via p65/NF-κB pathway. The inflammatory environment suppresses adipocyte browning to reduce lipid metabolism (via activation of uncoupling protein 1 and PGE2 receptor 4 (EP4) activates the cyclooxygenase 2 (COX2)/prostaglandin E2 (PGE2) axis, resulting in conversion of white to brown adipose tissue to generate heat by excessive energy expenditure   . Nlrp6 idiseases , by promus level . Obesityus level . Obesityus level . Apigenius level , 49.via a variety of mechanisms including inhibition of appetite, glucose signaling pathways and lipid metabolism. It also regulates the intestinal microbiome, enhances mitochondrial function and attenuates inflammation and oxidative stress. The aforementioned experimental approaches and mechanisms underlying the effects of apigenin on obesity are listed in in vivo and in vitro are also listed in In summary, apigenin alleviates obesity and its complications Diabetes also plays an important role in cardiometabolic disease –52. Sevevia following mechanisms:Insulin resistance plays a significant role in the pathophysiology of T2DM . InsulinInhibition of insulin receptor kinase. Apigenin inhibits tyrosine nitration of the insulin receptor kinase domain leading to alleviation of insulin resistance. Tyrosine nitration of IRβ (intracellular β subunits of the insulin receptor) may lead to decreased tyrosine phosphorylation, resulting in impaired insulin signal transduction in HFD mice  HFD mice . In vitrtyrosine .Regulation of miRNAs. Apigenin regulates miRNAs, which are associated with insulin resistance and glucose homeostasis. In vitro experiments involving Huh7 cells and in vivo studies investigating miR103 transgenic mice validates apigenin-mediated inhibition of the phosphorylation of transactivating response RNA-binding proteins (TRBP). Additionally, miRNA-generating complexes inhibited, leading to suppression of precursor miRNA103 maturation expressed in liver and fat, resulting in insulin resistance and impaired glucose metabolism and homeostasis ((2) eostasis , 60. ThuUpregulation of GLUT4/AMPK signaling. Apigenin extracted from Sophora davidii (Franch.) promotes glucose transporter 4 (GLUT4) expression and activates AMPK phosphorylation in L6 cells and insulin target tissues in KK-Ay mice ((3) -Ay mice . In insu-Ay mice , 63.Inhibition ofα-amylase. Several studies reported that apigenin decreases the inhibition of α-amylase in Kunming mice, thus reducing the digestion of dietary carbohydrates ((4) hydrates . The dighydrates . Inhibithydrates .via following mechanisms:Oxidative stress also triggers β-cell dysfunction, impaired glucose tolerance, and insulin resistance . The proDecreased ROS production. Apigenin pre-treatment of streptozocin (STZ)-treated RINm5F pancreatic β cells ameliorates STZ-induced intracellular ROS production, as well as DNA damage, lipid peroxidation, and apoptosis. Apigenin pre-treatment upregulates the expression of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) in RINm5F pancreatic β cells and diabetic rats (2–) to molecular oxygen (O2) and hydrogen peroxide (H2O2), resulting in a protective effect against ROS in cells ((1) tic rats . SOD catin cells . Catalasin cells . GSH-Px in cells . These tin cells .Inhibition of AGE. Apigenin inhibits the formation of advanced glycation end products (AGEs) and thereby alleviates oxidative stress (via cross-linking between molecules via the receptor for AGEs (RAGE). AGEs increase ROS formation and damage anti-oxidant systems (in vitro reduced the levels of AGEs ((2) e stress . AGE-med systems . Apigeni of AGEs .Regulation of Keap1-Nrf2 signaling. The anti-oxidant function of apigenin is mediated via the Kelch-like ECH-associated protein 1 (Keap1)-Nrf2 axis targeting liver tissues to alleviate oxidative stress ((3) e stress . Nrf2 ise stress , acting e stress , 76. Apie stress .In addition, persistent inflammation leads to pathogenesis of diabetes . Apigeniin vivo and in vitro are also listed in Currently, the apigenin-mediated regulation of blood glucose homeostasis can be summarized as follows: regulating the key enzymes and improving oxidative stress as well as inflammation. A detailed summary of the studies discussed above and the proposed mechanisms of apigenin-mediated effects in diabetes are presented in via attenuation of oxidative stress and recovery of mitochondrial dysfunction.Hypertension plays a central role in cardiometabolic diseases , which iApigenin plays a protective role in hypertension by alleviating oxidative stress. Apigenin can significantly restore normal blood pressure and reverse renal damage in cyclosporine-induced hypertensive Sprague-Dawley rats by decreasing lipid hydroperoxides and increasing anti-oxidant levels . Apigenivia hypoxia-inducible factor 1α (HIF-1α)-KV1.5 channel pathway. The inhibition of HIF-1α by apigenin upregulates the expression of KV1.5 channels to restore mitochondrial function, thereby attenuating PH . Mitochondrial dysfunction plays a vital role in PH, it may lead to the imbalance of ion homeostasis and downregulation of enzymes in apoptosis . Apigeniating PH .2+ influx is then promoted in vascular endothelium and smooth muscle to induce vasodilation . This inhibition stimulates cellular transformation into fibroblasts and increases the synthesis of matrix proteins to induce renal fibrosis  phosphorylation. PKCβII promotes oxidative stress, ROS production and mitochondria-dependent apoptosis in vascular endothelial dysfunction , glucose transporter 4 (GLUT-4), carnitine palmitoyltransferase 1 (CPT-1) and pyruvate dehydrogenase kinase 4 (PDK-4). Downregulation of GLUT4 and upregulation of PDK-4 can inhibit excessive glucose intake and oxidation, preventing abnormal glucose metabolism. Downregulation of GPAT and upregulation of CPT-1 decreases the rate of triglyceride synthesis and augments fatty acid oxidation, thereby improving lipid metabolism  is derived from celery and exhibits anti-adipogenic and anti-obesity effects in HFD mice via the AMPK/PPARγ axis  and G6Pase (glucose-6-phosphatase) in the liver, and promotion of glycogen synthesis via inhibition of glycogen synthase phosphorylation and induction of GSK3β (glycogen synthase kinase3β) phosphorylation ((1) ARγ axis , similarrylation .Apigetrin. Apigetrin (apigenin 7-glucoside) ameliorated pancreatic β cell damage via reduction of endoplasmic reticulum (ER) stress in RINm5F cells via the regulation of ER stress biomarkers, such as upregulation of CCAAT/enhancer-binding protein homologous protein (C/EBP), induction of spliced X-box binding protein 1 (XBP1), phosphorylation of protein kinase RNA-like ER kinase (PERK) and eukaryotic initiation factor 2α , and cleavage of caspase-12 ((2) spase-12 .Vitexin. Vitexin (apigenin 8-C-glucoside) regulates lipid metabolism via AMPK-mediated pathway in 3T3-L1 cells  ((3) L1 cells  in vitroHFD mice  in vivo  (HMGB1) , which iAcacetin. Acacetin  suppress adipogenesis in 3T3-L1 cells and HFD mice via upregulation of SIRT1 expression and AMPK phosphorylation  rylation . AcacetiG2 cells . In addi pathway .Apigenin 7, 4′-dimethyl ether. Apigenin 7, 4′-dimethyl ether (ADE) enhances glucose uptake in L6 cells and inhibits α-glucosidase enzyme (via hydrolyzation of α-glycosidic bonds from the non-reducing ends of oligosaccharide substrates and transfer of free glucose residues to another carbohydrate substrate.(5) e enzyme , which r(6) 8-(6″-umbelliferyl)-apigenin. 8-(6″-umbelliferyl)-apigenin promotes glucose uptake in 3T3-L1 cells, indicating improved glucose consumption .In summary, the potential signaling pathways mediated by apigenin resulting in alleviation of cardiometabolic diseases in different cell types are illustrated in In adipocytes, apigenin acts as a functional regulator of lipid metabolism to reduce fat accumulation. Apigenin downregulates PPARγ expression by inhibiting the STAT3/CD36 axis  and the via direct activation of Nrf2 and indirect activation of Nrf2 via the Keap1-Nrf2 pathway  was 3.9 ng h/mL in mice  also indicate that apigenin is one of the protective factors in cardiovascular diseases at the population level (In summary, the extensive review and validation of on level . FurtherXL designed this review, helped with writing and revising of the manuscript, and provided critical feedback. YX contributed to collecting and screening the literature, as well as summarizing the data, and then composed and revised the manuscript. HW reviewed the manuscript. All authors were involved in final approval of the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Dear Editor,1. In agreement, evidence is emerging that Omicron is inherently less pathogenic than previous SARS-CoV-2 variants. Omicron variant viruses cause less severe disease in animal studies2 and appear to display a lower capacity than other variants to replicate in the lower respiratory tract2. Additionally, initial clinical data indicated that the Omicron variant causes less severe disease than previous SARS-CoV-2 variants in unvaccinated individuals1.The SARS-CoV-2 Omicron variant (B.1.1.529) causes less severe disease than previous SARS-CoV-2 variants, although immune protection provided by vaccinations and previous infections is reduced against Omicron compared to previous variants3, which provides a mechanistic explanation for the reduced clinical severity of Omicron disease in individuals without pre-existing adaptive immunity1. Omicron virus replication was attenuated relative to Delta virus in interferon-competent Caco-2 and Calu-3 cells, but not in interferon-deficient Vero cells, and Omicron viruses caused enhanced interferon promoter activity compared to Delta viruses3. Additionally, depletion of the pattern recognition receptor MDA5, which plays a critical role in SARS-CoV-2 detection and interferon response initiation4, resulted in increased Omicron virus replication in interferon-competent cells3.We have most recently shown that Omicron variant viruses are less effective at antagonizing the host cell interferon response than Delta variant viruses5  replicate to lower titers Fig.  and indu3 see .Fig. 1IF6. However, when we compared interferon activity in the supernatants of SARS-CoV-2-infected cells in a HEK-Blue IFNα/β reporter cell assay, the supernatants of Omicron virus-infected RIG-I-knock out cells induced higher interferon promoter activation than the supernatants of Omicron virus-infected MDA5-knock out cells  and TMPRSS2 , the Omicron viruses also displayed alleviated infection capacity compared to the Delta virus Fig. . This dills Fig. . This is3 and that MDA5 is a major player in SARS-CoV-2 recognition6. Accordingly, elevated MDA5 levels were detected in the upper airways of SARS-CoV-2-infected individuals with mild or asymptomatic disease8. Since Delta has been found to display a similar level of interferon antagonism and sensitivity as previous SARS-CoV-2 variants10, the reduced interferon antagonism appears to be unique to Omicron.Taken together, these findings further confirm that Omicron viruses are less effective than Delta viruses in antagonizing the host cell interferon response11 indicated moderate synergism of interferon-β with remdesivir, synergism with nirmatrelvir, and strong synergism with EIDD-1931 (Fig. 15. Given the newly discovered substantially increased interferon sensitivity of Omicron viruses, interferons represent a promising option for the treatment of Omicron patients.Most notably, treatment with interferon-α, interferon-β, and interferon-γ revealed that the weaker interferon antagonism by Omicron virus isolates translates into a profoundly increased Omicron sensitivity to interferon treatment Fig. . Further931 Fig. . So far,In conclusion, we present further evidence that reduced interferon-antagonizing activity explains at least in part why Omicron variant infections are inherently less severe than infections with other SARS-CoV-2 variants. Sequence variations in the SARS-CoV-2 interferon antagonists nsp3, nsp12, nsp13, nsp14, M protein, the nucleocapsid protein, and/or ORF3a may contribute to these differences. Most importantly, this study shows that Omicron variant viruses display enhanced sensitivity to interferon treatment, which makes interferons promising therapeutic candidates for Omicron patients, in particular in combination with other antiviral agents.Supplementary Information