Human retinal pigment epithelium RPE-1 cells are immortalized diploid wild-type cells. RPE-1 is increasingly used for studies of spindle assembly dynamics and chromosome segregation. Here, we imaged living RPE-1 cells using the spinning disk confocal microscope and report their complete spindle assembly dynamic parameters. Live-cell experiments enabled ascribing precise timing of function of the kinesin-5 Eg5 and kinesin-14 HSET throughout different phases of mitosis. Eg5 functions at prophase and metaphase, to assemble and maintain spindle bipolarity, respectively. Eg5 inhibition results in spindle collapse during prophase and metaphase, resulting in monoastral/monopolar spindles. HSET functions throughout mitosis to maintain spindle length. HSET degradation results in shorter spindles through all phases of mitosis. Double-inhibition of Eg5 and HSET produces only monoastral/monopolar spindles, indicating that Eg5 and HSET may not be antagonistic in wild-type RPE-1 cells, contrary to previous studies using cancer cells. In the context of spindle assembly, our results highlight potential important differences between RPE-1 and other cancer-derived cell lines. Jiang et al., 1999). These cells are considered normal diploid cells, as they have the proper number of chromosomes and do not exhibit transformed phenotypes. As this cell line has gained prominence as a model system for research on spindle assembly and chromosome segregation, we sought to define its complete spindle assembly parameters throughout mitosis, as well as test the function of kinesin-5 and kinesin-14, two important motors involved in spindle assembly previously reported in cancer cells. Using CRISPR/Cas9 gene-editing, we created an RPE-1 cell line expressing at its genomic loci both alleles of auxin-inducible degron-degradable kinesin-14 eYFP-AID-HSET. We then performed confocal live-cell imaging to visualize HSET, as well as using the vital dye SiR-Tubulin and Hoechst to visualize the microtubules and DNA, respectively. Live-cell imaging enabled tracking mitosis from start to finish, with clear visualization of prophase, metaphase, anaphase and telophase spindles . The movies enabled the measurement of spindle length over time, where we observed three distinct spindle elongation phases, correlating with phase I/prophase, phase II/metaphase, and phase III/anaphase . The complete spindle elongation parameters for RPE-1 cells are reported .Human hTERT-RPE-1 cells were immortalized, via telomerase expression, from normal somatic retinal pigment epithelium cells , which inhibits motor binding to microtubules , creates monoastral/monopolar spindles and subsequent cell death . Live-cell imaging enabled precisely-timed introduction of STLC to cells. We observed that STLC-inhibition of Eg5 prevented spindle elongation at prophase, producing monoastral/monpolar spindles , indicating that Eg5 is required for bipolar spindle assembly, and consistent with known function of Eg5 . At metaphase, STLC-inhibition of Eg5 resulted in 78% of spindles collapsing from bipolar back to monoastral/monopolar state , consistent with previous reports , and indicating that Eg5 is required to maintain metaphase spindle bipolarity and length. In contrast, STLC-inhibition of Eg5 did not significantly alter anaphase spindle elongation , consistent with previous report , and indicating that Eg5 is not required for anaphase spindle elongation. Indeed, anaphase spindle elongation velocity showed no significant difference between control 0.76 ± 0.30 µm/min versus STLC (Eg5-) 0.56 ± 0.33 µm/min (p=0.12) .Kinesin-14 HSET is essential for cancer cells with supernumery centrosomes, to cluster their centrosomes and prevent multipolar spindles and cancer cell death . We tested the function of HSET in RPE-1 cells by degrading HSET via auxin addition. In control cells, HSET localized to the nucleus and prominently on the spindle during mitosis . Auxin-induced degron-degradation reduced the signal of HSET on spindles to 7% of control cells, i.e., we achieved 93% HSET degradation . Nevertheless, the overall spindle elongation dynamics did not significantly change compared to control cells . We observed a slight decrease in spindle length throughout mitosis in IAA (HSET-) cells, with a control metaphase length of 15.12 ± 1.51 µm versus IAA (HSET-) 13.47 ± 1.35 µm , indicating HSET helps maintain spindle length throughout mitosis, and consistent with a previous report .Finally, it has been reported that kinesin-5 and kinesin-14 double-inhibition can restore up to 20-40% spindle bipolarity in cancer cells CFPAC1  and HeLa , indicating that HSET can oppose the activity of Eg5. We tested this in RPE-1 cells by simultaneously degrading HSET and inhibiting Eg5. We observed only monoastral/monopolar spindles in STLC (Eg5-) cells (N=83 cells) . Similarly, we observed only monoastral/monopolar spindles in IAA/STLC (HSET-/Eg5-) cells (N=26 cells) . In contrast to previous report for some cancer cells, HSET cannot oppose the activity of Eg5 in non-cancer RPE-1 cells. We note that our experiments utilize HSET-degradation via degron and Eg5-inhibition via STLC drug. We speculate that alternative modes of inhibition such as gene-knockout or RNA-interference may produce different results. In summary, our work defines spindle elongation parameters for the different phases of mitosis in the wild-type human RPE-1 cells, and highlights potential important differences between wild-type and cancer cells in their utilization of kinesin motors for spindle assembly dynamics.Cell cultureBodnar et al., 1998), which has been modified into hTERT-RPE-1-TIR1 to include the F-box gene TIR1 that binds the degron (AID) domain upon auxin (IAA) induction . We introduced an auxin-inducible degron-degradable kinesin-14, eYFP-AID-HSET, into RPE-1-TIR1 at its genomic locus using CRISPR/Cas9 as described . This cell line was used for all experiments. Cell culture condition is well-established . Briefly, cells were maintained in DMEM/F-12 medium (GIBCO Cat# 21041-025) supplemented with 10% fetal calf serum (Eurobio Cat# CVFSVF-00-01), 1% penicillin-streptomycin (GIBCO Cat# 15140-22) in 5% CO2incubator at 37°C. For Eg5 inhibition, STLC was added to cells 10 min prior to imaging. For degron-induced HSET degradation, cells were treated with IAA 16-20 hr prior to imaging.We used human hTERT-RPE-1 (RRID:CVCL_4388) cells (DrugsS-trityl-L-cysteine (STLC) (Sigma Cat# 164739) was used at 10 µM concentration.Indole-3-acetic acid (IAA) (Sigma Cat# I5148) was used at 500 µM concentration.Live-cell imagingApproximately 16-20 hr prior to imaging, 10 µM Verapamil + 5nM SiR-tubulin (Spirochrome Cat# CY-SC002), and 1 hr prior to imaging, 10 µM Hoechst 33342 (Invitrogen Cat# H3570) dyes were added to the Fluorodish (World Precision Instruments Cat# FD35-100) containing 2 mL of RPE-1 cells. Cells were imaged using the spinning disk confocal microscope. Briefly, the Nikon Eclipse Ti-E perfect focus inverted microscope, with 40X/1.3 NA Plan Apo oil immersion objective lens and Mad City Piezo stepper stage, coupled to the Yokogawa CSU-X1 spinning disk confocal unit, the Photometrics Cascade EM-CCD camera, the Gataca Systems laser unit with Cy5/561 nm (100 mW), GFP/491 nm (100 mW) and UV/405 nm (100 mW) lines, controlled by Molecular Devices software MetaMorph 7.8, enclosed within a thermal box to keep stable temperatures of 37°C. Movies were made with the following parameters: laser power 5%, EM-gain 300, Bin 1X, exposure time 100-200 ms, 13 optical z-sections, 2 µm spacing per 3D stack, 3 min time interval between stacks, and 3 hr movies.Plasmid to generate eYFP-AID: AddGene #47329Plasmid to generate eYFP-AID-HSET with Cas9 and single guide RNA background: AddGene #42230Primers to generate eYFP-AID:sense : ATGGTGAGCAAGGGCGAGGantisense : GCTCGAAGCTCTGCTCTTPrimers to generate eYFP-AID-HSET:500 bp before HSET start:sense : CCCTTCTCCTCCGGCCGantisense : GTCCACCTCGGTCCCAC500 bp after HSET start:sense : GATCCGCAGGTGAGTAGGantisense : AGATTACCCTACTCTCCTTCsingle RNA-guide sequence:CTGCATTCCCCCGGCGCGTGTGGCell line used :RPE-1-TIR1 RPE1-1-eYFP-AID-HSET (this study)


Pseudomonas sp. strains MWU12-2029, MWU12-3088, and MWU12-3091 were isolated from wild and cultivated cranberry bog soils in southeastern Massachusetts. The three isolates are closely related to Pseudomonas kribbensis, a not validly published member of the P. fluorescens group, and contain three putative insecticidal protein genes, including the toxin complex A gene (tcaC). Pseudomonas spp. comprise a considerable component of bacteria isolated from cultivated and wild cranberry bogs during a multiyear culture-dependent survey conducted at the Cape Cod National Seashore (MWU12-2029)  and the University of Massachusetts State Bog (MWU12-3088 and MWU12-3091)   isolation. All kits described below were used according to the manufacturers’ instructions. gDNA was extracted with a DNeasy blood and tissue kit , and Kapa Biosystems Hyperplus library preparation kits  were used to generate Illumina-compatible genomic DNA libraries: DNA was enzymatically sheared to ~500 bp, end repaired, and A tailed; Illumina-compatible adapters with unique indexes  were then ligated to each sample; and adapter-ligated molecules were cleaned using Kapa pure beads  and amplified with Kapa Hifi enzyme . Library fragment sizes were determined on an Agilent TapeStation system and quantified by quantitative PCR (qPCR)  on a QuantStudio 5 system . Samples were multiplex pooled and sequenced on an Illumina MiSeq platform in a 2-by-250 flow cell. The software was set to default settings except as indicated below. Raw reads were assembled with Unicycler v0.4.8 (Pseudomonas (Pseudomonas kribbensis 46-2T (GenBank accession number CP029608) but fell below the 70% digital DNA-DNA hybridization (dDDHd4) (TYGS v342) (–shore MWU2-2029 4.66842W) 8. Althou42W 8. Alr v0.4.8 . The comshore MWU-2029 (42r v0.4.8  for the udomonas . All thrGS v342) –18 or 95S v342) –, 20 cutoS v342) –.tcaC)   and two PRJNA691338 with the accession numbers in JALJDY000000000.1 (MWU12-2029), JALJEA000000000.1 (MWU12-3088), and JALJET000000000.1 (MWU12-3091). RASTtk annotations are available under an open license at Zenodo .The whole-genome shotgun sequencing project has been deposited in the DDBJ/EMBL/GenBank database under BioProject accession number


Gallic acid is a phenolic acid present in various plants, nuts, and fruits. It is well known for its anti-oxidative and anti-inflammatory properties. The phenethyl ester of gallic acid (PEGA) was synthesized with the aim of increasing the bioavailability of gallic acid, and thus its pharmacological potential. Here, the effects of PEGA on encephalitogenic cells were examined, and PEGA was found to modulate the inflammatory activities of T cells and macrophages/microglia. Specifically, PEGA reduced the release of interleukin (IL)-17 and interferon (IFN)-γ from T cells, as well as NO, and IL-6 from macrophages/microglia. Importantly, PEGA ameliorated experimental autoimmune encephalomyelitis, an animal model of chronic inflammatory disease of the central nervous system (CNS)—multiple sclerosis. Thus, PEGA is a potent anti-inflammatory compound with a perspective to be further explored in the context of CNS autoimmunity and other chronic inflammatory disorders. Autoimmune reactivity against the central nervous system (CNS) plays a dominant role in the pathogenesis of multiple sclerosis—a chronic inflammatory, demyelinating, and neurodegenerative disease of the CNS . Accordi+ T cells, including Th17 . The absorbance was measured at 540 nm with a correction at 670 nm on an automated microplate reader .Cytokine concentration in cell culture supernatants was determined by sandwich ELISA using MaxiSorp plates (Nunc). For cytokine detection, anti-cytokine paired antibodies were used according to the manufacturer’s instructions . The antibodies were as follows: anti-rat IFN-γ purified mouse monoclonal (DB1), anti-rat IFN-γ biotinylated rabbit polyclonal, anti-mouse/rat IL-17A purified rat monoclonal (eBio17CK15A5), anti-mouse/rat IL-17A biotinylated rat monoclonal (eBio17B7), anti-mouse IL-6 purified mouse monoclonal (MP5-20F3), anti-mouse IL-6 biotinylated mouse monoclonal (MP5-32C11), anti-rat IL-6 purified mouse monoclonal (#53325), anti-rat IL-6 biotinylated goat polyclonal IgG, anti-mouse TNF purified rabbit monoclonal (EPR16803-2), anti-mouse TNF biotinylated rabbit monoclonal (EPR16803-84), anti-rat TNF purified Armenian hamster monoclonal (TN3-19.12), and anti-rat TNF biotinylated rabbit polyclonal IgG. The absorbance was recorded at 450 nM with a correction at 670 nm using a multiplate reader Synergy H1. Samples were analyzed in duplicates and the results were calculated using standard curves based on known concentrations of the recombinant rat IFN-γ and rat IL-17 (Peprotech), mouse IL-6 and rat TNF (Thermo Fisher), rat IL-6 (R&D Systems), and mouse TNF (Abcam). For all ELISA tests, the lower limit of detection was 30 pg/mL, while the upper limit of detection was 10 ng/mL.DAF-FM staining was used for the determination of NO production. The cells were treated with DAF-FM acetate (Thermo Fisher Scientific) in the absence or presence of PEGA for 20 h (DAF-FM). Fluorescence intensity (f.i.) was detected with a multiplate reader Synergy H1, using excitation at 510 nm and emission at 540 nm for DAF-FM.PLNC and/or SCIC were stained with the following antibodies: FITC- or PE-conjugated anti-CD4 , PE-conjugated anti-CD25 , APC-conjugated anti-CD134 , PerCP-Cy5.5-conjugated anti-IL-17 , FITC-conjugated anti-IFN-γ , PerCP-Cy5.5-conjugated anti-Foxp3 , and FITC-conjugated anti-Ki-67 . Intracellular staining for cytokines, FoxP3 and Ki67, was performed according to the procedure suggested by the manufacturer (eBioscience), using Foxp3/transcription factor fixation/permeabilization concentrate and diluent, intracellular fixation & permeabilization buffer set, and permeabilization buffer , as appropriate. Appropriate isotype control antibodies were used as needed to set gates for cell marker positivity. Typically, the proportion of isotype control antibody-stained cells was < 1%. FITC-conjugated annexin V was used for the detection of apoptotic cells in accordance with the manufacturer’s instructions (eBioscience). The acquisition of the samples was performed on a BD FACS Aria III cell sorter (BD Biosciences) and analyzed by FlowJo software v.10 (BD Biosciences). Results of cytofluorimetry are presented as the proportion of cells bound by an appropriate antibody.Total RNA was isolated from cells using a mi-Total RNA Isolation Kit  and reverse transcribed using random hexamer primers and MMLV (Moloney murine leukemia virus) reverse transcriptase according to the manufacturer’s instructions . Prepared cDNAs were amplified by using Maxima SYBR Green/ROX qPCR Master Mix  according to the recommendations of the manufacturer in a QuantStudio 3 real-time PCR system . Thermocycler conditions comprised an initial step at 50 °C for 5 min, followed by a step at 95 °C for 10 min, and a subsequent two-step PCR program at 95 °C for 15 s and 60 °C for 60 s for 40 cycles. The PCR primers  were as follows: IL-6: 5′-GCC CTT CAG GAA CAG CTA TGA-3′; 5′-TGT CAA CAA CAT CAG TCC CAA GA-3′; p19: 5′-GGG AGA CTC AAC AGA TGC CT-3′; 5′-GCA CTA AGG GCT CAG TCA GA-3′; p35: 5′-CAT CAC ACG GGA CAA AAC CA-3′; 5′-AGG CAC AGG GTC ATC ATC AA-3′; p40: 5′-CAC ATC TGC TGC TCC ACA AG-3′; 5′-CAA GTC CGT GTT TCT GTG CA-3′; transforming growth factor-beta (TGF-β): 5′-CCC TGC CCC TAC ATT TGG A-3′; 5′-ACG GTG ATG CGG AAG CAC-3′; GAPDH: 5′- ACA TCA TCC CTG CAT CCA CT-3′; 5′-GGG AGT TGC TGT TGA AGT CA-3′. Accumulation of PCR products was detected in real-time, and the results were analyzed with QuantStudio 3 software. Relative RNA expression is presented as 2−dCt, where dCt is the difference between Ct values of a gene of interest and the endogenous control (β-actin).® power calculator (http://biomath.info/power). Statistical analysis was performed using GraphPad Prism 8 software . The significance of the differences between the groups was determined using a two-tailed Student’s t-test or one-way ANOVA followed by Tukey’s post hoc test as indicated in the figure legends. A p-value less than 0.05 was considered statistically significant.Sample size for each of the analyses was selected based on our previous studies, using the Biomath


Scientific Reportshttps://doi.org/10.1038/s41598-021-04445-8, published online 10 January 2022Addendum to: Following publication, concerns have been raised that annotations in some figures depicting magnetic resonance images mask important features. A supplementary information file containing un-annotated images corresponding to Figures 1-4 is linked to this notice.Supplementary Information.


Escherichia coli, methicillin-resistant Staphylococcus aureus, and Pseudomonas aeruginosa biofilms. CdTe-2.4 eV QD versatility, safety, and ability to potentiate antibiotics makes them a potential treatment strategy for biofilm-associated infections.CdTe-2.4 eV quantum dots (QDs) show excellent efficacy due to their tunability and photo-potentiation for sterilizing drug-resistant planktonic cultures without harming mammalian cells but this QD fabrication has not been tested against biofilms. While the QD attack mechanism—production of superoxide radicals—is known to stimulate biofilm formation, here we demonstrate that CdTe-2.4 eV QD-antibiotic combination therapy can nearly eradicate  CdTe-2.4 eV quantum dots (QDs) show excellent efficacy due to their tunability and photo-potentiation for sterilizing drug-resistant planktonic cultures without harming mammalian cells but this QD fabrication has not been tested against biofilms. Resident bacteria diversify their gene expression—improving their response to environmental stressors such as antibiotic treatment through horizontal gene transfer of resistance genes—and surround themselves with an extracellular polymeric matrix, which may impede diffusion.Escherichia coli (E. coli) MG1655, methicillin-resistant Staphylococcus aureus (MRSA), and Pseudomonas aeruginosa (PAO1) biofilms. Used separately, effective concentrations of CdTe-2.4 eV QDs and antibiotics enhanced biofilm growth in clinical isolate strains compared to no treatment controls. Each strain, however, showed susceptibility to at least one CdTe-2.4 eV QD-antibiotic combination treatment (QD-ABX).We demonstrate synergy between 2.4 eV CdTe QDs and sub-Clinical & Laboratory Standards Institute (CLSI) breakpoint antibiotic treatments to early-stage, static 5 overnight cultures for 48 hours in 96-well U-bottom plates as described by O'Toole29 in conditions ideal for each strain (ESI Table S1via (1) crystal violet (CV) staining, (2) 2 hour incubation with Resazurin metabolic assay, or (3) counting colony forming units (CFU) (Biofilms were grown from 1 : 1 × 10ts (CFU) . NormaliE. coli MG1655—a well-established model strain—to inspect correlation among the 3 biofilm viability assays. A significant correlation  between normalized RFU and CFU measurements for increasing CdTe-2.4 eV QD doses, provides evidence for the use of Resazurin as an accurate, high-throughput assay of biofilm viability .30–34We used iability . While CE. coli MG1655 showing dose-dependent effects (−1 kanamycin. Though low doses of CdTe-2.4 eV QD (100–200 nM) and antibiotic  monotherapies achieved little killing  and PAO1 .35,36 Bacteria establish biofilms in response to environmental stressors, including ROS. MRSA established significantly larger biofilms with CdTe-2.4 eV QD or sub-breakpoint gentamicin treatments compared to the untreated control  showed dramatic variation , suggesting that effects of antibiotic monotherapy may vary with the bacterial population, making their efficacy difficult to predict. QD-ABX showed far more consistent results, even at mild concentrations, with reasonably high S-values.CdTe-2.4 eV QD-monotherapy showed no significant variation in PAO1 biofilms . FurtherS-values decreased as antibiotic monotherapies were sufficient for biofilm clearance. For both clinical isolates, CdTe-2.4 eV QDs potentiated lower concentrations of antibiotics to eradicate established biofilms. Similar to E. coli MG1655, we confirmed killing rather than dispersal of biofilm bacteria by measuring the viability of post-treatment media  and holistic disruption of cellular function (slowing MDR development). The versatility, safety, and ability to potentiate antibiotics makes CdTe-2.4 eV QDs a prime therapeutic candidate for persistent bacterial biofilm-associated infections.CdTe-2.4 eV QDs show potential not only for alternative therapies but also sterilization of surfaces prone to biofilm growth . This research lays a foundation for future work in treating late-stage biofilms with flow . CdTe-2.4 eV QDs are activated by visible light, which limits their application to surface infections. Future work will explore the application of near-infrared light-activated indium phosphide quantum dots to establish similar foundational work for the treatment of deep-tissue biofilm-associated infections.D. F. S. conducted all experiments. A. C. provided samples of MRSA and MG1655. D. F. S. and A. C. analyzed the results and wrote the manuscript. All authors edited the manuscript and have given final approval to the final version of the manuscript.A. C. and P. N. have a patent on QD technology. D. F. S. declares no competing interests.NA-003-D1NA00056J-s001


Scientific Reports 10.1038/s41598-022-19401-3, published online 02 September 2022Correction to: The Acknowledgments section in the original version of this Article was omitted. The Acknowledgments section now reads:“This work is partially funded by NIH the National Center for Advancing Translational Sciences (NCATS) UCLACTSI (UL1 TR001881-01), NCI grant P01CA233452-02, and U01 grantCA232859-01.”The original Article has been corrected.


RFP/IL-10GFP dual reporter mouse model along with intravascular fluorescent in vivo labeling, we characterized the TR-Treg cell responses to repetitive heterosubtypic influenza infections. We found lung tissue resident Treg cells accumulated and expressed high levels of co-inhibitory and co-stimulatory receptors post primary and secondary infections. Blockade of PD-1 or ICOS signaling reveals that PD-1 and ICOS signaling pathways counter-regulate TR-Treg cell expansion and IL-10 production, during secondary influenza infection. Furthermore, the virus-specific TR-Treg cell response displayed distinct kinetics, when compared to conventional CD4+ tissue resident memory T cells, during secondary flu infection. Our results provide insight into the tissue resident Foxp3+ regulatory T cell response during repetitive flu infections, which may be applicable to other respiratory infectious diseases such as tuberculosis and COVID.Regulatory T cells that express the transcription factor Foxp3 (Treg cells) are a highly heterogenous population of immunoregulatory cells critical for maintaining immune homeostasis and preventing immunopathology during infections. Tissue resident Treg (TR-Treg) cells are maintained within nonlymphoid tissues and have been shown to suppress proinflammatory tissue resident T cell responses and promote tissue repair. Human populations are repetitively exposed to influenza infections and lung tissue resident effector T cell responses are associated with flu-induced long-term pulmonary sequelae. The kinetics of TR-Treg cell development and molecular features of TR-Treg cells during repeated and/or long-term flu infections are unclear. Utilizing a Foxp3 We founGFP (B6(Cg)-Il10tm1.1Karp/J; 014530)    and Foxpescribed . All expAll influenza infections were performed intranasally under light sedation with isoflurane as described previously . Mouse avia in vivo fluorescent staining, mice were lightly anesthetized with isoflurane five minutes prior to sacrifice, and immediately injected via retroorbital intravenous injection (IV) with 1.5 μg allophycocyanin (APC)-conjugated anti-CD45.2 antibody in 200 μl sterile PBS as preciously described  of InVivoMAb anti-mouse ICOSL (CD275)  or InVivoMAb anti-mouse PD-L1 (B7-H1)  in 200 μl sterile PBS. Rat IgG2a isotypes were purchased from BioXCell as recommended by the product data sheets and used as controls. Treatment antibodies (or isotypes) were given to mice at 500 μg at the first timepoint, and 250 μg at the subsequent timepoints.Mice were injected intravenously via grinding against strainers, followed by filtering through the strainers. Red blood cells were lysed (RBC lysis buffer from Tonbo) before analysis. Cellular portion was resuspended in full RPMI media for staining and analysis via flow cytometry.Cells from various organs were isolated as we recently described . Lungs, All fluorescent antibodies are listed in “fluorochrome-target ” format below. Pacific Blue (PB)-CD103 (2E7), PerCP/Cy5.5-CD11a (M17/4), PerCP/Cy5.5-CD25 (3C7), PE-CD39 (DuHa59), Alexa Fluor (AF) 700-CD4 (GK1.5), PE-Cy7-CD69 (H12F3), BV421-CXCR6 (SA051D1), PE-Cy7-GITR (DTA-1), BV421-ICOS (C398.4A), PE-Cy7-LAG-3 (C9B7W), APC-Cy7-PD-1 (29F.1A12), APC-Cy7-TCRβ (H57-597) and APC-CD45.2 (104) were from BioLegend. PerCP/Cy5.5-T-bet (O4-46) was from BD Pharmingen. violetFluor (vF) 450-CD25 (PC61.5), PE-CD25 (PC61.5), APC-Cy7-CD4 (GK1.5), FITC-CD4 (RM4-5), PerCP/Cy5.5-CD8α (53-6.7), APC-Cy7-CD8α (53-6.7), PE-CTLA-4 (UC10-4F10-11), and APC-TIGIT (1G9) were purchased from Tonbo Biosciences. PE-eFluor610-Foxp3 (FJK-16s) and AF700-Ki-67 (SolA15) were from ThermoFisher Scientific.Other reagents: Ghost Dye™ Violet 510 viability dye was from Tonbo Biosciences. The following reagent(s) was obtained through the NIH Tetramer Core Facility: PE-labeled I-A(b) Influenza A nucleocapsid (NP) 311-325 QVYSLIRPNENPAHK loaded class II tetramer for NP-specific CD4+ T cell detection.via lymphocyte/single cells/live cells/TCRβ+ gating. CD4+Foxp3RFP+ T cells were defined as Foxp3+ gating Treg cells, while CD4+Foxp3RFP- T cells were defined as conventional CD4+ T cells. Tissue resident or circulating status was then determined via fluorescent in vivo IV staining of CD45.2 (CD45.2IV- for tissue resident cells and CD45.2IV+ for circulating cells respectively). IL-10GFP was utilized to analyze ex vivo IL-10 production by T cells during influenza infections.Surface staining of live cells were done in the presence of Fc block and fixable viability dye. For intracellular cytokine staining, cells were stimulated with cell stimulation cocktail (Tonbo Biosciences) for 4-5 hours, subjected to surface marker staining in the presence of Fc block, fixed in 4.2% paraformaldehyde, permeabilized and stained with the indicated anti-cytokine antibodies in intracellular perm/wash buffer (BioLegend) for 2 hours at 4 °C. Nuclear staining was performed with Foxp3 staining buffer kit (eBioscience). T cells were identified Left lung lobes were collected and fixed in 10% formalin. Lung sections were stained with hematoxylin and eosin (H&E) and scored by a board-certified pathologist at the Louisiana Animal Disease Diagnostic Laboratory, in a blinded manner. The scoring interactively evaluates the histopathologic levels of bronchiolar necrosis, alveolar bronchiolization/squamous metaplasia, peribronchiolar/perivascular cuffing, and alveolar inflammation. Scores were added together to achieve a cumulative score, at the range of 0 to 24.post hoc test were performed with GraphPad Prism 8 with p ≤ 0.05 considered statistically significant. “NS” refers to “No Significance”.T-test, one-way ANOVA and two-way ANOVA with Tukey’s + T cells. To determine whether and to what scale TR-Treg cells expand in the lungs of mouse model of primary acute influenza infections, we used Foxp3RFP/IL-10GFP dual reporter mice, infected them intranasally (IN) with the mouse-adapted WSN (H1N1) strain of influenza A and analyzed 7 days post infection (dpi) with in vivo fluorescent anti-CD45.2 antibody staining via intravenous (IV) injection to label the circulating T cells in the lungs  counterparts expanded ~12.63 and ~103.85 folds, respectively  cells in the lungs during acute primary influenza infection is not as well characterized as the tissue resident memory CD8RFP+ CD4+ T cells in these compartments and analyzed their levels of expression of CD25, Foxp3, CD103, CD69 and CD11a. The lung TR-Treg cells expressed higher levels of the high affinity IL-2 receptor, CD25, compared to their circulating counterpart in the lung of mice with acute influenza infection, but this level of CD25 expression in lung TR-Treg is comparable to that observed in splenic Treg cells  and and spleen of the same animals, we gated on the Foxp3eg cells , 35. In RFP/IL-10GFP dual reporter mouse model, we tracked the levels of IL-10 production in Treg cells during acute influenza infection and found that the lung tissue resident Foxp3+ Treg cells, along with the conventional CD4+ and CD8+ tissue resident T cells are the major producers of IL-10 during acute flu in mice . In contrast, their circulating counterparts contracted modestly following secondary influenza infection  cells that were lung tissue residents exhibited the highest percentages of NP311-325 tetramer bound fractions  cells , while TToxoplasma gondii infection, the PD-1hi eTreg cell population rapidly contracts and could be rescued via PD-L1 blockade  and T follicular helper (Tfh) cells in lymphoid tissues. ICOS and PD-1 signaling have been shown to counter-regulate effector Treg abundance at homeostasis in the spleen. ICOSL blockade reduces the numbers of effector Treg cells, while both PD-L1 blockade and Treg specific deletion of PD-1 results in effector Treg cell accumulation (fr cells while PD-1 inhibits Tfr differentiation in lymph nodes (LNs) and circulating in blood . MCM is a fellow recipient of the Careers in Immunology Fellowship from the American Association of Immunologists. WH is a fellow recipient of the Research Publication Grant in Engineering, Medicine, and Science from the American Association of University Women.We thank the NIH Tetramer Core Facility (contract number 75N93020D00005) for providing influenza NP peptide loaded tetramers. We also thank Ms. Qingxia Wang for technical assistance, Drs. David Topham and Gary Whittaker for mouse adapted WSN and X31 viral stocks, and Drs. Jie Sun and Gus Kousoulas for advice and helpful discussions.WH received research support from MegaRobo Technologies Co., Ltd., which was not used in this study.The remaining 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.


L) and apoptosis upon glutamine deprivation are inhibited in the presence of a membrane-permeable α-ketoglutarate. Collectively, our data support a model in which limiting glutamine utilization in glutamine-addicted tumor cells triggers a previously unknown cell death mechanism regulated by GCN2 that involves the TRAIL-R2-mediated activation of the extrinsic apoptotic pathway.Oncogenic transformation leads to changes in glutamine metabolism that make transformed cells highly dependent on glutamine for anabolic growth and survival. Herein, we investigated the cell death mechanism activated in glutamine-addicted tumor cells in response to the limitation of glutamine metabolism. We show that glutamine starvation triggers a FADD and caspase-8-dependent and mitochondria-operated apoptotic program in tumor cells that involves the pro-apoptotic TNF-related apoptosis-inducing ligand receptor 2 (TRAIL-R2), but is independent of its cognate ligand TRAIL. In glutamine-depleted tumor cells, activation of the amino acid-sensing general control nonderepressible-2 kinase (GCN2) is responsible for TRAIL-R2 upregulation, caspase-8 activation, and apoptotic cell death. Interestingly, GCN2-dependent ISR signaling induced by methionine starvation also leads to TRAIL-R2 upregulation and apoptosis. Moreover, pharmacological inhibition of transaminases activates a GCN2 and TRAIL-R2-dependent apoptotic mechanism that is inhibited by non-essential amino acids (NEAA). In addition, metabolic stress upon glutamine deprivation also results in GCN2-independent FLICE-inhibitory protein (FLIP) downregulation facilitating caspase-8 activation and apoptosis. Importantly, downregulation of the long FLIP splice form (FLIP The so-called extrinsic pathway of apoptosis utilizes membrane-localized death receptors of the tumor necrosis factor (TNF) receptor superfamily to activate the caspases cascade and apoptosis upon ligand binding . Tumor nThe increased uptake and metabolism of glutamine in tumor cells provide these cells with intermediates for nucleotide and protein synthesis, redox homeostasis, and mitochondrial energy metabolism required for proliferation . IncreasIn response to amino acid starvation cells can activate the integrated stress response (ISR) aiming at resolving stress and reestablishing homeostasis to maintain cell viability and proliferation . The iniHere, we have examined the role of GCN2 in cell fate decisions after either glutamine starvation or pharmacological inhibition of transaminases in glutamine-addicted tumor cell lines. We demonstrate that restricting glutamine utilization in tumor cells induces a GCN2-dependent signaling pathway leading to TRAIL-R2 upregulation that together with GCN2-independent FLIP downregulation results in caspase-8 activation and apoptotic cell death.Tumor cells heavily rely on extracellular glutamine to support cell survival and growth . HoweverIn response to different environmental and pathological stress conditions eukaryotic cells activate an adaptive pathway, called the integrated stress response (ISR), to restore cellular homeostasis , 29. TheTo further demonstrate the role of GCN2 in the stress response activated by glutamine deprivation, we genetically depleted GCN2 by RNA interference with two different siRNA oligonucleotides. GCN2 knockdown attenuated eIF2α phosphorylation and prevented ATF4 and CHOP induction in cells deprived of glutamine Fig. .Expression of pro-apoptotic receptor TRAIL-R2 is up-regulated in cells undergoing different forms of stress through a signaling pathway involving CHOP, a transcription factor for TRAIL-R2 gene expression , 34, 35.Involvement of GCN2 in TRAIL-R2 upregulation and apoptosis upon glutamine limitation was also investigated in the triple-negative breast tumor cell line MDA-MB468. Interestingly, glutamine deprivation-induced cell death was significantly inhibited by GCN2 knockdown  , to disrThere is a growing body of evidence indicating that feedback amplification of apical apoptotic caspases by effector caspases is required for efficient apoptosis . Thus, wCollectively, the above results demonstrated that in glutamine-addicted tumor cells depletion of glutamine triggers an apoptotic response mediated by the GCN2/TRAIL-R2/Caspase-8 signaling pathway. To ascertain whether sustained activation of the ISR by amino acid deprivation is sufficient to elicit an apoptotic response involving this pathway, we examined whether methionine deprivation in glutamine-replete medium may also activate a GCN2-dependent TRAIL-R2 increase and apoptosis in HCT-116 cells. Data shown in Fig. SL and FLIPS are key regulators of caspase-8 activation upon TRAIL receptors clustering by TRAIL [L expression was markedly down-regulated in both HCT116 and MDA-MB468 cells upon glutamine removal from the culture medium. Regarding FLIPs isoform, there were clear differences between HCT116 and MDA-MB468 cells in their response to glutamine deprivation, with a strong downregulation in MDA-MB468 cells and less pronounced effects in HCT116 cells. To assess whether the decrease in FLIP levels could be involved in cell death induced by glutamine deprivation, we generated bulk populations of HCT116  may prevent FLIPL loss and cell death by apoptosis in glutamine-starved tumor cells. Interestingly, addition of dmαKG to cultures of glutamine-starved HCT116 cells markedly inhibited FLIPL downregulation  to produce glutamate, a critical product of glutamine catabolism which is used for driving the biosynthesis of NEAA, through the activity of transaminases. In this respect, highly proliferative human breast tumors display high transaminase expression, coupling glutamine consumption to NEAA synthesis to boost protein biosynthesis required for tumor cells survival and growth . MoreoveL downregulation and GCN2-mediated TRAIL-R2 upregulation, mainly due to impairment of NEAA biosynthesis.To get further insight into the mechanism underlying apoptosis induction upon NEAA limitation we examined the role of TRAIL-R2-regulated extrinsic apoptotic pathway by first analyzing TRAIL-R2 expression, FLIP levels and caspase-8 activation in HCT116 cells treated with AOA. Interestingly, AOA treatment led to a marked upregulation of TRAIL-R2 expression Fig. S that werDespite the fact that glutamine is a non-essential amino acid different types of cancer cells exhibit a marked dependence on glutamine for cell survival and proliferation , 43–47. Ligation of pro-apoptotic TRAIL receptors by their ligand TRAIL leads to death-inducing signaling complex (DISC) formation and caspase-8 activation to trigger the extrinsic pathway of apoptosis . Our resSince the DISC components may colocalize in an intracellular membrane fraction in the absence of TRAIL , the incL levels that in cooperation with TRAIL-R2 accumulation will result in caspase-8 activation and apoptosis , and streptomycin (50 μg/ml). RPMI 1640 medium lacking methionine (Sigma-Aldrich) with 10% dialyzed fetal bovine serum, glutamine, penicillin and streptomycin was used in experiments to assess the response of tumor cells to methionine starvation.HEK293, HCT116 and HCT116 Bax/Bak K.O. cell lines were kindly provided by Dr. A. Rodriguez , Dr. J.A. Pintor-Toro  and Dr. C. Muñoz-Pinedo , respectively. MDA-MB468 cells were a gift from Dr. J. Arribas . MDA-MB468 and HEK293 cells were maintained in DMEM medium supplemented with 10% fetal bovine serum (Gibco), 2 mM L-glutamine, 50 U of penicillin/ml and 50 µg of streptomycin/ml. HCT116 and HCT116 Bax/Bak K.O. cells, in McCoy’s medium supplemented with 10% fetal bovine serum (Gibco), 2 mM L-glutamine, 50 U of penicillin/ml and 50 µg of streptomycin/ml. Cells were grown at 37 °C in a 5% COMedia supplements and chemical reagents for molecular biology and buffer preparation were from Sigma-Aldrich, . Anti-Hsp70 (H5147) antibody, propidium iodide, dimethyl α-ketoglutarate, doxycycline and puromycin were obtained from Sigma-Aldrich. PE-IgG Isotype control (554680), anti-Bcl-xL (556361) and anti-procaspase-3 (C-31720) antibodies, were obtained from BD Biosciences . Anti-human PE-TRAIL-R2 antibody (307405) was from Biolegend . Anti-procaspase-8 antibody 04-574) was from Upstate Millipore . Anti-α-tubulin (SC-23948), anti-GAPDH (SC-47724), anti-GCN2 (SC-374609) and anti-ATF4 (D4B8) antibodies were from Santa Cruz Biotechnology . Anti-TRAIL-R1 (AF347), anti-TRAIL-R2 (AF631) and Recombinant TRAIL-R2-Fc chimeric protein (631-T2) were from R&D Systems . Anti-c-FLIP monoclonal antibody 7F10 (ALX-804-961-0100) was from Enzo Life Sciences . Anti-p-eIF2α (S51) (3597), anti-eIF2α (D7D3) (5324), anti-CHOP (D46F1) (5554), anti-caspase 8 (1C12) (9746), anti-cleaved caspase-8 (18C8) (9496), anti-cleaved caspase-9 (9501) and anti-cleaved caspase-3 (9661) antibodies were purchased from Cell Signaling Technology . Anti-proaspase-9 (M054-3) was from MBL . Horseradish peroxidase-conjugated secondary antibodies were obtained from DAKO  . Recombinant human TRAIL was produced as previously described [4 was fro5/well) were treated in six-well plates as indicated in the figure legends. After treatment, hypodiploid apoptotic cells were detected by flow cytometry according to published procedures [Cells (3 × 10ocedures . Quantit5/well) were treated in 6-well plates as indicated in the figure legends. After treatment, cells are washed twice with PBS + 0.1% BSA, and incubated for 15 min on ice in the dark in the same buffer containing propidium iodide (2 μg/ml). Quantitative analysis of propidium iodide uptake was performed in a FACSCalibur cytometer using the Cell Quest Software .Cells (3 × 105) were washed with phosphate-buffered saline (PBS) and lysed in TR3 buffer . Protein content was measured with the Bradford reagent , before adding Laemmli sample buffer. Proteins were resolved on SDS-polyacrylamide minigels and detected as described previously [Cells  reagent, following the manufacturer’s instructions. mRNA expression was analyzed in triplicate by RT-qPCR on the ABI Prism7500 sequence detection system using predesigned Assay-on-demand primers and probes (Applied Biosystems). Hypoxanthine-guanine phosphoribosyltransferase (HPRT1 Hs01003267_m1) was used as an internal control and mRNA expression levels of TRAIL-R2 and TRAIL were given as fraction of mRNA levels in control cells. Primers and probes used were: TRAIL-R2 (TNFRSF1 Hs 00366278_m1) and TRAIL (TNFSF10 Hs00921974_m1).L and non-targeting scrambled oligonucleotide were synthesized by Sigma . HCT116 cells were transfected with siRNAs using jetPRIME (Polyplus Transfection) following manufacturer instructions. After 24 h, transfection medium was replaced with regular medium and cells were incubated for 48 h before further analysis. For siRNA transfection in MDA-MB468 cells, Dharmafect-1 (Dharmacon) was used as described by the manufacturer. After 6 h, transfection medium was replaced with regular medium and cells were further incubated for 48 h before analysis.siRNAs against GCN2, caspase-8, FLIP2-humidified, 95% air incubator), cells were washed with ice-cold phosphate-buffered saline (PBS) and re-suspended in PBS. Cells were then labeled either with 5 µg/ml of anti-TRAIL-R2-PE, or an IgG-PE control antibody (BD Bioscience) for 30 min on ice and darkness. Analysis of the receptor cell surface expression was carried out in a FACSCalibur cytometer using the Cell Quest Software .Cells were detached with trypsin solution and re-suspended in growth media. After incubation for 15 min under cell culture conditions , dnFADD and Bcl-xL retroviral vectors for stable gene expression have been described previously , 58. Forl vector . LentiviStable populations of HCT116 and MDA-MB468 cell lines infected with retroviruses were obtained after selection in culture medium containing puromycin (1.5 µg/ml) during 48 h. Tumor cells infected with GFP-expressing lentiviruses were detected by flow cytometry.TRAIL-R2 cloned into pCW57-MCS1-P2A-MCS2  as reported [HCT116 and MDA-MB468 cells stably overexpressing human TRAILR-2 were generated by lentiviral transduction of full length human reported . Cells tP < 0.05 was considered significant. n.s. non-significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.All data are presented as the mean ± standard deviation (SD) of at least three independent experiments. Statistical analysis was performed using GraphPAD Prism 9 . The differences among different groups were determined by the two-way ANOVA, Tukey’s multiple comparison test. Supplementary figure legendsFigure S1Figure S2Figure S3Figure S4Original Data FileReproducibility checklist


HER2-low breast cancers were reported to have distinct clinicopathological characteristics from HER2-zero; however, the difference in their genetic features remains unclear. This study investigated the clinical and molecular features of breast tumors according to HER2 status.n = 90), HER2-low (n = 231), and HER2-positive (n = 202) according to their immunohistochemistry and fluorescence in situ hybridization results.We analyzed the clinicopathological and genomic data of 523 Chinese women with breast cancer. Genomic data was generated by targeted next-generation sequencing (NGS) of breast tumor samples using a commercial 520 gene panel. The cohort was stratified according to HER2 status as HER2-zero (p < 0.001) and HER2-zero breast tumors (p < 0.01). HER2-zero tumors had more mutations in checkpoint factors (p < 0.01), Fanconi anemia (p < 0.05), and p53 signaling and cell cycle pathway (p < 0.05) compared to HER2-low breast tumors. Compared with HER2-zero tumors, HER2-low tumors had significantly lower pathological complete response rates after neoadjuvant therapy  and proportion of relapsed/progressed patients across follow-up time points (p = 0.031), but had comparable disease-free survival (p = 0.271).HER2-low breast tumors were enriched with hormone receptor-positive tumors, and who had lower Ki67 expression levels. Genes were differentially mutated across HER2 subgroups. HER2-low tumors had significantly more mutations involved in PI3K-Akt signaling than HER2-positive (Our results demonstrate the distinct clinical and molecular features and clinical outcomes of HER2-low breast tumors.The online version contains supplementary material available at 10.1186/s12916-022-02346-9. The overexpression of the human epidermal growth factor receptor 2 (HER2) is associated with shorter overall survival and higher risk of disease recurrence. HER2 overexpression is detected in 15–40% of unselected patients with breast cancer . These HHER2 gene amplification by fluorescence in situ hybridization (FISH). The results from these molecular methods were assessed based on the algorithm recommended by an expert panel from the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP) [Currently, HER2 status is assayed using standard molecular methods based on the assessment of HER2 protein overexpression by immunohistochemistry (IHC) and estimation of ts (CAP) –14. The ts (CAP) , 16.However, the clinical implications of classifying HER2 equivocal breast tumors as HER2-negative remain unclear. Some studies have demonstrated similar clinical and survival outcomes between HER2 equivocal and HER2-negative/zero breast tumors , 16, wheHER2 gene amplification status and other concomitant gene alterations.At present, novel HER2-targeting therapies with mechanisms independent of overexpressed HER2 protein have been developed. These include neratinib, a pan-EGFR tyrosine kinase inhibitor (TKI) , and traWe conducted this study to investigate the genomic characteristics of breast tumors with HER2-zero (IHC 0), HER2-low (IHC 1+ or IHC 2+/FISH-negative), and HER2-positive (IHC 3+ or IHC 2+/FISH-positive) status, by retrospectively analyzing the molecular, clinical, and survival data from 523 Chinese women with breast cancer. All breast tumor tissues were subjected to targeted NGS using a large gene panel to comprehensively elucidate their somatic mutational landscape. We then compared the breast cancer subtypes, molecular features, treatment outcome, and disease-free survival data according to HER2 expression status.Women diagnosed with breast cancer at Guangdong Provincial People’s Hospital (GDPH) were prospectively recruited for genomic studies and voluntarily submitted samples for NGS as previously described . The stuHER2 amplification positivity was defined as HER2 to chromosome 17 centromere (CEP17) signal ratio of ≥2.0 with HER2 signals exceeding 6.0 per cell. The slides were scored by experienced pathologists and/or technicians according to the 2018 ASCO/CAP guidelines [Thin FFPE sections of breast tumor samples from all patients were subjected to initial HER2 testing with IHC using HercepTest . HER2-positivity was determined as an IHC result of 3+, defined as having >10% intact membrane staining. With an IHC result of 2+ or upon patient request, the slides containing a thin section from the same breast tumor sample of the patient were submitted for subsequent FISH-based HER2 testing using HER2 IQFISH pharmDx . FISH-based idelines .p-value of <0.005 and a ratio of >0.5. The normal copy number is set at 2 as per the normal diploid genome, copy number ≤1.5 is considered copy number deletion, and copy number ≥2.75 is considered copy number amplification.Genomic DNA was extracted from FFPE breast tumor tissue with at least 10% tumor content using QIAamp DNA FFPE tissue kit as per the manufacturer’s instructions . DNA concentration was determined using Qubit dsDNA high-sensitivity quantification assay on the Qubit fluorometer according to the manufacturer’s instructions . Target capture of the tissue DNA samples was performed using a 520-gene panel  following previously published protocols , 31, 32.TMB per patient was computed as a ratio between the total number of non-synonymous mutations detected and the total coding region size of the panel using the equation below. The mutation count included non-synonymous SNVs and Indels detected within the coding region and ±2 bp upstream or downstream region and does not include hot mutation events, CNVs, SVs, and germline single-nucleotide polymorphisms (SNPs). Only mutations with allelic fractions (AF) of ≥ 2% for tissue samples and ≥ 0.2% for plasma samples were included in the mutation count. For accurate TMB calculation, maximum AF (maxAF) was calculated as ≥ 5% for tissue samples and ≥ 1% for plasma samples. The total size of the coding region for estimating TMB was 1.003 Mb for the 520-gene OncoScreen Plus panel.Neoadjuvant regimens were administered as various combinations of systemic chemotherapy/targeted inhibitors according to the patient’s HER2 and HR status and the physician’s decision. Regimens included were taxanes (T), epirubicin (E), doxorubicin (A), cyclophosphamide (C), 5-fluorouracil (F), trastuzumab (T), pertuzumab (P), carboplatin (Cb), and capecitabine (X). All combination regimens were administered at standard dosing schedules. Pathological complete response (pCR) was assessed using the completely resected breast specimen and all regional lymph nodes sampled after neoadjuvant therapy. pCR was defined as the absence of residual invasive cancer cells on hematoxylin and eosin evaluation of all specimens.Disease-free survival (DFS) was defined as the time in months from the date of diagnosis until the date of either disease relapse/disease progression or last follow-up. The proportion of patients who had radiologically confirmed disease relapse (PD ratio) was calculated as the ratio of patients with relapse to the total number of patients who remained disease-free at that particular year of follow-up, excluding the number of patients who were lost to follow-up. The data cut-off date was October 15, 2021.P < 0.05.The R statistics package  was used to analyze the data. Patient characteristics and sequencing results were summarized with descriptive statistics. Mutation profiles of HER2-positive, HER2-low, and HER2-zero groups in the GDPH cohort were compared using Fisher’s exact test and Wilcoxon signed-rank test, as deemed appropriate. Data clustering was performed using non-negative matrix factorization (NMF) based on Euclidean distance. R package NMF was implemented to estimate the best rank using Lee’s algorithm with the following initial parameters: 2:8 for rank, and numeric random seed (seed = 123456). The most ideal values for cophenetic consensus were observed at ranks 3–4; hence, we selected rank 3 to cluster our data into three subgroups. Kaplan-Meier survival curves with log-rank statistics were plotted to compare DFS among subgroups. All statistical tests were performed as two-sided test and significance level was set at n = 378), with invasive carcinoma of no special type (NST) , and HR-positive breast cancer . The cohort was further stratified according to HER2 status based on the 2018 ASCO/CAP guidelines. A total of 321 patients were HER2-negative as per the guidelines, with 90 patients (17.2%) having IHC 0, defined as HER2-zero, and 231 patients (44.2%) having either IHC 1+ or IHC 2+ with negative FISH results, defined as HER2-low. The remaining 202 patients (38.6%) had either IHC 3+ or IHC 2+ with positive FISH results, defined as HER2-positive. Table Figure p < 0.001) and more than HER2-positive   and HER2-positive patients  subgroups  and the HER2-positive patients (p < 0.001), HR status (p = 0.196), and Ki67 expression level (p = 0.28). We also used a median cut-off of 30% to stratify the Ki67 as low (<30%) and high (≥30%). Among the HR-negative tumors, the HER2-zero subgroup had a significantly higher proportion of tumors with high Ki67 expression than the HER2-positive subgroup . Among the HR-positive tumors, the HER2-positive subgroup had a significantly higher proportion of tumors with high Ki67 expression than HER2-low  and HER2-zero subgroup    and HER2-positive (p < 0.001) subgroups , and a significantly lower proportion of TNBC tumors . It should be noted that all luminal B tumors in HER2-low and HER2-zero subgroups were HER2-negative. Taken together, these findings indicate the difference in clinical features among HER2-zero, HER2-low, and HER2-positive breast tumors.The HER2-low subgroup were distinctive based on three criteria: (1) the subgroup exhibited more histological grade 2 than HER2-zero  and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), respectively, were detected in 54% and 46% of the cohort and were the two most frequently mutated genes. Copy number amplifications in cyclin-dependent kinase 12 (CDK12) were only exclusively detected together with copy number amplifications in HER2 , retinoic acid receptor alpha , and speckle-type POZ protein  and significantly higher TP53 mutation rates (p < 0.001) as compared with HER2-zero , GATA binding protein 3 , core-binding factor subunit beta , and AKT serine/threonine kinase 1  than HER2-positive breast tumors , PIK3CA (p < 0.05), mitogen-activated protein kinase kinase kinase 1 , and AT-rich interaction domain 1A  as compared with HER2-zero breast tumors , telomerase reverse transcriptase , polypeptide N-acetylgalactosaminyltransferase 12 , caspase recruitment domain family member 11 , and transformation/transcription domain-associated protein  than HER2-low tumors  for HER2-zero, 26.0% (60/231) for HER2-low, and 67.8% (137/202) for HER2-positive breast tumors. PIK3CA mutations were distributed across the functional domains  and H1047R/L (n = 11) were the most common PIK3CA mutations in HER2-zero. PIK3CA H1047R/L was the most common in HER2-low (n = 71) and HER2-positive (n = 46) breast tumors.Of the .0% 60/23 for HER2TP53 and PIK3CA  and HR-positive status   and HER2-low , but not among HER2-positive breast tumors  , luminal A , and luminal B , luminal A , and luminal B  , GALNT12 , and RICTOR  were significantly higher for HER2-zero than HER2-low subgroup , homologous recombination (p < 0.001), checkpoint factors (p < 0.01), and p53 signaling and cell cycle (p < 0.01) as compared with HER2-zero   , Fanconi anemia (p < 0.05), and p53 signaling and cell cycle pathway (p < 0.05) than HER2-low breast tumors , ERBB2 (or HER2), ERBB3, and ERBB4. Additional file ERBB2 gene, such as missense mutations and splice-site mutations, were detected in two HER2-zero patients . ERBB3 mutations were detected in four patients (4.4%) with HER2-zero tumors. ERBB4 mutations were detected in four patients each with HER2-low (1.7%) and HER2-positive tumors (2.0%).We also explored the mutation frequency of other members of the ErbB/HER family. The HER family consists of four receptor tyrosine kinases, epidermal growth factor receptor , fibroblast growth factor (FGF) 3 (FGF3), FGF4, FGF19, ribosomal protein S6 kinase B2 (RPS6KB2), P21 activated kinase 1 (PAK1), and EMSY BRCA2-interacting transcriptional repressor (EMSY), which are associated with chromosome 11q13 loci amplification, as well as FGF receptor 1 (FGFR1) and adhesion G protein-coupled receptor A2 (ADGRA2), which are both located in chromosome 8p12.Unsupervised clustering of the mutation profile of the cohort revealed three molecularly distinct clusters as shown by the heat map in Fig. Pathway analysis demonstrated that genes in Cluster 1 were involved in base excision repair, homologous recombination, p53 signaling and cell cycle, checkpoint factor, ErbB signaling, and Notch signaling. Cluster 2 was comprised of genes involved in PI3K/Akt pathway. Cluster 3 was comprised of genes involved in DNA damage response, receptor tyrosine kinase, and Wnt signaling pathway Fig. C.According to HER2 subgrouping, Cluster 1 comprised of 55.6% (168/302) HER2-positive, 27.2%% (82/302) HER2-low, and 17.2% (52/302) HER2-zero breast tumors. Of them, 178 patients (58.9%) were HR-positive. Cluster 2 comprised of 78.3% (108/138) HER2-low, 13.7% (19/139) HER2-zero, and 8.0% (11/138) HER2-positive breast tumors. Cluster 3 comprised of 49.4% (41/83) HER2-low, 27.7% (23/83) HER2-positive, and 22.9% (19/83) HER2-zero breast tumors. A majority of the patients in Cluster 2  and Cluster 3  were HR-positive  and HER2-positive subgroups  , HR-negative , TNBC , and luminal B , but was numerically higher in luminal A tumors ; however, statistical analysis showed no significant difference between HER2-low and HER2-zero subgroups across HR status and IHC-based molecular subtypes possibly due to lower sample size in some subgroups  according to HER2 subgroups. As expected, women with HER2-zero  and HER2-low  tumors had significantly shorter DFS than HER2-positive tumors (n = 20); however, DFS was comparable between HER2-zero and HER2-low subgroups . Despite the inconclusive DFS due to the largely immature survival data from our cohort, our findings still showed that a significantly lesser proportion of women with HER2-low breast tumors experienced disease relapse up to 6 years of follow-up. This finding suggest that women with HER2-low are expected to have longer DFS and better survival outcomes than HER2-zero breast tumors.TP53 mutations but more PIK3CA mutation in the HER2-low tumors than in the HER2-zero tumors. The enriched mutations in genes involved in the PI3K-Akt pathway suggest a potential therapeutic strategy of combining chemotherapy and PI3K-related targeted therapy to improve the pCR rate in women with HER2-low tumors, which have been successfully demonstrated in various clinical trials [The observed differences in clinical outcomes regarding pCR rates and survival trends could be attributed to differences in IHC-based molecular subtype distribution and molecular features, such as more luminal B and fewer TNBC subtypes, less l trials –36. It ml trials , 33, 37.TP53 mutations, CDK12 amplifications, and RARA amplifications. It should be noted that in our cohort, 81% of HER2-positive tumors were detected with TP53 mutations while all CDK12 amplifications were concurrently detected with HER2 amplification. TP53 mutations, particularly in the DNA-binding domain spanning exons 5–8, results in the malfunctioning of DNA damage repair pathways and are the most frequently reported mutation in solid tumors, including breast cancer [CDK12 is located in chromosome 17q12, which is a neighboring gene of HER2, and hence is frequently amplified with HER2 [Consistent with our previous study and other existing reports , 38, 39,t cancer , 40. CDKith HER2 , 42. CDKith HER2 . ConsistTP53 mutations. Cluster 2 was enriched in PIK3CA mutation but lack TP53 mutation. Cluster 3 was enriched in copy number amplifications of various genes associated with the chromosome 11q13 loci, wherein coamplification of CCND1 and FGF3/4/19 was the most common. Chromosome 11q13 locus is considered gene-rich and is one of the most commonly aberrant chromosome loci in breast cancer [CCND1 amplification has been associated with immunosuppression of the tumor microenvironment and poor outcomes with immune checkpoint inhibitors [FGFR1, and appear to be mutually exclusive from HER2 amplification [Our findings from the molecular data clustering showed that our breast cancer cohort can be classified into three clusters of differential gene mutation. Cluster 1 was enriched in t cancer . Amplifit cancer –46. CCNDhibitors . Furtherfication .n = 12,467) have reported an average HER2-positive rate of 24.7%, ranging 13.7 to 35.7% across 19 different institutions from China [Our study has some inherent limitations. Our study was carried out in a single institution, which could introduce patient selection bias. Our study only included tumor samples sequenced using a 520-gene panel, which might introduce patient sampling bias, such as the higher HER2 positive rate of our cohort despite being unselected for clinical or pathological features (~38.6%). However, a study with a larger cohort of unselected invasive breast cancer n = 12,67 have rOur study demonstrates the complex genetic heterogeneity across breast tumors with varied HER2 status and identifies potential oncogenic mechanism that drives HER2-low breast tumor. Our findings indicate that patients with either IHC 1+ or IHC 2+ with FISH-negative results have distinct clinical and mutational features as compared with HER2-zero and HER2-positive breast tumors. Moreover, the accumulating evidence that HER2-low is clinically and biologically distinct from HER2-zero raises the need to explore diagnostic techniques and personalized therapeutic approaches that could identify and potentially benefit the subset of patients with HER2-low breast cancer.Additional file 1: Table S1. Mutations in other members of the HER family detected from our cohort. Figure S1. Somatic mutation landscape of the cohort. Figure S2. Comparison of mutational profile of the HER2 subgroups. Figure S3. Distribution of TP53 mutations among the HER2 subgroups. Figure S4. Distribution of PIK3CA mutations among the HER2 subgroups. Figure S5. Genes frequently mutated across HER2 subgroups according to hormone receptor (HR) status. Figure S6. Comparison of TP53 (A) and PIK3CA (B) mutation rates across HER2 subgroups according to hormone receptor (HR) status. Figure S7. Genes frequently mutated across immunohistochemistry-based molecular subgrouping between HER2-low and HER2-zero subgroups. TNBC, triple negative breast cancer. Figure S8. Comparison of TP53 (A) and PIK3CA (B) mutation rates between HER2-zero and HER2-low subgroups according to immunohistochemistry-based molecular subgrouping. Figure S9. Comparison of genes (top 11) and signaling pathways (top 5) differentially mutated between HER2-low and HER2-zero subgroups according to immunohistochemistry-based molecular subgrouping: A. triple negative breast cancer (TNBC), B. luminal A, and C. luminal B. Figure S10. Tumor mutational burden (TMB) were comparable across HER2 subgroups. TMB were not statistically different among patients with HER2-zero (IHC 0), HER2-low (IHC1+ or IHC 2+/FISH-negative), and HER2-positive (IHC 3+ or IHC 2+/FISH-positive) breast tumors. Figure S11. Distribution of ERBB/HER family mutations according to HER2 status. Figure S12. Molecular clustering across hormone receptor (HR) status (A) and immunohistochemistry-based molecular subgrouping (B). Figure S13. Pathological complete response (pCR) rate is comparable between HER2-low and HER2-zero subgroups across hormone receptor (HR) status (A-B) and immunohistochemistry-based molecular subgrouping (C-E). Figure S14. Disease-free survival (DFS) was comparable between women with HER2-low and HER2-zero tumors as shown by Kaplan-Meier survival plot of the whole cohort (A) and when considering only the relapsed patients per group (B). Figure S15. Disease-free survival (DFS) was comparable between HER2-zero and HER2-low subgroups regardless of pathological complete response (pCR) status. Figure S16. Disease-free survival (DFS) was comparable between HER2-zero and HER2-low subgroups regardless of hormone receptor (HR) status. Figure S17. Disease-free survival (DFS) was comparable between HER2-zero and HER2-low subgroups across immunohistochemistry-based molecular subgrouping.


Correction to: Harm Reduction Journal (2022) 19:129 10.1186/s12954-022-00715-4 Following publication of the original article [1], a text under Funding section has been inserted.The original article has been corrected.


Azomethine ylides are nitrogen-based three-atom components commonly used in [3+2]-cycloaddition reactions with various unsaturated 2π-electron components. These reactions are highly regio- and stereoselective and have attracted the attention of organic chemists with respect to the construction of diverse heterocycles potentially bearing four new contiguous stereogenic centers. This review article complies the most important [3+2]-cycloaddition reactions of azomethine ylides with various olefinic, unsaturated 2π-electron components  reported over the past two decades. The three-atom component (TAC) is an organic species that is represented by zwitterionic octet structures and undergoes 3+2]-cycloadditions with an unsaturated 2π-electron component in a one-step reaction, often in an asynchronous and symmetry-conducive fashion, via a thermal six-electron Hückel aromatic transition state. The formal charges are lost in the [3+2→5] cycloaddition  1]. Rec. Rec1]. -cycloaddα-amino acids -Cycloadditions of azomethine ylide with homomultiple and heteromultiple unsaturated 2π-electron components have been extensively used to produce a wide range of heterocycles -cycloaddition of an azomethine ylide in 1991 , there hThis review article deals with the [3+2]-cycloaddition reaction of azomethine ylides with an unsaturated carbon–carbon bond  that leads to the formation of pyrrolidinyl-containing analogs reported in the last two decades and their biological applications. This review article is intended to be a critical resource for the researchers involved or interested in azomethine ylides-mediated heterocyclic synthesis. It is also hoped that this review article will inspire chemists in this area of research.2 derived from benzyl(methoxymethyl)(trimethylsilylmethyl)amine 1 undergoes a [3+2]-cycloaddition reaction with electron-deficient alkenes 3 under continuous flow conditions in the presence of catalytic trifluoroacetic acid, thereby affording the corresponding pyrrolidines 4  as an oxidizing agent. The transformation was assumed to take place through [3+2]-cycloaddition of N-substituted carbonylmethyleneisoquinolinium bromide (formed via the reaction of isoquinoline 11 and 12) with arylidene cyanoacetamide  isoquinolines 17 was observed probably due to DDQ oxidation  . In the xidation  23]..a]isoqui21 were prepared by the [3+2]-cycloaddition reaction of benzoimidazol-2-yl-3-phenylacrylonitriles 18 with azomethine ylides, which was generated in situ from the condensation of isatin 19 and sarcosine 20 in refluxing ethanol. Similarly, spirothiazoles] 23 were formed by using thioproline 22 as a secondary amino acid  with α-amino acids (sarcosine 20 and proline 27) and Knoevenagel adducts 24 . Quantum chemical calculations ) explained the reaction pathway benzylamine 1, which is an azomethine ylide equivalent, in the presence of trifluoroacetic acid in dichloromethane. Some of the synthesized 34 revealed promising inhibitory properties as Na+ channel blockers, which are useful in the treatment of ischemic stroke -nitroalkenes 36  of the electron-poor nitroalkenes avorable  30]..36 with 38 (major product) and 39 (minor product) were obtained from the cycloaddition reaction of azomethine ylides, generated in situ from isatin 19, with proline 27 and (E)-ß-nitrostyrene 32 (E)-1-phenyl-2-nitropropene.Spirooxindolo-nitropyrrolizines yrene 32  31]. A . A 38 (mS-shaped ylide with a cycloaddition via the endo-transition state (pathway B), yielding cycloadducts 38, and not the exo-transition state (pathway A). Computational studies (Gaussian 03) of the transition states , B3LYP, and 6-31G basis set) confirmed these assumptions  [42 were obtained by the reaction of tyrosine 41 in an ionic liquid [bmim]Br at 100 °C. Promising antiproliferation properties were observed for some of the synthesized compounds (42) against human A549  and Jurkat (T-cell lymphoma) cell lines (MTT assay) using Camptothecin as a positive control; the compounds exhibited a safe response against the non-cancer cell lines MCF-10  and PCS-130-010 (lung smooth muscle). Caspase-dependent apoptosis  was mentioned as the mode of action for the observed antiproliferative activity  . Analogoactivity  33]..40 with b]quinoxaline-11,2′-pyrrolidines] 47 by the cycloaddition reaction of nitroalkenes 36 with azomethine ylide  in an ionic liquid [bmim]Br. Some of the synthesized agents revealed antimycobacterial properties (Mycobacterium tuberculosis H37Rv) with an efficacy comparable to that of ethambutol (reference standard) [49 were obtained by using L-histidine 48 instead of L-phenylalanine 46 in this reaction. Some of the synthesized compounds revealed cholinesterase (acetylcholinesterase and butyrylcholinesterase)-inhibitory properties with considerable efficiencies relative to Galantamine  . Similarantamine  35]..b]quinoxß-lactams 52 were prepared as single diastereomers by the reaction of azomethine ylides 51, generated from β-lactam imines of α-amino ester 50, with nitrostyrenes 36 in the presence of silver acetate and triethylamine caprolactamate [Rh2(cap)4] catalyzed oxidation of tetrahydroisoquinoline 53 -3-aryl-1-(thiophen-2-yl)-prop-2-en-1-ones 55 with azomethine ylide generated in situ from the condensation of isatin 19 with L-proline 27  and antifungal activity against Candida albicans (relative to Amphotericin B)  osine 20 . Some ofT assay) ,42 and s66 were prepared in appreciable yields by the cycloaddition reaction of the unsaturated 2π-electron component (E)-2-(1H-indole-3-carbonyl)-3-phenylacrylonitrile 65 and azomethine ylides obtained from the condensation of isatin 19 and sarcosine 20 . Among all the synthesized compounds, some showed antimicrobial properties against Gram-positive and Gram-negative bacterial as well as fungal strains using Streptomycin and Ketconazole as standard references methyl]acrylonitrile 71, was synthesized by the Baylis–Hillman reaction of chromene-3-aldehyde, treated with the azomethine ylides (from isatin 19 and sarcosine 20), which afforded the corresponding regioselective spiro[pyrrolidine-oxindoles] 72 and 73 as major and minor products, respectively  and diastereoselectivity . Some of the synthesized cycloadducts 79 displayed cholinesterase-inhibitory properties (acetylcholinesterase and butyrylcholinestrase) with potency relative to Galantamine ..19 and L81 with azomethine ylide formed by the condensation of ninhydrin 44 and amino acids (sarcosine 20/L-proline 27) afforded the corresponding spiroindanopyrrolidines 82 and spiroindanopyrrolizines 83  ..84 with 88 were obtained by reacting β,γ-unsaturated α-keto esters of type 87 with proline 27 in a 2:1 molar ratio. The reaction was assumed to proceed via the formation of azomethine ylides by the condensation of the starting unsaturated esters of type 87 with amino acid 27, which, in turn, interacted with another molecule of 87 to ultimately yield pyrrolizidines of type 88 , afforded the corresponding spiro-pyrrolidines 90 and spiro-pyrrolizidines 91 (The reaction of dines 91  52]..O-acrylo94–97 were obtained via the reaction of methyl 2-(1H-inden-2-yl)acrylate 92 with azomethine ylides generated in situ by reacting ketones  with sarcosine 20 [(trimethylsilyl)methyl]amine 1 in the presence of a catalytic amount of trifluoroacetic acid, afforded the corresponding trans pyrrolidine derivatives 101  underwent intramolecular [3+2]-cycloaddition under microwave conditions, affording the 8-phenyldiazenylchromenopyrrolidines 104  and Gram-negative bacteria , fungi , and mycobacteria (M. Tuberculosis H37RV) relative to the antibacterial , antifungal , and antimycobacterial (Metronidazole) standard references used pyrroles 111  generated azomethine ylides, which underwent an intramolecular cycloaddition reaction yielding the corresponding polycyclic compounds 123 and 124 -2-{[allyl(benzyl)amino]methyl}cinnamaldehydes 130 with proline methyl ester hydrochloride 131 under microwave conditions, which afforded the pyridopyrrolizines 132 (Another example of intramolecular cycloaddition was the reaction of (ines 132  65]..E)-2-{oxathiinopyrroles 136. However, the reaction of derivative 135 with L-proline 27 gave the corresponding oxathiinopyrrolizines 137 as trans–trans (major) and cis–trans (minor) isomers -2-phe139 formation via the intramolecular cycloaddition of an azomethine ylide generated from a triazole-linked glycol-nitroalkenyl aldehyde derivative 138 and sarcosine 20 [osine 20 .b]pyrano-pyrrolizidine and indolizidine derivatives 141 and 143 were synthesized by the intramolecular [3+2]-cycloaddition of azomethine ylides generated from naphtho-O-alkenyl aldehydes 140 and α-amino acids (L-proline 27 or DL-pipecolinic acid 142)   69]..b]pyrano144 that led to the in situ formation of azomethine ylides, which underwent intramolecular cycloaddition, thus affording N-phthalimidopyrrolidine derivatives 145 as a mixture of two diastereoisomers [oisomers .γ-lactone 147 was created by the intramolecular [3+2]-cycloaddition of azomethine ylide generated via the thermolysis of aziridine derivative 146 in refluxing toluene (Another bicyclic system of  toluene  71]..γ-lacton19 and sarcosine 20) with 2-arylidene-1-cyclopentanones 148 in the presence of bentonite clay under microwave conditions afforded dispiropyrrolidinyl-oxindoles 149  with 2E,6E-bis(arylidene)-1-cyclohexanones 150 cyclohexanones 150 under solvent-free conditions using microwave irradiation, thereby affording the dispiro-oxindole 153 with high regioselectivity  afforded the corresponding spiro-cyclohexanones 155–158 upon reaction with 2,6-bis(ylidene)cyclohexanones 150 in refluxing methanol [Mycobacterium tuberculosis H37Rv (MTB) relative to Ethambutol and Pyrazinamide [Azomethine ylides derived from acenaphthenequinone methanol   with bis(ylidene)cycloalkanones 159 in the presence of hydrazine hydrate 160 in refluxing methanol regioselectively afforded the corresponding spiro-indenoquinoxaline-pyrrolidines 161 at a high yield -ylidene]-1,4-dioxa-spirodecane-8-ones 162 and azomethine ylides (formed from isatin 19 and sarcosine 20 or thioproline 22) in 2,2,2-trifluoroethanol (TFE)  and antimycobacterial properties against M. tuberculosis H37Rv relative to the standard references Nysyatin, Greseofulvin , and Isoniazid  ..165 were167 were regioselectively synthesized by the cycloaddition of 2-(ylidene)-1-indanones 166 with azomethine ylides (formed from isatin derivatives 19 with sarcosine 20) in refluxing ethanol. Promising anti-inflammatory properties were exhibited by the synthesized compounds (via a rat carrageenan paw edema assay) relative to Indomethacin (standard reference drug) –silica as a catalyst in refluxing acetonitrile, afforded the dispiroindenoquinoxaline-pyrrolizidines 180  with 2-malononitrile 181 (Dispiro compounds of type rile 181  43]..182 wereE)-arylidenefluorenes 183 with isatin 19 and sarcosine 20 or proline 27, under microwave conditions, afforded the corresponding dispiro-oxindoles 184 and 185, respectively (The solvent-free reaction of (ectively  94]..E)-aryli186 with azomethine ylides formed from the condensation of isatin 19 or acenaphthenequinone 25 and secondary amino acids (sarcosine 20 or L-proline 27) in refluxing methanol afforded the corresponding spirooxindoles 187–190 (The reaction of acenaphthenone-2-ylidene ketones of type  187–190  95]..186 withH)-acenaphthylen-1-ylidene-malononitrile 191 afforded the corresponding dispiropyrrolidine-oxindoles 192 by its reaction with isatin 19 and sarcosine 20 in refluxing toluene -acena194 and 195 were synthesized via the cycloaddition of (E)-1-naphthylidene-1-tetralone 193 with the corresponding azomethine ylides generated from isatin 19 and sarcosine 20 or L-proline 27 (Dispiro-oxindolopyrrolidine/pyrrolizidines oline 27  97]..194 and 196 with azomethine ylides (from isatin 19 and sarcosine 20) in a 1:2 molar ratio afforded the corresponding tetraspiro-bisoxindolopyrrolidine 198. With a 1:1 ratio of the reactants, mono derivatives of type 197 were formed, which, in the presence of an excess of isatin 19 and sarcosine 20, afforded bisoxindolopyrrolidines 198 -1,2:5,6-di-O-isopropylidene-α-D-glucofuranose 205 with isoquinoline-based azomethine ylide formed from isoquinolines 11 and alkyl bromoacetates or 2-bromoacetophenones 204 in the presence of Cu(OTf)2–Et3N as a catalyst  afforded the corresponding dispiropyrrolidine containing-thiophenones 208–210 in good yields (The reaction of 2-(ylidene)thiophen-3-ones d yields  103]..207 withH)-oxazolones 211 with azomethine ylide derived from cis-4-formyl-2-azetidinone 212 and sarcosine 20 or pyrrolidine 27, in the presence of camphor sulphonic acid (CSA) as a catalyst, afforded the corresponding spiro[3.4′]-(oxazol-5′-one)-pyrrolidines 213 and spiro[3.4′]-(oxazol-5′-one)-pyrrolizidines 214, respectively (The reaction of 4-ylidene-5-(4ectively  104]..H)-oxazo216–219 were obtained via the cycloaddition reaction of 4-ylidene-5-oxazolones 211 and azomethine ylides (generated from isatin 19 and the appropriate α-amino acids). Some of the synthesized compounds showed considerable antitumor properties against breast cancer  and hepatocellular  cell lines (MTT assay) relative to Gefitinib and Sorafenib (standard references) (Spiro-compounds of types erences)  105]..216–219 19 and sarcosine 20) underwent cycloaddition with 3-aroylmethyleneindol-2-ones 220 under green chemistry conditions in an ionic liquid  to afford the corresponding dispiropyrrolidine-bisoxindoles 221 –silica and afforded the corresponding dispiroindenoquinoxaline-pyrrolizidine 233 —with 2-cyano-2-(2-oxoindolin-3-ylidene)acetate 234 yielded the corresponding dispiropyrrolidine-bisoxindole 235 and dispiropyrrolizidine-bisoxindole 236, respectively (The reaction of azomethine ylides—formed from the condensation of isatin ectively  111]..19 and s238–241 were synthesized by a multi-component cycloaddition reaction of 2-oxo-(3H)-indol-3-ylidine-malononitrile 237 with azomethine ylides  in refluxing toluene containing molecular sieves (3 Å) (Spiropyrrolidine/spiropyrrolizine-oxindoles es (3 Å)  112]..238–241 242 were obtained from a three-atom component reaction of isatin 19, sarcosine 20, and isatylidene malononitrile 237 with high regioselectivity  via A+B pathways. X-Ray studies supported the postulated structures of type 243 (Dispiro-oxindoles of type type 243  113]..243 were245 and dispiro-oxindolopyrrolothiazole 246 were obtained by a multi-component reaction of 3-(ylidene)benzofuran-2-one 244 with isatin 19 and the appropriate α-amino acid  in refluxing methanol. Some of the synthesized compounds revealed promising antimycobacterial (M. tuberculosis H37Rv) properties relative to pyrazinamide (standard reference) (Dispiro-oxindolopyrrolidine ference)  114]..245 and 248 were synthesized regio- and stereoselectively by the reaction of 2-ylidene-1H-carbazol-1-one 247 and in situ-generated azomethine ylide (formed from isatin 19 and benzylamine 57). Some of the obtained compounds revealed antiproliferative properties (MTT assay) via apoptosis induction against MCF7 (breast) and A-549 (lung) cancer cell lines relative to Cisplatin -2-arylidine-1-ketocarbazole 247 with various azomethine ylides generated from sarcosine 20 and di/tri ketone  under microwave irradiation afforded the corresponding ketocarbazolodispiropyrrolidines 249–251 -2-arylidine-1-ketocarbazole 247 with the azomethine ylide formed from isatin 19 and proline 27  relative to Tetracycline and acted as inhibitors of plant fungal pathogen mycelial growth (Fusarium oxysporum and Macrophomena phaseolina) relative to the standard reference—Carbendazim Br generated the corresponding azomethine ylides that was added to 3-(arylidene)-4-piperidones 253 to form the corresponding spiropiperido-pyrrolizines/pyrrolidines 254–256 (Mycobacterium tuberculosis H37Rv (MTB), multi-drug resistant M. tuberculosis (MDR-TB), and Mycobacterium smegmatis (MC2) relative to ethambutol and pyrazinamide (standard references) -4-piperidone 257, ninhydrin 44, o-phenylenediamine 43, and α-amino acid (sarcosine 20 or L-tryptophan 269) in 1-butyl-3-methylimidazoliumbromide ([BMIm]Br) used as an ionic liquid produced the corresponding dispiro compounds 270 and 271 ..279 and E)-3-arylidene-4-chromanone of type 281 with azomethine ylides formed from acenaphthenequinone 25 and sarcosine 20 or proline 27 afforded spiropyrrolidines 282 and 283, respectively, with high regioselectivity (The reaction of (ectivity  136]..E)-3-ary284 or (E)-3-ylidene-4-chromanone 281 with azomethine ylide formed from indenoquinoxaline-11-one (generated from ninhydrin 44 and o-phenylenediamine 43) and L-proline 27 afforded, in the presence of heteropolyacid H4[Si(W3O10)3]‒silica as a catalyst in refluxing acetonitrile, the corresponding dispiroindenoquinoxaline-pyrrolizidines 285 and 286, respectively  in refluxing methanol. When thioproline 22 was used instead of sarcosine 20, the corresponding dispiro derivatives of type 289 were obtained. Some of the synthesized pyrrolizidines revealed antimycobacterial properties (Mycobacterium tuberculosis H37Rv) relative to Cycloserine and Pyrimethamine (standard references). Additionally, mild antiproliferative properties against CCRF-CEM (leukemia), HT29 (ovarian), and MCF7 (breast) cancer cell lines relative to Doxorubicin (MTT assay) were observed -2-(arylidene)-3,4-dihydro-1(2H)-acridinones 290 with azomethine ylides formed from the condensation of isatin 19 and sarcosine 20 or thioproline 22 in refluxing dioxane/methanol afforded the corresponding dispirooxindolyl--1′-ones 291 and 292, respectively (The reaction of (ectively  138]..E)-2--2-(arylidene)-3,4-dihydro-1(2H)-acridinone 290 with azomethine ylides generated from acenaphthoquinone 25 with sarcosine 20 or thioproline 22 in refluxing toluene, respectively , were obtained through the cycloaddition of azomethine ylide (generated from glycine methyl ester 295 and isatin 19) and 5-arylidine-2-thioxothiazolidin-4-one 296 methylidene]-thiazolidin-2,4-dione/2-thioxothiazolidin-4-one 298 with various azomethine ylides formed from isatin 19 and different amino acids  (Another group of benzo[line 27)  141]..h]quinolE)-arylmethylene-octahydro/decahydro cycloalka[d]thiazolopyrimidine-3-ones of type 302 reacted smoothly with azomethine ylides formed from isatin 19 and sarcosine 20 or thioproline 22 in a refluxing methanol-dioxane (1:1) mixture, thereby affording the corresponding spiro-oxindoles 303 and 304, respectively (Various (ectively  142]..E)-arylm306 and 307 and spiro-acenaphthylen-1-ones 308 and 309 were synthesized via a multicomponent reaction of 2-(4-methylbenzylidene)-4H-benzothiazin-3-one 305 and azomethine ylides derived from isatin 19 or acenaphthenequinone 25 with sarcosine 20 or L-proline 27 in refluxing toluene (trimethylsilylmethyl)amine smitters  144]..1 with c1,4-Naphthoquinone313 were obtained by the cycloaddition of azomethine ylides, formed from isatin 19 and sarcosine 20, with 1,4-naphthoquinone 312 in refluxing ethanol , Enterobacter aerogens, Micrococcus luteus, Proteus vulgaris, Klebsiella pneumonia, Salmonella typhimurium, and Salmonella paratyphi-B, and antifungal activities against Malassesia pachydermatis, Candida albicans, and Botyritis cinerea relative to Streptomycin and Ketoconazole (standard references) [Spirooxindoles of type  ethanol . Some oferences) .f]isoindole-1,3′-indolines] of type 315 were synthesized by the cycloaddition of azomethine ylides (formed from isatin 19 and 2-(3-methyl-5-styrylisoxazol-4-ylamino)acetic acids 314) and 1,4-naphthoquinone 312 using ceric ammonium nitrate (CAN) as a catalyst methyl iminium salt 319 in the presence of a catalytic amount of phosphoric acid in DMF as a solvent (The azatricyclic [6-5-7] ring system  solvent  148]..320 was Azomethine ylide’s cycloaddition to aromatic 2π-electron components (aromatic or heteroaromatic) was reviewed in .322 were obtained through thermal azomethine ylide-based cycloaddition of benzaldehydes bearing 3,5-dinitrophenyl 321 and N-substituted α-amino acids. The reaction was assumed to proceed through a regioselective dearomatizing [3+2] cycloaddition with the removal of HNO2, thus yielding the aromatic final product 322 (A series of benzoazepine-fused isoindolines of type duct 322  150]..322 were323–329 underwent [3+2] cycloaddition of azomethine ylide derived from (N-(methoxymethyl)-N-(trimethylsilyl-methyl)-benzylamine) 1, affording the pyrrolidinyl cycloadducts 330–337  cycloaddition with 3-nitropyridines 338 in refluxing toluene -N-(trimethylsilyl-methyl)-benzylamine) 1, affording the pyrrolidinyl-containing analogs 346–351 and 357–361  with meta-dinitro-containing nitrogenous heterocycles 362 in refluxing toluene  cycloaddition in refluxing benzene, affording the corresponding tetrahydro-5aH-oxa-diazoloisoindole 365. Alternatively, conducting the reaction in refluxing MeCN afforded a mixture of 366 and 367. Similarly, analogs with a pyrrolidinyl function (366 and 367) were obtained upon reacting the appropriate analog of 365 in MeCN at room temperature (in the darkness)   155]..364 undeN-(methoxymethyl)-N-(trimethylsilyl-methyl)-benzylamine) with 4-nitrobenzofuroxan 368 afforded either mono 369 or bis 370 cycloadducts based via the substitution of the starting benzofuroxan at the 7-position -pyrrolopyrroles] of type 372 were obtained in good yields by the cycloaddition of azomethine ylide (formed from sarcosine 20 and isatin 19) to the C3-C4 unsaturated bond of maleimide 371. Some of the synthesized compounds revealed promising to moderate antiproliferative properties against HEPG2 (liver), HCT116 (colon), and MCF7 (breast) cancer cell lines (SRB technique) relative to Doxorubicin (standard reference drug) (Spiro[3ce drug)  157]..H-indole371 with azomethine ylide produced from sarcosine 20 and ninhydrin 44 stereoselectively afforded spiropyrroles] 373. Some of the products showed promising antimycobacterial (M. tuberculosis H37Rv) properties relative to Cycloserine  (Tetracyclic pyrroloisoquinolines of type e (CTAB)  160]..378 werec]pyrrole-1-carboxylates 380 and 381 were obtained by reacting N-phenylmaleimide 371 with a series of azomethine ylides generated in situ from sulfanyl-substituted imines of glycine esters 379 acrylonitriles of type 385 in refluxing benzene caprolactamate [Rh2(cap)4] in the presence of tert-butyl hydroperoxide (TBHP)   37]..a]isoquib]thiophene-1,1-dioxide 390 with a thermally generated azomethine ylide from aziridines 391 in refluxing dry benzene afforded the cycloadducts of type 392  and paraformaldehyde 316  in refluxing dry toluene under N2 (inert atmosphere) to produce glucosylspiro-oxindoles 406–408 in a highly regio- and stereoselective manner  in refluxing toluene to afford the corresponding spiropyrrolidine/spiro-pyrrolizidine/spiroindolizidine-oxindoles 410 and 411 -trifluoroacetate (AgTFA) in tetrahydrofuran at room temperature  from stabilized azomethine ylides of type 430 and sugar-derived enones (429a and 429b) through the [3+2]-cycloaddition reaction in the presence of silver acetate (AgOAc) and DBU in acetonitrile. The cycloadducts were further used to synthesize enantiomeric polyhydroxyalkylpyrrolidines as potential β-galactofuranosidase inhibitors -N-(trimethylsilyl-methyl)-benzylamine) 1 cycloaddition to isatoic anhydride 432 in trifluoroacetic acid in the presence of molecular sieves (4 Å)   179]..433 wereAmong various methods, the [3+2]-cycloaddition reaction of azomethine ylides is one of the most adopted protocols for the formation of pyrrolidine and pyrrole systems. The chemistry of azomethine ylides has progressed significantly in the last two decades. Azomethine ylides have been used for the synthesis of many stereoselective natural products, core ring systems of natural products, and several bioactive molecules containing multiple chiral centers. The cycloaddition of a three-atom component to an appropriate unsaturated substrate, namely, the unsaturated 2π-electron component, is the most embraced approach to the synthesis of five-membered heterocyclic compounds. By using various unsaturated 2π-electron components in reaction with in situ-generated azomethine ylides, a plethora of pyrrolidinyl-containing heterocycles can be obtained in a highly regio- and stereoselective manner. As a result of intermolecular cycloadditions, one new ring with a defined stereochemistry is formed; however, when the three-atom component and the substrate are part of the same molecule, the cycloaddition is intramolecular and leads to a more complex molecular architecture that is difficult to access by other routes, namely, through the use of new bicyclic systems.This review summarizes the synthesis of some of the most important compounds resulting from the [3+2]-cycloaddition reactions of azomethine ylides with various olefinic  unsaturated 2π-electron components and highlights their potential therapeutic significance. We believe the compiled subject will develop interest within this field among the research community and encourage them to develop a wider variety of asymmetric [3+2]-cycloaddition reaction strategies for the synthesis of complex molecules.