Mapping enzyme activity in living systems by real-time mid-infrared photothermal imaging of nitrile chameleons.

Simultaneous spatial mapping of the activity of multiple enzymes in a living system can elucidate their functions in health and disease. However, methods based on monitoring fluorescent substrates are limited. Here, we report the development of nitrile (C≡N)-tagged enzyme activity reporters, named nitrile chameleons, for the peak shift between substrate and product. To image these reporters in real time, we developed a laser-scanning mid-infrared photothermal imaging system capable of imaging the enzymatic substrates and products at a resolution of 300 nm. We show that when combined, these tools can map the activity distribution of different enzymes and measure their relative catalytic efficiency in living systems such as cancer cells, Caenorhabditis elegans, and brain tissues, and can be used to directly visualize caspase-phosphatase interactions during apoptosis. Our method is generally applicable to a broad category of enzymes and will enable new analyses of enzymes in their native context.

Accelerating Cellular Uptake with Unnatural Amino Acid for Inhibiting Immunosuppressive Cancer Cells.

Targeting immunosuppressive metastatic cancer cells is a key challenge in therapy. We recently have shown that a rigid-rod aromatic, pBP-NBD, that responds to enzymes and kill immunosuppressive metastatic osteosarcoma (mOS) and castration resistant prostate cancer (CRPC) cells in mimetic bone microenvironment. However, pBP-NBD demonstrated moderate efficacy against CRPC cells. To enhance activity, we incorporated the unnatural amino acid L- or D-4,4'-biphenylalanine (L- or D-BiP) into pBP-NBD, drastically increasing cellular uptake and CRPC inhibition. Specifically, we inserted BiP into pBP-NBD to target mOS (Saos2 and SJSA1) and CRPC (VCaP and PC3) cells with overexpressed phosphatases. Our results show that the D-peptide backbone with an aspartate methyl diester at the C-terminal offers the highest activity against these immunosuppressive mOS and CRPC cells. Importantly, imaging shows that the peptide assemblies almost instantly enter the cells and accumulate primarily within the endoplasmic reticulum of Saos2, SJSA1, and PC3 cells and at the lysosomes of VCaP cells. By using BiP to boost cellular uptake and self-assembly within cancer cells, this work illustrates an unnatural hydrophobic amino acid as a versatile and effective residue to boost endocytosis of synthetic peptides for intracellular self-assembly.

Enzyme Responsive Rigid-Rod Aromatics Target "Undruggable" Phosphatases to Kill Cancer Cells in a Mimetic Bone Microenvironment.

Bone metastasis remains a challenge in cancer treatment. Here we show enzymatic responsive rigid-rod aromatics acting as the substrates of "undruggable" phosphatases to kill cancer cells in a mimetic bone microenvironment. By phosphorylation and conjugating nitrobenzoxadiazole (NBD) to hydroxybiphenylcarboxylate (BP), we obtained pBP-NBD (1P) as a substrate of both acid and alkaline phosphatases. 1P effectively kills both metastatic castration-resistant prostate cancer cells (mCRPCs) and osteoblast mimic cells in their coculture. 1P enters Saos2 almost instantly to target the endoplasmic reticulum (ER) of the cells. Co-culturing with Saos2 cells boosts the cellular uptake of 1P by mCRPCs. Cryo-EM reveals the nanotube structures of both 1P (2.4 Å resolution, pH 5.6) and 1 (2.2 Å resolution, pH 7.4). The helical packing of both nanotubes is identical, held together by strong pi-stacking interactions. Besides reporting the atomistic structure of nanotubes formed by the assembly of rigid-rod aromatics, this work expands the pool of molecules for designing EISA substrates that selectively target TME.

Enzymatic Noncovalent Synthesis.

Enzymatic reactions and noncovalent (i.e., supramolecular) interactions are two fundamental nongenetic attributes of life. Enzymatic noncovalent synthesis (ENS) refers to a process where enzymatic reactions control intermolecular noncovalent interactions for spatial organization of higher-order molecular assemblies that exhibit emergent properties and functions. Like enzymatic covalent synthesis (ECS), in which an enzyme catalyzes the formation of covalent bonds to generate individual molecules, ENS is a unifying theme for understanding the functions, morphologies, and locations of molecular ensembles in cellular environments. This review intends to provide a summary of the works of ENS within the past decade and emphasize ENS for functions. After comparing ECS and ENS, we describe a few representative examples where nature uses ENS, as a rule of life, to create the ensembles of biomacromolecules for emergent properties/functions in a myriad of cellular processes. Then, we focus on ENS of man-made (synthetic) molecules in cell-free conditions, classified by the types of enzymes. After that, we introduce the exploration of ENS of man-made molecules in the context of cells by discussing intercellular, peri/intracellular, and subcellular ENS for cell morphogenesis, molecular imaging, cancer therapy, and other applications. Finally, we provide a perspective on the promises of ENS for developing molecular assemblies/processes for functions. This review aims to be an updated introduction for researchers who are interested in exploring noncovalent synthesis for developing molecular science and technologies to address societal needs.

Intranuclear Nanoribbons for Selective Killing of Osteosarcoma Cells.

Herein, we show intranuclear nanoribbons formed upon dephosphorylation of leucine-rich L- or D-phosphopeptide catalyzed by alkaline phosphatase (ALP) to selectively kill osteosarcoma cells. Being dephosphorylated by ALP, the peptides are first transformed into micelles and then converted into nanoribbons. The peptides/assemblies first aggregate on cell membranes, then enter cells via endocytosis, and finally accumulate in nuclei (mainly in nucleoli). Proteomics analysis suggests that the assemblies interact with histone proteins. The peptides kill osteosarcoma cells rapidly and are nontoxic to normal cells. Moreover, the repeated stimulation of the osteosarcoma cells by the peptides sensitizes the cancer cells rather than inducing resistance. This work not only illustrates a novel mechanism for nucleus targeting, but may also pave a new way for selectively killing osteosarcoma cells and minimizing drug resistance.

[Prevalence and risk factors of obesity in children with Diamond-Blackfan anemia]. / å ˆå¤©æ€§çº¯çº¢ç»†èƒžå†ç”Ÿéšœç¢æ€§è´«è¡€æ‚£å„¿è‚¥èƒ–å‘ç”Ÿæƒ å†µåŠå½±å“å› ç´ åˆ†æž.

OBJECTIVES: To investigate the distribution of body mass index (BMI) and risk factors for obesity in children with Diamond-Blackfan Anemia (DBA). METHODS: The children with DBA who attended National Clinical Research Center for Blood Diseases, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, from January 2003 to December 2020 were enrolled as subjects. The related clinical data and treatment regimens were recorded. The height and weight data measured within 1 week before or after follow-up time points were collected to calculate BMI. The risk factors for obesity were determined by multivariate regression analysis in children with DBA. RESULTS: A total of 129 children with DBA were enrolled, among whom there were 80 boys (62.0%) and 49 girls (38.0%), with a median age of 49 months (range 3-189 months). The prevalence rate of obesity was 14.7% (19/129). The multivariate logistic regression analysis showed that the absence of ribosomal protein gene mutation was closely associated with obesity in children with DBA (adjusted OR=3.63, 95%CI: 1.16-11.38, adjusted P=0.027). In children with glucocorticoid-dependent DBA, obesity was not associated with age of initiation of glucocorticoid therapy, duration of glucocorticoid therapy, and maintenance dose of glucocorticoids (P>0.05). CONCLUSIONS: There is a high prevalence rate of obesity in children with DBA, and the absence of ribosomal protein gene mutation is closely associated with obesity in children with DBA.

Enzymatically Forming Intranuclear Peptide Assemblies for Selectively Killing Human Induced Pluripotent Stem Cells.

Tumorigenic risk of undifferentiated human induced pluripotent stem cells (iPSCs), being a major obstacle for clinical application of iPSCs, requires novel approaches for selectively eliminating undifferentiated iPSCs. Here, we show that an l-phosphopentapeptide, upon the dephosphorylation catalyzed by alkaline phosphatase (ALP) overexpressed by iPSCs, rapidly forms intranuclear peptide assemblies made of α-helices to selectively kill iPSCs. The phosphopentapeptide, consisting of four l-leucine residues and a C-terminal l-phosphotyrosine, self-assembles to form micelles/nanoparticles, which transform into peptide nanofibers/nanoribbons after enzymatic dephosphorylation removes the phosphate group from the l-phosphotyrosine. The concentration of ALP and incubation time dictates the morphology of the peptide assemblies. Circular dichroism and FTIR indicate that the l-pentapeptide in the assemblies contains a mixture of an α-helix and aggregated strands. Incubating the l-phosphopentapeptide with human iPSCs results in rapid killing of the iPSCs (=<2 h) due to the significant accumulation of the peptide assemblies in the nuclei of iPSCs. The phosphopentapeptide is innocuous to normal cells (e.g., HEK293 and hematopoietic progenitor cell (HPC)) because normal cells hardly overexpress ALP. Inhibiting ALP, mutating the l-phosphotyrosine from the C-terminal to the middle of the phosphopentapeptides, or replacing l-leucine to d-leucine in the phosphopentapeptide abolishes the intranuclear assemblies of the pentapeptides. Treating the l-phosphopentapeptide with cell lysate of normal cells (e.g., HS-5) confirms the proteolysis of the l-pentapeptide. This work, as the first case of intranuclear assemblies of peptides, not only illustrates the application of enzymatic noncovalent synthesis for selectively targeting nuclei of cells but also may lead to a new way to eliminate other pathological cells that express a high level of certain enzymes.

Long-term outcomes of 172 children with severe aplastic anemia treated with rabbit antithymocyte globulin and cyclosporine.

This study retrospectively analyzed the clinical outcome of 172 children with newly diagnosed severe aplastic anemia (SAA) between January 2008 and April 2018, who received rabbit antithymocyte globulin (ATG) and cyclosporine (CsA) as first-line treatment. The median age at diagnosis was 5 years (range, 1-14). The overall response rates were 22.7%, 45.3%, and 61% at 40 days, 3 months, and 6 months, respectively, after rabbit ATG. In multivariate analysis, mild disease severity was the only predictor of favorable response at 6 months (P = 0.006). In the present study, median follow-up period was 63 months (range, 1-135). The 5-year overall survival (OS) and failure-free survival (FFS) rates were 90.5% and 70.4%. Multivariate analysis showed that erythroid burst-forming units (BFU-E) > 2/105 bone marrow mononuclear cell (BMMNC) (P = 0.037) and time interval before IST ≤ 30 days (P = 0.017) were independent positive predictors for OS, meanwhile BFU-E > 2/105BMMNC (P = 0.029) was the only favorable prognostic factor for FFS.

Low absolute neutrophil count during induction therapy is an adverse prognostic factor in childhood acute lymphoblastic leukaemia.

Variation in normal blood cells during chemotherapy has not been recognised as a risk factor guiding chemotherapy in childhood acute lymphoblastic leukaemia (ALL). This study aims to explore whether variations in normal haematopoiesis determine prognosis as well as to improve risk-stratified treatment in childhood ALL. A retrospective study of 279 cases of ALL treated with the CCCG-ALL-2015 regimen in the Division of Pediatric Blood Diseases Center, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, from May 2015 to January 2017 was performed to analyse the prognostic impact of blood cell levels on day 19 of induction therapy by Kaplan-Meier method. Patients with childhood ALL with absolute neutrophil count (ANC) ≤ 90 cells/µl, absolute monocyte count (AMC) ≤ 10 cells/µl or absolute lymphocyte count (ALC) ≤ 1000 cells/µl on day 19 of induction therapy had a lower event-free survival (EFS) rate than those with higher values (all P < 0.05). Multivariate analysis confirmed that ANC ≤ 90 cells/µl and ALC ≤ 1000 cells/µl were independent adverse prognostic factors (HR = 1.981 and 2.162, respectively, both P < 0.05). Among patients with minimal residual disease (MRD) < 1% on day 19 of induction therapy, those with ANC ≤ 90 cells/µl had lower EFS than those with ANC > 90 cells/µl (70.8 ± 6.1% vs 86.4 ± 3.1%, P = 0.001). In the subgroup with the BCR/ABL1 fusion gene, patients with ANC ≤ 90 cells/µl on day 19 of induction therapy also had lower EFS than those with ANC > 90 cells/µl (34.4 ± 25.2% vs 25.0 ± 21.7%, P = 0.041). ANC and ALC during induction therapy are independent prognostic factors for childhood ALL. ANC contributes to guiding the prognosis of patients with low-level MRD or the BCR/ABL1 fusion gene.

Phosphobisaromatic motifs enable rapid enzymatic self-assembly and hydrogelation of short peptides.

Enzyme-instructed self-assembly (EISA) and hydrogelation is a versatile approach for generating soft materials. Most of the substrates for alkaline phosphatase catalysed EISA utilize phosphotyrosine (pTyr) as the enzymatic trigger for EISA and hydrogelation. Here we show the first example of phosphonaphthyl (pNP) and phosphobiphenyl (pBP) motifs acting as faster enzymatic triggers than phosphotyrosine for EISA and hydrogelation. This work illustrates novel enzyme triggers for rapid enzymatic self-assembly and hydrogelation.

Combination of umbilical cord mesenchymal stem cells and standard immunosuppressive regimen for pediatric patients with severe aplastic anemia.

BACKGROUND: Defects of bone marrow mesenchymal stem cells (BM-MSCs) in proliferation and differentiation are involved in the pathophysiology of aplastic anemia (AA). Infusion of umbilical cord mesenchymal stem cells (UC-MSCs) may improve the efficacy of immunosuppressive therapy (IST) in childhood severe aplastic anemia (SAA). METHODS: We conducted an investigator-initiated, open-label, and prospective phase IV trial to evaluate the safety and efficacy of combination of allogenic UC-MSCs and standard IST for pediatric patients with newly diagnosed SAA. In mesenchymal stem cells (MSC) group, UC-MSCs were injected intravenously at a dose of 1 × 106/kg per week starting on the 14th day after administration of rabbit antithymocyte globulin (ATG), for a total of 3 weeks. The clinical outcomes and adverse events of patients with UC-MSCs infusion were assessed when compared with a concurrent control group in which patients received standard IST alone. RESULTS: Nine patients with a median age of 4 years were enrolled as the group with MSC, while the data of another 9 childhood SAA were analysed as the controls. Four (44%) patients in MSC group developed anaphylactic reactions which were associated with rabbit ATG. When compared with the controls, neither the improvement of blood cell counts, nor the change of T-lymphocytes after IST reached statistical significance in MSC group (both p > 0.05) and there were one (11%) patient in MSC group and two (22%) patients in the controls achieved partial response (PR) at 90 days after IST. After a median follow-up of 48 months, there was no clone evolution occurring in both groups. The 4-year estimated overall survival (OS) rate in two groups were both 88.9% ± 10.5%, while the 4-year estimated failure-free survival (FFS) rate in MSC group was lower than that in the controls (38.1% ± 17.2% vs. 66.7% ± 15.7%, p = 0.153). CONCLUSIONS: Concomitant use of IST and UC-MSCs in SAA children is safe but may not necessarily improve the early response rate and long-term outcomes. This clinical trial was registered at ClinicalTrials.gov, identifier: NCT02218437 (registered October 2013).

Enzymatic Assemblies of Thiophosphopeptides Instantly Target Golgi Apparatus and Selectively Kill Cancer Cells*.

Changing an oxygen atom of the phosphoester bond in phosphopeptides by a sulfur atom enables instantly targeting Golgi apparatus (GA) and selectively killing cancer cells by enzymatic self-assembly. Specifically, conjugating cysteamine S-phosphate to the C-terminal of a self-assembling peptide generates a thiophosphopeptide. Being a substrate of alkaline phosphatase (ALP), the thiophosphopeptide undergoes rapid ALP-catalyzed dephosphorylation to form a thiopeptide that self-assembles. The thiophosphopeptide enters cells via caveolin-mediated endocytosis and macropinocytosis and instantly accumulates in GA because of dephosphorylation and formation of disulfide bonds in Golgi by themselves and with Golgi proteins. Moreover, the thiophosphopeptide potently and selectively inhibits cancer cells (HeLa) with the IC50 (about 3 µM), which is an order of magnitude more potent than that of the parent phosphopeptide.

Enzymatic Insertion of Lipids Increases Membrane Tension for Inhibiting Drug Resistant Cancer Cells.

Although lipids contribute to cancer drug resistance, it is challenging to target diverse range of lipids. Here, we show enzymatically inserting exceedingly simple synthetic lipids into membranes for increasing membrane tension and selectively inhibiting drug resistant cancer cells. The lipid, formed by conjugating dodecylamine to d-phosphotyrosine, self-assembles to form micelles. Enzymatic dephosphorylation of the micelles inserts the lipids into membranes and increases membrane tension. The micelles effectively inhibit a drug resistant glioblastoma cell (T98G) or a triple-negative breast cancer cell (HCC1937), without inducing acquired drug resistance. Moreover, the enzymatic reaction of the micelles promotes the accumulation of the lipids in the membranes of subcellular organelles (e.g., endoplasmic reticulum (ER), Golgi, and mitochondria), thus activating multiple regulated cell death pathways. This work, in which for the first time membrane tension is increased to inhibit cancer cells, illustrates a new and powerful supramolecular approach for antagonizing difficult drug targets.

The ratio of hydrogelator to precursor controls the enzymatic hydrogelation of a branched peptide.

Here, we report an apparently counterintuitive observation, in which a lower volume fraction of a branched peptide forms a stronger hydrogel after an enterokinase (ENTK) cleaves off the branch from the peptide. By varying the ratios of the branched peptide and ENTK and analysing the ratio of hydrogelator to precursor (H/P) in the enzymatic proteolysis reaction, our study shows that the H/P ratio controls the critical strain of the hydrogel formed, through enzymatic cleavage of the branch from the peptide. This work demonstrates that emergent properties (e.g., hydrogelation) of peptide assemblies, resulting from enzymatic noncovalent synthesis (ENS), are context-dependent, while also providing insights for developing dynamic soft materials via ENS.

[Clinical features and prognosis of core binding factor acute myeloid leukemia in children].

OBJECTIVE: To study the clinical features and prognosis of core binding factor acute myeloid leukemia (CBF-AML) in children. METHODS: A retrospective analysis was performed from the chart review data of children who were newly diagnosed with CBF-AML in the Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences, from August 2009 to November 2015. According to the type of fusion gene, the children were divided into CBFB-MYH11 and AML1-ETO groups. Clinical features and prognosis were analyzed and compared between the two groups. RESULTS: A total of 91 children with CBF-AML were enrolled in this study, among whom there were 74 (81%) in the AML1-ETO group and 17 (19%) in the CBFB-MYH11 group. Additional chromosomal abnormalities were observed in 38 children (42%), and deletion of sex chromosome was the most common abnormality and was observed in 28 children (31%). After the first course of induction treatment, the complete remission rate was 97% (88/91), the recurrence rate was 29% (26/91), the 5-year event-free survival (EFS) rate was 65%±6%, and the 5-year overall survival (OS) rate was 75%±5%. There were no significant differences between the AML1-ETO and CBFB-MYH11 groups in 5-year EFS rate (62%±7% vs 77%±11%, P>0.05) or 5-year OS rate (72%±6% vs 88%±9%, P>0.05). CONCLUSIONS: AML1-ETO is the main type of fusion gene in children with CBF-AML, and deletion of sex chromosome is the most common type of additional chromosomal abnormalities. Children with CBF-AML often have a good prognosis, and the children with AML1-ETO have a similar prognosis to those with CBFB-MYH11.

Predicting response to porcine antilymphocyte globulin plus cyclosporine A in children with acquired severe aplastic anemia.

BACKGROUND: In severe aplastic anemia (SAA), predictive markers of response to immunosuppressive therapy (IST) of porcine antilymphocyte globulin (pALG) have not been well defined. We investigated whether clinical and laboratory findings before treatment could predict response in a pediatric cohort. METHODS: In this study, we included 70 newly diagnosed SAA children and treated them with pALG. The response rate was documented during follow-up. The log-rank test compared response rates between the potential predictive factors. RESULTS: The response rate was 57.1% at 24 months follow-up. In log-rank test, mild disease severity was the most significant predictive marker of better response (P < 0.001); SAA patients with higher absolute reticulocyte count (ARC) and platelet level showed a higher response rate (both P < 0.001). Although insignificantly, elderly children and male sex show better response rate after treatment. The response rate worsened when the time interval before IST was more than 60 days. CONCLUSION: Modified IST with pALG was suitable for SAA children, and favorable response correlates with mild disease severity was identified. ARC and platelet status also appeared to be a reproducible prognostic model for response rate. IST should be started as soon as possible, given that the response rate worsens as the interval between diagnosis and treatment increases.

[Clinical features of Wiskott-Aldrich syndrome: an analysis of 13 cases].

OBJECTIVE: To study the clinical features of Wiskott-Aldrich syndrome (WAS) in children. METHODS: A retrospective analysis was performed for the clinical data of 13 children with WAS. RESULTS: All 13 children were boys, with a median age of onset of 3 months (range 1-48 months) and a median age of 24 months (range 1-60 months) at the time of diagnosis. Of the 13 children, only 3 had typical WAS and the remaining 10 children had X-linked thrombocytopenia (XLT). The mean WAS score was 2 (range 1-3), the mean platelet count was 20.5×109/L [range (13-46)×109/L], and the mean platelet volume was 8.1 fl (range 6.7-12.1 fl). Lymphocyte subsets and immunoglobulins were measured for 4 children, among whom 1 (25%) had a reduction in both the percentage of CD3+T cells per lymphocyte and lymphocyte per nuclear cells, 1(25%) had a reduction in CD3-CD56+ NK cells. Among these 4 children, 1 (25%) had an increase in IgG, 2 (50%) had a reduction in IgM, 1 (25%) had a reduction in IgA, and 4 (100%) had an increase in IgE. A total of 14 gene mutations belonging to 13 types were found in 13 children, among which there were 9 missense mutations (65%), 2 splicing mutations (14%), 2 nonsense mutation (14%), and 1 frameshift mutation (7%). The median follow-up time was 39 months (range 3-62 months), and all 13 children survived. CONCLUSIONS: Children with WAS often have a young age of onset, and most of them are boys. Major clinical features include thrombocytopenia with a reduction in platelet volume. Missense mutation is the main type of gene mutation.

The elevation of red blood cell distribution width is an independent prognostic factor for juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia (JMML) is a disorder characterized by the simultaneous presence of myeloproliferative and myelodysplastic features, primarily affecting infants and young children. Due to the heterogeneous genetic background among patients, the current clinical and laboratory prognostic features are insufficient for accurately predicting outcomes. Thus, there is a pressing need to identify novel prognostic indicators. Red cell distribution width (RDW) is a critical parameter reflecting the variability in erythrocyte size. Recent studies have emphasized that elevated RDW serves as a valuable predictive marker for unfavorable outcomes across various diseases. However, the prognostic role of RDW in JMML remains unclear. Patients with JMML from our single-center cohort between January 2008 and December 2019 were included. Overall, 77 patients were eligible. Multivariate Cox proportional hazard models showed that patients with red cell distribution width coefficient of variation (RDW-CV) >17.35% at diagnosis were susceptible to much worse overall survival rate (hazard ratio [HR] = 5.22, confidence interval [CI] = 1.50-18.21, P = .010). Besides, the combination of RDW elevation and protein phosphatase non-receptor type 11 (PTPN11) mutation was likely to predict a subgroup with the worst outcomes in our cohort. RDW is an independent prognostic variable in JMML subjects. RDW may be regarded as an inexpensive biomarker to predict the clinical outcome in patients with JMML.

Evaluating Alkaline Phosphatase-Instructed Self-Assembly of d-Peptides for Selectively Inhibiting Ovarian Cancer Cells.

Cancer is a major public health concern requiring novel treatment approaches. Enzyme-instructed self-assembly (EISA) provides a unique approach for selectively inhibiting cancer cells. However, the structure and activity correlation of EISA remains to be explored. This study investigates new EISA substrates of alkaline phosphatase (ALP) to hinder ovarian cancer cells. Analogues 2-8 were synthesized by modifying the amino acid residues of a potent EISA substrate 1 that effectively inhibits the growth of OVSAHO, a high-grade serous ovarian cancer (HGSOC) cell line. The efficacy of 2-8 against OVSAHO was assessed, along with the combination of substrate 1 with clinically used drugs. The results reveal that substrate 1 displays the highest cytotoxicity against OVSAHO cells, with an IC50 of around 8 µM. However, there was limited synergism observed between substrate 1 and the tested clinically used drugs. These findings indicate that EISA likely operates through a distinct mechanism that necessitates further elucidation.

Cell spheroid creation by transcytotic intercellular gelation.

Cell spheroids bridge the discontinuity between in vitro systems and in vivo animal models. However, inducing cell spheroids by nanomaterials remains an inefficient and poorly understood process. Here we use cryogenic electron microscopy to determine the atomic structure of helical nanofibres self-assembled from enzyme-responsive D-peptides and fluorescent imaging to show that the transcytosis of D-peptides induces intercellular nanofibres/gels that potentially interact with fibronectin to enable cell spheroid formation. Specifically, D-phosphopeptides, being protease resistant, undergo endocytosis and endosomal dephosphorylation to generate helical nanofibres. On secretion to the cell surface, these nanofibres form intercellular gels that act as artificial matrices and facilitate the fibrillogenesis of fibronectins to induce cell spheroids. No spheroid formation occurs without endo- or exocytosis, phosphate triggers or shape switching of the peptide assemblies. This study-coupling transcytosis and morphological transformation of peptide assemblies-demonstrates a potential approach for regenerative medicine and tissue engineering.