S1. The western diet extensively reprograms Lgr5hi stem cells that expands with time and is reversible. This includes the TCA cycle and Oxphos pathways.S2. The western diet elevates and suppresses multiple pathways by 3 months, most persisting to 12 months, and reversible by switching to control diet.S3. The western diet suppresses expression of a large number of genes encoding subunits of each of the 5 complexes of mitochondrial electron transport.S4. Mouse genotyping identified presence of the wild-type, or heterozygous or homozygous conditionally targeted Ppargc1a allele.S5. Rapid dietary cross-over experiment of Fig 3.S6. scRNAseq analysis of Bmi1+ derived cells.S7. Inflammatory response to NWD1.

Single cell analyses of cell progeny of the alternate stem cells mobilized by the western diet to maintain the mucosa.

The western diet causes chronic pro-tumorigenic inflammation.

The western diet elevates and suppresses multiple pathways by 3 months, most persisting to 12 months, and reversible by switching to control diet.

Targeted knockout of Ppargc1a in Lgr5hi cells was confirmed, and trajectory analyses of dietary and genetic alterations confirmed by marker expression.

The western diet suppresses expression of a large number of genes encoding subunits of each of the 5 complexes of mitochondrial electron transport.

Single cell RNAsequencing following 4 days of dietary switch, or then switch back and comparison to pathways altered in stem cells by Ppargc1a genetic inactivation.

Six Schiff bases (H₂L1-H₂L6) were synthesized by the reaction of 3,5-substituted salicylaldehyde (F/Cl/Br) with 2-amino-4-substituted phenol (F/Cl/Br), and their Ni(II) complexes (C1-C6) were obtained with NiCl₂·6H₂O, respectively. All complexes were characterized via infrared spectroscopy, NMR spectra analysis, UV-visible spectroscopy, thermal stability analysis, elemental analysis, X-ray powder diffraction, single-crystal X-ray diffraction, ICP-OES analysis, and lipophilicity assay. Single crystal X-ray analysis reveals that complexes C1, C4, C5, and C6 crystallize in a monoclinic system with space group I41/a, while complexes C2 and C3 crystallize in the trigonal system with space group P-1. The cytotoxicity of polyhalogenated 2-amino-phenol Schiff base ligands and their metal complexes on breast cancer cells (SKBR3), nasopharyngeal carcinoma cells (CNE-2Z), lung cancer cells (A549), rectal cancer cells (HCT-116) and two types of normal human cells (IOSE 80 and MCF-10 A) was determined by MTT assay. The results showed that C1-C6 complexes exhibited stronger anticancer efficacy on SKBR3 cells than ligands and cisplatin. Among them, C5, C6 and C4 could increase intracellular reactive oxygen species levels, reduce the mitochondrial membrane potential, arrest cells in the G0/G1 phase, induce SKBR3 cell apoptosis in a dose-dependent manner and promote Bax protein expression and inhibit Bcl-2 protein expression in vitro experiments. In vivo experiments showed that the C5 complex significantly suppressed SKBR3 tumor growth with low toxicity to major organs in the nude mice model.

The western diet extensively reprograms Lgr5hi stem cells that expands with time and is reversible. This includes the TCA cycle and Oxphos pathways

A non-platinum-metal decanuclear complex [Ni10L4(CH3COO)8 (C2H5OH)8]·8(C2H5OH) (Ni10 complex) has been developed with a tri-dentate 2,3-dihydroxybenzaldehyde-2-aminophenol Schiff base ligand (H3L). Single crystal X-ray analysis reveals that the Ni10 complex displays a sandwich loaf-shaped decanuclear structure and its anticancer activity was evaluated. The cell cytotoxicity results indicating that the Ni10 complex is most effective to human breast cancer cells MDA-MB-231 and its mechanism were further investigated. Flow cytometry analysis showed that the Ni10 complex triggered cell cycle arrest and induced apoptosis of MDA-MB-231 cells. Western blot analysis of the changes of intracellular protein expression showed that Ni10 triggers MDA-MB-231 apoptosis through mitochondrial mediated apoptosis signaling pathways. In vivo experiments showed that the Ni10 complex significantly suppressed breast tumor growth with low toxicity against major organs in a nude mice model. The good treatment effect, low toxicity and pharmacological mechanisms of the decanuclear NiII complex may provide a clue for the research and development of non-platinum multinuclear based chemotherapeutic drugs.

Three metal complexes [Cu(HL)Cl](C2H5OH)2] (complex 1), [Ni4L2(CH3COO)4(C2H5OH)4] (complex 2) and [CoHL2] (complex 3) based on 2,3-dihydroxybenzaldehyde-2-(2-aminophenyl)benzimidazole Schiff base (H2L) were synthesized by solvothermal reaction. The three complexes were characterized by FT-IR spectrometer, elemental analysis, thermogravimetric analysis, and X-ray single-crystal diffraction. X-ray single-crystal diffraction analysis confirmed that complex 1 belongs to monoclinic crystal system with P21/c space group, and complex 3 crystallizes in trigonal crystal system with R-3 space group. However, complex 2 crystallized in triclinic crystal system with P-1 space group and exhibits centrosymmetric tetranuclear crystallographic structure. Besides, MTT assay was studied antitumor activities against different tumor cells. The results show that the three complexes all have fine anticancer activities. Among them, complex 2 has significantly better anticancer properties against CNE-2Z cells than that of cisplatin. Finally, apoptosis and cell migration were studied to prove that complex 2 could inhibit tumor cells from proliferating in the manner of apoptosis and migration.

The intestinal epithelium consists of cells derived from continuously cycling Lgr5hi intestinal stem cells (Lgr5hi ISCs) that mature developmentally in an ordered fashion as the cells progress along the crypt-luminal axis. Perturbed function of Lgr5hi ISCs with aging is documented, but the consequent impact on overall mucosal homeostasis has not been defined. Using single-cell RNA sequencing, the progressive maturation of progeny was dissected in the mouse intestine, which revealed that transcriptional reprogramming with aging in Lgr5hi ISCs retarded the maturation of cells in their progression along the crypt-luminal axis. Importantly, treatment with metformin or rapamycin at a late stage of mouse lifespan reversed the effects of aging on the function of Lgr5hi ISCs and subsequent maturation of progenitors. The effects of metformin and rapamycin overlapped in reversing changes of transcriptional profiles but were also complementary, with metformin more efficient than rapamycin in correcting the developmental trajectory. Therefore, our data identify novel effects of aging on stem cells and the maturation of their daughter cells contributing to the decline of epithelial regeneration and the correction by geroprotectors.

Abstract New Western-style diet 1 (NWD1), a purified diet establishing mouse exposure to key nutrients recapitulating levels that increase human risk for intestinal cancer, reproducibly causes mouse sporadic intestinal and colonic tumors reflecting human etiology, incidence, frequency, and lag with developmental age. Complex NWD1 stem cell and lineage reprogramming was deconvolved by bulk and single-cell RNA sequencing, single-cell Assay for Transposase-Accessible Chromatin using sequencing, functional genomics, and imaging. NWD1 extensively, rapidly, and reversibly, reprogrammed Lgr5hi stem cells, epigenetically downregulating Ppargc1a expression, altering mitochondrial structure and function. This suppressed Lgr5hi stem cell functions and developmental maturation of Lgr5hi cell progeny as cells progressed through progenitor cell compartments, recapitulated by Ppargc1a genetic inactivation in Lgr5hi cells in vivo. Mobilized Bmi1+, Ascl2hi cells adapted lineages to the nutritional environment and elevated antigen processing and presentation pathways, especially in mature enterocytes, causing chronic, protumorigenic low-level inflammation. There were multiple parallels between NWD1 remodeling of stem cells and lineages with pathogenic mechanisms in human inflammatory bowel disease, also protumorigenic. Moreover, the shift to alternate stem cells reflects that the balance between Lgr5-positive and -negative stem cells in supporting human colon tumors is determined by environmental influences. Stem cell and lineage plasticity in response to nutrients supports historic concepts of homeostasis as a continual adaptation to environment, with the human mucosa likely in constant flux in response to changing nutrient exposures. Implications: Although oncogenic mutations provide a competitive advantage to intestinal epithelial cells in clonal expansion, the competition is on a playing field dynamically sculpted by the nutritional environment, influencing which cells dominate in mucosal maintenance and tumorigenesis.

Retrotransposons are a class of transposable elements capable of self-replication and insertion into new genomic locations. Across species, the mobilization of retrotransposons in somatic cells has been suggested to contribute to the cell and tissue functional decline that occurs during aging. Retrotransposons are broadly expressed across cell types, and de novo insertions have been observed to correlate with tumorigenesis. However, the extent to which new retrotransposon insertions occur during normal aging and their effect on cellular and animal function remains understudied. Here, we use a single nucleus whole genome sequencing approach in Drosophila to directly test whether transposon insertions increase with age in somatic cells. Analyses of nuclei from thoraces and indirect flight muscles using a newly developed pipeline, Retrofind, revealed no significant increase in the number of transposon insertions with age. Despite this, reducing the expression of two different retrotransposons, 412 and Roo, extended lifespan, but did not alter indicators of health such as stress resistance. This suggests a key role for transposon expression and not insertion in regulating longevity. Transcriptomic analyses revealed similar changes to gene expression in 412 and Roo knockdown flies and highlighted changes to genes involved in proteolysis and immune function as potential contributors to the observed changes in longevity. Combined, our data show a clear link between retrotransposon expression and aging.

The Schiff base ligand (5-bromosalicylaldehyde)-2-aminophenol (H 2 L) and its complex were synthesized and characterized. The crystal structure was established by single-crystal X-ray diffraction, which showed that the monomer [Ni 4 L 4 (CH 3 CH 2 OH) 4 ]·1.46(CH 3 CH 2 OH) (the Ni(II) complex) crystallized in the tetragonal structure with space group I 4 1 / a . IR, elemental analysis, and thermogravimetric analysis were used to further confirm the structure of the Ni(II) complex. Its independent unit consists of four Ni(II) atoms, four L 2− Schiff base ligands, and four coordinated ethanol molecules. In vitro anticancer activities against the four human cell lines, A-549, MDA-MB-231, SMMC-7721, and CNE-2Z, revealed that the Ni(II) complex exhibited significant anticancer activity. Furthermore, the anticancer activities of the Ni(II) complex on SMMC-7721 and CNE-2Z cells were much better than that of cisplatin. In addition, migration and the Transwell assay illustrated that the Ni(II) complex could inhibit the invasion and cell migration of tumor cells CNE-2Z and SMMC-7721.

Three metal complexes [Ni-2 (HL)(2)(C2H5OH)(2)Cl-2]center dot 2C(2)H(5)OH (complex 1), [Co (HL) (L)] (complex 2) and [Zn-2(HL)(2) (CH3COO)(2)] (complex 3) derived from 5-bromosalicylaldehyde-2-(2-aminophenyl)benzimidazole Schiff base ligand (H2L) were synthesized and characterized by infrared (IR) spectroscopy, thermogravimetric (TG) analysis and powder X-ray diffraction (XRD). Single crystal X-ray diffraction analysis confirmed that complex 1 crystallized in binuclear octahedral structure with space group P2(1)/C and complex 2 displayed in mononuclear octahedral structure with space group P 121/c 1. While complex 3 produced binuclear pyramid structure with space group P 121/c 1. Herein, cobalt (II) salt was oxidized to form Schiff base cobalt (III) complex by coordinating three deprotonated groups. The MTT method was employed to determine the three complexes in vitro cytotoxicity effects against human lung cancer cells (A-549), liver cancer cells (SMMC-7721), breast cancer cells (MDA-MB-231) and nasopharyngeal carcinoma cells (CNE-2Z). The results showed that Co (III) complex has significant anticancer activity against human breast cancer MDA-MB-231 cells compared with cisplatin. The further study of its anticancer mechanisms exhibited that Co (III) complex could inhibit MDA-MB-231 cells proliferation through both apoptosis and DNA replication inhibition.

Two dinuclear complexes [Cu2(LO)2] and [Ni2L2Cl2(C2H5OH)2] derived from (5‐chlorosalicylaldehyde)‐4‐aminoantipyrine (HL) were synthesized and characterized by infrared (IR) spectroscopy，thermogravimetric analysis, and powder X‐ray diffraction. Molecular structures of the Cu (II) and Ni (II) complexes were determined by single‐crystal X‐ray diffraction. Their cytotoxicity was evaluated by methyl thiazolyl tetrazolium assay against human tumor cells, such as lung carcinoma A549, lung adenocarcinoma H1975, breast carcinoma MDA‐MB‐231, and nasopharyngeal carcinoma CNE‐2Z cell lines. The results showed that the Cu (II) complex had much better anticancer activity than the Ni (II) complex, especially for MDA‐MB‐231 and CNE‐2Z cells. The reason may be that they have different structures. The methyl group on the (5‐chlorosalicylaldehyde)‐4‐aminoantipyrine (HL) was oxidized to the hydroxymethyl group, herein, namely LO, which is difficult to achieve in the normal chemical synthesis process. However, we did it by a solvothermal method at low temperature. It provides a new way of thinking and synthesis. The anticancer activity of the copper complex was next only to cisplatin, and the copper complex was nontoxic for the human normal cell lines. The experiment results also showed that the copper complex could inhibit tumor cell proliferation through the cell cycle and cell apoptosis.

Dietary patterns are major determinants of sporadic colon cancer incidence, the vast majority of the disease. NWD1 is a unique purified rodent diet that establishes mouse exposure to key nutrients recapitulating levels linked to higher risk in human, and is the only mouse model that develops sporadic intestinal and colon tumors, reflecting the etiology, incidence, frequency and lag with developmental age of the human disease. NWD1 fed mice reflect human metabolic syndrome with chronic inflammation, exhibit elevated Wnt signaling throughout the small and large intestinal mucosa and altered lineage marker expression, but Lgr5hi cells are suppressed in their lineage tracing, accumulation of mutations, and efficiency in tumor initiation. Alternate cells are mobilized to maintain the mucosa with increased efficiency to cause tumors. Deconvolving impact of NWD1 using scRNAseq, scATACseq and functional genomics provided novel insight into mechanisms elevating risk for sporadic tumors. NWD1 extensively, rapidly, and reversibly reprogrammed Lgr5hi stem cells, down-regulating Ppargc1a expression that altered mitochondrial structure and function and suppressed Oxphos and the TCA cycle. Ppargc1a genetic knockout in Lgr5hi cells recapitulated the dietary effects, and NWD1 or Ppargc1a inactivation repressed developmental maturation of Lgr5hi cell progeny as cells progressed through progenitor cell compartments. In compensation, mobilized Bmi1+, Ascl2hi cells maintained and adapted the mucosa. In parallel, NWD1 altered Ppargc1a chromatin structure in stem cells, and also for genes adapting enterocytes to the diet, independently confirming NWD1 epigenetic reprogramming of stem and differentiated cells. Importantly, pathways of antigen processing and presentation were elevated, especially in mature enterocytes, a pathogenic mechanism causing human chronic low-level pro-tumorigenic inflammation in IBD, also present in NWD1 fed mice. Fundamental conclusions: 1) plasticity of cells to function as stem cells mediates physiological adaptation of the mucosa. Thus, the human mucosa is in constant flux in response to its environment, supporting historic concepts of homeostasis by Claude Bernard and Walter Cannon as a continual process of tissue adaptation to its environment, not a single optimized state. 2) our data and the literature suggest that failure to recapitulate key human nutritional exposures contributes to why human stem cells reach crypt clonality in 6 years, 50-fold longer than the 6 weeks in the mouse. Thus, better modeling of human nutritional exposures in the mouse more accurately reflects human mechanisms of homeostasis and pathogenesis. 3) Complexity of cell reprogramming reported for the earliest premalignant human colon tumors is already present in the mucosa at nutritional risk for tumor development. 4) Oncogenic mutations provide a competitive advantage to intestinal stem cells in tumorigenesis, but the competition takes place on a playing field sculpted by the nutritional environment, a major determinant of who wins. Citation Format: Jiahn Choi, Xusheng Zhang, Wenge Li, Michele Houston, Karina Peregrina, Robert Dubin, Kenny Ye, Leonard H. Augenlicht. Deconvolution by scRNAseq, scATACseq and functional genomics of impact of a relevant high risk diet on stem cells and homeostasis [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B024.

Two metal-organic complexes of [CuLCl] (1), and [Ni2L2(C2H5OH)(2)Cl-2]center dot 2(C2H5OH)(2)(2) (HL= 2-hydroxy-1-naphthaldehyde-4-aminoantipyrine) were solvothermally synthesized from Schiff-base ligands. Single-crystal X-ray diffraction analysis shows that the mononuclear complex 1 is monoclinic while the dinuclear complex 2 belongs to triclinic crystal structure and the two complexes are both monomer molecules. Additionally, the cancer cytotoxicity, cell migration, and apoptosis properties of both complexes have been investigated. Owing to different coordination behavior between Ni(II) and Cu(II), it was found that com-plex 1 obviously displayed cytotoxicity and decreased cell migration rate by inducing apoptosis of SMMC-7721 cells (hepatoma carcinoma cells). (c) 2022 Published by Elsevier B.V.

NWD1, a mouse purified western diet uniquely relevant to human nutritional exposures elevating risk for human sporadic colon cancer rapidly and reversibly altered the stem cell signature and transcriptome of Lgr5hi intestinal stem cells. Down-regulated Ppargc1a expression altered mitochondrial structure and function, metabolically reprogramming Lgr5hi cells, suppressing developmental maturation of their progeny as they progress through progenitor cell compartments. This recruited Bmi1+, Ascl2 cells, remodeling the mucosa, including cell adaptation to the altered nutritional environment and elevated pathways of antigen processing and presentation, especially in mature enterocytes, a pathogenic mechanism in human Inflammatory Bowel Disease resulting in chronic low-level pro-tumorigenic inflammation. Plasticity of intestinal cells to function as stem-like cells encompasses a physiological and continual response to changing nutritional environments, supporting historic concepts of tissue homeostasis as a process of continual adaptation to the environment, with important consequences for mechanisms establishing probability for tumor development. The data emphasize importance of better reflecting human nutritional exposures in mouse models of development and disease.