CDC42 missense variant  resulting in infection and HPV-driven carcinogenesis in the mosaic mother and impaired thymopoiesis and profound T cell lymphopenia in the heterozygous daughter identified through newborn screening for SCID. We found that surface expression of IL-7Rα (CD127) was decreased, consistent with reduced IL-7-induced STAT5 phosphorylation and accelerated apoptotic T cell death. Consistent with the vital role of IL-7 in regulating thymopoiesis, both patients displayed reduced T cell receptor CDR3 repertoires. Moreover, the CDC42 variant prevented binding to the downstream effector, p21-activated kinase (PAK)1, suggesting this impaired interaction to underlie reduced IL-7Rα expression and signaling. Here, we provide the first report of severely compromised thymopoiesis and perturbed IL-7Rα signaling caused by a novel CDC42 variant and presenting with diverging clinical and immunological phenotypes in patients.Genetic variants in cell division cycle 42 (CDC42) can manifest with dysmorphic features, autoinflammation, hemophagocytic lymphohistiocytosis, and thrombocytopenia, whereas defective thymopoiesis is a rare disease manifestation. We report a novel The online version contains supplementary material available at 10.1007/s10875-023-01561-0. Cell division cycle 42 (CDC42) belongs to the RAS superfamily of GTPases. In its active, GTP-bound, state, CDC42 regulates vital cellular processes, including adhesion, migration, proliferation, and survival as well as actin dysregulation and cytoskeletal abnormalities . These fCDC42-associated diseases are rare and due to heterozygous missense variants in relation to the five G1–G5) functional CDC42 domains. CDC42-related phenotypes fall into four different groups, of which group I is characterized by thrombocytopenia, intellectual delay, growth retardation, and dysmorphic facial features (Takenouchi–Kosaki syndrome); group II includes developmental disorders with immunodeficiency, autoinflammation, hemophagocytic lymphohistiocytosis (HLH), and malignant lymphoproliferation; group III gives rise to Noonan syndrome ; and group IV causes autoinflammation with neonatal onset pancytopenia, rash, and HLH , 23, 24 –G5 functCDC42 plays a fundamental role in T cell homeostasis by linking cytokine responsiveness, TCR signaling and apoptosis, which is critical for both protective immunity and limitation of autoimmunity. CDC42 is critical for human T cell development prior to and after rearrangement of the pre-T cell receptor . Hence, CDC42 missense variant but presenting with diverging clinical and immunological phenotypes.Here, we provide the first report of severely compromised thymic output and perturbed IL-7Rα expression and signaling, the latter secondary to impaired CDC42-PAK1 interaction, in an infant (P2) and her mother (P1), both harboring the same novel monoallelic N-terminal Condylomata acuminata became clinical concerns. During her latest pregnancy (with daughter P2), HPV-associated vulva cancer with involvement of local lymph nodes was diagnosed. After primary resection, the vulva cancer relapsed and was complicated by skin and urinary tract-derived sepsis. Recurrent fevers, bleeding, HLH, or thrombocytopenia were not reported. P1 followed the national vaccination program for children including vaccination with morbilli, mumps, and rubella.Aged 29 years, P1 displays characteristic facial features with long palpebral fissures, broad nasal tip, slightly low hanging columella, short philtrum and a long neck, height (166.5 cm), and did not report learning disabilities. Early childhood pneumonias, skin infections, gastroenteritis, and urinary tract infections accompanied treatment-resistant acral warts. Following sexual debut, genital herpes virus infections and human papilloma virus (HPV)–associated P2, the daughter of P1, was born preterm . She is currently (19 months of age) thriving and appears without clear syndromic features but manifests a slight retardation in her gross motor function as she only started walking at the age of 18 months. She receives antiviral (acyclovir), antibacterial (sulfamethoxazole with trimetroprim), and antifungal (fluconazole) prophylaxis. During the first 18 months, she has experienced minor upper respiratory infections not necessitating additional antibiotic treatment. P2’s blood tests showed severe CD4 T cell lymphopenia but otherwise normal levels of total leukocytes, platelets, and immunoglobulins (except slightly elevated IgA)  and CD8 +  T cell lymphopenia, prompting whole genome sequencing of proband (P2), parents (mother (P1) and father) and maternal grandparents, supplemented by a panel of 365 PID genes. A missense variant resulting in a substitution of a positively charged lysine with a negatively charged glutamic acid  was identified in P2 in exon 2 of the CDC42 gene  in the exome, corresponding to mosaicism of 46.2%. In contrast, P1’s isolated CD3 + T cell (99.9% purity) allele frequency is 9.2% (read depth 141) in the genome, corresponding to mosaicism of 18.4%. Importantly, maternal DNA was not detected in the infant DNA sample (detection limit 10–15%)  motifs [CDC42 variant leads to CDC42 expression comparable to that of healthy controls as evidenced by western blotting of PBMC lysates  motifs . The var5 × 10 mo5 cells [5 cells) were only 4% of normal, corresponding to > 25 times lower levels than expected for her age [5 cells, similar to those observed in SCID (< 50 copies/1 × 105 cells) [In her historic dried blood spot sample (DBSS), P1 displayed normal amounts of sjTRECs at a number of 1742 copies/10 her age . Recent CDC42 have been shown to cause severe autoinflammation with increased production of proinflammatory cytokines in response to various innate immune stimuli [C-terminal variants in  stimuli , 23. To Evidence suggests TCR diversity to be prognostic in cervical carcinogenesis  and predNext T cell proliferation and death as well as NK cell activity was accessed. When stimulated with anti-CD3/CD28 beads for 48 h, CFSE stained patient CD4 + and CD8 + T cells exhibited modestly reduced percentage of proliferating cells but with a proliferation index similar to healthy controls  and repression of IL-7Rα expression [Based on previous findings that deletion of pression , we thenCDC42-induced responses are dependent on interaction between activated CDC42 and downstream mediators such as the kinase PAK1, and it has been demonstrated that PAK1 is necessary and sufficient for CDC42-regulated T cell homeostasis . We therCDC42 variant to be associated with severely compromised thymic output, markedly narrowed TCR repertories, decreased IL-7Rα expression and signaling, and abolished binding to PAK1 in humans. This is also the first report associating an N-terminal CDC42 variant with severe T cell immunodeficiency, mimicking the one encountered in T-B + SCID. The missense variant in exon 2 of CDC42, resulting in a substitution of a positively charged lysine with a negatively charged glutamic acid at position 16, was initially identified in P2 during newborn screening for SCID, and subsequently in the mother P1, in the latter de novo and as mosaic of 50% in peripheral leukocytes and 20% in T cells. P1’s Noonan-like features  and the absence of autoinflammation, HLH and thrombocytopenia in both patients accorded with the location of this variant which, like other N-terminal CDC42 variants, was predicted to interfere with binding to downstream substrates, including PAK1 and WASp [According to our knowledge, this is the first and WASp . The incCdc42 deficient mice with Cdc42 mutants, displaying impaired PAK1 binding, did not rescue T cell IL-7R expression, homeostasis, and survival in mice [Cdc42-deficient murine T cells [CDC42 governs homeostasis and apoptosis by integrating cellular signaling derived from IL-7R and TCR activation . Hence,  in mice , thereby in mice  as well  in mice . Hence,  in mice . Compare in mice . In cont T cells , we did  T cells , was not T cells , 20. HowCDC42 variant (reflecting her 60% leukocyte mosaicism). Due to compromised IL-7R signaling, these CLP, when entering her intrauterine/neonate thymus, would be homeostatically disadvantaged. As a consequence, preferentially thymocytes, carrying two WT CDC42 alleles, would multiply and sustain P1’s normal sjTRECs concentrations at birth although at the price of reduced TCR diversity). Nevertheless, due to P1’s mosaic CDC42 carrier state, increased thymocyte apoptosis likely accelerated age dependent thymic decline, leaving her with virtually extinguished sjTRECs formation at the age of 29. Contributing to P1’s current 20% peripheral CD3 + T cell mosaicism was probably death of predominantly variant positive naïve T cells, consistent with the patient’s markedly reduced fractions of naïve CD4 + and CD8 + T cells, as only naïve T cells are non-redundantly dependent upon IL-7 for homeostatic sustenance [CDC42 allele. However, we cannot entirely exclude the possibility of P1 harboring the CDC42 variant as a germline mutation with partially genetic reversal in the CD4 T cell compartment with mosaicism as the result.Interestingly, while P2 was born with SCID-like sjTRECs levels, P1 had normal sjTRECs levels at birth. We hypothesize that the majority of P1´s common lymphoid progenitors (CLP) carried the stenance . Selectistenance . P1’s T IL-7 receptor signaling confers the ability of CD8 + T cell to develop into long-lived memory T cells that will retain functional responsiveness . TherefoConsistent with the attenuated thymic function in both patients, their TCR CDR3 repertoires were markedly narrowed as reflected by elevated Gini coefficients. The abundance distribution also demonstrated that both P1 and P2 have reduced frequencies of unique CDR3 sequences compared to those of the adult controls. As the normal naïve TCR repertoire is characterized by a high proportion of unique CDR3 sequences, the abundance distributions, found in P2 (and P1), were consistent with clonal expansions of T cells with markedly reduced TCR repertoires . For botDue to the clinical and immunological heterogenicity of the two patients, P2’s prognosis is difficult to ascertain. So far, she appears without major developmental delays and has remained without any severe acute or chronic infections in the context of broad antiviral, antibacterial, and antifungal prophylaxis. However, given the development of malignancy and severe infectious history of her mosaic mother, allogeneic bone marrow transplantation is considered as the treatment of choice. Clearly, the full spectrum of disease-causing variants in CDC42 and the clinical somatic, infectious, autoinflammatory, and malignant presentation of CDC42 deficiency is highly heterogeneous and remains to be fully clarified.Whole exome sequencing (WES) was performed for P2, P1, the father, the maternal grandmother, and the maternal grandfather. DNA was extracted from whole blood with Maxwell® RSC Blood DNA Kit , or Tecan Freedom EVO® . After quantification, 1 μg of DNA was used for library preparation using NimbleGen SeqCap EZ Library SR . Validated libraries were pooled and paired-end sequenced on Illumina NovaSeq 6000 platform  following Illumina’s recommended protocol. Reads were mapped to the reference genome (hg19) using BWA-MEM and genotypes of targeted bases were called with GATK . Variant5 cells.For P1, sjTREC concentrations were determined in a recent blood sample and in a birth sample. For the recent sample, DNA was extracted from whole blood, whereas the newborn screening dried blood spot sample (DBSS) was used as birth sample. The birth sample was retrieved from the Danish Neonatal screening biobank. For P2, sjTRECs were determined as part of the routine neonatal screening. DNA was extracted from the dried bloodspot samples of both P1 and P2 using the Eonis DNA extraction kit . All samples were analyzed with the EonisPCR kit . The kit is a multiplex RealtimePCR assay with primers and TaqMan probes for TREC and a two copy reference gene, RPP30. RealtimePCR was performed on a Quantstudio7DX . TREC concentrations were calculated based on ∆Ct values between TREC and RPP30. The calculation formula is described in detail in . SCID cuhttps://www.ncbi.nlm.nih.gov/sra with accession to the SRA data).PBMC DNA was processed as previously described . In essePBMCs were lysed in RIPA buffer  with HALT protease/phosphatase inhibitors  and Benzonase . Protein concentration was measured by Pierce BCA assay , and 5-ug protein was separated by SDS electrophoresis on 4–20% gradient gels . Proteins were transferred to PVDF membranes  using the Trans-blot Turbo transfer system. Membranes were blocked in 5% skimmed-milk PBS-T for 1 h and washed in PBS-T before incubation overnight at 4 °C with primary antibodies. Membranes were subsequently washed and incubated with secondary antibodies for 1 h at room temperature. Finally, proteins were visualized by chemiluminescence. For STAT5 westerns, membranes blotted for pSTAT5 were stripped for 10 min in Restore western blot stripping buffer , washed in PBS-T, and re-blocked in skimmed-milk before incubation with STAT5 antibody similar to above. Primary antibodies: Mouse anti-STAT5  1:1000, mouse anti-STAT5 phospho (Y694) (BD) 1:1000, mouse anti-GAPDH , rabbit anti-CDC42  1:1000, mouse anti-FLAG  1:1000, rabbit anti-PAK1  1:1000. Secondary antibodies: Donkey anti-mouse  1:1000, Donkey anti-Rabbit .6 HEK293T cells were seeded in 10-cm dishes and transfected with 10 ug of pCCL/PGK-FLAG-CDC42 WT, pCCL/PGK-FLAG-CDC42 K16E, and pcDNA3.1-HA-PAK1 plasmids using PEI  as indicated for 24 h. Cells were lysed in Pierce IP lysis buffer  with Halt protease and phosphatase inhibitors  and 500 ug of total protein lysates were incubated with anti-FLAG magnetic agarose  for 2 h at 4 °C while rotating. The magnetic agarose was washed twice in lysis buffer and once in water. Bound protein was eluted by 5 min incubation in Pierce IgG elution buffer  and subsequently neutralized by adding 1 M Tris–HCl pH 8.5. Total protein lysates and eluted protein samples were investigated by western blotting as described above.3 × 10Unstained and CFSE stained PBMCs were left unstimulated or stimulated with anti-CD3/CD28 Dynabeads  for 48 h according to manufacturer’s instructions. Proliferation or CD25 and CD69 surface expression for both CD4 + and CD8 + T cells derived from separate 96 well plates (2–4 wells containing 100.000 cells/well were pooled) was ascertained.PBMCs were isolated from heparinized blood samples by density centrifugation and resuspended in complete RPMI1640 medium. Degranulation was assessed by incubating PBMCs with (and without) K562 target cells at a 1:1 ratio for 120 min. Degranulation was expressed as the percentage of CD107a positive cells among the classical NK cell population minus the corresponding percentage in the unstimulated control sample. NK cell cytotoxicity was assessed by incubating CellTrace Violet–labeled K562 target cells with (and without) PBMCs at an effector-to-target ratio of 50:1 for 24 h. The absolute count of fully viable target cells  in cultures with added effector cells was compared with the corresponding number in control cultures without added effector cells. NK cytotoxicity was expressed as the median percentage of viable target cells “missing” in cultures with added effector cells (median of triplicate measurements).T cell apoptosis and cell death was detected by flow cytometry as previously described . BrieflyFrozen PBMCs were thawed and rested overnight in RPMI1640 with 10% FBS, 1% penicillin–streptomycin. PBMCs were stimulated for 0, 15, or 30 min with 10 ng/mL human IL-7 . Cells were harvested, washed once with PBS and lysed in RIPA buffer for western blotting as described above.Frozen PBMCs were thawed and rested overnight in RPMI1640 with 10% FBS, 1% penicillin–streptomycin. PBMCs were then stimulated with LPS , R848 , CpG ODN 2006  and Pam3CSK4 , or left untreated for 6 h, and subsequently lysed for RNA purification. RNA was isolated using the NucleoSpin 96 RNA kit  following the instructions of the manufacturer. cDNA was synthesized using iScript gDNA Clear cDNA Synthesis kit . Expression levels of IL-6, TNF-α, IFN-β1, IFN-α2, and TBP was measured by qPCR using TaqMan Fast Advanced Master Mix  and the following TaqMan probes: TBP (Hs00427620_m1), IFN-β1 (Hs01077958_s1), IFN-α2 (Hs00265051_s1), TNF (Hs01113624_g1), and IL-6 (Hs00985639_m1) (All from Thermo Fischer Scientific).U test was used for comparison between two variables and Kruskal–Wallis test was used for comparison between more than two variables.The Mann–Whitney Supplementary file1 (PDF 396 KB)Below is the link to the electronic supplementary material.


Gli1+ stromal cells, and epithelial cells are required for ISC proliferation and villus homeostasis. Intestinal stromal cells are heterogeneous and single-cell profiling has divided them into telocytes/subepithelial myofibroblasts, myocytes, pericytes, trophocytes, and Pdgfralow stromal cells. Yet, the niche function of these stromal populations remains incompletely understood.The nutrient-absorbing villi of small intestines are renewed and repaired by intestinal stem cells (ISCs), which reside in a well-organized crypt structure. Genetic studies have shown that Wnt molecules secreted by telocytes, Twist2 stromal lineage, which constitutes the Pdgfralow stromal cell and trophocyte subpopulations, maintains the crypt structure to provide an inflammation-restricting niche for regenerating ISCs. Ablating Twist2 lineage cells or deletion of one Wntless allele in these cells disturbs the crypt structure and impairs villus homeostasis. Upon radiation, Wntless haplo-deficiency caused decreased production of anti-microbial peptides and increased inflammation, leading to defective ISC proliferation and crypt regeneration, which were partially rescued by eradication of commensal bacteria. In addition, we show that Wnts secreted by Acta2+ subpopulations also play a role in crypt regeneration but not homeostasis.We show here that a Twist2 lineage cells may help to maintain a microbe-restricted environment to allow ISC-mediated crypt regeneration.These findings suggest that ISCs may require different niches for villus homeostasis and regeneration and that the The online version contains supplementary material available at 10.1186/s12915-023-01667-2. Lgr5+ intestinal stem cells (ISCs) located at the crypt base [2–107 cells/g in small intestines in human) [The small intestinal villi, tissue responsible for nutrient absorption, are renewed every 3–5 days by ypt base . ISCs anypt base –4. Panetn human) . The viln human) . Some inn human) . The ISCn human) . Althougn human) , 15, then human) –19.Pdgfralow stromal cells [Pdgfra, Gli1, Foxl1, Ng2, or Gremlin 1 [Porcn, which encodes an enzyme required for palmitoylation and secretion of Wnts, in Pdgfra+ stromal cells leads to neonatal lethality with decreases in villus size and density [Porcn in Foxl1+ telocytes, gigantic cells that each encompasses dozens of villus/crypt cells, leads to villus and crypt collapse within 3 days of induction [Wls)  or killing stromal cells marked by Gli1, which is required for Foxl1 expression [Pdgfra is expressed in telocytes, trophocytes, and Pdgfralow stromal cells, Gli1 is expressed in some telocytes, myocytes, trophocytes, and Pdgfralow stromal cells, Foxl1 is expressed in telocytes, myocytes, and some epithelial cells, and Acta2 (encoding αSMA) is expressed in telocytes, myocytes, and pericytes [Recent studies suggest that intestinal stromal cells/myofibroblasts provide Wnt molecules to support ISC proliferation and organiod growth –23. Thesal cells –27. The remlin 1 , 25, 28. density . Inducibpression , takes 1pression , 30. Onepression –33. Intrericytes , 35. Nevericytes .Prrx1, Twist2, Nestin, and Acta2, labeled intestinal stromal cells [Twist2 lineage cells represent the largest among the four lineages examined. Single-cell profiling suggests that Twsit2 is expressed in portions of the trophocyte and Pdgfralow stromal cell subpopulations, but not Acta2+ subpopulations. In contrast to the findings that deletion of Wls in Ng2- or Gli1-marked stromal cells do not affect the intestinal villus [Wls allele in Twist2 lineage cells, but not Prrx1, Nestin, or Acta2 lineage cells, disturbed the crypt structure and villus homeostasis. Moreover, upon IR-induced injury, Wls haplo-deficiency resulted in decreases in the number of Paneth cells and the production of antimicrobial proteins, accompanied by increased inflammation and impaired crypt regeneration. These defects were largely rescued by the eradication of commensal bacteria with antibiotics. In addition, Acta2-marked telocytes, myocytes, and pericytes, but not Prrx1 or Nestin lineage cells, play a niche role in crypt regeneration but not homeostasis. This study thus uncovered ISC niche components with previously unidentified functions.Herein, we tested whether genetic markers for bone marrow mesenchymal stem/stromal cells (BM-MSCs), al cells . We showl villus , 25, ablPrrx1, Twist2, and Nestin marked intestinal stromal cells [ROSA-tdTomato reporter mice to the Cre mouse lines driven by the promoter of these genes to perform genetic tracing. We showed that Prrx1, Twist2, and Nestin marked 32.6%, 38.1%, and 11.8% of non-epithelial intestinal villus cells, respectively in adult mice, and some of these cells were located at the base of the crypts . These results indicate that Twist2 marks the largest portion of intestinal stromal/myofibroblast cells (38.1%) among the 4 marker genes.To understand the roles of various intestinal stromal subpopulations in villus homeostasis and regeneration, we tested whether BM-MSC markers, al cells –41. We ceostasis , 43. We Pdgfralow stromal cells  and PdgfralowCD81− stromal cells, but not much in telocytes/subepithelial myofibroblasts, myocytes, or pericytes  and PdgfralowCD81− stromal cells and found that their expression showed minimal overlapping , althouiewed in , 25. We ROSA26-iDTR mouse) to transiently deplete each of these populations [Prrx1-Cre; iDTR mice, Nestin-Cre; iDTR and Twist2-Cre; iDTR mice  play a role in villus homeostasis, we used an inducible diphtheria toxin receptor system  molecules are critical niche signals for ISCs. However, the Wnt expression pattern did not entirely overlap with the intestinal mesenchymal mixtures [Twist2 lineage does not represent all intestine stromal cells.The niche cells provide signaling molecules including Wnts to support ISC proliferation , 28. Folmixtures , consistTwist2 lineage cells, we ablated Wls, which encodes a sorting receptor for Wnt secretion [Twist2-Cre mouse to Wlsf/f mouse that also carries a Lgr5-GFP allele to label ISCs. Homozygous deletion of Wls in Twist2 lineage cells led to embryonic lethality at E18.5 whereas Wls heterozygous mice were viable. We found that deletion of even one Wls allele in Twist2 lineage led to a reduction of Wls expression and a decrease in β-Catenin, a downstream target stabilized by Wnt activation  impaired crypt regeneration but not villus homeostasis, this is inconsistent with the finding that deletion of Porcn in Foxl1+ telocytes causes collapse of the villi [Ng2+ cells-secreted Wnt molecules play more important roles in crypt regeneration than in homeostasis [Findings of this study support the notion that crypt regeneration requires a more demanding niche. We show that he villi . One exphe villi –33. Receeostasis  and thateostasis . Overalleostasis .Twist2 lineage stromal cells provide the Wnt molecules required for the activities of Paneth cells and imply that the interaction between Twist2 stromal cells and Paneth cells may generate a microbe-restricted and inflammation-restricted environment to support ISCs and crypt regeneration. Certainly, this warrants further investigation.During crypt regeneration, newly formed Paneth cells appear to shape the crypt morphogenesis and secrete anti-microbial peptides . Its impPrrx1 and Nestin lineage BM-MSCs constitute niches for hematopoietic stem cells, we show that Twist2 lineage stromal cells act as a niche for ISCs during villus homeostatic and regeneration via secretion of Wnt molecules. We also show that Twist2 stromal cells help control the structure of the crypts and may provide a unique niche for ISCs during regeneration.While ROSA-iDTR mouse was generated in Waisman’s laboratory and Prrx1-Cre, Twist2-Cre, Nestin-Cre, Acta2-Cre, Gli1-Cre/ERT, Lgr5-GFP-Cre/ERT, and ROSA-tdTomato mouse lines were purchased from The Jackson Laboratory.All animal work was in compliance with the recommendations in the National Research Council Guide for the Care and Use of Laboratory Animals, with the protocols approved. The iDTR mice expressing Cre were injected intraperitoneally with 100 ng of DT (Sigma) twice per day for several consecutive days. To irradiate the mice, lead plates were used to protect other parts of the mice, only the exposed belly received radiation with RAD SOURCE RS 2000 irradiator. Antibiotics treatment  of the mice was started two days before IR and sustained until the mice were sacrificed. LPS (2.5 mg/kg) was given at the same time of IR by intraperitoneal injection.To deplete a given type of cells, 2-month-old To generate the villus/crypt regeneration model, adult male mice were irradiated at 6.5 Gy (only to the abdomen region) and the mice were euthanized after different periods of time. The wildtype mice survived well and the body weight was maintained, as previously reported .For antibiotic treatment, the combined antibiotics  were added to drinking water till the end of the experiment.After the mice were sacrificed, the full intestine was removed and the mesenteric fat was cleaned. The length of the intestine was measured. The upper part of the intestine (1 cm) was removed and fixed in 4% paraformaldehyde overnight. Samples were then dehydrated, embedded in paraffin, sectioned at 4 μm through a longitudinal crevice, and prepared for staining. For the cryostat section, intestine samples were embedded in OCT  and frozen in liquid nitrogen. Sections were cut at 6 μm in thickness at − 20℃. To trace tomato-positive cells, cryostat sections were washed, counterstained with mixed DAPI & Mounting solution (1:1), and covered glasses for observation (Upright Microscope Nikon ECLIPSE 80i). H/E staining and ALP staining  were carried out following the standard protocols.The frozen sections were warmed at room temperature after being taken from the − 20℃ fridge. Samples were washed and permeabilized with Triton X100 (0.1%) in PBS for 30 min, blocked with goat serum (10%) at RT for 1 h, and incubated with primary antibody at 4℃ overnight. The next day, after washing, the samples were incubated with a secondary antibody at 37℃ for 1 h in darkness, counterstained with mixed DAPI & Mounting solution (1:1), and mounted for observation (Nikon ECLIPSE 80i). The following antibodies were used: αSMA  and lysozyme . CRS4C (1:100) was generated in Dr. Junping Wang’s lab.2O2 for 20 min to quench the endogenous peroxidase activity, permeabilized with Triton X100 (0.1%) in PBS for 30 min. Antigen retrieval was performed with heated 1X sodium citrate. The samples were blocked, incubated with primary antibodies and secondary antibodies that were conjugated to horseradish peroxidase (HRP) as described for immunofluorescent staining. Afterwards, the samples were incubated with a DAB stain kit (Boster), washed, and incubated with the SABC solution at 37℃ for 1 h in darkness. The slides were then dehydrated through 2 changes of 95% alcohol and absolute alcohol, cleared in two changes of xylene, and mounted with resin for observation. Antibodies against β-Catenin , Ki67 , lysozyme , and GFP  were used.The paraffin sections were dewaxed and rehydrated. The samples were treated with 3% HThe paraffin sections were deparaffinized with xylene and rehydrated through graded alcohol to deionized water. The samples were stained in Alcian Blue solution for 30 min, rinsed in running tap water for 5 min, placed in periodic acid solution (Periodic Acid-Schiff (PAS) Kit, Sigma) for 5 min, rinsed in several changes of deionized water, placed in Schiff Reagent for 15 min, and washed in lukewarm tap water for 10 min. The samples were then dehydrated through two changes of 95% alcohol and absolute alcohol, 2 min each, cleared in 2 changes of xylene, and mounted with resin for observation.For intestinal stromal cell isolation, mouse intestines were cut into 0.5–1 cm pieces. Tissues were rinsed with cold PBS + P/S for 2 min, immersed into 10 ml 1 mM DTT solution (Sigma), and rocked at 200 rpm for 10 min. The tissues were rinsed again for 2 min, incubated in 10 ml 3 mM EDTA/1 mM HEPES Buffer with rocking at 200 rpm for 10 min at 37℃, transferred into new 6 cm plates, and incubated with digestion solution (100U/ml collagenase VIII and 100U/ml DNase I) for 30 min. The samples were pipetted up and down to break up the tissue. The isolated cells were centrifuged with a low speed at 250 g for 5 min and single-cell suspension was obtained using a 70 μm strainer .The isolated intestinal stromal cells were analyzed for MSC markers using flow cytometry using a mouse MSC marker antibody panel kit , following the protocol provided by the manufacturer. Analysis was done using a BD FACSCalibur. For cell sorting, the isolated cells were resuspended with Flow Cytometry Staining Buffer. Flow cytometry was performed using Beckman MoFlo™ XDP high-speed cell sorter.Tissue samples or sorted cells were frozen in liquid nitrogen for storage and total RNA was isolated using TRIzol (Invitrogen). RNA was reverse transcribed to cDNA with PrimeScript™ RT reagent Kit (Takara. Code No. RR037A). Quantitative PCR was carried out in triplicate on LightCycler® 96 Real-Time PCR System (Roche) with a FastStart Universal SYBR Green Master (Roche), using the cDNA samples as templates. The primers for each gene of interest were shown in Additional file: Table S6 cells bone marrow cells were plated in 3.5 cm dishes and allowed to grow in α-MEM medium containing 10% fetal bovine serum, 1% L-glutamine, and 1% penicillin–streptomycin . After 3 days, the non-adherent cells were removed and the adherent cells were sub-cultured. Medium was replaced every 3 days. After 7 days, colonies were stained.The bone marrow was flushed out with α-MEM culture medium from the femurs and tibiae. Red blood cells were cracked with red blood cell lysis buffer. Single-cell suspensions were prepared by filtration through a 40-μm strainer. 1 × 10Raw reads were demultiplexed and mapped to the mouse reference genome with the Cell Ranger version 3.0.1 (10X Genomics) pipeline using the default parameters. The generated gene-cell expression matrix was used for subsequent analysis in R version 3.6.1 using Seurat version 3.1.5.t-test. Two-way ANOVA was performed to compare two corresponding data points. P < 0.05 is defined as being significantly different. *P < 0.05, **P < 0.01, and ***P < 0.001.To quantitate the cells in the villi, cells on ten views of three sections from at least 3 mice were counted. Data are given as mean and standard error of mean (SEM) of the results from > 3 samples in each experiment. Statistical analysis and plotting were processed with Graphpad Prism5. Differences between the two groups were measured by the Student’s Additional file 1:Fig. S1. tSNE analysis of intestinal cells of mice. Fig. S2. The intestinal Twist2 and Gli1 lineage cells expressed different surface markers. Fig. S3. Effects of depletion of Twist2, Prrx1, or Nestin lineage cells on the villi. Fig. S4. Depletion of Twist2 lineage cells impaired crypt regeneration. Fig. S5. tSNE analysis of Wnt and Rspo expression in intestinal cells. Fig. S6. Ablation of one Wls allele led to a decrease in β-Catenin in epithelial cells. Fig. S7. Effects of deletion of one copy of Wls in Prrx1 or Acta2 lineages on villus homeostasis. Fig. S8. Deletion of one Wls allele in Prrx1 lineage cells did not affect villus regeneration. Fig. S9. IR induced expression of inflammation-related cytokines and AMPs. Fig. S10. LPS-induced inflammation impaired ISC regeneration. Table S1. Primer sequences used for quantitative PCR.Additional file 2: The individual data values for Figs. 1-6 and S3, 4, 7, 8, 9, 10.


For the past 20 years, the majority of cell culture studies reported that increasing cholesterol level increases amyloid-β (Aβ) production. Conversely, other studies and genetic evidences support that cellular cholesterol loss leads to Aβ generation. As a highly controversial issue in Alzheimer’s disease pathogenesis, the apparent contradiction prompted us to again explore the role of cellular cholesterol in Aβ production. Here, we adopted new neuronal and astrocytic cell models induced by 3β-hydroxysterol-Δ24 reductase (DHCR24), which obviously differ from the widely used cell models with overexpressing amyloid precursor protein (APP) in the majority of previous studies. In neuronal and astrocytic cell model, we found that deficiency of cellular cholesterol by DHCR24 knockdown obviously increased intracellular and extracellular Aβ generation. Importantly, in cell models with overexpressing APP, we found that APP overexpression could disrupt cellular cholesterol homeostasis and affect function of cells, coupled with the increase of APP β-cleavage product, 99-residue transmembrane C-terminal domain. Therefore, we suppose the results derived from the APP knockin models will need to be re-evaluated. One rational explanation for the discrepancy between our outcomes and the previous studies could be attributed to the two different cell models. Mechanistically, we showed that cellular cholesterol loss obviously altered APP intracellular localization by affecting cholesterol-related trafficking protein of APP. Therefore, our outcomes strongly support cellular cholesterol loss by DHCR24 knockdown leads to Aβ production. A tight link between Alzheimer’s disease (AD) pathology and lipids was already observed more than a century ago by Alois Alzheimer, who described a higher occurrence of “lipoid granules” in postmortem AD-brain tissue as a third pathological hallmark of the disease , 2. MoreIntriguingly, a series of evidence demonstrated that in the brain of hereditary AD animals and patients, there are susceptible genes and gene polymorphism in the molecules responsible for the transport, uptake, and intracellular trafficking of cholesterol, including apolipoprotein E, ATP binding cassette transporters, low density lipoprotein receptor family, and Niemann-Pick Type C 1/2 (NPC1 and NPC2). Furthermore, these genic alterations easily lead to the decrease of cholesterol transport or uptake and blockade of intracellular cholesterol trafficking, resulting in cellular cholesterol deficiency/loss , 15, 16.In addition, the source of Aβ deposition remains a controversial issue in AD brain , 20, 21.Most notably, supporting the view that high cellular cholesterol increases amyloid beta production, nearly completely is derived from the cell or animal models carrying a mutant human APP , 7, 8, 9A rabbit antibody against C terminus of APP  was used to detect APP-C83 and APP-C99; a mouse antibody against amino acids 66–81 of the N terminus of APP  was used to detect full-length APP.Rabbit anti-BACE-1(ab10716), anti-Rab5(ab218624), anti-LAMP1(ab20894), and anti-Flotillin1(ab133497) were purchased from Abcam (British). Rabbit anti-DHCR24 (#2033), anti-SorLA (#79322), and mouse anti-GAPDH (#2118) were obtained from Cell Signaling Tech.Mouse hippocampal neuronal cell line (HT22) was obtained from Fu Heng (Cat# FH1027), and Astrocyte Type I clone cell line (C8D1a) was obtained from ZQXZbio (Cat# ZQ0566) and were grown in 4.5 g/L glucose Dulbecco’s Modified Eagle Medium , supplemented with 10% fetal bovine serum (Gibco). In addition, cells were cultured at 37°C with 5% CO2 according to the manufacturer’s instructions.Cells were transfected by lentivirus-mediated DHCR24 cDNA and DHCR24-shRNA or empty vectors, respectively . Firstly, these cells were transfected with lentivirus for 24 h when cells density was about 50%. Afterwards, we continued to cultivate the cells for 48 h in fresh medium. Subsequently, to screen out cells which had successfully transfected lentivirus, we cultivated cells in fresh medium supplemented with an appropriate concentration of puromycin (2 μg/ml). The shRNA sequence of DHCR24 is as follow: (5′-GCGAGGAATTCTGGGAGATGTTCGA-3′). Additionally, the method for APP overexpressing cell model construction was the same as above.Total RNA was extracted from cells using RNAiso Plus Kits  according to protocol recommended. Reverse transcription of mRNA was performed by PrimeScript™ RT Master Mix kits (Takara). Gene expression was quantitatively measured using TB Green Premix Ex Taq II kits (Takara) with specific primers . All expFilipin staining is widely used as a method to measure unesterified cholesterol levels . FirstlyFirst, cells were seeded in plates and cultivated for 12–18 h. In the next day, all plates were washed three times by PBS, and then cells were collected into Eppendorf tubes separately and immersed in appropriate amount of lysis solution in 10 min. Only the supernatants were collected and measured by a free cholesterol quantitative kit  according to protocol recommended by manufacturer. The concentration of protein in supernatants also was measured by BCA kits  to normalized free cholesterol content.All proteins are extracted from cells using SDS lysis buffer (Merk) supplemented with both phosphatase inhibitor  and protease inhibitor (1:100 Millipore). After removing cell debris by centrifugation at 12,000 × rpm, the concentration of protein was measured by BCA assay kits (Millipore). Subsequently, an equal amount (20 ug) of proteins was loaded on 10% polyacrylamide gels  according to their concentrations. Afterward, proteins were transferred onto polyvinylidene fluoride membranes, and the membranes were blocked with 5% skimmed milk at room temperature for 1 h. Besides, the membranes were incubated with primary antibodies at 4°C overnight, such as anti-BACE-1(1:1000), anti-Flotillin-1 (1:5000), anti-APP (1:1000), anti-SorLA (1:1000), anti-CTF (1:1000), anti-DHCR24 (1:1000), and anti-GAPDH . In the next day, the membranes were incubated with horseradish peroxidase–conjugated secondary antibodies  for 1 h at room temperature, and the immunoreactive bands were detected by Immobilon ECL Ultra Western HRP . All results had been repeated at least three times.To detect the amount of secreted Aβ40 and Aβ42 in neuronal and astrocytic cell lines, we seeded an equal number of cells in vessels and cultured cells for 12 h. After checking that the cell density of each group is roughly 50%, we removed culture medium completely and gently washed cells by PBS three times. Afterwards, 4 ml fresh culture medium was added to each culture flask, and then, we cultured cells for another 48 h. Subsequently, the mediums were collected and were centrifugated at 1000 rpm for 5 min to remove cell debris. Ultimately, the supernatants were measured by specific ELISA kits  according to protocol recommended by manufacturer.Additionally, to detect the amount of intracellular Aβ40 and Aβ42, firstly we extracted proteins from cells and measured the concentration of protein extraction via BCA kits. Subsequently, all samples were measured by ELISA kits according to protocol recommended by manufacturer. Most notably, the amount of intracellular Aβ40 and Aβ42 should were normalized by total protein concentration, which was measured by BCA kits. These experiments duplicated at least three times, and the data were acquired from independent experiments.Firstly, cells were seeded in coverslips and cultivated for 12–18 h. And then, cells were washed three times by PBS before being fixed via 4% paraformaldehyde for 20 min and exposed to 0.5% Triton X-100 for 30 min at room temperature. Afterward, cells were incubated with anti-APP antibody (1:500) in combination of specific marker proteins antibodies, including anti-Rab5 (1:100), anti-TGN46 (1:100), and anti-LAMP1 (1:100) overnight. After another wash, cells were incubated with both Alexa Fluor 488 and 594-conjugated secondary antibody (1:1000) for an hour at room temperature. After being washed, the cells were exposed with DAPI  about 5 min and were measured by confocal laser scanning microscope .Images were obtained using ×63 and ×100 oil immersion objectives; the colocalization of APP with specific marker proteins was quantified using Coloc2 plugin of ImageJ and presented using Mander’s correlation coefficient. To analyzed the size distribution of early endosomes puncta, we firstly converted picture unite from pixels into microns via Spatial calibration plugin of Image Pro Plus and counted the numbers of early endosomes within limited sizes and the areas of early endosomes, respectively, using Count/Size plugin of Image-Pro Plus software. These experiments duplicated at least three times, and the data were acquired from independent experiments.P > 0.05; In addition, ∗P < 0.05, ∗∗P < 0.01; ∗∗∗P < 0.001; and ∗∗∗∗P < 0.0001 were all considered statistically significant.All values were presented as mean ± standard deviation (mean ± SD) of at least three experiments, and analysis of variance was performed using GraphPad Prism 8 . To compare statistical differences among each group, one-way ANOVA was used. The definition of statistical no significance (ns) is To detect the effect of cholesterol mediated by DHCR24 on Aβ production, we knockin or knockdown DHCR24 to construct cell models with different cholesterol levels via lentiviral vectors in neuronal and astrocytic cell lines. Compared to blank and vector cells, expression level of DHCR24 gene and protein were significantly decreased in lower expressing DHCR24 cells, whereas were significantly elevated in overexpressing DHCR24 cells A–D, H–K.Although DHCR24 could not influence the expression of APP and BACE-1 C, G. ImpBased on the discrepancy between our outcomes and the previous results derived from the APP knockin models, we hypothesize that the different results may attribute to the two kinds of different cell models and APP’s effect on cellular cholesterol homeostasis. Because several studies found that APP and its fragments affect cellular cholesterol biosynthesis , 25, 26.Additionally, the mRNA expressions of major cholesterol synthetases, such as cholesterol regulatory element binding protein 2 (SREBP2), hydroxymethyl glutaryl-CoA reductase (HMGCR), squalene epoxidase (SQLE), 7-dehydrocholesterol reductase (DHCR7), and DHCR24, were significantly decreased in APP overexpressing neuronal and astrocytic cell lines E. FurtheIntriguingly, APP trafficking in endosomal recycling pathway has always been a focus in the research about the regulation of cellular cholesterol on Aβ generation , 35. To In our study, we found that in DHCR24 knockdown neuronal cell lines, the amount of APP in early endosomes was slightly reduced, by analyzing the colocalization of fluorescence signals of APP and Rab5, a specific early endosome marker. However, in DHCR24 knockin neuronal cell lines, the amount of APP in early endosomes had not significantly changed A, C. MorTo sum up, our data suggest that cellular cholesterol deficiency could lead to disrupt a normal APP trafficking by impaired early endosome’s functions and hindered the transport of APP through early endosome to TGN.Cholesterol as an important component of membrane, extensively participates in vesicles trafficking. Moreover, several cholesterol-associated proteins have been found to play an important role in APP vesicles trafficking. To examine the effect of cholesterol on membrane-dependent endosomal recycling pathway for APP, we found the expressions of vesicles trafficking–associated proteins Flotillin-1 and sortilin-related receptor 1 (SORL1/SorLA) were altered in HT22 neuronal cell lines. Flotillin-1, as a critical constitutive protein of lipid rafts, played an essential role in vesicles trafficking, including APP intracellular trafficking , 41. In SorLA is a sorting receptor involved in retrograding APP from early endosome into TGN , 43, 44.Although the majority of the cell culture studies suggest that elevated cholesterol level increases Aβ production, a consensus has not yet been reached , 7, 8, 9Srebp2, Hmgcr, Dhcr24, Dhcr7, and Sqle, resulting in drastically decrease of cellular cholesterol level  cellular APP expression level was not altered in the new cell models; (2) cellular cholesterol homeostasis also was not changed or disrupted in the new cell models before DHCR24 knockdown or knockin; and (3) cellular cholesterol levels could be effectively modulated by DHCR24 knockin or knockdown in the new cell models, which is consistent with our previous studies , 29, 30.It was reported that treating astrocytes with proinflammatory cytokine or Aβ42 oligomers can increase APP and BACE-1 expressions in astrocytes and then made astrocytes secret more Aβ40 , 52. In Mechanistically, the molecular control of the initiation of APP processing and Aβ generation in endolysosomal dysfunction is poorly understood in AD , 35. MorThere is growing interest in the potential role of cholesterol in the pathogenesis of AD. Very importantly, genetic evidences from the inherited AD animals and patients, such as APOE4 allele, obviously supports that cellular cholesterol deficiency is associated with Aβ production and accumulation , 58, 59)In summary, in new neuronal and astrocytic model systems mediated by DHCR24, we conclude that the deficiency of cellular cholesterol clearly plays an influential role in promoting Aβ production, at least partly by mediating cholesterol-dependent endosomal recycling pathway for APP . BesidesAll data supporting the conclusions of this article are available from the corresponding author on reasonable request.This article contains The authors have no competing interests.


Introduction: Colorectal cancer is one of the most prevalent life-threatening malignant tumors with high incidence and mortality. However, the efficacy of current therapeutic regimens is very limited. Regorafenib has been approved for second- or third-line treatment of patients who are refractory to standard chemotherapy diagnosed with metastatic colorectal cancer, but its clinical efficacy needs to be further improved. Accumulating evidence demonstrates that statins also possess potent anticancer activities. However, whether regorafenib and statins pose synergistic anticancer effects in colorectal cancer is still unclear.Methods: Sulforhodamine B (SRB) assays were applied to evaluate the anti-proliferative activity of regorafenib or/and rosuvastatin in vitro, and immunoblotting analysis were applied to detect the effects of regorafenib/rosuvastatin combined treatment on mitogen-activated protein kinase (MAPK) signaling and apoptosis-related proteins. MC38 tumors were applied to investigate the synergistic anticancer effects of regorafenib in combination with rosuvastatin in vivo.Results: We found that regorafenib in combination with rosuvastatin exerted significant synergistic inhibition against colorectal cancer growth in vitro and in vivo. Mechanistically, regorafenib and rosuvastatin combination synergistically suppressed MAPK signaling, a crucial signaling pathway promoting cell survival, as indicated by the reduction of phosphorylated MEK/ERK. In addition, regorafenib in combination with rosuvastatin synergistically induced the apoptosis of colorectal cancer in vitro and in vivo.Discussion: Our study demonstrated the synergistic anti-proliferative and pro-apoptotic effects of regorafenib/rosuvastatin combined treatment in colorectal cancer in vitro/vivo and might potentially be evaluated as a novel combination regimen for clinical treatment of colorectal cancer. Colorectal cancer (CRC) has emerged as the third most common cancer and the second leading cause of cancer-related deaths throughout the population worldwide . Current®), a novel diphenylurea multi-kinase inhibitor, is capable of blocking activities of multiple protein kinases, including VEGFR1-3, TIE2, PDGFR-β, FGFR, RAF, etc. , an enzyme accountable for the rate-limiting step of cholesterol synthesis . Statinsy in CRC .et al. demonstrated that simvastatin-mediated oxidative stress generation enhanced cellular apoptosis induced by mitogen-activated protein kinase (MEK) inhibitors in pancreatic cancer cells  via mevalonate pathway/YAP axis  signaling and promoting the apoptosis of colorectal cancer 2.Cell lines SW620 and MC38 were purchased from the Cell Bank of the Chinese Academy of Sciences . Both SW620 and MC38 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 U/mL). All cells were maintained at 37°C in a humidified atmosphere containing 5% CORegorafenib (HY-10331), Rosuvastatin Calcium (HY-17504) and Z-VAD-FMK (HY-16658B) were purchased from MedChemExpress. Both regorafenib and rosuvastatin were dissolved in sterile dimethyl sulfoxide (DMSO). Stock solutions (50 mM) were kept at −80°C and diluted in culture medium to working concentration before experiment. The antibodies against phospho-MEK (#9154), MEK (4694S), phospho-p44/42 MAPK (#4370) and p44/42 MAPK (#4695) were purchased from Cell Signaling Technology. The antibodies against cleaved PARP (ET1608-10) were purchased from HuaBio Bioscience, the antibodies against GAPDH (db 106) were purchased from Diagbio Bioscience.6 cells/well. For cellular apoptosis experiment, adherent cells were incubated with indicated drugs for 24 h, while for signaling pathway detection, cells were exposed to drugs for 4 h and subsequently stimulated with 500 ng/mL EGF for 30 min or not. Then, cells were harvested and lysed in 2.5× loading buffer. Lysates were boiled at 95°C for 25 min and size-fractionated using SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) membrane . Having blocked with 5% milk dissolved in phosphate-buffered saline with Tween 20 (PBS-T) for 1 h, membranes were incubated with primary antibodies: PBS-T  at 4°C overnight. Then membranes were washed by PBS-T for three times (10 min/time), followed by incubation with secondary antibodies: PBS-T  at room temperature for 1 h. Then membranes were washed by PBS-T for three times (10 min/time) again. Finally, the membranes were incubated using enhanced chemiluminescence (ECL) substrate , exposed and visualized on AmershamTM ImageQuant 800.CRC cells were seeded into 12-well plates at a density of 10SRB = 100% - / × 100%. The coefficient of drug interaction (CDI) analysis was conducted to quantitatively evaluate drug interaction, as synergism (CDI <1), additivity (CDI = 1) and antagonism (CDI >1), and especially, CDI <0.7 represents a significantly synergistic effect  assays. Inhibition ratio (IR) of SRB: IR c effect . CDI = X5. When MC38 tumors reached a mean size about 70 mm3, tumor-bearing mice were randomized into four groups (n = 7), with the difference of mean tumor volume between groups less than 1 mm3. Each group accepted intragastric administration of saline, regorafenib (10 mg/kg/day), rosuvastatin (50 mg/kg/day) and combination treatment, respectively. Tumor volume and body weight of each mouse were monitored every 2 days. Calculation was carried out as follows: Tumor volume (TV)= (length × width2)/2; Relative Tumor volume (RTV) = TV day n/TV day 1 × 100%; Inhibition ratio (IR) of Tumor: IR Tumor = 100% - Tumor weight treatment group/Tumor weight control group × 100%; T/C (Relative tumor proliferation rate) = RTV treatment group/RTV control group.Female C57BL/6 mice were purchased from National Rodent Laboratory Animal Resource . All animal experiments were conducted in accordance with the regulations of Institutional Animal Care and Use Committee (IACUC). The protocols for the animal study were approved by the Animal Care and Use committee of Zhejiang University, with the ethical approval numbers IACUC-s22-013. C57BL/6 mice were subcutaneously injected with MC38 cells at the density of 2 × 10About 0.01 g tumor tissue was weighed into centrifuge tubes with a small steel ball per tube, and 100 μL 1% NP40 lysate with phosphatase and proteasome inhibitors were added. Grind in a tissue grinder , and then centrifuge at 15,000 rpm for 30 min at 4°C. Supernatant was removed into a new centrifugal tube and protein concentration was measured. Remove 200 μg protein from the supernatant, add 100 μL 5× loading buffer, and fix the volume to 200 μL with ddH2O. Samples were heated at 95°C for 30 min, and subsequently immunoblotting analysis was performed.t-test were used to analyze comparisons between groups and p < 0.05 were considered statistically significant .All experiments were repeated at least three times in our study, Data was analyzed by GraphPad Prism 8.0 and presented as mean ± SD. Two-tail Student’s 50) values of 3.20 ± 0.46 μM and 1.07 ± 0.16 μM respectively, while rosuvastatin posed little effects on SW620 and MC38 cells growth, with IC50 values of 83.15 ± 20.75 μM and 72.11 ± 21.90 μM respectively  or rosuvastatin  for 72 h, then the cell viabilities were assessed by SRB assay. As shown in ectively .). Taken together, co-treatment with rosuvastatin and regorafenib induced a more pronounced growth inhibition in colorectal cancer cells compared to single-agent treatments in vitro, supporting the synergistic antitumor potential of regorafenib/rosuvastatin combination.To further investigate whether regorafenib and rosuvastatin posed synergistic antitumor effects in colorectal cancer cells, CRC cells were exposed with increasing concentrations of regorafenib and rosuvastatin for indicated times. As shown in As regorafenib was found to induce apoptosis of CRC cells, we next asked whether regorafenib/rosuvastatin co-treatment made difference in cellular apoptosis beyond proliferative inhibition . The clain vitro  signaling pathway, thus inhibiting tumor progression. Therefore, we speculated regorafenib/rosuvastatin co-treatment might exert a synergistic antitumor effect by amplifying inhibitory effect on MAPK signaling.First, we evaluated cellular MAPK pathway in response to regorafenib and/or rosuvastatin treatment in SW620 cells. As shown in in vitro, so we next attempted to investigate whether regorafenib and rosuvastatin co-treatment significantly inhibited colorectal cancer progression in vivo. To determine the doses of regorafenib and rosuvastatin in animal studies, we referred to previous reports and chose the suitable doses with regorafenib as 10 mg/kg, and rosuvastatin as 50 mg/kg. MC38 cells were subcutaneously injected into C57BL/6 mice, after tumor volume reached ∼70 mm3, these mice were divided into four groups randomly (n = 7/group), and daily intragastrical administered with regorafenib (10 mg/kg/day) and/or rosuvastatin (50 mg/kg/day), the tumor volume and body weight were evaluated and recorded every 2 days.Aforementioned data has demonstrated that regorafenib in combination with rosuvastatin synergistically inhibited colorectal cancer growth in vivo. To reflect the potential impact on animal survival from the side, we evaluated the tumor growth delay effect by calculating the average tumor growth days to reach approximately 4 times in the initial tumor volume for each group. As shown in As shown in in vitro, we next examined intratumor MAPK signaling and apoptosis related protein levels in tumor samples. As shown in in vivo. Meanwhile, we also investigated intratumor MAPK signaling related protein levels and found regorafenib and rosuvastatin co-treatment synergistically suppressed intratumor MAPK signaling, which was consistent with results of cellular experiments in vitro  were 13.2 months and 12 months respectively  mediating production of mevalonate therefore instrumentally interfering with endogenous cholesterol synthesis . Mountinin vitro and in vivo.Multiple studies have revealed that MAPK signaling pathway is widely involved in the process of cellular apoptosis and the activation of MAPK signaling inhibits apoptosis through complicated mechanisms. On the one hand, it suppresses of the function of pro-apoptotic proteins. For example, MEK1/2 or ERK1/2 activation inhibits Bim activity by phosphorylating Bim at Ser109/Thr110 and downregulates its expression at the same time . Moreovein vivo. The toxicity of statins includes elevated liver enzymes, acute kidney injury, hepatotoxicity, etc (According to pharmacokinetics studies, most statins are the substrates of CYP3A4, P-glycoprotein and/or OATP1B1 transporters, whose combined utilization with the inducers or inhibitors of these transporters would result in changing plasma concentrations and inducing toxicity, but rosuvastatin is not included . So, altity, etc . Some stity, etc , suggestity, etc , but thein vitro and in vivo. Mechanically, regorafenib in combination with rosuvastatin synergistically suppressed MAPK signaling, thus suppressing cell proliferation. In addition, regorafenib and rosuvastatin combination therapy synergistically induced the apoptosis of colorectal cancer in vitro and in vivo. Our study clarified that non-clinical studies demonstrated synergy between regorafenib and rosuvastatin. Therefore, it may potentially be evaluated as a novel combination regimen for clinical treatment of colorectal cancer.Our findings demonstrated the synergistic antitumor activity of regorafenib and rosuvastatin combination therapy in clinical colorectal cancer


Staphylococcus aureus biofilms produced by both areference strain and a clinical strain isolated from a diabetic footinfection, mainly by the killing of biofilm-embedded bacteria. Thedirect effect of vCPP2319 on bacterial cells was imaged using atomicforce and confocal laser scanning microscopy, showing that the peptideinduces morphological changes in bacterial cells and membrane disruption.Importantly, vCPP2319 exhibits low toxicity toward human cells andhigh stability in human serum. Since vCPP2319 has a limited effecton the biofilm EPS matrix itself, we explored a combined effect withα-amylase (EC 3.2.1.1), an EPS matrix-degrading enzyme. In fact,α-amylase decreases the density of S. aureus biofilms by 2.5-fold. Nonetheless, quantitative analysis of bioimagingdata shows that vCPP2319 partially restores biofilm compactness afterdigestion of the polysaccharides, probably due to electrostatic cross-bridgingof the matrix nucleic acids, which explains why α-amylase failsto improve the antibacterial action of the peptide.The formation of biofilms is a common virulence factorthat makesbacterial infections difficult to treat and a major human healthproblem. Biofilms are bacterial communities embedded in a self-producedmatrix of extracellular polymeric substances (EPS). In this work,we show that vCPP2319, a polycationic peptide derived from the capsidprotein of Torque teno douroucouli virus, is active against preformed  To worsenmatters, the dormancy of biofilm-embedded bacteria renders uselessantibiotics that target metabolic processes. Antibiotic-based treatmentof biofilms often fails despite prolonged use of high dosages. Thus,novel antimicrobial agents and strategies designed to target bacterialbiofilms are urgently needed.Biofilm-relatedinfections are difficult to treat because biofilm-embeddedbacteria can evade host immune defenses and are less susceptible toconventional antibiotics than bacteria in the planktonic (“freefloating”) form.18 based on theirunique properties. Molecules of this class are present in all organismsas part of the host defense machinery, and are active against a widerange of microbial pathogens.20 AMPs usually have anoverall cationic charge and a high content of hydrophobic amino acidresidues, which allow them to target bacterial membranes that areanionic. Unlike conventional antibiotics, most AMPs kill bacteriafast upon contact with external membranes,23 hence bactericidalactivity is independent of the metabolic status of target cells, givingAMPs the potential to kill slow-growing and nongrowing bacteria inbiofilms. However, being polyelectrolytes, AMP diffusion through thebiofilm matrix can be hindered due to charge interactions with theEPS components, slowing their availability at the inner layers ofthe biofilms.24 As such, the use of EPSmatrix-degrading agents is a particularly attractive strategy to accelerateAMP penetration in biofilms,25 as a combinationof molecules targeting biofilm components (EPS matrix and bacteria)is more likely to succeed in biofilm elimination than single-agenttherapies.26In recent years, antimicrobialpeptides (AMPs) have emerged aspromising candidates for antibiofilm agents27 It has cell-penetrating and anticancer properties,27 and it is also known for its broad-spectrumactivity against bacteria in the planktonic form.28 Here, we investigated the activity of vCPP2319 against S. aureus biofilms formed by a reference strainand by a clinical strain isolated from a diabetic foot infection,of utmost medical importance.9 We used quantitativeimaging methods to study the individual mechanism of action and theeffect on biofilm structure of vCPP2319, and its combined effect withthe matrix-degrading enzyme α-amylase from Bacillus sp. (EC 3.2.1.1), in search of an improved accessibility of thepeptide to bacterial cells that would increase its activity.vCPP2319 is a cationicpeptide reproducing residues 16–36of the capsid protein of Torque teno douroucouli virus.27 Previous studies have demonstrated its broad-spectrum activityagainst both Gram-positive and -negative bacteria in the planktonicform, acting through the disruption of bacterial cell membranes.28 Here, we first focused on studying its serum stability and toxicityagainst human cells, and then on characterizing its activity againstbiofilms of S. aureus, a pathogenthat is commonly involved in biofilm infections.7vCPP2319 is 20-residue, highlycationic peptide derived from thecapsid protein of Torque teno douroucouli virus.Wefirst evaluated the hemolytic effect of vCPP2319. Although the peptidehas a mild effect on hRBCs, in a time- and concentration-dependentmanner, it is clearly much less pronounced when compared with melittin,a gold-standard cytotoxic membrane-active peptide 1A. Thus,Figure S1), for all vCPP2319 concentrations studied,after 1 h incubation with human serum, more than 60% of the peptideremains intact. The corresponding t1/2 values ranged from 207 min at 50 μM to 627 min at 500 μM,revealing high stability, even surpassing the t1/2 ∼ 112 min predicted by a software recently developedby us to estimate peptide half-lives.29 Interestingly, the stability of vCPP2319 increases with concentration,most likely due to the ability to self-assemble into nanoparticles that protect the peptide fromproteolytic degradation.31The stability of peptides is an important factorto take into accountbecause their degradation in serum can reduce the concentration ofthe peptide and thus compromise its activity. As shown in S. aureus strains: a reference strain (S. aureus ATCC 6538) and a clinical strain isolated from a diabetic foot infection.On a previous study9 the virulence determinantsof this clinical isolate were investigated, revealing the presenceof genes involved in biofilm formation and development such as intracellularadhesin genes icaA and icaD, quorumsensing gene agrI, coagulase gene coa, protein A gene spa, and clumping factor clfa.The antibiofilm activity of vCPP2319 was testedagainst 24 h-preformed biofilms produced by two S. aureus ATCC 6538. Treatment with increasing concentrations of peptide for4 h  caused a significant reductionin the metabolic activity of biofilm-embedded cells, in a dose-dependentmanner, and irrespective of incubation time. In contrast, when thepeptide was used to treat preformed biofilms produced by the clinicalisolate for 4 h, only at 25 and 50 μM was the metabolic activitysignificantly reduced . As cells usually account for lessthan 10% of biofilm biomass, while the EPS matrix can represent over90% of the dry mass,3 these results demonstratethat vCPP2319 does not affect the matrix. However, for clinical isolatebiofilms  are shown in xy plane images). This indicatesthat the peptide damages the membrane of bacteria, enabling TO-PRO-3iodide to displace, at least partially, SYTO 9 bound to iDNA, whichresults in bacteria stained orange in the xy and xz overlay images.To further investigate the ability of vCPP2319 to directlyinduce membrane permeation of biofilm-embedded bacterial cells, alive/dead CLSM-based assay was performed. Twenty-four hour-preformed z = 2 μm) biofilm layer, an enzyme with nohemolytic activity up to 0.5 mg/mL. As shown in Figure S6, α-amylase had a significant concentration-dependenteffect on the total biomass of 24 h-preformed clinical isolate biofilms,with ca. 80% reduction at 0.5 mg/mL, the highest concentration tested,confirming its ability to degrade the biofilm matrix. Since biofilmmass reached a plateau, the remaining mass must be assigned to nonsaccharideEPS components, namely eDNA and proteins, and to biofilm-embeddedcells. From this dose–response assay, a concentration of 0.13mg/mL was chosen for further assays as the highest not affecting metabolicactivity of embedded cells (results not shown), thus only targetingthe matrix.As polysaccharides are a major component of the EPSmatrix of mostbacterial biofilms, we selected the glycoside hydrolase α-amylasefrom z = 2μm plane, Figure S6). The peptide vCPP2319 (25 μM) has a similar effectbut not as pronounced, and the sequential addition of α-amylaseand vCPP2319 has an intermediate effect  matrix; a similar effectis not detected with the peptide alone (Video S3). Intriguingly, biofilm height is more affected by the peptidethan by α-amylase; the combined effect is stronger  using The total (instead of polysaccharide-only) mass anddensity variationsof the biofilm can be calculated from the results of the crystal violetstaining assay  bydecreasing it. The peptide has a different effect, as it densifiesthe biofilm: the reduction in height balances the reduction in mass.4.When the peptide acts afterbiofilmdigestion by α-amylase, compactness is partially restored despitepolysaccharide loss. The cationic peptide probably crossbridges anionicnucleic acids in the matrix, increasing its density.5.43The compaction effect of the peptidecompensates the digesting effect of α-amylase abrogating synergyin antimicrobial activity 5D. This Biofilm-related infections are extremelychallenging to treat,making the development of new and effective therapeutic strategiesurgently needed. Here, we showed that the viral-derived peptide vCPP2319,known for its activity against bacteria in planktonic form, is alsoable to act against biofilms by killing bacterial cells, highlightingits potential as a dual-action AMP. Using quantitative imaging methods,we also demonstrated that the peptide acts by targeting and disruptingthe membrane of biofilm-embedded cells. Importantly, we showed thatvCPP2319 has remarkable stability in human serum as well as low hemolyticand cytotoxic activities. Moreover, sequential treatment of 28 was synthesized by Bachem AG  with a purityof >95%. The peptide has an amidated C-terminus and a free amineN-terminus.To prepare stock solutions, lyophilized peptide was weighed out ona high precision analytical microbalance, dissolved in sterile Milli-Qwater, and stored at −20 °C.vCPP2319, WRRRYRRWRRRRRWRRRPRR-amide,g for 10 min at 4 °C, washed three times, and resuspended insterile phosphate buffered saline (PBS) (pH 7.4) to a final concentrationof 0.25% (v/v). hRBCs suspensions were incubated with vCPP2319, rangingfrom 0.05 to 50 μM, for 4 or 24 h, with α-amylase from Bacillus sp. , ranging from 0.0078 to 0.5 mg/mL, for 1 h, orwith the hemolytic peptide melittin (positive control), ranging from0.05 to 50 μM, for 1 h at 37 °C, with gentle swirling,in a sterile 96-well microtiter round-bottomed polypropylene plate. After incubation, the plate was centrifuged at1000g for 5 min at 4 °C, and supernatants weretransferred to a sterile 96-well microtiter clear flat-bottomed polystyreneplate (Corning). Hemoglobin release from lysed cells was quantifiedby measuring the absorbance at 415 nm in an infinite M200 microplatereader . Samples incubated withPBS were used as negative control, and samples incubated with TritonX-100 at 1% (v/v in H2O) were used as a control for membrane-disruption.44 Hemolytic activity (%) was determined usingthe following equation:treated is the absorbanceof peptide-treated cells, Absuntreated is the absorbanceof samples incubated with PBS, and AbsTriton is the absorbanceof samples incubated with Triton X-100. HC50 values wereinferred from sigmoidal dose–response (variable slope) curves,using GraphPad Prism 7.0 software . Experiments were performed on different days using differenthuman blood samples.Fresh human blood was obtained fromhealthy donors after written informed consent. To isolate human redblood cells (hRBCs), samples were centrifuged at 1000l-glutamine) with phenol red .The medium was supplemented with 10% (v/v) fetal bovine serum (FBS),1% (v/v) penicillin/streptomycin , 10 ng/mLepidermal growth factor (EGF), 1 μg/mL hydrocortisone , and 10 mM glutamine (ATCC), according to themanufacturer’s instructions. Cells were grown in a humidifiedatmosphere of 5% CO2 at 37 °C. The cytotoxicity ofvCPP2319 toward HMEC-1 cells was determined using CellTiter-Blue CellViability Assay , according to the manufacturer’sinstructions. HMEC-1 cells were seeded into a sterile 96-well clearflat-bottomed polystyrene plate (Corning) at 2.5 × 104 cells/well and incubated for 24 h at 37 °C. After medium removal,cells were washed twice with PBS. Then, 100 μL of previouslydiluted vCPP2319 (0.01–50 μM) in MCDB-131 medium wasadded to the wells. After 24 h of incubation with vCPP2319, cellswere washed twice with PBS and 20 μL of CellTiter-Blue reagentin 100 μL of MCDB-131 medium was added to each well and incubatedfor 3 h in a humidified atmosphere of 5% CO2 at 37 °C.The fluorescence intensity was measured with excitation and emissionat 560 and 590 nm, respectively, using the infinite M200 microplatereader (Tecan). Medium and 1% (v/v) Triton X-100-containing mediumwere used as positive control and negative control, respectively.HMEC-1 cells metabolic activity (%) was determined using the followingequation:FP is thefluorescence intensity of peptide-treated cells, FNC is the fluorescence intensity of negative controls,and FPC is the fluorescence intensityof positive controls. Experiments were performed on different daysusing independently grown cell cultures.Human dermal microvascularendothelium cells  were cultured as a monolayer using MCDB-131 medium (without 2O) human serum (Sigma-Aldrich) at 37 °C, with gentleswirling. At different time points , 120 μL aliquots were taken and treated with 20μL of 5% perchloric acid (v/v in H2O) for 30 minat 4 °C, to stop the proteolytic reaction. To determine the maximumpeptide intensity , serum proteins were first precipitated, followed by the additionof the peptide. Controls of serum without peptide and peptide notincubated in serum were also included in the assay. Samples were nextcentrifuged at 13 000g for 10 min to remove serumproteins. The supernatants were analyzed by reversed-phase HPLC (RP-HPLC)and LC–mass spectrometry (LC-MS). The percentage of intactpeptide was calculated by RP-HPLC peak integration, expressed as percentof the amount at t0, and data were fittedto a monoexponential decay model using GraphPad Prism 7.0 to estimatethe peptide half-life (t1/2). AnalyticalRP-HPLC was performed on a LC-20AD instrument equipped with a Luna C18 column  using linear gradients of solvent B (0.036% (v/v)trifluoroacetic acid (TFA) in acetonitrile (ACN)) into A (0.045% (v/v)TFA in H2O) over 15 min, at a flow rate of 1 mL/min andUV detection at 220 nm. MS analysis was performed on an LC-MS 2010EVinstrument (Shimadzu) fitted with an XBridge C18 column , elutingwith linear gradients of F (0.08% (v/v) formic acid [FA] in ACN) intoE (0.1% (v/v) FA in H2O) over 15 min at 1 mL/min flow rate.45vCPP2319 at final concentrationsof 50, 100, 200, and 500 μM was incubated with 25% . vCPP2319, in PBS, was preincubated for 4 h, at 25 °C, atdifferent concentrations . The Z-averagehydrodynamic diameter was measured using DLS in particle size analysismode. For each experiment, a single measurement, corresponding toan autocorrelation curve averaged from a minimum of 20 runs, was performed.All measurements were performed at 25 °C. Data were obtainedfrom two independent experiments.S. aureus ATCC 6538 was purchased from ATCC. S. aureus clinical strain was previously isolatedfrom patients with infected foot ulcers, genotyped and screened forvirulence and antimicrobial resistance traits.9 Bacteria in the planktonic form were grown in Mueller Hinton Broth(MHB) , for 18 h at 37 °C. S. aureus bacterial biofilms were grown in trypticsoy broth (TSB) (BD) containing 0.25% (w/v) glucose (TSBG) (Sigma-Aldrich)for 24 h at 37 °C, to allow biofilm formation.47 as previously described.28 Briefly, S. aureus suspensions were prepared in MHB to a final concentration of 1 ×106 CFU/mL and incubated for 18 h at 37 °C in a sterile96-well microtiter round-bottomed polypropylene plate (Corning), containing2-fold dilutions of vCPP2319. Final bacterial concentration was 5× 105 CFU/mL whereas peptide concentration rangedfrom 0.78 to 100 μM. The MIC was defined as the lowest peptideconcentration required to inhibited visible bacterial growth.The ability ofvCPP2319 to inhibit bacterial growth was evaluated by determiningthe minimal inhibitory concentration (MIC) using a standard brothmicrodilution procedure,S. aureus biofilmswas evaluated using three distinct methods, as previously described:24 the metabolic activity and viability of biofilm-embeddedcells was determined using a resazurin reduction fluorometric assayand a colony count assay, respectively. The total biofilm biomasswas quantified by using a crystal violet assay.The effect of vCPP2319on 24 h-preformed 48S. aureus suspensions were preparedto a final concentration of 1 × 106 CFU/mL and incubatedin a sterile 96-well microtiter black flat-bottomed polystyrene plate(Corning) for 24 h at 37 °C. Nonadherent bacteria were removedafter a washing step with MHB, and preformed biofilms were incubatedin the absence or presence of 2-fold serial dilutions of vCPP2319,ranging from 0.78 to 50 μM, for 4 or 24 h at 37 °C. Untreatedbiofilms were used as a control. After washing the biofilms twicewith MHB, alamarBlue reagent was added at a final concentration 10%(v/v in MHB) to each sample and its reduction was monitored by measuringthe fluorescence intensity  of the samples every 5 min during 2h at 37 °C, in an infinite M200 microplate reader (Tecan). Fullyreduced resazurin in MHB, obtained after autoclaving the sample for15 min, was used as positive control. The percentage of resazurinreduction was determined relatively to the control, after blank (10%(v/v) alamarBlue reagent in MHB) correction.Resazurin, theactive compound in alamarBlue reagent (Invitrogen), is a blue dyethat can be reduced to a pink fluorescent intermediate, resorufin,as a result of cells metabolic activity.S. aureus suspensions at 1 × 106 CFU/mL were incubated ina sterile 96-well microtiter clear flat-bottomed polystyrene plate(Corning) for 24 h at 37 °C. Nonadherent bacteria were removedafter a washing step with MHB, and preformed biofilms were incubatedin the absence or presence of vCPP2319 at 25 or 50 μM, for 4h at 37 °C. Untreated biofilms were used as a control. To evaluatethe effect of vCPP2319 redosing on biofilm-embedded bacteria, 24 h-preformedbiofilms were incubated in the absence or presence of vCPP2319 at25 μM, for 4 h at 37 °C. After a washing step with MHB,biofilms were further incubated in the absence or presence of an additionaldose of vCPP2319 at 25 or 50 μM, for 4 h at 37 °C. In bothexperimental settings, after two washing steps with PBS, the biofilmswere resuspended in PBS, followed by cell scrapping with a pipet tipas described elsewhere.50 To evaluate the combinedeffect of vCPP2319 with α-amylase from Bacillus sp. (EC 3.2.1.1) on biofilm-embedded bacteria, 24 h-preformed biofilmswere incubated with 0.13 mg/mL α-amylase for 4 h, followed bythe addition of 25 μM vCPP2319, without washing, for another4 h at 37 °C. Controls of untreated biofilms and biofilms treatedwith α-amylase or vCPP2319 individually were prepared on thesame conditions as the ones used for the combination of the moleculesto have the same total time of incubation. After incubation, the biofilmswere removed by cell scrapping with a pipet tip as described elsewhere.50 In all experimental settings, aliquots were vortexed at high speed,serially diluted in PBS and plated on nutrient-rich trypcase soy agar(TSA) plates . Afterincubation for 24 h at 37 °C, bacterial colonies were counted,and viable bacteria (in CFU/mL) were reported as percentage of thecontrol.51S. aureus suspensions at 1 ×106 CFU/mL were incubated in a 96-well microtiter clearflat-bottomed polystyrene plate (Corning) for 24 h at 37 °C.After a washing step with MHB to remove nonadherent bacteria, preformedbiofilms were incubated in the absence or presence of 2-fold serialdilutions of vCPP2319, ranging from 0.78 to 50 μM, for 4 or24 h at 37 °C, or with α-amylase from Bacillus sp. (EC 3.2.1.1), ranging from 0.0078 to 0.5 mg/mL, for 4 h at 37°C. Untreated biofilms were used as a control. Biofilms werewashed twice with MHB and incubated with 0.25% (v/v in sterile Milli-Qwater) crystal violet (Sigma-Aldrich) for 30 min at room temperature.After each sample was washed three times with MHB, crystal violetwas solubilized with 95% (v/v) ethanol  by repeated pipetting. Biofilm biomass was quantified by measuringthe absorbance at 590 nm of each sample in an infinite M200 microplatereader (Tecan). The percentage of crystal violet staining was determinedrelative to the control after blank (0.25% (v/v) crystal violet insterile Milli-Q water) correction.Crystal violet is a basicdye that binds to negatively charged molecules, such as those in biofilmsEPS matrix and on bacterial membrane surface.S. aureus biofilms in the absence and presenceof vCPP2319. Bacterial suspensions at 1 × 106 CFU/mLwere incubated in a sterile slide with a removable 12 well siliconechamber (Ibidi) for 24 h at 37 °C. After a washing step withMHB, preformed biofilms were incubated in the absence or presenceof vCPP2319 at 25 or 50 μM, for 4 h at 37 °C. Untreatedand treated biofilms were then washed once with sterile Milli-Q waterand incubated with 0.1% (v/v) glutaraldehyde (Sigma-Aldrich) for 4h at room temperature. Afterward, each sample was washed twice withsterile Milli-Q water and allowed to dry in air for 1 h at room temperature.AFM images were acquired by using a JPK NanoWizard IV mounted on a Zeiss Axiovert 200 inverted microscope .The AFM head was equipped with a 15 μm z-range linearized piezoelectricscanner and an infrared laser. Images were acquired in air and inintermittent contact mode using uncoated silicon ACL cantilevers fromAppNano  with typical resonance frequenciesof 200–400 kHz and spring constant of 13–77 N/m. Scanspeeds ranged between 0.2 and 0.4 Hz and total scan areas of 5 ×5 μm were imaged. Height and error images were recorded. Heightimages reflect the topography of the sample. Error images are generatedby the deflection of the cantilever as it bends while interactingwith the sample and are adequate to improve resolution on the edgesof objects imaged by AFM. For each experiment, three different areaswere imaged. The surface roughness was defined as the root-mean-squareroughness (Rrms)52 and it was obtained from AFM height images usingGwyddion software version 2.56, based on the following equation aftercompensation for bacterial curvature:N is the number ofdata points and zi isthe height deviation of i-th point from a mean line.53 For the biofilm surface roughness, Rrms values were calculated from the totalimaged area of the biofilm, while for the cell surface roughness, Rrms values were calculatedfrom a line of cells in three different regions of each image of untreatedand peptide-treated biofilms. The final Rrms values are the averages of all measurements.AFM was used to visualize the topography and morphology of 24S. aureus suspensionsat 1 × 106 CFU/mL were incubated in a sterile μ-Slide8-well plate (Ibidi) for 24 h at 37 °C. Preformed biofilms werewashed once with PBS and incubated with vCPP2319 at 25 or 50 μM,in PBS, for 4 h at 37 °C. Untreated biofilms were used as a control.After a washing step with PBS, biofilms were sequentially stainedwith 3 μM SYTO 9 for 30 min and 4 μM TO-PRO-3 iodide for15 min at room temperature.To visualize the effect of vCPP2319 on membrane integrity of biofilm-embeddedbacteria, an adapted live/dead assay using SYTO 9 and TO-PRO-3 iodide(Invitrogen) was performed, as previously described.Bacillus sp. (EC 3.2.1.1) on the EPS matrixof S. aureus biofilms was evaluatedusing a CLSM-based assay. Briefly, the bacterial cells and the matrixcomponents of S. aureus biofilmswere imaged using SYTO 9 and Wheat Germ Agglutinin (WGA) Alexa Fluor633 Conjugate . Preformed biofilms(see above) were washed once with PBS and stained with 3 μMSYTO 9 and 5 μg/mL WGA Alexa Fluor 633, for 15 min at room temperature.After an additional washing step with PBS, biofilms were first incubatedwith 0.13 mg/mL α-amylase for 4 h, followed by a washing stepwith PBS, and then further incubated with 25 μM vCPP2319, foranother 4 h at 37 °C. Controls of untreated biofilms and biofilmstreated with α-amylase or vCPP2319 individually were preparedunder the same conditions used in the combined assay to have the samenumber of washing steps and total time of incubation.The effect of vCPP2319 and/or α-amylasefrom 54 TO-PRO-3 mean fluorescence intensity over the image was quantifiedfrom the xy plane of an inner layer  using Fiji’s incorporated plug-ins.54 WGA mean fluorescence intensity over the imagewas quantified from the xy plane of an inner layer and also from a z-projectionof the sum of the z-stack slices, using Fiji’s incorporatedplug-ins.54 Biofilm height was determinedfrom the xz orthogonal images using ZEN lite software(Carl Zeiss MicroImaging).SYTO 9was excited using the Argon laser (488 nm), and both TO-PRO-3iodide and WGA Alexa Fluor 633 were excited with the Helium–Neonlaser (633 nm), respectively. Image acquisition was performed on aZeiss LSM 710 confocal laser point-scanning inverted microscope  equipped with a Plan-ApochromatDIC 63× oil immersion objective . Foreach experiment, three different areas were imaged. All images wereanalyzed with the image processor Fiji.xy at a specific depth, z, If,WGA,xy, is proportional to the mass of polysaccharides in that plane. i  relativeto control.zz covering the total height (h) of thebiofilm, If, WGA,xyz, is proportional to the total mass (m) of the polysaccharide of the biofilm, and i relativeto control.i relative to thecontrol can be calculated using If,WGA,i,xyz and hi.fi is the fraction of crystal violet absorbance retained in condition i relative to control and Vi is the volume of the biofilm in condition i. fi equalsthe global mass ratio of the biofilm is condition i relative to control.fi valueas the same as that obtained for α-amylase alone.The mean emission fluorescence intensity of WGA integratedover a plane p < 0.05.Data are described as mean ±standard deviation (SD) of three independent experiments, unless otherwisestated. Statistical significance was assessed by a one-way ANOVA testfollowed by Tukey’s multiple comparison test and was consideredfor


Here, we used a N-rich nucleobase coordination self-assembly strategy to precisely regulate the dispersion of metal atoms by controlling the metal ratio. Meanwhile, the elimination of Zn during pyrolysis produced porous carbon microspheres with a specific surface area of up to 1151 m2 g−1, allowing maximum exposure of Co−N4 sites and facilitating charge transport in the oxygen reduction reaction (ORR) process. Thereby, the monodispersed cobalt sites (Co−N4) in N-rich (18.49 at%) porous carbon microspheres (CoSA/N−PCMS) displayed excellent ORR activity under alkaline conditions. Simultaneously, the Zn−air battery (ZAB) assembled with CoSA/N−PCMS outperformed Pt/C+RuO2-based ZABs in terms of power density and capacity, proving that they have good prospects for practical application.Metal/nitrogen-doped carbon single-atom catalysts (M−N−C SACs) show excellent catalytic performance with a maximum atom utilization and customizable tunable electronic structure. However, precisely modulating the M−N In mostguration ,22. To aguration . Howeveroduction .x sites. Therefore, incorporating additional nitrogen-rich sources  into the synthetic approach has been widely reported, but is considered a relatively cumbersome solution, which probably leads to buried M−Nx sites in the SACs [The well-defined metal-organic frameworks (MOFs) and other supramolecular coordination composites can potentially be used as desirable precursors for SACs by altering the metal nodes and organic ligands ,26. For the SACs ,32,33.4) on N-rich porous carbon microspheres (CoSA/N−PCMS) after pyrolysis was ensured by rationally adjusting the Zn/Co−N coordination and Zn/Co ratio in the Ad−Zn/Co SCMS precursor. Meanwhile, the elimination of Zn during pyrolysis produced porous carbon microspheres with a specific surface area up to 1151 m2 g−1, allowing maximum exposure of Co−N4 sites and facilitating charge transport for the ORR process. The ultra-high N doping (18.49 at%) promoted the efficient utilization of Co single atoms, which could significantly improve the activity of ORR [onset) and half-wave (E1/2) potential of CoSA/N−PCMS under alkaline conditions are 0.99 V and 0.87 V, respectively. Furthermore, CoSA/N−PCMS as a cathode material for Zn-air batteries (ZAB) exxhibits high opeN−Circuit voltage, power density, specific capacity, and charge/discharge cycle stability.In this study, adenine−Zn/Co supramolecular coordination microspheres (Ad−Zn/Co SCMS) were successfully prepared by a competitive coordination strategy. Adenine, as a nitrogen-rich (51.8 wt%) ligand, provides a large amount of reaction sites for metal ions. The creation of monodispersed cobalt sites . N, N-dimethylformamide (DMF), Co(NO3)2·6H2O, and Zn(NO3)2·6H2O were obtained from Macklin . Nafion solution (5%) was obtained from Macklin.Adenine was received from Sigma-Aldrich . Pt/C (20 wt%) and RuO3)2·6H2O (3.996 mmol) and Co(NO3)2·6H2O (0.004 mmol) were added to the adenine solution, which was stirred continuously for 4 h. After the reaction, Ad−Zn/Co SCMS was collected by centrifugation and then dried in an oven. CoSA/N−PCMS was obtained by directly calcining Ad−Zn/Co SCMS in a tube furnace at 1000 °C for 2 h under an Ar atmosphere (ramping rate of 5 °C min−1).Adenine (1.08 g) was dissolved in 400 mL DMF at 140 °C. Zn(NO3)2·6H2O or Zn(NO3)2·6H2O needs to be added. The calcined products of Ad−Co SCMS and Ad−Zn SCMS are denoted as Co/N−PCMS and N−PCMS, respectively.For comparison, the preparation method of Ad−Zn SCMS and Ad−Co SCMS is similar to that of Ad−Zn/Co SCMS, with the exception that only 0.4 mmol of Co and transmission electron microscopy  were applied to observe the morphology of the samples. The images of annular bright-field scanning transmission electron microscopy (ABF-STEM) and aberratioN−Corrected high-angle dark-field scanning transmission electron microscopy (HAADF-STEM) of CoSA/N−PCMS were collected on a JEM-GRAND ARM300F . X−ray diffraction (XRD) spectroscopy of samples was performed on a Bruker D8 Advance A25 diffractometer . The surface valence state and elemental composition of the catalyst were obtained by XPS . The Brunauer-Emmett-Teller (BET) specific surface area and pore size distribution were acquired on a gas analyzer ASAP 2460 . A Raman spectrometer  was used to determine the extent of defects of the catalysts. X−ray absorption spectroscopy (XAS) of the catalysts was performed on the 1W1B beamline at BSRF .2) or rotating ring disk electrode  surface with a loading of 0.4 mg cm−2. Linear scanning voltammetry (LSV) and cyclic voltammetry (CV) were carried out in O2-saturated 0.1 M KOH solution at a scanning rate of 10 mV s−1. In addition, for the accelerated durability test (ADT), the RDE was subjected to potential cycling. Specifically, the electrode was scanned from −0.4 to 0 V at a rate of 100 mV s−1. CV and ORR curves were recorded at each set of 1000 (1 k) cycles.ORR electrocatalytic tests were performed in a three-electrode system with platinum foil and Ag/AgCl as the counter and reference electrodes, respectively. All tests were performed on a CHI 760E electrochemical workstation. The catalyst sample (5 mg), Nafion , and ethanol (490 μL) were mixed, then sonicated for more than 1 h to obtain a homogeneous ink. Then, 8 μL or 10 μL of ink was dipped onto the rotating disk electrode  by the following equation:kJ) and electron transfer number (n) were calculated from the Koutecky-Levich (K-L) equation:ω is the electrode rotation rate, kJ is the kinetic current density, dJ is the diffusion-limiting current density, J is the measured current density, B is the inverse of the slope of K-L equation, F is the Faraday constant, C0 is the oxygen concentration in 0.1 M KOH solution, D is the diffusion coefficient of O2, and ν is the kinematic viscosity of the electrolyte.The kinetic current density  and the number of electron transfers (n) were calculated using the following equations:rI is the ring current, dI is the disk current, and N = 0.37 is the collection efficiency.The hydrogen peroxide yield (H2 (loading: 2 mg cm−2) was used as the cathode (air electrode), and the electrolyte consisted of a mixture of 6 M KOH/0.2 M zinc acetate. The battery was tested using a CHI 760E electrochemical workstation.In the Zn−air battery test, a polished Zn sheet (8 cm × 3.2 cm) was used as the anode, a carbon cloth coated with CoSA/N−PCMS or Pt/C+RuO−1 and the stretching vibrations of an amine (-NH2) in the band range of 3300−3100 cm−1, which are noticeably attenuated in the assembled Ad−Zn/Co SCMS, Ad−Zn SCMS, and Ad−Co SCMS.The synthetic route for CoSA/N−PCMS is illustrated in Furthermore, XAS was used to characterize the chemical states and local structure of Co and Zn in Ad−Zn/Co SCMS. From the X−ray absorption near-edge structure (XANES) spectra , the neaD/IG ratio, related to the promotion of defects by Co doping and Zn volatilization. Then, the porosity character of the CoSA/N−PCMS was investigated using N2 physisorption measurements . According to the pore size distribution curves  and half-wave potential (E1/2 = 0.87 V). onset and E1/2 of the four samples [−1) was superior to Pt/C (81 mV dec−1), indicating excellent reaction kinetics. To further evaluate the ORR of CoSA/N−PCMS, Koutecky-Levich (K-L) curves . After ADT testing, CoSA/N−PCMS was analyzed by XRD to investigate whether metal aggregation occurred. As shown in 4 active sites compared to N−PCMS, and possesses a considerably larger specific surface area and abundant micropores in contrast to Co/N−PCMS, thus exhibiting excellent ORR performance. Moreover, CoSA/N−PCMS also has superior catalytic activity compared to those of recently reported Co SACs with less N doping (The cyclic voltammetry (CV) curves under alkaline conditions are shown in  samples . Then, t) curves  were obt) curves e,f show N doping .2 (1.40 V). The ZAB with a CoSA/N−PCMS air cathode exhibits a maximum power density of 168.7 mW cm−2, which is better than the ZAB assembled with Pt/C+RuO2 (146.3 mW cm−2) (−1 outperforms that of Pt/C+RuO2 (698 mA h g−1) at a current density of 10 mA cm−2  was assembled a. CoSA/NmW cm−2) c. Moreov mA cm−2 d. As sho the ZAB f. The in4 sites result in CoSA/N−PCMS exhibiting excellent ORR properties . In particular, a ZAB based on CoSA/N−PCMS exhibits a maximum power density of 168.7 mW cm−2, an impressive specific capacity of 796 mA h g−1, and excellent cycling durability over 90 h. This work aims to regulate the coordination environment of metal atoms by using a nitrogen-rich molecule to improve the catalytic activity of the M−N−C SACs.In summary, highly active Co monoatomic catalysts with a high nitrogen content were successfully prepared by a N-rich nucleobase-engaged competitive coordination self-assembly strategy. The N originating from Ad−Zn/Co SCMS promotes the coordination of Co during pyrolysis. The large specific surface area, abundant micropores, and the wealth of atomically dispersed Co−N