Wnt/β-catenin signal transduction is implicated in initiation, propagation, and poor prognosis in AML. Epigenetic inactivation is central to Wnt/β-catenin hyperactivity, and Wnt/β-catenin inhibitors are being investigated as targeted therapy. Dysregulated Wnt/β-catenin signaling has also been linked to accelerated aging. Since AML is a disease of old age (>60 yrs), we hypothesized age-related differential activity of Wnt/β-catenin signaling in AML patients. We probed Wnt/β-catenin expression in a series of AML in the elderly (>60 yrs) and compared it to a cohort of pediatric AML (<18 yrs). RNA from diagnostic bone marrow biopsies (n = 101) were evaluated for key Wnt/β-catenin molecule expression utilizing the NanoString platform. Differential expression of significance was defined as >2.5-fold difference (p < 0.01). A total of 36 pediatric AML (<18 yrs) and 36 elderly AML (>60 yrs) were identified in this cohort. Normal bone marrows (n = 10) were employed as controls. Wnt/β-catenin target genes  showed upregulation, while Wnt/β-catenin inhibitors  were suppressed in elderly AML compared to pediatric AML and controls. Our data denote that suppressed inhibitor expression (through mutation or hypermethylation) is an additional contributing factor in Wnt/β-catenin hyperactivity in elderly AML, thus supporting Wnt/β-catenin inhibitors as potential targeted therapy.Dysregulated  Acute myeloid leukemia (AML) is a malignant neoplasm of myeloid stem cells (MSCs) . The patWnt)/β-catenin pathways are a group of signal transduction pathways that carefully coordinate critical cellular functions such as cell proliferation, differentiation, and migration. These effects are executed through three distinct cascades, namely, canonical and two noncanonical pathways [Wnt/β-catenin hyperactivity through epigenetic dysregulation, resulting in an imbalance in regulatory molecules and upregulating activity of Wnt intermediaries [Wnt/β-catenin signaling provides therapeutic options in AML pts [Signal transduction pathways are crucial cellular processes that modulate genetic expression for physiological functions to maintain homeostasis. However, aging is linked with aberrant function of multiple signaling pathways and other host of factors that maintain cellular health . Winglespathways . Extracepathways . AML patediaries . These eediaries . Gene ex AML pts .Wnt/β-catenin inhibitors are a group of molecules that attenuate signal transduction pathways by saturating Wnt/β-catenin receptors and degrading cytosolic β-catenin [Wnt/β-catenin inhibitors as well as Wnt/β-catenin-targeted therapies interfered with AML progression in cell lines, yet none explored whether they could be used for reducing the progression of AML [Wnt/β-catenin-targeted therapies have also been observed in colorectal cancer [Wnt/β-catenin intermediaries pertain to AML pathogenesis in elderly patients remains unknown.-catenin . Recent n of AML . The effl cancer ; howeverWnt/β-catenin activity via mRNA expression in diagnostic bone marrow biopsy samples of older AML patients while employing normal bone marrow and pediatric AML patient samples as controls. Our results authenticate Wnt/β-catenin overexpression in AML patients, as previously reported [Wnt/β-catenin inhibitor suppression contributes to Wnt/β-catenin hyperactivity. We believe these data could provide bases for future insights into the molecular mechanisms of Wnt/β-catenin overexpression for developing Wnt/β-catenin-related targeted therapies suited towards older AML patients.In this study, we conducted a comparative analysis of reported . Importan = 36) from older AML patients (>60 yrs) (2011–2015). A cohort of pediatric AML (<18 yrs) (n = 36) and age-matched (>60 yrs) normal bone marrow samples (n = 10) were used as controls. The normal control group (n = 10) comprised lymphoma staging bone marrow in patients >60 years, who were negative for lymphoma or any other pathology. Diagnosis and classification were reviewed  according to the 2016 WHO classification system [http://bioinformatics.mdanderson.org/Microarray/SampleSize, accessed on 17 July 2022). FLT3 (ITD and/or TDK) and NPM1 mutation results, where available, were recorded from clinical files. We employed standard criteria for differential expression  of <0.05). The institutional ethics committee approved this study (HREBA.CC-16-0771-MODI dated 14 June 2022).This retrospective study utilized formalin-fixed, paraffin-embedded (FFPE) diagnostic bone marrow (BM) biopsy samples  utilizing multiple cores (1 mm) harvested off areas with maximum tumor concentration. The RNA concentration was quantified using a Nanodrop UV-VIS spectrophotometer  and integrity was assessed using a Bio-analyzer 2100 and RNA Nano Chip assay . Total RNA samples were processed according to the manufacturer’s protocol for the nCounter gene expression PAN-cancer pathway code set containing 770 key genes related to major pathways in cancer biology . Briefly, using nCounterTM technology, mRNA expression analysis was conducted for each sample using specific probes. Probes were hybridized to 300 ng of total RNA for 20 h at 65 °C and applied to the nCounterTM Prep Station for automated removal of excess probe and immobilization of probe–transcript complexes on a streptavidin-coated cartridge. Data were collected using the nCounterTM Digital Analyzer by counting the individual barcodes. mRNA analysis and normalization of the raw data were conducted using nSolver Analysis Software v3.0 (NanoString Technologies). mRNA raw counts were normalized to internal levels of 40 reference genes. Normalized data were log2-transformed and then used as input for further analysis.p-value < 0.05 were considered significant.We used nSolver software v3.0 (NanoString Technologies) for the normalization of raw counts for various genes as determined by the NanoString nSolver platform. SPSS software v24.0  was utilized for other statistical evaluations. Hierarchical clustering and principal component analyses were performed employing Qlucore Omics Explorer v3.2 . Results with fold change ≥2.0 and Wnt/β-catenin pathway molecules in this group ; p = 0.31). A total of 12/36 (33%) patients were positive for both FLT3 and NPM1 mutations. The FLT3+/NPM1− mutation was noted in only 4/36 (11%), while 10/36 (28%) patients were positive for the NPM1 mutation only. In total, 10/36 (28%) were negative for both FLT3 and NPM1 mutations. No distinct differential expression was noted between FLT3 and/or NPM1 mutational status for Wnt/β-catenin molecules. The pediatric group comprised 36 pediatric AML patients . The median bone marrow blast count in this cohort was 53% (range 42–98%). The median expression counts for Wnt/β-catenin pathway molecules did not relate to blast counts ; p = 0.73). In this group, 08/36 (22%) patients were positive for both FLT3 and NPM1 mutations. The FLT3+/NPM1− mutation was noted in only 1/36 (3%), while 07/36 (19%) patients were positive for the NPM1 mutation only. A total of 11/36 (30%) were negative for both FLT3 and NPM1 mutations, while in 9/36 (25%) patients, the FLT3/NPM1 mutational status was unknown. GEP by hierarchical clustering revealed a distinct pattern in older AML as compared to pediatric AML or normal controls  were included. The median bone marrow blast count was 61% (range 46–90%). All patients were investigated on the basis of a standardized protocol. No significant correlation was observed between blast count and median counts for the expression of controls . We idenpatients . We note samples .Wnt/β-catenin signaling contributes towards AML promotion by enhancing cellular growth through several mechanisms, including mutations and epigenetic variations of effectors that moderate these processes [Wnt/β-catenin inhibitors by attenuating Wnt/β-catenin signaling are thought to coordinate tissue growth and specialization during embryonic development and tissue homeostasis [Wnt/β-catenin signaling in various other cancers and physiological processes, the role of aging on Wnt/β-catenin inhibitor activity has not been investigated [Wnt/β-catenin expression in detail could help in devising additional therapeutic options in elderly AML patients. To expand our understanding, we performed a comprehensive comparative GEP analysis in elderly AML against matched normal controls and compared it with pediatric AML patients. We captured holistic data related to tumor cells as well as the influence of the tumor microenvironment across several signal transduction pathways. Our study reported increased Wnt/β-catenin signaling in the elderly AML cohort compared to the pediatric AML cohort. We also ascertained that several Wnt/β-catenin inhibitors were significantly repressed amid elderly AML patients.AML in elderly patients is a distinct disease due to its discrete biology and poor prognosis . The conrocesses . Wnt/β-ceostasis , but theeostasis . Althougstigated ,22. HencWnt/β-catenin pathway expression and FLT3/NPM1 mutations in AML [Wnt/β-catenin pathway expression and FLT3/NPM1 mutational status either in the elderly AML or pediatric AML group. This may be attributed to the small sample size and there being several intricate molecules within a complex network. There was no further attempt made to collectively analyze FLT3/NPM1 mutation status within Wnt/β-catenin pathway molecules.The mutational landscape in AML has a discrete impact on clinical progression, and the WHO classification system has defined distinct AML subtypes based on FLT3 and NPM1 mutations . It is es in AML ,24. HoweWnt/β-catenin inhibitors consists of a broad group of ligands released by a subset of cellular elements for mediating homeostasis and modeling the microenvironments of vascular tissues [Wnt/β-catenin signaling remains unknown [Wnt/β-catenin signaling for elderly AML patients to enhance remission.The secreted frizzled related family (SFRP) of  tissues . However tissues ,27. We f tissues . The inf tissues . Moreove tissues . Like SR unknown . SFRP1 h unknown . SFRP1 i unknown . Hence, Wnt/β-catenin inhibitors and are associated in AML progression inferring oncogenic and tumor-suppressive properties depending on the tumor and its microenvironment [Wnt/β-catenin inhibitor. Furthermore, DKK1 is also secreted by malignant bone marrow stromal cells for inhibiting the adaptive immune response and expansion of myeloid-derived suppressor cells (MDSC), hence permitting immune evasion [Additionally, our study detected a distinct difference in the expression profile of Dikkop (DKK) family molecules between the two age groups within AML. These molecules mediate several signal transduction pathways for coordinating homeostasis . Selectiironment . In thisironment . In AML,ironment . This ef evasion . In this evasion .Wnt/β-catenin inhibitors. They are implicated in the neoplastic processes with or without DKK1 expression, indicating they can mediate an independent impact on AML progression [We also found DKK2 and DKK4 were downregulated in elderly patients, but to a lesser degree than the other gression . Howevergression . AlthougWnt/β-catenin inhibitors in AML are sketchy. Hypermethylation, being the most rationale mechanism, has been reported in a subset (8% to 54%) of AML patients [Wnt/β-catenin inhibitors in AML. This is further supported by epigenetic influence through high expression of micro-RNA impacting Wnt/β-catenin inhibitor (DKK3) as reported in adult B-cell lymphoblastic leukemia [Wnt/b-catenin inhibitors are providing benefits in solid cancers, such agents may have therapeutic potential in elderly AML as well [Wnt/β-catenin transcripts with protein expression, either through immunohistochemistry or Western blotting in solid cancers and other pathological processes [The insight into the molecular mechanisms leading to the downregulation of patients . This imleukemia . These aleukemia . In solileukemia . Hence, leukemia . Since s as well . It willrocesses ,48,49,50Wnt/β-catenin signaling is implicated in clinical biopsies of elderly AML at diagnosis. Some effector molecules are repressed to a greater extent than others, suggesting the disease-founding mechanisms related to Wnt/β-catenin signaling are distinct in elderly AML and are independent of the normal aging process. Furthermore, the GEPs indicate re-routing of other signal transduction pathways, emphasizing the inclusion of expression profile into the genetic analysis to explore biology and prospects of target discovery. The restoration of suppressed Wnt/β-catenin inhibitors may offer a novel and less toxic strategy in the management of elderly AML, although such an approach mandates harnessing DKK1 suppression of host immunity.Taken together, our study demonstrates that elevated


In both systems, poor condition hens had lower DCX+ cell densities, exhibited elevated splenic expression of interleukin-6 (IL6) mRNA, and had a higher relative caecal abundance of methanogenic archea Methanomethylophilaceae. The findings suggest poor body condition is an indicator that individual hens have experienced a comparatively greater degree of cumulative chronic stress, and that a survey of the proportion of hens with poor body conditions might be one way to evaluate the impact of housing systems on hen welfare.It is still unclear which commercial housing system provides the best quality of life for laying hens. In addition, there are large individual differences in stress levels within a system. Hippocampal neurogenesis or plasticity may provide an integrated biomarker of the stressors experienced by an individual. We selected 12 adult hens each with good and poor body condition  from a multi-tier free range system containing H&N strain hens, and from an enriched cage system containing Hy-Line hens . Immature neurons expressing doublecortin (DCX) were quantified in the hippocampus, contents of the caecal microbiome were sequenced, and expression of inflammatory cytokines was measured in the spleen. DCX Non-cage systems comprise a litter floor and either single-tier structures , or multi-tier structures with resources spread across several levels. The systems differ in parameters including average group sizes, environmental complexity, exposure to parasites and disease, challenges to bone health/integrity, opportunities for a naturalistic behavioural repertoire  and incidences of deleterious behaviours 2. Average mortality is higher in non-cage  systems than in cages4. Behaviourally, hens from conventional and enriched cages appear to be more acutely fearful than non-cage birds5, though hens kept in floor pens, single-bird and 5-bird cages displayed comparable durations of tonic immobility, shorter than for 17-bird cages6, implying lower anxiety levels for the smaller groups. Physiologically, observed differences in basal levels of plasma corticosterone (Cort) between cage and non-cage systems are not consistent in their direction between studies12. Heterophil:lymphocyte (H:L) white blood cell ratios are lower in hens provided experimentally with range access13, suggesting an anti-stress effect, but observational studies have not found a difference in H:L ratios between enriched cages and free-range systems15. The majority of assessments have focused upon measures of acute stress. Application of an integrated biomarker to compare the collective longer-term stressful experience associated with each type of housing would provide further insight into the welfare qualities of the different housing systems.While conventional cages are no longer permitted in the European Union (EU Council Directive 1999/74/EC), current types of commercial housing for laying hens still differ in notable ways, and comparisons of relative stressful experience for birds within them have yielded mixed results. Laying hens are housed either in enriched cages, or in non-cage systems, which may provide outdoor access16, but differences in the experiences of individual hens within systems may also have significant implications. Within genetic strains, hens differ in terms of intrinsic traits  and their non-shared experiences within production systems . Whether such intra-flock variation influences the cumulative stressful experience of hens within specific systems, and contributes to whether individuals have lives worth living, warrants further exploration. Observable phenotypic characteristics are generally easier to assess than behaviour or physiology, particularly in commercial settings. Studies suggest that physical and psychological stress are associated with hens exhibiting lower body weights30 and poorer feathering37, while birds that are fearful38 and subordinate40 tend to have paler combs. However, it is not clear whether the overall cumulative welfare of these poor condition hens is worse than that of flock-mates in good conditions at the end of lay. If physical body condition can be associated with a validated marker of long-term stress, it may be employed as an easily assessable proxy for welfare status. How individual factors compare to shared effects of the housing environment in magnitude is also not known, and production systems may differ in the extent to which they favour certain characteristics over others. For example, individual differences in immunity may be more consequential where pathogen exposure is greater, and the importance of socially relevant traits may differ with group size.Consumer concern often focuses on the consequences of housing structure (particularly use of cages and range access) for group welfare41, intermediate (and comparable to control conditions) following combined positive and negative experiences47, and lowest following multiple negative experiences48. In rodents, new hippocampal neurons are often identified by their expression of plasticity marker doublecortin (DCX), which labels a mixed population of immature neurons. In the rodent hippocampus, staining for DCX has been reported to produce similar estimates of cell numbers to BrdU labelling49, including with respect to changes in neurogenesis rates that occur in response to behavioural experiences50. DCX has also been used in birds, most notably in the song system, where it labels neurons ranging from 3 to 30 days old52. However, in birds, there is still a debate about whether DCX only marks new neurons, or more generally neurons with high plasticity55. We therefore err on the side of caution and refer to DCX staining as representing neural plasticity, which includes newly-generated neurons and potentially also other neurons expressing morphological plasticity. Recently, DCX+ neurons in the hippocampus have been employed as a biomarker of chronic stressful experience in chickens, being quantitatively suppressed by experimental unpredictable stress56, severe keel bone fractures57 and long-term food restriction58. In terms of negative experience, inflammation is a further physiological sign of chronic stress and depressive-like mood59, and challenges to health and immunity60, across species. In chickens, quantitative PCR has been used to measure the effect of Cort administration on the expression of cytokines, chemokines, and their receptors, providing a marker of the effects of stress on the immune system61. Composition of the intestinal microbiome (the population of microbes that make up an ecosystem within the gut) is also responsive to physical and psychological stress63, and has been linked to health and welfare in chickens, in particular through microbial dysbiosis65. Interactions between the brain, gut and microbiome influence laying hen behaviour66, as chicken lines displaying high and low levels of feather pecking are characterised by divergent microbiota68. Research on the effect of housing system on the microbiome in hens is currently limited, though one descriptive study indicated differences between caged and free-range systems69.Studies in rats and mice suggest that adult hippocampal neurogenesis provides an integrated measure of positive and negative experiences: levels are highest following multiple positive experiencesBy measuring biomarkers of longer-term stress in hens with good and poor physical body conditions  sampled from both a commercial enriched cage system and a multi-tier aviary with outdoor range, we sought to determine whether: (1) one housing system offers superior welfare for birds at both extremes of the body condition spectrum, (2) there is overlap in experience between systems relating to physical individual differences, or (3) birds in similar conditions have comparable experiences in different housing environments. In terms of outcome measures, adult hippocampal plasticity was assessed by quantifying densities of immature neurons expressing differentiation-marker doublecortin (DCX) in serial hippocampal sections, contents of the caecal microbiome were sequenced to determine relative abundance of different bacterial clades, and inflammatory gene expression was measured in the spleen.The study was approved by the Animal Welfare and Ethical Review Body at Newcastle University (Project ID #702), and all methods complied with UK regulations regarding the treatment of animals. Birds were housed and managed according to RSPCA-Assured standards and DEFRA guidelines on farm, and the Home Office Code of Practice while at the University. A Home Office Schedule 1 method of euthanasia was used. Reporting of the study follows the recommendations in the ARRIVE guidelines.Gallus gallus domesticus) were reared in two floor-based systems with litter at commercial farms in the Midlands, UK. Both sites were operated by the same pullet rearing company, Country Fresh Pullets, according to RSPCA-Assured standards, and arrived at an egg production farm, operated by a different company, in northern England in October 2017. The H&N pullets were 16 weeks of age when introduced to a multi-tier free range adult housing system, whilst the Hy-Line birds were 17 weeks of age upon introduction to an enriched cage system on the adjacent site. At the time of sampling in October 2018, both groups had spent almost a year living in their respective systems, and hens were 65 weeks of age in the multi-tier system and 66 weeks of age in the enriched cage system.From one day old, H&N and Hy-Line Brown pullets  × 150 (w) × 55 (h) cm in dimension and held 50 birds. Cages were arranged into four banks of three vertical tiers with 22 cages per tier, repeated over two floors. Enrichment provided in each cage consisted of perches, a nest box, a scratch mat and a grit auger to drop feed onto the scratch mat. The average temperature in the unit was 19–23 °C. Birds received 15.5 h of artificial light per day, from 01:45 to 17:15. Water was provided through nipple-drinkers, with one nipple for every 10 birds. Layer feed was circulated six times a day through a conveyor belt system. Birds in both systems experienced an identical programme of vaccinations. Hens in the multi-tier system were given a wormer at four intervals during lay. Due to poor laying performance, enriched cage birds alone were given a course of antibiotics at 39 weeks of age . Feed intake was typically 20 g per bird higher per day in multi-tier system than in the enriched cages. At 65 weeks of age (for comparability), average bird mass was 1544 g in the multi-tier aviary compared to 1967 g in the enriched cage system. Production rates were 90.3% in the multi-tier and 81.6% in the enriched cage system, and cumulative mortality had reached 2.50 and 2.74% respectively.The enriched cage housing unit contained 33,120 Hy-Line strain birds, with a stocking density of 15 birds/mWhilst the multi-tier free-range and enriched cage housing systems were operated by separate personnel, they were overseen by the same Production Manager, whose role was to promote the efficient and sustainable production of eggs for retail sale.57, candidate hens were palpated by the same researcher (EAA), trained to an agreed standard70, and those displaying signs of damage were rejected. A total of 12 hens with good body condition and 12 hens with poor body condition were sampled from both the multi-tier and enriched cage system, equating to a total sample size of 48 birds. This group size has proved sufficient to detect experience-based differences in our primary outcome measure (DCX+ cell densities) in previous studies57. While the intention had been to sample hens of the same strain from the two housing systems, this was not logistically possible. Sampling occurred over four successive days, upon each of which 12 birds  were selected from the farm and transported in carry boxes to Newcastle University for processing on the same day. In order to capture extremes of experience in the multi-tier system, good condition birds were selected directly from the range, while poor condition birds were selected from the top inside tier. This meant that the sampled good condition birds ventured outside at least some of the time, whilst the sampled poor condition birds may or may not have chosen to range. To allow birds time to go outside, there was a 30-min delay between the opening of pop-holes in the morning and sampling of birds from the range. Hens were sampled from a different internal colony of the shed on each of the four days, along with the area of the range proximal to these colonies. To ensure independence in the enriched cage system, no two birds were sampled from the same cage, but to avoid a potential confound of cage height, a good and poor condition bird were always selected from different cages on the same tier. To collect a sample representative of the whole housing system, birds were sampled from each tier  in both an inside and outside bank on each of the two floors. Representative images of good and poor physical condition hens sampled from each housing system are displayed in Fig. Within each housing system, birds with good and poor external indicators of physical body condition were selected. Hens were chosen by the farm’s Production Manager, who was familiar with the conditions of the birds. The a priori criteria employed were: 1) body size, (2) feather coverage, and (3) redness of the comb, wherein a high level of each factor represented good physical condition. Because keel bone damage was previously found to be associated with reduced adult hippocampal plasticity body siz71. The chicks originated from separate (hybrid-specific) hatcheries, each in the west of England. Both housing sheds contained litter in the form of wood shavings , which were bedded to a depth of 7.6 cm at the point chicks were introduced. Raised slatted areas were provided for perching, with access assisted by ramps placed every 7.6 m along their length. The H&N shed had a total area of 1187.92 m2 (including the floor and raised slatted areas) and contained 17,085 pullets, which equated to a stocking density of 14.4 birds/m2. Though similar in design, the Hy-Line shed had a larger total area of 1722.12 m2 and housed 25,236 pullets, with a stocking density of 14.7 birds/m2. Water was provided via nipple drinkers, with 12.2 and 12.4 birds per drinker in the H&N and Hy-Line sheds respectively. Both sets of birds had access to feed at all times via a chain feeder, but the quantity of feed in the feeder and the number of times it was topped up per day was adjusted throughout rearing, in order to maintain target body mass for each flock. The H&N shed housed 39 birds per metre of chain feeder, compared to 37 birds in the Hy-Line shed. Both strains received 10 h of artificial light per day from six weeks of age onwards, with a light intensity of 10 lx at bird height. Ambient temperatures were 32–33 °C for at the introduction of day-old chicks and were gradually reduced by 0.5 °C per day, before being maintained at 20 °C. Both strains experienced an identical program of vaccinations, administered from day-old to 13 weeks. At the time of sampling, average bird mass was 1203 g for the H&N flock and 1239 g for the Hy-Line flock. Both flocks had a cumulative mortality of 1.9%. Sampling of pullets occurred during a single day, when both strains of bird were 14 weeks and 3 days old. Twelve birds of average size and body condition were selected from each rearing barn by a senior Production Manager  and manually palpated by EAA to determine if keel bone fractures were present. Individuals exhibiting damage were rejected. Animals were placed in carry boxes and transported to Newcastle University, where they were housed in two pens  with ad libitum feed and water, prior to tissue collection over the following two days (12 birds/day).As different strains of brown hens were sampled from the two adult housing systems, thereby confounding strain and housing type, a sample of pullets of the same genotypes were also taken directly from the rearing farm, to provide a baseline for possible genetic influences on the other measures taken. This sampling occurred after that of the adult hens, in January 2019. H&N and Hy-Line pullets were housed from one day-old in adjacent barns of a commercial rearing farm in Shropshire, England, operated by Country Fresh Pullets according to RSPCA-Assured standardsCollection of tissue from the adult hens and pullets occurred in two separate phases . Animals were weighed before being killed with an intravenous injection of pentobarbital , according to a schedule that alternated between housing system/strain and body condition for the adult hens (n = 48) and strain for the pullets (n = 24). Blood samples were collected for analysis of DNA methylation (results to be reported elsewhere). The spleen was removed and weighed before a sample was placed on ice in a tube containing 1 ml RNAlater® Stabilization Solution . The caecum was dissected from the gut and placed into a 15 ml Falcon tube before freezing on dry ice. Simultaneously, the brain was removed from the skull, placed into 0.1 M phosphate-buffered saline (PBS) in a Petri dish and divided along the longitudinal fissure with a scalpel. The forebrain hemisphere collected for immunohistochemical analysis alternated between left and right, in a manner that was balanced within groups of hens of each body condition and from each housing system. This tissue was immersion fixed for 44–48 h in 4% paraformaldehyde in 0.5 M PBS at 4 °C. Samples were then cryoprotected in a solution of 30% sucrose in 0.5 M PBS, before being embedded in OCT . Coronal Sects. (50 μm) were cut on a cryostat  and stored in cryoprotectant solution  at − 20 °C. Serial sections taken at 400 μm intervals were processed for immunohistochemistry.57, hippocampal formation (HF) tissue sections were stained using an antibody to doublecortin (DCX), to allow quantification of immature neurons. Free-floating sections from the adult hens were stained over six batches, each of which contained tissue from eight birds . Sections from the pullets were stained over three batches, each of which contained tissue from eight birds, wherein four were of the H&N strain and four were Hy-Line. Staining was conducted according to the protocol detailed in Armstrong et al.57. The primary antibody was rabbit polyclonal to doublecortin , incubated at a concentration of 1:1000 for 18 h (4 °C). Secondary antibody incubation utilised biotinylated antirabbit IgG secondary antibody made in goat , at concentration 1:500 for 120 min (room temperature). 1:250 Horse Radish Streptavidin  was used for conjugate enzyme incubation, and 3,3′-Diaminobenzidine (DAB) SIGMAFAST tablets were dissolved in pure water  for chromogen incubation.As previously42, stained DCX+ cells were quantified in the rostral  and caudal 72 HF. An optical microscope  equipped with a digital video camera  and motorized stage system  was connected to a computer running Stereo Investigator software . HF borders were outlined at 2.5X magnification  according to the chick stereotaxic atlas72, and cell counting performed at 100X magnification  according to the Optical Fractionator method. Stereological parameters were set to an optical fractionator grid of 120 × 120 µm for rostral HF and 240 × 240 µm for caudal HF, a counting frame of 50 × 50 µm for both regions and a mounted thickness of 20 µm. For each animal, 4 to 6 hippocampal Sects. 800 μm apart were systematically analysed, starting with the rostral-most section bearing hippocampal tissue. Quantification was performed blind to housing and body condition groups. DCX+ cells of multipolar and bipolar (or fusiform) morphologies were counted separately . The bipolar/fusiform neurons  are assumed to be younger and still migrating, while the multipolar neurons  are assumed to be more mature and settling73. Photomicrographs of example cells with both morphologies are displayed in Fig. + cells per cubic millimetre of sampled tissue were calculated by dividing the number of counted cells of each type by the area of the counting frame (2500 µm2), multiplied by both the number of counting sites sampled in that brain and the section thickness (50 µm), and then multiplying the resulting density per µm3 by 109 to obtain a density per mm3. Densities for rostral and caudal HF were calculated separately.As previously74. Primer sequences for the quantified target genes involved in the inflammatory response  are displayed in Table 74, with LBR utilised as a control gene for normalisation. The 48 samples from the adult hens were processed in a single batch. Analyses were performed blind to the group status of the samples. Splenic gene expression was not explored in the sampled pullets. Gene expression values were log(10)- transformed for statistical analysis.The methods used to extract RNA from the sampled spleen tissue, reverse-transcribe it into cDNA, and produce gene-specific templates for standard curves are detailed in Armstrong et al.For each of the 48 adult hens, DNA was extracted from a 200 mg section of intestinal tissue and caecal content using ZymoBIOMICS DNA minikits  according to the manufacturer’s instructions. The section was cut using a sterile scalpel blade to expose the mucosa and luminal contents to bead-beating with a Qiagen TissueLyser at 30 Hz for 10 min. At each extraction, two controls were included, a blank extraction kit to control for contamination and 75 μl of ZymoBIOMICS standard bacterial community  to control for variations in DNA extraction efficacy. Extracted DNA was quantified using a NanoDrop 2000 spectrophotometer (NanoDrop Technologies).75. Library preparation was performed using a universal tailed tag design with subsequent amplification performed using a two-step PCR with a HiFi Hot Start polymerase (Kapa)76. The first round of PCR was performed using the primers 5′ ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNGTGCCAGCMGCCGCGGTAA-3′ (forward) and 5′- GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGACTACHVGGGTWTCTAAT-3′ (reverse)76. The raw Fastq files were trimmed for the presence of Illumina adapter sequences using Cutadapt version 1.2.1. The reads were further trimmed using Sickle version 1.200 with a minimum window quality score of 20. Reads shorter than 10 base pairs after trimming were removed.Extracted DNA was sent for paired-end sequencing of the 16S rRNA gene at the Centre for Genomic Research (University of Liverpool) using an Illumina MiSeq run. The V4 hypervariable region (515F/R806) was amplified to yield an amplicon of 254 base pairs77. Amplicon sequence variant (ASV) assignment was completed using the dada2 plugin78 and a feature table produced using the feature-table plugin79. Taxonomy was assigned using a pre-trained NaiveBayes classifier based on the SILVA 132 database of the 515F/R806 region of the 16S rRNA gene80, available for download at https://docs.qiime2.org/2018.11/data-resources/, using the q2-feature-classifier plugin81.QIIME2 version 2020.2.0 was used for analysis of the Illumina data+ multipolar and bipolar cell densities. These included staining batch as a random factor, HF subregion  as a repeated fixed factor, and strain (H&N/Hy-Line) as between-subject fixed factor. The interaction between subregion and strain was also included. A univariate ANOVA indicated that DCX+ cell densities  did not significantly differ between staining batches for the pullet samples, F1,44 = 2.98, p = 0.061 . Other pairwise comparisons were non-significant.Analysis of body and spleen mass, adult hippocampal plasticity and splenic mRNA expression was performed in IBM SPSS Statistics v.25). Data was confirmed to meet the assumptions of the statistical approaches employed . In the sample of pullets, body masses of the two strains were compared through an independent samples t-test. For spleen mass, univariate ANOVAs were conducted with body mass as a covariate and strain as a between-subject fixed factor. In the adult hens, a univariate ANOVA was used to compare body mass, with housing system and body condition as fixed factors and together in an interaction term. Spleen mass was explored in a similar model, but which also included body mass as a covariate. To explore adult hippocampal plasticity in the pullets, separate linear mixed models (LMMs) with unstructured covariance were conducted for raw DCX5. Data w+ multipolar and bipolar cell densities as the dependent variables. Models included staining batch as a random factor, HF subregion as a repeated fixed factor and housing system/strain (multi-tier free range/H&N vs. enriched cage/Hy-Line) and body condition (good vs. poor) as between-subject fixed factors. All interactions between HF subregion, housing system and body condition were included. For the purpose of figures, cell densities were normalised (z-scored) within their staining batch and, due to the consistently higher cell densities quantified in the caudal HF, also within the rostral or caudal subregion. This means the figures don’t allow for a comparison across subregions, but the statistical analysis does.To analyse cell counts for the adult hens, separate LMMs were again conducted with DCXLBR mRNA expression in the same sample and log-transformed. A series of univariate ANOVAs were conducted with target transcript expression ratios  as dependent variables, with housing system/strain and body condition as between-subject fixed factors and in an interaction term.For each inflammatory gene quantified in the spleen, measured molar mRNA was converted to a ratio of 82, phylogeny83 and diversity (https://github.com/qiime2/q2-diversity) plugins. Alpha diversity was measured using Faith’s phylogenetic diversity (FPD) index84 to assess species richness and a Shannon diversity (SD) index to assess species evenness. Alpha diversity was compared between sample groups using a Kruskal Wallis test with a false discovery rate (FDR) correction. For numerical data, a Pearson rank correlation was used to assess relationships of other key outcome variables (DCX+ cell densities & splenic IL-6 expression) and alpha diversity. Taxa plots were produced using the q2-taxa plugin (https://github.com/qiime2/q2-taxa). Beta diversity, a metric used to compare species diversity and abundance between samples, was calculated with a robust Aitchison PCA metric85 using the DEICODE plugin (https://library.qiime2.org/plugins/deicode). The beta diversity matrix was used to draw principal coordinate analysis (PCoA) plots and an ANOSIM test over 999 permutations were used to determine the significance of differences in beta diversity between groups. Songbird was chosen to analyse differential abundance of ASVs between groups (–p-formula “Body_Condition + Housing_System”) since it overcomes challenges created by the compositional nature of microbiota data86. Results from both DEICODE and Songbird were visualised through Qurro to identify the taxonomy of ASVs (features) contributing to differences between housing system/strain and body condition groups87.Alpha and beta diversity analyses of extracted caecal DNA were performed at a sampling depth of 13,000 using the alignmentBody masses of the 24 sampled pullets in the strain-baseline group were consistent with flock averages at the time of sampling. Mean spleen masses are also displayed in Table t22 = 1.77, p = 0.090). Body mass did not predict spleen mass on an individual basis , and there was no difference in spleen mass between the two strains . One H&N bird had a particularly large spleen, weighing 6.46 g. However, after removal of this outlier, neither body mass nor strain came to predict spleen mass .There was a trend towards the sampled Hy-Line pullets being heavier than their H&N counterparts . The effect of housing system/strain on body mass was also significant, with birds from the multi-tier free-range (H&N) having lower mass than birds from the enriched cages . According to breed standards which describe typical performance, Hy-line birds are expected to be heavier than H&N birds89. There was no interaction between body condition and housing system/strain . In the adult hens, body mass significantly predicted spleen mass as a covariate . There was no main effect of body condition on spleen mass , but a significant effect of housing system/strain, whereby caged birds (Hy-Line) had heavier spleens . This could be ascribed to the poor body condition birds alone . Specifically, within the enriched cage system (Hy-Line), poor condition birds had heavier spleens than good condition birds (p = 0.018), whilst the former group also had higher spleen mass than hens of poor condition in the multi-tier system . In contrast, no difference in spleen mass was present between housing systems/strains for birds in good condition (p = 0.895).For the sampled adult hens, H&N strain birds were taken from the multi-tier free-range system and Hy-Line birds were taken from the enriched cage system, Body and spleen masses of good and poor body condition hens are displayed in Fig. + cell densities in the two sampled strains of 14-week-old laying hen pullet are displayed in Table + multipolar  and bipolar  cells were quantified in the caudal HF than in the rostral subregion. The H&N and Hy-Line pullet strains did not differ from each other in their densities of DCX+ multipolar cells over the whole HF . Densities of DCX+ bipolar cells also did not differ between the two strains .Descriptive statistics for subregional DCX+ cell densities in the sampled groups of adult birds are displayed in Table + multipolar neurons was again found in the caudal HF than in the rostral subregion . There was no main effect of housing system/strain  on multipolar cell density , but hens with poor body condition had lower multipolar cell densities over the whole HF than hens with good body condition , nor did HF subregion interact with housing system/strain  or body condition . Finally, there was no three-way interaction between HF subregion, housing system/strain and body condition for multipolar cell densities . The density of DCX+ bipolar cells was also higher at the caudal HF pole than in the rostral region . Housing system/strain also had no main effect on bipolar cell densities . There was a trend towards hens with poor body condition having a lower density of DCX+ bipolar cells than their good condition counterparts , and HF subregion did not interact with housing system/strain  or body condition . Also, there was no three-way interaction between HF subregion, housing system/strain and body condition on bipolar cell densities .Descriptive statistics for DCXLBR in the spleens of the adult hens did not differ with housing system/strain  or body condition , nor did these factors interact . Inflammatory gene expression also did not differ between the two housing systems/strains  for IL-10 , IL-1β , IL-6 , IL-8 , or TGFβ . There was no effect of body condition on expression of IL-10 , IL1β , IL-8 , or TGFβ . However, splenic expression of IL-6 mRNA was higher in hens with poor body condition  than in hens with good body condition .Expression of control gene IL-6 expression ratio did not correlate with DCX+ multipolar cell densities in the HF of individual hens . The same was true for bipolar cells .When included as a covariate in the previous model, splenic Sufficient DNA for 16S rRNA sequencing was extracted from all samples. A total of 7,443,396 reads were obtained. After filtering, merging of paired reads and chimera removal, a total of 4,988,403 reads remained  giving a mean of 69,283  reads per sample. The median number of reads per sample was 69,415.p = 0.0002) and Shannon Diversity  with higher FPD and SD in chickens from multi-tier housing (H&N). There was no significant difference in mean FPD index  or SD  between good and poor body condition chickens. Other variables included in the analysis were not correlated with alpha diversity: DCX+ cell counts in the caudal  and rostral  HF; splenic IL-6 mRNA expression (r = 0.20 p = 0.19).Housing system/strain  had a significant impact on mean Faith’s Phylogenetic Diversity , but body condition did not . A PCoA plot of robust Aitchison distance between samples showed some clustering of samples by housing system/strain and body condition  but not in the enriched cages . Beta diversity was not significantly predicted by DCX+ cell densities in the caudal  or rostral  HF, or by IL-6 expression .Housing system/strain had a significant effect on beta diversity  chickens  indicating a higher proportion of Firmicutes compared to Bacteroidetes in these animals. From a plot of differential rankings of features  birds. To explore other taxa contributing to differences between housing systems, the top and bottom 10% of discriminating features (n = 87) between housing systems were selected as the numerator and denominator features. The taxonomic family of these features is shown in Fig. p < 0.001), indicating a higher proportion of numerator features, with a higher proportion of denominator features in multi-tier (H&N) samples. A greater number of Cyanobacteria were present in the numerator features (higher in enriched-cage samples) while a higher number of Spirochaetes were present in the denominator features (higher in multi-tier samples). Within the features assigned to Spirochaetes, those further identified as Treponema were only identified as differential in multi-tier (Hy-Line) samples.Songbird analysis included 878 features. First the effect of housing system/strain was considered. A common dichotomy in the chicken caecal microbiome is the balance between Bacteroidetes:Firmicutes ratio has previously been postulated to affect host metabolism91, the log ratio of these taxa was explored for an effect of body condition. There was no significant difference in the log ratio of Bacteroidetes and Firmicutes between good condition and poor condition hens . The top and bottom 5% of discriminating features (n = 43) between body condition groups were selected as the numerator and denominator features. The taxonomic family of these features is shown in Fig. p < 0.001), indicating an increased proportion of numerator features, with a higher proportion of denominator features in good body condition samples. Features assigned to Cyanobacteria, Euryarchaeota, Spirochaetes and Lentisphaerae were associated with poor body condition while features assigned to Firmicutes and Bacteroidetes were more associated with good condition samples. The taxonomy of features associated with poor body condition were explored further. Those assigned to Cyanobacteria could only be assigned to the level of order as Gastranaerophilales. The four features assigned to Euryarchaeota all belonged to the family Methanomethylophilaceae and two of the features assigned to Spirochaetes were assigned to the genus Brachyspira. Further exploration of the taxonomy of features associated with good body condition showed a high proportion of features assigned to the family Clostridiaceae vadinBB60 and genus Bacteroides. The distribution of these taxa within the feature rankings is shown in Fig. Features that were differentially abundant between chickens of different body conditions were explored further. Given that the IL-6, and a higher abundance of caecal microbiota such as Methanomethylophilaceae. Microbial differences were also observed between hens from the two housing systems, potentially relating to exposure to a greater diversity of microbes occurring within the multi-tier free-range system, but these groups did not differ in their average DCX+ cell densities or inflammatory gene expression. The results suggest that, within laying hen flocks, poor condition hens experience relatively more chronic stress than their good condition counterparts. In contrast, stress profiles appear comparable between housing systems for hens at both the top and bottom of the body condition spectrum, suggesting that the range of stressful experience may be similar for flocks housed in enriched cages and multi-tier free-range systems. Because the two sampled housing systems contained adult hens of different strains (H&N and Hy-Line), a potential strain difference in adult hippocampal plasticity was assessed in a separate group of 14-week-old pullets from the rearing farm. As baseline DCX+ cell densities were comparable, it is unlikely that a genetic difference in initial hippocampal plasticity obscured a subsequent disparity in adult stress.Within both enriched cage and multi-tier aviary housing, all measures of chronic stress taken differed between adult hens with good and poor physical body conditions. Poor condition hens exhibited lower densities of multipolar neurons expressing DCX, elevated splenic expression of inflammatory cytokine 92, and a contribution of ill health to the observed suppression in adult hippocampal plasticity is supported by the other measures taken. In the enriched cages, poor condition hens had enlarged spleens, consistent with the presence of acute infection94. Poor condition hens from both types of housing also presented elevated expression of IL-6 mRNA. IL-6 is one of the primary proinflammatory cytokines driving activation of the HPA-axis in response to immune challenge95, and chronically elevated expression of such cytokines is associated with inflammation and poor welfare59. In mice, IL-6 expression is also upregulated by repeated social stress, in a response blocked by anxiolytic treatment96. In chickens, IL-6 expression is known to be increased by heat97 and cold98 stress, Cort administration61 and infection99. While this finding supports the contribution of illness to poor body condition, social stressors such as the receipt of aggressive pecks, reflected by poor feathering37, might also add to inflammation.Within housing and strains, results of the body condition comparison indicate either that prolonged stress results in a phenotype characterised by low body mass, sparse feathering, and a pale comb, and/or that being in a poor physical state is chronically stressful for commercial hens. The correlational nature of the study precludes a definitive conclusion about the causal direction of this association, though it is likely that cumulative chronic stress has a detrimental impact on both body condition and adult hippocampal plasticity. Immune responses (e.g. to infectious agents) activate the hypothalamic–pituitary–adrenal (HPA) stress axis100. Microbiome composition is altered by stress101 and has reciprocal influences on stress-related behaviour and physiology104. Significant differences in caecal microbiota were present between hens with good and poor body conditions. Most amplicon sequence variants that were more abundant in poor condition birds were assigned to Gastranaerophilales, a poorly characterised taxon that makes interpretation of the result difficult. However, in both types of housing, poor body condition hens also had a higher abundance of methanogenic archea Methanomethylophilaceae. In ruminants, production of methane represents an energy loss of up to 12% of gross energy intake105, which in theory could be used for growth and production. Given that the bovine rumen and chicken caecum are functionally analogous bacterial fermentation chambers for plant material, the presence of methanogenic archea may also represent a net energy loss to the chicken. If this is the case, as further studies of chicken host metabolism may determine, then higher levels of methanogens likely contribute to poorer body condition. Furthermore, the identification of Brachyspira in the microbiome of some poor body condition birds may also represent a direct cause of this state, as a number of species of this genus lead to intestinal spirochaetosis, with associated weight loss, splenomegaly and loss of body condition in laying hens106.Generally, gut microbiota interact in complex, bidirectional ways with the nervous and immune systems2. Notably, Rodenburg et al.1 compared flocks from several enriched cage and non-cage systems and found no difference in an integrated welfare score that collated multiple indicators, including fearfulness, plumage, body and bone condition, dust levels and mortality. As with the sampled flocks in the present study, group size usually differs dramatically between enriched cage and aviary systems, but it is not clear which social system hens would prefer108. Multi-tier  systems are more complex and facilitate greater activity2, but cannibalism and other deleterious behaviours (e.g. piling and smothering) may be more prevalent and difficult to control than in enriched cages109. Non-cage systems are generally associated with an increased incidence of keel bone fractures2, but birds exhibiting such damage were excluded from the present sample due to thorough exploration of this welfare issue in our previous study57. Though not true for the flocks sampled here, greater mortality has been observed in litter-based (i.e. non-cage) housing than enriched cages110 and associated with higher airborne concentrations of dust and bacteria1 and greater average prevalence of bacterial and parasitic diseases111 in the former. Contact with infectious microorganisms in free-range systems is further increased by exposure to soil and transfer from wild animals2. Overall, trade-offs between different positive and negative aspects of both systems may lead to little net difference in chronic stressful experience for hens. In the case of hippocampal plasticity levels, cognitive stimulation113 and stress56 have opposing directional influences, so comparable DCX+ cell densities may reflect the overall balance between environmental complexity and negative experience in each housing system, rather than relative stress alone.There was no main effect of housing system on adult hippocampal plasticity or inflammatory gene expression. This result is consistent with previous conclusions that no single type of commercial housing system is ideal from the perspective of hen welfare, as each is associated with unique limitationsBacteroidetes in the multi-tier free-range and increased Firmicutes in the enriched cages. Because most Firmicutes genomes contain genes enabling sporulation, whereas Bacteroidetes are not spore-forming but microaerotolerant (able to survive up to 24 h)114, there is a relative time limit on the survival of Bacteroidetes within a system. An experiment observing bacterial colonisation of chicks housed with an adult hen further demonstrated that Bacteroidetes were passed from the hen to the chicks, but Firmicutes such as Lachnospiraceae and Ruminococcaceae were derived from the environment115. It is therefore logical that taxa such as Bacteroidetes sustain a greater diversity and abundance within the microbiota in housing systems such as the multi-tier free-range because their design permits contact between larger numbers of individuals. In the present study, differences in caecal microbiota composition cannot be attributed solely to housing system design, as uncontrolled factors such as local exposure to different bacterial profiles116 will have doubtless contributed. Though potential effects of strain should also not be discounted, it has been concluded that environmental exposure, diet and management practices are more influential than genetics in shaping the caecal microbiota117.Observed differences in composition of the microbiome between the sampled housing systems appear to reflect these environments. The broad pattern was for increased 56, it is theoretically possible that the influence of existing individual differences in adult hippocampal neurogenesis on personality and behaviour manifested in differences in external physical condition between hens.A limitation of the present comparison is that the multi-tier housing contained H&N strain hens, while the enriched cage system housed Hy-Line birds. Though no baseline difference in adult hippocampal plasticity was observed between pullets of the two strains, differences in age-dependent adult hippocampal plasticity and/or in lifetime stress resilience cannot be ruled out. The lack of observed difference between multi-tier H&N hens and enriched-cage Hy-Line hens suggests an overall difference in hippocampal neurogenesis may not exist between the two strains later in life, but the design of the study does not permit assessment of possible interactions between strain and other factors (e.g. body condition). Inflammatory gene expression was also not measured in the pullets, though unlike the adult birds, they exhibited no group difference in relative spleen mass. Given that only one flock was sampled from each type of housing system, it is also possible that the flocks sampled were relatively good or poor welfare examples of enriched cage and multi-tier free range systems. Future studies should assess differences between housing systems over a larger number of representative flocks. Additionally, as no experimental manipulations were conducted, the causal relationships cannot be conclusively determined. While it is presumed that changes in adult hippocampal plasticity between the body condition groups are a result of prolonged experience, as in Gualtieri et al.118. Because the distribution of hens with different body conditions may not be comparable across systems, population level stressful experience may still differ between the sampled housing types. Importantly, our results suggest that future assessment of comparative welfare between housing systems, husbandry practices and flocks may be facilitated by systematic sampling to categorise birds according to external body condition. Measuring the body condition distribution associated with particular housing systems could provide a corresponding proxy “stress profile”.A threshold for the requisite level of stress to elicit a measurable change in adult hippocampal plasticity may exist. On a technical level, variability between staining batches in the present study may also have introduced some ceiling effects in terms of the proportion of cells labelled, possibly limiting power to detect a housing system effect. Nevertheless, at the extremes of the body condition distribution, individual variation within systems appears to exceed shared inter-system effects of the housing environment on cumulative chronic stress. This finding has several implications. Interventions targeted at improving flock body condition, particularly for those birds in the poorest physical states, may have the greatest benefit for overall welfare. Controlled experiments to determine which factors are most associated with poor body condition and may therefore contribute most to long-term stress may further inform these measures. Selection for innate immunity and stress resilience may also have advantages for the subjective experience of laying hens, improving their collective chances of having a “life worth living”IL-6, differing microbial compositions (including an increased abundance of Methanomethylophilaceae and presence of Brachyspira), and enlarged spleens in the enriched cages. These factors point to associations between physical health, inflammation, chronic stress and body condition. A lack of main effect of housing type on adult hippocampal plasticity suggests that the range of stressful experience for hens in the multi-tier aviary and enriched cage housing is similar, or at least below the threshold for detectable differences, but only one replicate house per system was used here. Assessing whether experience for an average hen in each system differs will require systematic sampling of body condition distribution. Overall, our findings suggest that housing systems (and husbandry practices) which produce the lowest proportion of birds with poor body conditions may be associated with the “best” relative overall welfare. Measures to promote and maintain good body condition will have positive consequences for hen welfare.In an enriched cage and non-cage (multi-tier aviary) commercial housing system, hens in poor physical condition exhibit a lower level of adult hippocampal plasticity than hens in good condition. This suggests that the cumulative chronic stress experienced by these individual hens over the laying period is greater. Poor condition hens also displayed elevated splenic expression of


This review aims to present the current state of WNT signaling in chronic lymphocytic leukemia. First, we briefly summarize WNT pathways in physiological conditions and cancer. Then, we focus on WNT pathways in CLL with an emphasis on ROR1. The problem of limited data on canonical WNT signaling in CLL is also highlighted. In conclusion, we suggest that further research should focus on understanding the role of β-catenin signaling in CLL.WNT pathways play an important role in cancer development and progression, but WNT pathways can also inhibit growth in melanoma, prostate, and ovarian cancers. Chronic lymphocytic leukemia (CLL) is known for its overexpression of several WNT ligands and receptors. Canonical WNT signaling is β-catenin-dependent, whereas non-canonical WNT signaling is β-catenin-independent. Research on WNT in CLL focuses mainly on non-canonical signaling due to the high expression of the WNT-5a receptor ROR1. However, it was also shown that mutations in canonical WNT pathway genes can lead to WNT activation in CLL. The focus of this review is β-catenin-independent signaling and β-catenin-dependent signaling within CLL cells and the role of WNT in the leukemic microenvironment. The major role of WNT pathways in CLL pathogenesis also makes WNT a possible therapeutic target, directly or in combination with other drugs.  In humans, the WNT family of secreted glycoproteins consists of 19 members  and 10 dWNT pathways are central to tumor development and progression due to their involvement in proliferation, but usually only one of the two pathways is overactive in each tumor type ,3. For eChronic lymphocytic leukemia cells overexpress ROR1, and β-catenin-independent signaling is crucial for CLL development and survival ,6. HowevAPC is mutated and β-catenin can no longer be degraded. FAP is characterized by the formation of polyps in the large intestine, a pathologic state that can be a precursor of colorectal cancer (CRC) [Aberrant activation of the canonical WNT pathway is linked to several pathologic entities. The high occurrence of neurological disorders such as schizophrenia, depression or bipolar disorder was linked to the chromosomal translocation of the disrupted in schizophrenia 1 (DISC1) gene, whose protein product inhibited GSK-3β and subsequently activated β-catenin . Other der (CRC) .APC is considered to be one of the most frequently mutated genes in human cancers [CTNNB1 gene can lead to the disease progression of CRC, which is identical to what happens when the APC gene is mutated, underlining the functional connection of the two genes [CTNNB1 gene are found in around 30% of cases of hepatocellular carcinoma (HCC). Another gene regulating the WNT pathway, AXIN1, accounts for mutations in 15% of cases of HCC, while APC is mutated in only 1.6% of HCC cases [In fact,  cancers . The APC cancers . Intereswo genes . Gain-ofCC cases . HoweverCC cases . This suCC cases . Thus, tCC cases . BCL2L1 gene [The reliance of cancers on the canonical WNT pathway is not surprising, as it drives proliferation and blocks differentiation . From th2L1 gene .Inhibition of PORCN to disrupt ligand secretion;Antibodies blocking FZD or ROR receptors;Inhibitors of positive regulators of the WNT pathway, such as RSPO3 or tankyrase;Compounds directly targeting β-catenin and β-catenin-mediated transcription;Activation of the WNT pathway by inhibition of negative regulators; this strategy was developed for tumors such as leukemia, prostate cancer or lymphoma.The involvement of the canonical WNT pathway in the regulation of not only the proliferation but also the apoptosis of tumors makes the WNT pathway an attractive target for therapy. Although there are concerns about the toxicity that arises when inhibiting the WNT pathway, particularly for developing tissues, several compounds and antibodies targeting β-catenin-dependent signaling have entered clinical trials. Strategies to clinically target WNT signaling can be divided into five categories :InhibitiAlthough none of these compounds has yet been approved by the FDA for clinical application, several of these compounds have entered phase II clinical trials. Compared to canonical WNT-signaling, much less is known about non-canonical WNT signaling. β-catenin-independent signaling of the non-canonical WNT pathway plays a role in planar cell polarity, tissue patterning, cell migration via modulation of the cytoskeleton, embryonic development and axon guidance, amongst others ,9. For t3, FZD6 and FZD7 [2+ pathway. The WNT/Ca2+ pathway is responsible for the regulation of calcium levels in the cytoplasm, which triggers gene expression and further enhances cellular migration. Upon stimulation of the WNT/Ca2+ pathway, the intracellular calcium concentration increases and activates calcineurin, which in turn triggers the expression of NFAT-controlled genes [WNT ligands that are linked to β-catenin-independent signaling are WNT4, WNT5a/b and WNT11 . Receptoand FZD7 . The setand FZD7 . Due to ed genes . The WNT3 and FZD7, are correlated with poor prognosis in B-cell malignancies [Since the non-canonical WNT pathway is involved in cell migration, it follows that in several types of tumors, dysregulation of non-canonical WNT signaling is associated with metastasis. Overexpression of WNT5a in melanoma, gastric and pancreatic cancer increases metastasis ,11. HoweIt should be noted that very little is known about mutations of non-canonical WNT pathway genes in cancer. Rather, the activation of non-canonical WNT signaling is achieved by the overexpression of its components, which may make it easier to therapeutically target them. Currently, clinical trials are testing monoclonal antibodies, inhibitors of ROR1 and ROR2 and WNT5a mimetics to modulate non-canonical WNT signaling .10, WNT7a stimulation leads to the activation of JNK kinases that are targets of non-canonical signaling [6 receptor [The canonical and non-canonical WNT signaling pathways are often viewed as independent entities sharing only ligands and receptors. This perspective is outdated, and, in fact, both pathways are intertwined. First of all, receptors and ligands are not branch-specific. WNT3a is often viewed as a classic canonical ligand. However, WNT3a can also activate the PLC pathway in the colon cancer RKO cell line . WNT7a wreceptor ,14,15. Breceptor . 2+ pathway can suppress canonical signaling on a transcriptional level by inhibiting the β-catenin complex via NLK kinase, which is activated by calcineurin [BCL2L11 in melanoma, and the downregulation of cyclin D1 in neuroblastoma [The relationship between CTNNB1-dependent and -independent signaling also includes the interregulation of their activity. It was shown that non-canonical pathways can inhibit canonical WNT signaling on several levels. ROR1 and ROR2 induce the expression of E3 ligase Siah2, which ubiquitinates β-catenin via interaction with APC. This mechanism was reported in hematopoietic stem cells and colon cancer ,16,17. Mcineurin . WNT5a cblastoma ,19,20. FTumor cells often activate WNT pathways in an autocrine manner, which is highlighted by the fact that the inhibition of porcupine—an enzyme required for the posttranslational processing of WNT ligands—results in the apoptosis of cancer cells, pointing to a crucial role of porcupine for tumor survival ,4,8. In Tumor cells communicate with their microenvironment via several signaling molecules, including WNT. In addition, signaling from the microenvironment can also activate WNT pathways in tumor cells . It was Overall, the modulation of the WNT pathway for cancer therapy may not only be used to inhibit proliferation and/or induce apoptosis in cancer cells but might also be beneficial for immunotherapy.+CD23+ B lymphocytes per ml in peripheral blood. The five-year survival rate for CLL is 87% [CLL is the most common leukemia in the western world . It is aL is 87% . HoweverL is 87% . MYC proto-oncogene [Both ibrutinib and idelalisib target the B-cell receptor (BCR) signaling pathway, which is central to the pathomechanism of CLL. BCR stimulation with IgM results in the activation of the MAPK-, PI3K- and NF-κB pathways and leads to cell cycle progression, cellular adhesion and the homing of CLL cells to secondary lymphoid organs such as the lymph nodes and bone marrow . Anotheroncogene . It is iMutations in both the BCR- and NOTCH1 pathways are of prognostic value in CLL. Immunoglobulin heavy-chain genes (IGHV) constitute the BCR receptor and can be mutated in the development of non-malignant B-cells to increase the affinity of the BCR for antigens. CLL cells with a non-mutated IGHV have a worse prognosis, possibly because they reflect an earlier developmental state . MutatioROR1 is highly expressed on CLL cells, induced by STAT3 signaling [FZD3, FZD6, WNT5b and PRICKLE-1 [6 was shown to be crucial for CLL development in a murine CLL model that overexpressed TCL1 under the control of the B-cell specific Eµ promoter. 6FZD-null Eµ-TCL1 mice developed less severe leukemia than those with physiological levels of 6FZD [ROR1 is expressed at high levels only during the early stages of B-cell development in the bone marrow and is therefore not present on the surfaces of mature B-cells ,29. Howeignaling ,30. MoreRICKLE-1 . OverexpRICKLE-1 . FurtherIL-6, IL-8, CCL2, CCL3, CCL4 and CXCL1 [ROR1 signaling is involved in several processes that are central to the pathomechanism of CLL . Upon stnd CXCL1 . IL-6 acnd CXCL1 . Finallynd CXCL1 .BCL2L1—another anti-apoptotic member of the BCL2 family [WNT/PCP signaling is a highly interconnected network and may interact with other key pathways for CLL. Interestingly, WNT/PCP signaling is connected to the BCR pathway. As mentioned above, BCR is involved in the regulation of cell adhesion and proliferation via the activation of cortactin and ERK1/2 ,33. The 2 family ,38. Due CYLD gene, which possibly acts as a tumor suppressor [The role of β-catenin-dependent signaling in CLL is much more elusive. β-catenin-dependent signaling is undoubtedly active in CLL compared to non-malignant B-cells . Moreoveppressor . In the ppressor . These d5, RYK), cytoplasmic factors  and nuclear factors that modulate TCF/LEF  [CSNK1E, WNT1 and FZD5 resulted in canonical WNT pathway repression, while mutations in BCL9, DKK2 and RYK caused canonical WNT pathway activation. Interestingly, BCL9 acts as an enhancer of β-catenin signaling in colon cancer and is considered to be an oncogene [The particular role of canonical WNT signaling in CLL is underscored by genetic mutations: mutations in WNT pathway genes occur in approximately 14% of CLL patients . MutatioP, BCL9) . It shouoncogene , and theoncogene . Mutatiooncogene . Mantle cell lymphoma (MCL) can also help us to understand the role of canonical WNT signaling in hematologic malignancies. The much more aggressive MCL tumors are genetically very similar to CLL, differing mostly in the translocation of Cyclin D1 in MCL, which induces the cell cycle and drives the proliferation of MCL cells. In MCL cell lines, stimulation of the BCR receptor with anti-IgM resulted in the inhibition of GSK-3β and subsequent stabilization of β-catenin within 60 min . ActivatSimilar to other malignancies, CLL cells also interact with their microenvironment and this may affect the WNT pathway.ROR1 is a receptor that is specific to WNT5a; hence, the activation of non-canonical WNT signaling in CLL cells may depend on the WNT5a ligand provided by the microenvironment. The WNT5a ligand can be provided by mesenchymal stromal cells (MSCs) in the bone marrow or by ROR (NLCs) and thereby mobilize CLL cells, as well as contributing to the resistance of CLL to venetoclax ,38.WNT3 (WNT3hi) have a much better prognosis than WNT3low patients. In addition, decreased expression of WNT3 is a hallmark of disease progression [The situation is more complex with the canonical WNT pathway. As mentioned above, CLL cells overexpress several canonical ligands, including WNT3. Thus far, attempts to detect the activation of β-catenin by canonical ligands via TCF reporter assays have been unsuccessful . The mosgression . This suOn the other hand, the activation of β-catenin in CLL may not be limited only to WNT ligands . CLL celThe WNT pathway’s role in the CLL microenvironment is undoubtedly complex and has positive and negative aspects, which should be explored further.ROR1 plays a major role in the pathophysiology of CLL and is therefore a target for therapy. Indeed, the anti-ROR1 humanized monoclonal antibody cirmtuzumab is currently in several clinical trials for CLL and other B-cell malignancies. In a phase 1 clinical trial in 26 progressive or R/R CLL patients, cirmtuzumab was shown to be well tolerated and safe. Most of the adverse events, such as anemia, thrombocytopenia and neutropenia, were directly attributable to CLL. Treatment with cirmtuzumab resulted in a 33% reduction in the surface level of ROR1 and the reduced expression of genes associated with stemness, RhoA and Rac1 pathways. None of the patients met complete or partial response criteria as the treatment lasted only for 4 weeks. However, the median time to next treatment was 262 days. Interestingly, this corresponds with the time at which cirmtuzumab becomes undetectable in plasma .Further clinical trials of cirmtuzumab are focusing on its combination with drugs that are already approved for the treatment of CLL, such as ibrutinib and venetoclax. Since both the B-cell receptor and WNT/PCP pathways regulate the migration and proliferation of CLL cells, and the BTK inhibitor ibrutinib can only block signals from the B-cell receptor, it was reasonable to therapeutically pair ibrutinib with anti-ROR1 mAb. Ongoing clinical trials are testing the combination of ibrutinib and cirmtuzumab vs. ibrutinib alone in treatment-naïve or R/R CLL and R/R MCL patients. The combination of cirmtuzumab and ibrutinib was well tolerated in both diseases and showed superior efficacy over ibrutinib alone. For MCL, the overall response rate (ORR) was 82% and median progression-free survival (mPFS) was not reached after 14.6 months, whereas mPFS for ibrutinib alone was 12.8 months. In CLL, mPFS was also not reached at 14.6 months and the ORR was 91% .WNT-based therapy in CLL is not limited to anti-ROR1 Abs. Currently in development are several small-molecule inhibitors of ROR1: VLS-101, KAN 0439834 and ES425, amongst others . AnotherThere is no doubt that the WNT/PCP pathway plays a major role in CLL. This important role is highlighted by the involvement of WNT/PCP signaling in several key processes in CLL, including adhesion, migration and proliferation, as well as interactions with other pathways, such as BCR, the intrinsic apoptotic pathway and canonical WNT signaling. This involvement makes ROR1 an attractive target for the treatment of CLL, and the anti-ROR1 mAb cirmtuzumab is a promising agent with favorable clinical characteristics. Targeting WNT signaling in CLL patients could open up another targeted therapeutic route for the treatment of CLL.Canonical WNT signaling in CLL, on the other hand, remains a puzzle. Current data suggest that β-catenin signaling is important in CLL as the treatment of leukemic cells with WNT inhibitors induces apoptosis in CLL cells. However, the exact functions of canonical WNT signaling and its gene targets remain unknown. CLL cells overexpress several canonical WNT ligands, yet, so far, there is no evidence for canonical WNT ligands activating specific pathways within CLL cells. Taking into account the fact that WNT3 expression correlates with the clinical course of the patients, it is, however, very reasonable to assume that CLL cells use this ligand to communicate with their environment. Therefore, future research should focus on finding target cells for WNT3 and other WNT signaling ligands and understanding their role in the pathophysiology of the disease. It should also not be forgotten that although CLL in general depends on WNT/PCP signaling, there might be a subset of patients in whose CLL cells β-catenin signaling is upregulated. An example is CLL cells harboring mutated RYK or BCL9 genes, or, possibly, ROR1neg patients . Such CLLastly, it is known that β-catenin can interact with other signaling pathways, such as YAP1 in CRC or NF-κB in MCL, thereby leading to unique transcriptional programs ,44. It wCanonical WNT signaling in CLL is still mostly unexplored and holds many secrets, and therefore it should be a major focus of further research.


E and Z forms, the structure of the peptide-based matrix can be modulated, by which the catalytic activity of the multienzyme complex can be further controlled using UV and visible light. This study provides a new approach for constructing a stimuli-responsive multienzyme complex based on an adjustable material platform.In this study, a light-responsive multienzyme complex (GOx&hemin@PepM) was developed by incorporating glucose oxidase (GOx) and hemin within a peptide-based matrix. An azobenzene group (Azo) was linked to the N-terminucs of glycine-phenylalanine-glycine tripeptide (GFG) to facilitate the formation of the supramolecular peptide-based matrix. Due to the proximity effect of GOx and hemin in the matrix as well as the biomimetic microenvironment of the peptide-based material, the transfer of intermediates can be enhanced and the catalytic activity of this multienzyme complex was greatly improved over free enzymes for catalyzing a cascade reaction. In addition, based on the light-responsive conformational switching of azobenzene between  A light-responsive multienzyme complex was developed by incorporating glucose oxidase and hemin within a peptide-based matrix, in which the catalytic activity of multienzyme complex can be controlled using UV and visible light. Sun et al. developed a multienzyme catalyst by immobilizing an elastin-like polypeptide (ELP)-modified d-amino acid oxidase (DAAO) on hematin-functionalized multiwalled carbon nanotubes (MWCNTs), which exhibited higher catalytic efficiency than free ELP-DAAO.7Cascade reactions are common in all living organisms; a series of chemical reactions is carried out in an order such that the product of one reaction is the substrate for another.8,9 Peptide molecules can interact with each other through hydrophobic, π–π stacking, hydrogen bonding or ionic interactions, leading to the formation of various structures, such as nanofibrils, nanoparticles or nanotubes.10,11 In addition, the obtained supramolecular structure also provides a protein-like microenvironment that can facilitate biomimetic applications.12–14Recently, the self-assembly of short peptides has attracted growing attention in constructing micro- and nanostructural matrices due to its favorable stability, suitable variability and good biocompatibility.15–18 In this study, we created a novel light-responsive multienzyme complex (GOx&hemin@PepM) by incorporating glucose oxidase (GOx) and hemin within the azobenzene modified peptide-based matrix (PepM). Hemin is the prosthetic group of catalase, which is an iron-containing porphyrin and can be used individually as a molecular catalyst. The glycine-phenylalanine-glycine tripeptide (GFG) was designed with an aromatic azobenzene group (Azo) linked to the N-terminus to facilitate π–π stacking and the formation of the supramolecular matrix  and catalases are widely used in disease diagnoses, food processing and chemical synthesis.r matrix . The obtby hemin . Specifiiscussed . To our −1) and hemin (98%) were purchased from Aladdin Reagent Co. . All other chemicals were of analytical grade and obtained from commercial sources. Ultrapure water was used throughout the experiments.Azo-Gly-Phe-Gly (Azo-GFG) was purchased from Wuxi AppTec . Glucose oxidase  was dissolved in 200 μL of methanol as the stock solution. For the preparation of PepM, 200 μL of stock solution was dropped into 1 mL of phosphate-buffered solution . After vortexing for 2 min and incubating for 24 h at room temperature without disturbance, PepM was obtained.−1) and 200 μL of stock solution were added into 1 mL of PBS (pH 7.4) and vortexed for 2 min. The mixture was incubated at room temperature for 24 h without disturbance, leading to the formation of GOx&hemin@PepM.For the preparation of GOx&hemin@PepM, a hemin solution was prepared by adding hemin  and sodium carbonate  into 1 mL of ultrapure water and heating at 60 °C until the solution became clear. Then, 80 μL of as-prepared fresh hemin solution, 30 μL of GOx solution  with a 2 mm path length quartz cell and a wavelength range of 220–700 nm.The morphologies were characterized using an S-4800 field-emission scanning electron microscope  at an acceleration voltage of 3 kV. All samples were sputter-coated with platinum on an E1045 Pt-coater  before evaluation. The zeta potential values were measured on a Malvern Nano ZS . Transmission electron microscopy  images were recorded on JEOL 100CX-II at an acceleration voltage of 200 kV. Circular dichroism (CD) spectra were obtained on a J-810 CD spectropolarimeter  with a 0.2 mm path length quartz cell at room temperature. The wavelength range was set to 190–500 nm, and the scan speed was 100 nm min420 nm) was monitored by a spectrometer  to determine the release of purpurogallin. The activity was defined as the initial production rate of purpurogallin (μM min−1) catalyzed by GOx&hemin@PepM.The release rate of purpurogallin was used to represent the activity of GOx&hemin@PepM in the cascade reaction. In a typical experiment, 300 μL of GOx&hemin@PepM was added into 300 μL of a reaction solution containing 9 μmol of glucose and 4 μmol of pyrogallol in 50 mM PBS, pH 7.4. The reaction was maintained at 25 °C and the absorbance at 420 nm  containing different concentration of substrates. To investigate the kinetic mechanism of the multiple substrate cascade reaction, the assays were performed with a fixed concentration of one substrate and varying the concentration of the other. As comparison, the kinetic analysis of free enzymes was also carried out with the same amount of free GOx and free hemin to that in GOx&hemin@PepM.e.g., Fmoc, naphthalene or pyrene) is a consistent facilitator for peptide self-assembly.19,20 Herein, a functional aromatic group, azobenzene, was attached to the N-terminus with glycine (G) as a flexible linker. Phenylalanine (F) is a commonly used amino acid in peptide assembly materials because its aromatic side-chain can also form inter-molecular π–π stacking. The glycine at the C-terminus provides a –COOH tail to enhance the hydrophilicity of the peptide and facilitate the formation of hydrogen bonds.The importance of aromatic stacking has been illustrated for many peptide assembly systems, where the aromatic group that linked to the N-terminus  were used. As shown in 2HPO4/NaH2PO4 affects the formation of the hydrogel. When the peptide stock solution was dropped into 20 mM PBS , the strong absorption at 330 nm was retained, indicating that the incorporation of GOx and hemin did not affect the configuration of azobenzene. The shoulder at approximately 400 nm appearing in GOx&hemin@PepM comes from the hemin molecules. The direct application of hemin in aqueous solution is a significant challenge because hemin will aggregate into catalytically inactive dimers and self-destruct in oxidizing media.26 However, the hemin that dissolves in organic reagents, such as methanol or dimethyl sulfoxide, or that exists in natural catalase maintains its active monomeric form. To investigate the molecular form of hemin in GOx&hemin@PepM, the interferential spectrum of PepM was subtracted from that of GOx&hemin@PepM  was used to investigate the morphology of GOx&hemin@PepM. As shown in 2O2. Then, H2O2 was transferred to hemin and began the second oxidation reaction toward pyrogallol to produce chromogenic purpurogallin:To evaluate the catalytic activity of GOx&hemin@PepM, a cascade reaction was performed under mild conditions. In the reaction 420 nm value increased rapidly in the first 1.5 h but remained constant afterwards. This indicates that although the catalysts in CONTROL 1 displayed favorable activity (4.33 μM min−1) in the first 1.5 h, they were easily inactivated in the long-time reaction. As discussed above, free hemin is in a low-activity dimer form when dissolved in aqueous solution, and it can be easily disassembled in the oxidizing media.26 In the solution environment of CONTROL 1, GOx and hemin are distributed freely in the medium, and there was no spatial confinement of catalysts to provide a close distance for H2O2 transfer. Therefore, large amount of H2O2 was released from GOx and accumulated in the reaction medium, further inhibiting the activity of hemin. In the case of CONTROL 2, hemin was incorporated into PepM (hemin@PepM), which ensures the catalytic activity of hemin. However, there is also a serious mass-transfer limitation for H2O2 to move from the bulk solution into the matrix. Therefore, although the catalysts were active throughout the whole reaction, the activity was still in a low level (2.99 μM min−1). For CONTROL 3, pure PepM was mixed with substrates and the unchanged absorbance indicated no instinct catalytic ability of peptide matrix. Compared with the controls, GOx&hemin@PepM exhibited excellent catalytic performance. The OD420 nm value increased rapidly during the whole reaction, implying the H2O2 could be efficiently transferred and consumed within GOx&hemin@PepM. This comes from the synergism of the proximity effect and the highly active hemin, which were provided by the peptide-based matrix. As a result, the catalytic activity of GOx&hemin@PepM is 6.67 μM min−1.The increased absorbance at 420 nm can be monitored over time to evaluate the catalytic activity of GOx&hemin@PepM in the cascade reaction. As controls, the same amount of GOx and hemin in the form of free GOx + free hemin (CONTROL 1), free GOx + hemin@PepM (CONTROL 2) and pure PepM (CONTROL 3) were also studied, as shown in Km value than free enzymes, implying the stronger affinity towards pyrogallol. When at the same pyrogallol concentration, GOx&hemin@PepM showed higher Vmax value, indicating the higher conversion efficiency of multienzyme complex.The reaction kinetic for the two substrates cascade reaction catalysed by GOx&hemin@PepM was investigated. The typical Michaelis–Menten curves were found to each substrate over a large concentration range Fig. S4. The linE/Z conformational switches that can be modulated by UV and visible light. In this study, light was also found to affect the formation of the peptide-based matrix and the catalytic activity of GOx&hemin@PepM. When GOx&hemin@PepM was exposed to UV light, the hydrogel began to disintegrate and completely turned into solution within 12 h. The UV-vis spectrum was used to investigate the conformational change of Azo-GFG during the gel–sol transition process. It can be seen from Z-azobenzene, respectively. This confirmed the conformational switch of the azobenzene moiety in Azo-GFG from the E to the Z-form. The secondary structure of the Azo-GFG backbone was analyzed by CD, as shown in E-azobenzene. Additionally, for GOx&hemin@PepM before UV irradiation, the strong negative peaks at 205 nm and 222 nm were assigned to the α-helix structure of the peptide backbone. After UV irradiation, the characteristic structure of the α-helix faded. Since the primary driving force of the Azo-GFG assembly is π–π stacking among E-azobenzene groups, the configuration change of azobenzene directly affects the molecule arrangement in GOx&hemin@PepM, destroying the supramolecular α-helical structure and resulting in the disassembly of the peptide-based matrix. The light-responsive property of azobenzene was exploited due to the 2O2 efficiently. Given that UV irritation destroys the supramolecular structure of PepM, GOx and hemin will be released from the matrix and act as free catalysts, similar to in CONTROL 1. As a result, the catalytic activity of GOx&hemin@PepM was clearly decreased to 4.48 μM min−1 after UV irradiation. Due to the reversibility of E- and Z-azobenzene under UV and visible irradiation, the catalytic activity under repeated UV-visible irradiation was also investigated. For each cycle, GOx&hemin@PepM was processed with 12 h UV irradiation to turn into solution, followed by a 24 h visible irradiation for reassembly into a hydrogel (see ESI, Fig. S5Z-azobenzene slowly turns back into the E form, driving the free Azo-GFG to reassemble into the fibrous matrix and, at the same time, reincorporates GOx and hemin. However, we also observed that the reversible activity declined during repeated UV-visible light switches. In the third cycle, the reversible activity of GOx&hemin@PepM already dropped to 3.96 μM min−1, which was only 60% that of the initial value. This might be caused by the increasingly fragile structure of PepM and the deactivation of GOx and hemin during repeated irradiation processes.As mentioned above, the high catalytic activity of GOx&hemin@PepM depends largely on the supramolecular structure of PepM to suppress dimeric hemin and enable hemin and GOx to approach each other and transfer HIn summary, a multienzyme complex was developed by incorporating GOx and hemin within a peptide-based matrix. Upon the self-assembly of Azo-GFG, the peptide-based matrix displayed a fibrous network structure, which provides a confined space for the efficient transfer of intermediates between two catalysts and provides a biomimetic environment for hemin to retain high activity. Compared with the cascade reaction catalyzed by free GOx and hemin, the obtained GOx&hemin@PepM exhibits excellent activity during the whole reaction. In addition, based on the light-responsive property of azobenzene, the activity of GOx&hemin@PepM can be modulated by UV and visible light. This study provides a new strategy for constructing a multienzyme complex and also opens a novel approach for developing an adjustable material platform for enzyme activity control.There are no conflicts to declare.RA-008-C7RA10372G-s001