In vivo, shRNA-mediated knockdown of BRG1 significantly ameliorated renal fibrosis, improved tubular senescence, and inhibited UUO-induced activation of Wnt/β-catenin pathway. In mouse renal tubular epithelial cells (mTECs) and primary renal tubular cells, inhibition of BRG1 diminished transforming growth factor-β1 (TGF-β1)-induced cellular senescence and fibrotic responses. Correspondingly, ectopic expression of BRG1 in mTECs or normal kidneys increased p16INK4a, p19ARF, and p21 expression and senescence-associated β-galactosidase  activity, indicating accelerated tubular senescence. Additionally, BRG1-mediated pro-fibrotic responses were largely abolished by small interfering RNA (siRNA)-mediated p16INK4a silencing in vitro or continuous senolytic treatment with ABT-263 in vivo. Moreover, BRG1 activated the Wnt/β-catenin pathway, which further inhibited autophagy. Pharmacologic inhibition of the Wnt/β-catenin pathway (ICG-001) or rapamycin (RAPA)-mediated activation of autophagy effectively blocked BRG1-induced tubular senescence and fibrotic responses, while bafilomycin A1 (Baf A1)-mediated inhibition of autophagy abolished the effects of ICG-001. Further, BRG1 altered the secretome of senescent tubular cells, which promoted proliferation and activation of fibroblasts. Taken together, our results indicate that BRG1 induces tubular senescence by inhibiting autophagy via the Wnt/β-catenin pathway, which ultimately contributes to the development of renal fibrosis.Although accelerated cellular senescence is closely related to the progression of chronic kidney disease (CKD) and renal fibrosis, the underlying mechanisms remain largely unknown. Here, we reported that tubular aberrant expression of Brahma-related gene 1 (BRG1), an enzymatic subunit of the SWItch/Sucrose Non-Fermentable complex, is critically involved in tubular senescence and renal fibrosis. BRG1 was significantly up-regulated in the kidneys, predominantly in tubular epithelial cells, of both CKD patients and unilateral ureteral obstruction (UUO) mice.  Chronic kidney disease (CKD) is increasingly regarded as a global public health problem, as it affects approximately 10–15% adults worldwide . ClinicaINK4a-retinoblastoma (Rb) pathways [The mechanistic basis of kidney ageing is cellular senescence, which is an irreversible state of cell-cycle arrest accompanied by a series of changes in cell morphology and epigenetics, induced by various forms of stress . Mechanipathways . Despitepathways . The clapathways ,10. In apathways .INK4a and/or p21, has been reported to be accelerated in multiple experimental animal models and human kidney diseases, which correlates with renal dysfunction and disease progression [Cellular senescence, identified by senescence markers SA-β-Gal, p16gression . During gression ,20. Tubugression . Therefogression ,13. Recegression . Thus, aSMARCA4 gene, is a core catalytic ATPase subunit of the SWItch/Sucrose Non-Fermentable chromatin remodelling complex, which performs fundamental roles in gene regulation and cell lineage specification by altering chromatin structure using energy from ATP hydrolysis [Brahma-related gene 1 (BRG1), encoded by the drolysis ,27. Yangdrolysis . The fibdrolysis ,30. In kdrolysis  found thdrolysis  demonstrBRG1 has been implicated in cell cycle regulation and ageing process of cancer cells in very different ways. Hendricks et al.  and NapoWnt/β-catenin pathway is an evolutionarily conserved pathway, which plays critical roles in various biological processes, such as embryonic development, tumorigenesis and tissue repair ,37. Aberin vivo and in vitro and further explored its underlying mechanisms. Our results establish a critical role for BRG1 in the pathogenesis of renal fibrosis.In the present study, we demonstrated that BRG1 was induced in the kidneys, specifically in tubular epithelial cells, of CKD patients and UUO mice. We revealed the potential role of BRG1 in tubular senescence and renal fibrosis Male C57BL/6J mice (weighing 20–23 g) were obtained from Southern Medical University Animal Center . Mice were raised in a standard environment on a regular light/dark cycle with free access to chow and water. All animal experimental protocols were approved by the Southern Medical University Ethics Committee  and conducted in Southern Medical University.In vivo knockdown or expression of BRG1 in mice was performed by a hydrodynamic-based gene delivery approach, as described previously [n=6 per group): (1) sham control, (2) UUO mice injected with empty shRNA plasmid pGPH1-vector, and (3) UUO mice injected with pGPH1-shBRG1. Mice were killed by exsanguination under anaesthesia with inhaled 5% isoflurane in room air at day 10 after UUO. Kidneys were collected for further analysis. In the second set of experiments, after unilateral nephrectomy (UNx) under anaesthesia, the mice were divided into four groups as follows (n=6 per group): (1) mice injected with empty vector pReceiver-M14 and treated with vehicle   by gavage, (2) mice injected with pReceiver-M14 vector and treated with ABT-263   by gavage, as described previously [The UUO mouse model was established by double-ligating the left ureter using a 4-0 silk after a midline abdominal incision under anaesthesia, as described previously . For shaeviously ,46. Brieeviously , (3) micINK4a , and anti-TGFβ1 .The renal tissue was fixed in 4% paraformaldehyde, and paraffin-embedded kidney sections (4-μm-thickness) were prepared by a routine procedure. Masson’s trichrome staining was performed according to the manufacturer’s procedures . For each animal, ten randomly selected fields of Masson’s trichrome staining were observed under a light microscope (×400). To examine tubulointerstitial collagen deposition, the blue-stained area was semiquantitatively calculated as the fibrotic area using Image-Pro Plus 6.0 software . Immunohistochemical staining was conducted as previously described . The prig at 4°C for 15 min. Cytoplasmic and nuclear proteins from mouse renal tubular epithelial cells (mTECs) were separated by a commercial protein separation kit  according to the manufacturer’s procedures. The protein concentrations were quantified using the BCA Protein Assay Kit . Protein expression was analysed by Western blot analysis as described previously [INK4a , anti-p21 , anti-p19ARF , anti-β-catenin , anti-non-phospho (active) β-catenin  anti-MMP7 , anti-Snail-1 , anti-PAI-1 , anti-Histone H3 , anti-TGFβ1 , anti-LC3A/B , anti-SQSTM1 , and anti-Beclin-1 .Total protein sample from kidney tissues or cells were extracted with RIPA lysis buffer  on ice. The supernatants were then collected after centrifugation at 10000×eviously . The primTECs were provided by Dr Jeffrey B. Kopp  and cultured as described previously . Normal INK4a-specific siRNA  using the Lipofectamine 2000 reagent  according to the manufacturer’s protocol, and HEK-293T cells were transfected with pReceiver-M14 or pReceiver-M14-BRG1. For some experiments, mTECs were transfected with BRG1-specific siRNA (or control siRNA) or pReceiver-M14-BRG1 (or pReceiver-M14), followed by incubation with TGF-β1 (5 ng/ml) or ICG-001 (5 µM), respectively. To assess autophagy, pReceiver-M14-BRG-tranfected mTECs were stimulated with ICG-001 (5 µM), followed by treatment with RAPA  or Baf A1 , as indicated. The Ctrl-CM and BRG1-CM were prepared by transfecting mTECs with pReceiver-M14 and pReceiver-M14-BRG1, respectively. NRK-49F cells were treated with Ctrl-CM and BRG1-CM in different proportions for the indicated time period.For transfection, mTECs were transiently transfected with control vector (pReceiver-M14), BRG1 expression vector (pReceiver-M14-BRG1), BRG1-specific small interfering RNA (siRNA) or p16Primary tubular epithelial cells were isolated from C57BL/6J mice according to procedures as previously described, with some modifications . BrieflyRenilla luciferase reporter plasmid  , and BRG1 expression vector (pReceiver-M14-BRG1) or control vector (pReceiver-M14) using Lipofectamine 2000 reagent. Cells were harvested 48 h after transfection, Firefly and Renilla luciferase activity were measured using a dual-luciferase reporter assay kit according to the manufacturer’s recommendations . The relative luciferase activity represents the ratio of the firefly luciferase activity to the Renilla luciferase activity.mTECs and HEK-293T cells were co-transfected with TOPFlash TCF reporter plasmid, mTECs or NRK-49F cells grown on coverslips were fixed with 4% formaldehyde and permeabilised with 1% Triton X-100. After blocking with 10% goat serum for 1 h, the cells were incubated with primary antibodies against E-cadherin , β-catenin , or fibronectin . After washing, the cells were stained with DyLight 594-conjugated goat anti-rabbit or goat anti-mouse IgG , and the nuclei were counterstained with DAPI . Images were captured by an inverted confocal microscope .Total RNA in mTECs was extracted using RNAiso reagent  and converted into cDNA using a Reverse Transcription System kit according to the manufacturer’s protocols (Takara). Quantitative real-time PCR was conducted using SYBR Green Premix (Takara) on the ABI PRISM 7500 Real-Time PCR System  as previously described . PrimersFrozen kidney sections (4 µm) or primary tubular epithelial cells were stained to detect SA-β-gal activity according to the manufacturer’s protocol .To detect mTECs-secreted cytokines, the Mouse XL Cytokine Array Kit  was used following the manufacturer’s instructions. The signals were detected by chemiluminescence and measured by Image-Pro Plus 6.0 software .Mouse TGF-β1, IL-6, TNF-α, and CCL2 levels in the supernatants of mTECs were measured using enzyme-linked immunosorbent assay (ELISA) kits  in accordance with manufacturer’s instructions.t test was used to analyse the differences between two groups, and one-way analysis of variance was used for comparisons across multiple groups, followed by either a Bonferroni’s post hoc test or Dunnett’s T3 test based on the results of a homogeneity of variance test. P<0.05 was considered to indicate statistical significance.All data are presented as the mean ± SD. Statistical analysis was performed with SPSS 20.0 . Student’s In the present study, we first examined the expression and localisation of BRG1 in various types of nephropathies. To this end, we performed immunostaining in kidney biopsy specimens from CKD patients with minimal change disease (MCD), IgA nephropathy (IgAN), focal segmental glomerular sclerosis (FSGS), membranous nephropathy (MN), and lupus nephritis (LN). As shown in To ascertain the pro-fibrotic role of BRG1 in renal fibrosis, mice were intravenously injected with an shRNA vector encoding the interference sequence targeting BRG1 (pGPH1-shBRG1) through a hydrodynamic-based gene delivery approach ,46 FiguA. We obsin vivo. As shown in INK4a, p21 and p19ARF in the UUO kidneys. This observation was supported by immunohistochemical staining results, which showed that in vivo knockdown of BRG1 significantly suppressed UUO-induced SA-β-gal activity and p16INK4a expression in tubular cells  or empty vector (pReceiver-M14) for 4 weeks to induce tubular senescence and fibrotic response. Subsequently, these mice were treated with senolytic agent ABT-263 or vehicle for two cycles A. ABT-26pression B,D,E andpression I after Bpression I and deced group B,D,E. Noed group B,F–H. InWe further investigated the role of BRG1 on the fibrotic response in renal tubular epithelial cells. To this end, mTECs were transiently transfected with BRG1 expression vector (pReceiver-M14-BRG1) or empty vector (pReceiver-M14), and the overexpression efficacy was confirmed by western blot A,B. The in vitro. Results of Western blot analysis showed that TGF-β1 substantially increased the expression of ECM proteins fibronectin, collagen I, and myofibroblast marker α-SMA in mTECs, which indicates the activation of EMT [Because TGF-β1 is identified as a core regulator of fibrosis , we thenn of EMT . Notablyin vitro. Renal tubules were isolated from mouse kidneys and cultivated for use as primary tubular cells . As shown in Supplementary Figure S2A,D–F, knockdown of p16INK4a significantly reduced the expression of fibronectin, collagen I, and α-SMA induced by BRG1 overexpression. Taken together, these findings revealed that BRG1 promotes the fibrotic response possibly through activation of the p16INK4a signalling pathway in tubular cells.To further clarify the causality between BRG1-induced pro-fibrotic responses and tubular senescence, mTECs were co-transfected with BRG1 expression vector (pReceiver-M14-BRG1) and p16Renilla luciferase reporter experiment to evaluate the effect of BRG1 on the Wnt/β-catenin-mediated transcription activity in vitro. As shown in in vitro. Since activation of the Wnt/β-catenin pathway requires binding of Wnt ligands and Wnt receptors, we next examined the effect of BRG1 overexpression on the expression of Wnt ligands and Wnt receptors by real-time PCR. We found that BRG1 overexpression increased the expression levels of various Wnt ligands  and several Wnt receptors , but had no effect on Axin1, DVL1/2, and GSK3β mRNA levels .We further explored whether the activation of Wnt/β-catenin signalling is required for BRG1 in mediating tubular senescence and fibrotic response in tubular epithelial cells. We first performed a TOPFlash/INK4a, p21, p19ARF, and TGF-β1 in mTECs. Further, the expression of fibrotic markers up-regulated by BRG1 overexpression, including fibronectin, collagen I, PAI-1 and α-SMA, were reversed after ICG-001 intervention . Together, these results suggested that BRG1 mediates tubular senescence and fibrotic responses dependent on the activation of Wnt/β-catenin signalling pathway.To further detect the role of Wnt/β-catenin pathway in BRG1-induced tubular senescence and fibrotic responses, we used a pharmacologic approach to inhibit Wnt/β-catenin pathway by treating mTECs with a small molecule inhibitor, ICG-001 . As showThe above findings indicated that BRG1 promotes tubular senescence through the activation of Wnt/β-catenin signalling. Next, we investigated the underlying mechanism by which BRG1/Wnt/β-catenin modulates tubular senescence. As accumulating evidence has proposed the association between autophagy deficiency and accelerated senescence ,65, we aINK4a and p21 expression  as an inducer of autophagy. RAPA treatment markedly increased the expression of LC3-II and decreased SQSTM1 levels C–E, suggpression C,F,G. Innescence H–L. ThusInterestingly, we observed that the inhibition of Wnt/β-catenin signalling by ICG-001 significantly decreased the protein level of BRG1 but increased its mRNA level M–O, indiINK4a knockdown (INK4a-Rb pathway in BRG1-induced SASP formation. Treatment of fibroblasts (NRK-49F) with BRG1-CM concentration-dependently induced the secretion of fibronectin and collagen I, as detected by Western blot analysis and immunofluorescence . Simultaneously, the secretion of various pro-inflammatory cytokines induced by BRG1 was also inhibited by the knockdown of p16INK4a, which further illustrated that BRG1-mediated transition of tubular secretome phenotype is closely related to cellular senescence (Our data also established a strong causative link of BRG1-induced tubular senescence and renal fibrosis. We found that BRG1-mediated pro-fibrotic responses were largely abolished by continuous senolytic treatment with ABT-263 in vivo  or siRNAnescence . Collectin vivo and in vitro studies showed that TGF-β1 expression could be up-regulated by BRG1 overexpression (TGF-β1 has been identified as a key regulator of fibrosis  as well pression  and 6. Tin vivo data demonstrated that shRNA-mediated knockdown of BRG1 efficiently inhibited the Wnt/β-catenin signalling activation induced by UUO (in vitro was evidenced by the enhanced relative luciferase activity of TOPFlash reporter, the increased nuclear accumulation of β-catenin, as well as the up-regulation of active-β-catenin and several classical target genes of Wnt/β-catenin, including MMP7, snail-1, and PAI-1 (in vitro diminished BRG1-induced tubular senescence (Previous studies have implicated that Wnt/β-catenin signalling pathway could be activated by BRG1 in some biological systems . Indeed,d by UUO . In addind PAI-1 . Furthernescence  and fibrWe also revealed the mechanism underlying BRG1/Wnt/β-catenin axis in inducing tubular senescence could be related to the inhibition of autophagy. Previous studies have provided evidence that Wnt/β-catenin signalling acts as a crucial negative regulator of autophagy activation . In lineALP, one of the major protein degradation pathways, has a critical role in maintaining cellular homoeostasis . We demoINK4a, suggesting a p16INK4a-dependent role of BRG1 in SASP induction in tubular senescence.Another important finding in the present study is that BRG1 promotes the communication between senescent tubular cells and interstitial fibroblasts. As shown by our results, overexpression of BRG1 significantly changed the secretion profile of tubular cells to a pro-inflammatory and pro-fibrotic secretome phenotype, which further promoted proliferation and transdifferentiation of fibroblasts . This fiIn summary, our data demonstrated that tubular aberrant expression of BRG1 is a common pathological feature of CKD and critically involved in tubular senescence through the inhibition of autophagy via activating Wnt/β-catenin pathway, which further promotes fibrotic responses through a vicious positive feedback with TGF-β1. BRG1 also induces fibroblast activation indirectly through tubule-derived SASP factors. Although further studies are warranted, these findings undoubtedly provide a new determinant pathological factor and potential target for the prevention of renal fibrosis.in vivo and in vitro and its underlying mechanisms were explored.The potential role of BRG1 in tubular senescence and renal fibrosis Aberrant up-regulation of BRG1, a common pathological feature of CKD, inhibits tubular autophagy through the activation of Wnt/β-catenin pathway, which accelerates tubular senescence and ultimately promotes the development of renal fibrosis.Our findings establish a critical role for BRG1 in the pathogenesis of renal fibrosis and provide a new determinant pathological factor and potential target for the prevention of renal fibrosis.Click here for additional data file.


Aberrant activation of Wnt/β-catenin signaling and dysregulation of metabolism have been frequently observed in lung cancer. However, the molecular mechanism by which Wnt/β-catenin signaling is regulated and the link between Wnt/β-catenin signaling and cancer metabolism are not fully understood. In this study, we showed that the loss of dual serine/threonine tyrosine protein kinase (DSTYK) led to the activation of Wnt/β-catenin signaling and upregulation of its target gene, lactate dehydrogenase (LDHA), and thus the elevation of lactate. DSTYK phosphorylated the N-terminal domain of β-catenin and inhibited Wnt/β-catenin signaling, which led to the inhibition of cell growth, colony formation and tumorigenesis in a lung adenocarcinoma mouse model. DSTYK was downregulated in lung cancer tissues, and its expression was positively correlated with the survival of patients with lung adenocarcinoma. Taken together, these results demonstrate that the loss of DSTYK activates Wnt/β-catenin/LDHA signaling to promote the tumorigenesis of lung cancer and that DSTYK may be a therapeutic target. Wnt/β-catenin, TGF β, and Hedgehog pathways [Lung cancer is one of the most common malignant cancers , 2, and pathways –6; an abpathways . TherefoAxin, APC, GSK3β, and CK1α within the cytoplasm phosphorylates β-catenin [33, 37, 41, and 45 of its N-terminal domain is the core event in β-catenin regulation [45 before GSK3β phosphorylates residues 33, 37, and 41 [The Wnt/β-catenin pathway plays an important role in the progression of lung cancer , 9. A co-catenin . Phosphogulation . Current, and 41 . The novDual serine/threonine and tyrosine protein kinase (DSTYK) encodes a protein product consisting of 850-931 amino acids . The N-tmTORC1, accelerated TFEB entry into the nucleus, and activated the expression of genes related to lysosome synthesis [The deletion of DSTYK in zebrafish via positional cloning was found by CRISPR-Cas 9 screening to cause notochord development defects , 16. Morynthesis , 16.Met296Ile) in intracranial solitary fibrous tumors promoted tumor metastasis by activating the ERK pathway [Whole-exon sequencing showed that DSTYK exhibits multiple mutations in a variety of pathological conditions. DSTYK gene mutations have also been observed in unilateral renal agenesis . In addi pathway . To dateIn this study, the expression pattern of DSTYK in lung adenocarcinoma was detected and the function and expression pattern of DSTYK in lung cancer were evaluated. Moreover, the biological function mechanism of DSTYK was investigated.2 incubator. Cells were transfected with Lipofectamine 8000 (Invitrogen) according to manufacturer instructions. Stable cell lines were selected using puromycin  for one week. Cell lines were authenticated using STR profile analysis and used within 3–20 passages of thawing the original stocks.Normal bronchial epithelial cell line Bease-2B and lung cancer cell lines were obtained from the Cell Bank of the Chinese Academy of Science  and maintained in DMEM (Gibco) containing 10% FBS (Gibco) and antibiotics in a 5% CO-ΔΔCt method. The primer sequences were as follows: F, 5’-ttgcatactgatcctcgg-3’; R, 5’-tgtgcactagttcatact-3’.Total RNA was extracted from cells or tissues using TRIzol and reverse transcribed to cDNA using a kit (Takara). RT-PCR analysis was performed using an Applied Biosystems Prism 7900 HT sequence detection system with Universal SYBR qPCR Master Mix . The expression levels of each mRNA relative to the β-actin expression level were calculated based on the 230 clinical samples were collected from Shanghai Chest Hospital after the informed consent was obtained was obtained from all subject. This study was approved by the ethical committee of the Shanghai Chest Hospital. The clinical information was listed in Table p53f/f; LSL-KrasG12D (KP) model C57BL/6 mice were housed under standard conditions with 12 h dark and 12 h light cycles.9 viruses  were administered per mouse (8 weeks old) using an intranasal/orthotropic infection protocol as described [G12D. The administration of pSECC/Sg DSTYK viruses led to the deletion of P53 and DSTYK and activation of KrasG12D. Twelve weeks later, the lungs were harvested, and western blot assays were performed.To study the roles of DSTYK in the tumorigenesis in vivo, the sg con or sg DSTYK were inserted into the cassette of pSECC lentivirus vector which expressed cas 9 enzyme and Cre recombinase. To induce lung cancer, the 8-week-old male KP mice were randomly divided into two groups (5 mice in each group) and anesthetized with 2.5% avertin; full anesthetization and absence of reaction to pain were ensured. A total of 1 × 10escribed . The admFor the examination of DSTYK protein levels in the KP tumors, the mice were treated with/without Ad-Cre virus (adenovirus). This study was approved by the ethical committee of the Shanghai Chest Hospital, and complied with the ethical regulations of ethical committee of the Shanghai Chest Hospital.Cells were harvested with RIPA buffer. The protein concentration was determined using BCA. Then, SDS-PAGE was performed, and the proteins were transferred to a PVDF membrane. After blocking with 5% BSA at room temperature for 1 h, the membrane was incubated with the primary antibody for 4 h and sequentially with the secondary antibody for 1 h. Then, the signals were examined using an ECL kit. The information about the antibodies were: Anti-DSTYK , anti-GAPDH , ant-β-catenin , anti-phosphorylated β-catenin , anti-GST , anti-Flag , anti-Cyclin D1 , anti-c-Jun , anti-c-Myc , anti-LDHA , anti-Tubulin .An MTT  kit was used. Cells were plated in 96-well plates at a density of 1000 cells per well, and each well was loaded with 200 µl of medium. On days 1, 3, 5, and 7, 20 µl of MTT solution were added to each well, and the plates were incubated for 4 h. Then, supernatants were removed, precipitates were dissolved in 200 µl of DMSO, and absorbance was measured.6 cells/L. A bottom agar layer was prepared with an equal volume of 1.2% agar and 2× DMEM containing antibiotics and 20% FBS, and 3 ml of the mixture was added to a 6 cm dish. Thirty minutes later, a bottom agar layer was prepared with an equal volume of 1.2% agar and 2× DMEM with an additional 0.2 ml of the cell suspension. After softly mixing, the mixture was added to the dish with the bottom agar layer. Fourteen days later, colonies were counted.Cells were suspended in 2× DMEM at a density of 104 viable cells per well were counted and seeded in low-adherent 6-well plates  in serum-free F12/MEM containing B27, EGF (20 ng/ml) and FGF (40 ng/ml). After incubation at 37 °C for 14 days, pictures were taken under a microscope, and tumor spheres in five separate fields were counted. EGF , FGF , and B27  were obtained from R&D.Cells from each group were collected and digested into single cells. Subsequently, for each group, 2 × 10Cells were scraped and proteins were extracted from the cells with 1 ml RIPA buffer. After centrifugation  for 20 min, supernatant was divided into two tubes and immunoprecipitated with the indicated antibody and IgG as control at 4 °C. On the next day, protein A beads  were added to the supernatant and incubated for another 4 h. Then, the beads were collected through centrifugation and washed three times. After as much wash buffer as possible was removed, the beads were mixed with 30 µl of loading buffer and boiled for 5 min at 100 °C. The precipitates were examined using western blot.Cells were scraped, and proteins were extracted from the cells with 1 ml RIPA buffer. After centrifugation  for 20 min, the supernatants were divided into two tubes and immunoprecipitated with 5 µg of GST or GST-DSTYK fusion protein at 4 °C. The next day, Sepharose 4B beads  were added to the supernatants and incubated for another 4 h. Then, the beads were collected through centrifugation and washed three times. After as much wash buffer as possible was removed, the beads were mixed with 30 µl of loading buffer and boiled for 5 min at 100 °C. The precipitates were then examined using western blot.Cells at 70% confluence were plated in a 24-well plate. For each well, the cells were transfected with 0.01 µg of TOPFlash, 0.01 µg of TK Renilla, and 0.25 µg of DSTYK expression vector or empty vector. Forty-eight hours later, the cells were lysed with RIPA buffer, and reporter activity was examined using a dual reporter assay kit (Promega).Fusion protein GST-β-catenin (N)  was purified and incubated with Flag-DSTYK protein immunoprecipitated from a stable A549 cell line in the presence of kinase assay buffer and ATP . The reaction was performed at 32 °C for 30 min and stopped by the addition of loading buffer. The phosphorylation of β-catenin was examined using western blot.ChIP was performed using a kit  according to manufacturer instructions. The primers for qPCR were as follows: F, 5’-TCAAAACCAAGAAACTCAG-3’; F, 5’-GGGAAAGACCAAATGATTA-3’. qPCR was performed using a 2× SYBR qPCR mixture according to manufacturer instructions.A lactate assay was performed using a kit . Briefly, cell culture medium was collected. The culture medium and standards were added to the wells of a plate, after which reaction mixture was added and incubated for 30 min at room temperature. The samples were analyzed with a microplate reader according to manufacturer instructions.For RNA sequencing, A549 control cells and cells with the knockdown of DSTYK were harvested. The libraries were performed by Novogene , and an Illumina HiSeqX10  was used for high-throughput sequencing. Quality control and removal of adapters of raw paired-end reads were performed using fastp (version 0.20.1). The gene expression counts were analyzed using HTseq-count software. The differently expressed genes were identified using DEseq2 (version 3.12) in R environment (version 3.6.3). Data were uploaded, and the GEO number is GSE178487.t-tests analysis. Data were expressed as mean ± S.D, P < 0.05 was considered a significant difference .All experiments in this study were performed in triplicate and error bars represent the standard deviation (±S.D) of triplicate samples. Statistical analysis was conducted using GraphPad Prism (version 7.0). Comparisons between groups were performed using two-tailed independent sample Student’s LSL-KrasG12D;P53loxp/loxp) mouse model is a classic model for investigating lung cancer. The western blot  was found to be a downstream target gene of the DSTYK/Wnt/β-catenin signaling pathway; thus, the biological function of DSTYK was determined to be exerted through the activation of Wnt/β-catenin/LDHA signaling. This study reveals the function and mechanism of DSTYK in lung cancer and suggests the importance of DSTYK agonists in the treatment of lung cancer.The phosphorylation of β-catenin by DSTYK is one of the most novel findings in this study. Kinases are important targets for tumor therapy. Previous studies have shown that GSK3β and CK1α are the most important kinases in the Wnt/β-catenin signaling pathway during embryonic development . The bioThe regulation of LDHA expression through the Wnt/β-catenin signaling pathway was also found in this study. LDHA catalyzes lactate production, and glycolysis is one of the basic characteristics of tumor cell metabolism , 24. UpoMultiple studies have demonstrated the roles of LDHA in the progression of lung cancer. The expression level of LDHA has been negatively correlated with the survival of lung cancer patients . LDHA haIn summary, this study revealed the expression pattern of DSTYK in lung cancer, clarified its function and mechanism in lung cancer, and provided a new target for the treatment of lung cancer.The clinical information of the patients


Mitral valve prolapse (MVP) is a common and progressive cardiovascular disease with developmental origins. How developmental errors contribute to disease pathogenesis are not well understood.DZIP1S24R variant resulted in reduced DZIP1 and CBY1 stability and increased β‐catenin activities. The β‐catenin target gene, MMP2 was up‐regulated in the Dzip1S14R/+ valves and correlated with loss of collagenous ECM matrix and myxomatous phenotype.A multimeric complex was identified that consists of the MVP gene Dzip1, Cby1, and β‐catenin. Co‐expression during valve development revealed overlap at the basal body of the primary cilia. Biochemical studies revealed a DZIP1 peptide required for stabilization of the complex and suppression of β‐catenin activities. Decoy peptides generated against this interaction motif altered nuclear vs cytosolic levels of β‐catenin with effects on transcriptional activity. A mutation within this domain was identified in a family with inherited non‐syndromic MVP. This novel mutation and our previously identified Dzip1 functions to restrain β‐catenin signaling through a CBY1 linker during cardiac development. Loss of these interactions results in increased nuclear β‐catenin/Lef1 and excess MMP2 production, which correlates with developmental and postnatal changes in ECM and generation of a myxomatous phenotype. DZIP1, CBY1, and β‐catenin form a complex which localizes to the basal body. This complex sequesters β‐catenin and downstream signaling is suppressed. In the context of Dzip1 mutations or loss of members of this complex, β‐catenin is released and transported into the nucleus. While in the nucleus, β‐catenin along with Lef1 increase transcription of various target genes including MMP2. Genetic perturbation of Dzip1 or Cby1 can affect primary ciliogenesis. However, regulation of β‐catenin in the valves is independent of the presence of primary cilia.Identified a novel molecular interaction between Dzip1‐Cby1 and b‐catenin signalingIdentified a new mutation in DZIP1 that is involved in MVPIdentified how DZIP1 mutations have a damaging impact on b‐catenin signalingShow that b‐catenin signaling is independent of primary cilia function in the valves Recent genetic studies have revealed a role for primary cilia during valvulogenesis, indicating a potential unifying pathway for disease initiation.To investigate the mechanism of DZIP1 in MVP, we identified Chibby‐1 (CBY1) a binding partner of DZIP1 thorough proteomics analyses. CBY1 is not only a ciliary gene essential for both motile and primary cilia assembly,22.1). Whereas Dzip1 is observed both at the basal body and in the nucleus as we previously reported,To identify direct binding partners for DZIP1 in the heart, yeast two‐hybrid screens were performed where full length DZIP1 was used as the bait. Of note, 113 million clones from a human embryonic and adult heart library were screened. CBY1 was identified as one of the most prevalent interactors with 14 total clones being identified from the screen. Co‐immunoprecipitations (Co‐IPs) confirmed an interaction between DZIP1 and CBY1 in HEK293T cells Figure . Co‐exprCby1+/− and wild‐type P0 mouse anterior leaflets were quantified  failed to interact with CBY1. Likewise, constructs only expressing the amino acids 613‐to the C‐terminus (613‐867) were unable to interact with CBY1. However, amino acids 357 to 612 revealed a positive interaction between DZIP1 and CBY1 Figure . This re2.3As Cby1 was previously shown to directly interact with β‐catenin,2.4Dzip1f/f; NfatC1Cre(+)) results in a statistically significant increase in activated, nuclear β‐catenin (P = .0002) compared to control littermates at P0, as detected by a phospho‐specific β‐catenin antibody (pβ‐cateninS552). This finding correlates with a significant increase in the β‐catenin co‐factor, Lef1 (P < .01), which is required for its transcriptional regulatory function. Previous reports have indicated that β‐catenin activities may be dependent on the presence of primary cilia.NfatC1Cre+;Ift88f/f) conditional knockout mitral valves failed to reveal a statistically significant change in activated β‐catenin protein expression  resulted in decreased protein stability and likely loss of function.Dzip1S14R/+ and wild‐type littermates revealed a similar finding of increased nuclear β‐catenin and reduced fraction of cytosolic β‐catenin  and a P5 reverse peptide were synthesized with a cell‐penetrating TAT peptide sequence and a 5‐FAM moiety for fluorescent visualization at the amino terminus. Peptides were also biotinylated to be able to confirm interaction with CBY1 and β‐catenin. These wild‐type peptides were able to transverse the cell membrane Figure  and remaTo test whether the peptides function to disrupt β‐catenin activities within the cell, VICs were transfected with the TOP flash β‐catenin reporter plasmids. Forty‐eight hours post‐transfection, valve fibroblasts were stimulated with P5 , P5' (1μM) or reverse peptide (high dose 10 μM). Controls included cells that were transfected with the TOP flash reporter and no peptide as well as cells that were not transfected with DNA constructs or subjected to peptide treatment (untransfected + no peptide). Luciferase readouts from the TOP flash reporter confirmed a significant reduction in β‐catenin activities when treated with both the P5 and P5' mutant peptides at 1 μM dose Figure . P5 trea2.9Dzip1S14R/+) that was previously validated as having myxomatous valves and MVP.Cby1+/−) mitral valves by postnatal day 0. This finding of increased MMP2 was also observed in the Dzip1S14R/+ model of MVP. Although our previous RNAseq dataCby1+/− valves, which is consistent with findings in human myxomatous valves and in hyperactive β‐catenin murine models.Enhanced Wnt/β‐catenin signaling has been observed in human myxomatous valves.3Dzip1S14R/+). Utilizing a combination of proteomic based approaches with in vivo and in vitro studies, our findings reveal a mechanism by which myxomatous degeneration may occur through altered developmental pathways that invoke both ciliogenesis and β‐catenin signaling. Previous studies have indicated that primary cilia can regulate canonical Wnt/β‐catenin signaling.The genetic causes of mitral valve prolapse are only now beginning to be discovered. Once disease genes are identified, it will be important to identify the role that these genes play in disease initiation and progression. We recently identified mutations in the DZIP1 gene in multiple families with inherited non syndromic mitral valve prolapse.Our recent studies have highlighted a dynamic expression pattern between nuclear and membrane bound β‐catenin.C585W mutation results in a profound reduction in protein half‐life and premature degradation of CBY1 protein. Thus, although the DZIP1C585W mutation appears to enhance an interaction with the complex, the reduced protein expression results in an overall loss of function, consistent with our other MVP‐identified mutation, DZIP1S24R and our Dzip1 conditional knockout mice.Whole exome sequencing identified a multi‐generational family with a rare, damaging variant in the DZIP1‐CBY1 interaction motif. Peptide analyses and Co‐IP studies revealed that this mutation altered the 3D structure of the protein and conferred a paradoxical increase in Cby1‐β‐catenin interactions. This should theoretically result in an increase in the stability of the complex and a reduction in β‐catenin activity. However, this is confounded by our data showing that the Dzip1Dzip1 and Cby1 deficient mitral valves is significant since MMP2 is a direct target of β‐catenin/Lef1 transcriptional regulation. Major substrates for this enzyme are collagens I, III, IV, V, VII, elastin, and TGF‐β. Transcripts for each of these ECM genes are increased in both of our Dzip1 and Ift88 cilia deficient mice.Ift88 conditional knockout (NfatC1Cre(+); Ift88f/f) mice show robust increases in collagen I transcript and protein at P0. The study presented in this report combined with our previous study paradoxically shows that that while Dzip1S14R/+ and Dzip1 conditional knockout mice (NfatC1Cre(+); Dzip1f/f) have increased transcripts for collagen and elastin genes, overall valvular collagen I protein levels are lower. A potential explanation for this finding is that in a mutated Dzip1 tissue, increased β‐catenin activity leads to upregulation of MMPs and subsequent proteolysis of collagens and elastin. Our study highlights that Dzip1 mutations result in a shift in the balance of ECM synthesis and destruction through loss of cilia and/or increased β‐catenin signaling. This altered ECM homeostasis eventually leads to the generation of a myxomatous phenotype that is incompatible with normal valvular function. Thus, the molecular interaction between Dzip1 and Cby1 suppresses β‐catenin signaling. This suppressive activity appears critical during development to ensure proper synthesis and organization of the valvular ECM.How altered β‐catenin signaling may lead to a myxomatous valve and mitral valve prolapse is poorly understood. Previous reports have shown that either loss or gain of β‐catenin function can result in a myxomatous phenotype.44.1targettm Mammalian Expression Vector System . The Dzip1 mutation was incorporated through a QuickChange II XL Site‐Directed Mutagenesis Kit (Agilent Technologies) based on the manufacturer's recommendations and had a HA‐epitope tag at the C terminus. CBY1 plasmid with a HA‐epitope tag at C terminus was purchased from Sino Biological (clone ID: BC016139). CBY1 plasmid with a Flag‐epitope tag at the carboxy terminus was a kind gift from Dr.Ken‐Ichi Takemaru and described previously.Control human DZIP1‐Myc‐DDK plasmid was purchased from Origene (clone ID: 198968). Truncated DZIP1 plasmids were sub‐cloned with a HA tag sequence at the C terminus using p4.2Dzip1S14R/+ or wild‐type mouse embryonic fibroblast(MEFs) were generated as previously detailedHuman valvular cells  were cultured in DMEM with 15% fetal calf serum and antibiotics as described previously.4.3Dzip1 conditional mice and Dzip1 knock in mice were genotyped and generated as previously described.Cby1 mice were a kind gift from Dr. Ken‐Ichi Takemaru. All animal experiments were performed under protocols approved by the Institutional Animal Care and Use Committees at the Medical University of South Carolina. Before cardiac resection, mice were euthanized by isoflurane  induction, followed by cervical dislocation in accordance with the Guide for the Care and Use of Laboratory Animals . Comparisons of the data generated for both male and female sexes showed no appreciable differences. As such, combined data for both sexes are shown.4.4S552 . Primary antibodies were placed in blocking buffer overnight at 4°C. Following primary antibody incubations, specimens were washed five times in PBS and incubated at room temperature with Alexa Fluor goat α‐rabbit 568 and goat α‐mouse 488  diluted 1:100 in PBS. Nuclei were stained with Hoechst dye  in PBS for 5 minutes prior to the final washes in PBS. All samples were cover‐slipped using Dabco mounting medium (Sigma). Images of immunostained sections were captured with SP5 confocal microscopy . Z‐stacks were set by finding the highest and lowest depth with visible fluorescence and using the system optimized setting to determine steps. Z‐stacks were then compiled to form maximum projection images.Immunohistochemistry (IHC) was performed as we have previously reported.Cby1 control (Cby1+/+) vs Cby1+/−, values were plotted every 0.5 μm to assess cilia length distribution differences between the two genotypes at P0. Three slides from a minimum of three animals/genotype were imaged at three different parts on anterior leaflets . Cilia length measurements were performed using Z‐stack images of anterior leaflet stained with acetylated alpha tubulin or arl13b, gamma tubulin and counterstained with Hoechst. Z‐stack images were then imported into Imaris software and measurements were taken from the base to the tip of the axoneme. All cilia in the field of view were measured. For control mitral leaflets, a total of n = 1266 cilia lengths quantified. For Cby1+/− mitral leaflets a total of n = 497 cilia lengths were quantified. Total cell numbers from each image were counted blindly by two people. n = 26 images were counted from wild‐type and n = 25 images were counted from Cby1+/−.For measuring cilia length in the 4.5HEK293T cells were seeded at 5X104.6cVICs were seeded at 2X104.7de novo peptide tertiary structure prediction from amino acid sequences.RaptorX Structure Alignment was used to align the protein structures of interest: P5 wild‐type (WT), P5 C585W mutant, P2 WT.4.85/well on six‐well plate. On the next day, cells were co‐transfected with wild‐type DZIP1‐Flag or DZIP1C585W‐Flag and CBY1‐HA constructs using FuGENE HD transfection Reagent . Cycloheximide was added 48 hours post transfection at concentration of 100 ng/mL. Cells were lysed with RIPA buffer at 0, 4, 8, 16, 32 hours after cycloheximide treatment. Dzip1S14R/+ and wild‐type MEFs were seeded at 2 × 105/well on six‐well plate and treated with cycloheximide at concentration of 100 ng/mL the next day. Cell lysates were harvested using RIPA buffer at 0 and 48 hours post cycloheximide treatment. Immunoblotting was performed as described previously.HEK293T cells were seeded at 5 × 104.9Dzip1S14R/+ and wild‐type MEFs from E13.5 embryos were plated at 2 × 105 /well on six‐well plates. On the following day, cells were released with trypsin and washed with 1× PBS twice and fragmentation was done following the instruction of NE‐PER Nuclear and Cytoplasmic Extraction Reagents . N = 3/genotype. Nuclear and cytoplasmic extractions were collected for Western Blotting. β‐Catenin was normalized by total protein loading by Ponceau S.4.10t‐test.All data are presented as means ± SD from two‐tailed Students 4.11All studies involving human research were approved by the Institutional Review Board Institut du Thorax, Nantes, France and all participants provided written informed consent.4.12Exome sequencing was performed on the proband (II.4). Exome capture was carried out using the SureSelect Human All Exon System using the manufacturer's protocol version 1.0 (Agilent Inc.) that is compatible with Illumina paired‐end sequencing. Exome‐enriched genomes were multiplexed by flow cell for 101‐bp paired‐end read sequencing according to the protocol for the HiSeq 2000 sequencer  to allow a minimum coverage of 30×. Reads were aligned to the human reference genome (UCSC NCBI36/hg19) using the Burrows‐Wheeler Aligner (version 0.5.9). Quality control to determine sample and genotyping quality and to potentially remove poor SNPs and/or samples was performed as we have previously published.Lilong Guo: Conceptualization; data curation; formal analysis; investigation; methodology; writing‐original draft; writing‐review & editing. Tyler Beck: Data curation; formal analysis. Diana Fulmer: Conceptualization; data curation. Sandra Ramos‐Ortiz: Data curation; validation. Janiece Glover: Data curation; formal analysis; methodology. Christina Wang: Data curation; methodology. Kelsey Moore: Data curation; formal analysis; investigation; methodology. Cortney Gensemer: Data curation; formal analysis; investigation; methodology. Jordan Morningstar: Data curation; investigation; methodology. Reece Moore: Data curation; formal analysis; investigation; methodology. Jean‐Jacques Schott: Data curation; formal analysis; funding acquisition; investigation; methodology. Thierry leTourneau: Data curation; formal analysis; funding acquisition; investigation. Natalie Koren: Methodology; project administration. Russell Norris: Conceptualization; formal analysis; funding acquisition; project administration; resources; supervision; writing‐original draft; writing‐review & editing.


The pace-of-life hypothesis is a socio-psychological theory postulating that citizens of different cities transact the business of life at varying paces, and this pace is associated with a number of population level variables. Here we apply the pace-of-life hypothesis to a hospital context to empirically test the association between pace and patient and staff outcomes. As pressure on hospitals grow and pace increases to keep up with demand, is there empirical evidence of a trade-off between a rapid pace and poorer outcomes? We collected data from four large Australian hospitals, inviting all staff  to complete a survey, and conducted a series of observations of hospital staff’s walking pace and transactional pace. From these data we constructed three measures of pace: staff perception of pace, transactional pace, and walking pace. Outcome measures included: hospital culture, perceived patient safety, and staff well-being outcomes of job satisfaction and burnout. Overall, participants reported experiencing a “fast-paced” “hurried” and “rapid” pace-of-life working in the Australian hospital sector. We found a significant difference in perceived pace across four hospital sites, similar to trends observed for transactional pace. This provides support that the pace-of-life hypothesis may apply to the hospital context. We tested associations between faster perceived pace, hospital culture, staff well-being and patient safety. Results revealed perceived faster pace significantly predicted negative perceptions of organizational culture, greater burnout and lower job satisfaction. However, perceived pace did not predict perceptions of patient safety. Different perceptions of hospital pace-of-life were found between different clinical settings and the type of care delivered; staff working in emergency departments reported significantly “faster-paced” work environments than staff working in palliative, aged care, or rehabilitation wards. The pace-of-life hypothesis is a socio-psychological theory suggesting that different cities and countries vary in the speed at which their residents experience and interact with the world  . The resThe pace-of-life hypothesis has recently been theoretically applied to the context of hospitals , but to There is evidence to suggest that the time and activity patterns of health professionals, such as the way nurses distribute their time, the increasingly fragmentary nature of task switching, and the number of interruptions they receive during work activities, play an integral role in shaping organisational culture , 5. FurtTheories of pace-of-life may hold considerable promise to shed light on the relationship between the hospital environment, patient outcomes and staff well-being. The pace of hospital life may vary depending on context and the type of care delivered , however this relationship is yet to be tested empirically . HospitaAs hospital staff face pressures to deliver care at a faster pace there is also a potential risk to staff well-being , which can be detrimental to patient safety . WorkloaWe are the first to apply the theory of pace-of-life and the relationship to culture, patient outcomes and staff well-being to the hospital context. We anticipate the data will allow for insights into hospital organisational culture that are new and important, with potential implications for improving the delivery of patient care in the longer term through interventions targeted at modifying hospital pace. While there may be less conspicuous aspects of organisational cultural context, our study of these constructs reflects a growing recognition that taking care of the “small things”  might make it easier to keep the “big things” in check  .The aim of this study was to empirically test Braithwaite and colleagues’ theoretical findings regarding how the pace-of-life hypothesis applies to the hospital context   consistently.There is a relationship between faster pace and a negative workplace hospital culture.Faster hospital pace is linked to lower job satisfaction.Faster hospital pace is linked to higher burnout.Faster hospital pace is predictive of poor patient safety outcomes.Hospital pace differs based on clinical settings.The researcher team employed a convergent, parallel mixed methods design . Data coThe study was conducted at four public hospitals in metropolitan Sydney, Australia. The four hospitals were of similar size , and varying geographic locations and socio-economic disadvantage across greater metropolitan Sydney . All hosQualtrics –that’s spelt [––––] was admitted today?” We consistently used the name of one of the authors and provided their date of birth if requested. The timing ended when the response “No, she wasn’t admitted” was given and the call terminated. We calculated an average transactional pace score . Outliers were considered based on standard deviation and removed from the dataset.Walking pace was measured using observational data. Following the methodology of Levine and Norenzayan’s  study, wHospital culture was measured using the Safety Attitudes Questionnaire (SAQ)  as part Culture was also operationalised as ‘staff demeanour’ and captured in the observational data. When measuring transactional pace, we assessed the demeanour of hospital staff (who answered the question) on a five-point Likert-type scale (1 –surly/unpleasant to 5 –pleasant).Patient safety was assessed with a single-item measure from the Hospital Survey on Patient Safety Culture (HSOPS)  includedExisting validated scales were selected to measure job satisfaction and burnout in the survey. Job Satisfaction was assessed using five items from the Job Diagnostic Survey (JDS), for example: “In general, I like working here”. Responses were rated on a five-point Likert scale (1 –strongly disagree to 5 –strongly agree), with an option for ‘neutral’. Scores were summed, with higher scores indicating a greater degree of job satisfaction. Bowling and Hammond  reportedBurnout was measured using a 10-item version of the Maslach Burnout Inventory MBI –30). Due. Due30])Three open-ended questions were included in the survey. Questions were phrased in a general way to allow participants to reflect on the influence of pace in their work. For example, “Are there other things you notice about the behaviour of people in this hospital that negatively affect peace, cooperation and well-coordinated work?”Survey responses and observational pace data were analysed using descriptive statistics followed by regression analyses. All data were analysed via IBM’s SPSS Statistics Version 25  and condF = 0.88, p = .467. For perception of pace measured in the survey, hospital staff across all four sites perceived their work as fast-paced, rushed, and time-poor. Free text answers, especially from nurses validated this perception: “very rushed, running out of time, sometimes there’s no time for morning tea break, lunch can be rushed, interruptions, exhausted by mid-day” . A one-way ANOVA revealed a significant difference in perceptions of pace based on hospital site, F = 3.86, p = .010. Trends of which hospital had a faster pace-of-life were consistent for perceptions of pace and transactional pace, providing partial support for Hypothesis 1. Hospitals are ranked by their pace-of-life in Pace of hospital life was measured in four hospital sites using three measures: perception of pace, transactional pace, and walking pace. For walking pace, we observed 439 staff . Observational data showed it took an average walking pace of 14 seconds  for hospital staff to traverse 20 meters of a corridor. There was minimal variation in this data, across and within hospitals. The average transactional pace across all four hospital was 40 seconds  for staff to answer a simple patient enquiry on the main switch phone, with no significant differences across sites, t(317) = -2.04, p = .042), indicating that clinical staff reported higher scores on the pace scale  compared to non-clinical staff  as shown in t(337) = -1.10, p = .274), overall patient safety grade (t(343) = 1.59, p = .123), patient safety issues reported (t(335) = -1.53, p = .126), job satisfaction (t(345) = 0.68, p = .496) and burnout (t(343) = 1.23, p = .220). Hereafter, results are presented in relation to hypotheses.We compared clinical  and non-clinical staff  and found significant differences based on perceptions of pace  = 3.44, p = .005. The analysis showed that perceived pace (t(295) = -3.57, p < .05) significantly predicted hospital culture, supporting Hypothesis 2. That is, perceiving daily work tasks in a hospital as more “fast-paced”, “racing”, “quick”, significantly predicted a negative organizational culture. Quantitative results were supported with responses to open-ended questions. Participants suggested that, in the context of a “fast-paced” working environment, there are increases in work pressure and environmental constraints which negatively impact on teamwork culture: “When we are overwhelmed, especially with no sick leave, annual leave or maternity leave, we all work under strain and can be "bitchy" ; “Peace, good cooperation and coordination can only happen if there is sufficient staff given sufficient time” .A multiple linear regression examined if staff perceptions of pace predicted organizational culture, considering age, duration at hospital, hours worked per week, and interaction with patients as confounding variables. The model was statistically significant, p = .278) and transactional pace did not predict demeanour, while holding time of day constant (p = .545).Further, when measuring transactional pace, we also rated the demeanour of hospital staff that answered the main switch phone as an additional measure of culture. We investigated whether the demeanour of hospital staff was a predictor of transactional pace, keeping time of day  constant. Demeanour and transactional pace were not significantly related  = 3.35, p = .006. The analysis showed that perceived pace (t (301) = -4.07, p < .05) significantly predicted job satisfaction. That is, perceiving daily tasks at work in a hospital as more “fast-paced” and “quick”, significantly predicted low scores of job satisfaction. Thus, our findings support Hypothesis 3.A multiple linear regression was run to predict job satisfaction from perceived pace, considering age, duration at hospital, hours worked per week, and interaction with patients as confounding variables. The model was statistically significant, F = 7.75, p < .05. Scoring higher on the pace (t (303) = 5.82, p < .05) significantly predicted burnout. Thus, in support of Hypothesis 4, we found that perceiving daily tasks at work in a hospital as more “fast-paced”, significantly predicted higher levels of burnout in hospital staff. For some staff, “fast-paced” patient care resulted in issues that increased workload and potentially, burnout: “Staff, both from my department and others, are focused on how to transfer/discharge the patients in the quickest way possible. This means that issues that should have been addressed are left for the next person to have to deal with” .A multiple linear regression was run to examine whether perceived pace predicted burnout in hospital staff, treating age, duration at hospital, hours worked per week, and interaction with patients as confounding variables. The model was statistically significant, F = 1.60, p = .160. Therefore, our findings do not support Hypothesis 5. Responses to open-ended questions suggested that a relationship may still exist between pace of hospital life and safety of patient care. For some staff, the “fast-paced” nature of the hospital brought on a sense of pressure that could lead to negative ramifications for the safety of care delivery: “Pressure to admit and discharge inappropriately and quickly” .We tested whether perceived pace of hospital life predicted patient safety, treating age, duration at hospital, hours worked per week, and interaction with patients as confounding variables. The model was not statistically significant, n = 42) compared to presumably slower-paced wards . Across the four hospitals, analysis revealed that staff working in the emergency department perceived their work as significantly more “fast-paced”, compared to staff working in palliative care, aged care, and rehabilitation wards, F = 14.27, p < .05. This provides support for Hypothesis 6. While staff working in the emergency department had lower mean scores of job satisfaction  and higher mean scores of burnout  compared to staff working in palliative care, aged care, and rehabilitation wards , there were no significant differences in staff well-being (job satisfaction or burnout) based on clinical setting.To examine differences in staff perceptions of pace based on clinical settings, we conducted a one-way ANCOVA to examine if differences in pace existed dependent on the type of care delivered, keeping age, duration at hospital, hours worked per week, and interaction with patients constant. As theorised in previous work , we examThe study applied the pace-of-life hypothesis to hospitals, examining if pace in a hospital is related to organisational culture, staff well-being, and the safety of patient care; and whether pace is different in different clinical settings. There were differences in the perceived pace of hospital life for different hospitals. Faster perceived workplace pace significantly predicted negative organizational culture, higher burnout, and lower job satisfaction; however, pace did not significantly predict patient safety. Lastly, perceptions of pace significantly differed depending on the clinical setting.The study, provides empirical support for the applicability of the pace-of-life hypothesis  to hospiOur study provides empirical support to the Goldilocks hypothesis . We founThe pace subscale from Ballard and Seibold (6) did not assign a positive or negative sentiment to the words used to describe the pace of work. While “hurried” or “racing” are most likely perceived as negative, it is possible that “fast-paced” could be seen as a neutral or positive attribute. Saying your job is “fast-paced” may indicate an exciting, active and desirable place to work. Together, the items in the perceived pace scale showed high inter-item reliability suggesting “fast-paced” was seen as either negative or neutral. Our study also provided some qualitative evidence to support a faster pace of work in the hospital being a negative construct.A rapid transactional pace is generally perceived by the person receiving the service as positive, so long as it is done with care and efficiency. This is the Goldilocks zone , that isThis paper is the first to investigate the relationship between pace, organizational culture, staff well-being and patient outcomes. It is also the first study to consider the socio-psychological theory of pace-of-life in the hospital context. Our paper adds to the knowledge base about the relationships and interactions between perceptions of pace, culture and staff and patient outcomes by applying some novel tools and methods to collect data. Having shown the feasibility and limitations of the methods, the next step is to refine these so that more robust testing of the Goldilocks effect is possible, i.e., what is the sweet spot for pace in hospitals?S1 Appendix(DOCX)Click here for additional data file. This paper was transferred from another journal. As a result, its full editorial history  may not be present.4 Jun 2021PONE-D-20-33878The Pace of Hospital Life: A mixed methods studyPLOS ONEDear Dr. Long,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.The authors have assumed that all fast-pace hospital activity would not only lead to work dissatisfaction but also, as a consequence, would lead to less effective patient care. It is not a logical conclusion that fast-paced hospital activity would damage patient health care. This is because faster paced, and potentially more attentive patient care could very well lead to a higher standard of patient care and decreased patient illness and mortality. The authors do not mention this major counter hypothesis. But the broadest finding in the literature of hospital care is that more extensive and repetitive medical and surgical procedures are associated with beneficial patient outcomes. The authors should explore this counter hypothesis, both in terms of the actual intensiveness of work and the higher stress levels it tends to produce. Stress levels in the working staff could as easily indicate a high level of tension-related performance.plosone@plos.org. 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The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: NoReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: At first I did not quite understand the purpose of the paper. At least it seemed somewhat obvious regarding the fast pace of hospitals. As I read further, it seems that the methods used were rigorous. Specifically the authors went beyond mere survey data and conducted direct observation. It would be good to make this clearer. The casual reader may not be aware that the methods went beyond just taking a survey.It was unclear to me from the paper why people are rushing around so much in the hospital. Is there a reason for this - beyond the obvious need to keep pace with the workload? And does all this rushing around actually achieve anything? I would like to see some attempt to explain why some hospitals are working at more of a burnout pace. One metric that is often used are nurse-to-patient ratios. If hospitals do not have enough nurses - then they may have to rush around more. However, there must be a limit to this. The ability of one nurse to do the work of two nurses is finite. This is a huge problem in the US and has been made exceedingly worse by the current pandemic. There are just not enough nurses and in spite of their best efforts much work never gets done and patients die in part due to a shortage of nurses.Reviewer #2: This paper, on the “Pace of Hospital Life” in Australian hospitals finds that a “fast-paced” “hurried” and “rapid” pace of life leads to negative perceptions of organizational culture, higher burn-out and lower job satisfaction. However, perceived pace did not predict patient safety.The authors have assumed that all fast-pace hospital activity would not only lead to work dissatisfaction but also, as a consequence, would lead to less effective patient care. This did not occur.On the other hand, it is not a logical conclusion that fast-paced hospital activity would damage patient health care. This is because faster paced, and potentially more attentive patient care could very well lead to a higher standard of patient care and decreased patient illness and mortality. The authors do not mention this major counter hypothesis. But the broadest finding in the literature of hospital care is that more extensive and repetitive medical and surgical procedures are associated with beneficial patient outcomes. The authors should explore this counter hypothesis, both in terms of the actual intensiveness of work and the higher stress levels it tends to produce. Stress levels in the working staff could as easily indicate a high level of tension-related performance.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  1 Jul 2021Please see our uploaded Response Table where each comment has been answered. 26 Jul 2021The Pace of Hospital Life: A mixed methods studyPONE-D-20-33878R1Dear Dr. Long,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,M. Harvey Brenner, PhDAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 10 Aug 2021PONE-D-20-33878R1 The Pace of Hospital Life:  A mixed methods study Dear Dr. Long:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofProfessor M. Harvey Brenner Academic EditorPLOS ONE