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Dooley, S.

Publications and source records attributed to Dooley, S..

16 recordsLinked to original sources

Artificial light at night and concealment level affect detection probability of stranded seabirds: a study with 3D-printed puffin decoys

Fledglings of burrow-nesting seabirds are vulnerable to disorientation by artificial light at night (ALAN) and grounding in coastal human settlements, where most individuals presumably perish if not rescued. Accurate estimates of the effects of stranding on seabird populations remain elusive due to several levels of uncertainty, such as the number of stranded seabirds estimated to remain undiscovered by rescuers. One species subjected to stranding is the Atlantic puffin (Fratercula arctica). We hypothesized that the detection probability of puffin fledglings by the Puffin Patrol rescue program, operating in Newfoundland, Canada, is affected by light level, concealment level, and search effort. We deployed realistic puffin decoys in locations of varying light and concealment levels throughout two sites that are searched nightly by rescuers and measured the amount of time and search effort required until each decoy was found. Approximately 78% more search effort was required to find a decoy in darkness (32.0 min) compared with illuminated conditions (18.0 min), and up to 100% more search effort was required to find a decoy placed in concealed conditions (35.5 min) than one in the open (17.5 min). After two hours of searching, on average over 10% of decoys were still not found, although each additional minute of search effort increased detection rate of an individual decoy by 0.6%. In combination with recent advances in our knowledge of seabird post-stranding behaviour, our study offers a first step in the development of correction factors which can be adapted to site- and species-specific requirements of other rescue programs around the world that rely on volunteers to search for stranded birds. Application of such correction factors will hopefully result in better estimation of mortality rates and their resultant effects on populations.

zoology↗

Transient suppression of the ECM1 gatekeeper is essential for HGF/c-MET-driven liver regeneration

Hepatocyte growth factor (HGF) is a multifunctional cytokine stored in the extracellular matrix as an inactive precursor and is essential for tissue repair. How HGF activity is dynamically regulated during regeneration remains unclear. Here, we identify extracellular matrix protein 1 (ECM1) as a physiological inhibitor of active HGF during liver regeneration. Following 70% partial hepatectomy, active HGF rapidly increases in parallel with a sharp decline in ECM1, and preventing this downregulation delays liver mass recovery. Mechanistically, ECM1 directly binds the active HGF -subunit through a mechanism dependent on residue R392, thereby suppressing c-MET-ERK-MYC signaling and hepatocyte proliferation. Loss of ECM1 permits activation of this pathway, whereas MYC overexpression rescues ECM1-mediated growth inhibition. In patients, proliferative hepatocytes localize to ECM1-negative regions. Supported by transcriptomic analyses and computational modeling, these findings identify ECM1 as an extracellular gatekeeper whose transient downregulation enables HGF-driven tissue repair. HighlightsO_LIECM1 is temporally downregulated during liver regeneration, while forced ECM1 expression delays liver mass recovery after 70% PHx by suppressing hepatocyte proliferation. C_LIO_LIRapid and transient ECM1 downregulation permits HGF-c-Met-ERK-MYC signaling to drive hepatocyte cell-cycle entry and expansion. C_LIO_LIECM1 directly binds the active HGF -subunit with residue R392 playing a critical role, revealing a novel extracellular mechanism for growth factor inhibition. C_LIO_LILoss of ECM1 marks proliferative hepatocytes in human liver repair, identifying ECM1 as a tunable regulator of regenerative capacity. C_LI

cell biology↗

Immunoglobulin G binding as a quantitative marker of hepatocellular death across acute and chronic liver injury

Background & AimsAccurate detection of hepatocellular death is fundamental for understanding liver injury, intoxication, regeneration, and fibrosis. Conventional markers, such as serum transaminases and histopathological scoring, suffer from limited temporal resolution, high variability, and observer dependence. We evaluated immunoglobulin G (IgG) binding as a quantitative and spatially resolved marker of hepatocyte death in acute and chronic liver injury models. MethodsMale C57BL/6 mice were subjected to acute carbon tetrachloride (CCl) intoxication (1600 mg/kg, single dose), dose-escalation (0-800 mg/kg), and chronic injury paradigms including Western diet (WD), WD+CCl, and Mdr2-/- mice with or without a single CCl challenge. The serum ALT and AST levels were measured. Liver sections were stained with IgG, Hematoxylin and Eosin (H&E), bromodeoxyuridine (BrdU), glutamine synthetase (GS), CD26, and alpha-smooth muscle actin (Acta2). Spatial and integrative transcriptomic analyses were performed to characterize the IgG hepatocyte dead regions. ResultsHepatocellular IgG labeling emerged as early as 6h post-CCl, peaked at 72-96h, and declined during regeneration. IgG-positive areas correlated strongly with Ishak necroinflammatory score (r=0.70) and serum transaminase levels (p=0.74-0.85), surpassing both in Receiver Operating Characteristic (ROC) analyses (AUC=0.92-0.95). IgG bound to both apoptotic (TUNEL) and necrotic (TUNEL-) hepatocytes. In chronic liver injury models, IgG deposition was localized to the injury zones and correlated with ALT/AST, irrespective of etiology. Multiplex imaging revealed IgG-positive necrotic cores surrounded by proliferating hepatocytes and Acta2+ myofibroblasts. Spatial transcriptomics identified immune cell enrichment, Fc{gamma}R-mediated signaling, phagocytosis, and vascular remodeling within the IgG-marked regions. ConclusionsIgG immunostaining provides a robust, quantitative, and pathologist-independent readout of hepatocellular death, which scales with injury severity, delineates necrotic zones, and reveals immune-active microenvironments. These findings establish IgG-based detection as a versatile, high-resolution tool for assessing liver injury, regeneration, and fibrosis. Conflict of Interest declarationThe authors declare that they have no affiliations with or involvement in any organization or entity with any financial interest in the subject matter or materials discussed in this manuscript. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/690787v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1d9b05eorg.highwire.dtl.DTLVardef@ca2ad8org.highwire.dtl.DTLVardef@c2d384org.highwire.dtl.DTLVardef@cca46d_HPS_FORMAT_FIGEXP M_FIG C_FIG Liver injury induced by toxins, dietary stress, or genetic knockout provokes chemokine release (i.e. CXCL1, CXCL2, and CCL3) and recruitment of immune cells, including T cells, NK cells (NKs), and dendritic cells (DCs). Activated immune cells secrete IgG, which binds to damaged hepatocytes, leading to IgG deposition and opsonization. Opsonized hepatocytes expose "eat me" signals, promoting their phagocytic clearance and contributing to the resolution of liver injury and restoration of hepatic homeostasis.

cell biology↗

The interplay between biomechanics and cell kinetics explains the spatial pattern in liver fibrosis

The formation of liver fibrosis patterns, characterized by excess extracellular matrix (ECM), is a complex process that is difficult to investigate experimentally. To complement experimental approaches, we developed a digital twin (DT) model to simulate the pattern formation of septal and biliary fibrosis, the two common forms of liver fibrosis. This model is based on iterative calibration with experiments from animal models treated with the hepatotoxic substance CCl4 (septal form) and Abcb4-knockout mice (biliary form). Septal fibrosis is characterized by ECM accumulation along the connective line between the central veins of neighboring liver lobules, while biliary fibrosis is marked by a scattered ECM pattern within the portal fields. This mechanistic DT model includes the components of hepatocytes (Heps{spadesuit}), hepatic stellate cells (HSCs), macrophages (Mphs), bile duct (BD) cells, collagen fibers secreted by activated HSCs, blood vessels, and cell-cell communication. It allows for the integration and simultaneous modulation of multiple hypothesized mechanisms underlying fibrotic wall formation. The model simulates the formation of liver fibrosis pattern and demonstrates that ECM distribution results from the pattern of cell death zones and biomechanical compression due to cell proliferation. "Healthy" Heps proliferate to compensate for cell loss. In septal fibrosis, where the cell death zones are several cells thick, the proliferating Heps surrounding a zone mechanically compress the deposited collagen network. After a transient phase of collagen scattered between/around Heps, the ECM eventually adopts a sharp, "wall"-like structure. Whereas, in biliary fibrosis, the pattern of cell death is more scattered, leading to a corresponding scattered ECM pattern. In this case, a pattern of scattered distributed collagen forms without transitioning to a sharp wall. Notably, the failure of fibrotic wall formation in endothelial cell-specific GATA4LSEC-KO mice, due to the disrupted pattern of CYP2E1-expressing Heps, validates our DT model. In conclusion, the DT model provided a deeper understanding of liver fibrosis pattern formation. It enabled comparison between simulated outcomes of hypothesized mechanisms and experimental data. Additionally, it guided the design of validation experiments and enabled the identification of optimal strategies for drug testing and extrapolation to humans.

systems biology↗

Regulation of essential hepatocyte functions and identity by super-enhancers in health and disease

Physiologically, hepatocytes perform an estimated 500 essential liver functions to ensure systemic homeostasis. The mechanisms how hepatocytes maintain these numerous crucial functions in response to physiological and pathophysiological challenges are largely unknown. In this study, we found that vital liver function genes (e.g., HNF4A, ALB, coagulation factors, and SLC2A2) in hepatocytes possess super-enhancers. Interfering with the super-enhancers by dCas9-sgRNA remarkably decreases the expression of these genes. Quiescent liver progenitor cells (LPCs) do not have active super-enhancers in vital liver function genes. In acute liver failure (ALF) caused by massive hepatic necrosis, activated LPCs perform liver function and differentiate into hepatocytes to rescue patients lives. We show that activated LPCs gradually form super-enhancers in liver function genes (e.g., HNF4A, ALB, and coagulation factors) during proliferation and differentiation. These super-enhancers are regulated by FOXA2 through the recruitment of HNF4 a, p300 and Mediators in most liver function genes, such as ALB and coagulation factors. However, super-enhancers in some genes (e.g., SLC2A2) are FOXA2-indenpendent. Hepatic FOXA2 expression is closely associated with the clinical outcome of ALF. In a cohort of ALF patients, robust FOXA2 is observed only in the surviving patients. Collectively, super-enhancers represent a critical epigenetic regulatory mechanism that enables rapidly enhanced transcription of liver function genes in response to pathophysiological challenges. In urgent clinical syndrome such as ALF, LPCs rescue liver function by rapid formation of super-enhancers in vital genes.

molecular biology↗

TGF-β serves as a critical signaling determinant of liver progenitor cell fate and function

Liver progenitor cells (LPCs) are the smallest cholangiocytes that perform hepatocyte functions to rescue the lives of patients suffering from acute liver failure (ALF) caused by massive hepatic necrosis (MHN). To date, it remains largely unknown how LPCs remain quiescent and become activated following MHN. This study elucidates the essential role of TGF-{beta} in regulating LPC quiescence and activation. Spatial transcriptomics analysis of liver tissues from four MHN-ALF patients revealed that LPCs receive multiple active signals from surrounding macrophages and hepatic stellate cells, including TGF-{beta}, HGF, and EGF. Physiologically, TGF-{beta} inhibits LPC proliferation by impeding G1-S phase transition. Ectopic Smad7 expression remarkably increased LPC proliferation in 3,5-diethoxycarbonyl-1,4-dihydrocollidine-fed mice. Intriguingly, extensive LPC proliferation was observed in ALF patients despite robust TGF-{beta}-p-SMAD2 signaling in activated LPCs. Immunohistochemistry and immunofluorescence revealed significantly elevated expression of p-MET, p-STAT3, p-EGFR, and p-ERK in LPCs, indicating active HGF and EGF signaling. In vitro, either HGF or EGF promoted LPC proliferation despite the presence of TGF-{beta}. Beyond acting as mitogens, HGF and EGF regulate master hepatocyte genes (e.g., HNF4) and cholangiocyte genes (e.g., SOX9) in LPCs. Notably, HGF-dependent HNF4 required TGF-{beta}-activated SMADs. Collectively, TGF-{beta} serves as a critical signaling determinant of LPC fate and function.

cell biology↗

CD271 sorting for improved liver cell isolation: Semiautomated and simultaneous preparation of parenchymal and non-parenchymal cells from mouse and human livers

BackgroundA detailed understanding of the dynamic fate changes of hepatocytes, hepatic stellate cells (HSC), Kupffer cells (KC), and liver sinusoidal endothelial cells (LSEC) is critical for studying liver (patho)physiology during disease progression. Current isolation methods often focus on single cell types, limiting utility in comprehensive research. AimTo develop a novel, semi-automated protocol for the simultaneous isolation of hepatocytes and non-parenchymal cells (NPCs), including HSC, KC, and LSEC, from mouse and human, with high yield, purity, and viability from healthy and diseased livers. MethodThe protocol employs a two-step EGTA and collagenase II perfusion for tissue digestion. Hepatocytes were isolated by low-speed centrifugation and a Percoll gradient. Subsequently, magnetic-activated cell separation, using CD271 as a selective surface marker for HSC, CD11b for KC and CD146 for LSEC) was performed. Validation was achieved with immunofluorescence staining, flow cytometry, RT-PCR, and UV fluorescence, whereby yield, purity, and viability were assessed. ResultsWith our method, yield of hepatocytes, HSC, KC, and LSEC, is 33.4{+/-}5.5x10, 5.2{+/-}6.3x10, 12.4{+/-}4.8x10 and 18.2{+/-}8.9x10 cells per healthy mouse liver, respectively, with cell viabilities exceeding 89%, and purity surpassing 90%. CD271 was validated as an effective marker for purifying HSC in healthy and diseased human (n=4-6) and mouse livers. Compared to microfluidic and organ-on-a-chip approaches, with our protocol, we achieve higher yield and purity values while enabling the simultaneous isolation of multiple cell types from a single sample. ConclusionOur semi-automated protocol offers a scalable, reliable, and versatile solution for isolating main liver cell types with high yield, purity, and viability from both healthy and diseased tissues, advancing liver research and facilitating downstream investigations. Impact and implicationsO_LIBroad applicability: The CD271-based method efficiently isolates key liver cell types (Hepatocyte, HSC, KC, LSEC) simultaneously. C_LIO_LIVersatility in disease models: Effective for studying healthy, fibrotic, and damaged liver tissues. C_LIO_LIRobust across variability: Works reliably across different mouse strains, age groups, and conditions. C_LIO_LIHuman research potential: Scalable for high-purity isolation from human liver tissue, enabling translational studies. C_LIO_LIHigh-quality results: Ensures >85% viability and >90% purity, supporting reproducible liver research applications. C_LI

cell biology↗

EGF/STAT1 signals to maintain ECM1 expression in hepatic homeostasis are disrupted by IFNγ/NRF2 in chronic liver disease

In healthy livers, extracellular matrix protein 1 (ECM1) is essential for liver homeostasis by keeping latent transforming growth factor-{beta} (LTGF-{beta}) quiescent. Upon hepatocyte damage, ECM1 is downregulated, facilitating LTGF-{beta} activation and fibrogenesis. However, little is known about how hepatic ECM1 is regulated. Here we found in healthy hepatocytes, EGF/EGFR signaling sustains ECM1 expression through phosphorylating STAT1 at S727, enhancing its binding to the ECM1 promoter and boosting gene transcription. During liver inflammation, accumulating IFN{gamma} disrupts this process by downregulating EGFR and inhibiting EGF/EGFR/STAT1-mediated ECM1 promoter binding. Mechanistically, IFN{gamma}-induced STAT1 phosphorylation at Y701 impairs the binding of p-STAT1 S727 to the ECM1 promoter. Additionally, IFN{gamma} induces NRF2 nuclear translocation, which repressively binds to the ECM1 promoter, further reducing its expression. These findings were confirmed in several chronic liver disease (CLD) mouse models. Moreover, AAV8-ECM1 significantly attenuates liver fibrosis and injuries in Western diet (WD)-fed mice. Notably, in patients with CLD, ECM1 levels align with EGFR expression, while NRF2 and LTGF-{beta} activation show a negative correlation with both.

cell biology↗

Retinoic acid generates a beneficial microenvironment for liver progenitor cell activation in acute liver failure

ObjectiveWhen massive necrosis occurs in acute liver failure (ALF), rapid expansion of hepatic stem cells called liver progenitor cells (LPC) in a process called ductular reaction (DR) is required for survival. The exact underlying mechanisms of this process are not known to date. In ALF, high levels of retinoic acid (RA), a molecule known for its pleiotropic roles in embryonic development, are secreted by activated hepatic stellate cells (HSCs). We hypothesized that RA plays a key role during DR in ALF. MethodsRNA-Seq was performed to identify molecular signaling pathways affected by all-trans retinoid acid (atRA) treatment in HepaRG LPC cells. Functional assays for RA were performed in HepaRG cells with atRA treatment as well as co-culture with LX-2 cells in vitro, and liver tissue of patients suffering from ALF in vivo. ResultsUnder ALF conditions, activated HSCs secreted RA, inducing RAR nuclear translocation in LPCs. RNA-seq data and investigations in HepaRG cells revealed that atRA treatment activated the WNT-{beta}-Catenin pathway, enhanced stemness genes (SOX9, AFP, et.al), promoted energy storage, and elevated the expression of ATP-binding cassette (ABC) transporters depending on RAR nuclear translocation. Further, atRA treatment-induced pathways were confirmed in a co-culture system of HepaRG with LX-2 cells. Patients with ALF who displayed RAR nuclear translocation in LPC had significantly better MELD scores than those without. ConclusionIn ALF, RA secreted by activated hepatic stellate cells promotes LPC activation, a prerequisite for subsequent LPC-mediated liver regeneration.

cell biology↗

Extracellular Matrix Protein 1 Attenuates Hepatic Fibrosis by Inhibiting TSP-, ADAMTS-, and MMP-Mediated Latent TGF-β1 Activation

ObjectiveExtracellular Matrix Protein 1 (ECM1) serves as a gatekeeper of hepatic fibrosis by maintaining transforming growth factor-{beta}1 (TGF-{beta}1) in its latent form. ECM1 knockout (KO) causes latent (L) TGF-{beta}1 activation, resulting in hepatic fibrosis with rapid mortality. In chronic liver disease (CLD), ECM1 decreases with increasing CLD severity. We investigate the regulatory role of ECM1 in TGF-{beta}1 bioavailability and its impact on CLD progression. DesignRNAseq was performed to analyze hepatic gene expression. Functional assays were performed using hepatic stellate cells (HSCs), Ecm1-KO and Fxr-KO mice, patient liver tissue, and computer simulations. ResultsExpression of LTGF-{beta}1 activators, including thrombospondins (TSPs), ADAMTS proteases, and matrix metalloproteinases (MMPs) increased along with pro-fibrotic gene expression in liver tissue of Ecm1-KO mice. In HSCs, overexpression of ECM1 prevented TSP-1-, ADAMTS1-, and MMP-2/9-mediated LTGF-{beta}1 activation. In vitro interaction assays demonstrated that ECM1 inhibited LTGF-{beta}1 activation by interacting with TSP-1 and ADAMTS1 via their respective, intrinsic KRFK or KTFR amino acid sequences, and by suppressing MMP-2/9 proteolytic activity. In mice, ECM1 overexpression attenuated KRFK-induced LTGF-{beta}1 activation, while KTFR treatment reversed Ecm1-KO- and Fxr-KO-mediated liver injury. In patients with CLD, ECM1 expression was inversely correlated with TSP-1, ADAMTS1, MMP-2/9 expression and LTGF-{beta}1 activation. And these results were complemented by a computational compartment model representing the key network of cellular phenotypes and predicted interactions in liver fibrogenesis. ConclusionOur findings underscore the hepatoprotective effect of ECM1, which interferes with mediators of LTGF-{beta}1 activation, suggesting ECM1 or its representative peptide as potential anti-fibrotic therapies in CLD. What is already known on this topic?[tpltrtarr] ECM1 expression is negatively correlated with CLD progression. [tpltrtarr]ECM1 maintains liver homeostasis by keeping TGF-{beta}1 latency. What this study adds?[tpltrtarr] ECM1 inhibits LTGF-{beta}1 activation through interfering with key activators, including TSP-1, ADAMTS1, MMP-2, and MMP-9. [tpltrtarr]ECM1 interacts with TSP-1 and ADAMTS1 via their respective, intrinsic KRFK or KTFR amino acid motifs, and suppresses MMP-2/9 proteolytic activity. [tpltrtarr]In vivo, ECM1 overexpression mitigates KRFK peptide-induced LTGF-{beta}1 activation, while KTFR peptide rescues Ecm1-KO- and Fxr-KO-induced liver injury. [tpltrtarr]ECM1 expression inversely correlates with TSP-1, ADAMTS1, MMP-2/9 expression and LTGF-{beta}1 activation in CLD patients. How might this study affect research, practice or policy?[tpltrtarr] Considering severe adverse effects associated with anti-fibrotic treatments utilizing TGF-{beta}1 receptor inhibitors, our findings indicate that restoration of ECM1 expression or phenocopying peptides might represent a novel and safe route to urgently needed anti-fibrotic therapies in CLD.

cell biology↗

FOXA2 is essential for maintaining the urea cycle in acute liver failure

Hepatic encephalopathy is a lethal complication of acute liver failure (ALF), and is caused by hyperammonemia. Ammonia clearance by the liver requires an intact and complete urea cycle comprising six enzymes, including the rate-limiting enzyme carbamoyl phosphate synthetase I (CPS1). To date, the detailed regulation of CPS1 transcription in order to maintain urea cycle in physiological condition and ALF remains largely unknown. This study scrutinizes the role of pioneer factor forkhead box A 2 (FOXA2) in the regulation of CPS1 transcription, urea cycle performance and hyperammonemia. Physiologically, CPS1 transcription requires FOXA2 to maintain chromatin accessibility on its enhancers, which is essential for CCAAT enhancer-binding protein-alpha (C/EBP) binding to activate gene transcription. In ALF, hepatic C/EBP expression is inhibited by inflammatory mediators such as TGF-{beta} and TNF-. In this setting, retinoic acid receptor synergizes with FOXA2 to maintain CPS1 transcriptions. Once ALF patients suffer from massive hepatic necrosis, liver progenitor cells initiate a transcription network comprising FOXA2 and C/EBP to perform the urea cycle and prevent hyperammonemia. In ALF, hepatic encephalopathy occurs in patients lacking hepatic FOXA2 expression. In mice with acetaminophen-induced ALF, injection of Foxa2-AAV8 maintains urea cycle and prevents hyperammonemia. Taken together, FOXA2 is essential for maintaining the urea cycle. Pharmaceutical induction of hepatic FOXA2 expression might represent a novel approach to treat hepatic encephalopathy in ALF. One Sentence SummaryPioneer factor FOXA2 synergizes with C/EBP or RAR to maintain urea cycle in acute liver failure

molecular biology↗

Rapid dexamethasone treatment inhibits LPS-induced cytokine storm in mice

Severe infection-induced cytokine storm is an urgent medical syndrome with high mortality. To date, no therapy is available. This study shows that high concentrations of lipopolysaccharide (LPS) induce cytokine storm within 48h and thus kill most experimental mice. Rapid, but not late dexamethasone administration remarkably inhibits cytokine storm and rescues LPS-treated mice. Monocytes and macrophages are the major source of cytokine storm. In these cells, pro-inflammatory genes (i.e., Tnf, Il6 and Il1{beta}) have preassembled RNA polymerase II (RNA Pol II), but stay at the pause stage of transcriptional elongation in the absence of stimulation. LPS rapidly activates transcription of these "pre-loaded" genes within 2h. Administration of dexamethasone within this time window inhibits RNA Pol II ser2 binding to the core promoters of pro-inflammatory genes and thus reduces LPS-induced cytokine transcription. Therefore, rapid utilization of dexamethasone might be efficacious to prevent severe bacterium-induced cytokine storm in clinical practice.

molecular biology↗

TGF-β1 inhibits cholesterol metabolism in hepatocytes to facilitate cell death, EMT and signals for HSC activation.

Background and Aims: Transforming growth factor-{beta}1 (TGF-{beta}1) plays important roles in chronic liver diseases, including metabolic dysfunction-associated steatotic liver disease (MASLD). MASLD involves various biological processes including dysfunctional cholesterol metabolism and contributes to progression to metabolic dysfunction-associated steatohepatitis (MASH) and hepatocellular carcinoma (HCC). However, the reciprocal regulation of TGF-{beta}1 signaling and cholesterol metabolism in MASLD is yet unknown. Methods: Changes in transcription of genes associated with cholesterol metabolism were assessed by RNA-Seq of murine hepatocyte cell line (AML12) and mouse primary hepatocytes (MPH) treated with TGF-{beta}1. Functional assays were performed on AML12 cells (untreated, TGF-{beta}1 treated, or subjected to cholesterol enrichment (CE) or depletion (CD)), and on mice injected with adeno-associated virus 8 (AAV8)-Control/TGF-{beta}1. Results: TGF-{beta}1 inhibited mRNA expression of several cholesterol metabolism regulatory genes, including rate-limiting enzymes of cholesterol biosynthesis in AML12 cells, MPHs, and AAV8-TGF-{beta}1-treated mice. Total cholesterol levels and lipid droplet accumulation in AML12 cells and liver tissue were also reduced upon TGF-{beta}1 treatment. Smad2/3 phosphorylation following 2 h TGF-{beta}1 treatment persisted after CE or CD and was mildly increased following CD, while TGF-{beta}1-mediated AKT phosphorylation (30 min) was inhibited by CE. Furthermore, CE protected AML12 cells from several effects mediated by 72 h incubation with TGF-{beta}1, including EMT, actin polymerization, and apoptosis. CD mimicked the outcome of long term TGF- {beta}1 administration, an effect that was blocked by an inhibitor of the type I TGF-{beta} receptor. Additionally, the supernatant of CE- or CD-treated AML12 cells inhibited or promoted, respectively, the activation of LX-2 hepatic stellate cells. Conclusions: TGF-{beta}1 inhibits cholesterol metabolism while cholesterol attenuates TGF-{beta}1 downstream effects in hepatocytes.

cell biology↗

Insulin determines the effects of TGF-beta on HNF4alpha transcription and epithelial-to-mesenchymal transition in hepatocytes

To date, epithelial-to-mesenchymal transition (EMT) has been observed in cultured hepatocytes, but not in vivo. TGF-{beta} is supposed to initiate EMT in hepatocytes by inhibiting HNF4 through the SMAD2/3 complex. We report that TGF-{beta} does not directly inhibit HNF4, but contributes to its transcriptional regulation by SMAD2/3 recruiting acetyltransferase CBP/p300 to the HNF4 promoter. The recruitment of CBP/p300 is indispensable for C/EBPa binding, another essential requirement for constitutive HNF4 expression in hepatocytes. In contrast to the observed induction of HNF4, SMAD2/3 inhibits C/EBP transcription. Therefore, long-term TGF-{beta} incubation results in C/EBP depletion, which abrogates HNF4 expression. Intriguingly, SMAD2/3 inhibitory binding to the C/EBP promoter is abolished by insulin. Thus, maintaining a high insulin concentration in culture medium ensures constitutive HNF4 and thereby prevents TGF-{beta}-induced hepatocyte EMT. Furthermore, insulin inhibits TGF-{beta}-induced SMAD2/3 binding to the promoters of core EMT transcription factors e.g., SNAI1. SNAI1 transcription requires both SMAD2/3 and FOXO1 in nuclei. Insulin inhibits SNAI1 transcription through impeding SMAD2/3 binding to its promoter and inducing FOXO1 phosphorylation. Hence, insulin is the key factor that prevents TGF-{beta}-induced EMT in hepatocytes.

cell biology↗

Training induced improvements in knee extensor force accuracy are associated with reduced vastus lateralis motor unit firing variability

BackgroundMuscle force output during sustained submaximal isometric contractions fluctuates around an average value and is known to be influenced by variation in motor unit (MU) firing rates. MU firing rate variability seemingly reduces following exercise training interventions, however, much less is known with respect to peripheral MU properties. We therefore investigated whether targeted force accuracy training could lead to improved muscle functional capacity and control, in addition to determining any alterations of individual MU features. MethodsTen healthy participants (7 females, 3 males, 27{+/-}6 years, 170{+/-}8 cm, 69{+/-}16kg) underwent a 4-week supervised, unilateral, force accuracy training intervention. The coefficient of variation for force (FORCECoV) and sinusoidal wave force tracking accuracy (FORCESinu) were determined at 25% maximal voluntary contraction (MVC) pre- and post-training. Intramuscular electromyography was utilised to record individual MU potentials from the vastus lateralis (VL) muscles at 25% MVC during sustained contractions, pre- and post-training. ResultsKnee extensor muscle strength remained unchanged following training, with no improvements in unilateral leg-balance. FORCECoV and FORCESinu significantly improved in only the trained knee extensors by ~13% (p=0.01) and ~30% (p<0.0001) respectively. MU firing rate variability significantly reduced in the trained VL by ~16% (n=8; p=0.001), with no further alterations to MU firing rate or neuromuscular junction transmission instability. ConclusionOur results suggest muscle force control and tracking accuracy is a trainable characteristic in the knee extensors, which is likely explained by the reduction in MU firing rate variability apparent in the trained limb only.

physiology↗

Repeated toxic injuries of murine liver are tolerated through microsteatosis and mild inflammation

The liver has a remarkable capacity to regenerate and thus compensates for repeated injuries through toxic chemicals, drugs, alcohol or malnutrition for decades. However, largely unknown is how and when alterations in the liver occur due to tolerable damaging insults. To that end, we induced repeated liver injuries over ten weeks in a mouse model injecting carbon tetrachloride (CCl4) twice a week. We lost 10% of the study animals within the first six weeks, which was accompanied by a steady deposition of extracellular matrix (ECM) regardless of metabolic activity of the liver. From week six onwards, all mice survived, and in these mice ECM deposition was rather reduced, suggesting ECM remodeling as a liver response contributing to better coping with repeated injuries. The data of time-resolved paired transcriptome and proteome profiling of 18 mice was subjected to multi-level network inference, using Knowledge guided Multi-Omics Network inference (KiMONo), identified multi-level key markers exclusively associated with the injury-tolerant liver response. Interestingly, pathways of cancer and inflammation were lighting up and were validated using independent data sets compiled of 1034 samples from publicly available human cohorts. A yet undescribed link to lipid metabolism in this damage-tolerant phase was identified. Immunostaining revealed an unexpected accumulation of small lipid droplets (microvesicular steatosis) in parallel to a recovery of catabolic processes of the liver to pre-injury levels. Further, mild inflammation was experimentally validated. Taken together, we identified week six as a critical time point to switch the liver response program from an acute response that fosters ECM accumulation to a tolerant "survival" phase with pronounced deposition of small lipid droplets in hepatocytes potentially protecting against the repetitive injury with toxic chemicals. Our data suggest that microsteatosis formation plus a mild inflammatory state represent biomarkers and probably functional liver requirements to resist chronic damage. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=163 HEIGHT=200 SRC="FIGDIR/small/476054v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@11ecc7aorg.highwire.dtl.DTLVardef@1027df5org.highwire.dtl.DTLVardef@9ba0f7org.highwire.dtl.DTLVardef@164f80a_HPS_FORMAT_FIGEXP M_FIG C_FIG The datasets generated via transcriptomics, proteomics as well as blood, histopathological and biochemical analysis were analyzed in an independent and integrative manner. The independent analysis was performed via state-of-the-art statistical approaches i.e. differential and consistently regulated genes and proteins. Combining the results identified three fibrosis phases. Using the KiMONo algorithm, a fibrosis specific multi-omic network was inferred. Within this network we identified several nodes connecting phase III specific features forming 13 distinct multi-omic modules suggesting a tolerance scheme. Some of these modules were experimentally validated and compared to 11 independent human studies of various liver diseases.

systems biology↗