Mechanical Intercellular Communication via Matrix-Borne Cell Force Transmission During Vascular Network Formation.

Intercellular communication is critical to the formation and homeostatic function of all tissues. Previous work has shown that cells can communicate mechanically via the transmission of cell-generated forces through their surrounding extracellular matrix, but this process is not well understood. Here, mechanically defined, synthetic electrospun fibrous matrices are utilized in conjunction with a microfabrication-based cell patterning approach to examine mechanical intercellular communication (MIC) between endothelial cells (ECs) during their assembly into interconnected multicellular networks. It is found that cell force-mediated matrix displacements in deformable fibrous matrices underly directional extension and migration of neighboring ECs toward each other prior to the formation of stable cell-cell connections enriched with vascular endothelial cadherin (VE-cadherin). A critical role is also identified for calcium signaling mediated by focal adhesion kinase and mechanosensitive ion channels in MIC that extends to multicellular assembly of 3D vessel-like networks when ECs are embedded within fibrin hydrogels. These results illustrate a role for cell-generated forces and ECM mechanical properties in multicellular assembly of capillary-like EC networks and motivates the design of biomaterials that promote MIC for vascular tissue engineering.

Matrix architecture and mechanics regulate myofibril organization, costamere assembly, and contractility of engineered myocardial microtissues.

The mechanical function of the myocardium is defined by cardiomyocyte contractility and the biomechanics of the extracellular matrix (ECM). Understanding this relationship remains an important unmet challenge due to limitations in existing approaches for engineering myocardial tissue. Here, we established arrays of cardiac microtissues with tunable mechanics and architecture by integrating ECM-mimetic synthetic, fiber matrices and induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), enabling real-time contractility readouts, in-depth structural assessment, and tissue-specific computational modeling. We find that the stiffness and alignment of matrix fibers distinctly affect the structural development and contractile function of pure iPSC-CM tissues. Further examination into the impact of fibrous matrix stiffness enabled by computational models and quantitative immunofluorescence implicates cell-ECM interactions in myofibril assembly and notably costamere assembly, which correlates with improved contractile function of tissues. These results highlight how iPSC-CM tissue models with controllable architecture and mechanics can inform the design of translatable regenerative cardiac therapies.

Direct comparison of angiogenesis in natural and synthetic biomaterials reveals that matrix porosity regulates endothelial cell invasion speed and sprout diameter.

Vascularization of large, diffusion-hindered biomaterial implants requires an understanding of how extracellular matrix (ECM) properties regulate angiogenesis. Sundry biomaterials assessed across many disparate angiogenesis assays have highlighted ECM determinants that influence this complex multicellular process. However, the abundance of material platforms, each with unique parameters to model endothelial cell (EC) sprouting presents additional challenges of interpretation and comparison between studies. In this work we directly compared the angiogenic potential of commonly utilized natural (collagen and fibrin) and synthetic dextran vinyl sulfone (DexVS) hydrogels in a multiplexed angiogenesis-on-a-chip platform. Modulating matrix density of collagen and fibrin hydrogels confirmed prior findings that increases in matrix density correspond to increased EC invasion as connected, multicellular sprouts, but with decreased invasion speeds. Angiogenesis in synthetic DexVS hydrogels, however, resulted in fewer multicellular sprouts. Characterizing hydrogel Young's modulus and permeability (a measure of matrix porosity), we identified matrix permeability to significantly correlate with EC invasion depth and sprout diameter. Although microporous collagen and fibrin hydrogels produced lumenized sprouts in vitro, they rapidly resorbed post-implantation into the murine epididymal fat pad. In contrast, DexVS hydrogels proved comparatively stable. To enhance angiogenesis within DexVS hydrogels, we incorporated sacrificial microgels to generate cell-scale pores throughout the hydrogel. Microporous DexVS hydrogels resulted in lumenized sprouts in vitro and enhanced cell invasion in vivo. Towards the design of vascularized biomaterials for long-term regenerative therapies, this work suggests that synthetic biomaterials offer improved size and shape control following implantation and that tuning matrix porosity may better support host angiogenesis. STATEMENT OF SIGNIFICANCE: Understanding how extracellular matrix properties govern angiogenesis will inform biomaterial design for engineering vascularized implantable grafts. Here, we utilized a multiplexed angiogenesis-on-a-chip platform to compare the angiogenic potential of natural (collagen and fibrin) and synthetic dextran vinyl sulfone (DexVS) hydrogels. Characterization of matrix properties and sprout morphometrics across these materials points to matrix porosity as a critical regulator of sprout invasion speed and diameter, supported by the observation that nanoporous DexVS hydrogels yielded endothelial cell sprouts that were not perfusable. To enhance angiogenesis into synthetic hydrogels, we incorporated sacrificial microgels to generate microporosity. We find that microporosity increased sprout diameter in vitro and cell invasion in vivo. This work establishes a composite materials approach to enhance the vascularization of synthetic hydrogels.

Engineered Extracellular Matrices with Integrated Wireless Microactuators to Study Mechanobiology.

Mechanobiology explores how forces regulate cell behaviors and what molecular machinery are responsible for the sensing, transduction, and modulation of mechanical cues. To this end, probing of cells cultured on planar substrates has served as a primary experimental setting for many decades. However, native extracellular matrices (ECMs) consist of fibrous protein assemblies where the physical properties spanning from the individual fiber to the network architecture can influence the transmission of forces to and from the cells. Here, a robotic manipulation platform that allows wireless, localized, and programmable deformation of an engineered fibrous ECM is introduced. A finite-element-based digital twin of the fiber network calibrated against measured local and global parameters enables the calculation of deformations and stresses generated by different magnetic actuation schemes across a range of network properties. Physiologically relevant mechanical forces are applied to cells cultured on the fiber network, statically or dynamically, revealing insights into the effects of matrix-borne forces and deformations as well as force-mediated matrix remodeling on cell migration and intracellular signaling. These capabilities are not matched by any existing approach, and this versatile platform has the potential to uncover fundamental mechanisms of mechanobiology in settings with greater relevance to living tissues.

Phylogenetics and comparative plastome genomics of two of the largest genera of angiosperms, Piper and Peperomia (Piperaceae).

Biological radiations provide unique opportunities to understand the evolution of biodiversity. One such radiation is the pepper plant family Piperaceae, an early-diverging and mega-diverse lineage that could serve as a model to study the diversification of angiosperms. However, traditional genetic markers lack sufficient variation for such studies, and testing hypotheses on poorly resolved phylogenetic frameworks becomes challenging. Limited genomic data is available for Piperaceae, which contains two of the largest genera of angiosperms, Piper (>2100 species) and Peperomia (>1300 species). To address this gap, we used genome skimming to assemble and annotate whole plastomes (152-161kbp) and >5kbp nuclear ribosomal DNA region from representatives of Piper and Peperomia. We conducted phylogenetic and comparative genomic analyses to study plastome evolution and investigate the role of hybridization in this group. Plastome phylogenetic trees were well resolved and highly supported, with a hard incongruence observed between plastome and nuclear phylogenetic trees suggesting hybridization in Piper. While all plastomes of Piper and Peperomia had the same gene content and order, there were informative structural differences between them. First, ycf1 was more variable and longer in Piper than Peperomia, extending well into the small single copy region by thousands of base pairs. We also discovered previously unknown structural variation in 14 out of 25 Piper taxa, tandem duplication of the trnH-GUG gene resulting in an expanded large single copy region. Other early-diverging angiosperms have a duplicated trnH-GUG, but the specific rearrangement we found is unique to Piper and serves to refine knowledge of relationships among early-diverging angiosperms. Our study demonstrates that genome skimming is an efficient approach to produce plastome assemblies for comparative genomics and robust phylogenies of species-rich plant genera.

Microenvironmental determinants of organized iPSC-cardiomyocyte tissues on synthetic fibrous matrices.

Cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) show great potential for engineering myocardium to study cardiac disease and create regenerative therapies. However, iPSC-CMs typically possess a late embryonic stage phenotype, with cells failing to exhibit markers of mature adult tissue. This is due in part to insufficient knowledge and control of microenvironmental cues required to facilitate the organization and maturation of iPSC-CMs. Here, we employed a cell-adhesive, mechanically tunable synthetic fibrous extracellular matrix (ECM) consisting of electrospun dextran vinyl sulfone (DVS) fibers and examined how biochemical, architectural, and mechanical properties of the ECM impact iPSC-CM tissue assembly and subsequent function. Exploring a multidimensional parameter space spanning cell-adhesive ligand, seeding density, fiber alignment, and stiffness, we found that fibronectin-functionalized DVS matrices composed of highly aligned fibers with low stiffness optimally promoted the organization of functional iPSC-CM tissues. Tissues generated on these matrices demonstrated improved calcium handling and increased end-to-end localization of N-cadherin as compared to micropatterned fibronectin lines or fibronectin-coated glass. Furthermore, DVS matrices supported long-term culture (45 days) of iPSC-CMs; N-cadherin end-to-end localization and connexin43 expression both increased as a function of time in culture. In sum, these findings demonstrate the importance of recapitulating the fibrous myocardial ECM in engineering structurally organized and functional iPSC-CM tissues.

Microengineered 3D pulmonary interstitial mimetics highlight a critical role for matrix degradation in myofibroblast differentiation.

Fibrosis, characterized by aberrant tissue scarring from activated myofibroblasts, is often untreatable. Although the extracellular matrix becomes increasingly stiff and fibrous during disease progression, how these physical cues affect myofibroblast differentiation in 3D is poorly understood. Here, we describe a multicomponent hydrogel that recapitulates the 3D fibrous structure of interstitial tissue regions where idiopathic pulmonary fibrosis (IPF) initiates. In contrast to findings on 2D hydrogels, myofibroblast differentiation in 3D was inversely correlated with hydrogel stiffness but positively correlated with matrix fibers. Using a multistep bioinformatics analysis of IPF patient transcriptomes and in vitro pharmacologic screening, we identify matrix metalloproteinase activity to be essential for 3D but not 2D myofibroblast differentiation. Given our observation that compliant degradable 3D matrices amply support fibrogenesis, these studies demonstrate a departure from the established relationship between stiffness and myofibroblast differentiation in 2D, and provide a new 3D model for studying fibrosis and identifying antifibrotic therapeutics.

Fiber Crimp Confers Matrix Mechanical Nonlinearity, Regulates Endothelial Cell Mechanosensing, and Promotes Microvascular Network Formation.

Mechanical interactions between cells and their surrounding extracellular matrix (ECM) guide many fundamental cell behaviors. Native connective tissue consists of highly organized, 3D networks of ECM fibers with complex, nonlinear mechanical properties. The most abundant stromal matrix component is fibrillar type I collagen, which often possesses a wavy, crimped morphology that confers strain- and load-dependent nonlinear mechanical behavior. Here, we established a new and simple method for engineering electrospun fibrous matrices composed of dextran vinyl sulfone (DexVS) with controllable crimped structure. A hydrophilic peptide was functionalized to DexVS matrices to trigger swelling of individual hydrogel fibers, resulting in crimped microstructure due to the fixed anchorage of fibers. Mechanical characterization of these matrices under tension confirmed orthogonal control over nonlinear stress-strain responses and matrix stiffness. We next examined ECM mechanosensing of individual endothelial cells (ECs) and found that fiber crimp promoted physical matrix remodeling alongside decreases in cell spreading, focal adhesion area, and nuclear localization of Yes-associated protein (YAP). These changes corresponded to an increase in migration speed, along with evidence for long-range interactions between neighboring cells in crimped matrices. Interestingly, when ECs were seeded at high density in crimped matrices, capillary-like networks rapidly assembled and contained tube-like cellular structures wrapped around bundles of synthetic matrix fibers due to increased physical reorganization of matrix fibers. Our work provides an additional level of mechanical and architectural tunability to synthetic fibrous matrices and implicates a critical role for mechanical nonlinearity in EC mechanosensing and network formation.

Spatiotemporal control of micromechanics and microstructure in acoustically-responsive scaffolds using acoustic droplet vaporization.

Acoustically-responsive scaffolds (ARSs), which are composite fibrin hydrogels, have been used to deliver regenerative molecules. ARSs respond to ultrasound in an on-demand, spatiotemporally-controlled manner via a mechanism termed acoustic droplet vaporization (ADV). Here, we study the ADV-induced, time-dependent micromechanical and microstructural changes to the fibrin matrix in ARSs using confocal fluorescence microscopy as well as atomic force microscopy. ARSs, containing phase-shift double emulsion (PSDE, mean diameter: 6.3 µm), were exposed to focused ultrasound to generate ADV - the phase transitioning of the PSDE into gas bubbles. As a result of ADV-induced mechanical strain, localized restructuring of fibrin occurred at the bubble-fibrin interface, leading to formation of locally denser regions. ADV-generated bubbles significantly reduced fibrin pore size and quantity within the ARS. Two types of ADV-generated bubble responses were observed in ARSs: super-shelled spherical bubbles, with a growth rate of 31 µm per day in diameter, as well as fluid-filled macropores, possibly as a result of acoustically-driven microjetting. Due to the strain stiffening behavior of fibrin, ADV induced a 4-fold increase in stiffness in regions of the ARS proximal to the ADV-generated bubble versus distal regions. These results highlight that the mechanical and structural microenvironment within an ARS can be spatiotemporally modulated using ultrasound, which could be used to control cellular processes and further the understanding of ADV-triggered drug delivery for regenerative applications.

Myofibroblast activation in synthetic fibrous matrices composed of dextran vinyl sulfone.

Mechanical interactions between fibroblasts and their surrounding extracellular matrix (ECM) guide fundamental behaviors such as spreading, migration, and proliferation that underlie disease pathogenesis. The challenges of studying ECM mechanics in vivo have motivated the development of in vitro models of the fibrous ECM in which fibroblasts reside. Natural materials such as collagen hydrogels bear structural and biochemical resemblance to stromal ECM, but mechanistic studies in these settings are often confounded by cell-mediated material degradation and the lack of structural and mechanical tunability. Here, we established a new material system composed of electrospun dextran vinyl sulfone (DexVS) polymeric fibers. These fibrous matrices exhibit mechanical tunability at both the single fiber (80-340 MPa) and bulk matrix (0.77-11.03 kPa) level, as well as long-term stability in mechanical properties over a two-week period. Cell adhesion to these matrices can be either user-defined by functionalizing synthetic fibers with thiolated adhesive peptides or methacrylated heparin to sequester cell-derived ECM proteins. We utilized DexVS fibrous matrices to investigate the role of matrix mechanics on the activation of fibroblasts into myofibroblasts, a key step of the fibrotic progression. In contrast to previous findings with non-fibrous hydrogel substrates, we find that fibroblasts in soft and deformable matrices exhibit increased spreading, focal adhesion formation, proliferation, and myofibroblast activation as compared to cells on stiffer matrices with equivalent starting architecture. STATEMENT OF SIGNIFICANCE: Cellular mechanosensing of fibrillar extracellular matrices plays a critical role in homeostasis and disease progression in stromal connective tissue. Here, we established a new material system composed of electrospun dextran vinyl sulfone polymeric fibers. These matrices exhibit architectural, mechanical, and biochemical tunability to accurately model diverse tissue microenvironments found in the body. In contrast to previous observations with non-fibrous hydrogels, we find that fibroblasts in soft and deformable fibrous matrices exhibit increased spreading and focal adhesion formation as compared to those in stiffer matrices with equivalent architecture. We also investigated the role of matrix stiffness on myofibroblast activation, a critical step in the fibrotic cascade, and find that low stiffness matrices promote increased myofibroblast activation.

Exercise Mediated Nrf2 Signaling Protects the Myocardium From Isoproterenol-Induced Pathological Remodeling.

Although exercise derived activation of Nrf2 signaling augments myocardial antioxidant signaling, the molecular mechanisms underlying the benefits of moderate exercise training (MET) in the heart remain elusive. Here we hypothesized that exercise training stabilizes Nrf2-dependent antioxidant signaling, which then protects the myocardium from isoproterenol-induced damage. The present study assessed the effects of 6 weeks of MET on the Nrf2/antioxidant function, glutathione redox state, and injury in the myocardium of C57/BL6J mice that received isoproterenol (ISO; 50 mg/kg/day for 7 days). ISO administration significantly reduced the Nrf2 promoter activity (p < 0.05) and downregulated the expression of cardiac antioxidant genes (Gclc, Nqo1, Cat, Gsr, and Gst-µ) in the untrained (UNT) mice. Furthermore, increased oxidative stress with severe myocardial injury was evident in UNT+ISO when compared to UNT mice receiving PBS under basal condition. Of note, MET stabilized the Nrf2-promoter activity and upheld the expression of Nrf2-dependent antioxidant genes in animals receiving ISO, and attenuated the oxidative stress-induced myocardial damage. Echocardiography analysis revealed impaired diastolic ventricular function in UNT+ISO mice, but this was partially normalized in the MET animals. Interestingly, while there was a marginal reduction in ubiquitinated proteins in MET mice that received ISO, the pathological signs were attenuated along with near normal cardiac function in response to exercise training. Thus, moderate intensity exercise training conferred protection against ISO-induced myocardial injury by augmentation of Nrf2-antioxidant signaling and attenuation of isoproterenol-induced oxidative stress.

Actomyosin contractility-dependent matrix stretch and recoil induces rapid cell migration.

Cells select from a diverse repertoire of migration strategies. Recent developments in tunable biomaterials have helped identify how extracellular matrix properties influence migration, however, many settings lack the fibrous architecture characteristic of native tissues. To investigate migration in fibrous contexts, we independently varied the alignment and stiffness of synthetic 3D fiber matrices and identified two phenotypically distinct migration modes. In contrast to stiff matrices where cells migrated continuously in a traditional mesenchymal fashion, cells in deformable matrices stretched matrix fibers to store elastic energy; subsequent adhesion failure triggered sudden matrix recoil and rapid cell translocation. Across a variety of cell types, traction force measurements revealed a relationship between cell contractility and the matrix stiffness where this migration mode occurred optimally. Given the prevalence of fibrous tissues, an understanding of how matrix structure and mechanics influences migration could improve strategies to recruit repair cells to wound sites or inhibit cancer metastasis.

Cell force-mediated matrix reorganization underlies multicellular network assembly.

Vasculogenesis is the de novo formation of a vascular network from individual endothelial progenitor cells occurring during embryonic development, organogenesis, and adult neovascularization. Vasculogenesis can be mimicked and studied in vitro using network formation assays, in which endothelial cells (ECs) spontaneously form capillary-like structures when seeded in the appropriate microenvironment. While the biochemical regulators of network formation have been well studied using these assays, the role of mechanical and topographical properties of the extracellular matrix (ECM) is less understood. Here, we utilized both natural and synthetic fibrous materials to better understand how physical attributes of the ECM influence the assembly of EC networks. Our results reveal that active cell-mediated matrix recruitment through actomyosin force generation occurs concurrently with network formation on Matrigel, a reconstituted basement membrane matrix regularly used to promote EC networks, and on synthetic matrices composed of electrospun dextran methacrylate (DexMA) fibers. Furthermore, modulating physical attributes of DexMA matrices that impair matrix recruitment consequently inhibited the formation of cellular networks. These results suggest an iterative process in which dynamic cell-induced changes to the physical microenvironment reciprocally modulate cell behavior to guide the formation and stabilization of multicellular networks.

Guidelines on the management of atrial fibrillation in the emergency department: a critical appraisal.

Several guidelines often exist on the same topic, sometimes offering divergent recommendations. For the clinician, it can be difficult to understand the reasons for this divergence and how to select the right recommendations. The aim of this study is to compare different guidelines on the management of atrial fibrillation (AF), and provide practical and affordable advice on its management in the acute setting. A PubMed search was performed in May 2014 to identify the three most recent and cited published guidelines on AF. During the 1-week school of the European School of Internal Medicine, the attending residents were divided in five working groups. The three selected guidelines were compared with five specific questions. The guidelines identified were: the European Society of Cardiology guidelines on AF, the Canadian guidelines on emergency department management of AF, and the American Heart Association guidelines on AF. Twenty-one relevant sub-questions were identified. For five of these, there was no agreement between guidelines; for three, there was partial agreement; for three data were not available (issue not covered by one of the guidelines), while for ten, there was complete agreement. Evidence on the management of AF in the acute setting is largely based on expert opinion rather than clinical trials. While there is broad agreement on the management of the haemodynamically unstable patient and the use of drugs for rate-control strategy, there is less agreement on drug therapy for rhythm control and no agreement on several other topics.

The Emergence of Earliest Angiosperms may be Earlier than Fossil Evidence Indicates.

Gaps between molecular ages and fossils undermine the validity of time-calibrated molecular phylogenies. An example of the time gap surrounds the age of angiosperm's origin. We calculate molecular ages of the earliest flowering plant lineages using 22 fossil calibrations (101 genera, 40 families). Our results reveal the origin of angiosperms at the late Permian, ~275 million years ago. Different prior probability curves of molecular age calculations on dense calibration point distributions had little effect on overall age estimates compared to the effects of altered calibration points. The same is true for reasonable root age constraints. We conclude that our age estimates based on multiple datasets, priors, and calibration points are robust and the true ages are likely between our extremes. Our results, when integrated with the ecophysiological evolution of early angiosperms, imply that the ecology of the earliest angiosperms is critical to understand the pre-Cretaceous evolution of flowering plants.

A biphasic effect of TNF-α in regulation of the Keap1/Nrf2 pathway in cardiomyocytes.

Antagonizing TNF-α signaling attenuates chronic inflammatory disease, but is associated with adverse effects on the cardiovascular system. Therefore the impact of TNF-α on basal control of redox signaling events needs to be understand in more depth. This is particularly important for the Keap1/Nrf2 pathway in the heart and in the present study we hypothesized that inhibition of a low level of TNF-α signaling attenuates the TNF-α dependent activation of this cytoprotective pathway. HL-1 cardiomyocytes and TNF receptor1/2 (TNFR1/2) double knockout mice (DKO) were used as experimental models. TNF-α (2-5ng/ml, for 2h) evoked significant nuclear translocation of Nrf2 with increased DNA/promoter binding and transactivation of Nrf2 targets. Additionally, this was associated with a 1.5 fold increase in intracellular glutathione (GSH). Higher concentrations of TNF-α (>10-50ng/ml) were markedly suppressive of the Keap1/Nrf2 response and associated with cardiomyocyte death marked by an increase in cleavage of caspase-3 and PARP. In vivo experiments with TNFR1/2-DKO demonstrates that the expression of Nrf2-regulated proteins (NQO1, HO-1, G6PD) were significantly downregulated in hearts of the DKO when compared to WT mice indicating a weakened antioxidant system under basal conditions. Overall, these results indicate that TNF-α exposure has a bimodal effect on the Keap1/Nrf2 system and while an intense inflammatory activation suppresses expression of antioxidant proteins a low level appears to be protective.

Surgical management of acute cholecystitis.

BACKGROUND: Acute cholecystitis occurs in approximately 1% of patients with known gallstones. It presents as a surgical emergency and usually requires hospitalisation for treatment. It is associated with significant morbidity and mortality, particularly in the elderly. Cholecystectomy is advocated for acute cholecystitis; however, the timing of cholecystectomy and the value of the additional treatments have been a matter of debate. This review examines the available evidence regarding the optimal surgical management of patients with acute cholecystitis. METHODS: A literature search was performed on the MEDLINE, EMBASE and WHO International Clinical Trials Registry Platform, databases for English language publications. The MeSH headings 'cholecystitis', 'acute', 'gallbladder', 'inflammation', 'surgery', 'cholecystectomy', 'laparoscopic', 'robotic', 'telerobotic' and 'computer-assisted' were used. RESULTS: Data from eight randomised controlled trails and three population-based analyses show that early cholecystectomy for acute cholecystitis performed on the index admission is safe and not associated with increased conversion rates or morbidity in comparison to conservative treatment followed by elective cholecystectomy. Delaying cholecystectomy increases readmissions for gallstone-related events, complications, hospital stay and mortality in the elderly. Early cholecystectomy is also more cost-effective. Randomised trials addressing antibiotic use in acute cholecystitis suggest that antibiotics should be stopped on the day of cholecystectomy. Insufficient trials have been performed to address the optimal analgesia regime post cholecystectomy. Similarly, a lack of trials on intraoperative cholangiography and management of common bile duct stones in patients with acute cholecystitis means that treatment of concomitant bile duct stones should be based on institutional expertise and resource availability. As regards acute cholecystitis in elderly and high-risk patients, case series and retrospective studies would suggest that cholecystectomy is more effective and of lower mortality than percutaneous cholecystostomy. There is not enough evidence to support the routine use of robotic surgery, single-incision laparoscopic cholecystectomy or natural orifice transluminal endoscopic surgery (NOTES) in the treatment of acute cholecystitis. CONCLUSIONS: Trial evidence would favour a policy of early laparoscopic cholecystectomy following admission with acute cholecystitis. The optimal approach to support early cholecystectomy is suggested but requires evidence from further randomised trials.

Nrf2 deficiency promotes apoptosis and impairs PAX7/MyoD expression in aging skeletal muscle cells.

Skeletal muscle redox homeostasis is transcriptionally regulated by nuclear erythroid-2-p45-related factor-2 (Nrf2). We recently demonstrated that age-associated stress impairs Nrf2-ARE (antioxidant-response element) transcriptional signaling. Here, we hypothesize that age-dependent decline or genetic ablation of Nrf2 leads to accelerated apoptosis and skeletal muscle degeneration. Under basal-physiological conditions, disruption of Nrf2 significantly downregulates antioxidants and causes oxidative stress. Surprisingly, Nrf2-null mice had enhanced antioxidant capacity identical to wild-type (WT) upon acute endurance exercise stress (AEES), suggesting activation of Nrf2-independent mechanisms (i.e., PGC1α) against oxidative stress. Analysis of prosurvival pathways in the basal state reveals decreased AKT levels, whereas p-p53, a repressor of AKT, was increased in Nrf2-null vs WT mice. Upon AEES, AKT and p-AKT levels were significantly (p < 0.001) increased (>10-fold) along with profound downregulation of p-p53 (p < 0.01) in Nrf2-null vs WT skeletal muscle, indicating the onset of prosurvival mechanisms to compensate for the loss of Nrf2 signaling. However, we found a decreased stem cell population (PAX7) and MyoD expression (differentiation) along with profound activation of ubiquitin and apoptotic pathways in Nrf2-null vs WT mice upon AEES, suggesting that compensatory prosurvival mechanisms failed to overcome the programmed cell death and degeneration in skeletal muscle. Further, the impaired regeneration was sustained in Nrf2-null vs WT mice after 1 week of post-AEES recovery. In an age-associated oxidative stress condition, ablation of Nrf2 results in induction of apoptosis and impaired muscle regeneration.