The Carboxyl-Terminal Amino Acids Render Pro-Human LC3B Migration Similar to Lipidated LC3B in SDS-PAGE

LC3 is widely used marker for macroautophagy assays. After translation pro-LC3 is processed by Atg4 to expose C-terminal glycine residue for downstream conjugation reactions to accomplish the conversion of LC3-I to LC3-II. SDS-PAGE based Western blot (Wb) is generally utilized to quantify LC3-II levels where the LC3-I band migrates slower than LC3-II. We found that pro-human LC3B migrated at similar rate as LC3B-II in SDS-PAGE. The carboxyl-terminal five amino acids, particularly Lysine122 and Leucine123 of human LC3B play a major role in the faster migration of unprocessed LC3B, rendering it indistinguishable from LC3B-II in Wb assays. The unique faster migration of unprocessed LC3B than LC3B-I is also revealed in mouse LC3B, rat LC3B and rat LC3 but not in human LC3C. Our findings for the first time define pro-LC3 migration patterns for LC3 family member from human, mouse and rat species in SDS-PAGE. These findings provide a reference for pro-LC3 band patterns when Atg4 function is inhibited. © 2013 Wang et al

Displacement Across a Fracture Gap with Axial Loading of Far Cortical Locking Constructs

Purpose: Far cortical locking has been proposed for reducing stiffness and promoting greater dynamic stability in locked plating constructs. Prior studies have shown reduced stiffness with axial loading of these constructs, leading to a theoretical increase in inter-fragmentary motion and secondary bone healing. The purpose of this study was to examine strain across a fracture gap using far cortical locking constructs in a biomechanical model of distal femoral fractures. Methods: Fourth generation sawbones were cut transversely along the distal diaphysis and plated with distal femoral buttress plates and cortical locking screws. Far cortical locking (FCL) specimens were predrilled in the lateral cortex and control specimens were plated with a standard locked plating construct. The constructs were loaded sequentially with 100, 200, and 400 lbs of force on a mechanical test frame. Displacement across the fracture gap measured in pixels using an optical system. Results: Strain across the fracture gap increased with progressive loading from zero to 400 lbs in both groups. Strain also decreased in a linear fashion from medial to lateral across the fracture gap in both constructs (Figure 1). Standard locking constructs exhibited an average 28% greater strain than the far cortical locking constructs at all loading forces. Control specimens exhibited greater lateral displacement of the distal segment relative to the plate (Figure 2), consistent with higher shear forces compared to FCL specimens. Conclusions: In all specimens, there was considerable strain seen with loading that increased in characteristic fashion from lateral to medial. Overall, FCL constructs exhibited both lower strain, and importantly, lower shear, than measured in controls. This biomechanical model suggests that FCL changes loading across the femoral diaphysis in complex ways, and that assumptions about strain approaching zero on the lateral side of the distal femur with conventional locking or FCL may be incorrect

The HMGB1/RAGE inflammatory pathway promotes pancreatic tumor growth by regulating mitochondrial bioenergetics

Tumor cells require increased adenosine triphosphate (ATP) to support anabolism and proliferation. The precise mechanisms regulating this process in tumor cells are unknown. Here, we show that the receptor for advanced glycation endproducts (RAGE) and one of its primary ligands, high-mobility group box 1 (HMGB1), are required for optimal mitochondrial function within tumors. We found that RAGE is present in the mitochondria of cultured tumor cells as well as primary tumors. RAGE and HMGB1 coordinately enhanced tumor cell mitochondrial complex I activity, ATP production, tumor cell proliferation and migration. Lack of RAGE or inhibition of HMGB1 release diminished ATP production and slowed tumor growth in vitro and in vivo. These findings link, for the first time, the HMGB1-RAGE pathway with changes in bioenergetics. Moreover, our observations provide a novel mechanism within the tumor microenvironment by which necrosis and inflammation promote tumor progression

Effect of rejection on electrophysiologic function of canine intestinal grafts: Correlation with histopathology and na-k-ATPase activity

To investigate whether electrophysiologic changes can detect the early onset and progress of intestinal rejection, changes in in vitro electrophysiologic function, intestinal histopathology, and Na-K-ATPase activity were studied in dogs. Adult mongrel dogs of both sexes, weighing 18-24 kg, were used for auto and allo small bowel transplantation. The entire small bowels, except for short segments at the proximal and distal ends, were snitched between a pair of dogs (allograft). Animals receiving intestinal autotransplantation were used as controls. AIIograji recipients were sacrificed 3, 4, 5, 7, or 9 days after transplantation, and autograft recipients were sacrificed 3, 7, or 14 days afier transplantation. Immunosuppression was not used. Electrophysiologic measurements were done with an Ussing chamber. Histological analysis was performed blindly using whole thickness sections. Na-K-ATPase activity in the mucosal tissue, which is said to regulate the potential difference, was also measured. Potential difference, resistance, and Na-K-ATPase activity of the allografi intestine decreased with time and were significantly lower 7 and 9 days after transplantation compared to host intestine, normul intestine, and graft intestine of controls (autograft). Potential difference, resistance, and Na-K-ATPase activity of the native intestinal tissue and the autografts did not decrease with time. Detection of histologically mild rejection of the intestine, which is important for appropriate immunosup-pressive treatment in clinical cases, could not be achieved based on electrophysiology or Na-K-ATPase activity. Deterioration of electrophysiologic function during rejection correlated with the histological rejection process and Na-K-ATPase activity; however, electrophysiology my not be a reliable tool for monitoring grafrs, since it cannot detect early intestinal rejection. © 1995 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted

PKM2 regulates the Warburg effect and promotes HMGB1 release in sepsis

Increasing evidence suggests the important role of metabolic reprogramming in the regulation of the innate inflammatory response, but the underlying mechanism remains unclear. Here we provide evidence to support a novel role for the pyruvate kinase M2 (PKM2)-mediated Warburg effect, namely aerobic glycolysis, in the regulation of high-mobility group box 1 (HMGB1) release. PKM2 interacts with hypoxia-inducible factor 1 alpha (HIF1 alpha) and activates the HIF-1 alpha-dependent transcription of enzymes necessary for aerobic glycolysis in macrophages. Knockdown of PKM2, HIF1 alpha and glycolysis-related genes uniformly decreases lactate production and HMGB1 release. Similarly, a potential PKM2 inhibitor, shikonin, reduces serum lactate and HMGB1 levels, and protects mice from lethal endotoxemia and sepsis. Collectively, these findings shed light on a novel mechanism for metabolic control of inflammation by regulating HMGB1 release and highlight the importance of targeting aerobic glycolysis in the treatment of sepsis and other inflammatory diseases

The HIV protease inhibitor saquinavir inhibits HMGB1 driven inflammation by targeting the interaction of cathepsin V with TLR4/MyD88

Extracellular HMGB1 (disulfide form), via activation of Toll-Like-Receptor (TLR4)-dependent signaling, is a strong driver of pathologic inflammation in both acute and chronic conditions. Identification of selective inhibitors of HMGB1-TLR4 signaling could offer novel therapies that selectively target proximal endogenous activators of inflammation. A cell-based screening strategy led us to identify first generation HIV-protease inhibitors (PI) as potential inhibitors of HMGB1-TLR4 driven cytokine production. Here we report, that the first-generation HIV-PI saquinavir (SQV), as well as a newly identified mammalian protease inhibitor STO33438 (334), potently block disulfide HMGB1 induced TLR4 activation, as assayed by the production of TNF-alpha by human monocyte-derived macrophages (THP-1). We further report on the identification of mammalian cathepsin V, a protease, as a novel target of these inhibitors. Cellular as well as recombinant protein studies show that the mechanism of action involves a direct interaction between cathepsin V with TLR4 and its adaptor protein MyD88. Treatment with SQV, 334, or the known cathepsin inhibitor SID26681509 (SID) significantly improved survival in murine models of sepsis and reduced liver damage following warm liver I/R, models both characterized by strong HMGB1-TLR4 driven pathology. The current study demonstrates a novel role for cathepsin V in TLR4 signaling and implicates cathepsin V as a novel target for first-generation HIV-PI compounds. The identification of cathepsin V as a target to block HMGB1-TLR4 driven inflammation could allow for a rapid transition of the discovery from the bench to the bedside

The nuclear factor HMGB1 mediates hepatic injury after murine liver ischemia-reperfusion

High-mobility group box 1 (HMGB1) is a nuclear factor that is released extracellularly as a late mediator of lethality in sepsis as well as after necrotic, but not apoptotic, death. Here we demonstrate that in contrast to the delayed role of HMGB1 in the systemic inflammation of sepsis, HMGB1 acts as an early mediator of inflammation and organ damage in hepatic ischemia reperfusion (I/R) injury. HMGB1 levels were increased during liver I/R as early as 1 h after reperfusion and then increased in a time-dependent manner up to 24 h. Inhibition of HMGB1 activity with neutralizing antibody significantly decreased liver damage after I/R, whereas administration of recombinant HMGB1 worsened I/R injury. Treatment with neutralizing antibody was associated with less phosphorylation of c-Jun NH2-terminal kinase and higher nuclear factor–κB DNA binding in the liver after I/R. Toll-like receptor 4 (TLR4)-defective (C3H/Hej) mice exhibited less damage in the hepatic I/R model than did wild-type (C3H/HeOuj) mice. Anti-HMGB1 antibody failed to provide protection in C3H/Hej mice, but successfully reduced damage in C3H/Ouj mice. Together, these results demonstrate that HMGB1 is an early mediator of injury and inflammation in liver I/R and implicates TLR4 as one of the receptors that is involved in the process

Cellular and Matrix Mechanics of Bioartificial Tissues During Continuous Cyclic Stretch

Bioartificial tissues are useful model systems for studying cell and extra-cellular matrix mechanics. These tissues provide a 3D environment for cells and allow tissue components to be easily modified and quantified. In this study, we fabricated bioartificial tissue rings from a 1 ml solution containing one million cardiac fibroblasts and 1 mg collagen. After 8 days, rings compacted to <1% of original volume and cell number increased 2.4 fold. We initiated continuous cyclic stretching of the rings after 2, 4, or 8 days of incubation, while monitoring the tissue forces. Peak tissue force during each cycle decreased rapidly after initiating stretch, followed by further slow decline. We added 2 μM Cytochalasin-D to some rings prior to initiation of stretch to determine the force contributed by the matrix. Cell force was estimated by subtracting matrix force from tissue force. After 12 h, matrix force-strain curves were highly nonlinear. Cell force-strain curves were linear during loading and showed hysteresis indicating viscoelastic behavior. Cell stiffness increased with stretching frequency from 0.001–0.25 Hz. Cell stiffness decreased with stretch amplitude (5–25%) at 0.1 Hz. The trends in cell stiffness do not fit simple viscoelastic models previously proposed, and suggest possible strain-amplitude related changes during cyclic stretch