Cell confluence regulates claudin-2 expression: possible role for ZO-1 and Rac.

Claudin-2 (Cldn-2) is a channel-forming tight junction (TJ) protein in the proximal tubules that mediates paracellular Na+ transport and has also emerged as a regulator of proliferation and migration. Expression of Cldn-2 is altered by numerous stimuli, but the underlying mechanisms remain incompletely understood. Here we show that Cldn-2 protein and mRNA expression were low in subconfluent tubular cells and increased during junction maturation. Cldn-1 or occludin did not exhibit similar confluence-dependence. Conversely, disruption of TJs by Ca2+ removal or silencing of zonula occludens-1 (ZO-1) or ZO-2 induced a large drop in Cldn-2 abundance. Immunofluorescent staining revealed a more uneven Cldn-2 staining in nascent, Cldn-1-positive TJs. Subconfluence and ZO-1 silencing augmented Cldn-2 degradation and reduced Cldn-2 promoter activity, suggesting that insertion into the TJs slows Cldn-2 turnover. Indeed, blocking endocytosis or lysosomal degradation increased Cldn-2 abundance. Cell confluence increased expression of the junctional adapters ZO-1 and -2, and the small GTPase Rac, and elevated Rac activity and p21-activated kinase (Pak) phosphorylation, suggesting that they might mediate confluence-dependent Cldn-2 regulation. Indeed, Rac silencing or Pak inhibition strongly reduced Cldn-2 protein abundance, which was likely the combined effect on turnover, as these interventions reduced Cldn-2 promoter activity and augmented Cldn-2 degradation. Taken together, our data suggest that TJ integrity and maturity, ZO-1 expression/TJ localization, and Rac/Pak control Cldn-2 degradation and synthesis. A feedback mechanism connecting Cldn-2 expression with junction remodeling, e.g., during wound healing, epithelial-mesenchymal transition, or tumor metastasis formation, may have important downstream effects on permeability, proliferation, and migration.

Remote Ischemic Conditioning Prevents Lung and Liver Injury After Hemorrhagic Shock/Resuscitation: Potential Role of a Humoral Plasma Factor.

OBJECTIVE: To evaluate the efficacy of remote ischemic conditioning (RIC) on organ protection after hemorrhagic shock/resuscitation (S/R) in a murine model. BACKGROUND: Ischemia/reperfusion resulting from S/R contributes to multiple organ dysfunction in trauma patients. We hypothesized that RIC before shock (remote ischemic preconditioning), during shock (remote ischemic "PER"conditioning), or during resuscitation (remote ischemic "POST"conditioning) could confer organ protection. We also tested the effect of ischemic conditioned plasma on neutrophil migration in vivo using transgenic zebrafish models. METHODS: C57Bl/6 mice were subjected to S/R with or without hindlimb RIC. Serum levels of alanine aminotransferase and tumor necrosis factor-alpha, and liver tumor necrosis factor-alpha and interleukin 1ß mRNA were evaluated. In some experiments, lung protein leakage, cytokines, and myeloperoxidase activity were investigated. Plasma from mice subjected to RIC was microinjected into zebrafish, and neutrophil migration was assessed after tailfin transection or copper sulfate treatment. RESULTS: In mice subjected to S/R, remote ischemic preconditioning, remote ischemic "PER"conditioning, and remote ischemic "POST"conditioning each significantly reduced serum alanine aminotransferase and liver mRNA expression of tumor necrosis factor-alpha and interleukin 1ß and improved liver histology compared with control S/R mice. Lung injury and inflammation were also significantly reduced in mice treated with remote ischemic preconditioning. Zebrafish injected with plasma or dialyzed plasma (fraction >14 kDa) from ischemic conditioned mice had reduced neutrophil migration toward sites of injury compared with zebrafish injected with control plasma. CONCLUSIONS: RIC protects against S/R-induced organ injury, in part, through a humoral factor(s), which alters neutrophil function. The beneficial effects of RIC, performed during the S/R phase of care, suggest a role for its application early in the posttrauma period.

Augmented Parkin-dependent mitophagy underlies the hepatoprotective effect of remote ischemic conditioning used prior to hemorrhagic shock.

BACKGROUND AND AIMS: Hemorrhagic shock-resuscitation (HSR) following trauma contributes to organ dysfunction by causing ischemia-reperfusion injury (IRI). We previously showed that 'remote ischemic preconditioning' (RIPC) exerted multi-organ protection from IRI. Maintenance of mitochondrial quality by clearance of dysfunctional mitochondria via mitophagy is vital in restoring organ integrity. We hypothesized that parkin-dependent mitophagy played a role in RIPC-induced hepatoprotection following HSR. METHODS: The hepatoprotective effect of RIPC in a murine model of HSR-IRI was investigated in wild type and parkin-/- animals. Mice were subjected to HSR ± RIPC and blood and organs were collected, followed by cytokine ELISAs, histology, qPCR, Western blots, and transmission electron microscopy. RESULTS: HSR increased hepatocellular injury, as measured by plasma ALT and liver necrosis, while antecedent RIPC prevented this injury; in parkin-/- mice, RIPC failed to exert hepatoprotection. The ability of RIPC to lessen HSR-induced rises in plasma IL-6 and TNFα, was lost in parkin-/- mice. While RIPC alone did not induce mitophagy, the application of RIPC prior to HSR caused a synergistic increase in mitophagy, this increase was not observed in parkin-/- mice. RIPC induced shifts in mitochondrial morphology favoring mitophagy in WT but not in parkin-/- animals. CONCLUSIONS: RIPC was hepatoprotective in WT mice following HSR but not in parkin-/- mice. Loss of protection in parkin-/- mice corresponded with the failure of RIPC plus HSR to upregulate the mitophagic process. Improving mitochondrial quality by modulating mitophagy, may prove to be an attractive therapeutic target in disease processes caused by IRI.

ACTIVATION OF THE MITOCHONDRIAL ANTIVIRAL SIGNALING PROTEIN (MAVS) FOLLOWING LIVER ISCHEMIA/REPERFUSION AND ITS EFFECT ON INFLAMMATION AND INJURY.

ABSTRACT: Resuscitation of trauma patients after hemorrhagic shock causes global I/R, which may contribute to organ dysfunction. Oxidative stress resulting from I/R is known to induce signaling pathways leading to the production of inflammatory molecules culminating in organ dysfunction/injury. Our recent work demonstrated that oxidative stress was able to induce activation of the mitochondrial antiviral signaling protein (MAVS), a protein known to be involved in antiviral immunity, in an in vitro model. We therefore hypothesized that the MAVS pathway might be involved in I/R-induced inflammation and injury. The present studies show that MAVS is activated in vivo by liver I/R and in vitro in RAW 264.7 cells by hypoxia/reoxygenation (H/R). We utilized both in vivo (liver I/R in MAVS knockout mice) and in vitro (MAVS siRNA in RAW 264.7 cells followed by H/R) models to study the role of MAVS activation on downstream events. In vivo , we demonstrated augmented injury and inflammation in MAVS knockout mice compared with wild-type animals; as shown by increased hepatocellular injury, induction of hepatocyte apoptosis augmented plasma TNF-α levels. Further, in vitro silencing of MAVS by specific siRNA in RAW 264.7 and exposure of the cells to H/R caused activation of mitophagy. This may represent a compensatory response to increased liver inflammation. We conclude that activation of MAVS by hypoxia/reoxygenation dampens inflammation, potentially suggesting a novel target for intervention.

Improved SARS-CoV-2 PCR detection and genotyping with double-bubble primers.

A new approach for improved RT-PCR is described. It is based on primers designed to form controlled stem-loop and homodimer configurations, hence the name 'double-bubble' primers. The primers contain three main regions for efficient RT-PCR: a 3' short overhang to allow reverse transcription, a stem region for hot start and a template-specific region for PCR amplification. As proof of principle, GAPDH, SARS-CoV-2 synthetic RNA and SARS-CoV-2 virus-positive nasopharyngeal swabs were used as templates. Additionally, these primers were used to positively confirm the N501Y mutation from nasopharyngeal swabs. Evidence is presented that the double-bubble primers offer fast, specific, robust and cost-effective improvement in RT-PCR amplification for detection of gene expression in general and for diagnostic detection and genotyping of SARS-CoV-2 in particular.

Nuclear Factor (Erythroid-Derived 2)-Like 2 Regulates the Hepatoprotective Effects of Remote Ischemic Conditioning in Hemorrhagic Shock.

AIMS: Remote ischemic conditioning (RIC) protects against organ ischemia/reperfusion injury in experimental and clinical settings. We have demonstrated that RIC prevents liver and lung inflammation/injury after hemorrhagic shock/resuscitation (S/R). In this study, we used a murine model of S/R to investigate the role of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in mediating hepatoprotection. RESULTS: The combination of RIC with S/R caused a synergistic rise in Nrf2 and its translocation to the nucleus in the liver. Increased activation of Nrf2 by RIC augmented heme oxygenase-1 (HO-1) and autophagy and exerted hepatoprotection, concurrent with reductions in S/R-induced TNF-α (tumor necrosis factor alpha) and IL-6 (interleukin-6). In Nrf2 knockout (KO) animals, RIC did not exert hepatoprotection, and it failed to upregulate HO-1 and autophagy. Further, resuscitating wildtype (WT) animals with blood from donor WT animals undergoing RIC was hepatoprotective, but not in Nrf2 KO recipient animals. Interestingly, RIC blood from Nrf2 KO donor animals was also not protective when used to resuscitate WT animals, suggesting a role for Nrf2 both in the afferent arm of RIC where protective factors are generated and also in the efferent arm where organ protection is exerted. Finally, RIC plasma prevented oxidant-induced zebrafish mortality, but not in Nrf2a morpholino knockdown fish. INNOVATION: Activation of Nrf2 is an essential mechanism underlying the hepatoprotective effects of RIC. Nrf2 appears to play a role in the afferent limb of RIC protection, as its absence precludes the generation of the protective humoral factors induced by RIC. CONCLUSION: Our studies demonstrate the critical role of Nrf2 in the ability of RIC to prevent organ injury after S/R.

Hemorrhagic Shock/Resuscitation Reduces the M2 Phenotype of Alveolar Macrophages: A Potential Mechanism Contributing to Increased LPS-Induced Lung Injury.

Major hemorrhage is a significant contributor to the morbidity and mortality resulting from traumatic injury. In addition to its role in in early mortality, hemorrhagic shock followed by resuscitation (HS/R) is known to initiate immunological events that contribute to the development of organ dysfunction. The pathogenesis of acute lung injury following HS/R involves macrophage activation. Recent studies have shown that macrophage function may in part be regulated by polarization toward classical M1 pro-inflammatory cells or alternatively activated anti-inflammatory M2 cells. We hypothesized that alteration in the M1/M2 phenotypic balance of alveolar macrophages in the lung may contribute to a pro-inflammatory state following HS/R. Using a murine model, we show that HS/R causes a rapid reduction in surface cluster of differentiation (CD)206 and CD36, markers of M2 cells, as well as in CD206 messenger ribonucleic acid (mRNA). M1 markers including surface CD80 and tumour necrosis factor alpha and inducible nitric oxide synthase mRNA were increased, albeit in a somewhat delayed time course. The prostaglandin 5-deoxyDelta12,14 prostaglandin J2 (15d-PGJ2), known to polarize cells toward M2, restored levels of M2 macrophages toward control and prevented lung injury, as assessed by bronchoalveolar protein content. Adoptive cell transfer of in vitro M2 polarized macrophages also reduced lung inflammation/injury following hemorrhagic shock. Together, these studies demonstrate that HS/R increases M1/M2 ratio, predominantly by lowering M2 cells, and thus enhances the proinflammatory state. Various strategies aimed at promoting M2 polarization may lessen the magnitude of inflammation and injury. This represents a novel approach to the prevention/treatment of lung injury in critically ill trauma patients.

Cytochalasin D disruption of actin filaments in 3T3 cells produces an anti-apoptotic response by activating gelatinase A extracellularly and initiating intracellular survival signals.

Disruption of actin filaments affects multiple cell functions including motility, signal transduction and cell division, ultimately culminating in cell death. Although this is the usual sequence of events, we have made the interesting observation that disruption of actin filaments by the potent toxin cytochalasin D (Cyto D) causes one cell type, mouse mesangial cells (MMC), to undergo apoptosis, while in another cell type (NIH 3T3), it has the opposite effect, resulting in production of survival signals. The purpose of this study was to investigate the molecular basis for these observed differences. In the present communication, we demonstrate that exposure to Cyto D induces the pro-apoptotic pathways, p38 and stress-activated protein kinase (SAPK)/jun amino-terminal kinase (JNK), in both cell types. However, in 3T3, but not MMC, the extracellular signal regulated kinase (ERK) 1/2 pathway is protected from inhibition following treatment with Cyto D-leading to phosphorylation of Bclxi/Bcl 2-associated death promoter (BAD). Inhibition of Cyto D-induced secretion and activation of gelatinase A in 3T3 cells reverses the production of survival signals by Cyto-D. To investigate this effect further we employed CS-1 cells, a well-characterized melanoma cell line that lacks integrin beta3, and also does not secrete gelatinase A. Co-transfection of CS-1 cells with integrin beta3 and a gelatinase A transgene, which enables the cells to secrete constituitively active gelatinase A, enhances CS-1 cell survival signals. Together, our findings suggest that extracellularly activated gelatinase A, through interaction with integrin alphaVbeta3, elicits survival signals mediated through ERK 1/2 that override activation of p38 and SAPK/JNK stress pathways.

Description of a PCR-based technique for DNA splicing and mutagenesis by producing 5' overhangs with run through stop DNA synthesis utilizing Ara-C.

BACKGROUND: Splicing of DNA molecules is an important task in molecular biology that facilitates cloning, mutagenesis and creation of chimeric genes. Mutagenesis and DNA splicing techniques exist, some requiring restriction enzymes, and others utilize staggered reannealing approaches. RESULTS: A method for DNA splicing and mutagenesis without restriction enzymes is described. The method is based on mild template-dependent polymerization arrest with two molecules of cytosine arabinose (Ara-C) incorporated into PCR primers. Two rounds of PCR are employed: the first PCR produces 5' overhangs that are utilized for DNA splicing. The second PCR is based on polymerization running through the Ara-C molecules to produce the desired final product. To illustrate application of the run through stop mutagenesis and DNA splicing technique, we have carried out splicing of two segments of the human cofilin 1 gene and introduced a mutational deletion into the product. CONCLUSION: We have demonstrated the utility of a new PCR-based method for carrying out DNA splicing and mutagenesis by incorporating Ara-C into the PCR primers.

Oxidative Stress Increases Surface Toll-Like Receptor 4 Expression in Murine Macrophages Via Ceramide Generation.

Multiorgan failure is a major cause of late mortality following trauma. Oxidative stress generated during shock/resuscitation contributes to tissue injury by priming the immune system for an exaggerated response to subsequent inflammatory stimuli, such as lipopolysaccharide (LPS). We recently reported that oxidative stress causes rapid recruitment of the LPS receptor Toll-like receptor 4 (TLR4) to membrane lipid rafts, thus increasing LPS responsiveness and cellular priming. We hypothesized that activation of Src family kinases by oxidants might contribute to these events. We utilized microscopy, flow cytometry, Western blotting, and thin-layer chromatography methods. Using hydrogen peroxide in vitro and hemorrhagic shock/resuscitation in vivo, oxidant-induced TLR4 translocation in macrophages occurred in an Src-dependent manner. Approaches supporting this conclusion included pharmacologic inhibition of the Src family kinases by PP2, Src inhibition by a molecular approach of cell transfection with Csk, and genetic inhibition of all Src kinases relevant to the monocyte/macrophage lineage in hckfgrlyn triple knockout mice. To evaluate the upstream molecules involved in Src activation, we evaluated the ability of oxidative stress to activate the bioactive lipid molecule ceramide. Oxidants induced ceramide generation in macrophages both in vitro and in vivo, an effect that appears to be due to activation of the acid sphingomyelinase. Using pharmacological approaches, ceramide was shown to be both necessary and sufficient to mediate TLR4 translocation to the plasma membrane in an Src-dependent manner. This study identifies a hierarchy of signaling molecules following oxidative stress that might represent novel targets for therapy in critical illness and organ injury.

Altered inhibitory κBα expression in LPS-stimulated alveolar macrophages following resuscitated hemorrhagic shock.

Patients resuscitated from hemorrhagic shock are at increased risk for the development of organ dysfunction, particularly acute respiratory distress syndrome. The "two-hit hypothesis" wherein shock/resuscitation (S/R) renders the immune system more responsive to subsequent inflammatory stimuli has been suggested as a major mechanism contributing to organ injury. Previous work has shown that S/R primes alveolar macrophages for increased nuclear factor κB (NF-κB) translocation in response to LPS, culminating in increased lung cytokine and chemokine production. Inhibitory κB (IκB) is known to be an important regulator of NF-κB activity. In this article, we investigated the effect of S/R on regulation of IκBα expression in response to LPS both in vitro and in vivo. Two discrete effects on IκB regulation were observed after S/R, which served to augment NF-κB activity. First, antecedent exposure of alveolar macrophages to S/R resulted in increased LPS-induced IκBα degradation through activation of upstream signaling, an effect that resulted in increased NF-κB translocation and cytokine-induced neutrophil chemoattractant gene expression. Second, cells recovered from rodents after S/R had reduced levels of IκB mRNA in response to LPS compared with sham/LPS treatment. This effect was primarily due to the ability of S/R to reverse the prolongation of IκB mRNA stability observed after LPS-alone treatment. Together, these effects on the important regulatory molecule IκB in the macrophage may contribute to the heightened inflammatory response observed after S/R.

An improved Huffman coding method for archiving text, images, and music characters in DNA.

An improved Huffman coding method for information storage in DNA is described. The method entails the utilization of modified unambiguous base assignment that enables efficient coding of characters. A plasmid-based library with efficient and reliable information retrieval and assembly with uniquely designed primers is described. We illustrate our approach by synthesis of DNA that encodes text, images, and music, which could easily be retrieved by DNA sequencing using the specific primers. The method is simple and lends itself to automated information retrieval.

Effect of a matrix metalloproteinase sequestering biomaterial on Caco-2 epithelial cell barrier integrity in vitro.

A novel matrix metalloproteinase sequestering biomaterial (MI Theramer beads) restored the epithelial barrier in a double chamber in vitro test system after disruption by Cytochalasin D and the secretion of the metalloproteinase MMP-2. MI beads are chemically modified (hydroxamated) poly(methacrylic acid-co-methyl methacrylate). We are exploring the utility of this material in inflammatory bowel disease (IBD), in which one manifestation is a compromised intestinal epithelial barrier. In a first step towards this goal we incubated MI beads (or polymethyl methacrylate control beads) with Caco-2 epithelial cells and mesenchymal 3T3 fibroblasts on two sides of a Matrigel((R))-coated filter and used Cytochalasin D (Cyto D) to activate MMP-2 (secreted by the 3T3 cells), disrupt actin filaments of Caco-2 cells and render the epithelial barrier leaky, as measured by dextran fluorescein equilibration. Addition of MI beads to Cyto D-treated cells inhibited active MMP-2 and prevented equilibration of dextran fluorescein. This study is the first step in showing a potential benefit to local (as opposed to systemic) inhibition of metalloproteinases in IBD or other intestinal inflammatory diseases.

Trichostatin A-histone deacetylase inhibitor with clinical therapeutic potential-is also a selective and potent inhibitor of gelatinase A expression.

Modulation of histone acetylation is currently being explored as a therapeutic strategy in treatment of cancer. Specifically, inhibition of histone deacetylase by trichostatin A (TSA) has been shown to prevent tumorigenesis and metastasis. In the present paper we demonstrate that increased histone acetylation by TSA-treated 3T3 cells decreases mRNA as well as zymographic activity of gelatinase A, a matrix metalloproteinase, which is itself, implicated in tumorigenesis and metastasis. Furthermore, TSA inhibits cytochalasin D-induced activation of gelatinase A, but TSA does not affect other members of the gelatinase A activation complex, MT1-MMP and TIMP-2. Thus, TSA is a selective and potent inhibitor of expression and activation of gelatinase A. This finding not only strengthens the rationale for continuing to investigate the therapeutic utility of TSA in cancer, but also, provides evidence that TSA inhibition of gelatinase A expression and activation can be used as a biological marker to monitor and determine end-points of clinical trials involving TSA.

Differential effects of trichostatin A on gelatinase A expression in 3T3 fibroblasts and HT-1080 fibrosarcoma cells: implications for use of TSA in cancer therapy.

Trichostatin A (TSA) is a histone deacetylase (HDAC) inhibitor with potential in cancer therapeutics. In a recent communication, we demonstrated that TSA is a selective, potent inhibitor of gelatinase A in 3T3 fibroblasts. In the present study, we extend these observations and examine the effects of TSA in 3T3 fibroblasts compared to HT-1080 fibrosarcoma cells with respect to gelatinase A expression, cell viability, and apoptosis. We find that while expression of gelatinase A in 3T3 fibroblasts is exquisitely sensitive to inhibition by TSA, expression of this enzyme in HT-1080 cells is minimally affected by this compound. Moreover, we show that TSA is pro-apoptotic in HT-1080 cells, but is anti-apoptotic in 3T3 cells. We propose a two-pronged model for the therapeutic action of TSA. On the one hand TSA selectively decreases cancer cell viability, while enhancing the viability of stromal cells. On the other hand, by selectively decreasing gelatinase A expression in stromal but not cancer cells, TSA acts to control metastatic potential by reducing the ability of metastatic cells to recruit stromal cells to secrete gelatinase A.