Cyclic stretch of alveolar epithelial cells alters cytoskeletal micromechanics

Cytoplasmic transport of large molecules such as plasmid DNA (pDNA) has been shown to increase when cells are subjected to mild levels of cyclic stretch for brief periods. In the case of pDNA, this is in part due to the increased active transport of pDNA along stabilized, acetylated microtubules in the cytoplasm, whose levels are increased in response to stretch. It also has been shown that disruption of the dense actin network leads to increased pDNA and macromolecule diffusion as well. We hypothesize that stretch not only increases active transport of pDNA but also, similar to actin disrupting drugs, decreases cytoplasmic stiffness leading to a less restive pathway for macromolecules to diffuse. To test this we used particle tracking microrheology to measure cytoplasmic mechanics. We conclude that while cyclic stretch transiently decreases cytoplasmic stiffness and increases diffusivity, stretch‐independent modulation of the levels of acetylated, stable microtubules has no effect on cytoplasmic stiffness. Furthermore, stretching cells that have maximally acetylated microtubules increases cytoplasmic trafficking of pDNA, without increasing levels of acetylated microtubules. These findings suggest that stretch‐enhanced gene transfer may occur by two independent mechanisms: increased levels of acetylated microtubules for directed active transport, and reduced cytoplasmic stiffness for increased diffusion. Biotechnol. Bioeng. 2011;108: 446–453. © 2010 Wiley Periodicals, Inc.     

The Impact of Isolated Baseline Cannabis Use on Outcomes Following Thoracolumbar Spinal Fusion: A Propensity Score-Matched Analysis

BackgroundThere is limited literature evaluating the impact of isolated cannabis use on outcomes for patients following spinal surgery. This study sought to compare 90-day complication, 90-day readmission, as well as 2-year revision rates between baseline cannabis users and non-users following thoracolumbar spinal fusion (TLF) for adult spinal deformity (ASD).MethodsThe New York Statewide Planning and Research Cooperative System (SPARCS) database was queried between January 2009 and September 2013 to identify all patients who underwent TLF for ASD. Inclusion criteria were age ≥18 years and either minimum 90-day (for complications and readmissions) or 2-year (for revisions) follow-up surveillance. Cohorts were created and propensity score-matched based on presence or absence of isolated baseline cannabis use. Baseline demographics, hospital-related parameters, 90-day complications and readmissions, and two-year revisions were retrieved. Multivariate binary stepwise logistic regression identified independent outcome predictors.Results704 patients were identified (n=352 each), with comparable age, sex, race, primary insurance, Charlson/Deyo scores, surgical approach, and levels fused between cohorts (all, p>0.05). Cannabis users (versus non-users) incurred lower 90-day overall and medical complication rates (2.4% vs. 4.8%, p=0.013; 2.0% vs. 4.1%, p=0.018). Cohorts had otherwise comparable complication, revision, and readmission rates (p>0.05). Baseline cannabis use was associated with a lower risk of 90-day medical complications (OR=0.47, p=0.005). Isolated baseline cannabis use was not associated with 90-day surgical complications and readmissions, or two-year revisions.ConclusionIsolated baseline cannabis use, in the absence of any other diagnosed substance abuse disorders, was not associated with increased odds of 90-day surgical complications or readmissions or two-year revisions, though its use was associated with reduced odds of 90-day medical complications when compared to non-users undergoing TLF for ASD. Further investigations are warranted to identify the physiologic mechanisms underlying these findings. Level of Evidence: III     

Identification and Molecular Mechanisms of the Rapid Tonicity-induced Relocalization of the Aquaporin 4 Channel

The aquaporin family of integral membrane proteins is composed of channels that mediate cellular water flow. Aquaporin 4 (AQP4) is highly expressed in the glial cells of the central nervous system and facilitates the osmotically driven pathological brain swelling associated with stroke and traumatic brain injury. Here we show that AQP4 cell surface expression can be rapidly and reversibly regulated in response to changes of tonicity in primary cortical rat astrocytes and in transfected HEK293 cells. The translocation mechanism involves PKA activation, influx of extracellular calcium, and activation of calmodulin. We identify five putative PKA phosphorylation sites and use site-directed mutagenesis to show that only phosphorylation at one of these sites, serine 276, is necessary for the translocation response. We discuss our findings in the context of the identification of new therapeutic approaches to treating brain edema.     

Kinetics and thermodynamics of salt-dependent T7 gene 2.5 protein binding to single- and double-stranded DNA

Bacteriophage T7 gene 2.5 protein (gp2.5) is a single-stranded DNA (ssDNA)-binding protein that has essential roles in DNA replication, recombination and repair. However, it differs from other ssDNA-binding proteins by its weaker binding to ssDNA and lack of cooperative ssDNA binding. By studying the rate-dependent DNA melting force in the presence of gp2.5 and its deletion mutant lacking 26 C-terminal residues, we probe the kinetics and thermodynamics of gp2.5 binding to ssDNA and double-stranded DNA (dsDNA). These force measurements allow us to determine the binding rate of both proteins to ssDNA, as well as their equilibrium association constants to dsDNA. The salt dependence of dsDNA binding parallels that of ssDNA binding. We attribute the four orders of magnitude salt-independent differences between ssDNA and dsDNA binding to nonelectrostatic interactions involved only in ssDNA binding, in contrast to T4 gene 32 protein, which achieves preferential ssDNA binding primarily through cooperative interactions. The results support a model in which dimerization interactions must be broken for DNA binding, and gp2.5 monomers search dsDNA by 1D diffusion to bind ssDNA. We also quantitatively compare the salt-dependent ssDNA- and dsDNA-binding properties of the T4 and T7 ssDNA-binding proteins for the first time.     

The effects of trifluoperazine on brain edema,aquaporin-4 expression and metabolic markers during the acute phase of stroke using photothrombotic mouse model

Stroke is the second leading cause of death and the third leading cause of disability globally. Edema is a hallmark of stroke resulting from dysregulation of water homeostasis in the central nervous system (CNS) and plays the major role in stroke-associated morbidity and mortality. The overlap between cellular and vasogenic edema makes treating this condition complicated, and to date, there is no pathogenically oriented drug treatment for edema. Water balance in the brain is tightly regulated, primarily by aquaporin 4 (AQP4) channels, which are mainly expressed in perivascular astrocytic end-feet. Targeting AQP4 could be a useful therapeutic approach for treating brain edema; however, there is no approved drug for stroke treatment that can directly block AQP4. In this study, we demonstrate that the FDA-approved drug trifluoperazine (TFP) effectively reduces cerebral edema during the early acute phase in post-stroke mice using a photothrombotic stroke model. This effect was combined with an inhibition of AQP4 expression at gene and protein levels. Importantly, TFP does not appear to induce any deleterious changes on brain electrolytes or metabolic markers, including total protein or lipid levels. Our results support a possible role for TFP in providing a beneficial extra-osmotic effect on brain energy metabolism, as indicated by the increase of glycogen levels. We propose that targeting AQP4-mediated brain edema using TFP is a viable therapeutic strategy during the early and acute phase of stroke that can be further investigated during later stages to help in developing novel CNS edema therapies.