ABCB5+ mesenchymal stromal cells therapy protects from hypoxia by restoring Ca2+ homeostasis in vitro and in vivo

Background: Hypoxia in ischemic disease impairs Ca2+ homeostasis and may promote angiogenesis. The therapeutic efficacy of mesenchymal stromal cells (MSCs) in peripheral arterial occlusive disease is well established, yet its influence on cellular Ca2+ homeostasis remains to be elucidated. We addressed the influence of ATP-binding cassette subfamily B member 5 positive mesenchymal stromal cells (ABCB5+ MSCs) on Ca2+ homeostasis in hypoxic human umbilical vein endothelial cells (HUVECs) in vitro and in vivo. Methods: Hypoxia was induced in HUVECs by Cobalt (II) chloride (CoCl2) or Deferoxamine (DFO). Dynamic changes in the cytosolic- and endoplasmic reticulum (ER) Ca2+ and changes in reactive oxygen species were assessed by appropriate fluorescence-based sensors. Metabolic activity, cell migration, and tube formation were assessed by standard assays. Acute-on-chronic ischemia in Apolipoprotein E knock-out (ApoE−/−) mice was performed by double ligation of the right femoral artery (DFLA). ABCB5+ MSC cells were injected into the ischemic limb. Functional recovery after DFLA and histology of gastrocnemius and aorta were assessed. Results: Hypoxia-induced impairment of cytosolic and ER Ca2+ were restored by ABCB5+ MSCs or their conditioned medium. Similar was found for changes in intracellular ROS production, metabolic activity, migratory ability and tube formation. The restoration was paralleled by an increased expression of the Ca2+ transporter Sarco-/endoplasmic reticulum ATPase 2a (SERCA2a) and the phosphorylation of Phospholamban (PLN). In acute-on-chronic ischemia, ABCB5+ MSCs treated mice showed a higher microvascular density, increased SERCA2a expression and PLN phosphorylation relative to untreated controls. Conclusions: ABCB5+ MSCs therapy can restore cellular Ca2+ homeostasis, which may beneficially affect the angiogenic function of endothelial cells under hypoxia in vitro and in vivo

Fluorescent Protein-Based Methods for On-Plate Screening of Gene Insertion

Unlike the commonly used method of blue-white screening for gene insertion, a fluorescent protein-based screening method offers a gain-of-function screening process without using any co-factors and a gene fusion product with a fluorescent protein reporter that is further useful in cell imaging studies. However, complications related to protein-folding efficiencies of the gene insert in fusion with fluorescent protein reporters prevent effective on-plate bacterial colony selection leading to its limited use.Here, we present three methods to tackle this problem. Our first method promotes the folding of the gene insert by using an N-terminal protein such as calmodulin that is well folded and expressed. Under this method, fluorescence was increased more than 30x over control allowing for enhanced screening. Our second method creates a fluorescent protein that is N-terminal to the gene upon insertion, thereby reducing the dependency of the fluorescent protein reporter on the folding of the gene insert. Our third method eliminates any dependence of the fluorescent protein reporter on the folding of the gene insert by using a stop and start sequence for protein translation.The three methods together will expand the usefulness of fluorescence on-plate screening and offer a powerful alternative to blue-white screening

Prostate transglutaminase (TGase-4) antagonizes the anti-tumour action of MDA-7/IL-24 in prostate cancer

Background Transglutamiase-4 (TGase-4), also known as prostate transglutaminase, belongs to the TGase family and is uniquely expressed in the prostate gland. The functions of this interesting protein are not clearly defined. In the present study, we have investigated an unexpected link between TGase-4 and the melanoma differentiation-associated gene-7/interleukin-24 (MDA-7/IL-24), a cytokine known to regulate the growth and apoptosis of certain cancer and immune cells. Methods Frozen sections of normal and malignant human prostate tissues and human prostate cancer (PCa) cell lines PC-3 and CA-HPV-10, cell lines expressing low and high levels of TGase-4, and recombinant MDA-7/IL-24 (rhMDA-7/IL-24) were used. Expression construct for human TGase-4 was generated using a mammalian expression vector with full length human TGase-4 isolated from normal human prostate tissues. PC-3 cells were transfected with expression construct or control plasmid. Stably transfected cells for control transfection and TGase-4 over expression were created. Similarly, expression of TGase-4 in CA-HPV-10 cells were knocked down by way of ribozyme transgenes. Single and double immunofluorescence microscopy was used for localization and co-localization of TGase-4 and MDA-7/IL-24 in PCa tissues and cells with antibodies to TGase-4; MDA-7/IL-24; IL-20alpha; IL-20beta and IL-22R. Cell-matrix adhesion, attachment and migration were by electric cell substrate impedance sensing and growth by in vitro cell growth assay. A panel of small molecule inhibitors, including Akt, was used to determine signal pathways involving TGase-4 and MDA-7/IL-24. Results We initially noted that MDA-7 resulted in inhibition of cell adhesion, growth and migration of human PCa PC-3 cells which did not express TGase-4. However, after the cells over-expressed TGase-4 by way of transfection, the TGase-4 expressing cells lost their adhesion, growth and migratory inhibitory response to MDA-7. On the other hand, CA-HPV-10 cells, a cell type naturally expressing high levels of TGase-4, had a contrasting response to MDA-7 when compared with PC-3 cells. Inhibitor to Akt reversed the inhibitory effect of MDA-7, only in PC-3 control cells, but not the TGase-4 expressing PC-3 cells. In human prostate tissues, TGase-4 was found to have a good degree of co-localization with one of the MDA-7 receptor complexes, IL-20Ra. Conclusion The presence of TGase-4 has a biological impact on a prostate cancer cell's response to MDA-7. TGase-4, via mechanism(s) yet to be identified, blocked the action of MDA-7 in prostate cancer cells. This has an important implication when considering the use of MDA-7 as a potential anticancer cytokine in prostate cancer therapies

Analysis of the anti-tumor effect of cetuximab using protein kinetics and mouse xenograft models

<p>Abstract</p> <p>Background</p> <p>The binding of EGFR and its ligands leads to autophosphorylation of receptor tyrosine kinase as well as subsequent activation of signal transduction pathways that are involved in regulating cellular proliferation, differentiation, and survival. An EGFR inhibitor, cetuximab binds to EGFR and consequently blocks a variety of cellular processes. <it>KRAS</it>/<it>BRAF </it>mutations are known to be associated with a low response rate to cetuximab. In the present study, to clarify the anti-tumor mechanisms of cetuximab, we evaluated the <it>KRAS</it>/<it>BRAF </it>status, phosphorylation level of the EGFR pathway, and the tumor suppression effect in vivo, using a human colon cancer cell line HT29, which exhibited the highest EGFR expression in response to the cetuximab therapy among the 6 colorectal cancer cell lines tested.</p> <p>Findings</p> <p>The conventional growth suppression assay did not work efficiently with cetuximab. EGF, TGF-α, and IGF activated the EGFR/MAPK cell signaling pathway by initiating the phosphorylation of EGFR. Cetuximab partially inhibited the EGFR/MAPK pathway induced by EGF, TGF-α, and IGF. However, cetuximab exposure induced the EGFR, MEK, and ERK1/2 phosphorylation by itself. Mouse xenograft tumor growth was significantly inhibited by cetuximab and both cetuximab-treated and -untreated xenograft specimens exhibited phosphorylations of the EGFR pathway proteins.</p> <p>Conclusions</p> <p>We have confirmed that cetuximab inhibited the EGFR/MAPK pathway and reduced tumor growth in the xenografts while the remaining tumor showed EGFR pathway activation. These results suggest that: ( i ) The effect of cetuximab in growth signaling is not sufficient to induce complete growth suppression in vitro; ( ii ) time-course monitoring may be necessary to evaluate the effect of cetuximab because EGFR signaling is transmitted in a minute order; and ( iii ) cetuximab treatment may have cells acquired resistant selectively survived in the heterogeneous cancer population.</p

The double [3+2] photocycloaddition reaction

One of a synthetic organic chemists‟ greatest challenges is to create step-efficient routes toward compounds with high molecular complexity. Therefore, reactions such as the meta photocycloaddition of an olefin to a benzene derivative, which provide more than one bond in a single step are of significant importance. It this remarkable reaction three new σ bonds, three new rings and up to six new stereocenters are formed simultaneously. Additional complexity can be added by tethering the two reacting partners together and this form of the reaction has found many uses in natural product synthesis. In this work a remarkable double [3+2] photocycloaddition reaction is reported that results in the formation of a complex cis, cis, cis, trans-[5, 5, 5, 5] fenestrane derivative from a simple flat aromatic acetal with two branching alkenes. During this dramatic transformation four carboncarbon bonds, five new rings and seven new stereocenters are created in a single one-pot process using only UV light. The reaction occurs in a sequential manner from the linear meta photocycloadduct, via a secondary [3+2] addition of the alkene across the cyclopropane of the adduct. In addition, an angular meta photocycloadduct also produced in the initial addition step, undergoes an alternative fragmentation-translocation photoreaction to afford a silphinene-like angular tricyclic compound. In this work the investigation of this newly discovered process is discussed via the synthesis and subsequent irradiation of a series of photosubstrates containing different functional groups in the arene-alkene tether. In addition, attempts toward the synthesis of alternative structures using the same double [3+2] photocycloaddition are reported

Microscopy of bacterial translocation during small bowel obstruction and ischemia in vivo – a new animal model

BACKGROUND: Existing animal models provide only indirect information about the pathogenesis of infections caused by indigenous gastrointestinal microflora and the kinetics of bacterial translocation. The aim of this study was to develop a novel animal model to assess bacterial translocation and intestinal barrier function in vivo. METHODS: In anaesthetized male Wistar rats, 0.5 ml of a suspension of green fluorescent protein-transfected E. coli was administered by intraluminal injection in a model of small bowel obstruction. Animals were randomly subjected to non-ischemic or ischemic bowel obstruction. Ischemia was induced by selective clamping of the terminal mesenteric vessels feeding the obstructed bowel loop. Time intervals necessary for translocation of E. coli into the submucosal stroma and the muscularis propria was assessed using intravital microscopy. RESULTS: Bacterial translocation into the submucosa and muscularis propria took a mean of 36 ± 8 min and 80 ± 10 min, respectively, in small bowel obstruction. Intestinal ischemia significantly accelerated bacterial translocation into the submucosa (11 ± 5 min, p < 0.0001) and muscularis (66 ± 7 min; p = 0.004). Green fluorescent protein-transfected E. coli were visible in frozen sections of small bowel, mesentery, liver and spleen taken two hours after E. coli administration. CONCLUSIONS: Intravital microscopy of fluorescent bacteria is a novel approach to study bacterial translocation in vivo. We have applied this technique to define minimal bacterial transit time as a functional parameter of intestinal barrier function

Identifying core features of adaptive metabolic mechanisms for chronic heat stress attenuation contributing to systems robustness

The contribution of metabolism to heat stress may play a significant role in defining robustness and recovery of systems; either by providing the energy and metabolites required for cellular homeostasis, or through the generation of protective osmolytes. However, the mechanisms by which heat stress attenuation could be adapted through metabolic processes as a stabilizing strategy against thermal stress are still largely unclear. We address this issue through metabolomic and transcriptomic profiles for populations along a thermal cline where two seagrass species, Zostera marina and Zostera noltii, were found in close proximity. Significant changes captured by these profile comparisons could be detected, with a larger response magnitude observed in northern populations to heat stress. Sucrose, fructose, and myo-inositol were identified to be the most responsive of the 29 analyzed organic metabolites. Many key enzymes in the Calvin cycle, glycolysis and pentose phosphate pathways also showed significant differential expression. The reported comparison suggests that adaptive mechanisms are involved through metabolic pathways to dampen the impacts of heat stress, and interactions between the metabolome and proteome should be further investigated in systems biology to understand robust design features against abiotic stress

A Method for the Simultaneous Estimation of Selection Intensities in Overlapping Genes

Inferring the intensity of positive selection in protein-coding genes is important since it is used to shed light on the process of adaptation. Recently, it has been reported that overlapping genes, which are ubiquitous in all domains of life, seem to exhibit inordinate degrees of positive selection. Here, we present a new method for the simultaneous estimation of selection intensities in overlapping genes. We show that the appearance of positive selection is caused by assuming that selection operates independently on each gene in an overlapping pair, thereby ignoring the unique evolutionary constraints on overlapping coding regions. Our method uses an exact evolutionary model, thereby voiding the need for approximation or intensive computation. We test the method by simulating the evolution of overlapping genes of different types as well as under diverse evolutionary scenarios. Our results indicate that the independent estimation approach leads to the false appearance of positive selection even though the gene is in reality subject to negative selection. Finally, we use our method to estimate selection in two influenza A genes for which positive selection was previously inferred. We find no evidence for positive selection in both cases