Electrical synapses interconnecting axons revealed in the optic nerve head – a novel model of gap junctions’ involvement in optic nerve function

Abstract Purpose To characterize newly discovered electrical synapses, formed by connexin (Cx) 36 and 45, between neighbouring axons within the optic nerve head. Methods Twenty-five Wistar rats were killed by CO2 inhalation. Proximal and distal optic nerve (ON) stumps were collected and processed for immunostainings, electron microscopy (EM) with immunogold labelling, PCR and Western blots (WB). Additional 15 animals were deeply anaesthetized, and flash visual evoked potentials (fVEP) after retrobulbar injection of saline (negative control) or 100 ?m meclofenamic acid solution (gap junctions? blocker) were recorded. Human paraffin cross-sections of eyeballs for immunostainings were obtained from the Human Eye Biobank for Research. Results Immunostainings of both rat and human ON revealed the presence of Cx45 and 36 colocalizing with ?3-tubulin, but not with glial fibrillary acidic protein (GFAP). In WB, Cx36 content in optic nerve was approximately halved when compared with retina (0.58 ± 0.005 in proximal stump and 0.44 ± 0.02 in distal stump), Cx45 showed higher levels (0.68 ± 0.01 in proximal stump and 0.9 ± 0.07 in distal stump). In immunogold-EM of optic nerve sections, we found electric synapses (formed mostly by Cx45) directly coupling neighbouring axons. In fVEP, blocking of gap junctions with meclofenamic acid resulted in significant prolongation of the latency of P1 wave up to 160% after 30 min (p Peer reviewe

Autonomic Business Processes

Business processes in large organisations are typically poorly understood and complex in structure. Adapting such a business process to changing internal and external conditions requires costly and time consuming investigative work and change management. In contrast autonomic systems are able to adapt to changing environments and continue to function without external intervention. Enabling business processes to adapt to changing conditions in the same way would be extremely valuable. This work investigates the potential to self-heal individual business process executions in generic business processes. Classical and Immune-inspired classification algorithms are tested for their predictive utility with Decision Trees augmented with MetaCost and Immunos 99 exhibiting the best performance respectively. An approach to deriving recovery strategies from historical process data in the absence of a process model is presented and tested for suitability. Also presented is an approach to selecting the best of the determined recovery strategies for application to a business process execution, which is then tested to determine the impact of its parameters on the quality of selected recoveries

Immunohistochemical and biochemical assay of versican in human sound predentine/dentine matrix

Aim of this study was to investigate the distribution of versican proteoglycan within the human dentine organic matrix by means of a correlative immunohistochemical analysis with field emission in-lens scanning electron microscope (FEI-SEM), transmission electron microscope (TEM), fluorescence microscope (FM) and biochemical assay. Specimens containing dentine and predentine were obtained from non carious human teeth and divided in three groups: 1) FEI-SEM group: sections were exposed to a pre-embedding immunohistochemical procedure; 2) TEM group: specimens were fixed, demineralised, embedded and submitted to a post-embedding immunohistochemical procedure; 3) FM group: sections mineralised and submitted to a pre-embedding immunohistochemical procedure with fluorescence labelling. Specimens were exposed to two different antibodies to assay distribution of versican fragments and whole versican molecule. Western Blotting analysis of dentine and pulp extracts was also performed. The correlative FEI-SEM,TEM and FM analysis revealed positive immunoreaction for versican fragments both in predentine and dentine, while few gold particles identifying the whole versican molecule were found in predentine only under TEM. No labelling of versican whole molecule was detected by FEI-SEM and FM analysis. The immunoblotting analysis confirmed the morphological findings. This study suggests that in fully developed human teeth versican fragments are significant constituents of the human dentine and predentine organic matrix, while versican whole molecule can be visualised in scarce amount within predentine only. The role of versican fragments within human dentine organic matrix should be further elucidated

Pericyte FAK negatively regulates Gas6/Axl signalling to suppress tumour angiogenesis and tumour growth

The overexpression of the protein tyrosine kinase, Focal adhesion kinase (FAK), in endothelial cells has implicated its requirement in angiogenesis and tumour growth, but how pericyte FAK regulates tumour angiogenesis is unknown. We show that pericyte FAK regulates tumour growth and angiogenesis in multiple mouse models of melanoma, lung carcinoma and pancreatic B-cell insulinoma and provide evidence that loss of pericyte FAK enhances Gas6-stimulated phosphorylation of the receptor tyrosine kinase, Axl with an upregulation of Cyr61, driving enhanced tumour growth. We further show that pericyte derived Cyr61 instructs tumour cells to elevate expression of the proangiogenic/protumourigenic transmembrane receptor Tissue Factor. Finally, in human melanoma we show that when 50% or more tumour blood vessels are pericyte-FAK negative, melanoma patients are stratified into those with increased tumour size, enhanced blood vessel density and metastasis. Overall our data uncover a previously unknown mechanism of tumour growth by pericytes that is controlled by pericyte FAK

Misfolded Mutant SOD1 Directly Inhibits VDAC1 Conductance in a Mouse Model of Inherited ALS

SummaryMutations in superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by loss of motor neurons. With conformation-specific antibodies, we now demonstrate that misfolded mutant SOD1 binds directly to the voltage-dependent anion channel (VDAC1), an integral membrane protein imbedded in the outer mitochondrial membrane. This interaction is found on isolated spinal cord mitochondria and can be reconstituted with purified components in vitro. ADP passage through the outer membrane is diminished in spinal mitochondria from mutant SOD1-expressing ALS rats. Direct binding of mutant SOD1 to VDAC1 inhibits conductance of individual channels when reconstituted in a lipid bilayer. Reduction of VDAC1 activity with targeted gene disruption is shown to diminish survival by accelerating onset of fatal paralysis in mice expressing the ALS-causing mutation SOD1G37R. Taken together, our results establish a direct link between misfolded mutant SOD1 and mitochondrial dysfunction in this form of inherited ALS

Peroxisome Proliferator-Activated Receptor β/δ in the Brain: Facts and Hypothesis

peroxisome proliferator-activated receptors (PPARs) are nuclear receptors acting as lipid sensors. Besides its metabolic activity in peripheral organs, the PPAR beta/delta isotype is highly expressed in the brain and its deletion in mice induces a brain developmental defect. Nevertheless, exploration of PPARβ action in the central nervous system remains sketchy. The lipid content alteration observed in PPARβ null brains and the positive action of PPARβ agonists on oligodendrocyte differentiation, a process characterized by lipid accumulation, suggest that PPARβ acts on the fatty acids and/or cholesterol metabolisms in the brain. PPARβ could also regulate central inflammation and antioxidant mechanisms in the damaged brain. Even if not fully understood, the neuroprotective effect of PPARβ agonists highlights their potential benefit to treat various acute or chronic neurological disorders. In this perspective, we need to better understand the basic function of PPARβ in the brain. This review proposes different leads for future researches

A link between the accumulation of DNA damage and loss of multi-potency of human mesenchymal stromal cells

Human mesenchymal stromal cells (hMSCs) represent an attractive cell source for clinic applications. Besides being multi-potent, recent clinical trials suggest that they secrete both trophic and immunomodulatory factors, allowing allogenic MSCs to be used in a wider variety of clinical situations. The yield of prospective isolation is however very low, making expansion a required step toward clinical applications. Unfortunately, this leads to a significant decrease in their stemness. To identify the mechanism behind loss of multi-potency, hMSCs were expanded until replicative senescence and the concomitant molecular changes were characterized at regular intervals. We observed that, with time of culture, loss of multi-potency was associated with both the accumulation of DNA damage and the respective activation of the DNA damage response pathway, suggesting a correlation between both phenomena. Indeed, exposing hMSCs to DNA damage agents led to a significant decrease in the differentiation potential. We also showed that hMSCs are susceptible to accumulate DNA damage upon in vitro expansion, and that although hMSCs maintained an effective nucleotide excision repair activity, there was a progressive accumulation of DNA damage. We propose a model in which DNA damage accumulation contributes to the loss of differentiation potential of hMSCs, which might not only compromise their potential for clinical applications but also contribute to the characteristics of tissue agein

Selection of reference genes for quantitative real-time PCR in a rat asphyxial cardiac arrest model

<p>Abstract</p> <p>Background</p> <p>Cardiac arrest, and the associated arrest of blood circulation, immediately leads to permanent brain damage because of the exhaustion of oxygen, glucose and energy resources in the brain. Most hippocampal CA1 neurons die during the first week post the insult. Molecular data concerning the recovery after resuscitation are sparse and limited to the early time period. Expression analysis of marker genes via quantitative real-time RT-PCR enables to follow up the remodeling process. However, proper validation of the applied normalization strategy is a crucial prerequisite for reliable conclusions.</p> <p>Therefore, the present study aimed to determine the expression stability of ten commonly used reference genes (<it>Actb</it>, actin, beta; <it>B2m</it>, beta-2 microglobulin;<it>CypA</it>, cyclophilin A; <it>Gapdh</it>, glyceraldehyde-3-phosphate dehydrogenase; <it>Hprt</it>, hypoxanthine guanine phosphoribosyl transferase; <it>Pgk1</it>, phosphoglycerate kinase 1; <it>Rpl13a</it>, ribosomal protein L13A; <it>Sdha</it>, succinat dehydrogenase complex, subunit a, flavoprotein (Fp); <it>Tbp</it>, TATA box binding protein; <it>Ywhaz</it>, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide) in the rat hippocampus four, seven and twenty-one days after cardiac arrest. Moreover, experimental groups treated with the anti-inflammatory and anti-apoptotic drug minocycline have been included in the study as well.</p> <p>Results</p> <p>The microglial marker <it>Mac-1</it>, used as a target gene to validate the experimental model, was found to be upregulated about 10- to 20-fold after cardiac arrest.</p> <p>Expression stability of candidate reference genes was analyzed using geNorm and NormFinder software tools. Several of these genes behave rather stable. <it>CypA </it>and <it>Pgk1 </it>were identified by geNorm as the two most stable genes 4 and 21 days after asphyxial cardiac arrest, <it>CypA </it>and <it>Gapdh </it>at 7 days post treatment. <it>B2m </it>turned out to be the most variable candidate reference gene, being about 2-fold upregulated in the cardiac arrest treatment groups.</p> <p>Conclusion</p> <p>We have validated endogenous control genes for qRT-PCR analysis of gene expression in rat hippocampus after resuscitation from cardiac arrest. For normalization purposes in gene profiling studies a combination of <it>CypA </it>and <it>Pgk1 </it>should be considered 4 and 21 days post injury, whereas <it>CypA </it>and <it>Gapdh </it>is the best combination at 7 days. <it>CypA </it>is most favorable if restriction to a single reference gene for all time points is required.</p