Bothnia dystrophy is caused by domino-like rearrangements in cellular retinaldehyde-binding protein mutant R234W

Cellular retinaldehyde-binding protein (CRALBP) is essential for mammalian vision by routing 11-cis-retinoids for the conversion of photobleached opsin molecules into photosensitive visual pigments. The arginine-to-tryptophan missense mutation in position 234 (R234W) in the human gene RLBP1 encoding CRALBP compromises visual pigment regeneration and is associated with Bothnia dystrophy. Here we report the crystal structures of both wild-type human CRALBP and of its mutant R234W as binary complexes complemented with the endogenous ligand 11-cis-retinal, at 3.0 and 1.7 A resolution, respectively. Our structural model of wild-type CRALBP locates R234 to a positively charged cleft at a distance of 15 A from the hydrophobic core sequestering 11-cis-retinal. The R234W structural model reveals burial of W234 and loss of dianion-binding interactions within the cleft with physiological implications for membrane docking. The burial of W234 is accompanied by a cascade of side-chain flips that effect the intrusion of the side-chain of I238 into the ligand-binding cavity. As consequence of the intrusion, R234W displays 5-fold increased resistance to light-induced photoisomerization relative to wild-type CRALBP, indicating tighter binding to 11-cis-retinal. Overall, our results reveal an unanticipated domino-like structural transition causing Bothnia-type retinal dystrophy by the impaired release of 11-cis-retinal from R234W.

Potentiometric platform for the quantification of cellular potassium efflux

Renewed interest in the measurement of cellular K(+) effluxes has been prompted by the observation that potassium plays an active and important role in numerous key cellular events, in particular cell necrosis and apoptosis. Although necrosis and apoptosis follow different pathways, both induce intracellular potassium effluxes. Here, we report the use of potassium-selective microelectrodes located in a microfluidic platform for cell culture to monitor and quantify such effluxes in real time. Using this platform, we observed and measured the early signs of cell lysis induced by a modification of the extracellular osmolarity. Furthermore, we were able to quantify the number of dying cells by evaluating the extracellular potassium concentration. A comparison between the potentiometric measurement with a fluorescent live-dead assay performed under similar conditions revealed the delay between potassium effluxes and cell necrosis. These results suggest that such platforms may be exploited for applications, such as cytotoxicological screening assays or tumor cell proliferation assays, by using extracellular K(+) as cell death marker.

Detection of type III secretion genes as a general indicator of bacterial virulence

Type III secretion systems of Gram-negative bacteria are specific export machineries for virulence factors which allow their translocation to eukaryotic cells. Since they correlate with bacterial pathogenicity, their presence is used as a general indicator of bacterial virulence. By comparing the genetic relationship of the major type III secretion systems we found the family of genes encoding the inner-membrane channel proteins represented by the Yersinia enterocolitica lcrD (synonym yscV) and its homologous genes from other species an ideal component for establishing a general detection approach for type III secretion systems. Based on the genes of the lcrD family we developed gene probes for Gram-negative human, animal and plant pathogens. The probes comprise lcrD from Y. enterocolitica, sepA from enteropathogenic Escherichia coli, invA from Salmonella typhimurium, mxiA from Shigella sonnei, as well as hrcV from Erwinia amylovora. In addition we included as a control probe the flhA gene from E. coli K-12 to validate our approach. FlhA is part of the flagellar export apparatus which shows a high degree of similarity with type III secretions systems, but is not involved in pathogenicity. The probes were evaluated by screening a series of pathogenic as well as non-pathogenic bacteria. The probes detected type III secretion in pathogens where such systems were either known or were expected to be present, whereas no positive hybridization signals could be found in non-pathogenic Gram-negative bacteria. Gram-positive bacteria were devoid of known type III secretion systems. No interference due to the genetic similarity between the type III secretion system and the flagellar export apparatus was observed. However, potential type III secretion systems could be detected in bacteria where no such systems have been described yet. The presented approach provides therefore a useful tool for the assessment of the virulence potential of bacterial isolates of human, animal and plant origin. Moreover, it is a powerful means for a first safety assessment of poorly characterized strains intended to be used in biotechnological applications.

CCN2/CTGF is required for matrix organization and to protect growth plate chondrocytes from cellular stress

CCN2 (connective tissue growth factor (CTGF/CCN2)) is a matricellular protein that utilizes integrins to regulate cell proliferation, migration and survival. The loss of CCN2 leads to perinatal lethality resulting from a severe chondrodysplasia. Upon closer inspection of Ccn2 mutant mice, we observed defects in extracellular matrix (ECM) organization and hypothesized that the severe chondrodysplasia caused by loss of CCN2 might be associated with defective chondrocyte survival. Ccn2 mutant growth plate chondrocytes exhibited enlarged endoplasmic reticula (ER), suggesting cellular stress. Immunofluorescence analysis confirmed elevated stress in Ccn2 mutants, with reduced stress observed in Ccn2 overexpressing transgenic mice. In vitro studies revealed that Ccn2 is a stress responsive gene in chondrocytes. The elevated stress observed in Ccn2-/- chondrocytes is direct and mediated in part through integrin α5. The expression of the survival marker NFκB and components of the autophagy pathway were decreased in Ccn2 mutant growth plates, suggesting that CCN2 may be involved in mediating chondrocyte survival. These data demonstrate that absence of a matricellular protein can result in increased cellular stress and highlight a novel protective role for CCN2 in chondrocyte survival. The severe chondrodysplasia caused by the loss of CCN2 may be due to increased chondrocyte stress and defective activation of autophagy pathways, leading to decreased cellular survival. These effects may be mediated through nuclear factor κB (NFκB) as part of a CCN2/integrin/NFκB signaling cascade.

Cellular Photo Digital Breathalyzer for Monitoring Alcohol Use: A Pilot Study

Background: Monitoring alcohol use is important in numerous situations. Direct ethanol metabolites, such as ethyl glucuronide (EtG), have been shown to be useful tools in detecting alcohol use and documenting abstinence. For very frequent or continuous control of abstinence, they lack practicability. Therefore, devices measuring ethanol itself might be of interest. This pilot study aims at elucidating the usability and accuracy of the cellular photo digital breathalyzer (CPDB) compared to self-reports in a naturalistic setting. Method: 12 social drinkers were included. Subjects used a CPDB 4 times daily, kept diaries of alcohol use and submitted urine for EtG testing over a period of 5 weeks. Results: In total, the 12 subjects reported 84 drinking episodes. 1,609 breath tests were performed and 55 urine EtG tests were collected. Of 84 drinking episodes, CPDB detected 98.8%. The compliance rate for breath testing was 96%. Of the 55 EtG tests submitted, 1 (1.8%) was positive. Conclusions: The data suggest that the CPDB device holds promise in detecting high, moderate, and low alcohol intake. It seems to have advantages compared to biomarkers and other Monitoring devices. The preference for CPDB by the participants might explain the high compliance. Further studies including comparison with biomarkers and transdermal devices are needed.

Mobile Cloud Networking: Virtualisation of Cellular Networks

Virtualisation of cellular networks can be seen as a way to significantly reduce the complexity of processes, required nowadays to provide reliable cellular networks. The Future Communication Architecture for Mobile Cloud Services: Mobile Cloud Networking (MCN) is a EU FP7 Large-scale Integrating Project (IP) funded by the European Commission that is focusing on cloud computing concepts to achieve virtualisation of cellular networks. It aims at the development of a fully cloud-based mobile communication and application platform, or more specifically, it aims to investigate, implement and evaluate the technological foundations for the mobile communication system of Long Term Evolution (LTE), based on Mobile Network plus Decentralized Computing plus Smart Storage offered as one atomic service: On-Demand, Elastic and Pay-As-You-Go. This paper provides a brief overview of the MCN project and discusses the challenges that need to be solved.

Cardiac phenotype of Duchenne Muscular Dystrophy: Insights from cellular studies

Dilated cardiomyopathy is a serious and almost inevitable complication of Duchenne Muscular Dystrophy, a devastating and fatal disease of skeletal muscle resulting from the lack of functional dystrophin, a protein linking the cytoskeleton to the extracellular matrix. Ultimately, it leads to congestive heart failure and arrhythmias resulting from both cardiac muscle fibrosis and impaired function of the remaining cardiomyocytes. Here we summarize findings obtained in several laboratories, focusing on cellular mechanisms that result in degradation of cardiac functions in dystrophy. This article is part of a Special Issue entitled "Calcium Signaling in Heart".

THE EXPRESSION AND CELLULAR LOCALIZATION OF CC-CHEMOKINE RECEPTOR 5 (CCR5) AFTER TRAUMATIC BRAIN INJURY

Traumatic brain injury results from a primary insult and secondary events that together result in tissue injury. This primary injury occurs at the moment of impact and damage can include scalp laceration, skull fraction, cerebral contusions and lacerations as well as intracranial hemorrhage. Following the initial insult, a delayed response occurs and is characterized by hypoxia, ischemia, cerebral edema, and infection. During secondary brain injury, a series of neuroinflammatory events are triggered that can produce additional damage but may also help to protect nervous tissue from invading pathogens and help to repair the damaged tissue. Brain microglia and astrocytes become activated and migrate to the site of injury where these cells secrete immune mediators such as cytokines and chemokines. CC-chemokine receptor 5 (CCR5) is a member of the CC chemokine receptor family of seven transmembrane G protein coupled receptors. CCR5 is expressed in the immune system and is found in monocytes, leukoctyes, memory T cells, and immature dendritic cells. Upon binding to its ligands, CCR5 functions in the chemotaxis of these immune cells to the site of inflammation. In the CNS, CCR5 and its ligands are expressed in multiple cell types. In this study, I investigated whether CCR5 expression is altered in brain after traumatic brain injury. I examined the time course of CCR5 protein expression in cortex and hippocampus using quantitative western analysis of tissues from injured rat brain after mild impact injury. In addition, I also investigated the cellular localization of CCR5 before and after brain injury using confocal microscopy. I have observed that after brain injury CCR5 is upregulated in a time dependent manner in neurons of the parietal cortex and hippocampus. The absence of CCR5 expression in microglia and its delayed expression in neurons after injury suggests a role for CCR5 in neuronal survival after injury.

Convergent synthesis and cellular uptake of multivalent cell penetrating peptides derived from Tat, Antp, pVEC, TP10 and SAP

Cell penetrating peptides (CPP) are peptides of 10 to 30 residues derived from natural translocating proteins. Multivalency is known to enhance cellular uptake for the Tat peptide and closely related polycationic sequences. To test whether multivalency effects on cellular uptake might also occur with other CPP types, we prepared multivalent versions of the strongly cationic Tat, the amphipathic sequences Antp, pVEC and TP10, and the polyproline helix SAP by convergent thioether ligation of the linear CPP onto multivalent scaffolds, and evaluated their uptake in HeLa and CHO cells, intracellular localization, cytotoxicity and hemolysis. While multivalency did not increase the cellular uptake of pVEC or SAP, multivalency effects on uptake comparable to Tat were observed with TP10 and Antp, which are attributable to their polycationic nature. The efficient synthetic protocol for these divalent CPP and their localization in the cytoplasm suggest that CPP might be useful for application in cargo delivery into cells.

THE EXPRESSION AND CELLULAR LOCALIZATION OF CC-CHEMOKINE RECEPTOR 5 (CCR5) AFTER TRAUMATIC BRAIN INJURY

Traumatic brain injury results from a primary insult and secondary events that together result in tissue injury. This primary injury occurs at the moment of impact and damage can include scalp laceration, skull fraction, cerebral contusions and lacerations as well as intracranial hemorrhage. Following the initial insult, a delayed response occurs and is characterized by hypoxia, ischemia, cerebral edema, and infection. During secondary brain injury, a series of neuroinflammatory events are triggered that can produce additional damage but may also help to protect nervous tissue from invading pathogens and help to repair the damaged tissue. Brain microglia and astrocytes become activated and migrate to the site of injury where these cells secrete immune mediators such as cytokines and chemokines. CC-chemokine receptor 5 (CCR5) is a member of the CC chemokine receptor family of seven transmembrane G protein coupled receptors. CCR5 is expressed in the immune system and is found in monocytes, leukoctyes, memory T cells, and immature dendritic cells. Upon binding to its ligands, CCR5 functions in the chemotaxis of these immune cells to the site of inflammation. In the CNS, CCR5 and its ligands are expressed in multiple cell types. In this study, I investigated whether CCR5 expression is altered in brain after traumatic brain injury. I examined the time course of CCR5 protein expression in cortex and hippocampus using quantitative western analysis of tissues from injured rat brain after mild impact injury. In addition, I also investigated the cellular localization of CCR5 before and after brain injury using confocal microscopy. I have observed that after brain injury CCR5 is upregulated in a time dependent manner in neurons of the parietal cortex and hippocampus. The absence of CCR5 expression in microglia and its delayed expression in neurons after injury suggests a role for CCR5 in neuronal survival after injury.

Cellular uptake and localization of inhaled gold nanoparticles in lungs of mice with chronic obstructive pulmonary disease

BACKGROUND: Inhalative nanocarriers for local or systemic therapy are promising. Gold nanoparticles (AuNP) have been widely considered as candidate material. Knowledge about their interaction with the lungs is required, foremost their uptake by surface macrophages and epithelial cells.Diseased lungs are of specific interest, since these are the main recipients of inhalation therapy. We, therefore, used Scnn1b-transgenic (Tg) mice as a model of chronic obstructive pulmonary disease (COPD) and compared uptake and localization of inhaled AuNP in surface macrophages and lung tissue to wild-type (Wt) mice. METHODS: Scnn1b-Tg and Wt mice inhaled a 21-nm AuNP aerosol for 2 h. Immediately (0 h) or 24 h thereafter, bronchoalveolar lavage (BAL) macrophages and whole lungs were prepared for stereological analysis of AuNP by electron microscopy. RESULTS: AuNP were mainly found as singlets or small agglomerates of <= 100 nm diameter, at the epithelial surface and within lung-surface structures. Macrophages contained also large AuNP agglomerates (> 100 nm). At 0 h after aerosol inhalation, 69.2+/-4.9% AuNP were luminal, i.e. attached to the epithelial surface and 24.0+/-5.9% in macrophages in Scnn1b-Tg mice. In Wt mice, 35.3+/-32.2% AuNP were on the epithelium and 58.3+/-41.4% in macrophages. The percentage of luminal AuNP decreased from 0 h to 24 h in both groups. At 24 h, 15.5+/-4.8% AuNP were luminal, 21.4+/-14.2% within epithelial cells and 63.0+/-18.9% in macrophages in Scnn1b-Tg mice. In Wt mice, 9.5+/-5.0% AuNP were luminal, 2.2+/-1.6% within epithelial cells and 82.8+/-0.2% in macrophages. BAL-macrophage analysis revealed enhanced AuNP uptake in Wt animals at 0 h and in Scnn1b-Tg mice at 24 h, confirming less efficient macrophage uptake and delayed clearance of AuNP in Scnn1b-Tg mice. CONCLUSIONS: Inhaled AuNP rapidly bound to the alveolar epithelium in both Wt and Scnn1b-Tg mice. Scnn1b-Tg mice showed less efficient AuNP uptake by surface macrophages and concomitant higher particle internalization by alveolar type I epithelial cells compared to Wt mice. This likely promotes AuNP depth translocation in Scnn1b-Tg mice, including enhanced epithelial targeting. These results suggest AuNP nanocarrier delivery as successful strategy for therapeutic targeting of alveolar epithelial cells and macrophages in COPD.