Discovery of novel arenavirus receptors and entry factors

Arenaviruses are enveloped negative-strand RNA viruses that contain a bi-segmented genome. They are rodent-borne pathogens endemic to the Americas and Africa, with the exception of lymphocytic choriomeningitis virus (LCMV) that is world-wide distributed. The arenaviruses include numerous important human pathogens including the Old World arenavirus Lassa virus (LASV), the causative agent of a severe viral hemorrhagic fever in humans with several hundred thousand infections per year in Africa and thousands of deaths. Viruses are obligatory intracellular parasites, strictly depending on cellular processes and factors to complete their replication cycle. The binding of a virus to target cells is the first step of every viral infection, and is mainly mediated by viral proteins that can directly engage cellular receptors, providing a key determinant for viral tropism. This early step of infection represents a promising target to block the pathogen before it can take control over the host cell. Old World arenaviruses, such as LASV and LCMV, bind to host cells via attachment to their main receptor, dystroglycan (DG), an ubiquitous receptor for extracellular matrix proteins. The engagement of DG by LASV results in a fast internalization and transfer the virus to late endosomal compartment suggesting that the virus binding to DG causes marked changes in the dynamics of the receptor. These events could result in the clustering of the receptor and subsequent induction of signaling that could be modulated by the virus. Recently, numerous findings also suggest the presence of alternative receptor(s) for LASV in absence of the main DG receptor. In my first project, I was interested to investigate the effects of virus-receptor binding on the tyrosine phosphorylation of the cytoplasmic domain of DG and to test if this post-translational modification was crucial for the internalization of the LASV-receptor complex. We found that engagement of cellular DG by a recombinant LCMV expressing the envelope GP of LASV in human epithelial cells induced tyrosine phosphorylation of the cytoplasmic domain of DG. LASV GP binding to DG further resulted in dissociation of the adapter protein utrophin from virus-bound DG. Virus-induced dissociation of utrophin and consequent virus internalization were affected by the broadly specific tyrosine kinase inhibitor genistein. We speculate that the detachment of virus- bound DG from the actin-based cytoskeleton following DG phosphorylation may facilitate subsequent endocytosis of the virus-receptor complex. In the second project, I was interested to characterize the newly indentified LASV alternative receptor Axl in the context of productive arenavirus infection. In a first step, we demonstrated that Axl supports productive infection by rLCMV-LASVGP in a DG-independent manner. In line with previous studies, cell entry of rLCMV-LASVGP via Axl was less efficient when compared to functional DG. Interestingly, Axl-mediated infection showed rapid kinetics similar to DG-dependent entry. Using a panel of inhibitors, we found that Axl-mediated cell entry of rLCMV-LASVGP involved a clathrin-independent pathway that critically depended on actin and dynamin and was sensitive to EIPA but not to PAK inhibitors, compatible with a macropinocytosis-like mechanism of entry. In a next step, we aimed to investigate the molecular mechanism by which rLCMV-LASVGP recognizes Axl. Phosphatidylserine (PS) is the natural ligand of Axl via the adaptor protein Gas6. We detected the presence of PS in the envelope of Old World arenaviruses, suggesting that PS could mediate Axl-virus binding, in a mechanism of apoptotic mimicry already described for other viruses. Whether envelope PS and/or the GP of LASV plays any role in virus entry via Axl is still an open question. The molecular mechanisms underlying host cell-virus interaction are of particular interest to answer basic scientific questions as well as to apply key findings to translational research. Understanding pathogen induced-signaling and its link to invasion of the host cell is of great importance to develop drugs for therapeutic intervention against highly pathogenic viruses like LASV. - Les Arenavirus sont des virus enveloppés à ARN négatifs organisés sous forme de génome bisegmenté. Ils sont véhiculés par les rongeurs et se retrouvent de manière endémique aux Amériques et en Afrique avec l'exception du virus de la chorioméningite lymphocytaire (LCMV) qui lui est distribué mondialement. De nombreux pathogènes humains font parti de la famille des Arenavirus dont le virus de l'Ancien Monde Lassa (LASV), un agent responsable de fièvres hémorragiques sévères chez les humains. Le virus de Lassa cause plusieurs centaines de milliers d'infections par année en Afrique ainsi que des milliers de morts. De manière générale, les virus sont des parasites intracellulaires obligatoires qui dépendent strictement de processus et facteurs cellulaires pour clore leur cycle de réplication. L'attachement d'un virus à sa cellule cible représente la première étape de chaque infection virale et est principalement dirigée par des protéines virales qui interagissent directement avec leur récepteurs cellulaires respectifs fournissant ainsi un indicateur déterminant pour le tropisme d'un virus. Cette première étape de l'infection représente aussi une cible prometteuse pour bloquer le pathogène avant qu'il ne puisse prendre le contrôle de la cellule. Les Arenavirus de l'Ancien Monde comme LASV et LCMV s'attachent à la cellule hôte en se liant à leur récepteur principal, le dystroglycan (DG), un récepteur ubiquitaire pour les protéines de la matrice extracellulaire. La liaison du DG par LASV résulte en une rapide internalisation transférant le virus aux endosomes tardifs suggérant ainsi que l'attachement du virus au DG peut provoquer des changements marqués dans la dynamique moléculaire du récepteur. Ces événements sont susceptibles d'induire un regroupement du récepteur à la surface cellulaire, ainsi qu'une induction subséquente qui pourrait être, par la suite, modulée par le virus. Récemment, plusieurs découvertes suggèrent aussi la présence d'un récepteur alternatif pour LASV en l'absence du récepteur principal, le DG. Concernant mon premier projet, j'étais intéressée à étudier les effets de la liaison virus- récepteur sur la phosphorylation des acides aminés tyrosines se trouvant dans la partie cytoplasmique du DG, le but étant de tester si cette modification post-translationnelle était cruciale pour Γ internalisation du complexe LASV-DG récepteur. Nous avons découvert que l'engagement du récepteur DG par le virus recombinant LCMV, exprimant la glycoprotéine de LASV, dans des cellules épithéliales humaines induit une phosphorylation de résidu(s) tyrosine se situant dans le domaine cytoplasmique du DG. La liaison de la glycoprotéine de LASV au DG induit par la suite la dissociation de la protéine adaptatrice utrophine du complexe virus-DG récepteur. Nous avons observé que cette dissociation de l'utrophine, induite par le virus, ainsi que son internalisation, sont affectées par l'inhibiteur à large spectre des tyrosines kinases, la génistéine. Nous avons donc supposé que le détachement du virus, lié au récepteur DG, du cytosquelette d'actine suite à la phosphorylation du DG faciliterait l'endocytose subséquente du complexe virus-récepteur. Dans le second projet, j'étais intéressée à caractériser le récepteur alternatif Axl qui a été récemment identifié dans le contexte de l'infection productive des Arenavirus. Dans un premier temps, nous avons démontré que le récepteur alternatif Axl permet l'infection des cellules par le virus LCMV recombinant LASV indépendamment du récepteur DG. Conformément aux études publiées précédemment, nous avons pu observer que l'entrée du virus recombinant LASV via Axl est moins efficace que via le récepteur principal DG. De façon intéressante, nous avons aussi remarqué que l'infection autorisée par Axl manifeste une cinétique virale d'entrée similaire à celle observée avec le récepteur DG. Utilisant un éventail de différents inhibiteurs, nous avons trouvé que l'entrée du virus recombinant rLCMV-LASVGP via Axl implique une voie d'entrée indépendante de la clathrine et dépendant de manière critique de l'actine et de la dynamine. Cette nouvelle voie d'entrée est aussi sensible à l'EIPA contrairement aux inhibiteurs PAK indiquant un mécanisme d'entrée compatible avec un mécanisme de macropinocytose. L'étape suivante du projet a été d'investiguer le mécanisme moléculaire par lequel le virus recombinant rLCMV-LASVGP reconnaît le récepteur alternatif Axl. La phosphatidylsérine (PS) se trouve être un ligand naturel pour Axl via la protéine adaptatrice Gas6. Nous avons détecté la présence de PS dans l'enveloppe des Arenavirus du Vieux Monde suggérant que la PS pourrait médier la liaison du virus à Axl dans un mécanisme de mimétisme apoptotique déjà observé et décrit pour d'autres virus. Cependant, il reste encore à déterminer qui de la PS ou de la glycoprotéine de l'enveloppe virale intervient dans le processus d'entrée de LASV via le récepteur alternatif Axl. Les mécanismes moléculaires à la base de l'interaction entre virus et cellule hôte sont d'intérêts particuliers pour répondre aux questions scientifiques de base ainsi que dans l'application de découvertes clés pour la recherche translationnelle. La compréhension de la signalisation induite par les pathogènes ainsi que son lien à l'invasion de la cellule hôte est d'une importance considérable pour le développement de drogues pour l'intervention thérapeutique contre les virus hautement pathogènes comme LASV.

Non integrative lentiviral vactors for gene transfer in the retina

Purpose:Lentiviral vectors are among the most efficient gene transfer tools for both dividing and non dividing cells, including pigmented epithelial cells of the retina. One of the latest developments in the field, which represents a significant advance in biosafety, consists in the use of non integrative lentiviral vectors (NILVs). These newly described tools were already shown to be efficient in various tissues, such as the retina. They allow prolonged transgene expression as long as the transduced cells do not divide or divide slowly. However, they were also shown to induce transgene expression less efficiently than their integrative counterparts. Further investigations are thus needed to improve their potential. To this aim, different strategies are under evaluation. In this study, we focused on using different integrase mutations. Methods:We considered different integrase mutations, including modifications in the catalytic site and in the C-terminal domain of the enzyme. Lentiviral vectors bearing these mutant integrases and allowing expression of various transgenes were produced and characterized in vitro and in vivo. In particular, we evaluated their transgene expression capability. Influence of integrase mutation on the residual integration activity was also investigated. Results:In line with the fact that the lentiviral integrase is involved in several steps of the replication cycle of lentiviruses, we observed that integrase mutations can modify lentiviral vector features, resulting in different transduction efficiencies as well as modulation of the integration activity. Conclusions:NILVs appear as suitable tools for gene transfer in the retina, particularly to transduce RPE cells. They can be advantageously used, for instance, to develop neuroprotective strategies aimed at rescuing photoreceptors from death in various retinal diseases.

Quantitative impact of thymic clonal deletion on the T cell repertoire.

Interactions between major histocompatibility complex (MHC) molecules expressed on stromal cells and antigen-specific receptors on T cells shape the repertoire of mature T lymphocytes emerging from the thymus. Some thymocytes with appropriate receptors are stimulated to undergo differentiation to the fully mature state (positive selection), whereas others with strongly autoreactive receptors are triggered to undergo programmed cell death before completing this differentiation process (negative selection). The quantitative impact of negative selection on the potentially available repertoire is currently unknown. To address this issue, we have constructed radiation bone marrow chimeras in which MHC molecules are present on radioresistant thymic epithelial cells (to allow positive selection) but absent from radiosensitive hematopoietic elements responsible for negative selection. In such chimeras, the number of mature thymocytes was increased by twofold as compared with appropriate control chimeras This increase in steady-state numbers of mature thymocytes was not related to proliferation, increased retention, or recirculation and was accompanied by a similar two- to threefold increase in the de novo rate of generation of mature cells. Taken together, our data indicate that half to two-thirds of the thymocytes able to undergo positive selection die before full maturation due to negative selection.

Antiviral antibodies target adenovirus to phagolysosomes and amplify the innate immune response.

Adenovirus is a nonenveloped dsDNA virus that activates intracellular innate immune pathways. In vivo, adenovirus-immunized mice displayed an enhanced innate immune response and diminished virus-mediated gene delivery following challenge with the adenovirus vector AdLacZ suggesting that antiviral Abs modulate viral interactions with innate immune cells. Under naive serum conditions in vitro, adenovirus binding and internalization in macrophages and the subsequent activation of innate immune mechanisms were inefficient. In contrast to the neutralizing effect observed in nonhematopoietic cells, adenovirus infection in the presence of antiviral Abs significantly increased FcR-dependent viral internalization in macrophages. In direct correlation with the increased viral internalization, antiviral Abs amplified the innate immune response to adenovirus as determined by the expression of NF-kappaB-dependent genes, type I IFNs, and caspase-dependent IL-1beta maturation. Immune serum amplified TLR9-independent type I IFN expression and enhanced NLRP3-dependent IL-1beta maturation in response to adenovirus, confirming that antiviral Abs specifically amplify intracellular innate pathways. In the presence of Abs, confocal microscopy demonstrated increased targeting of adenovirus to LAMP1-positive phagolysosomes in macrophages but not epithelial cells. These data show that antiviral Abs subvert natural viral tropism and target the adenovirus to phagolysosomes and the intracellular innate immune system in macrophages. Furthermore, these results illustrate a cross-talk where the adaptive immune system positively regulates the innate immune system and the antiviral state.

Ocular biocompatibility of novel Cyclosporin A formulations based on methoxy poly(ethylene glycol)-hexylsubstituted poly(lactide) micelle carriers.

Topical ocular drug delivery has always been a challenge for pharmaceutical technology scientists. In the last two decades, many nano-systems have been studied to find ways to overcome the typical problems of topical ocular therapy, such as difficult corneal penetration and poor drug availability. In this study, methoxy poly(ethylene glycol)-hexylsubstituted poly(lactides) (MPEG-hexPLA) micelle formulations, which are promising nanocarriers for poorly water soluble drugs, were investigated for the delivery of Cyclosporin A (CsA) to the eye. As a new possible pharmaceutical excipient, the ocular compatibility of MPEG-hexPLA micelle formulations was evaluated. An in vitro biocompatibility assessment on human corneal epithelial cells was carried out using different tests. Cytotoxicity was studied by using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT), and clonogenic tests and revealed that the CsA formulations and copolymer solutions were not toxic. After incubation with MPEG-hexPLA micelle formulations, the activation of caspase-dependent and -independent apoptosis as well as autophagy was evaluated using immunohistochemistry by analyzing the localization of four antibodies: (1) anti-caspase 3; (2) anti-apoptotic inducing factor (AIF); (3) anti-IL-Dnase II and (4) anti-microtubule-associated protein 1 light chain 3 (LC3). No apoptosis was induced when the cells were treated with the micelle solutions that were either unloaded or loaded with CsA. The ocular tolerance was assessed in vivo on rabbit eyes by Confocal Laser Scanning Ophthalmoscopy (CLSO), and very good tolerability was seen. The observed corneal surface was comparable to a control surface that was treated with a 0.9% NaCl solution. In conclusion, these results demonstrate that MPEG-hexPLA micelles are promising drug carriers for ocular diseases involving the activation of cytokines, such as dry eye syndrome and autoimmune uveitis, or for the prevention of corneal graft rejection.

Alzheimer disease: curly fibers and tangles in organs other than brain.

The filamentous brain lesions that define Alzheimer disease (AD) consist of senile plaques and neurofibrillary tangles. Undulated pathological filaments--curly fibers or neuropil threads--also occur in the neuropil. Beta-amyloid precursor proteins are synthesized by many cells outside the central nervous system and recently, deposition of beta-amyloid-protein was reported to occur in non-neuronal tissues. In addition, increasing data claim the importance of chronic inflammation in the pathogenesis of AD. These observations suggest that AD may be a widespread systemic disorder. Here we report that pathological argyrophilic filaments with histochemical properties of amyloid showing striking morphological similarity to curly fibers and/or tangles accumulate not only in ependymal layer and in epithelial cells of choroid plexus, but also in several other organs (e.g. liver, pancreas, ovary, testis, thyroid) in AD. The ependyma, choroid plexus, and various organs of 39 autopsy cases were analyzed. In search of curly fiber and tangle-like changes in organs other than brain, 395 blocks from 21 different tissues of 24 AD cases, 5 cases with discrete or moderate AD-type changes, and 10 control cases were investigated. We found in non-neuronal cells "curly fibers" or "tangles" immunoreactive with antibodies to P component, Tau-protein, ubiquitin, fibronectin, and Apolipoprotein-E, but lacking immunoreactivity with antibodies to neurofilament proteins. Ultrastructurally they consist of densely packed straight and paired helical filaments and closely resemble neurofibrillary tangles and neuropil threads. These observations indicate that the formation of "curly fibers" and "tangles" is not unique to the central nervous system. The results suggest that AD might be a systemic disorder or that similar fibrillary changes to tangles and curly fibers may also be associated with other amyloidosis than beta-amyloidosis. Further investigations are necessary to understand the pathogenetic interest of these fibrillary changes outside the CNS.

Cycle length of spermatogenesis in shrews (mammalia: soricidae) with high and low metabolic rates and different mating systems.

The aim of the present study was to establish and compare the durations of the seminiferous epithelium cycles of the common shrew Sorex araneus, which is characterized by a high metabolic rate and multiple paternity, and the greater white-toothed shrew Crocidura russula, which is characterized by a low metabolic rate and a monogamous mating system. Twelve S. araneus males and fifteen C. russula males were injected intraperitoneally with 5-bromodeoxyuridine, and the testes were collected. For cycle length determinations, we applied the classical method of estimation and linear regression as a new method. With regard to variance, and even with a relatively small sample size, the new method seems to be more precise. In addition, the regression method allows the inference of information for every animal tested, enabling comparisons of different factors with cycle lengths. Our results show that not only increased testis size leads to increased sperm production, but it also reduces the duration of spermatogenesis. The calculated cycle lengths were 8.35 days for S. araneus and 12.12 days for C. russula. The data obtained in the present study provide the basis for future investigations into the effects of metabolic rate and mating systems on the speed of spermatogenesis.

Importance of ENaC-mediated sodium transport in alveolar fluid clearance using genetically-engineered mice.

The lung possesses specific transport systems that intra- and extracellularly maintain salt and fluid balance necessary for its function. At birth, the lungs rapidly transform into a fluid (Na(+))-absorbing organ to enable efficient gas exchange. Alveolar fluid clearance, which mainly depends on sodium transport in alveolar epithelial cells, is an important mechanism by which excess water in the alveoli is reabsorbed during the resolution of pulmonary edema. In this review, we will focus and summarize on the role of ENaC in alveolar lung liquid clearance and discuss recent data from mouse models with altered activity of epithelial sodium channel function in the lung, and more specifically in alveolar fluid clearance. Recent data studying mice with hyperactivity of ENaC or mice with reduced ENaC activity clearly illustrate the impaired lung fluid clearance in these adult mice. Further understanding of the physiological role of ENaC and its regulatory proteins implicated in salt and water balance in the alveolar cells may therefore help to develop new therapeutic strategies to improve gas exchange in pulmonary edema.

Potentiation of polarized intestinal Caco-2 cell responsiveness to probiotics complexed with secretory IgA.

The precise mechanisms underlying the interaction between intestinal bacteria and the host epithelium lead to multiple consequences that remain poorly understood at the molecular level. Deciphering such events can provide valuable information as to the mode of action of commensal and probiotic microorganisms in the gastrointestinal environment. Potential roles of such microorganisms along the privileged target represented by the mucosal immune system include maturation prior, during and after weaning, and the reduction of inflammatory reactions in pathogenic conditions. Using human intestinal epithelial Caco-2 cell grown as polarized monolayers, we found that association of a Lactobacillus or a Bifidobacterium with nonspecific secretory IgA (SIgA) enhanced probiotic adhesion by a factor of 3.4-fold or more. Bacteria alone or in complex with SIgA reinforced transepithelial electrical resistance, a phenomenon coupled with increased phosphorylation of tight junction proteins zonula occludens-1 and occludin. In contrast, association with SIgA resulted in both enhanced level of nuclear translocation of NF-κB and production of epithelial polymeric Ig receptor as compared with bacteria alone. Moreover, thymic stromal lymphopoietin production was increased upon exposure to bacteria and further enhanced with SIgA-based complexes, whereas the level of pro-inflammatory epithelial cell mediators remained unaffected. Interestingly, SIgA-mediated potentiation of the Caco-2 cell responsiveness to the two probiotics tested involved Fab-independent interaction with the bacteria. These findings add to the multiple functions of SIgA and underscore a novel role of the antibody in interaction with intestinal bacteria.

Intestinal antiinflammatory effect of 5-aminosalicylic acid is dependent on peroxisome proliferator-activated receptor-gamma.

5-aminosalicylic acid (5-ASA) is an antiinflammatory drug widely used in the treatment of inflammatory bowel diseases. It is known to inhibit the production of cytokines and inflammatory mediators, but the mechanism underlying the intestinal effects of 5-ASA remains unknown. Based on the common activities of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligands and 5-ASA, we hypothesized that this nuclear receptor mediates 5-ASA therapeutic action. To test this possibility, colitis was induced in heterozygous PPAR-gamma(+/-) mice and their wild-type littermates, which were then treated with 5-ASA. 5-ASA treatment had a beneficial effect on colitis only in wild-type and not in heterozygous mice. In epithelial cells, 5-ASA increased PPAR-gamma expression, promoted its translocation from the cytoplasm to the nucleus, and induced a modification of its conformation permitting the recruitment of coactivators and the activation of a peroxisome-proliferator response element-driven gene. Validation of these results was obtained with organ cultures of human colonic biopsies. These data identify PPAR-gamma as a target of 5-ASA underlying antiinflammatory effects in the colon.

Global phylogenomic analysis of nonencapsulated Streptococcus pneumoniae reveals a deep-branching classic lineage that is distinct from multiple sporadic lineages.

The surrounding capsule of Streptococcus pneumoniae has been identified as a major virulence factor and is targeted by pneumococcal conjugate vaccines (PCV). However, nonencapsulated S. pneumoniae (non-Ec-Sp) have also been isolated globally, mainly in carriage studies. It is unknown if non-Ec-Sp evolve sporadically, if they have high antibiotic nonsusceptiblity rates and a unique, specific gene content. Here, whole-genome sequencing of 131 non-Ec-Sp isolates sourced from 17 different locations around the world was performed. Results revealed a deep-branching classic lineage that is distinct from multiple sporadic lineages. The sporadic lineages clustered with a previously sequenced, global collection of encapsulated S. pneumoniae (Ec-Sp) isolates while the classic lineage is comprised mainly of the frequently identified multilocus sequences types (STs) ST344 (n = 39) and ST448 (n = 40). All ST344 and nine ST448 isolates had high nonsusceptiblity rates to β-lactams and other antimicrobials. Analysis of the accessory genome reveals that the classic non-Ec-Sp contained an increased number of mobile elements, than Ec-Sp and sporadic non-Ec-Sp. Performing adherence assays to human epithelial cells for selected classic and sporadic non-Ec-Sp revealed that the presence of a integrative conjugative element (ICE) results in increased adherence to human epithelial cells (P = 0.005). In contrast, sporadic non-Ec-Sp lacking the ICE had greater growth in vitro possibly resulting in improved fitness. In conclusion, non-Ec-Sp isolates from the classic lineage have evolved separately. They have spread globally, are well adapted to nasopharyngeal carriage and are able to coexist with Ec-Sp. Due to continued use of PCV, non-Ec-Sp may become more prevalent.

Fas (CD95/APO-1) limits the expansion of T lymphocytes in an environment of limited T-cell antigen receptor/MHC contacts.

Fas-deficient mice (Fas(lpr/lpr)) and humans have profoundly dysregulated T lymphocyte homeostasis, which manifests as an accumulation of CD4(+) and CD8(+) T cells as well as an unusual population of CD4(-)CD8(-)TCRαβ(+) T cells. To date, no unifying model has explained both the increased T-cell numbers and the origin of the CD4(-)CD8(-)TCRαβ(+) T cells. As Fas(lpr/lpr) mice raised in a germ-free environment still manifest lymphadenopathy, we considered that this process is primarily driven by recurrent low-avidity TCR signaling in response to self-peptide/MHC as occurs during homeostatic proliferation. In these studies, we developed two independent systems to decrease the number of self-peptide/MHC contacts. First, expression of MHC class I was reduced in OT-I TCR transgenic mice. Although OT-I Fas(lpr/lpr) mice did not develop lymphadenopathy characteristic of Fas(lpr/lpr) mice, in the absence of MHC class I, OT-I Fas(lpr/lpr) T cells accumulated as both CD8(+) and CD4(-)CD8(-) T cells. In the second system, re-expression of β(2)m limited to thymic cortical epithelial cells of Fas(lpr/lpr) β(2)m-deficient mice yielded a model in which polyclonal CD8(+) thymocytes entered a peripheral environment devoid of MHC class I. These mice accumulated significantly greater numbers of CD4(-)CD8(-)TCRαβ(+) T cells than conventional Fas(lpr/lpr) mice. Thus, Fas shapes the peripheral T-cell repertoire by regulating the survival of a subset of T cells proliferating in response to limited self-peptide/MHC contacts.

Rapid fluidic exchange microsystem for recording of fast ion channel kinetics in Xenopus oocytes.

We present a new lab-on-a-chip system for electrophysiological measurements on Xenopus oocytes. Xenopus oocytes are widely used host cells in the field of pharmacological studies and drug development. We developed a novel non-invasive technique using immobilized non-devitellinized cells that replaces the traditional "two-electrode voltage-clamp" (TEVC) method. In particular, rapid fluidic exchange was implemented on-chip to allow recording of fast kinetic events of exogenous ion channels expressed in the cell membrane. Reducing fluidic exchange times of extracellular reagent solutions is a great challenge with these large millimetre-sized cells. Fluidic switching is obtained by shifting the laminar flow interface in a perfusion channel under the cell by means of integrated poly-dimethylsiloxane (PDMS) microvalves. Reagent solution exchange times down to 20 ms have been achieved. An on-chip purging system allows to perform complex pharmacological protocols, making the system suitable for screening of ion channel ligand libraries. The performance of the integrated rapid fluidic exchange system was demonstrated by investigating the self-inhibition of human epithelial sodium channels (ENaC). Our results show that the response time of this ion channel to a specific reactant is about an order of magnitude faster than could be estimated with the traditional TEVC technique.

Postmortem diagnosis of hypothermia.

The identification of hypothermia as the cause of death has always been somewhat problematic in forensic pathology because of unspecific, inconstant, or even negative macroscopic and microscopic findings. Though the simultaneous presence of frost erythema, Wischnewski spots, hemorrhages into the synovial membrane, bloody discoloration of synovial fluid of the knee, and basal vacuolization of the renal tubular epithelial cells has been indicated as strongly supportive of fatal hypothermia, their absence does not allow the diagnosis of hypothermia to be ruled out. Postmortem biochemical investigations are valuable in detecting adaptation responses to cold stress and metabolic changes that occur following cold exposure. However, ethanol intoxication prevents appearance of adaptation responses to cold, rendering the diagnosis less obvious. Immunohistochemistry, postmortem imaging, and molecular pathology have shown promising results, although at present, they do not provide pathognomonic signs of fatal hypothermia. The aim of this article is to present a review of the literature covering the significance of different postmortem investigations that are associated with hypothermia fatalities.

Defining the genomic signature of the parous breast.

ABSTRACT: BACKGROUND: It is accepted that a woman's lifetime risk of developing breast cancer after menopause is reduced by early full term pregnancy and multiparity. This phenomenon is thought to be associated with the development and differentiation of the breast during pregnancy. METHODS: In order to understand the underlying molecular mechanisms of pregnancy induced breast cancer protection, we profiled and compared the transcriptomes of normal breast tissue biopsies from 71 parous (P) and 42 nulliparous (NP) healthy postmenopausal women using Affymetrix Human Genome U133 Plus 2.0 arrays. To validate the results, we performed real time PCR and immunohistochemistry. RESULTS: We identified 305 differentially expressed probesets (208 distinct genes). Of these, 267 probesets were up- and 38 down-regulated in parous breast samples; bioinformatics analysis using gene ontology enrichment revealed that up-regulated genes in the parous breast represented biological processes involving differentiation and development, anchoring of epithelial cells to the basement membrane, hemidesmosome and cell-substrate junction assembly, mRNA and RNA metabolic processes and RNA splicing machinery. The down-regulated genes represented biological processes that comprised cell proliferation, regulation of IGF-like growth factor receptor signaling, somatic stem cell maintenance, muscle cell differentiation and apoptosis. CONCLUSIONS: This study suggests that the differentiation of the breast imprints a genomic signature that is centered in the mRNA processing reactome. These findings indicate that pregnancy may induce a safeguard mechanism at post-transcriptional level that maintains the fidelity of the transcriptional process.