NGS Nominated CELA1, HSPG2, and KCNK5 as Candidate Genes for Predisposition to Balkan Endemic Nephropathy

Balkan endemic nephropathy (BEN) is a familial chronic tubulointerstitial disease with insidious onset and slow progression leading to terminal renal failure. The results of molecular biological investigations propose that BEN is a multifactorial disease with genetic predisposition to environmental risk agents. Exome sequencing of 22 000 genes with Illumina Nextera Exome Enrichment Kit was performed on 22 DNA samples (11 Bulgarian patients and 11 Serbian patients). Software analysis was performed via NextGene, Provean, and PolyPhen. The frequency of all annotated genetic variants with deleterious/damaging effect was compared with those of European populations. Then we focused on nonannotated variants (with no data available about them and not found in healthy Bulgarian controls). There is no statistically significant difference between annotated variants in BEN patients and European populations. From nonannotated variants with more than 40% frequency in both patients' groups, we nominated 3 genes with possible deleterious/damaging variants-CELA1, HSPG2, and KCNK5. Mutant genes (CELA1, HSPG2, and KCNK5) in BEN patients encode proteins involved in basement membrane/extracellular matrix and vascular tone, tightly connected to process of angiogenesis. We suggest that an abnormal process of angiogenesis plays a key role in the molecular pathogenesis of BEN.

The pre- and post-somatic segments of the human type I spiral ganglion neurons--structural and functional considerations related to cochlear implantation.

Human auditory nerve afferents consist of two separate systems; one is represented by the large type I cells innervating the inner hair cells and the other one by the small type II cells innervating the outer hair cells. Type I spiral ganglion neurons (SGNs) constitute 96% of the afferent nerve population and, in contrast to other mammals, their soma and pre- and post-somatic segments are unmyelinated. Type II nerve soma and fibers are unmyelinated. Histopathology and clinical experience imply that human SGNs can persist electrically excitable without dendrites, thus lacking connection to the organ of Corti. The biological background to this phenomenon remains elusive. We analyzed the pre- and post-somatic segments of the type I human SGNs using immunohistochemistry and transmission electron microscopy (TEM) in normal and pathological conditions. These segments were found surrounded by non-myelinated Schwann cells (NMSCs) showing strong intracellular expression of laminin-β2/collagen IV. These cells also bordered the perikaryal entry zone and disclosed surface rugosities outlined by a folded basement membrane (BM) expressing laminin-β2 and collagen IV. It is presumed that human large SGNs are demarcated by three cell categories: (a) myelinated Schwann cells, (b) NMSCs and (c) satellite glial cells (SGCs). Their BMs express laminin-β2/collagen IV and reaches the BM of the sensory epithelium at the habenula perforata. We speculate that the NMSCs protect SGNs from further degeneration following dendrite loss. It may give further explanation why SGNs can persist as electrically excitable monopolar cells even after long-time deafness, a blessing for the deaf treated with cochlear implantation.

The heparan sulfate proteoglycan agrin contributes to barrier properties of mouse brain endothelial cells by stabilizing adherens junctions

Barrier characteristics of brain endothelial cells forming the blood-brain barrier (BBB) are tightly regulated by cellular and acellular components of the neurovascular unit. During embryogenesis, the accumulation of the heparan sulfate proteoglycan agrin in the basement membranes ensheathing brain vessels correlates with BBB maturation. In contrast, loss of agrin deposition in the vasculature of brain tumors is accompanied by the loss of endothelial junctional proteins. We therefore wondered whether agrin had a direct effect on the barrier characteristics of brain endothelial cells. Agrin increased junctional localization of vascular endothelial (VE)-cadherin, β-catenin, and zonula occludens-1 (ZO-1) but not of claudin-5 and occludin in the brain endothelioma cell line bEnd5 without affecting the expression levels of these proteins. This was accompanied by an agrin-induced reduction of the paracellular permeability of bEnd5 monolayers. In vivo, the lack of agrin also led to reduced junctional localization of VE-cadherin in brain microvascular endothelial cells. Taken together, our data support the notion that agrin contributes to barrier characteristics of brain endothelium by stabilizing the adherens junction proteins VE-cadherin and β-catenin and the junctional protein ZO-1 to brain endothelial junctions.

deltaNp63 has a role in maintaining epithelial integrity in airway epithelium.

The upper airways are lined with a pseudostratified bronchial epithelium that forms a barrier against unwanted substances in breathing air. The transcription factor p63, which is important for stratification of skin epithelium, has been shown to be expressed in basal cells of the lungs and its ΔN isoform is recognized as a key player in squamous cell lung cancer. However, the role of p63 in formation and maintenance of bronchial epithelia is largely unknown. The objective of the current study was to determine the expression pattern of the ΔN and TA isoforms of p63 and the role of p63 in the development and maintenance of pseudostratified lung epithelium in situ and in culture. We used a human bronchial epithelial cell line with basal cell characteristics (VA10) to model bronchial epithelium in an air-liquid interface culture (ALI) and performed a lentiviral-based silencing of p63 to characterize the functional and phenotypic consequences of p63 loss. We demonstrate that ΔNp63 is the major isoform in the human lung and its expression was exclusively found in the basal cells lining the basement membrane of the bronchial epithelium. Knockdown of p63 affected proliferation and migration of VA10 cells and facilitated cellular senescence. Expression of p63 is critical for epithelial repair as demonstrated by wound healing assays. Importantly, generation of pseudostratified VA10 epithelium in the ALI setup depended on p63 expression and goblet cell differentiation, which can be induced by IL-13 stimulation, was abolished by the p63 knockdown. After knockdown of p63 in primary bronchial epithelial cells they did not proliferate and showed marked senescence. We conclude that these results strongly implicate p63 in the formation and maintenance of differentiated pseudostratified bronchial epithelium.

Late Quaternary history of the Vakinankaratra volcanic field (central Madagascar): insights from luminescence dating of phreatomagmatic eruption deposits

The Quaternary Vakinankaratra volcanic field in the central Madagascar highlands consists of scoria cones, lava flows, tuff rings, and maars. These volcanic landforms are the result of processes triggered by intracontinental rifting and overlie Precambrian basement or Neogene volcanic rocks. Infrared-stimulated luminescence (IRSL) dating was applied to 13 samples taken from phreatomagmatic eruption deposits in the Antsirabe–Betafo region with the aim of constraining the chronology of the volcanic activity. Establishing such a chronology is important for evaluating volcanic hazards in this densely populated area. Stratigraphic correlations of eruption deposits and IRSL ages suggest at least five phreatomagmatic eruption events in Late Pleistocene times. In the Lake Andraikiba region, two such eruption layers can be clearly distinguished. The older one yields ages between 109 ± 15 and 90 ± 11 ka and is possibly related to an eruption at the Amboniloha volcanic complex to the north. The younger one gives ages between 58 ± 4 and 47 ± 7 ka and is clearly related to the phreatomagmatic eruption that formed Lake Andraikiba. IRSL ages of a similar eruption deposit directly overlying basement laterite in the vicinity of the Fizinana and Ampasamihaiky volcanic complexes yield coherent ages of 68 ± 7 and 65 ± 8 ka. These ages provide the upper age limit for the subsequently developed Iavoko, Antsifotra, and Fizinana scoria cones and their associated lava flows. Two phreatomagmatic deposits, identified near Lake Tritrivakely, yield the youngest IRSL ages in the region, with respective ages of 32 ± 3 and 19 ± 2 ka. The reported K-feldspar IRSL ages are the first recorded numerical ages of phreatomagmatic eruption deposits in Madagascar, and our results confirm the huge potential of this dating approach for reconstructing the volcanic activity of Late Pleistocene to Holocene volcanic provinces.

Primary human lung pericytes support and stabilize in-vitro perfusable microvessels.

The formation of blood vessels is a complex tissue-specific process that plays a pivotal role during developmental processes, in wound healing, cancer progression, fibrosis and other pathologies. To study vasculogenesis and vascular remodeling in the context of the lung, we developed an in-vitro microvascular model that closely mimics the human lung microvasculature in terms of 3D architecture, accessibility, functionality and cell types. Human pericytes from the distal airway were isolated and characterized using flow cytometry. To assess their role in the generation of normal microvessels, lung pericytes were mixed in fibrin gel and seeded into well-defined microcompartments together with primary endothelial cells (HUVEC). Patent microvessels covering an area of 3.1 mm2 formed within 3-5 days and were stable for up to 14 days. Soluble signals from the lung pericytes were necessary to establish perfusability, and pericytes migrated towards endothelial microvessels. Cell-cell communication in the form of adherens and tight junctions, as well as secretion of basement membrane was confirmed using transmission electron microscopy and immunocytochemistry on chip. Direct co-culture of pericytes with endothelial cells decreased the microvascular permeability by one order of magnitude from 17.8∙10-6 cm/s to 2.0∙10-6 cm/s and led to vessels with significantly smaller and less variable diameter. Upon phenylephrine administration, vasoconstriction was observed in microvessels lined with pericytes but not in endothelial microvessels only. Perfusable microvessels were also generated with human lung microvascular endothelial cells and lung pericytes. Human lung pericytes were thus shown to have a prominent influence on microvascular morphology, permeability, vasoconstriction and long-term stability in an in-vitro microvascular system. This biomimetic platform opens new possibilities to test functions and interactions of patient-derived cells in a physiologically relevant microvascular setting.

Kollektive Erinnerung. Topographie und Topik in Walthers ›Palästinalied‹

The French sociologist Maurice Halbwachs (1877–1945) conceived re- membrance as a product of ›collective memory‹ and explained this idea in his book on ›La Topographie légendaire des Évangiles en Terre sainte‹ (1941) showing that the topography of the Holy Land was predominantly an imaginary landscape construed by Christian communities. Following this concept, this article studies the ›Palästinalied‹, a text describing the arrival of a pilgrim in the Holy Land in the time of the crusades, abundantly transmitted under the name of Walther von der Vogelweide. The high degree of textual variance in the diverse manuscripts testifies the acting of ›collective memory‹ in the medieval poetic tradition. Of special interest in this context are the strophic arrangements, the variation of deictic markers, the reworking of melodic models documented in the manuscript transmission and the diatopic opposition existing between the emphasis of ›distant love‹ expressed in Jaufré Rudel’s Occitan song ›Lanqand li jorn son lonc en mai‹ (one of the named models) and the attitude of proximity prevailing in the ›Palästinalied‹.

The tectonometamorphic evolution of the Sesia–Dent Blanche nappes (internal Western Alps): review and synthesis

This study reviews and synthesizes the present knowledge on the Sesia–Dent Blanche nappes, the highest tectonic elements in the Western Alps (Switzerland and Italy), which comprise pieces of pre-Alpine basement and Mesozoic cover. All of the available data are integrated in a crustal-scale kinematic model with the aim to reconstruct the Alpine tectono-metamorphic evolution of the Sesia–Dent Blanche nappes. Although major uncertainties remain in the pre-Alpine geometry, the basement and cover sequences of the Sesia–Dent Blanche nappes are seen as part of a thinned continental crust derived from the Adriatic margin. The earliest stages of the Alpine evolution are interpreted as recording late Cretaceous subduction of the Adria-derived Sesia–Dent Blanche nappes below the South-Alpine domain. During this subduction, several sheets of crustal material were stacked and separated by shear zones that rework remnants of their Mesozoic cover. The recently described Roisan-Cignana Shear Zone of the Dent Blanche Tectonic System represents such a shear zone, indicating that the Sesia–Dent Blanche nappes represent a stack of several individual nappes. During the subsequent subduction of the Piemonte–Liguria Ocean large-scale folding of the nappe stack (including the Roisan-Cignana Shear Zone) took place under greenschist facies conditions, which indicates partial exhumation of the Dent Blanche Tectonic System. The entrance of the Briançonnais micro-continent within the subduction zone led to a drastic change in the deformation pattern of the Alpine belt, with rapid exhumation of the eclogite-facies ophiolite bearing units and thrust propagation towards the foreland. Slab breakoff probably was responsible for allowing partial melting in the mantle and Oligocene intrusions into the most internal parts of the Sesia–Dent Blanche nappes. Finally, indentation of the Adriatic plate into the orogenic wedge resulted in the formation of the Vanzone back-fold, which marks the end of the pervasive ductile deformation within the Sesia–Dent Blanche nappes during the earliest Miocene.

Geometry and kinematics of the Roisan-Cignana Shear Zone, and the orogenic evolution of the Dent Blanche Tectonic System (Western Alps)

The Dent Blanche Tectonic System (DBTS) is a composite thrust sheet derived from the previously thinned passive Adriatic continental margin. A kilometric high-strain zone, the Roisan-Cignana Shear Zone (RCSZ) defines the major tectonic boundary within the DBTS and separates it into two subunits, the Dent Blanche s.s. nappe to the northwest and the Mont Mary nappe to the southeast. Within this shear zone, tectonic slices of Mesozoic and pre-Alpine meta-sediments became amalgamated with continental basement rocks of the Adriatic margin. The occurrence of high pressure assemblages along the contact between these tectonic slices indicates that the amalgamation occurred prior to or during the subduction process, at an early stage of the Alpine orogenic cycle. Detailed mapping, petrographic and structural analysis show that the Roisan-Cignana Shear Zone results from several superimposed Alpine structural and metamorphic stages. Subduction of the continental fragments is recorded by blueschist-facies deformation, whereas the Alpine collision is reflected by a greenschist facies overprint associated with the development of large-scale open folds. The postnappe evolution comprises the development of low-angle brittle faults, followed by large-scale folding (Vanzone phase) and finally brittle extensional faults. The RCSZ shows that fragments of continental crust had been torn off the passive continental margin prior to continental collision, thus recording the entire history of the orogenic cycle. The role of preceding Permo-Triassic lithospheric thinning, Jurassic rifting, and ablative subduction processes in controlling the removal of crustal fragments from the reactivated passive continental margin is discussed. Results of this study constrain the temporal sequence of the tectono-metamorphic processes involved in the assembly of the DBTS, but they also show limits on the interpretation. In particular it remains difficult to judge to what extent precollisional rifting at the Adriatic continental margin preconditioned the efficiency of convergent processes, i.e. accretion, subduction, and orogenic exhumation.

Multiple metamorphic stages within an eclogite-facies terrane (Sesia Zone, Western Alps) revealed by Th-U-Pb petrochronology

Convergent plate margins typically experience a transition from subduction to collision dynamics as massive continental blocks enter the subduction channel. Studies of high-pressure rocks indicate that tectonic fragments are rapidly exhumed from eclogite facies to midcrustal levels, but the details of such dynamics are controversial.To understand the dynamics of a subduction channel we report the results of a petrochronological study from the central Sesia Zone, a key element of the internalWestern Alps.This comprises two polymetamorphic basement complexes (Eclogitic Micaschist Complex and Gneiss Minuti Complex) and a thin, dismembered cover sequence (Scalaro Unit) associated with pre-Alpine metagabbros and metasediments (Bonze Unit). Structurally controlled samples from three of these units (Eclogitic Micaschist Complex and Scalaro-Bonze Units) yield unequivocal petrological and geochronological evidence of two distinct high-pressure stages. Ages (U-Th-Pb) of growth zones in accessory allanite and zircon, combined with inclusion and textural relationships, can be tied to the multi-stage evolution of single samples.Two independent tectono-metamorphic ‘slices’ showing a coherent metamorphic evolution during a given time interval have been recognized: the Fondo slice (which includes Scalaro and Bonze rocks) and the Druer slice (belonging to the Eclogitic Micaschist Complex).The new data indicate separate stages of deformation at eclogite-facies conditions for each recognized independent kilometer-sized tectono-metamorphic slice, between ~85 and 60 Ma, with evidence of intermittent decompression (∆P~0.5 GPa) within only the Fondo slice. The evolution path of the Druer slice indicates a different P-T-time evolution with prolonged eclogite-facies metamorphism between ~85 and 75Ma. Our approach, combining structural, petrological and geochronological techniques, yields field-based constraints on the duration and rates of dynamics within a subduction channel.

Geological map of the Scalaro Valley – Sesia Zone (Italian Western Alps)

In the Sesia Zone (Italian Western Alps), slivers of continental crust characterised by an Alpine high-pressure imprint are intermingled with abundant mafic rocks and Mesozoic metasediments. An extensive study of the central Sesia Zone was undertaken to identify and reconstruct the lithological setting of the mono-cyclic sediments of the Scalaro Unit. A new geological map (1:5000) and schematic cross sections across the Scalaro Unit and the adjoining Eclogitic Micaschist Complex are presented here. In order to delimit the size and shape of the mono-metamorphic unit and understand its internal geometry with respect to the poly-metamorphic basement, an integrated approach was used. Linking observations and data across a range of scales, from kilometres in the field down to petrological and chronological data obtained at micrometre scale, we define for the first time the real size and internal geometry of the Scalaro Unit, as well as its large-scale structural context.

Quantitative Analysis of Mouse Retinal Layers Using Automated Segmentation of Spectral Domain Optical Coherence Tomography Images.

PURPOSE Quantification of retinal layers using automated segmentation of optical coherence tomography (OCT) images allows for longitudinal studies of retinal and neurological disorders in mice. The purpose of this study was to compare the performance of automated retinal layer segmentation algorithms with data from manual segmentation in mice using the Spectralis OCT. METHODS Spectral domain OCT images from 55 mice from three different mouse strains were analyzed in total. The OCT scans from 22 C57Bl/6, 22 BALBc, and 11 C3A.Cg-Pde6b(+)Prph2(Rd2) /J mice were automatically segmented using three commercially available automated retinal segmentation algorithms and compared to manual segmentation. RESULTS Fully automated segmentation performed well in mice and showed coefficients of variation (CV) of below 5% for the total retinal volume. However, all three automated segmentation algorithms yielded much thicker total retinal thickness values compared to manual segmentation data (P < 0.0001) due to segmentation errors in the basement membrane. CONCLUSIONS Whereas the automated retinal segmentation algorithms performed well for the inner layers, the retinal pigmentation epithelium (RPE) was delineated within the sclera, leading to consistently thicker measurements of the photoreceptor layer and the total retina. TRANSLATIONAL RELEVANCE The introduction of spectral domain OCT allows for accurate imaging of the mouse retina. Exact quantification of retinal layer thicknesses in mice is important to study layers of interest under various pathological conditions.

Angiogenesis-related ultrastructural changes to capillaries in human skeletal muscle in response to endurance exercise

The ultrastructure of capillaries in skeletal muscle was morphometrically assessed in vastus lateralis muscle (VL) biopsies taken before and after exercise from 22 participants of two training studies. In study 1 (8 wk of ergometer training), light microscopy revealed capillary-fiber (C/F) ratio (+27%) and capillary density (+16%) to be higher (P ≤ 0.05) in postexercise biopsies than in preexercise biopsies from all 10 participants. In study 2 (6 mo of moderate running), C/F ratio and capillary density were increased (+23% and +20%; respectively, P ≤ 0.05) in VL biopsies from 6 angiogenesis responders (AR) after training, whereas 6 nonangiogenesis responders (NR) showed nonsignificant changes in these structural indicators (-4%/-4%, respectively). Forty capillary profiles per participant were evaluated by point and intersection counting on cross sections after transmission electron microscopy. In study 1, volume density (Vv) and mean arithmetic thickness (T) of endothelial cells (ECs; +19%/+17%, respectively) and pericytes (PCs; +20%/+21%, respectively) were higher (P ≤ 0.05), whereas Vv and T of the pericapillary basement membrane (BM) were -23%/-22% lower (P ≤ 0.05), respectively, in posttraining biopsies. In study 2, exercise-related differences between AR and NR-groups were found for Vv and T of PCs (AR, +26%/+22%, respectively, both P ≤ 0.05; NR, +1%/-3%, respectively, both P > 0.05) and BM (AR, -14%/-13%, respectively, both P ≤ 0.05; NR, -9%/-11%, respectively, P = 0.07/0.10). Vv and T of ECs were higher (AR, +16%/+18%, respectively; NR, +6%/+6%, respectively; all P ≤ 0.05) in both groups. The PC coverage was higher (+13%, P ≤ 0.05) in VL biopsies of individuals in the AR group but nonsignificantly altered (+3%, P > 0.05) in those of the NR group after training. Our study suggests that intensified PC mobilization and BM thinning are related to exercise-induced angiogenesis in human skeletal muscle, whereas training per se induces EC-thickening.

Capillary growth, ultrastructure remodelling and exercise training in skeletal muscle of essential hypertensive patients

AIM The aim was to elucidate whether essential hypertension is associated with altered capillary morphology and density and to what extent exercise training can normalize these parameters. METHODS To investigate angiogenesis and capillary morphology in essential hypertension, muscle biopsies were obtained from m. vastus lateralis in subjects with essential hypertension (n = 10) and normotensive controls (n = 11) before and after 8 weeks of aerobic exercise training. Morphometry was performed after transmission electron microscopy, and protein levels of several angioregulatory factors were determined. RESULTS At baseline, capillary density and capillary-to-fibre ratio were not different between the two groups. However, the hypertensive subjects had 9% lower capillary area (12.7 ± 0.4 vs. 13.9 ± 0.2 μm(2)) and tended to have thicker capillary basement membranes (399 ± 16 vs. 358 ± 13 nm; P = 0.094) than controls. Protein expression of vascular endothelial growth factor (VEGF), VEGF receptor-2 and thrombospondin-1 were similar in normotensive and hypertensive subjects, but tissue inhibitor of matrix metalloproteinase was 69% lower in the hypertensive group. After training, angiogenesis was evident by 15% increased capillary-to-fibre ratio in the hypertensive subjects only. Capillary area and capillary lumen area were increased by 7 and 15% in the hypertensive patients, whereas capillary basement membrane thickness was decreased by 17% (P < 0.05). VEGF expression after training was increased in both groups, whereas VEGF receptor-2 was decreased by 25% in the hypertensive patients(P < 0.05). CONCLUSION Essential hypertension is associated with decreased lumen area and a tendency for increased basement membrane thickening in capillaries of skeletal muscle. Exercise training may improve the diffusion conditions in essential hypertension by altering capillary structure and capillary number.

Immune cell trafficking across the barriers of the central nervous system in multiple sclerosis and stroke

Each year about 650,000 Europeans die from stroke and a similar number lives with the sequelae of multiple sclerosis (MS). Stroke and MS differ in their etiology. Although cause and likewise clinical presentation set the two diseases apart, they share common downstream mechanisms that lead to damage and recovery. Demyelination and axonal injury are characteristics of MS but are also observed in stroke. Conversely, hallmarks of stroke, such as vascular impairment and neurodegeneration, are found in MS. However, the most conspicuous common feature is the marked neuroinflammatory response, marked by glia cell activation and immune cell influx. In MS and stroke the blood-brain barrier is disrupted allowing bone marrow-derived macrophages to invade the brain in support of the resident microglia. In addition, there is a massive invasion of auto-reactive T-cells into the brain of patients with MS. Though less pronounced a similar phenomenon is also found in ischemic lesions. Not surprisingly, the two diseases also resemble each other at the level of gene expression and the biosynthesis of other proinflammatory mediators. While MS has traditionally been considered to be an autoimmune neuroinflammatory disorder, the role of inflammation for cerebral ischemia has only been recognized later. In the case of MS the long track record as neuroinflammatory disease has paid off with respect to treatment options. There are now about a dozen of approved drugs for the treatment of MS that specifically target neuroinflammation by modulating the immune system. Interestingly, experimental work demonstrated that drugs that are in routine use to mitigate neuroinflammation in MS may also work in stroke models. Examples include Fingolimod, glatiramer acetate, and antibodies blocking the leukocyte integrin VLA-4. Moreover, therapeutic strategies that were discovered in experimental autoimmune encephalomyelitis (EAE), the animal model of MS, turned out to be also effective in experimental stroke models. This suggests that previous achievements in MS research may be relevant for stroke. Interestingly, the converse is equally true. Concepts on the neurovascular unit that were developed in a stroke context turned out to be applicable to neuroinflammatory research in MS. Examples include work on the important role of the vascular basement membrane and the BBB for the invasion of immune cells into the brain. Furthermore, tissue plasminogen activator (tPA), the only established drug treatment in acute stroke, modulates the pathogenesis of MS. Endogenous tPA is released from endothelium and astroglia and acts on the BBB, microglia and other neuroinflammatory cells. Thus, the vascular perspective of stroke research provides important input into the mechanisms on how endothelial cells and the BBB regulate inflammation in MS, particularly the invasion of immune cells into the CNS. In the current review we will first discuss pathogenesis of both diseases and current treatment regimens and will provide a detailed overview on pathways of immune cell migration across the barriers of the CNS and the role of activated astrocytes in this process. This article is part of a Special Issue entitled: Neuro inflammation: A common denominator for stroke, multiple sclerosis and Alzheimer's disease, guest edited by Helga de Vries and Markus Swaninger.