Electron tomographic analysis of cytoskeletal cross-bridges in the paranodal region of the node of Ranvier in peripheral nerves

The node of Ranvier is a site for ionic conductances along myelinated nerves and governs the saltatory transmission of action potentials. Defects in the cross-bridging and spacing of the cytoskeleton are a prominent pathological feature in diseases of the peripheral nerve. Electron tomography was used to examine cytoskeletal–cytoskeletal, membrane–cytoskeletal, and heterologous cell connections in the paranodal region of the node of Ranvier in peripheral nerves. Focal attachment of cytoskeletal filaments to each other and to the axolemma and paranodal membranes of the Schwann cell via narrow cross-bridges was visualized in both neuronal and glial cytoplasm. A subset of intermediate filaments associates with the cytoplasmic surfaces of supramolecular complexes of transmembrane structures that are presumed to include known and unknown junctional proteins. Mitochondria were linked to both microtubules and neurofilaments in the axoplasm and to neighboring smooth endoplasmic reticulum by narrow cross-bridges. Tubular cisternae in the glial cytoplasm were also linked to the paranodal glial cytoplasmic loop juxtanodal membrane by short cross-bridges. In the extracellular matrix between axon and Schwann cell, junctional bridges formed long cylinders linking the two membranes. Interactions between cytoskeleton, membranes, and extracellular matrix associations in the paranodal region are likely critical not only for scaffolding, but also for intracellular and extracellular communication.     

Efficient site-specific integration of large transgenes by an enhanced herpes simplex virus/adeno-associated virus hybrid amplicon vector

We previously demonstrated that a herpes simplex virus type 1 (HSV-1)/adeno-associated virus (AAV) hybrid amplicon vector constructed by inserting the sequences of regulatory protein (rep) and inverted terminal repeats of AAV into an HSV amplicon vector resulted in the enhanced stability of transgene expression compared to the original HSV-1 amplicon vector. However, problems related to the expression of Rep compromised its therapeutic applications. We report here a new HSV/AAV hybrid amplicon vector system that not only solved problems associated with Rep expression but also markedly improved the stable transduction efficiency of this vector. This new HSV/AAV vector is designed in a way that little or no Rep would be expressed in packaging cells, but it can be expressed in transduced cells if Cre recombinase is provided. Furthermore, Rep expression will be automatically suppressed as a consequence of Rep-mediated integration. Our results showed that the new hybrid amplicon vector yielded titers comparable to those of standard amplicon vectors. When Cre-expressing 293 cells were transduced, a low level of Rep expression was detected, and stable transduction was achieved in approximately 22% of transduced cells; of those cells, approximately 70% transduction was achieved by Rep-mediated site-specific integration. In the majority of the stably transduced cells, Rep expression was no longer observed. Our results also proved that this vector system is capable of efficiently accommodating and site-specifically integrating large transgenes, such as the full-length dystrophin expression cassette. Thus, the new HSV/AAV vector demonstrated unique advantages in safe and effective delivery of long-lasting transgene expression into human cells.     

Interplay of cysteinyl leukotrienes and TGF-β in the activation of hepatic stellate cells from Schistosoma mansoni granulomas

Hepatic stellate cells (HSCs) have a critical role in liver physiology, and in the pathogenesis of liver inflammation and fibrosis. Here, we investigated the interplay between leukotrienes (LT) and TGF-β in the activation mechanisms of HSCs from schistosomal granulomas (GR-HSCs). First, we demonstrated that GR-HSCs express 5-lipoxygenase (5-LO), as detected by immunolocalization in whole cells and confirmed in cell lysates through western blotting and by mRNA expression through RT-PCR. Moreover, mRNA expression of 5-LO activating protein (FLAP) and LTC₄-synthase was also documented, indicating that GR-HSCs have the molecular machinery required for LT synthesis. Morphological analysis of osmium and Oil-Red O-stained HSC revealed large numbers of small lipid droplets (also known as lipid bodies). We observed co-localization of lipid droplet protein marker (ADRP) and 5-LO by immunofluorescence microscopy. We demonstrated that GR-HSCs were able to spontaneously release cysteinyl-LTs (CysLTs), but not LTB_4, into culture supernatants. CysLT production was highly enhanced after TGF-β-stimulation. Moreover, the 5-LO inhibitor zileuton and 5-LO gene deletion were able to inhibit the TGF-β-stimulated proliferation of GR-HSCs, suggesting a role for LTs in HSC activation. Here, we extend the immunoregulatory function of HSC by demonstrating that HSC from liver granulomas of schistosome-infected mouse are able to release Cys-LTs in a TGF-β-regulated manner, potentially impacting pathogenesis and liver fibrosis in schistosomiasis.     

Speciation in the Artemia genus: Mitochondrial DNA analysis of bisexual and parthenogenetic brine shrimps

From the cloned mitochondrial DNAs (mtDNAs) isolated from two bisexual species, one Mediterranean, Artemia salina, and one American, Artemia franciscana, and two parthenogenetic (diploid and tetraploid) strains of Artemia parthenogenetica collected in Spain, physical maps have been constructed and compared. They are extremely different among themselves, much more than the differences between Drosophila melanogaster and D. yakuba and in the same range of different mammalian species such as mouse/rat or man/cow. The nucleotide sequences of two regions of mtDNA encoding parts of the cytochrome c oxidase subunit I (COI) and cytochrome b (Cytb) genes have been determined in the two bisexual species and the two parthenogenetic strains. Comparisons of these sequences have revealed a high degree of divergence at the nucleotide level, averaging more than 15%, in agreement with the differences found in the physical maps. The majority of the nucleotide changes are silent and there is a strong bias toward transitions, with the CT substitutions being highly predominant. The evolutionary distance between the two Artemia parthenogenetica is high and there is no clear relationship with any of the bisexual species, including the one present nowadays in Spain. Using a combination of molecular (mtDNA) and morphological markers it is possible to conclude that all of these Artemia isolates should be actually considered as belonging to different species, even the two Artemia parthenogenetica diploidica and tetraploidica.On sabbatical leave from Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense de Madridearly Italian artemiologists to designate the Medi-Beatriz Batuecas died in an accident during the Christmas holy days of 1988 after she had initiated this workCorrespondence to: R. Garesse     

Epidermal growth factor-like domain 7 protects endothelial cells from hyperoxia-induced cell death

Hyperoxia is one of the major contributors to the development of bronchopulmonary dysplasia (BPD), a chronic lung disease in premature infants. Emerging evidence suggests that the arrested lung development of BPD is associated with pulmonary endothelial cell death and vascular dysfunction resulting from hyperoxia-induced lung injury. A better understanding of the mechanism of hyperoxia-induced endothelial cell death will provide critical information for the pathogenesis and therapeutic development of BPD. Epidermal growth factor-like domain 7 (EGFL7) is a protein secreted from endothelial cells. It plays an important role in vascular tubulogenesis. In the present study, we found that Egfl7 gene expression was significantly decreased in the neonatal rat lungs after hyperoxic exposure. The Egfl7 expression was returned to near normal level 2 wk after discounting oxygen exposure during recovery period. In cultured human endothelial cells, hyperoxia also significantly reduced Egfl7 expression. These observations suggest that diminished levels of Egfl7 expression might be associated with hyperoxia-induced endothelial cell death and lung injury. When we overexpressed human Egfl7 (hEgfl7) in EA.hy926 human endothelial cell line, we found that hEgfl7 overexpression could partially block cytochrome c release from mitochondria and decrease caspase-3 activation. Further Western blotting analyses showed that hEgfl7 overexpression could reduce expression of a proapoptotic protein, Bax, and increase expression of an antiapoptotic protein, Bcl-xL. Theses findings indicate that hEGFL7 may protect endothelial cell from hyperoxia-induced apoptosis by inhibition of mitochondria-dependent apoptosis pathway.     

Factors associated with the 6-minute walk distance in patients with systemic sclerosis

Background

There is an ongoing debate regarding the relevance of the 6-minute walking distance (6MWD) in systemic sclerosis (SSc) assessment, widely used as a usual test in these patients as well as an outcome measure in clinical trials. In this work, we aimed to assess the associations between the 6MWD and various disease parameters in patients with SSc.

Methods

Consecutive patients followed in our SSc National Reference Centre were included in this cross-sectional study if they fulfilled the 2013 American College of Rheumatology/European League Against Rheumatism criteria for SSc. Data were systematically collected during a comprehensive standardized evaluation that included a 6-minute walk test, clinical assessment, biological results, pulmonary function tests, transthoracic echocardiography, composite scores (European Scleroderma Study Group Activity Index, Medsger severity score, Health Assessment Questionnaire–Disability Index (HAQ-DI)) and treatments.Associations of the 6MWD with various disease parameters were assessed by linear regression in univariate and multivariate analyses.

Results

The study population comprised 298 patients (females 81%; mean age 58.2?±?13.3 years; limited cutaneous SSc 82%; interstitial lung disease (ILD) 42%; pulmonary arterial hypertension (PAH) 6%). The 6MWD was significantly and independently associated with gender, age, body mass index, baseline heart rate (HR), HR variation during the test, PAH, history of arterial thrombosis and C-reactive protein levels, as well as with the HAQ-DI score in a sensitivity analysis. Muscle involvement, joint involvement and ILD were not independently associated with the 6MWD.

Conclusions

During SSc, the 6MWD is independently associated with initial HR and HR variation; with PAH but not ILD, suggesting that pulmonary vasculopathy may have a greater impact than parenchymal involvement on functional limitation; and with global markers of disease activity and patient disability. These results give clinicians further insight into how to interpret the 6MWD in the context of SSc.

     

The active component of ginseng,ginsenoside Rb1, improves erythropoiesis in models of Diamond–Blackfan anemia by targeting Nemo-like kinase

Nemo-like kinase (NLK) is a member of the mitogen-activated protein kinase family of kinases and shares a highly conserved kinase domain with other mitogen-activated protein kinase family members. The activation of NLK contributes to the pathogenesis of Diamond–Blackfan anemia (DBA), reducing c-myb expression and mechanistic target of rapamycin activity, and is therefore a potential therapeutic target. Unlike other anemias, the hematopoietic effects of DBA are largely restricted to the erythroid lineage. Mutations in ribosomal genes induce ribosomal insufficiency and reduced protein translation, dramatically impacting early erythropoiesis in the bone marrow of patients with DBA. We sought to identify compounds that suppress NLK and increases erythropoiesis in ribosomal insufficiency. We report that the active component of ginseng, ginsenoside Rb1, suppresses NLK expression and improves erythropoiesis in in vitro models of DBA. Ginsenoside Rb1–mediated suppression of NLK occurs through the upregulation of miR-208, which binds to the 3′-UTR of NLK mRNA and targets it for degradation. We also compare ginsenoside Rb1–mediated upregulation of miR-208 with metformin-mediated upregulation of miR-26. We conclude that targeting NLK expression through miRNA binding of the unique 3′-UTR is a viable alternative to the challenges of developing small-molecule inhibitors to target the highly conserved kinase domain of this specific kinase.     

Standing crop and aboveground biomass partitioning of a dwarf mangrove forest in Taylor River Slough,Florida

The structure and standing crop biomass of a dwarf mangrove forest, located in the salinity transition zone ofTaylor River Slough in the Everglades National Park, were studied. Although the four mangrove species reported for Florida occurred at the study site, dwarf Rhizophora mangle trees dominated the forest. The structural characteristics of the mangrove forest were relatively simple: tree height varied from 0.9 to 1.2 meters, and tree density ranged from 7062 to 23 778 stems ha^–1. An allometric relationship was developed to estimate leaf, branch, prop root, and total aboveground biomass of dwarf Rhizophora mangle trees. Total aboveground biomass and their components were best estimated as a power function of the crown area times number of prop roots as an independent variable (Y = B × X^–0.5083). The allometric equation for each tree component was highly significant (p<0.0001), with all r² values greater than 0.90. The allometric relationship was used to estimate total aboveground biomass that ranged from 7.9 to 23.2 ton ha^–1. Rhizophora mangle contributed 85% of total standing crop biomass. Conocarpus erectus, Laguncularia racemosa, and Avicennia germinans contributed the remaining biomass. Average aboveground biomass allocation was 69% for prop roots, 25% for stem and branches, and 6% for leaves. This aboveground biomass partitioning pattern, which gives a major role to prop roots that have the potential to produce an extensive root system, may be an important biological strategy in response to low phosphorus availability and relatively reduced soils that characterize mangrove forests in South Florida.     

Frictional properties of contacting surfaces in the hemelytra-hindwing locking mechanism in the bug<Emphasis Type="Italic"> Coreus marginatus</Emphasis> (Heteroptera,Coreidae)

The structure and function of the hemelytra-to-hindwing locking mechanism of the bug Coreus marginatus were analysed. The system consists of a cuticular protrusion in the ventral side of the hemelytra, which locks the subcostal border of the hindwing in flight. The speed and distance slid by both surfaces against one another during flight were assessed using a combination of high-speed video recordings and a 2D geometrical model. The friction coefficient between sliding surfaces was assessed using a micromanipulator, coupled with force transducers. This was done under three experimental conditions: freshly dissected, air dried and rehydrated ethanol preserved samples. The results showed a high speed of sliding, approximately 0.18 m s^–1, with a relatively low friction coefficient (0.2 ). There was no evident difference in the friction measured under the various treatments, with the exception of the rehydrated condition, which was lower. The surface morphology of the wing locking mechanism, namely outgrowths of one part having rounded edges, and completely flat surface on the counterpart, effectively aids in the reduction of friction at the microscopic level. The structure is effective even dry, and after being preserved in ethanol, suggesting that no cuticle secreted lubrication substance is responsible for its effectiveness. The ultrastructure presumably confers mechanical stability to the system under the high load it is subjected to in flight.     

Y-chromosomal diversity in Haiti and Jamaica: contrasting levels of sex-biased gene flow

Although previous studies have characterized the genetic structure of populations from Haiti and Jamaica using classical and autosomal STR polymorphisms, the patrilineal influences that are present in these countries have yet to be explored. To address this lacuna, the current study aims to investigate, for the first time, the potential impact of different ancestral sources, unique colonial histories, and distinct family structures on the paternal profile of both groups. According to previous reports examining populations from the Americas, island-specific demographic histories can greatly impact population structure, including various patterns of sex-biased gene flow. Also, given the contrasting autosomal profiles provided in our earlier study (Simms et al.: Am J Phys Anthropol 142 (2010) 49-66), we hypothesize that the degree and directionality of gene flow from Europeans, Africans, Amerindians, and East Asians are dissimilar in the two countries. To test this premise, 177 high-resolution Y-chromosome binary markers and 17 Y-STR loci were typed in Haiti (n = 123) and Jamaica (n = 159) and subsequently utilized for phylogenetic comparisons to available reference collections encompassing Africa, Europe, Asia (East and South), and the New World. Our results reveal that both studied populations exhibit a predominantly South-Saharan paternal component, with haplogroups A1b-V152, A3-M32, B2-M182, E1a-M33, E1b1a-M2, E2b-M98, and R1b2-V88 comprising 77.2% and 66.7% of the Haitian and Jamaican paternal gene pools, respectively. Yet, European derived chromosomes (i.e., haplogroups G2a*-P15, I-M258, R1b1b-M269, and T-M184) were detected at commensurate levels in Haiti (20.3%) and Jamaica (18.9%), whereas Y-haplogroups indicative of Chinese [O-M175 (3.8%)] and Indian [H-M69 (0.6%) and L-M20 (0.6%)] ancestry were restricted to Jamaica.