Structural insights into the cause of human RSPH4A primary ciliary dyskinesia

Cilia and flagella are evolutionarily conserved eukaryotic organelles involved in cell motility and signaling. In humans, mutations in Radial Spoke Head Component 4A (RSPH4A) can lead to primary ciliary dyskinesia (PCD), a life-shortening disease characterized by chronic respiratory tract infections, abnormal organ positioning, and infertility. Despite its importance for human health, the location of RSPH4A in human cilia has not been resolved, and the structural basis of RSPH4A^–/– PCD remains elusive. Here, we present the native three-dimensional structure of RSPH4A^–/– human respiratory cilia using samples collected noninvasively from a PCD patient. Using cryo–electron tomography (cryo-ET) and subtomogram averaging, we compared the structures of control and RSPH4A^–/– cilia, revealing primary defects in two of the three radial spokes (RSs) within the axonemal repeat and secondary (heterogeneous) defects in the central pair complex. Similar to RSPH1^–/– cilia, the radial spoke heads of RS1 and RS2, but not RS3, were missing in RSPH4A^–/– cilia. However, RSPH4A^–/– cilia also exhibited defects within the arch domains adjacent to the RS1 and RS2 heads, which were not observed with RSPH1 loss. Our results provide insight into the underlying structural basis for RSPH4A^–/– PCD and highlight the benefits of applying cryo-ET directly to patient samples for molecular structure determination.     

Insights in to the pathogenesis of axial spondyloarthropathy based on gene expression profiles

Introduction  

Axial spondyloarthropathy (SpA) is a group of inflammatory diseases, with ankylosing spondylitis as the prototype. SpA affects the axial skeleton, entheses, joints and, at times, the eyes. This study tested the hypothesis that SpA is characterized by a distinct pattern of gene expression in peripheral blood of affected individuals compared with healthy controls.     

Spermatozoon ultrastructure of Aponurus laguncula (Digenea: Lecithasteridae), a parasite of Aluterus monoceros (Pisces: Teleostei)

The mature spermatozoon of Aponurus laguncula, a parasite of the unicorn leatherjacket Aluterus monoceros, was studied by transmission electron microscopy. The spermatozoon possesses 2 axonemes of the 9 + “1” trepaxonematan pattern, attachment zones, a nucleus, a mitochondrion, external ornamentation of the plasma membrane and cortical microtubules. The major features are the presence of: 1) external ornamentation in the anterior part of the spermatozoon not associated with cortical microtubules; 2) one mitochondrion; and 3) cortical microtubules arranged as a single field in the ventral side. The maximum number of microtubules is in the nuclear region. The extremities of the axonemes are characterized by the disappearance of the central core and the presence of microtubule doublets or singlets. This study is the first undertaken with a member of the Lecithasteridae and exemplifies the sperm ultrastructure for the superfamily Hemiuroidea.     

Ultrastructural study of the spermatozoon of Heterolebes maculosus (Digenea,Opistholebetidae), a parasite of the porcupinefish Diodon hystrix (Pisces,Teleostei)

This paper describes the ultrastructure of the mature spermatozoon of Heterolebes maculosus. It is the first study of this kind concerning the Opistholebetidae (Platyhelminthes, Digenea). The ultrastructural elements observed in the spermatozoon are: two axonemes with 9+“1” pattern of Trepaxonemata and their attachment zones, two mitochondria, a nucleus, cortical microtubules, external ornamentation of the plasma membrane and spine-like bodies. The number and the disposition of cortical microtubules, the organisation of 11 cortical microtubules disposed in semi-circle around the first mitochondrion in the external ornamentation region and the organisation of the posterior part of the spermatozoon are discussed. Three principal types of posterior part of digenean spermatozoa are proposed. The similarity between the spermatozoon of the Opistholebetidae H. maculosus and Opecoelidae enables us to confirm that these two families are closely related.     

Fibulin-1 Is Increased in Asthma – A Novel Mediator of Airway Remodeling?

Background

The extracellular matrix is a dynamic and complex network of macromolecules responsible for maintaining and influencing cellular functions of the airway. The role of fibronectin, an extracellular matrix protein, is well documented in asthma. However, the expression and function of fibulin-1, a secreted glycoprotein which interacts with fibronectin, has not been reported. Fibulin-1 is widely expressed in basement membranes in many organs including the lung. There are four isoforms in humans (A–D) of which fibulin-1C and 1D predominate. The objective of this study was to study the expression of fibulin-1 in volunteers with and without asthma, and to examine its function in vitro.

Methodology/Principal Findings

We used immunohistochemistry and dot-blots to examine fibulin-1 levels in bronchial biopsies, bronchoalveolar lavage fluid and serum. Real-time PCR for fibulin-1C and 1D, and ELISA and western blotting for fibulin-1 were used to study the levels in airway smooth muscle cells. The function of fibulin-1C was determined by assessing its role, using an antisense oligonucleotide, in cell proliferation, migration and wound healing. A murine model of airway hyperresponsiveness (AHR) was used to explore the biological significance of fibulin-1. Levels of fibulin-1 were significantly increased in the serum and bronchoalveolar lavage fluid of 21 asthmatics compared with 11 healthy volunteers. In addition fibulin-1 was increased in asthma derived airway smooth muscle cells and fibulin-1C contributed to the enhanced proliferation and wound repair in these cells. These features were reversed when fibulin-1C was suppressed using an antisense oligomer. In a mouse model of AHR, treatment with an AO inhibited the development of AHR to methacholine.

Conclusions

Our data collectively suggest fibulin-1C may be worthy of further investigation as a target for airway remodeling in asthma.     

Mentha longifolia in vitro cultures as safe source of flavouring ingredients

In vitro plantlets and callus of M. longifolia were established and their volatile constituents characterized by GC-MS analysis of their headspaces (HSs) and essential oils (EOs). Significant quali-quantitative differences were found in the aromatic fingerprints in comparison with the M. longifolia parent plants. In fact, limonene and carvone were the main constituents in the EOs of the mother plants, while the aroma of the in vitro plant material were especially enriched in oxygenated terpenes. In particular, huge amounts of piperitenone and piperitenone oxide (75 %) were found for in vitro plantlets, while trans-carvone oxide (19 %) and trans-piperitone epoxide (9 %) were found in callus EO. However, the established in vitro plant material showed lack of pulegone and menthofurane, thus preserving an important feature observed in the volatile fingerprint of the parent plants. In fact, because of their well-known toxicity significant amounts of pulegone and menthofurane may compromise the safety using of mint essential oil. Therefore the in vitro M. longifolia plantlets and callus may be regarded as a potential source of a safe flavouring agent.     

Tropomyosin isoform 3 promotes the formation of filopodia by regulating the recruitment of actin-binding proteins to actin filaments

Tropomyosins are believed to function in part by stabilizing actin filaments. However, accumulating evidence suggests that fundamental differences in function exist between tropomyosin isoforms, which contributes to the formation of functionally distinct filament populations. We investigated the functions of the high-molecular-weight isoform Tm3 and examined the molecular properties of Tm3-containing actin filament populations. Overexpression of the Tm3 isoform specifically induced the formation of filopodia and changes in actin solubility. We observed alterations in actin-binding protein recruitment to filaments, co-incident with changes in expression levels, which can account for this functional outcome. Tm3-associated filaments recruit active actin depolymerizing factor and are bundled into filopodia by fascin, which is both up-regulated and preferentially associated with Tm3-containing filaments in the Tm3 overexpressing cells. This study provides further insight into the isoform-specific roles of different tropomyosin isoforms. We conclude that variation in the tropomyosin isoform composition of microfilaments provides a mechanism to generate functionally distinct filament populations.     

ZFPIP/Zfp462 is involved in P19 cell pluripotency and in their neuronal fate

The nuclear zinc finger protein ZFPIP/Zfp462 is an important factor involved in cell division during the early embryonic development of vertebrates. In pluripotent P19 cells, ZFPIP/Zfp462 takes part in cell proliferation, likely via its role in maintaining chromatin structure. To further define the function of ZFPIP/Zfp462 in the mechanisms of pluripotency and cell differentiation, we constructed a stable P19 cell line in which ZFPIP/Zfp462 knockdown is inducible.We report that ZFPIP/Zfp462 was vital for mitosis and self-renewal in pluripotent P19 cells. Its depletion induced substantial decreases in the expression of the pluripotency genes Nanog, Oct4 and Sox2 and was associated with the transient expression of specific neuronal differentiation markers. We also demonstrated that ZFPIP/Zfp462 expression appears to be unnecessary after neuronal differentiation is induced in P19 cells.Taken together, our results strongly suggest that ZFPIP/Zfp462 is a key chromatin factor involved in maintaining P19 pluripotency and in the early mechanisms of neural differentiation but that it is dispensable in differentiated P19 cells.     

c-Src inactivation reduces renal epithelial cell-matrix adhesion, proliferation, and cyst formation

c-Src is a non-receptor tyrosine kinase whose activity is induced by phosphorylation at Y418 and translocation from the cytoplasm to the cell membrane. Increased activity of c-Src has been associated with cell proliferation, matrix adhesion, motility, and apoptosis in tumors. Immunohistochemistry suggested that activated (pY(418))-Src activity is increased in cyst-lining autosomal dominant polycystic kidney disease (ADPKD) epithelial cells in human and mouse ADPKD. Western blot analysis showed that SKI-606 (Wyeth) is a specific inhibitor of pY(418)-Src without demonstrable effects on epidermal growth factor receptor or ErbB2 activity in renal epithelia. In vitro studies on mouse inner medullary collecting duct (mIMCD) cells and human ADPKD cyst-lining epithelial cells showed that SKI-606 inhibited epithelial cell proliferation over a 24-h time frame. In addition, SKI-606 treatment caused a striking statistically significant decrease in adhesion of mIMCD and human ADPKD to extracellular collagen matrix. Retained viability of unattached cells was consistent with a primary effect on epithelial cell anchorage dependence mediated by the loss of extracellular matrix (ECM)-attachment due to α(2)β(1)-integrin function. SKI-606-mediated attenuation of the human ADPKD hyperproliferative and hyper-ECM-adhesive epithelial cell phenotype in vitro was paralleled by retardation of the renal cystic phenotype of Pkd1 orthologous ADPKD heterozygous mice in vivo. This suggests that SKI-606 has dual effects on cystic epithelial cell proliferation and ECM adhesion and may have therapeutic potential for ADPKD patients.     

Identification of a novel neuropathogenic Theiler's murine encephalomyelitis virus

Theiler's murine encephalitis viruses (TMEV) are divided into two subgroups based on their neurovirulence. Persistent strains resemble Theiler's original viruses (referred to as the TO subgroup), which largely induce a subclinical polioencephalomyelitis during the acute phase of the disease and can persist in the spinal cord of susceptible animals, inducing a chronic demyelinating disease. In contrast, members of the neurovirulent subgroup cause an acute encephalitis characterized by the rapid onset of paralysis and death within days following intracranial inoculation. We report herein the characterization of a novel neurovirulent strain of TMEV, identified using pyrosequencing technology and referred to as NIHE. Complete coverage of the NIHE viral genome was obtained, and it shares <90% nucleotide sequence identity to known TMEV strains irrespective of subgroup, with the greatest sequence variability being observed in genes encoding the leader and capsid proteins. The histopathological analysis of infected brain and spinal cord demonstrate inflammatory lesions and neuronal necrosis during acute infection with no evidence of viral persistence or chronic disease. Intriguingly, genetic analysis indicates the putative expression of the L protein, considered a hallmark of strains within the persistent subgroup. Thus, the identification and characterization of a novel neurovirulent TMEV strain sharing features previously associated with both subgroups will lead to a deeper understanding of the evolution of TMEV strains and new insights into the determinants of neurovirulence.