Expression, purification, and projection structure by single particle electron microscopy of functional human TRPM4 heterologously expressed in Xenopus laevis oocytes

Despite efforts implicating the cationic channel transient receptor potential melastatin member 4 (TRPM4) to cardiac, nervous, and immunological pathologies, little is known about its structure and function. In this study, we optimized the requirements for purification and extraction of functional human TRPM4 protein and investigated its supra-molecular assembly. We selected the Xenopus laevis oocyte expression system because it lacks endogenous TRPM4 expression, it is known to overexpress functional human membrane channels, can be used for structure-function analysis within the same system, and is easily scaled to improve yield and develop moderate throughput capabilities through the use of robotics. Negative-stain electron microscopy (EM) revealed various sized low-resolution particles. Single particle analysis identified the majority of the projections represented the monomeric form with additional oligomeric structures potentially characterized as tetramers. Two-electrode voltage clamp electrophysiology demonstrated that human TRPM4 is functionally expressed at the oocyte plasma membrane. This study opens the door for medium-throughput screening and structure-function determination of this important therapeutically relevant target.

Test Facility to Study Surface-Interaction Processes for Particle Detection in Space

The Imager for Low Energetic Neutral Atoms test facility at the University of Bern was developed to investigate, characterize, and quantify physical processes on surfaces that are used to ionize neutral atoms before their analysis in neutral particle-sensing instruments designed for space research. The facility has contributed valuable knowledge of the interaction of ions with surfaces (e.g., fraction of ions scattered from surfaces and angular scattering distribution) and employs a novel measurement principle for the determination of secondary electron emission yields as a function of energy, angle of incidence, particle species, and sample surface for low particle energies. Only because of this test facility it was possible to successfully apply surface-science processes for the new detection technique for low-energetic neutral particles with energies below about 1 keV used in space applications. All successfully flown spectrometers for the detection of low-energetic neutrals based on the particle–surface interaction process use surfaces evaluated, tested, and calibrated in this facility. Many instruments placed on different spacecraft (e.g., Imager for Magnetopause-to-Aurora Global Exploration, Chandrayaan-1, Interstellar Boundary Explorer, etc.) have successfully used this technique.

Delta like ligand 4 is a critical regulator of bone marrow cell differentiation into pericytes/vascular smooth muscle cells and is essential for the vasculogenesis that supports the growth of Ewing’s sarcoma

We have previously shown that vasculogenesis, the process by which bone marrow-derived cells are recruited to the tumor and organized to form a blood vessel network de novo, is essential for the growth of Ewing’s sarcoma. We further demonstrated that these bone marrow cells differentiate into pericytes/vascular smooth muscle cells(vSMC) and contribute to the formation of the functional vascular network. The molecular mechanisms that control bone marrow cell differentiation into pericytes/vSMC in Ewing’s sarcoma are poorly understood. Here, we demonstrate that the Notch ligand Delta like ligand 4 (DLL4) plays a critical role in this process. DLL4 is essential for the formation of mature blood vessels during development and in several tumor models. Inhibition of DLL4 causes increased vascular sprouting, decreased pericyte coverage, and decreased vessel functionality. We demonstrate for the first time that DLL4 is expressed by bone marrow-derived pericytes/vascular smooth muscle cells in two Ewing’s sarcoma xenograft models and by perivascular cells in 12 out of 14 patient samples. Using dominant negative mastermind to inhibit Notch, we demonstrate that Notch signaling is essential for bone marrow cell participation in vasculogenesis. Further, inhibition of DLL4 using either shRNA or the monoclonal DLL4 neutralizing antibody YW152F led to dramatic changes in blood vessel morphology and function. Vessels in tumors where DLL4 was inhibited were smaller, lacked lumens, had significantly reduced numbers of bone marrow-derived pericyte/vascular smooth muscle cells, and were less functional. Importantly, growth of TC71 and A4573 tumors was significantly inhibited by treatment with YW152F. Additionally, we provide in vitro evidence that DLL4-Notch signaling is involved in bone marrow-derived pericyte/vascular smooth muscle cell formation outside of the Ewing’s sarcoma environment. Pericyte/vascular smooth muscle cell marker expression by whole bone marrow cells cultured with mouse embryonic stromal cells was reduced when DLL4 was inhibited by YW152F. For the first time, our findings demonstrate a role for DLL4 in bone marrow-derived pericyte/vascular smooth muscle differentiation as well as a critical role for DLL4 in Ewing’s sarcoma tumor growth.

Smooth Muscle Hyperplasia due to ACTA2/MYH11 Mutations: Identification of Novel Pathology and Pathways Leading to Aneurysms and Diverse Vascular Occlusive Diseases

Missense mutations in smooth muscle cell (SMC) specific ACTA2 (á-actin) and MYH11 (â-myosin heavy chain) cause diffuse and diverse vascular diseases, including thoracic aortic aneurysms and dissections (TAAD) and early onset coronary artery disease and stroke. The mechanism by which these mutations lead to dilatation of some arteries but occlusion of others is unknown. We hypothesized that the mutations act through two distinct mechanisms to cause varied vascular diseases: a loss of function, leading to decreased SMC contraction and aneurysms, and a gain of function, leading to increased SMC proliferation and occlusive disease. To test this hypothesis, ACTA2 mutant SMCs and myofibroblasts were assessed and found to not form á-actin filaments whereas control cells did, suggesting a dominant negative effect of ACTA2 mutations on filament formation. A loss of á-actin filaments would be predicted to cause decreased SMC contractility. Histological examination of vascular tissues from patients revealed SMC hyperplasia leading to arterial stenosis and occlusion, supporting a gain of function associated with the mutant gene. Furthermore, ACTA2 mutant SMCs and myofibroblasts proliferated more rapidly in static culture than control cells (p<0.05). We also determined that Acta2-/- mice have ascending aortic aneurysms. Histological examination revealed aortic medial SMC hyperplasia, but minimal features of medial degeneration. Acta2-/- SMCs proliferated more rapidly in culture than wildtype (p<0.05), and microarray analysis of Acta2-/- SMCs revealed increased expression of Actg2, 15 collagen genes, and multiple focal adhesion genes. Acta2-/- SMCs showed altered localization of vinculin and zyxin and increased phosphorylated focal adhesion kinase (FAK) in focal adhesions. A specific FAK inhibitor decreased Acta2-/- SMC proliferation to levels equal to wildtype SMCs (p<0.05), suggesting that FAK activation leads to the increased proliferation. We have described a unique pathology associated with ACTA2 and MYH11 mutations, as well as an aneurysm phenotype in Acta2-/- mice. Additionally, we identified a novel pathogenic pathway for vascular occlusive disease due to loss of SMC contractile filaments, alterations in focal adhesions, and activation of FAK signaling in SMCs with ACTA2 mutations.

Energy harvesting through arterial wall deformation: A FEM approach to fluid–structure interactions and magneto-hydrodynamics

Engineers are confronted with the energy demand of active medical implants in patients with increasing life expectancy. Scavenging energy from the patient’s body is envisioned as an alternative to conventional power sources. Joining in this effort towards human-powered implants, we propose an innovative concept that combines the deformation of an artery resulting from the arterial pressure pulse with a transduction mechanism based on magneto-hydrodynamics. To overcome certain limitations of a preliminary analytical study on this topic, we demonstrate here a more accurate model of our generator by implementing a three-dimensional multiphysics finite element method (FEM) simulation combining solid mechanics, fluid mechanics, electric and magnetic fields as well as the corresponding couplings. This simulation is used to optimize the generator with respect to several design parameters. A first validation is obtained by comparing the results of the FEM simulation with those of the analytical approach adopted in our previous study. With an expected overall conversion efficiency of 20% and an average output power of 30 μW, our generator outperforms previous devices based on arterial wall deformation by more than two orders of magnitude. Most importantly, our generator provides sufficient power to supply a cardiac pacemaker.

Multicentric hepatic EBV-associated smooth muscle tumors in an AIDS patient: a case report, investigation of mTOR activation and review of the literature.

Epstein-Barr virus (EBV) - associated smooth muscle tumors (EBV-SMT) are a rare, recently recognized distinct group of mesenchymal tumors that develop exclusively in patients with immunosuppression. It is believed that tumorigenesis is, at least in part, through the activation of the Akt/mammalian target of rapamycin (mTOR) signal pathway. We describe the clinicopathologic and immunohistochemical features of a multifocal hepatic EBV-SMT in a 34-year-old acquired immunodeficiency syndrome (AIDS) patient and investigate the activation status of the mTOR signal pathway in this tumor. In addition, we provide a review of the literature on the clinicopathologic findings of hepatic EBV-SMT in adult AIDS patients, and discuss their biologies and possible therapeutic strategies.

A genetically engineered, adherent smooth muscle cell lining for left ventricular assist devices

Due to the clinical success of left ventricular assist devices (LVADs) used for short term "bridge to transplant" and the limited availability of donor organs, heart assist devices are being considered for long term implantation as an alternative to heart transplantation. In an effort to improve biocompatibility, a nonthrombogenic cellular lining was developed from genetically engineered smooth muscle cells (GE-SMC) for the Thermocardiosystems Heartmate$\sp{\rm TM}$ LVAD. SMCs have been transduced with the genes for endothelial nitric oxide synthase (NOS III) and GTP cyclohydrolase (GTPCH) with subsequent stable expression of the NOS III protein via an Epstein Barr based DNA expression vector. Transduced SMCs produce nitric oxide at concentrations that reduce platelet deposition and smooth muscle cell proliferation when tested in vitro. In addition, the adhesive capabilities of GE-SMC linings were also examined, and optimized in physical environments mimicking typical in vivo LVAD operation. Preliminary investigations examining cell adhesion during constant shear stress exposure demonstrated an acute phase of cell loss corresponding to cytoskeletal F-actin rearrangement. Subsequently, an in vitro circulatory loop was designed to expose cell lined LVADs to in vivo operating conditions. Cumulative cell loss from cell lined LVADs was less than 10% after 24 hours of flow. Using a protocol for "preconditioning" the cell lining within the mock circulatory loop, the first implantation of an LVAD containing a genetically engineered SMC lining was successfully implemented in a bovine model. Results from this 24 hour study indicate that the flow-conditioned cellular lining remained intact with no evidence of thromboembolization and only minimal changes in coagulation studies. ^

Immunostaining of a heterodimeric dermatan sulphate proteoglycan is correlated with smooth muscles and some basement membranes.

A heterodimeric 760-kDa dermatan sulphate proteoglycan tentatively named PG-760 was characterized as a product of keratinocytes, endothelial cells, and fibroblasts. The two core proteins of 460 kDa and 300 kDa are linked by disulphide bridges, and both carry one or only very few dermatan sulphate chains. Different antisera against PG-760 were used in the present study to investigate the distribution in selected murine tissues by light and electron microscopy. PG-760 immunostaining was observed in cornea (epithelium including basement membrane, stroma, and Descemet's membrane), skin, mucosa of the small intestine, Engelbreth-Holm-Swarm (EHS)-tumour (matrix and cells), and the smooth muscle layers of uterus, small intestine, and blood vessels. No staining was observed in capillaries, striated muscles, and liver parenchyma including the central vein. The expression of PG-760 in EHS-tumour was also demonstrated after extraction with 4 M guanidine and partial purification by diethylaminoethyl (DEAE)-chromatography. We conclude that this novel proteoglycan exhibits a unique tissue distribution being a constituent of some but not all basement membranes, of some other extracellular matrices, and additionally, of all investigated smooth muscle layers.

Particulate Backscattering Ratio at Leo 15 and Its Use to Study Particle Composition and Distribution

Particulate scattering and backscattering are two quantities that have traditionally been used to quantify in situ particulate concentration. The ratio of the backscattering by particles to total scattering by particles (the particulate backscattering ratio) is weakly dependent on concentration and therefore provides us with information on the characteristics of the particulate material, such as the index of refraction. The index of refraction is an indicator of the bulk particulate composition, as inorganic minerals have high indices of refraction relative to oceanic organic particles such as phytoplankton and detrital material that typically have a high water content. We use measurements collected near the Rutgers University Long-term Ecosystem Observatory in 15 m of water in the Mid-Atlantic Bight to examine application of the backscattering ratio. Using four different instruments, the HOBILabs Hydroscat-6, the WETLabs ac-9 and EcoVSF, and a prototype VSF meter, three estimates of the ratio of the particulate backscattering ratio were obtained and found to compare well. This is remarkable because these are new instruments with large differences in design and calibration. The backscattering ratio is used to map different types of particles in the nearshore region, suggesting that it may act as a tracer of water movement. We find a significant relationship between the backscattering ratio and the ratio of chlorophyll to beam attenuation. This implies that these more traditional measurements may be used to identify when phytoplankton or inorganic particles dominate. In addition, it provides an independent confirmation of the link between the backscattering ratio and the bulk composition of particles.