Detection of six serotypes of botulinum neurotoxin using fluorogenic reporters

Methods that do not require animal sacrifice to detect botulinum neurotoxins (BoNTs) are critical for BoNT antagonist discovery and the advancement of quantitative assays for biodefense and pharmaceutical applications. Here we describe the development and optimization of fluorogenic reporters that detect the proteolytic activity of BoNT/A, B, D, E, F, and G serotypes in real time with femtomolar to picomolar sensitivity. Notably, the reporters can detect femtomolar concentrations of BoNT/A in 4 h and BoNT/E in 20 h, sensitivity that equals that of animal-based methods. The reporters can be used to determine the specific activity of BoNT preparations with intra- and inter-assay coefficients of variation of approximately 10%. Finally, we find that the greater sensitivity of our reporters compared with those used in other commercially available assays makes the former attractive candidates for high-throughput screening of BoNT antagonists

Homologous and heterologous desensitization of guanylyl cyclase-B signaling in GH3 somatolactotropes

The guanylyl cyclases, GC-A and GC-B, are selective receptors for atrial and C-type natriuretic peptides (ANP and CNP, respectively). In the anterior pituitary, CNP and GC-B are major regulators of cGMP production in gonadotropes and yet mouse models of disrupted CNP and GC-B indicate a potential role in growth hormone secretion. In the current study, we investigate the molecular and pharmacological properties of the CNP/GC-B system in somatotrope lineage cells. Primary rat pituitary and GH3 somatolactotropes expressed functional GC-A and GC-B receptors that had similar EC50 properties in terms of cGMP production. Interestingly, GC-B signaling underwent rapid homologous desensitization in a protein phosphatase 2A (PP2A)-dependent manner. Chronic exposure to either CNP or ANP caused a significant down-regulation of both GC-A- and GC-B-dependent cGMP accumulation in a ligand-specific manner. However, this down-regulation was not accompanied by alterations in the sub-cellular localization of these receptors. Heterologous desensitization of GC-B signaling occurred in GH3 cells following exposure to either sphingosine-1-phosphate or thyrotrophin-releasing hormone (TRH). This heterologous desensitization was protein kinase C (PKC)-dependent, as pre-treatment with GF109203X prevented the effect of TRH on CNP/GC-B signaling. Collectively, these data indicate common and distinct properties of particulate guanylyl cyclase receptors in somatotropes and reveal that independent mechanisms of homologous and heterologous desensitization occur involving either PP2A or PKC. Guanylyl cyclase receptors thus represent potential novel therapeutic targets for treating growth-hormone-associated disorders

Inhibition of Neuroblastoma Tumor Growth by Targeted Delivery of MicroRNA-34a Using Anti-Disialoganglioside GD2 Coated Nanoparticles

Neuroblastoma is one of the most challenging malignancies of childhood, being associated with the highest death rate in paediatric oncology, underlining the need for novel therapeutic approaches. Typically, patients with high risk disease undergo an initial remission in response to treatment, followed by disease recurrence that has become refractory to further treatment. Here, we demonstrate the first silica nanoparticle-based targeted delivery of a tumor suppressive, pro-apoptotic microRNA, miR-34a, to neuroblastoma tumors in a murine orthotopic xenograft model. These tumors express high levels of the cell surface antigen disialoganglioside GD2 (GD(2)), providing a target for tumor-specific delivery.Nanoparticles encapsulating miR-34a and conjugated to a GD(2) antibody facilitated tumor-specific delivery following systemic administration into tumor bearing mice, resulted in significantly decreased tumor growth, increased apoptosis and a reduction in vascularisation. We further demonstrate a novel, multi-step molecular mechanism by which miR-34a leads to increased levels of the tissue inhibitor metallopeptidase 2 precursor (TIMP2) protein, accounting for the highly reduced vascularisation noted in miR-34a-treated tumors.These novel findings highlight the potential of anti-GD(2)-nanoparticle-mediated targeted delivery of miR-34a for both the treatment of GD(2)-expressing tumors, and as a basic discovery tool for elucidating biological effects of novel miRNAs on tumor growth

Determination of the Young Modulus of Ti-TiAl 3 Metallic Intermetallic Laminate Composites by Nano-Indentation

Nano-indentation is an important technique to determine the Young modulus of multiphase materials where normal tensile tests are not appropriate. In this work, Ti-TiAl3 metallic-intermetallic laminate composites have been fabricated successfully in open atmosphere using commercial purity Al and Ti foils with 250 µm and 500 µm initial thicknesses, respectively. Sintering process was performed at 700 • C under 2 MPa pressure for 7.5 h. Mechanical properties including the Young modulus were determined after manufacturing. The Young moduli of metallic and intermetallic phases were determined as 89 GPa and 140 GPa, respectively. Microstructure analyses showed that aluminum foil was almost consumed by forming a titanium aluminide intermetallic compound. Titanium aluminides grow up through spherical shaped islands and metallic-intermetallic interface is a wavy form in Ti-Al system. Thus, the final microstructure consists of alternating layers of intermetallic compound and unreacted Ti metal. Microstructure and phase characterizations were performed by scanning electron microscopy, energy dispersive spectroscopy, and X-ray diffraction. Hardness of test samples was determined as 600 HV for intermetallic zone and 130 HV for metallic zone by the Vickers indentation method

Determination of the Young Modulus of Ti-TiAl3 Metallic Intermetallic Laminate Composites by Nano-Indentation

Nano-indentation is an important technique to determine the Young modulus of multiphase materials where normal tensile tests are not appropriate. In this work, Ti-TiAl3 metallic-intermetallic laminate composites have been fabricated successfully in open atmosphere using commercial purity Al and Ti foils with 250 mu m and 500 mu m initial thicknesses, respectively. Sintering process was performed at 700 degrees C under 2 MPa pressure for 7.5 h. Mechanical properties including the Young modulus were determined after manufacturing. The Young moduli of metallic and intermetallic phases were determined as 89 GPa and 140 GPa, respectively. Microstructure analyses showed that aluminum foil was almost consumed by forming a titanium aluminide intermetallic compound. Titanium aluminides grow up through spherical shaped islands and metallic-intermetallic interface is a wavy form in Ti-Al system. Thus, the final microstructure consists of alternating layers of intermetallic compound and unreacted Ti metal. Microstructure and phase characterizations were performed by scanning electron microscopy, energy dispersive spectroscopy, and X-ray diffraction. Hardness of test samples was determined as 600 HV for intermetallic zone and 130 HV for metallic zone by the Vickers indentation method