Investigating the interaction between osteoprotegerin and receptor activator of NF-kappaB or tumor necrosis factor-related apoptosis-inducing ligand: evidence for a pivotal role for osteoprotegerin in regulating two distinct pathways

Osteoprotegerin (OPG) binds the ligand for receptor activator of nuclear factor kappaB (RANKL) to prevent association with its receptor RANK and inhibit osteoclast-mediated bone resorption. OPG has been reported, recently, to inhibit tumor necrosis factor-related apoptosis-induced ligand (TRAIL)-induced tumor cell apoptosis. This raises the possibility that OPG may play a unique role in regulating these two signaling pathways. However, there are little data on the interactions between OPG, RANKL, and TRAIL, and the relative affinity of OPG for these two ligands is unknown. In the present study we examined the ability of OPG to bind native human TRAIL and RANKL under physiological conditions. Native TRAIL was expressed in Escherichia coli, purified to homogeneity, and shown to induce human myeloma cell apoptosis. OPG inhibited native TRAIL from binding the TRAILR1 at 37 degrees C in vitro. Similarly, OPG prevented RANKL from binding to RANK. TRAIL also prevented OPG-mediated inhibition of RANKL from binding RANK. The affinity of OPG for native TRAIL and RANKL at 37 degrees C was determined by plasmon surface resonance analysis. OPG had a binding affinity for TRAIL of 45 nM, whereas the affinity of OPG for RANKL was 23 nM. These data suggest that OPG can bind both RANKL and TRAIL and that the affinity of OPG for these two ligands is of a similar order of magnitude. Furthermore, OPG prevented TRAIL-mediated reductions in cell viability, whereas TRAIL inhibited OPG-mediated inhibition of osteoclastogenesis in vitro. This highlights the pivotal role of OPG in regulating the biology of both RANKL and TRAIL.     

SorCS2 Controls Functional Expression of Amino Acid Transporter EAAT3 and Protects Neurons from Oxidative Stress and Epilepsy-Induced Pathology

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Phosphorylation of the Pseudomonas aeruginosa response regulator AlgR is essential for type IV fimbria-mediated twitching motility

The response regulator AlgR is required for both alginate biosynthesis and type IV fimbria-mediated twitching motility in Pseudomonas aeruginosa. In this study, the roles of AlgR signal transduction and phosphorylation in twitching motility and biofilm formation were examined. The predicted phosphorylation site of AlgR (aspartate 54) and a second aspartate (aspartate 85) in the receiver domain of AlgR were mutated to asparagine, and mutant algR alleles were introduced into the chromosome of P. aeruginosa strains PAK and PAO1. Assays of these mutants demonstrated that aspartate 54 but not aspartate 85 of AlgR is required for twitching motility and biofilm initiation. However, strains expressing AlgR D85N were found to be hyperfimbriate, indicating that both aspartate 54 and aspartate 85 are involved in fimbrial biogenesis and function. algD mutants were observed to have wild-type twitching motility, indicating that AlgR control of twitching motility is not mediated via its role in the control of alginate biosynthesis. In vitro phosphorylation assays showed that AlgR D54N is not phosphorylated by the enteric histidine kinase CheA. These findings indicate that phosphorylation of AlgR most likely occurs at aspartate 54 and that aspartate 54 and aspartate 85 of AlgR are required for the control of the molecular events governing fimbrial biogenesis, twitching motility, and biofilm formation in P. aeruginosa.     

Transient Transfection Factors for High-Level Recombinant Protein Production in Suspension Cultured Mammalian Cells

The efficient transfection of cloned genes into mammalian cells system plays a critical role in the production of large quantities of recombinant proteins (r-proteins). In order to establish a simple and scaleable transient protein production system, we have used a cationic lipid-based transfection reagent-FreeStyle MAX to study transient transfection in serum-free suspension human embryonic kidney (HEK) 293 and Chinese hamster ovary (CHO) cells. We used quantification of green fluorescent protein (GFP) to monitor transfection efficiency and expression of a cloned human IgG antibody to monitor r-protein production. Parameters including transfection reagent concentration, DNA concentration, the time of complex formation, and the cell density at the time of transfection were analyzed and optimized. About 70% GFP-positive cells and 50-80 mg/l of secreted IgG antibody were obtained in both HEK-293 and CHO cells under optimal conditions. Scale-up of the transfection system to 1 l resulted in similar transfection efficiency and protein production. In addition, we evaluated production of therapeutic proteins such as human erythropoietin and human blood coagulation factor IX in both HEK-293 and CHO cells. Our results showed that the higher quantity of protein production was obtained by using optimal transient transfection conditions in serum-free adapted suspension mammalian cells.     

Adapting Stand‐Alone Renewable Energy Technologies for the Circular Economy through Eco‐Design and Recycling

Renewable energy (RE) technologies are looked upon favorably to provide for future energy demands and reduce greenhouse gas (GHG) emissions. However, the installation of these technologies requires large quantities of finite material resources. We apply life cycle assessment to 100 years of electricity generation from three stand‐alone RE technologies—solar photovoltaics, run‐of‐river hydro, and wind—to evaluate environmental burden profiles against baseline electricity generation from fossil fuels. We then devised scenarios to incorporate circular economy (CE) improvements targeting hotspots in systems’ life cycle, specifically (1) improved recycling rates for raw materials and (ii) the application of eco‐design. Hydro presented the lowest environmental burdens per kilowatt‐hour of electricity generation compared with other RE technologies, owing to its higher efficiency and longer life spans for main components. Distinct results were observed in the environmental performance of each system based on the consideration of improved recycling rates and eco‐design. CE measures produced similar modest savings in already low GHG emissions burdens for each technology, while eco‐design specifically had the potential to provide significant savings in abiotic resource depletion. Further research to explore the full potential of CE measures for RE technologies will curtail the resource intensity of RE technologies required to mitigate climate change.     

Species traits and climate velocity explain geographic range shifts in an ocean‐warming hotspot

Species' ranges are shifting globally in response to climate warming, with substantial variability among taxa, even within regions. Relationships between range dynamics and intrinsic species traits may be particularly apparent in the ocean, where temperature more directly shapes species' distributions. Here, we test for a role of species traits and climate velocity in driving range extensions in the ocean‐warming hotspot of southeast Australia. Climate velocity explained some variation in range shifts, however, including species traits more than doubled the variation explained. Swimming ability, omnivory and latitudinal range size all had positive relationships with range extension rate, supporting hypotheses that increased dispersal capacity and ecological generalism promote extensions. We find independent support for the hypothesis that species with narrow latitudinal ranges are limited by factors other than climate. Our findings suggest that small‐ranging species are in double jeopardy, with limited ability to escape warming and greater intrinsic vulnerability to stochastic disturbances.     

Synthesis and DNA binding properties of an amidine-linked and phenyl-containing analogue of distamycin A

The synthesis of a novel amidine-linked analogue 1 of the phenyl-containing congener 2 of distamycin A and its DNA binding properties are described. The amidine group in 1 improves its water solubility while retaining the minor groove and AT sequence binding selectively.A phenyl-containing and amidine-linked analogue 1 of distamycin A has improved water solubility while retaining the minor groove and AT sequence binding selectivity to DNA.     

Effect of Repeat Dosing of Engineered Oncolytic Herpes Simplex Virus on Preclinical Models of Rhabdomyosarcoma

Rhabdomyosarcoma (RMS), a tumor of skeletal muscle origin, is the most common sarcoma of childhood. Despite multidrug chemotherapy regimens, surgical intervention, and radiation treatment, outcomes remain poor, especially in advanced disease, and novel therapies are needed for the treatment of these aggressive malignancies. Genetically engineered oncolytic viruses, such as herpes simplex virus-1 (HSV), are currently being explored as treatments for pediatric tumors. M002, an oncolytic HSV, has both copies of the γ₁34.5 gene deleted, enabling replication in tumor cells but thwarting infection of normal, postmitotic cells. We hypothesized that M002 would infect human RMS tumor cells and lead to decreased tumor cell survival in vitro and impede tumor growth in vivo. In the current study, we demonstrated that M002 could infect, replicate in, and decrease cell survival in both embryonal (ERMS) and alveolar rhabdomyosarcoma (ARMS) cells. Additionally, M002 reduced xenograft tumor growth and increased animal survival in both ARMS and ERMS. Most importantly, we showed for the first time that repeated dosing of oncolytic virus coupled with low-dose radiation provided improved tumor response in RMS. These findings provide support for the clinical investigation of oncolytic HSV in pediatric RMS.     

Fluorescence polarization assay and inhibitor design for MDM2/p53 interaction

MDM2 is an important negative regulator of the tumor suppressor protein p53 which regulates the expression of many genes including MDM2. The delicate balance of this autoregulatory loop is crucial for the maintenance of the genome and control of the cell cycle and apoptosis. MDM2 hyperactivity, due to amplification/overexpression or mutational inactivation of the ARF locus, inhibits the function of wild-type p53 and can lead to the development of a wide variety of cancers. Thus, the development of anti-MDM2 therapies may restore normal p53 function in tumor cells and induce growth suppression and apoptosis. We report here a novel high-throughput fluorescence polarization binding assay and its application in rank ordering small-molecule inhibitors that block the binding of MDM2 to a p53-derived fluorescent peptide.     

ANTARCTIC DISTRIBUTION,PIGMENT AND LIPID COMPOSITION,AND MOLECULAR IDENTIFICATION OF THE BRINE DINOFLAGELLATE POLARELLA GLACIALIS (DINOPHYCEAE)1

Polarella glacialis (Montresor et al.) was identified in Davis Station sea ice by morphological and DNA sequence comparison of cultures with those of the authentic strain P. glacialis CCMP 1383 isolated from McMurdo Sound. Cells and cysts of the Davis isolate (FL1B) were morphologically indistinguishable from P. glacialis, and comparison of the large subunit rDNA of both cultures demonstrated only 0.2% sequence divergence over 1366 base pairs. The photosynthetic pigments of P. glacialis (strains FL1B and CCMP 1383) were typical of dinoflagellates, with peridinin (contributing up to 31%) as the major accessory pigment. Extremely high levels of polyunsaturated fatty acids (PUFA, up to 76.3%) were characteristic of P. glacialis isolate FL1B. The high PUFA concentration of this species is thought to be an adaptation to survive the cold temperatures of the upper fast ice. The sterol profile of FL1B was atypical of dinoflagellates, with 4‐desmethylsterols (up to 79%) in greater abundance than 4α‐methyl sterols (up to 24%). 27‐Nor‐24‐methylcholest‐5,22E‐dien‐3β‐ol was identified as the principle sterol in P. glacialis, contributing up to 64% of the total sterol composition.