Molecular tools for a molecular medicine: analyzing genes, transcripts and proteins using padlock and proximity probes

Procedures and reagents are needed to specifically detect all the macromolecules that are being identified in the course of genome projects. We discuss how this challenge may be met using a set of ligation-based reagents termed padlock probes and proximity ligation probes. These probes include elements with affinity for specific nucleic acid and protein molecules, respectively, along with unique identifier DNA sequence elements that encode the identity of the recognized target molecules. The information content of DNA strands that form in the detection reactions are recorded after amplification, allowing the recognized target molecules to be identified. The procedures permit highly specific solution-phase or localized analyses of large sets of target molecules as required in future molecular analyses.     

Synthesis of aldehydo-sugar derivatives of pyrazoloquinoline as inhibitors of herpes simplex virus type 1 replication

Synthesis of a novel series of structurally related pyrazoloquinoline nucleosides is described. All the newly synthesized compounds were examined for their in vitro antiviral activity against herpes simplex type-1 as shown by two different bioassays, namely; crystal violet staining or the MTS tetrazolium dye measurement. The acute toxicity (LD50) values of the biologically active compounds were determined.     

Acidosis has opposite effects on neuronal survival during hypoxia and reoxygenation

To study the effect of extracellular acidosis on apoptosis and necrosis during ischemia and reoxygenation, we exposed human post-mitotic NT2-N neurones to oxygen and glucose deprivation (OGD) followed by reoxygenation. In some experiments, pH of the cell medium was lowered to 5.9 during either OGD or reoxygenation or both. Staurosporine, used as a positive control for apoptosis, caused Poly(ADP-ribose)-polymerase (PARP) cleavage and nuclear fragmentation, but no PARP cleavage and little fragmentation were seen after OGD. Low molecular weight DNA fragments were found after staurosporine treatment, but not after OGD. No protective effect of caspase inhibitors was seen after 3 h of OGD and 21 h of reoxygenation, but after 45 h of reoxygenation caspase inhibition induced a modest improvement in 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) cleavage. While acidosis during OGD accompanied by neutral medium during reoxygenation protected the neurones (MTT: 228 +/- 117% of neutral medium, p < 0.001), acidosis during reoxygenation only was detrimental (MTT: 38 +/- 25%, p < 0.01). We conclude that apoptotic mechanisms play a minor role after OGD in NT2-N neurones. The effect of acidosis on neuronal survival depends on the timing of acidosis, as acidosis was protective during OGD and detrimental during reoxygenation.     

Sialoside specificity of the siglec family assessed using novel multivalent probes: identification of potent inhibitors of myelin-associated glycoprotein

Ten of the 11 known human siglecs or their murine orthologs have been evaluated for their specificity for over 25 synthetic sialosides representing most of the major sequences terminating carbohydrate groups of glycoproteins and glycolipids. Analysis has been performed using a novel multivalent platform comprising biotinylated sialosides bound to a streptavidin-alkaline phosphatase conjugate. Each siglec was found to have a unique specificity for binding 16 different sialoside-streptavidin-alkaline phosphatase probes. The relative affinities of monovalent sialosides were assessed for each siglec in competitive inhibition studies. The quantitative data obtained allows a detailed analysis of each siglec for the relative importance of sialic acid and the penultimate oligosaccharide sequence on binding affinity and specificity. Most remarkable was the finding that myelin-associated glycoprotein (Siglec-4) binds with 500-10,000-fold higher affinity to a series of mono- and di-sialylated derivatives of the O-linked T-antigen (Galbeta(1-3)-GalNAc(alpha)OThr) as compared with alpha-methyl-NeuAc.     

Environmental impacts of hybrid and electric vehicles—a review

Purpose

A literature review is undertaken to understand how well existing studies of the environmental impacts of hybrid and electric vehicles (EV) address the full life cycle of these technologies. Results of studies are synthesized to compare the global warming potential (GWP) of different EV and internal combustion engine vehicle (ICEV) options. Other impacts are compared; however, data availability limits the extent to which this could be accomplished.

Method

We define what should be included in a complete, state-of-the-art environmental assessment of hybrid and electric vehicles considering components and life cycle stages, emission categories, impact categories, and resource use and compare the content of 51 environmental assessments of hybrid and electric vehicles to our definition. Impact assessment results associated with full life cycle inventories (LCI) are compared for GWP as well as emissions of other pollutants. GWP results by life cycle stage and key parameters are extracted and used to perform a meta-analysis quantifying the impacts of vehicle options.

Results

Few studies provide a full LCI for EVs together with assessment of multiple impacts. Research has focused on well to wheel studies comparing fossil fuel and electricity use as the use phase has been seen to dominate the life cycle of vehicles. Only very recently have studies begun to better address production impacts. Apart from batteries, very few studies provide transparent LCIs of other key EV drivetrain components. Estimates of EV energy use in the literature span a wide range, 0.10?C0.24?kWh/km. Similarly, battery and vehicle lifetime plays an important role in results, yet lifetime assumptions range between 150,000?C300,000?km. CO₂ and GWP are the most frequently reported results. Compiled results suggest the GWP of EVs powered by coal electricity falls between small and large conventional vehicles while EVs powered by natural gas or low-carbon energy sources perform better than the most efficient ICEVs. EV results in regions dependant on coal electricity demonstrated a trend toward increased SO _x emissions compared to fuel use by ICEVs.

Conclusions

Moving forward research should focus on providing consensus around a transparent inventory for production of electric vehicles, appropriate electricity grid mix assumptions, the implications of EV adoption on the existing grid, and means of comparing vehicle on the basis of common driving and charging patterns. Although EVs appear to demonstrate decreases in GWP compared to conventional ICEVs, high efficiency ICEVs and grid-independent hybrid electric vehicles perform better than EVs using coal-fired electricity.     

Novel Zn2+ Modulated GPR39 Receptor Agonists Do Not Drive Acute Insulin Secretion in Rodents

Type 2 diabetes (T2D) occurs when there is insufficient insulin release to control blood glucose, due to insulin resistance and impaired β-cell function. The GPR39 receptor is expressed in metabolic tissues including pancreatic β-cells and has been proposed as a T2D target. Specifically, GPR39 agonists might improve β-cell function leading to more adequate and sustained insulin release and glucose control. The present study aimed to test the hypothesis that GPR39 agonism would improve glucose stimulated insulin secretion in vivo. A high throughput screen, followed by a medicinal chemistry program, identified three novel potent Zn²⁺ modulated GPR39 agonists. These agonists were evaluated in acute rodent glucose tolerance tests. The results showed a lack of glucose lowering and insulinotropic effects not only in lean mice, but also in diet-induced obese (DIO) mice and Zucker fatty rats. It is concluded that Zn²⁺ modulated GPR39 agonists do not acutely stimulate insulin release in rodents.     

Is population structure in the European white stork determined by flyway permeability rather than translocation history?

European white stork are long considered to diverge to eastern and western migration pools as a result of independent overwintering flyways. In relatively recent times, the western and northern distribution has been subject to dramatic population declines and country‐specific extirpations. A number of independent reintroduction programs were started in the mid 1950s to bring storks back to historical ranges. Founder individuals were sourced opportunistically from the Eastern and Western European distributions and Algeria, leading to significant artificial mixing between eastern and western flyways. Here we use mitochondrial and microsatellite DNA to test the contention that prior to translocation, eastern and western flyways were genetically distinct. The data show a surprising lack of structure at any spatial or temporal scale suggesting that even though birds were moved between flyways, there is evidence of natural mixing prior to the onset of translocation activities. Overall a high retention of genetic diversity, high N_ef, and an apparent absence of recent genetic bottleneck associated with early 20th century declines suggest that the species is well equipped to respond to future environmental pressures.