FMRFamide-Like Peptides (FLPs) Enhance Voltage-Gated Calcium Currents to Elicit Muscle Contraction in the Human Parasite Schistosoma mansoni

Schistosomes are amongst the most important and neglected pathogens in the world, and schistosomiasis control relies almost exclusively on a single drug. The neuromuscular system of schistosomes is fertile ground for therapeutic intervention, yet the details of physiological events involved in neuromuscular function remain largely unknown. Short amidated neuropeptides, FMRFamide-like peptides (FLPs), are distributed abundantly throughout the nervous system of every flatworm examined and they produce potent myoexcitation. Our goal here was to determine the mechanism by which FLPs elicit contractions of schistosome muscle fibers. Contraction studies showed that the FLP Tyr-Ile-Arg-Phe-amide (YIRFamide) contracts the muscle fibers through a mechanism that requires Ca²⁺ influx through sarcolemmal voltage operated Ca²⁺ channels (VOCCs), as the contractions are inhibited by classical VOCC blockers nicardipine, verapamil and methoxyverapamil. Whole-cell patch-clamp experiments revealed that inward currents through VOCCs are significantly and reversibly enhanced by the application of 1 µM YIRFamide; the sustained inward currents were increased to 190% of controls and the peak currents were increased to 180%. In order to examine the biochemical link between the FLP receptor and the VOCCs, PKC inhibitors calphostin C, RO 31–8220 and chelerythrine were tested and all produced concentration dependent block of the contractions elicited by 1 µM YIRFamide. Taken together, the data show that FLPs elicit contractions by enhancing Ca²⁺ influx through VOCC currents using a PKC-dependent pathway.     

Alcohol biosensing by polyamidoamine (PAMAM)/cysteamine/alcohol oxidase‐modified gold electrode

A highly stable and sensitive amperometric alcohol biosensor was developed by immobilizing alcohol oxidase (AOX) through Polyamidoamine (PAMAM) dendrimers on a cysteamine‐modified gold electrode surface. Ethanol determination is based on the consumption of dissolved oxygen content due to the enzymatic reaction. The decrease in oxygen level was monitored at ?0.7 V vs. Ag/AgCl and correlated with ethanol concentration. Optimization of variables affecting the system was performed. The optimized ethanol biosensor showed a wide linearity from 0.025 to 1.0 mM with 100 s response time and detection limit of (LOD) 0.016 mM. In the characterization studies, besides linearity some parameters such as operational and storage stability, reproducibility, repeatability, and substrate specificity were studied in detail. Stability studies showed a good preservation of the bioanalytical properties of the sensor, 67% of its initial sensitivity was kept after 1 month storage at 4°C. The analytical characteristics of the system were also evaluated for alcohol determination in flow injection analysis (FIA) mode. Finally, proposed biosensor was applied for ethanol analysis in various alcoholic beverage as well as offline monitoring of alcohol production through the yeast cultivation. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Chemical proteomic and bioinformatic strategies for the identification and quantification of vascular antigens in cancer

One avenue towards the development of more selective anti-cancer drugs consists in the targeted delivery of bioactive molecules to the tumor environment by means of binding molecules specific to tumor-associated markers. In this context, the targeted delivery of therapeutic agents to newly-formed blood vessels (“vascular targeting”) is particularly attractive, because of the dependence of tumors on new blood vessels to sustain growth and invasion, and because of the accessibility of neo-vascular structures for therapeutic agents injected intravenously. Ligand-based vascular targeting strategies crucially rely on good-quality vascular tumor markers. Here we describe a number of established technologies for the enrichment of accessible vascular proteins based on the isolation of glycoproteins, the in vivo coating of accessible cell surfaces with colloidal silica and the in vivo perfusion with reactive ester derivatives of biotin. Label-free as well as isotopic labeling based strategies for the subsequent MS-based protein quantification are outlined. Finally, bioinformatic workflows for protein quantification are depicted aiming at assisting in the evaluation of appropriate strategies for individual projects. This review gives an overview of current chemical proteomic strategies for the enrichment and quantification of the accessible vascular proteome and helps in selecting bioinformatic strategies for data analysis and validation.     

Experimental Infection of Rabbits with Rabbit and Genotypes 1 and 4 Hepatitis E Viruses

Background

A recent study provided evidence that farmed rabbits in China harbor a novel hepatitis E virus (HEV) genotype. Although the rabbit HEV isolate had 77–79% nucleotide identity to the mammalian HEV genotypes 1 to 4, their genomic organization is very similar. Since rabbits are used widely experimentally, including as models of infection, we investigated whether they constitute an appropriate animal model for human HEV infection.

Methods

Forty-two SPF rabbits were divided randomly into eleven groups and inoculated with six different isolates of rabbit HEV, two different doses of a second-passage rabbit HEV, and with genotype 1 and 4 HEV. Sera and feces were collected weekly after inoculation. HEV antigen, RNA, antibody and alanine aminotransferase in sera and HEV RNA in feces were detected. The liver samples were collected during necropsy subject to histopathological examination.

Findings

Rabbits inoculated with rabbit HEV became infected with HEV, with viremia, fecal virus shedding and high serum levels of viral antigens, and developed hepatitis, with elevation of the liver enzyme, ALT. The severity of disease corresponded to the infectious dose (genome equivalents), with the most severe hepatic disease caused by strain GDC54-18. However, only two of nine rabbits infected with HEV genotype 4, and none infected with genotype 1, developed hepatitis although six of nine rabbits inoculated with the genotype 1 HEV and in all rabbits inoculated with the genotype 4 HEV seroconverted to be positive for anti-HEV IgG antibody by 14 weeks post-inoculation.

Conclusions

These data indicate that rabbits are an appropriate model for rabbit HEV infection but are not likely to be useful for the study of human HEV. The rabbit HEV infection of rabbits may provide an appropriate parallel animal model to study HEV pathogenesis.     

Aptamer-based multiplexed proteomic technology for biomarker discovery

Background

The interrogation of proteomes (“proteomics”) in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology and medicine.

Methodology/Principal Findings

We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 µL of serum or plasma). Our current assay measures 813 proteins with low limits of detection (1 pM median), 7 logs of overall dynamic range (∼100 fM–1 µM), and 5% median coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding signature of DNA aptamer concentrations, which is quantified on a DNA microarray. Our assay takes advantage of the dual nature of aptamers as both folded protein-binding entities with defined shapes and unique nucleotide sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD). We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to rapidly discover unique protein signatures characteristic of various disease states.

Conclusions/Significance

We describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine.     

The Three Filament Model of Skeletal Muscle Stability and Force Production

Ever since the 1950s, muscle force regulation has been associated with the cross-bridge interactions between the two contractile filaments, actin and myosin. This gave rise to what is referred to as the "two-filament sarcomere model". This model does not predict eccentric muscle contractions well, produces instability of myosin alignment and force production on the descending limb of the force-length relationship, and cannot account for the vastly decreased ATP requirements of actively stretched muscles. Over the past decade, we and others, identified that a third myofilament, titin, plays an important role in stabilizing the sarcomere and the myosin filament. Here, we demonstrate additionally how titin is an active participant in muscle force regulation by changing its stiffness in an activation/force dependent manner and by binding to actin, thereby adjusting its free spring length. Therefore, we propose that skeletal muscle force regulation is based on a three filament model that includes titin, rather than a two filament model consisting only of actin and myosin filaments.     

Assessing the ecological risk posed by a recently established invasive alien predator: <Emphasis Type="Italic">Harmonia axyridis</Emphasis> as a case study

Invasive alien predators are a serious threat to biodiversity worldwide. However, there is no generic method for assessing which local species are most at risk following the invasion of a new predator. The harlequin ladybird, Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae), is an alien in Europe and many other parts of the world where it affects other species of ladybirds through competition for food and intra-guild predation (IGP). Here, we describe a method developed to assess which European ladybird species are most at risk following the invasion of H. axyridis. The three components of the risk assessment are: the likelihood that the assessed native species encounters H. axyridis in the field, the hazard of competition for food, and the IGP hazard. Thirty native European ladybird species were assessed through data obtained from field observations, laboratory experiments and literature reviews. The species that are considered most at risk are found on deciduous trees, have immature stages which are highly vulnerable to IGP by H. axyridis, and are primarily aphidophagous. These species should be the focus of specific studies and possibly conservation actions. The risk assessment method proposed here could be applied to other alien predators which are considered a threat to native species through competition and predation.