H11-induced immunoprotection is predominantly linked to N-glycan moieties during Haemonchus contortus infection

Nematodes are one of the largest groups of animals on the planet. Many of them are major pathogens of humans, animals and plants, and cause destructive diseases and socioeconomic losses worldwide. Despite their adverse impacts on human health and agriculture, nematodes can be challenging to control, because anthelmintic treatments do not prevent re-infection, and excessive treatment has led to widespread drug resistance in nematode populations. Indeed, many nematode species of livestock animals have become resistant to almost all classes of anthelmintics used. Most efforts to develop commercial anti-nematode vaccines (native or recombinant) for use in animals and humans have not succeeded, although one effective (dead) vaccine (Barbervax) has been developed to protect animals against one of the most pathogenic parasites of livestock animals – Haemonchus contortus (the barber’s pole worm). This vaccine contains native molecules, called H11 and H-Gal-GP, derived from the intestine of this blood-feeding worm. In its native form, H11 alone consistently induces high levels (75-95%) of immunoprotection in animals against disease (haemonchosis), but recombinant forms thereof do not. Here, to test the hypothesis that post-translational modification (glycosylation) of H11 plays a crucial role in achieving such high immunoprotection, we explored the N-glycoproteome and N-glycome of H11 using the high-resolution mass spectrometry and assessed the roles of N-glycosylation in protective immunity against H. contortus. Our results showed conclusively that N-glycan moieties on H11 are the dominant immunogens, which induce high IgG serum antibody levels in immunised animals, and that anti-H11 IgG antibodies can confer specific, passive immunity in naïve animals. This work provides the first detailed account of the relevance and role of protein glycosylation in protective immunity against a parasitic nematode, with important implications for the design of vaccines against metazoan parasites.Peer Reviewe

Production and purification of refolded recombinant Plasmodium falciparum β-ketoacyl-ACP reductase from inclusion bodies

A recombinant form of Plasmodium falciparum β -ketoacyl-ACP reductase (PfFabG) was overexpressed in Escherichia coli BL-21 codon plus (DE3). The resulting insoluble inclusion bodies were separated from cellular debris by extensive washing with buffer containing 0.05% Tween 20 and solubilized by homogenization with 8 M urea. Attempts to refold PfFabG from solubilized inclusion bodies employing Rotofor (separation based on different pIs of proteins in a mixture) followed by Ni<SUP>2+</SUP> or cation exchange chromatography were not successful either by bringing down the urea concentration instantaneously, stepwise, or by dialysis. Denatured PfFabG was therefore initially purified by cation exchange chromatography and was then correctly refolded at a final concentration of 100-200 μg/ml in a 20 mM Na-acetate buffer, pH 5.3, with 300 mM NaCl, 10% glycerol, and 0.05% Tween 20. The protein was found to be properly folded only in the presence of the cofactor NADPH and salt at a concentration ≤300 mM by drop dilution method at 2-8° C for 12 h. The purified final product was &gt;98% pure by denaturing gel electrophoresis. The purified protein was biologically active in a standard enzymatic assay using acetoacetyl-CoA as a substrate. The enzyme was found to be stable up to fourth day of purification and glycerol was found to stabilize enzyme activity for several weeks, during storage. This effort paves the way for elucidation of the structure-function correlations for PfFabG as well as exploration of the enzyme for developing inhibitors against it for combating malaria

Experimental validation of roughness power spectrum-based theory of anomalous cottrell response

We experimentally validate theoretical relation between the roughness power spectrum (PS) and electrochemical current transient for a reversible charge transfer system under a single potential step. Roughness features at the electrochemically roughened electrode are characterized using standard measurements such as scanning electron microscopy (SEM), atomic force microscopy (AFM) and cyclic voltammetry (CV). The PS obtained from AFM shows composite finite fractal and nonfractal nature in roughness, whereas the PS from SEM shows only a finite fractal nature. AFM or SEM measurements provide knowledge of fractal dimension (D_H) and two fractal cutoff lengths (&#x2113; and L). Topothesy length (&#x2113;_&#964;) or the related proportionality factor (&#956;&#8803; &#x2113;_&#964;^2D_H-3) from PS data of AFM requires extrapolation of data for unit wavenumber, but this method usually provides unphysical values of &#956;. We provide a novel method to determine the topothesy of electrodes from CV measurements of electroactive area in conjunction with SEM or AFM measurements. Chronoamperometric measurements were made on morphologically characterized Pt-electrodes for a solution of K₄[Fe(CN)₆] and K₃[Fe(CN)₆] in 3 M NaNO₃. The transient response observed experimentally is validated using the measured PS in the theoretical equation for the current. The transient response does not show contributions from Gaussian PS in the low wavenumber region; this is due to the fact that the effective lower cutoff wavenumber is usually limited up to the inverse of the width of roughness (or topothesy length). Fractal dimensions obtained through chronoamperometric measurement on electrodes using Pajkossy’s approach do not correspond to the one obtained from AFM and SEM measurements. Finally, the anomalous response in the Cottrell measurements can be understood through PS-based theory

The spinal muscular atrophy disease protein SMN is linked to the rho-kinase pathway via profilin

Spinal muscular atrophy (SMA), a frequent neurodegenerative disease, is caused by reduced levels of functional survival of motoneuron (SMN) protein. SMN is involved in multiple pathways, including RNA metabolism and splicing as well as motoneuron development and function. Here we provide evidence for a major contribution of the Rho-kinase (ROCK) pathway in SMA pathogenesis. Using an in vivo protein interaction system based on SUMOylation of proteins, we found that SMN is directly interacting with profilin2a. Profilin2a binds to a stretch of proline residues in SMN, which is heavily impaired by a novel SMN2 missense mutation (S230L) derived from a SMA patient. In different SMA models, we identified differential phosphorylation of the ROCK-downstream targets cofilin, myosin-light chain phosphatase and profilin2a. We suggest that hyper-phosphorylation of profilin2a is the molecular link between SMN and the ROCK pathway repressing neurite outgrowth in neuronal cells. Finally, we found a neuron-specific increase in the F-/G-actin ratio that further support the role of actin dynamics in SMA pathogenesis