Two novel dermonecrotic toxins LiRecDT4 and LiRecDT5 from brown spider (Loxosceles intermedia) venom: from cloning to functional characterization

Loxoscelism (the condition produced by the bite of brown spiders) has been reported worldwide, but especially in warmer regions. Clinical manifestations include skin necrosis with gravitational spreading while systemic loxoscelism may include renal failure, hemolysis and thrombocytopenia. The venom contains several toxins, of which the best biochemically and biologically studied is the dermonecrotic toxin, a phospholipase-D. Purified toxin induces cutaneous and systemic loxoscelism, especially necrotic lesions, hematological disturbances and renal failure. Herein, we describe cloning, heterologous expression and purification of two novel dermonecrotic toxins: LiRecDT4 and LiRecDT5. The recombinant proteins stably expressed in Escherichia coli cells were purified from culture supernatants in a single step using Ni(2+)-chelating chromatography producing soluble proteins of 34 kDa (LiRecDT4) and 37 kDa (LiRecDT5). Circular dichroism analysis evidenced correctly folding for toxins but differences in secondary structures. Both proteins were recognized by whole venom serum antibodies and by a specific antibody to dermonecrotic toxin. Also, recombinant toxins with phospholipase activity induced experimental skin lesions and caused a massive inflammatory response in rabbit skin dermis. Nevertheless, toxins displayed different effects upon platelet aggregation, increase in vascular permeability and not caused death in mice. These characteristics in combination with functional studies illustrates that a family of dermonecrotic toxins exists, and includes two novel members that are useful for future structural and functional studies. They will also be useful in biotechnological ends, for example, as inflammatory and platelet aggregating studies, as antigens for serum therapy source and for lipids biochemical research.     

Elevated [11C]-D-deprenyl uptake in chronic Whiplash Associated Disorder suggests persistent musculoskeletal inflammation

There are few diagnostic tools for chronic musculoskeletal pain as structural imaging methods seldom reveal pathological alterations. This is especially true for Whiplash Associated Disorder, for which physical signs of persistent injuries to the neck have yet to be established. Here, we sought to visualize inflammatory processes in the neck region by means Positron Emission Tomography using the tracer (11)C-D-deprenyl, a potential marker for inflammation. Twenty-two patients with enduring pain after a rear impact car accident (Whiplash Associated Disorder grade II) and 14 healthy controls were investigated. Patients displayed significantly elevated tracer uptake in the neck, particularly in regions around the spineous process of the second cervical vertebra. This suggests that whiplash patients have signs of local persistent peripheral tissue inflammation, which may potentially serve as a diagnostic biomarker. The present investigation demonstrates that painful processes in the periphery can be objectively visualized and quantified with PET and that (11)C-D-deprenyl is a promising tracer for these purposes.     

Behavioral transitions and weight change patterns within the PREMIER trial

Little is known about the transition in behaviors from short-term weight loss to maintenance of weight loss. We wanted to determine how short-term and long-term weight loss and patterns of weight change were associated with intervention behavioral targets. This analysis includes overweight/obese participants in active treatment (n = 507) from the previously published PREMIER trial, an 18-month, multicomponent lifestyle intervention for blood pressure reduction, including 33 intervention sessions and recommendations to self-monitor food intake and physical activity daily. Associations between behaviors (attendance, recorded days/week of physical activity, food records/week) and weight loss of ≥5% at 6 and 18 months were examined using logistic regression. We characterized the sample using 5 weight change categories (weight gained, weight stable, weight loss then relapse, late weight loss, and weight loss then maintenance) and analyzed adherence to the behaviors for each category, comparing means with ANOVA. Participants lost an average of 5.3 ± 5.6 kg at 6 months and 4.0 ± 6.7 kg (4.96% of body weight) by 18 months. Higher levels of attendance, food record completion, and recorded days/week of physical activity were associated with increasing odds of achieving 5% weight loss. All weight change groups had declines in the behaviors over time; however, compared to the other four groups, the weight loss/maintenance group (n = 154) had statistically less significant decline in number of food records/week (48%), recorded days/week of physical activity (41.7%), and intervention sessions attended (12.8%) through 18 months. Behaviors associated with short-term weight loss continue to be associated with long-term weight loss, albeit at lower frequencies. Minimizing the decline in these behaviors may be important in achieving long-term weight loss.     

Resilin-bearing wing vein joints in the dragonfly Epiophlebia superstes

In this study, we compared the dorsal and ventral patterns of three vein joint types and three types of resilin patches in the wings of the dragonfly Epiophlebia superstes. The joint types were classified according to their general structure and the resilin patch types according to their arrangement at joints and in the adjacent wing membrane. Resilin patches are found in both dorsal and ventral pleat valleys of the corrugated wings of E. superstes, which results in different patterns of resilin distribution on the dorsal and ventral sides of the wing. In addition to its probable function in conferring flexibility to stressed joints, resilin may also have a damping function. Our results suggest that resilin patches in the leading edge may be loaded in compression, whereas in the trailing area, they may be involved in angle widening and thus loaded in tension. Possible adaptations to the deformability of different areas of the wing, e.g. during the process of camber formation, are discussed.     

Mutational analysis of hepatitis C virus nonstructural protein 5A: potential role of differential phosphorylation in RNA replication and identification of a genetically flexible domain

Nonstructural protein 5A of the hepatitis C virus (HCV) is a highly phosphorylated molecule implicated in multiple interactions with the host cell and most likely involved in RNA replication. Two phosphorylated variants of NS5A have been described, designated according to their apparent molecular masses (in kilodaltons) as p56 and p58, which correspond to the basal and hyperphosphorylated forms, respectively. With the aim of identifying a possible role of NS5A phosphorylation for RNA replication, we performed an extensive mutation analysis of three serine clusters that are involved in phosphorylation and hyperphosphorylation of NS5A. In most cases, alanine substitutions for serine residues in the central cluster 1 that enhanced RNA replication to the highest levels led to a reduction of NS5A hyperphosphorylation. Likewise, several highly adaptive mutations in NS4B, which is also part of the replication complex, resulted in a reduction of NS5A hyperphosphorylation too, arguing that alterations of the NS5A phosphorylation pattern play an important role for RNA replication. On the other hand, a deletion encompassing all highly conserved serine residues in the C-terminal region of NS5A that are involved in basal phosphorylation did not significantly affect RNA replication but reduced formation of p56. This region was found to tolerate even large insertions with only a moderate effect on replication. Based on these results, we propose a model of the role of NS5A phosphorylation in the viral life cycle.     

MSight: an image analysis software for liquid chromatography-mass spectrometry

Images obtained from high-throughput mass spectrometry (MS) contain information that remains hidden when looking at a single spectrum at a time. Image processing of liquid chromatography-MS datasets can be extremely useful for quality control, experimental monitoring and knowledge extraction. The importance of imaging in differential analysis of proteomic experiments has already been established through two-dimensional gels and can now be foreseen with MS images. We present MSight, a new software designed to construct and manipulate MS images, as well as to facilitate their analysis and comparison.