Generalized mathematical representation of the soft tissue artefact

While reconstructing skeletal movement using stereophotogrammetry, the relative movement between a skin marker and the underlying bone is regarded as an artefact (soft tissue artefact: STA). Similarly, the consequent pose, size and shape variations that affect a cluster of markers associated with a bony segment, or any arbitrary change of configuration in the marker local positions as representative of the skin envelope shape variation, may also be looked upon as an STA. Bone pose estimators able to compensate for these artefacts must embed relevant a priori knowledge in the form of an STA mathematical model. Prior to tackling this modeling exercise, an appropriate definition and mathematical representation of the STA time histories must be accomplished. Relevant appropriateness is based on the degree of approximation of the STA reconstruction and on the number of parameters involved.     

The α5 Subunit Regulates the Expression and Function of α4*-Containing Neuronal Nicotinic Acetylcholine Receptors in the Ventral-Tegmental Area

Human genetic association studies have shown gene variants in the α5 subunit of the neuronal nicotinic receptor (nAChR) influence both ethanol and nicotine dependence. The α5 subunit is an accessory subunit that facilitates α4* nAChRs assembly in vitro. However, it is unknown whether this occurs in the brain, as there are few research tools to adequately address this question. As the α4*-containing nAChRs are highly expressed in the ventral tegmental area (VTA) we assessed the molecular, functional and pharmacological roles of α5 in α4*-containing nAChRs in the VTA. We utilized transgenic mice α5+/+(α4YFP) and α5-/-(α4YFP) that allow the direct visualization and measurement of α4-YFP expression and the effect of the presence (α5+/+) and absence of α5 (-/-) subunit, as the antibodies for detecting the α4* subunits of the nAChR are not specific. We performed voltage clamp electrophysiological experiments to study baseline nicotinic currents in VTA dopaminergic neurons. We show that in the presence of the α5 subunit, the overall expression of α4 subunit is increased significantly by 60% in the VTA. Furthermore, the α5 subunit strengthens baseline nAChR currents, suggesting the increased expression of α4* nAChRs to be likely on the cell surface. While the presence of the α5 subunit blunts the desensitization of nAChRs following nicotine exposure, it does not alter the amount of ethanol potentiation of VTA dopaminergic neurons. Our data demonstrates a major regulatory role for the α5 subunit in both the maintenance of α4*-containing nAChRs expression and in modulating nicotinic currents in VTA dopaminergic neurons. Additionally, the α5α4* nAChR in VTA dopaminergic neurons regulates the effect of nicotine but not ethanol on currents. Together, the data suggest that the α5 subunit is critical for controlling the expression and functional role of a population of α4*-containing nAChRs in the VTA.     

Akt regulates L-type Ca2+ channel activity by modulating Cavα1 protein stability

The insulin IGF-1–PI3K–Akt signaling pathway has been suggested to improve cardiac inotropism and increase Ca²⁺ handling through the effects of the protein kinase Akt. However, the underlying molecular mechanisms remain largely unknown. In this study, we provide evidence for an unanticipated regulatory function of Akt controlling L-type Ca²⁺ channel (LTCC) protein density. The pore-forming channel subunit Ca_vα1 contains highly conserved PEST sequences (signals for rapid protein degradation), and in-frame deletion of these PEST sequences results in increased Ca_vα1 protein levels. Our findings show that Akt-dependent phosphorylation of Ca_vβ2, the LTCC chaperone for Ca_vα1, antagonizes Ca_vα1 protein degradation by preventing Ca_vα1 PEST sequence recognition, leading to increased LTCC density and the consequent modulation of Ca²⁺ channel function. This novel mechanism by which Akt modulates LTCC stability could profoundly influence cardiac myocyte Ca²⁺ entry, Ca²⁺ handling, and contractility.     

Env-Expressing Autologous T Lymphocytes Induce Neutralizing Antibody and Afford Marked Protection against Feline Immunodeficiency Virus

The envelope (Env) glycoproteins of HIV and other lentiviruses possess neutralization and other protective epitopes, yet all attempts to induce protective immunity using Env as the only immunogen have either failed or afforded minimal levels of protection. In a novel prime-boost approach, specific-pathogen-free cats were primed with a plasmid expressing Env of feline immunodeficiency virus (FIV) and feline granulocyte-macrophage colony-stimulating factor and then boosted with their own T lymphocytes transduced ex vivo to produce the same Env and interleukin 15 (3 × 10⁶ to 10 × 10⁶ viable cells/cat). After the boost, the vaccinees developed elevated immune responses, including virus-neutralizing antibodies (NA). Challenge with an ex vivo preparation of FIV readily infected all eight control cats (four mock vaccinated and four naïve) and produced a marked decline in the proportion of peripheral CD4 T cells. In contrast, five of seven vaccinees showed little or no traces of infection, and the remaining two had reduced viral loads and underwent no changes in proportions of CD4 T cells. Interestingly, the viral loads of the vaccinees were inversely correlated to the titers of NA. The findings support the concept that Env is a valuable immunogen but needs to be administered in a way that permits the expression of its full protective potential.Despite years of intense research, a truly protective AIDS vaccine is far away. Suboptimal immunogenicity, inadequate antigen presentation, and inappropriate immune system activation are believed to have contributed to these disappointing results. However, several lines of evidence suggest that the control or prevention of infection is possible. For example, despite repeated exposures, some individuals escape infection or delay disease progression after being infected (1, 14, 15). Furthermore, passively infused neutralizing antibodies (NA) (28, 42, 51) or endogenously expressed NA derivatives (29) have been shown to provide protection against intravenous simian immunodeficiency virus challenge. On the other hand, data from several vaccine experiments suggest that cellular immunity is an important factor for protection (6, 32). Therefore, while immune protection against human immunodeficiency virus (HIV) and other lentiviruses appears feasible, the strategies for eliciting it remain elusive.Because of its crucial role in viral replication and infectivity, the HIV envelope (Env) is an attractive immunogen and has been included in nearly all vaccine formulations tested so far (28, 30, 31). Env surface (SU) and transmembrane glycoproteins (gp) are actively targeted by the immune system (9, 10, 47), and Env-specific antibodies and cytotoxic T lymphocytes (CTLs) are produced early in infection. The appearance of these effectors also coincides with the decline of viremia during the acute phase of infection (30, 32). Individuals who control HIV infection in the absence of antiretroviral therapy have Env-specific NA and CTL responses that are effective against a wide spectrum of viral strains (14, 23, 35, 52, 60). At least some of the potentially protective epitopes in Env appear to interact with the cellular receptors during viral entry and are therefore highly conserved among isolates (31, 33, 39, 63). However, these epitopes have complex secondary and tertiary structures and are only transiently exposed by the structural changes that occur during the interaction between Env and its receptors (10, 11, 28). As a consequence, these epitopes are usually concealed from the immune system, and this may explain, at least in part, why Env-based vaccines have failed to show protective efficacy. Indeed, data from previous studies suggested that protection may be most effectively triggered by nascent viral proteins (22, 28, 30, 48, 62).We have conducted a proof-of-concept study to evaluate whether presenting Env to the immune system in a manner as close as possible to what occurs in the context of a natural infection may confer some protective advantage. The study was carried out with feline immunodeficiency virus (FIV), a lentivirus similar to HIV that establishes persistent infections and causes an AIDS-like disease in domestic cats. As far as it is understood, FIV evades immune surveillance through mechanisms similar to those exploited by HIV, and attempts to develop an effective FIV vaccine have met with difficulties similar to those encountered with AIDS vaccines (25, 37, 66). In particular, attempts to use FIV Env as a protective immunogen have repeatedly failed (13, 38, 58). Here we report the result of one experiment in which specific-pathogen-free (SPF) cats primed with a DNA immunogen encoding FIV Env and feline granulocyte-macrophage colony-stimulating factor (GM-CSF) and boosted with viable, autologous T lymphocytes ex vivo that were transduced to express Env and feline interleukin 15 (IL-15) showed a remarkable level of protection against challenge with ex vivo FIV. Consistent with recent findings indicating the importance of NA in controlling lentiviral infections (1, 59, 63), among the immunological parameters investigated, only the titers of NA correlated inversely with protection. Collectively, the findings support the notion that Env is a valuable vaccine immunogen but needs to be administered in a way that permits the expression of its full protective potential.     

Mapping the domains of CD134 as a functional receptor for feline immunodeficiency virus

The feline homologue of CD134 is the primary binding receptor for feline immunodeficiency virus (FIV), targeting the virus preferentially to activated CD4+ helper T cells. However, strains of FIV differ in utilization of CD134; the prototypic strain PPR requires a minimal determinant in the first cysteine-rich domain (CRD1) of feline CD134 to confer near-optimal receptor function, while strains such as GL8 require additional determinants in the CD134 CRD2. We map this determinant to a loop in CRD2 governing the interaction between the receptor and its ligand; the amino acid substitutions S78N-S79Y-K80E restored full viral receptor activity to the CDR2 of human CD134 in the context of feline CD134, with tyrosine-79 appearing to be the critical residue for restoration of receptor function.     

Color Discrimination in the Tufted Capuchin Monkey,Sapajus spp

The present study evaluated the efficacy of an adapted version of the Mollon-Reffin test for the behavioral investigation of color vision in capuchin monkeys. Ten tufted capuchin monkeys (Sapajus spp., formerly referred to as Cebus apella) had their DNA analyzed and were characterized as the following: one trichromat female, seven deuteranope dichromats (six males and one female), and two protanope males, one of which was identified as an “ML protanope.” For their behavioral characterization, all of the subjects were tested at three regions of the Commission International de l''Eclairage (CIE) 1976 u′v′ diagram, with each test consisting of 20 chromatic variation vectors that were radially distributed around the chromaticity point set as the test background. The phenotypes inferred from the behavioral data were in complete agreement with those predicted from the genetic analysis, with the threshold distribution clearly differentiating between trichromats and dichromats and the estimated confusion lines characteristically converging for deuteranopes and the “classic” protanope. The discrimination pattern of the ML protanope was intermediate between protan and deutan, with confusion lines horizontally oriented and parallel to each other. The observed phenotypic differentiation confirmed the efficacy of the Mollon-Reffin test paradigm as a useful tool for evaluating color discrimination in nonhuman primates. Especially noteworthy was the demonstration of behavioral segregation between the “classic” and “ML” protanopes, suggesting identifiable behavioral consequences of even slight variations in the spectral sensitivity of M/L photopigments in dichromats.     

Yeast Killer Toxin-Like Candidacidal Ab6 Antibodies Elicited through the Manipulation of the Idiotypic Cascade

A mouse anti-anti-anti-idiotypic (Id) IgM monoclonal antibody (mAb K20, Ab4), functionally mimicking a Wyckerhamomyces anomalus (Pichia anomala) killer toxin (KT) characterized by fungicidal activity against yeasts presenting specific cell wall receptors (KTR) mainly constituted by β-1,3-glucan, was produced from animals presenting anti-KT Abs (Ab3) following immunization with a rat IgM anti-Id KT-like mAb (mAb K10, Ab2). MAb K10 was produced by immunization with a KT-neutralizing mAb (mAb KT4, Ab1) bearing the internal image of KTR. MAb K20, likewise mAb K10, proved to be fungicidal in vitro against KT-sensitive Candida albicans cells, an activity neutralized by mAb KT4, and was capable of binding to β-1,3-glucan. MAb K20 and mAb K10 competed with each other and with KT for binding to C. albicans KTR. MAb K20 was used to identify peptide mimics of KTR by the selection of phage clones from random peptide phage display libraries. Using this strategy, four peptides (TK 1-4) were selected and used as immunogen in mice in the form of either keyhole limpet hemocyanin (KLH) conjugates or peptide-encoding minigenes. Peptide and DNA immunization could induce serum Abs characterized by candidacidal activity, which was inhibited by laminarin, a soluble β-1,3-glucan, but not by pustulan, a β-1,6-glucan. These findings show that the idiotypic cascade can not only overcome the barrier of animal species but also the nature of immunogens and the type of technology adopted.     

Cantharidin content in two Mediterranean species of blister beetles,Lydus trimaculatus and Mylabris variabilis (Coleoptera: Meloidae)

Cantharidin (CA), a toxic terpene produced by blister beetles (Coleoptera: Meloidae), attracts the interest of many researchers for its renowned medical properties. The CA content in blister beetles has been mainly quantified in some Oriental species, due to their use in traditional Chinese medicine, or in few other species of toxicological importance. As CA quantification has been largely ignored in many other members of this family, we aimed at estimating CA content in natural populations of two of the most abundant species in central Italy, Mylabris variabilis and Lydus trimaculatus. Nearly 100 individuals for each species were collected in the field, identified and reared in fauna‐boxes until CA collection. Available protocols were optimized for CA extraction from both dried‐body tissues and exuded hemolymph collected from leg joints using capillary tubes. The CA content was quantified in a gas chromatography system coupled to a mass spectrometer using a calibration curve with diethyl‐ester of norcantharidin as the internal standard. We observed: (i) high variability in CA content among specimens, which was positively related to individual dry‐weight; and (ii) slightly larger (though non‐significant) amount of CA in males than in females for both species. Our data are consistent with the available published reports about CA content, transfer and distribution in blister beetles and suggest a conserved biological role of this terpene in Meloidae.