Infrared thermal imaging associated with pain in laboratory animals

The science of animal welfare has evolved over the years, and recent scientific advances have enhanced our comprehension of the neurological, physiological, and ethological mechanisms of diverse animal species. Currently, the study of the affective states (emotions) of nonhuman animals is attracting great scientific interest focused primarily on negative experiences such as pain, fear, and suffering, which animals experience in different stages of their lives or during scientific research. Studies underway today seek to establish methods of evaluation that can accurately measure pain and then develop effective treatments for it, because the techniques available up to now are not sufficiently precise. One innovative technology that has recently been incorporated into veterinary medicine for the specific purpose of studying pain in animals is called infrared thermography (IRT), a technique that works by detecting and measuring levels of thermal radiation at different points on the body’s surface with high sensitivity. Changes in IRT images are associated mainly with blood perfusion, which is modulated by the mechanisms of vasodilatation and vasoconstriction. IRT is an efficient, noninvasive method for evaluating and controlling pain, two critical aspects of animal welfare in biomedical research. The aim of the present review is to compile and analyze studies of infrared thermographic changes associated with pain in laboratory research involving animals.     

Advances in approaches to seagrass restoration in Australia

Three case studies involving two temperate Australian seagrass species – Pondweed (Ruppia tuberosa) and Ribbon Weed (Posidonia australis) – highlight different approaches to their restoration. Seeds and rhizomes were used in three collaborative programmes to promote new approaches to scale up restoration outcomes.     

Elastic modulus and stress-transfer properties of tunicate cellulose whiskers

Experimental deformation micromechanics of natural cellulose fibers using Raman spectroscopy and X-ray diffraction have been widely reported. However, little has been published on the direct measurements of the mechanical properties, and in particular the elastic modulus, of the highly crystalline material in the native state. Here we report on measurements of the elastic modulus of tunicate cellulose using a Raman spectroscopic technique. A dispersed sample of the material is deformed using a four-point bending test, and a shift in a characteristic Raman band (located at 1095 cm(-1)) is used as an indication of the stress in the material. Relatively little intensity change of the Raman band located at 1095 cm(-1) is shown to occur for samples oriented parallel and perpendicular to the polarization direction of the laser, as compared to a highly oriented flax sample. This indicates that the tunicate sample is a two-dimensional in-plane random network of fibers. By use of this result, the Raman shift, and calibrations with strain from other materials, it is shown that the modulus of the material is very high, at about 143 GPa, and a lack of Raman band broadening is thought to be due to the fact that there is pure crystalline deformation occurring without the effect of crystalline/amorphous fractions. A strain sensitivity of the shift in the 1095-cm(-1) Raman peak for this specimen is shown to be -2.4 +/- 0.2 cm(-1)/%. A molecular mechanics approach, using computer simulation and an empirical force field, was used to predict the modulus of a highly oriented chain of the material, and this is found to be 145 GPa, which is in agreement with the experimental data. However, by use of a normal-mode analysis, it is found that a number of modes have positions close to the central positions of the experimental Raman band. One in particular is found to shift at a rate of 2.5 cm(-1)/%, but due to the complex nature of the structure, it is not entirely conclusive that this band is representative of the experimental findings.     

Elongating modified conserved peptides eliminates their immunogenicity and protective efficacy against P. falciparum malaria

Plasmodium falciparum malaria protein peptides were synthesised in the search for more effective routes for inducing a protective immune response against this deadly parasite and this information has been associated with such molecules' three-dimensional structure. These peptides had high red blood cell binding activity and their carboxy- and amino-terminal extremes were elongated for determining their immunogenic and protection-inducing activity against this disease in the Aotus monkey experimental model. 1H-NMR was used for analysing their three-dimensional structure; FAST ELISA, immunofluorescence antibody test, and Western blot were used for identifying their antibody inducing capacity and these previously immunised Aotus were inoculated with a highly infective P. falciparum strain to determine whether these elongated peptides were able to induce protection. This was aimed at establishing an association or correlation between long peptides' three-dimensional structure and their immunogenic and protection-inducing response in these monkeys. Peptides 20026 (25 residue), 20028 (30 residue), and 20030 (35 residues) were synthesised based on elongating the amino-terminal region of the 10022 highly immunogenic and protection-inducing modified peptide. 1H-NMR studies revealed that the first three had Classical type III beta-turn structures, different from the 20-amino acid long modified peptide 10022 which had a distorted type III beta-turn. Humoral immune response analysis showed that even when some antibodies could be generated against the parasite, none of the immunised Aotus could be protected with elongated peptides suggesting that elongating them eliminated modified peptide 10022 immunogenic and protection-inducing capacity.     

Delineation of stage specific expression of Plasmodium falciparum EBA-175 by biologically functional region II monoclonal antibodies

Background

The malaria parasite Plasmodium falciparum EBA-175 binds its receptor sialic acids on glycophorin A when invading erythrocytes. The receptor-binding region (RII) contains two cysteine-rich domains with similar cysteine motifs (F1 and F2). Functional relationships between F1 and F2 domains and characterization of EBA-175 were studied using specific monoclonal antibodies (mAbs) against these domains.

Methods and Findings

Five mAbs specific for F1 or F2 were generated. Three mAbs specific for F2 potently blocked binding of EBA-175 to erythrocytes, and merozoite invasion of erythrocytes (IC₅₀ 10 to 100 µg/ml IgG in growth inhibition assays). A mAb specific for F1 blocked EBA-175 binding and merozoite invasion less effectively. The difference observed between the IC₅₀ of F1 and F2 mAbs was not due to differing association and disassociation rates as determined by surface plasmon resonance. Four of the mAbs recognized conformation-dependent epitopes within F1 or F2. Used in combination, F1 and F2 mAbs blocked the binding of native EBA-175 to erythrocytes and inhibited parasite invasion synergistically in vitro. MAb R217, the most potent, did not recognize sporozoites, 3-day hepatocyte stage parasites, nor rings, trophozoites, gametocytes, retorts, ookinetes, and oocysts but recognized 6-day hepatocyte stage parasites, and schizonts. Even though efficient at blocking binding to erythrocytes and inhibiting invasion into erythrocytes, MAb R217 did not inhibit sporozoite invasion and development in hepatocytes in vitro.

Conclusions

The role of the F1 and F2 domains in erythrocyte invasion and binding was elucidated with mAbs. These mAbs interfere with native EBA-175 binding to erythrocyte in a synergistic fashion. The stage specific expression of EBA-175 showed that the primary focus of activity was the merozoite stage. A recombinant RII protein vaccine consisting of both F1 and F2 domains that could induce synergistic activity should be optimal for induction of antibody responses that interfere with merozoite invasion of erythrocytes.     

Purification and some properties of an extracellular acid protease from <Emphasis Type="Italic">Aspergillus</Emphasis><Emphasis Type="Italic">clavatus</Emphasis>

Acid proteases represent an important group of enzymes, widely used in food, beverage and pharmaceutical industries. For most of these applications the enzymatic preparation must be at least partially purified and free of substances that could change the characteristics of the product or the process. Fungal proteases have replaced other sources because they are easily obtained mainly from Mucor, Rhizopus, Penicillium and Aspergillus species. A strain of Aspergillus clavatus was selected by producing high level of acid protease activity. An extracellular aspartatic protease from this strain was purified 37.2 times with 37% recovery using (NH₄)₂SO₄ fractionation and ion-exchange chromatography. The enzyme was found to be monomeric having a molecular mass of 30.4 kDa. The purified enzyme is an acid protease with optimum pH of 5.5 and temperature for optimum activity of 50 °C. Its high pH stability was verified in the range of 3.5–6.5. The acid protease was strongly inhibited by Hg⁺² and partially inhibited by Cu⁺², Zn⁺² and Mn⁺². The enzyme was sensitive to denaturing agent SDS and activated by thiol-containing reducing agent dithiotreitol (DTT). The protease activity was not influenced by iodoacetic acid, E-64 and PMSF, while it was lightly actived by EDTA and totally inhibited by pepstatin, with a K_i of 7.8 μM, indicating that is an aspartic protease. A. clavatus acid protease presents interesting characteristics for biotechnological process, such as cheese and flavor manufacture and dietary supplements, in which activity and stability in acid pH are required.     

IL-17RA Signaling Reduces Inflammation and Mortality during Trypanosoma cruzi Infection by Recruiting Suppressive IL-10-Producing Neutrophils

Members of the IL-17 cytokine family play an important role in protection against pathogens through the induction of different effector mechanisms. We determined that IL-17A, IL-17E and IL-17F are produced during the acute phase of T. cruzi infection. Using IL-17RA knockout (KO) mice, we demonstrate that IL-17RA, the common receptor subunit for many IL-17 family members, is required for host resistance during T. cruzi infection. Furthermore, infected IL-17RA KO mice that lack of response to several IL-17 cytokines showed amplified inflammatory responses with exuberant IFN-γ and TNF production that promoted hepatic damage and mortality. Absence of IL-17RA during T. cruzi infection resulted in reduced CXCL1 and CXCL2 expression in spleen and liver and limited neutrophil recruitment. T. cruzi-stimulated neutrophils secreted IL-10 and showed an IL-10-dependent suppressive phenotype in vitro inhibiting T-cell proliferation and IFN-γ production. Specific depletion of Ly-6G+ neutrophils in vivo during T. cruzi infection raised parasitemia and serum IFN-γ concentration and resulted in increased liver pathology in WT mice and overwhelming wasting disease in IL-17RA KO mice. Adoptively transferred neutrophils were unable to migrate to tissues and to restore resistant phenotype in infected IL-17RA KO mice but migrated to spleen and liver of infected WT mice and downregulated IFN-γ production and increased survival in an IL-10 dependent manner. Our results underscore the role of IL-17RA in the modulation of IFN-γ-mediated inflammatory responses during infections and uncover a previously unrecognized regulatory mechanism that involves the IL-17RA-mediated recruitment of suppressive IL-10-producing neutrophils.     

Digoxin Derivatives with Enhanced Selectivity for the α2 Isoform of Na,K-ATPase: EFFECTS ON INTRAOCULAR PRESSURE IN RABBITS*

In the ciliary epithelium of the eye, the pigmented cells express the α1β1 isoform of Na,K-ATPase, whereas the non-pigmented cells express mainly the α2β3 isoform of Na,K-ATPase. In principle, a Na,K-ATPase inhibitor with selectivity for α2 could effectively reduce intraocular pressure with only minimal local and systemic toxicity. Such an inhibitor could be applied topically provided it was sufficiently permeable via the cornea. Previous experiments with recombinant human α1β1, α2β1, and α3β1 isoforms showed that the classical cardiac glycoside, digoxin, is partially α2-selective and also that the trisdigitoxose moiety is responsible for isoform selectivity. This led to a prediction that modification of the third digitoxose might increase α2 selectivity. A series of perhydro-1,4-oxazepine derivatives of digoxin have been synthesized by periodate oxidation and reductive amination using a variety of R-NH₂ substituents. Several derivatives show enhanced selectivity for α2 over α1, close to 8-fold in the best case. Effects of topically applied cardiac glycosides on intraocular pressure in rabbits have been assessed by their ability to either prevent or reverse acute intraocular pressure increases induced by 4-aminopyridine or a selective agonist of the A3 adenosine receptor. Two relatively α2-selective digoxin derivatives efficiently normalize the ocular hypertension, by comparison with digoxin, digoxigenin, or ouabain. This observation is consistent with a major role of α2 in aqueous humor production and suggests that, potentially, α2-selective digoxin derivatives could be of interest as novel drugs for control of intraocular pressure.