Experimental ivermectin treatment of sarcoptic mange and establishment of a mange-free population of Spanish ibex.

Ivermectin was used to treat sarcoptic mange in Spanish ibex (Capra pyrenaica hispanica). Its therapeutic effectiveness was analyzed when it was administered through subcutaneous injection, to sick animals in the consolidation stage of mange (third phase) and, with double injections to chronically affected animals (fourth phase) at a dosage of 0.2 or 0.4 mg/kg body weight (bw). Three wk after treatment, the animals in the third phase of mange treated with a high dose (0.4 mg/kg bw) of ivermectin were completely cured. The same result was achieved after 4 wk of treatment in those animals in phase 3 of mange when 0.2 mg/kg body weight was used. Double injection with ivermectin, even at high doses, did not guarantee the complete cure of all cases of sarcoptic mange in the chronic stage (phase 4); only three of six animals were free of Sarcoptes scabiei. The second experiment consisted on the application of a sanitation program in order to obtain a population of Spanish ibex free from S. scabiei, starting with free-ranging animals, some of them healthy and others sick. After capture the animals were classified as chronically ill, in which case they were excluded from the program, mite carriers and healthy specimens. All the animals were treated first topically with foxim (500 mg/l) and subcutaneously with ivermectin (0.4 mg/kg bw). The infected animals were housed in the treatment pen, and received two doses of ivermectin (0.2 mg/kg bw) at an interval of 15 days, then spent 15 days in the quarantine pen, where they received a further dose before they were included in the pool of healthy animals, and immediately were placed in the quarantine phase. The sanitation we implemented was fully effective in curing the affliction of Spanish ibex affected by S. scabiei.     

Development of molecular techniques for detection of lymphocystis disease virus in different marine fish species

AIMS: The development and evaluation of a protocol based on polymerase chain reaction (PCR) and nucleic acid hybridization techniques for the specific detection of lymphocystis disease virus (LCDV) in several marine fish species. METHODS AND RESULTS: The pair of primers for PCR, OBL3 and OBL4, was designed based on published nucleotide sequence (LCDV-1) and amplifies a fragment within the major capsid protein. The sensitivity was evaluated using DNA from purified viral particles, as well as from cells inoculated with several viral concentrations. The PCR combined with slot blot was the most sensitive methodology, detecting 2.5 ng of viral DNA. Using this methodology LCDV was detected at 5 days postinoculation from SAF-1 cells initially inoculated with 10(-5) TCID(50) ml(-1). The combination of PCR with membrane hybridization has also been proved to be adequate to detect LCDV from apparently healthy carriers by means of caudal fin sample analysis. This asymptomatic infection was also demonstrated by classical virological methods (cell culture and immunoblot). CONCLUSIONS: The protocol described in this study allows the specific detection of LCDV, both in cell cultures and in fin homogenates from asymptomatic fish. SIGNIFICANCE AND IMPACT OF THE STUDY: The detection of asymptomatic carriers by a rapid molecular method using caudal fin sampling, which does not imply animal killing, could be an important tool to control epizootics caused by LCDV, as fish could be analysed before their introduction and/or mobilization in farm facilities.     

Monitoring of microbial adhesion and biofilm growth using electrochemical impedancemetry

Electrochemical impedance spectroscopy was tested to monitor the cell attachment and the biofilm proliferation in order to identify characteristic events induced on the metal surface by Gram-negative (Pseudomonas aeruginosa PAO1) and Gram-positive (Bacillus subtilis) bacteria strains. Electrochemical impedance spectra of AISI 304 electrodes during cell attachment and initial biofilm growth for both strains were obtained. It can be observed that the resistance increases gradually with the culture time and decreases with the biofilm detachment. So, the applicability of electric cell-substrate impedance sensing (ECIS) for studying the attachment and spreading of cells on a metal surface has been demonstrated. The biofilm formation was also characterized by the use of scanning electron microscopy and confocal laser scanning microscopy and COMSTAT image analysis. The electrochemical results roughly agree with the microscope image observations. The ECIS technique used in this study was used for continuous real-time monitoring of the initial bacterial adhesion and the biofilm growth. It provides a simple and non-expensive electrochemical method for in vitro assessment of the presence of biofilms on metal surfaces.     

An internal ribosome entry site element directs the synthesis of the 80 kDa isoforms of protein 4.1R

Background  

In red blood cells, protein 4.1 (4.1R) is an 80 kDa protein that stabilizes the spectrin-actin network and anchors it to the plasma membrane through its FERM domain. While the expression pattern of 4.1R in mature red cells is relatively simple, a rather complex array of 4.1R protein isoforms varying in N-terminal extensions, internal sequences and subcellular locations has been identified in nucleated cells. Among these, 135 kDa and 80 kDa isoforms have different N-terminal extensions and are expressed either from AUG1- or AUG2-containing mRNAs, respectively. These two types of mRNAs, varying solely by presence/absence of 17 nucleotides (nt) which contain the AUG1 codon, are produced by alternative splicing of the 4.1R pre-mRNA. It is unknown whether the 699 nt region comprised between AUG1 and AUG2, kept as a 5' untranslated region in AUG2-containing mRNAs, plays a role on 4.1R mRNA translation.     

A new role of diacylglycerol kinase alpha on the secretion of lethal exosomes bearing Fas ligand during activation-induced cell death of T lymphocytes

Exosomes are small membrane vesicles that intracellularly accumulate into late or multivesicular endosomes (multivesicular bodies, MVB). Exosomes have a particular lipid and protein content, reflecting their origin as intraluminal vesicles of late endosomes. The stimulation of several hematopoietic cells induces the fusion of the limiting membrane of the MVB with the plasma membrane, leading to the release of exosomes towards the extracellular environment. In T lymphocytes, stimulation of the T cell receptor (TCR) induces the fusion of the MVBs with the plasma membrane and exosomes carrying several bio-active proteins are secreted. Among these proteins, the pro-apoptotic protein Fas ligand (FasL) is released as a non-proteolysed form (mFasL), associated to the exosomes. These mFasL-bearing exosomes may trigger the apoptosis of T lymphocytes. Here, we present evidences supporting a role of diacylglycerol kinase alpha (DGKalpha), a diacylglycerol (DAG)-consuming enzyme, on the secretion of exosomes carrying mFasL, and the subsequent activation-induced cell death (AICD) on a T cell line and primary T lymphoblasts.     

Obesity-dependent metabolic signatures associated with nonalcoholic fatty liver disease progression

Our understanding of the mechanisms by which nonalcoholic fatty liver disease (NAFLD) progresses from simple steatosis to steatohepatitis (NASH) is still very limited. Despite the growing number of studies linking the disease with altered serum metabolite levels, an obstacle to the development of metabolome-based NAFLD predictors has been the lack of large cohort data from biopsy-proven patients matched for key metabolic features such as obesity. We studied 467 biopsied individuals with normal liver histology (n=90) or diagnosed with NAFLD (steatosis, n=246; NASH, n=131), randomly divided into estimation (80% of all patients) and validation (20% of all patients) groups. Qualitative determinations of 540 serum metabolite variables were performed using ultraperformance liquid chromatography coupled to mass spectrometry (UPLC-MS). The metabolic profile was dependent on patient body-mass index (BMI), suggesting that the NAFLD pathogenesis mechanism may be quite different depending on an individual's level of obesity. A BMI-stratified multivariate model based on the NAFLD serum metabolic profile was used to separate patients with and without NASH. The area under the receiver operating characteristic curve was 0.87 in the estimation and 0.85 in the validation group. The cutoff (0.54) corresponding to maximum average diagnostic accuracy (0.82) predicted NASH with a sensitivity of 0.71 and a specificity of 0.92 (negative/positive predictive values=0.82/0.84). The present data, indicating that a BMI-dependent serum metabolic profile may be able to reliably distinguish NASH from steatosis patients, have significant implications for the development of NASH biomarkers and potential novel targets for therapeutic intervention.     

Self-assembling systems comprising intrinsically disordered protein polymers like elastin-like recombinamers

Despite lacking cooperatively folded structures under native conditions, numerous intrinsically disordered proteins (IDPs) nevertheless have great functional importance. These IDPs are hybrids containing both ordered and intrinsically disordered protein regions (IDPRs), the structure of which is highly flexible in this unfolded state. The conformational flexibility of these disordered systems favors transitions between disordered and ordered states triggered by intrinsic and extrinsic factors, folding into different dynamic molecular assemblies to enable proper protein functions. Indeed, prokaryotic enzymes present less disorder than eukaryotic enzymes, thus showing that this disorder is related to functional and structural complexity. Protein-based polymers that mimic these IDPs include the so-called elastin-like polypeptides (ELPs), which are inspired by the composition of natural elastin. Elastin-like recombinamers (ELRs) are ELPs produced using recombinant techniques and which can therefore be tailored for a specific application. One of the most widely used and studied characteristic structures in this field is the pentapeptide (VPGXG)_n. The structural disorder in ELRs probably arises due to the high content of proline and glycine in the ELR backbone, because both these amino acids help to keep the polypeptide structure of elastomers disordered and hydrated. Moreover, the recombinant nature of these systems means that different sequences can be designed, including bioactive domains, to obtain specific structures for each application. Some of these structures, along with their applications as IDPs that self-assemble into functional vesicles or micelles from diblock copolymer ELRs, will be studied in the following sections. The incorporation of additional order- and disorder-promoting peptide/protein domains, such as α-helical coils or β-strands, in the ELR sequence, and their influence on self-assembly, will also be reviewed. In addition, chemically cross-linked systems with controllable order–disorder balance, and their role in biomineralization, will be discussed. Finally, we will review different multivalent IDPs-based coatings and films for different biomedical applications, such as spatially controlled cell adhesion, osseointegration, or biomaterial-associated infection (BAI).     

RESPONSIVE-TO-ANTAGONIST1, a Menkes/Wilson disease-related copper transporter, is required for ethylene signaling in Arabidopsis.

Ethylene is an important regulator of plant growth. We identified an Arabidopsis mutant, responsive-to-antagonist1 (ran1), that shows ethylene phenotypes in response to treatment with trans-cyclooctene, a potent receptor antagonist. Genetic epistasis studies revealed an early requirement for RAN1 in the ethylene pathway. RAN1 was cloned and found to encode a protein with similarity to copper-transporting P-type ATPases, including the human Menkes/Wilson proteins and yeast Ccc2p. Expression of RAN1 complemented the defects of a ccc2delta mutant, demonstrating its function as a copper transporter. Transgenic CaMV 35S::RAN1 plants showed constitutive expression of ethylene responses, due to cosuppression of RAN1. These results provide an in planta demonstration that ethylene signaling requires copper and reveal that RAN1 acts by delivering copper to create functional hormone receptors.