Mass spectrometry and animal science: protein identification strategies and particularities of farm animal species

Proteomic approaches are gaining increasing importance in the context of all fields of animal and veterinary sciences, including physiology, productive characterization, and disease/parasite tolerance, among others. Proteomic studies mainly aim the proteome characterization of a certain organ, tissue, cell type or organism, either in a specific condition or comparing protein differential expression within two or more selected situations. Due to the high complexity of samples, usually total protein extracts, proteomics relies heavily on separation procedures, being 2D-electrophoresis and HPLC the most common, as well as on protein identification using mass spectrometry (MS) based methodologies. Despite the increasing importance of MS in the context of animal and veterinary science studies, the usefulness of such tools is still poorly perceived by the animal science community. This is primarily due to the limited knowledge on mass spectrometry by animal scientists. Additionally, confidence and success in protein identification is hindered by the lack of information in public databases for most of farm animal species and their pathogens, with the exception of cattle (Bos taurus), pig (Sus scrofa) and chicken (Gallus gallus). In this article, we will briefly summarize the main methodologies available for protein identification using mass spectrometry providing a case study of specific applications in the field of animal science. We will also address the difficulties inherent to protein identification using MS, with particular reference to experiments using animal species poorly described in public databases. Additionally, we will suggest strategies to increase the rate of successful identifications when working with farm animal species.     

Investigation of trypanothione reductase inhibitory activity by 1,3,4-thiadiazolium-2-aminide derivatives and molecular docking studies

The biological activities of a series of mesoionic 1,3,4-thiadiazolium-2-aminide derivatives have been studied. The most active compounds (MI-HH; MI-3-OCH(3); MI-4-OCH(3) and MI-4-NO(2)) were evaluated to determine their effect on trypanothione reductase (TryR) activity in Leishmania sp. and Trypanosoma cruzi. Among the assayed compounds, only MI-4-NO(2) showed enzyme inhibition effect on extracts from different cultures of parasites, which was confirmed using the recombinant enzyme from T. cruzi (TcTryR) and Leishmania infantum (LiTryR). The enzyme kinetics determined with LiTryR demonstrated a non-competitive inhibition profile of MI-4-NO(2). A molecular docking study showed that the mesoionic compounds could effectively dock into the substrate binding site together with the substrate molecule. The mesoionic compounds were also effective ligands of the NADPH and FAD binding sites and the NADPH binding site was predicted as the best of all three binding sites. Based on the theoretical results, an explanation at the molecular level is proposed for the MI-4-NO(2) enzyme inhibition effect. Given TryR as a molecular target, it is important to continue the study of mesoionic compounds as part of a drug discovery campaign against Leishmaniasis or Chagas' disease.     

Human NK cells differ more in their KIR2DL1-dependent thresholds for HLA-Cw6-mediated inhibition than in their maximal killing capacity

In this study we have addressed the question of how activation and inhibition of human NK cells is regulated by the expression level of MHC class I protein on target cells. Using target cell transfectants sorted to stably express different levels of the MHC class I protein HLA-Cw6, we show that induction of degranulation and that of IFN-γ secretion are not correlated. In contrast, the inhibition of these two processes by MHC class-I occurs at the same level of class I MHC protein. Primary human NK cell clones were found to differ in the amount of target MHC class I protein required for their inhibition, rather than in their maximum killing capacity. Importantly, we show that KIR2DL1 expression determines the thresholds (in terms of MHC I protein levels) required for NK cell inhibition, while the expression of other receptors such as LIR1 is less important. Furthermore, using mathematical models to explore the dynamics of target cell killing, we found that the observed delay in target cell killing is exhibited by a model in which NK cells require some activation or priming, such that each cell can lyse a target cell only after being activated by a first encounter with the same or a different target cell, but not by models which lack this feature.     

Microbial Contaminants of Cord Blood Units Identified by 16S rRNA Sequencing and by API Test System,and Antibiotic Sensitivity Profiling

Over a period of ten months a total of 5618 cord blood units (CBU) were screened for microbial contamination under routine conditions. The antibiotic resistance profile for all isolates was also examined using ATB strips. The detection rate for culture positive units was 7.5%, corresponding to 422 samples.16S rRNA sequence analysis and identification with API test system were used to identify the culturable aerobic, microaerophilic and anaerobic bacteria from CBUs. From these samples we recovered 485 isolates (84 operational taxonomic units, OTUs) assigned to the classes Bacteroidia, Actinobacteria, Clostridia, Bacilli, Betaproteobacteria and primarily to the Gammaproteobacteria. Sixty-nine OTUs, corresponding to 447 isolates, showed 16S rRNA sequence similarities above 99.0% with known cultured bacteria. However, 14 OTUs had 16S rRNA sequence similarities between 95 and 99% in support of genus level identification and one OTU with 16S rRNA sequence similarity of 90.3% supporting a family level identification only. The phenotypic identification formed 29 OTUs that could be identified to the species level and 9 OTUs that could be identified to the genus level by API test system. We failed to obtain identification for 14 OTUs, while 32 OTUs comprised organisms producing mixed identifications. Forty-two OTUs covered species not included in the API system databases. The API test system Rapid ID 32 Strep and Rapid ID 32 E showed the highest proportion of identifications to the species level, the lowest ratio of unidentified results and the highest agreement to the results of 16S rRNA assignments. Isolates affiliated to the Bacilli and Bacteroidia showed the highest antibiotic multi-resistance indices and microorganisms of the Clostridia displayed the most antibiotic sensitive phenotypes.     

Neurodegeneration in an Aβ-induced model of Alzheimer's disease: the role of Cdk5

Cdk5 dysregulation is a major event in the neurodegenerative process of Alzheimer's disease (AD). In vitro studies using differentiated neurons exposed to Aβ exhibit Cdk5-mediated tau hyperphosphorylation, cell cycle re-entry and neuronal loss. In this study we aimed to determine the role of Cdk5 in neuronal injury occurring in an AD mouse model obtained through the intracerebroventricular (icv) injection of the Aβ_1–40 synthetic peptide. In mice icv-injected with Aβ, Cdk5 activator p35 is cleaved by calpains, leading to p25 formation and Cdk5 overactivation. Subsequently, there was an increase in tau hyperphosphorylation, as well as decreased levels of synaptic markers. Cell cycle reactivation and a significant neuronal loss were also observed. These neurotoxic events in Aβ-injected mice were prevented by blocking calpain activation with MDL28170 , which was administered intraperitoneally (ip). As MDL prevents p35 cleavage and subsequent Cdk5 overactivation, it is likely that this kinase is involved in tau hyperphosphorylation, cell cycle re-entry, synaptic loss and neuronal death triggered by Aβ. Altogether, these data demonstrate that Cdk5 plays a pivotal role in tau phosphorylation, cell cycle induction, synaptotoxicity, and apoptotic death in postmitotic neurons exposed to Aβ peptides in vivo , acting as a link between diverse neurotoxic pathways of AD.     

Yeast water channels: an overview of orthodox aquaporins

In yeast, the presence of orthodox aquaporins has been first recognized in Saccharomyces cerevisiae, in which two genes (AQY1 and AQY2) were shown to be related to mammal and plant water channels. The present review summarizes the putative orthodox aquaporin protein sequences found in available genomes of yeast and filamentous fungi. Among the 28 yeast genomes sequenced, most species present only one orthodox aquaporin, and no aquaporins were found in eight yeast species. Alignment of amino acid sequences reveals a very diverse group. Similarity values vary from 99% among species within the Saccharomyces genus to 34% between ScAqy1 and the aquaporin from Debaryomyces hansenii. All of the fungal aquaporins possess the known characteristic sequences, and residues involved in the water channel pore are highly conserved. Advances in the establishment of the structure are reviewed in relation to the mechanisms of selectivity, conductance and gating. In particular, the involvement of the protein cytosolic N‐terminus as a channel blocker preventing water flow is addressed. Methodologies used in the evaluation of aquaporin activity frequently involve the measurement of fast volume changes. Particular attention is paid to data analysis to obtain accurate membrane water permeability parameters. Although the presence of aquaporins clearly enhances membrane water permeability, the relevance of these ubiquitous water channels in yeast performance remains obscure.     

Genotoxic effect of formocresol pulp therapy of deciduous teeth

OBJECTIVE: To investigate whether formocresol, in Buckley's original formulation, used for pulp therapy of deciduous teeth, can have a genotoxic effect. Genotoxicity was tested in lymphocyte cultures from the peripheral blood of children aged 5-10y, in Recife, Pernambuco, Brazil. This was a case-control study. The sample comprised 40 children who had primary teeth with non-vital pulps. Two venous blood samples (6-8ml) were collected from each child, the first prior to pulp therapy (control group) and the second 24h after pulp therapy (experimental group). Lymphocyte cultures were grown in 78% RPMI 1640 medium, 20% fetal bovine serum, 2% phytohemagglutinin. The lymphocytes were assessed for chromosomal aberrations; each sample involved analysis of 100 metaphases. There was a statistically significant difference between the control and treated groups for the isochromatid gap (p<0.001), chromatid break (p<0.009), isochromatid break (p<0.046), other chromosomal alterations (p<0.001), and for total aberrations. In view of these results, caution in the use of formocresol in pediatric dentistry is recommended.