Quantitative proteomics by 2-DE, 16O/18O labelling and linear ion trap mass spectrometry analysis of lymph nodes from piglets inoculated by porcine circovirus type 2

Porcine circovirus type 2 (PCV2) has been identified as the essential causal agent of postweaning multisystemic wasting syndrome. However, little is known regarding the mechanism(s) underlying the pathogenesis of PCV2-induced disease and the interaction of the virus with the host immune system. Here, we present a proteomics study on inguinal lymph nodes of piglets inoculated with PCV2, in order to better understand the pathogenesis of postweaning multisystemic wasting syndrome and the pathways might be affected after infection. We used two proteomics strategies, 2-DE and 1-DE followed by (16)O/(18)O peptide labelling and peptide identification and quantification by MS. More than 100 proteins were found to be differentially regulated and the results obtained by the two strategies were fairly concordant but also complementary, the (18)O labelling approach being a more robust alternative. Analysis of these proteins by systems biology tools revealed the implication of acute phase response and NrF2-mediated oxidative stress, suggesting a putative role for these pathways in the pig immune response. Besides, CD81 was found to be up-regulated, suggesting a possible role in the internalization of the virus. The use of proteomics technologies together with biology analysis systems opens up the way to gain more exhaustive and systematic knowledge of virus-pathogen interactions.     

Endophytic and pathogenic isolates of the cacao fungal pathogen Moniliophthora perniciosa (Tricholomataceae) are indistinguishable based on genetic and physiological analysis

We evaluated the genetic and physiological variability of Moniliophthora perniciosa obtained from healthy and diseased branches of cacao (Theobroma cacao) plants. The diversity of the isolates was evaluated by RAPD technique and by studies of virulence and exoenzyme production. The genetic variability of endophytic and pathogenic M. perniciosa was evaluated in association with pathogenicity assays. RAPD analysis showed eight genetic groups, which were not related to plant disease status (healthy versus diseased branches). Isolates from cacao were included in three groups, excluding isolates from other host plants. Pathogenicity and enzyme analysis showed that the virulence of the isolates is not related to exoenzyme production. This is the first evidence that M. perniciosa colonizes healthy parenchymatic tissues, showing that endophytic behavior may occur in this species.     

Day-4 myeloid dendritic cells pulsed with whole tumor lysate are highly immunogenic and elicit potent anti-tumor responses

"Day-7" myeloid DCs are commonly used in the clinic. However, there is a strong need to develop DCs faster that have the same potent immunostimulatory capacity as "Day-7" myeloid DCs and at the same time minimizing time, labor and cost of DC preparations. Although "2 days" DCs can elicit peptide-specific responses, they have not been demonstrated to engulf, process and present complex whole tumor lysates, which could be more convenient and personalized source of tumor antigens than defined peptides. In this preclinical study, we evaluated the T-cell stimulatory capacity of Day-2, Day-4, and Day-7 cultured monocyte-derived DCs loaded with SKOV3 cell whole lysate prepared by freeze-thaw or by UVB-irradiation followed by freeze-thaw, and matured with lipopolysaccharide (LPS) and interferon (IFN)-gamma. DCs were evaluated for antigen uptake, and following maturation with LPS and IFN-gamma, DCs were assessed for expression of CD80, CD40, CD86, ICAM-1 and CCR7, production of IL-12p70 and IP-10, and induction of tumor-specific T-cell responses. Day-4 and Day-7 DCs exhibited similar phagocytic abilities, which were superior to Day-2 DCs. Mature Day-7 DCs expressed the highest CD40 and ICAM-1, but mature Day-4 DCs produced the most IL-12p70 and IP-10. Importantly, Day-4 and Day-7 DCs derived from ovarian cancer patients stimulated equally strongly tumor-specific T-cell responses. This is the first study demonstrating the highly immunogenic and strong T-cell stimulatory properties of Day-4 myeloid DCs, and provided important preclinical data for rapid development of potent whole tumor lysate-loaded DC vaccines that are applicable to many tumor types.     

Deletion of TAK1 in the Myeloid Lineage Results in the Spontaneous Development of Myelomonocytic Leukemia in Mice

Previous studies of the conditional ablation of TGF-β activated kinase 1 (TAK1) in mice indicate that TAK1 has an obligatory role in the survival and/or development of hematopoietic stem cells, B cells, T cells, hepatocytes, intestinal epithelial cells, keratinocytes, and various tissues, primarily because of these cells’ increased apoptotic sensitivity, and have implicated TAK1 as a critical regulator of the NF-κB and stress kinase pathways and thus a key intermediary in cellular survival. Contrary to this understanding of TAK1’s role, we report a mouse model in which TAK1 deletion in the myeloid compartment that evoked a clonal myelomonocytic cell expansion, splenomegaly, multi-organ infiltration, genomic instability, and aggressive, fatal myelomonocytic leukemia. Unlike in previous reports, simultaneous deletion of TNF receptor 1 (TNFR1) failed to rescue this severe phenotype. We found that the features of the disease in our mouse model resemble those of human chronic myelomonocytic leukemia (CMML) in its transformation to acute myeloid leukemia (AML). Consequently, we found TAK1 deletion in 13 of 30 AML patients (43%), thus providing direct genetic evidence of TAK1’s role in leukemogenesis.     

Tissue-engineered fetal dermal matrices

In the early to mid-gestation fetus, skin wounds heal with no scar formation and perfect restoration of dermal architecture. This phenomenon is intrinsic to fetal skin. The intrinsic phenotypic properties of the fetal fibroblast are believed to be ??the effector of scarless repair??. We sought to prepare dermal matrices with high similarity to the mid-gestation fetal dermis using the technology of ??self-assembly?? with fetal dermal cells of 18, 20, and 22?wk gestation. Comparison of these dermal constructs to those prepared with neonatal dermal cells, adult skin, neonatal foreskin, and mid-gestation fetal skin demonstrates that these fetal dermal matrices bear marked morphological and biochemical resemblance to the mid-gestation fetal dermis. In order to shed further light on the genes involved in scarless wound healing, we conducted a differential gene array analysis of the neonatal and fetal dermal matrices. Using a gene chip (GLYCOv4 gene chip) of approximately 1,260 human genes, we observed differential expression of 67 genes. A number of fibrotic genes were observed to be downregulated and anti-fibrotic genes upregulated.     

Evaluation of normalization methods in mammalian microRNA-Seq data

Simple total tag count normalization is inadequate for microRNA sequencing data generated from the next generation sequencing technology. However, so far systematic evaluation of normalization methods on microRNA sequencing data is lacking. We comprehensively evaluate seven commonly used normalization methods including global normalization, Lowess normalization, Trimmed Mean Method (TMM), quantile normalization, scaling normalization, variance stabilization, and invariant method. We assess these methods on two individual experimental data sets with the empirical statistical metrics of mean square error (MSE) and Kolmogorov-Smirnov (K-S) statistic. Additionally, we evaluate the methods with results from quantitative PCR validation. Our results consistently show that Lowess normalization and quantile normalization perform the best, whereas TMM, a method applied to the RNA-Sequencing normalization, performs the worst. The poor performance of TMM normalization is further evidenced by abnormal results from the test of differential expression (DE) of microRNA-Seq data. Comparing with the models used for DE, the choice of normalization method is the primary factor that affects the results of DE. In summary, Lowess normalization and quantile normalization are recommended for normalizing microRNA-Seq data, whereas the TMM method should be used with caution.     

Reliability and Discriminative Ability of a New Method for Soccer Kicking Evaluation

The study aimed to evaluate the test–retest reliability of a newly developed 356 Soccer Shooting Test (356-SST), and the discriminative ability of this test with respect to the soccer players'' proficiency level and leg dominance. Sixty-six male soccer players, divided into three groups based on their proficiency level (amateur, n = 24; novice semi-professional, n = 18; and experienced semi-professional players, n = 24), performed 10 kicks following a two-step run up. Forty-eight of them repeated the test on a separate day. The following shooting variables were derived: ball velocity (BV; measured via radar gun), shooting accuracy (SA; average distance from the ball-entry point to the goal centre), and shooting quality (SQ; shooting accuracy divided by the time elapsed from hitting the ball to the point of entry). No systematic bias was evident in the selected shooting variables (SA: 1.98±0.65 vs. 2.00±0.63 m; BV: 24.6±2.3 vs. 24.5±1.9 m s^-1; SQ: 2.92±1.0 vs. 2.93±1.0 m s^-1; all p>0.05). The intra-class correlation coefficients were high (ICC = 0.70–0.88), and the coefficients of variation were low (CV = 5.3–5.4%). Finally, all three 356-SST variables identify, with adequate sensitivity, differences in soccer shooting ability with respect to the players'' proficiency and leg dominance. The results suggest that the 356-SST is a reliable and sensitive test of specific shooting ability in men’s soccer. Future studies should test the validity of these findings in a fatigued state, as well as in other populations.     

Trypanosoma cruzi: treatment with the iron chelator desferrioxamine reduces parasitemia and mortality in experimentally infected mice

The effects of prolonged treatment with iron chelator (desferrioxamine) on the development of infection in mice inoculated with Y Trypanosoma cruzi were determined. Infected/treated mice presented lower levels of parasitemia and reduced mortality rate compared with infected/non-treated animals. The five out of twenty infected/treated mice that survived the acute phase of infection showed negative hemoculture and positive ELISA in the acute and chronic phases and positive PCR in the acute phase: in the chronic phase, three of the animals presented negative PCR. The single surviving infected/non-treated animal exhibited positive hemoculture, PCR and ELISA in both phases of infection. Infected groups presented lower levels of iron in the liver compared with treated/non-infected or non-treated/non-infected animals. The serum iron levels of the infected/non-treated group were higher on the 21st day post-infection in comparison with control and infected/treated groups. These results suggest that decrease of iron in the host leads to T. cruzi infection attenuation.