The Goat (Capra hircus) Mammary Gland Mitochondrial Proteome: A Study on the Effect of Weight Loss Using Blue-Native PAGE and Two-Dimensional Gel Electrophoresis

Seasonal weight loss (SWL) is the most important limitation to animal production in the Tropical and Mediterranean regions, conditioning producer’s incomes and the nutritional status of rural communities. It is of importance to produce strategies to oppose adverse effects of SWL. Breeds that have evolved in harsh climates have acquired tolerance to SWL through selection. Most of the factors determining such ability are related to changes in biochemical pathways as affected by SWL. In this study, a gel based proteomics strategy (BN: Blue-Native Page and 2DE: Two-dimensional gel electrophoresis) was used to characterize the mitochondrial proteome of the secretory tissue of the goat mammary gland. In addition, we have conducted an investigation of the effects of weight loss in two goat breeds with different levels of adaptation to nutritional stress: Majorera (tolerant) and Palmera (susceptible). The study used Majorera and Palmera dairy goats, divided in 4 sets, 2 for each breed: underfed group fed on wheat straw (restricted diet, so their body weight would be 15–20% reduced by the end of experiment), and a control group fed with an energy-balanced diet. At the end of the experimental period (22 days), mammary gland biopsies were obtained for all experimental groups. The proteomic analysis of the mitochondria enabled the resolution of a total of 277 proteins, and 148 (53%) were identified by MALDI-TOF/TOF mass spectrometry. Some of the proteins were identified as subunits of the glutamate dehydrogenase complex and the respiratory complexes I, II, IV, V from mitochondria, as well as numerous other proteins with functions in: metabolism, development, localization, cellular organization and biogenesis, biological regulation, response to stimulus, among others, that were mapped in both BN and 2DE gels. The comparative proteomics analysis enabled the identification of several proteins: NADH-ubiquinone oxidoreductase 75 kDa subunit and lamin B1 mitochondrial (up-regulated in the Palmera breed), Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-2 (up-regulated in the Majorera breed) and cytochrome b-c1 complex subunit 1, mitochondrial and Chain D, Bovine F1-C8 Sub-Complex Of Atp Synthase (down-regulated in the Majorera breed) as a consequence of weight loss.     

Modeling Staphylococcus epidermidis-Induced Non-Unions: Subclinical and Clinical Evidence in Rats

S. epidermidis is one of the leading causes of orthopaedic infections associated with biofilm formation on implant devices. Open fractures are at risk of S. epidermidis transcutaneous contamination leading to higher non-union development compared to closed fractures. Although the role of infection in delaying fracture healing is well recognized, no in vivo models investigated the impact of subclinical low-grade infections on bone repair and non-union. We hypothesized that the non-union rate is directly related to the load of this commonly retrieved pathogen and that a low-grade contamination delays the fracture healing without clinically detectable infection. Rat femurs were osteotomized and stabilized with plates. Fractures were infected with a characterized clinical-derived methicillin-resistant S. epidermidis (10³, 10⁵, 10⁸ colony forming units) and compared to uninfected controls. After 56 days, bone healing and osteomyelitis were clinically assessed and further evaluated by micro-CT, microbiological and histological analyses. The biofilm formation was visualized by scanning electron microscopy. The control group showed no signs of infection and a complete bone healing. The 10³ group displayed variable response to infection with a 67% of altered bone healing and positive bacterial cultures, despite no clinical signs of infection present. The 10⁵ and 10⁸ groups showed severe signs of osteomyelitis and a non-union rate of 83–100%, respectively. The cortical bone reaction related to the periosteal elevation in the control group and the metal scattering detected by micro-CT represented limitations of this study. Our model showed that an intra-operative low-grade S. epidermidis contamination might prevent the bone healing, even in the absence of infectious signs. Our findings also pointed out a dose-dependent effect between the S. epidermidis inoculum and non-union rate. This pilot study identifies a relevant preclinical model to assess the role of subclinical infections in orthopaedic and trauma surgery and to test specifically designed diagnostic, prevention and therapeutic strategies.     

Focus: The Aging Brain: The Significance of the Default Mode Network (DMN) in Neurological and Neuropsychiatric Disorders: A Review

The relationship of cortical structure and specific neuronal circuitry to global brain function, particularly its perturbations related to the development and progression of neuropathology, is an area of great interest in neurobehavioral science. Disruption of these neural networks can be associated with a wide range of neurological and neuropsychiatric disorders. Herein we review activity of the Default Mode Network (DMN) in neurological and neuropsychiatric disorders, including Alzheimer’s disease, Parkinson’s disease, Epilepsy (Temporal Lobe Epilepsy - TLE), attention deficit hyperactivity disorder (ADHD), and mood disorders. We discuss the implications of DMN disruptions and their relationship to the neurocognitive model of each disease entity, the utility of DMN assessment in clinical evaluation, and the changes of the DMN following treatment.     

Above and beyond state-of-the-art approaches to investigate sequence data: summary of methods and results from the population-based association group at the Genetic Analysis Workshop 19

This paper summarizes the contributions from the Population-Based Association group at the Genetic Analysis Workshop 19. It provides an overview of the new statistical approaches tried out by group members in order to take best advantage of population-based sequence data.Although contributions were highly heterogeneous regarding the applied quality control criteria and the number of investigated variants, several technical issues were identified, leading to practical recommendations. Preliminary analyses revealed that Hurdle-negative binomial regression is a promising approach to investigate the distribution of allele counts instead of called genotypes from sequence data. Convergence problems, however, limited the use of this approach, creating a technical challenge shared by environment-stratified models used to investigate rare variant-environment interactions, as well as by rare variant haplotype analyses using well-established public software. Estimates of relatedness and population structure strongly depended on the allele frequency of selected variants for inference. Another practical recommendation was that dissenting probability values from standard and small-sample tests of a particular hypothesis may reflect a lack of validity of large-sample approximations. Novel statistical approaches that integrate evolutionary information showed some advantage to detect weak genetic signals, and Bayesian adjustment for confounding was able to efficiently estimate causal genetic effects. Haplotype association methods may constitute a valuable complement of collapsing approaches for sequence data. This paper reports on the experience of members of the Population-Based Association group with several novel, promising approaches to preprocessing and analyzing sequence data, and to following up identified association signals.     

Potential use of high levels of vegetal proteins in diets for market-sized gilthead sea bream (Sparus aurata)

The effect of partial or total dietary substitution of fishmeal (FM) by vegetal protein sources on growth and feed efficiency was carried out in on-growing gilthead sea bream (mean initial weight 131 g). The Control diet (FM 100) contained FM as the primary protein source, while in Diets FM 25 and FM 0 the FM protein was replaced at 75% and 100%, respectively, by a vegetable protein mixture consisting of wheat gluten, soybean meal, rapeseed meal and crystalline amino acids. Diets FM 25 and FM 0 also contained krill meal at 47 g/kg in order to improve palatability. At the end of the trial (after 158 d), fish survival was above 90%. Final weight and the specific growth rate were statistically lower in fish fed the Control diet (361 g and 0.64%/d), compared with 390–396 g and 0.69–0.70%/d after feeding vegetal diets. No significant differences were found regarding feed intake and feed conversion ratio. The digestibility of protein and amino acids (determined with chromium oxide as indicator) was similar in all diets. The blood parameters were not significantly affected by treatments. The activity of trypsin and pepsin was significantly reduced after feeding Diet FM 0. In the distal intestine, the villi length in fish fed Diet FM 25 was significantly longer and the intestine of the fish fed the FM 100 diet showed a smaller number of goblet cells. In conclusion, a total FM substitution by a vegetal mix supplemented with synthetic amino acids in on-growing sea bream is feasible.     

Fluram-Kemptide-Lys8 Non-radioactive Assay for Protein Kinase A

The cAMP-dependent protein kinase (PKA) is the best understood member of the superfamily of serine–threonine protein kinases and is involved in controlling a variety of cellular processes. Measurements of PKA activity traditionally relied on the use of [³²P]-labeled ATP as the phosphate donor and a protein or peptide substrate as the phosphoaceptor. Recently non-isotopic assays for the PKA have been developed and this paper presents an improvement of a fluorometric assay for measuring the activity of PKA. Three peptides were synthesized with the following sequences: LRRASLG (Kemptide), LRRASLGK (Kemptide-Lys8) and LRRASLGGGLRRASLG (Bis-Kemptide), these have in common the substrate sequence recognized by the PKA (RRXS/TΨ), where X is any amino acid and Ψ is a hydrophobic amino acid. Optimal conditions were established for the non-radioactive assay to detect the PKA activity by phosphorylation of these three peptides that are covalently linked to fluorescamine at their N-terminus. The phosphorylated and non-phosphorylated peptides were easily separated by electrophoresis, identified and quantified with optical densitometry and ultraviolet light. The fluorescamine-labeled Kemptide-Lys8 substrate (Fluram-Kemptide-Lys8) was used to calculate the Km and V_max of the catalytic subunit of PKA from pig heart and showed a detection limit of 260 pmol, a linear range between 700 and 1150 pmol with a linear regression R ² = 0.956.     

Antioxidant Defenses in Wild Growing Halophyte Crithmum maritimum from Inland and Coastline Populations

Traditional Mediterranean diet includes the halophyte Crithmum maritimum L. (Apiaceae) which can be found in the coastline of the Balearic Islands but also inland. Both areas differed in the environmental conditions, mainly in salinity which can affect the oxidative status of this species. The aim was to evaluate the antioxidant enzyme activities, polyphenols and the lipid peroxidation in leaves of wild C. maritimum growing in a natural coastal area influenced by marine salinity and an inland area without marine influence. The activities of the antioxidant enzymes catalase, superoxide dismutase and glutathione peroxidase as well as polyphenol and reduced glutathione content were significantly higher in the samples from coastline population, whereas no significant differences were found in glutathione reductase activity and in malondialdehyde levels. The production of H₂O₂ was also significantly higher in the population from coastline. In conclusion, C. maritimum adapt their antioxidant defense machinery to the different salinity conditions, avoiding the instauration of oxidative stress.