1,721,042 research outputs found
Effects of ageing in muscle tissue: the contribution of proteomics
No full textAgeing induces a progressive functional decline affecting the entire organism, therefore, the loss of muscle mass and function (sarcopenia) contributes significantly to a loss of functional autonomy. In muscles, the age-related degenerative changes produce alteration of morphology associated to muscle fiber atrophy, loss of satellite cells, and remodelling of neuronal structures. Morphological changes induced by ageing indicate a decrement of muscle fiber size, a change in fiber type distribution and appearance of mitochondrial aggregates with an increment of lipofuscin. This functional and morphological changes are also observed in nerves, in which ageing induces biochemical, morphological and functional variations both in myelin and in axons. Beside that, mitochondrial metabolism plays a pivotal role in aging, as its alteration influences the muscle function. In particular, the two morphologically distinct mitochondrial sub-fractions, named sub-sarcolemmal (SS) and inter-myofibrillar (IMF) according to their diverse cellular localization, play different roles in muscle tissue. The modulation of the respiration rate, proteins and lipids composition, and the diverse biochemical (e.g. protein import) properties, contributes to muscle cell adaptation during ageing. The SS sub-fraction is more influenced by muscle changes and is more affected by ageing. Proteomics has been utilized to monitor, at the molecular level, the decline of muscle tissue, nerves innervating it and mitochondrial machiner
DIGE Analysis of Human Tissues
No full textTwo-dimensional difference gel electrophoresis (2-D DIGE) is an advanced and elegant gel electrophoretic analytical tool for comparative protein assessment. It is based on two-dimensional gel electrophoresis (2-DE) separation of fluorescently labeled protein extracts. The tagging procedures are designed to not interfere with the chemical properties of proteins with respect to their pI and electrophoretic mobility, once a proper labeling protocol is followed. The two-dye or three-dye systems can be adopted and their choice depends on specific applications. Furthermore, the use of an internal pooled standard makes 2-D DIGE a highly accurate quantitative method enabling multiple protein samples to be separated on the same two-dimensional gel. The image matching and cross-gel statistical analysis generates robust quantitative results making data validation by independent technologies successful
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
New evidence of specific synaptic remodelling by corticosterone-treated astrocytes conditioned medium
Full text linkMajor depressive disorder (MDD) is a debilitating multifactorial neuropsychiatric syndrome, affecting about 20% of the population and representing the leading cause of disability worldwide with severe social and economic consequences [1]. Stress exposure has been recognized as the main risk factor and more in detail, an individual’s ability to cope with stress in an adaptive way can determine their resilience or vulnerability to MDD development [2].
MDD is characterized by many alterations among which, in most patients, hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis and glucocorticoid resistance. In patients with severe depression, increased levels of serum and salivary cortisol have been found [3].
Astrocytes are glial cells fundamental for the central nervous system, functioning as neuronal support and participating in the regulation of ion homeostasis, neurotransmission, synaptic plasticity and neuroinflammation. Evidence from both human post-mortem brains and animal models indicate the involvement of astrocytes in MDD pathophysiology [4].
In literature the impact of glucocorticoids on astrocyte alone is still understudied and even less is known about how treated astrocytes can affect neuronal function.
Thus, to shed light on this topic, we treated mouse primary hippocampal astrocytes with corticosterone (CORT) at low concentration or DMSO twice a day for 3 days and then used their conditioned medium (ACM) to treat mouse primary hippocampal neurons at DIV 17 for either 1 or 24 hours. We investigated the impact of such treatment on neurons by (i) recording miniature post-synaptic currents (mEPSCs and mIPSCs) in whole-cell patch clamp configuration, and by (ii) performing immunofluorescence experiments targeting vGLUT1 and vGAT, markers of the excitatory and inhibitory pre-synaptic compartments respectively.
Interestingly, ACM-CORT-treated neurons displayed specific and persistent changes of both mEPSCs and mIPSCs frequencies compared to ACM-DMSO-treated neurons at both 1 and 24 hours whereas only minimal changes appeared in terms of amplitude. In parallel and in line, ACM-CORT-treated neurons showed changes in the expression of excitatory and inhibitory synaptic puncta. The TUNEL assay showed no changes in the number of apoptotic nuclei, indicating that the functional and structural variations in ACM-CORT-treated neurons were not related to neuronal loss.
We could conclude that the medium conditioned by astrocytes treated chronically with CORT has a toxic effect on neuron synapses, specifically affecting the pre-synaptic and post-synaptic compartment at 1 hour and then at 24 hours.
In order to pinpoint the mechanisms by which ACM-CORT and ACM-DMSO have different effects on neurons, ACMs were analyzed through liquid chromatography-tandem mass spectrometry (LC-MS/MS) with label-free quantification. Of all the 1193 proteins present in both conditions, a total of 636 were significantly varied: 451 were decreased and 221 were increased in ACM-CORT compared to ACM-DMSO. 16 proteins were present only in ACM-DMSO and 20 were present only in ACM-CORT.
In the next future, understanding exactly which factors released from CORT-treated astrocytes are impacting on neuronal function will be important to better understand how astrocytes and glucocorticoids can be involved in MDD pathophysiology and potentially be targeted to discover new treatments
Effects of the loss of triose phosphate isomerase activity on carbon metabolism in Kluyveromyces lactis
No full textThe effect of the loss of triose phosphate isomerase activity on carbon metabolism in Kluyveromyces lactis was studied in batch and in continuous cultures. The KltpiI mutant was able to grow on media containing glucose as the sole carbon source both in batch and in continuous culture, unlike the corresponding S. cerevisiae mutant. In K. lactis tpiI mutant no glycerol production was detected in chemostat cultivations. DHAP accumulation triggers glycerol production only when glucose is the sole carbon source in excess. The analysis of the activities of some key enzymes of carbon metabolism shows that in chemostat cultivations on mixed-substrates the activities of enzymes involved in ethanol assimilation are higher both in K. lactis wild type and mutant strains than in S. cerevisiae
How physiological and cultural conditions influence heterologous protein production in Kluyveromyces lactis
No full textThe optimization and scale-up of a specific protein production process have to take into account cultural conditions as well as cell physiology of growth and influence of foreign protein expression on host cell metabolism. Growth on cheap substrates, efficient secretion ability and a weaker tendency to hypermannosilate proteins than S. cerevisiae, make K. lactis an excellent and well-accepted host for heterologous protein production, even for human use. A fairly good heterologous glucoamylase yield and the setting of the optimal conditions to produce it were obtained expressing the Arxula adeninivorans glucoamylase in a strain of K. lactis and its isogenic mutant, which seems to have higher secretion ability. We performed batch cultures of both strains to analyze the influence of different physiological and environmental parameters on glucoamylase production/secretion. Interestingly, the maintenance of pH in the range of neutrality causes the consumption of a larger amount of carbon source, a longer time of production and a better stability of the active form of the enzyme, thus increasing biomass and glucoamylase production. Furthermore, the enrichment of the culture medium adds up to the action of pH control, forcing the mutant production/secretion to higher levels
Proteomic analysis for the study of vitality on human rib fractures: a pilot study
No full textIntroduction. Blunt force chest injuries with rib fractures are frequent in forensic caseworks. However, the possibility to set chronologically the traumatic mechanism on human bone is currently very limited. This study aims to a) identify the proteomic expression on bone marrow of human ribs that were damaged with fracture (vitality); b) evaluate the proteomic changes over different known survival times; c) assess proteomic differences among resuscitation fractures and other types of rib traumas (e.g., vehicle and train crashes, falling from heights).
Material and methods. 15 cases with rib fractures and different survival times, and 7 controls with different causes of deaths but no traumatic injuries. Specifically, the survival times of cases included (4) < 1 hour, (3) = 1 hour, (3) = 2 hours, (3) = 5-10 hours, and (2) = 24-72 hours; ratio men-to-women was 1,14:1 and the mean age 54,06. The selection of the case samples was based on the highest hemorrhagic infiltration which could be detected macroscopically; for controls, sampling was conventionally taken on the 4th right rib. Then, soft tissues around each sample were mechanically removed and frozen at -80°C. Each sample was further processed and dissected with a scalpel to collect the bone marrow (in the fracture foci for cases). Proteomic analyses were performed on bone marrows and results were expressed over logarithmic2 fold changes. Statistical tests included ANOVA applying the false discovery rate (q<0.05).
Results and discussion. The trauma group showed high expressions of acute-phase and inflammatory proteins (q<0.001) such as alpha-crystallin B chain, plasminogen, complement component C3, complement factor B and alpha-1 acid glycoprotein 1. Moreover, there was an increasing expression in the cases of both myosins (heavy and light chains) and different extracellular matrix proteins including the bone-specific collagen alpha-1 (XII) chain. Finally, carbonic anhydrase 2 (CA2) was detected as highly expressed and significative (q<0.001) in the trauma group. In the trauma group, inflammatory proteins showed a common increasing trend over the time whereas both myosins and alpha-crystallin B chain decreased progressively. Notably, the expression of CA2 increased linearly with high statistical significance (q<0.05) for all the survival subgroups of trauma. No statistical differences were noted among resuscitation fractures and the other types of rib traumas. These results provide that the bone marrow reacts to the trauma similarly to other tissues by recruiting cells in the fracture foci (evidence of myosins) to start the early inflammatory phase (evidence of inflammatory proteins). However, bone marrow also shows a particular response to trauma by increasing the expression of CA2 which is a very specific enzyme and fundamental for the process of bone remodeling. Therefore, this study provides specific forensic markers of vitality which could help to set chronologically the traumatic mechanism on human bone
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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