The Dendritic Cell Major Histocompatibility Complex II (MHC II) Peptidome Derives from a Variety of Processing Pathways and Includes Peptides with a Broad Spectrum of HLA-DM Sensitivity.

The repertoire of peptides displayed in vivo by MHC II molecules derives from a wide spectrum of proteins produced by different cell types. Although intracellular endosomal processing in dendritic cells and B cells has been characterized for a few antigens, the overall range of processing pathways responsible for generating the MHC II peptidome are currently unclear. To determine the contribution of non-endosomal processing pathways, we eluted and sequenced over 3000 HLA-DR1-bound peptides presented in vivo by dendritic cells. The processing enzymes were identified by reference to a database of experimentally determined cleavage sites and experimentally validated for four epitopes derived from complement 3, collagen II, thymosin ß4, and gelsolin. We determined that self-antigens processed by tissue-specific proteases, including complement, matrix metalloproteases, caspases, and granzymes, and carried by lymph, contribute significantly to the MHC II self-peptidome presented by conventional dendritic cells in vivo. Additionally, the presented peptides exhibited a wide spectrum of binding affinity and HLA-DM susceptibility. The results indicate that the HLA-DR1-restricted self-peptidome presented under physiological conditions derives from a variety of processing pathways. Non-endosomal processing enzymes add to the number of epitopes cleaved by cathepsins, altogether generating a wider peptide repertoire. Taken together with HLA-DM-dependent and-independent loading pathways, this ensures that a broad self-peptidome is presented by dendritic cells. This work brings attention to the role of "self-recognition" as a dynamic interaction between dendritic cells and the metabolic/catabolic activities ongoing in every parenchymal organ as part of tissue growth, remodeling, and physiological apoptosis.

Hydrogeochemical and biological processes affecting the long-term performance of an iron-based permeable reactive barrier.

Despite the wide diffusion of zero-valent iron (Fe(0)) permeable reactive barriers (PRBs), there is still a great uncertainty about their longevity and long-term performance. The aim of this study is to investigate the biological and the hydrogeochemical processes that take place at a Fe(0) installation located in Avigliana, Italy, and to derive some general considerations about long-term performance of PRBs.The examined PRB was installed in November 2004 to remediate a chlorinated solvents plume (mainly trichloroethene and 1,2-dichloroethene). The investigation was performed during the third year of operation and included: (1) groundwater sampling and analysis for chlorinated solvents, dissolved CH(4), dissolved H(2) and major inorganic constituents; (2) Fe(0) core sampling and analysis by SEM-EDS, XRD, and FTIR spectroscopy for the organic fraction; (3) in situ permeability tests and flow field monitoring by water level measurements.The study revealed that iron passivation is negligible, as the PRB is still able to effectively treat the contaminants and to reduce their concentrations below target values. Precipitation of several inorganic compounds inside the PRB was evidenced by SEM-EDS and XRD analysis conducted on iron samples. Groundwater sampling evidenced heavy sulfate depletion and the highest reported CH(4) concentration (>5,000 microg/L) at zero-valent iron PRB sites. These are due to the intense microbial activity of sulfate-reducers and methanogens, whose proliferation was most likely stimulated by the use of a biopolymer (i.e. guar gum) as shoring fluid during the excavation of the barrier. Slug tests within the barrier evidenced an apparent hydraulic conductivity two orders of magnitude lower than the predicted value. This occurrence can be ascribed to biofouling and/or accumulation of CH(4)(g) inside the iron filings.This experience suggests that when biopolymer shoring is planned to be used, long-term column tests should be performed beforehand with initial bacterial inoculation and organic substrate dosing, in order to predict the effects of bacterial overgrowth and gas generation. During construction particular care should be taken in order to minimize the amount of used biopolymer so that complete breakdown can be achieved.

Role of Carbonyl Modifications on Aging-Associated Protein Aggregation.

Protein aggregation is a common biological phenomenon, observed in different physiological and pathological conditions. Decreased protein solubility and a tendency to aggregate is also observed during physiological aging but the causes are currently unknown. Herein we performed a biophysical separation of aging-related high molecular weight aggregates, isolated from the bone marrow and splenic cells of aging mice and followed by biochemical and mass spectrometric analysis. The analysis indicated that compared to younger mice an increase in protein post-translational carbonylation was observed. The causative role of these modifications in inducing protein misfolding and aggregation was determined by inducing carbonyl stress in young mice, which recapitulated the increased protein aggregation observed in old mice. Altogether our analysis indicates that oxidative stress-related post-translational modifications accumulate in the aging proteome and are responsible for increased protein aggregation and altered cell proteostasis.

Annexin A2 promotes phagophore assembly by enhancing Atg16L⁺ vesicle biogenesis and homotypic fusion.

Plasma membrane budding of Atg-16L-positive vesicles represents a very early event in the generation of the phagophore and in the process of macroautophagy. Here we show that the membrane curvature-inducing protein annexin A2 contributes to the formation of these vesicles and their fusion to form phagophores. Ultrastructural, proteomic and FACS analyses of Atg16L-positive vesicles reveal that 30% of Atg16L-positive vesicles are also annexin A2-positive. Lipidomic analysis of annexin A2-deficient mouse cells indicates that this protein plays a role in recruiting phosphatidylserine and phosphatidylinositides to Atg16L-positive vesicles. Absence of annexin A2 reduces both vesicle formation and homotypic Atg16L vesicle fusion. Ultimately, a reduction in LC3 flux and dampening of macroautophagy are observed in dendritic cells from Anxa2(-/-) mice. Together, our analyses highlight the importance of annexin A2 in vesiculation of a population of Atg16L-positive structures from the plasma membrane, and in their homotypic fusion to form phagophore structures.

Hydrodynamic size-based separation and characterization of protein aggregates from total cell lysates.

Herein we describe a protocol that uses hollow-fiber flow field-flow fractionation (FFF) coupled with multiangle light scattering (MALS) for hydrodynamic size-based separation and characterization of complex protein aggregates. The fractionation method, which requires 1.5 h to run, was successfully modified from the analysis of protein aggregates, as found in simple protein mixtures, to complex aggregates, as found in total cell lysates. In contrast to other related methods (filter assay, analytical ultracentrifugation, gel electrophoresis and size-exclusion chromatography), hollow-fiber flow FFF coupled with MALS allows a flow-based fractionation of highly purified protein aggregates and simultaneous measurement of their molecular weight, r.m.s. radius and molecular conformation (e.g., round, rod-shaped, compact or relaxed). The polyethersulfone hollow fibers used, which have a 0.8-mm inner diameter, allow separation of as little as 20 µg of total cell lysates. In addition, the ability to run the samples in different denaturing and nondenaturing buffer allows defining true aggregates from artifacts, which can form during sample preparation. The protocol was set up using Paraquat-induced carbonylation, a model that induces protein aggregation in cultured cells. This technique will advance the biochemical, proteomic and biophysical characterization of molecular-weight aggregates associated with protein mutations, as found in many CNS degenerative diseases, or chronic oxidative stress, as found in aging, and chronic metabolic and inflammatory conditions.

Aging-related anatomical and biochemical changes in lymphatic collectors impair lymph transport, fluid homeostasis, and pathogen clearance.

The role of lymphatic vessels is to transport fluid, soluble molecules, and immune cells to the draining lymph nodes. Here, we analyze how the aging process affects the functionality of the lymphatic collectors and the dynamics of lymph flow. Ultrastructural, biochemical, and proteomic analysis indicates a loss of matrix proteins, and smooth muscle cells in aged collectors resulting in a decrease in contraction frequency, systolic lymph flow velocity, and pumping activity, as measured in vivo in lymphatic collectors. Functionally, this impairment also translated into a reduced ability for in vivo bacterial transport as determined by time-lapse microscopy. Ultrastructural and proteomic analysis also indicates a decrease in the thickness of the endothelial cell glycocalyx and loss of gap junction proteins in aged lymph collectors. Redox proteomic analysis mapped an aging-related increase in the glycation and carboxylation of lymphatic's endothelial cell and matrix proteins. Functionally, these modifications translate into apparent hyperpermeability of the lymphatics with pathogen escaping from the collectors into the surrounding tissue and a decreased ability to control tissue fluid homeostasis. Altogether, our data provide a mechanistic analysis of how the anatomical and biochemical changes, occurring in aged lymphatic vessels, compromise lymph flow, tissue fluid homeostasis, and pathogen transport.

Molecular analysis of chromium and cobalt-related toxicity.

Occupational and environmental exposure to Co and Cr has been previously linked to a wide array of inflammatory and degenerative conditions and cancer. Recently, significant health concerns have been raised by the high levels of Cr and Co ions and corrosion products released by biomedical implants. Herein, we set to analyze the biological responses associated with Co and Cr toxicity. Histological, ultrastructural, and elemental analysis, performed on Cr and Co exposed patients reveal the presence of corrosion products, metallic wear debris and metal ions at varying concentrations. Metallic ions and corrosion products were also generated in vitro following macrophage phagocytosis of metal alloys. Ex vivo redox proteomic mapped several oxidatively damaged proteins by Cr(III) and Co(II)-induced Fenton reaction. Importantly, a positive correlation between the tissue amounts of Cr(III) and Co(II) ions and tissue oxidative damage was observed. Immobilized- Cr(III) and Co(II) affinity chromatography indicated that metal ions can also directly bind to several metallo and non-metalloproteins and, as demonstrated for aldolase and catalase, induce loss of their biological function. Altogether, our analysis reveals several biological mechanisms leading to tissue damage, necrosis, and inflammation in patients with Cr and Co-associated adverse local tissue reactions.

Love me tender: an Omics window on the bovine meat tenderness network.

Meat tenderness prediction is a challenging task, especially in Maremmana, an Italian autochtonous and highly appreciated beef breed. In the present study we reported an integrated proteomics, phosphoproteomics and metabolomics overview of meat tenderness in longissimus dorsi from 15 male Maremmana individuals, through the discrimination of tender and tough groups via standard meat tenderness indicators (WBS, MFI(4 h), MFI(10 days), sarcomere length) and their correlation with results from Omics analyses. Results revealed that the tender meat group was characterized by higher levels of glycolytic enzymes, which were less phosphorylated and overall more active (lactate accumulation was higher in the tender group), than in tough counterparts. Additionally, we could observe a higher level of oxidative stress in the tender group. No proteomics nor phosphoproteomics result hinted at the widely accepted role of calpains and cathepsins, except for the indication of calcium homeostasis dysregulation. Nevertheless, myofibrillar degradation was monitored and related to structural protein fragmentations. Fragmentation of structural proteins and activities of glycolytic enzymes were inversely related to their phosphorylation levels, suggesting that PTMs might add further levels of complexity in the frame of meat tenderness.

Meat quality of the longissimus lumborum muscle of Casertana and Large White pigs: metabolomics and proteomics intertwined.

Longissimus lumborum muscles from high fat-deposing Casertana and lean meat Large White pigs were assayed for meat quality parameters, including early and ultimate post mortem pH, water holding capacity and Minolta L*a*b*values. These parameters were correlated to results from differential proteomic and targeted metabolomic analyses. Higher levels of glycolytic enzymes and lactate accumulation were related to slow pH drop in Casertana pigs, albeit not to rapid pH lowering in LW counterparts. On the other hand, the individuation of pyruvate kinaseM1 and tropomyosin levels in LW were related to water holding capacity and Minolta values at 24h after slaughter. Bioinformatic analyses strengthened the correlation between over-expression of structural proteins in LW and more accentuated growth aptitude in this breed. Conversely, enzymes taking part into branching glycolytic reactions, such as glycerol 3-phosphate and creatine kinase M, were related to accentuated lipogenesis and slower albeit prolonged glycolytic rate in Casertana, respectively. Breed-specific differences at the protein level were not only related to growth performances and fat accumulation tendency in vivo, but they also affected post mortem performances through a direct influence on the forcedly anaerobic behavior of pig muscles after slaughter.