Proteomics and transcriptomics investigation on longissimus muscles in Large White and Casertana pig breeds

Consumer complaints against the blandness of modern lean meat and the frequent reference to the more strongly flavored meat that was available years ago have prompted reconsideration of high fat-depositing typical pig breeds. Casertana and Large White pig breeds are characterized by a different tendency toward fat accumulation as they exhibit opposite genetic and physiological traits with respect to the energy metabolism. These physiological differences were investigated in longissimus lumborum muscles through proteomics (2-DE, MS/MS) and microarray approaches. Data were analyzed for pathway and network analyses, as well as GO term enrichment of biological functions. As a result, Casertana showed a greater amount of proteins involved in glycolitic metabolism and mainly rely on fast-mobilizable energy sources. Large White overexpressed cell cycle and skeletal muscle growth related genes. Metabolic behavior and other implications are discussed.     

Long-Term Structural and Functional Myocardial Adaptations in Healthy Living Kidney Donors: A Pilot Study

Background and Aims

Compensatory renal hypertrophy following unilateral nephrectomy (UNX) occurs in the remaining kidney. However, the long-term cardiac adaptive process to UNX remains poorly defined in humans. Our goal was to characterize myocardial structure and function in living kidney donors (LKDs), approximately 12 years after UNX.

Methods and Results

Cardiac function and structure in 15 Italian LKDs, at least 5 years after UNX (median time from donation = 8.4 years) was investigated and compared to those of age and sex matched U.S. citizens healthy controls (n = 15). Standard and speckle tracking echocardiography (STE) was performed in both LKDs and controls. Plasma angiotensin II, aldosterone, atrial natriuretic peptide (ANP), N terminus pro B-type natriuretic peptide (NT-proBNP), cyclic guanylyl monophosphate (cGMP), and amino-terminal peptide of procollagen III (PIIINP) were also collected. Median follow-up was 11.9 years. In LKDs, LV geometry and function by STE were similar to controls, wall thickness and volumes were within normal limits also by CMR. In LKDs, CMR was negative for myocardial fibrosis, but apical rotation and LV torsion obtained by STE were impaired as compared to controls (21.4 ± 7.8 vs 32.7 ± 8.9 degrees, p = 0.04). Serum creatinine and PIIINP levels were increased [1.1 (0.9–1.3) mg/dL, and 5.8 (5.4–7.6)] μg/L, respectively), while urinary cGMP was reduced [270 (250–355) vs 581 (437–698) pmol/mL] in LKDs. No LKD developed cardiovascular or renal events during follow-up.

Conclusions

Long-term kidney donors have no apparent structural myocardial abnormalities as assessed by contrast enhanced CMR. However, myocardial deformation of the apical segments, as well as apical rotation, and LV torsion are reduced. The concomitant increase in circulating PIIINP level is suggestive of fibrosis. Further studies, focused on US and EU patients are warranted to evaluate whether these early functional modifications will progress to a more compromised cardiac function and structure at a later time.     

Reinstitutionalisation in mental health care: comparison of data on service provision from six European countries

Objective To establish whether reinstitutionalisation is occurring in mental health care and, if so, with what variations between western European countries.Design Comparison of data on changes in service provision.Setting Six European countries with different traditions of mental health care that have all experienced deinstitutionalisation since the 1970s—England, Germany, Italy, the Netherlands, Spain, and Sweden.Outcome measures Changes in the number of forensic hospital beds, involuntary hospital admissions, places in supported housing, general psychiatric hospital beds, and general prison population between 1990-1 and 2002-3.Results Forensic beds and places in supported housing have increased in all countries, whereas changes in involuntary hospital admissions have been inconsistent. The number of psychiatric hospital beds has been reduced in five countries, but only in two countries does this reduction outweigh the number of additional places in forensic institutions and supported housing. The general prison population has substantially increased in all countries.Conclusions Reinstitutionalisation is taking place in European countries with different traditions of health care, although with significant variation between the six countries studied. The precise reasons for the phenomenon remain unclear. General attitudes to risk containment in a society, as indicated by the size of the prison population, may be more important than changing morbidity and new methods of mental healthcare delivery.     

Genetic metabolic polymorphisms and the risk of cancer: a review of the literature

The purpose of this paper is to systematically analyse the design and results of epidemiological studies on the association between various types of cancer (lung, bladder, breast, colon, stomach) and four genetically-based metabolic polymorphisms, involved in the metabolism of several carcinogens (glutathione-S-transferase M1, debrisoquine hydroxylase, N acetyltransferase, aryl hydrocarbon hydroxylase). These inherited polymorphisms usually cause modifications in the quality or quantity of the relevant enzymes. Such enzymes are involved in the activation/inactivation of known carcinogens and seem to modify the extent to which carcinogens interact with DNA in target tissues. Two enzymes, debrisoquine hydroxylase and aryl hydrocarbon hydroxylase, activate procarcinogens to carcinogens (phase I enzymes). The other two, glutathione-S-transferase M1 and N-acetyltransferase, mainly detoxity carcinogenic substances (phase II enzymes). Because of their role as host factors (modulating the action of carcinogens), it has been hypothesized that subjects presenting a specific phenotype for such polymorphisms could be at a greater risk of developing various types of cancer. A number of epidemiological studies have investigated such associations, often with discordant results. We examine and discuss the design of the studies, and present a meta-analysis of the available data.     

Protein aggregation and amyloid fibril formation by an SH3 domain probed by limited proteolysis

The SH3 domains are small protein modules of 60-85 amino acid residues that are found in many proteins involved in intracellular signal transduction. The SH3 domain of the p85alpha subunit of bovine phosphatidylinositol 3'-kinase (PI3-SH3) under acidic solution adopts a compact denatured state from which amyloid fibrils are readily formed. This aggregation process has been found to be modulated substantially by solution conditions. Here, we have analyzed the conformational features of the native and acid denatured states of PI3-SH3 by limited proteolysis experiments using proteinase K and pepsin, respectively. Moreover, we have analyzed the propensity of PI3-SH3 to be hydrolyzed by pepsin at different stages in the process of aggregation and amyloid formation at pH 1.2 and 2.0 and compared the sites of proteolysis under these conditions with the conformational features of both native and aggregated PI3-SH3. The results demonstrate that the denatured state of PI3-SH3 formed at low pH is relatively resistant to proteolysis, indicating that it is partially folded. The long loop connecting beta-strands b and c in the native protein is the region in this structure most susceptible to proteolysis. Remarkably, aggregates of PI3-SH3 that are formed initially from this denatured state in acid solution display enhanced susceptibility to proteolysis of the long loop, suggesting that the protein becomes more unfolded in the early stages of aggregation. By contrast, the more defined amyloid fibrils that are formed over longer periods of time are completely resistant to proteolysis. We suggest that the protein aggregates formed initially are relatively dynamic species that are able readily to reorganize their interactions to enable formation of very well ordered fibrillar structures. In addition, the disordered and non-native character of the polypeptide chains in the early aggregates could be important in determining the high cytotoxicity that has been revealed in previous studies of these species.     

Effect of 1-MCP and low-temperature storage on postharvest conservation of camu–camu

Camu–camu, a native fruit from the Amazon region, is a rich source of bioactive compounds. However, its intense metabolic activity and high-water content limit the fruit’s postharvest storage and marketing. The aim of this study, conducted in two parts, was to evaluate the effects of 1-MCP and storage temperature on the physiology and postharvest preservation of camu–camu fruit. In part 1 of the study, fruit harvested at maturity stage 3 were divided into groups: control, 1-methylcyclopropene (1-MCP; 900 nL L^?1; 12 h) and ethylene (1000 µL L^?1; 24 h) and were stored at 22?±?1 °C and 85?±?5% RH for 9 days. In part 2, fruit harvested at maturity stage 3 were stored at 5, 10, 15, 20, or 25?±?1 °C and 85?±?5% RH for 9 days. During storage, fruit were evaluated daily for decay, mass loss, respiratory activity, and ethylene production, and every 3 days they were evaluated for peel color, pulp firmness, soluble solids content, total titratable acidity, ascorbic acid, total chlorophyll, and total anthocyanins. Fruit treated with 1-MCP exhibited delayed ripening due to lower metabolic activity, as evidenced by delay to softening, reduced mass loss and no decay. Storage at 5 °C prevented ethylene production, mass loss, color changes, and maintained pulp firmness, while did not affect soluble solids content. The results indicated that storage of camu–camu fruit at 5 °C or at 25 °C following application of 900 nL L^?1 1-MCP were effective strategies to delay ripening and maintain fruit quality up to 9 days.     

Differential modification of seawater carbonate chemistry by major coral reef benthic communities

Ocean acidification (OA) resulting from uptake of anthropogenic CO₂ may negatively affect coral reefs by causing decreased rates of biogenic calcification and increased rates of CaCO₃ dissolution and bioerosion. However, in addition to the gradual decrease in seawater pH and Ω _a resulting from anthropogenic activities, seawater carbonate chemistry in these coastal ecosystems is also strongly influenced by the benthic metabolism which can either exacerbate or alleviate OA through net community calcification (NCC = calcification – CaCO₃ dissolution) and net community organic carbon production (NCP = primary production ? respiration). Therefore, to project OA on coral reefs, it is necessary to understand how different benthic communities modify the reef seawater carbonate chemistry. In this study, we used flow-through mesocosms to investigate the modification of seawater carbonate chemistry by benthic metabolism of five distinct reef communities [carbonate sand, crustose coralline algae (CCA), corals, fleshy algae, and a mixed community] under ambient and acidified conditions during summer and winter. The results showed that different communities had distinct influences on carbonate chemistry related to the relative importance of NCC and NCP. Sand, CCA, and corals exerted relatively small influences on seawater pH and Ω _a over diel cycles due to closely balanced NCC and NCP rates, whereas fleshy algae and mixed communities strongly elevated daytime pH and Ω _a due to high NCP rates. Interestingly, the influence on seawater pH at night was relatively small and quite similar across communities. NCC and NCP rates were not significantly affected by short-term acidification, but larger diel variability in pH was observed due to decreased seawater buffering capacity. Except for corals, increased net dissolution was observed at night for all communities under OA, partially buffering against nighttime acidification. Thus, algal-dominated areas of coral reefs and increased net CaCO₃ dissolution may partially counteract reductions in seawater pH associated with anthropogenic OA at the local scale.     

Nusinersen treatment and cerebrospinal fluid neurofilaments: An explorative study on Spinal Muscular Atrophy type 3 patients

The antisense oligonucleotide Nusinersen has been recently licensed to treat spinal muscular atrophy (SMA). Since SMA type 3 is characterized by variable phenotype and milder progression, biomarkers of early treatment response are urgently needed. We investigated the cerebrospinal fluid (CSF) concentration of neurofilaments in SMA type 3 patients treated with Nusinersen as a potential biomarker of treatment efficacy. The concentration of phosphorylated neurofilaments heavy chain (pNfH) and light chain (NfL) in the CSF of SMA type 3 patients was evaluated before and after six months since the first Nusinersen administration, performed with commercially available enzyme-linked immunosorbent assay (ELISA) kits. Clinical evaluation of SMA patients was performed with standardized motor function scales. Baseline neurofilament levels in patients were comparable to controls, but significantly decreased after six months of treatment, while motor functions were only marginally ameliorated. No significant correlation was observed between the change in motor functions and that of neurofilaments over time. The reduction of neurofilament levels suggests a possible early biochemical effect of treatment on axonal degeneration, which may precede changes in motor performance. Our study mandates further investigations to assess neurofilaments as a marker of treatment response.     

Preserved Proteins from Extinct Bison latifrons Identified by Tandem Mass Spectrometry; Hydroxylysine Glycosides are a Common Feature of Ancient Collagen

Bone samples from several vertebrates were collected from the Ziegler Reservoir fossil site, in Snowmass Village, Colorado, and processed for proteomics analysis. The specimens come from Pleistocene megafauna Bison latifrons, dating back ∼120,000 years. Proteomics analysis using a simplified sample preparation procedure and tandem mass spectrometry (MS/MS) was applied to obtain protein identifications. Several bioinformatics resources were used to obtain peptide identifications based on sequence homology to extant species with annotated genomes. With the exception of soil sample controls, all samples resulted in confident peptide identifications that mapped to type I collagen. In addition, we analyzed a specimen from the extinct B. latifrons that yielded peptide identifications mapping to over 33 bovine proteins. Our analysis resulted in extensive fibrillar collagen sequence coverage, including the identification of posttranslational modifications. Hydroxylysine glucosylgalactosylation, a modification thought to be involved in collagen fiber formation and bone mineralization, was identified for the first time in an ancient protein dataset. Meta-analysis of data from other studies indicates that this modification may be common in well-preserved prehistoric samples. Additional peptide sequences from extracellular matrix (ECM) and non-ECM proteins have also been identified for the first time in ancient tissue samples. These data provide a framework for analyzing ancient protein signatures in well-preserved fossil specimens, while also contributing novel insights into the molecular basis of organic matter preservation. As such, this analysis has unearthed common posttranslational modifications of collagen that may assist in its preservation over time. The data are available via ProteomeXchange with identifier PXD001827.During the last decade, paleontology and taphonomy (the study of decaying organisms over time and the fossilization processes) have begun to overlap with the field of proteomics to shed new light on preserved organic matter in fossilized bones (1–4). These bones represent a time capsule of ancient biomolecules, owing to their natural resistance to post mortem decay arising from a unique combination of mechanical, structural, and chemical properties (4–7).Although bones can be cursorily described as a composite of collagen (protein) and hydroxyapatite (mineral), fossilized bones undergo three distinct diagenesis pathways: (i) chemical deterioration of the organic phase; (ii) chemical deterioration of the mineral phase; and (iii) (micro)biological attack of the composite (6). In addition, the rate of these degradation pathways are affected by temperature, as higher burial temperatures have been shown to accelerate these processes (6, 8). Though relatively unusual, the first of these three pathways results in a slower deterioration process, which is more generally mitigated under (6) specific environmental constraints, such as geochemical stability (stable temperature and acidity) that promote bone mineral preservation. Importantly, slower deterioration results in more preserved biological materials that are more amenable to downstream analytical assays. One example of this is the controversial case of bone and soft-tissue preservation from the Cretaceous/Tertiary boundary (9–22). In light of these and other studies of ancient biomolecules, paleontological models have proposed that organic biomolecules in ancient samples, such as collagen sequences from the 80 million-year-(my)-old Campanian hadrosaur, Brachylophosaurus canadensis (16) or 68-my-old Tyrannosaurus rex, might be protected by the microenvironment within bones. Such spaces are believed to form a protective shelter that is able to reduce the effects of diagenetic events. In addition to collagen, preserved biomolecules include blood proteins, cellular lipids, and DNA (4, 5). While the maximum estimated lifespan of DNA in bones is ∼20,000 years (ky) at 10 °C, bone proteins have an even longer lifespan, making them an exceptional target for analysis to gain relevant insights into fossilized samples (6). Indeed, the survival of collagen, which is considered to be the most abundant bone protein, is estimated to be in the range 340 ky at 20 °C. Similarly, osteocalcin, the second-most abundant bone protein, can persist for ≈45 ky at 20 °C, thus opening an unprecedented analytical window to study extremely old samples (2, 4, 23).Although ancient DNA amplification and sequencing can yield interesting clues and potential artifacts from contaminating agents (7, 24–28), the improved preservation of ancient proteins provides access to a reservoir of otherwise unavailable genetic information for phylogenetic inference (25, 29, 30). In particular, mass spectrometry (MS)-based screening of species-specific collagen peptides has recently been used as a low-cost, rapid alternative to DNA sequencing for taxonomic attribution of morphologically unidentifiable small bone fragments and teeth stemming from diverse archeological contexts (25, 31–33).For over five decades, researchers have presented biochemical evidence for the existence of preserved protein material from ancient bone samples (34–36). One of the first direct measurements was by amino acid analysis, which showed that the compositional profile of ancient samples was consistent with collagens in modern bone samples (37–39). Preservation of organic biomolecules, either from bone, dentin, antlers, or ivory, has been investigated by radiolabeled ¹⁴C fossil dating (40) to provide an avenue of delineating evolutionary divergence from extant species (3, 41, 42). It is also important to note that these parameters primarily depend on ancient bone collagen as the levels remain largely unchanged (a high percentage of collagen is retained, as gleaned by laboratory experiments on bone taphonomy (6)). Additionally, antibody-based immunostaining methods have given indirect evidence of intact peptide amide bonds (43–45) to aid some of the first evidence of protein other than collagen and osteocalcin in ancient mammoth (43) and human specimens (46).In the past, mass spectrometry has been used to obtain MS signals consistent with modern osteocalcin samples (2, 47), and eventually postsource decay peptide fragmentation was used to confirm the identification of osteocalcin in fossil hominids dating back ∼75 ky (48). More recently, modern “bottom-up” proteomic methods were applied to mastodon and T. rex samples (10), complementing immunohistochemistry evidence (13, 17). The results hinted at the potential of identifying peptides from proteolytic digest of well-preserved bone samples. This work also highlighted the importance of minimizing sources of protein contamination and adhering to data publication guidelines (20, 21). In the past few years, a very well-preserved juvenile mammoth referred to as Lyuba was discovered in the Siberian permafrost and analyzed using high-resolution tandem mass spectrometry (29). This study was followed with a report by Wadsworth and Buckley (30) describing the analysis of proteins from 19 bovine bone samples spanning 4 ky to 1.5 my. Both of these groups reported the identification of additional collagen and noncollagen proteins.Recently, a series of large extinct mammal bones were unearthed at a reservoir near Snowmass Village, Colorado, USA (49, 50). The finding was made during a construction project at the Ziegler Reservoir, a fossil site that was originally a lake formed at an elevation of ∼2,705 m during the Bull Lake glaciations ∼140 ky ago (49, 51). The original lake area was ∼5 hectares in size with a total catchment of ∼14 hectares and lacked a direct water flow inlet or outlet. This closed drainage basin established a relatively unique environment that resulted in the exceptional preservation of plant material, insects (52), and vertebrate bones (49). In particular, a cranial specimen from extinct Bison latifrons was unearthed from the Biostratigraphic Zone/Marine Oxygen Isotope Stage (MIS) 5d, which dates back to ∼120 ky (53, 54).Here, we describe the use of paleoproteomics, for the identification of protein remnants with a focus on a particularly unique B. latifrons cranial specimen found at the Ziegler site. We developed a simplified sample processing approach that allows for analysis of low milligram quantities of ancient samples for peptide identification. Our method avoids the extensive demineralization steps of traditional protocols and utilizes an acid labile detergent to allow for efficient extraction and digestion without the need for additional sample cleanup steps. This approach was applied to a specimen from B. latifrons that displayed visual and mechanical properties consistent with the meninges, a fibrous tissue that lines the cranial cavity. Bioinformatics analysis revealed the presence of a recurring glycosylation signature in well-preserved collagens. In particular, the presence of glycosylated hydroxylysine residues was identified as a unique feature of bone fossil collagen, as gleaned through meta-analyses of raw data from previous reports on woolly mammoth (Mammuthus primigenius) and bovine samples (29, 30). The results from these meta-analyses indicate a common, unique feature of collagen that coincides with, and possibly contributes to its preservation.