Osteochondrosis in pigs diagnosed with computed tomography: heritabilities and genetic correlations to weight gain in specific age intervals

The aim of this study was to develop a method for scoring osteochondrosis (OC) by using information from computed tomography (CT), as well as to estimate the heritability for OC scored by means of CT (OCwCT) of the medial and lateral condyles at the distal end of the humerus or the femur of the right and left leg and the sum of these scores (OCT). In addition, we were aiming at revealing the genetic relationship between OCwCT traits and growth in different periods (days from birth to 30 kg (D30), days from 30 to 50 kg (D30_50), days from 50 to 70 kg (D50_70), days from 70 to 90 kg (D70_90), days from 90 to 100 kg (D90_100) and days from birth to 100 kg (D100)). The OCwCT was assessed for 1449 boars, and growth data were collected for these 1449 boars and additional 3779 boars tested in the same time period. All boars were tested as part of the Norsvin Landrace boar test and in the same test station. Heritabilities for OCwCT on anatomical locations varied from 0.21 (s.e. = 0.08) on the medial condyle of the right humerus to 0.06 (s.e. = 0.06) on the lateral condyle of the left femur, whereas OCT exhibited the highest heritability (h² = 0.31, s.e. = 0.09). Genetic correlations between OCT and OCwCT for the anatomical locations ranged from 0.94 (s.e. = 0.07) for OCT and OCwCT score for the medial condyle of the humerus right side to 0.26 (s.e. = 0.39) for OCT and the lateral condyle of the femur left side. Genetic correlations between D30 and OCT were medium high and unfavourable (r_g = −0.74). As the boar gain weight, the relationship between growth rate – expressed as number of days spent growing from one interval to the next – and OCT decreased to 0.12 (s.e. = 0.19, i.e. not significantly different from zero) for the trait D90_100 kg. These changes of genetic correlation coefficients coincide with the maturing of the joint cartilage and skeletal structures. In this study, we demonstrate that CT could be used for selection against OC in breeding programmes in pigs and that the genetic correlations between growth periods and OC are decreasing over time.     

Further records for Skye

As one of the most conspicuous boundaries between different types of ecosystem, the alpine timberline has attracted the interest of researchers in Innsbruck for more than 85 years. It is evident that the tree life form here reaches its absolute limit and is constrained by the harsh environment. However, the nature of constraints is less obvious and requires detailed ecophysiological analyses as exemplified in this review, which concentrates on the Central Tyrolean Alps. After focusing on the timberline environment, effects of elevation on the water relations and the CO₂ gas exchange of timberline-associated conifer species will be outlined towards conclusions on tree growth and treeline fluctuations. Presently, temperature is suggested to be the key environmental factor in determining the transition from forests to alpine shrub and grassland. As many physiological aspects are influenced directly or indirectly by the temperature regime at the alpine timberline, tree life at the timberline exists close to a number of physiological limits which interact to determine the position of the alpine timberline and modulate the upper boundary of tree life. Nevertheless, understanding of the altitude of the treeline must also consider seedling establishment, especially when evaluating treeline advances and global change effects in a changing environment.     

Trends and inequities in colorectal cancer screening participation in Ontario,Canada, 2005–2011

Background: Participation in screening tests for colorectal cancer (CRC) is generally low in Ontario, Canada. In addition, inequities in participation exist including lower participation among low-income individuals, males and individuals living in rural areas. In April 2008, Colon Cancer Check (CCC) program, the province-wide CRC screening program, was launched in Ontario. This study describes the trends and inequities in CRC screening participation three years before and three years after the CCC, and assesses the effect of the program on CRC screening participation, overall and among certain population groups. Methods: We used administrative data to identify cohorts of individuals eligible for CRC screening in fiscal years 2005–2011. We calculated the age-standardized percent of Fecal Occult Blood Test (FOBT) participation, large bowel endoscopy participation, and being ‘up-to-date’ with CRC screening tests. Results: From 2005 to 2011, FOBT participation increased from 7.6% to 14.8%, large bowel endoscopy participation from 3.4% to 5.7%, and ‘up-to-date’ with CRC screening from 27.2% to 41.3%. Before the launch of the CCC program, the quarterly increase in FOBT participation was 0.07% (p = 0.19), increased immediately after the launch (1.8%, p < 0.01), followed by a decline (?0.08%, p = 0.08), returning to its pre-program increase rate. We noted a significant decrease in FOBT participation every summer (?0.44%, p < 0.01). The CCC program had minimal effect on large bowel endoscopy participation. Before the launch of the CCC program, the quarterly increase in ‘up-to-date’ with CRC screening was 0.9% (p < 0.01), increased immediately after the launch (2.5%, p = 0.05), followed by a modest decline thereafter (?0.59%, p < 0.02). From 2005 to 2011, recent residents living in low-income neighborhoods were consistently and significantly less likely to have a FOBT and be ‘up-to-date’ with CRC screening than long-term residents living in high-income neighborhoods (2.9–4.5%; 14.7–17.3% respectively). Pre-CCC inequities in CRC participation persisted after the launch of the program. Conclusion: CRC testing was increasing in Ontario from 2005. An immediate increase in CRC testing, FOBT in particular, occurred after the launch of the CCC program, followed by a return to its pre-CCC increase rate thereafter. Future efforts are needed to improve screening participation and address inequities.     

Quantitative assessment of methyl‐esterification and other side reactions in a standard propionylation protocol for detection of histone modifications

Histone modifications play an important role in regulating chromatin stability and gene expression, but to date, investigating them remains challenging. In order to obtain peptides suitable for MS‐based analysis, chemical derivatization of N‐terminus and lysine residues by propionic anhydride is commonly performed. Several side reactions (methyl‐esterification, amidation, solvolysis, overpropionylation, and missed propionylation) during propionylation protocols have been described, yet their relative abundances remain vague. Because methyl‐esterification could interfere with correct interpretation of the modification pattern, it is essential to take measures to avoid it. Here we present in‐depth quantitative analyses of methyl‐esterification and the other side reactions in a standard propionylation protocol containing methanol, and when replacing methanol with isopropanol or acetonitrile. We show that the use of alternative solvents can eliminate methyl‐esterification and that even though other side reactions are not prevented, their contribution can be kept relatively small. We also show that replacing methanol can be of importance also in other proteomics methods, such as mixed cation exchange, using methanol under acidic conditions.     

A proof‐of‐concept study in engineering synthetic protein for selective recognition of substrate‐free polyubiquitin

Similar to substrate‐conjugated polyubiquitin, unanchored polyubiquitin chains are emerging as important regulators for diverse biological processes. The affinity purification of unanchored polyubiquitin from various organisms has been reported, however, tools able to distinguish unanchored polyubiquitin chains with different isopeptide linkages have not yet been described. Toward the goal of selectively identifying and purifying unanchored polyubiquitin chains linked through different Lysines, Scott et al. developed a novel strategy in their study [Proteomics 2016, 16, 1961–1969]. They designed a linker‐optimized ubiquitin‐binding domain hybrid (t‐UBD) containing two UBDs, a ZnFCUBP domain, and a linkage‐selective UBA domain, to specifically recognize unanchored Lys48‐linked polyubiquitin chains. Subsequently, a series of assays has proved the feasibility of this novel strategy for the purification of endogenous substrate‐free Lys48‐linked polyubiquitin chains from mammalian cell extracts. Their research not only provides a tool for purifying unanchored polyubiquitin with different isopeptide linkages, but also paves the way for generating reagents to study the function of unanchored polyubiquitin chains of different linkages in the future. The design of UBD hybrids for defined unanchored polyubiquitin (Lys48‐polyubiquitin) in this study also set an excellent example for future methodology studies regarding monitoring in vivo dynamic changes in the patterns of ubiquitination.     

Specific protein changes contribute to the differential muscle mass loss during ageing

In the skeletal muscle, the ageing process is characterized by a loss of muscle mass and strength, coupled with a decline of mitochondrial function and a decrease of satellite cells. This profile is more pronounced in hindlimb than in forelimb muscles, both in humans and in rodents. Utilizing light and electron microscopy, myosin heavy chain isoform distribution, proteomic analysis by 2D‐DIGE, MALDI‐TOF MS and quantitative immunoblotting, this study analyzes the protein levels and the nuclear localization of specific molecules, which can contribute to a preferential muscle loss. Our results identify the molecular changes in the hindlimb (gastrocnemius) and forelimb (triceps) muscles during ageing in rats (3‐ and 22‐month‐old). Specifically, the oxidative metabolism contributes to tissue homeostasis in triceps, whereas respiratory chain disruption and oxidative‐stress‐induced damage imbalance the homeostasis in gastrocnemius muscle. High levels of dihydrolipoyllysine‐residue acetyltransferase (Dlat) and ATP synthase subunit alpha (Atp5a1) are detected in triceps and gastrocnemius, respectively. Interestingly, in triceps, both molecules are increased in the nucleus in aged rats and are associated to an increased protein acetylation and myoglobin availability. Furthermore, autophagy is retained in triceps whereas an enhanced fusion, decrement of mitophagy and of regenerative potential is observed in aged gastrocnemius muscle.     

Phosphoproteome analysis of the MAPK pathway reveals previously undetected feedback mechanisms

The RAS‐RAF‐MEK‐ERK (MAPK) pathway is prevalently perturbed in cancer. Recent large‐scale sequencing initiatives profiled thousands of tumors providing insight into alterations at the DNA and RNA levels. These efforts confirmed that key nodes of the MAPK pathway, in particular KRAS and BRAF, are among the most frequently altered proteins in cancer. The establishment of targeted therapies, however, has proven difficult. To decipher the underlying challenges, it is essential to decrypt the phosphorylation network spanned by the MAPK core axis. Using mass spectrometry we identified 2241 phosphorylation sites on 1020 proteins, and measured their responses to inhibition of MEK or ERK. Multiple phosphorylation patterns revealed previously undetected feedback, as upstream signaling nodes, including receptor kinases, showed changes at the phosphorylation level. We provide a dataset rich in potential therapeutic targets downstream of the MAPK cascade. By integrating TCGA (The Cancer Genome Atlas) data, we highlight some downstream phosphoproteins that are frequently altered in cancer. All MS data have been deposited in the ProteomeXchange with identifier PXD003908 ( http://proteomecentral.proteomexchange.org/dataset/PXD003908 ).     

Design,synthesis, and characterization of new cyclic d,l-α-alternate amino acid peptides by capillary electrophoresis coupled to electrospray ionization mass spectrometry

The self-assembly of peptide nanotubes (PNTs) depends on the structure and chemistry of cyclic peptide (CP) monomers, having an impact on their properties, making the choice of their monomers and their characterization a great challenge. We synthesized for the first time a new set of eight original CP sequences of 8, 10, and 12 d,l-α-alternate amino acids with a controlled internal diameter from 7 to 13 Å. They present various properties (e.g., diameter, global surface charge, hydrophobicity) that can open the way to new applications. Their structure and purity were determined thanks to a capillary electrophoresis coupled to electrospray ionization mass spectrometry (CE–ESI–MS) methodology developed for the first time for this purpose. The CPs were successfully separated in a basic hydro-organic background electrolyte (BGE, pH 8.0, H₂O/EtOH 50:50, v/v) and analyzed in MS positive mode. The effect of CP structure on electrophoretic mobility was studied, and the mass spectra were deeply analyzed. This methodology allowed verifying their purity and the absence of linear peptide precursors as well as their stability when stored over several months. Therefore, we have developed a new CE–ESI–MS methodology for the structure and purity control of interesting potential precursors for PNTs that could be employed as nanoplatforms in diagnostics or as pseudo sieving tools for separative purposes.     

High-performance targeted mass spectrometry with precision data-independent acquisition reveals site-specific glycosylation macroheterogeneity

Protein glycosylation is a critical post-translational modification that regulates the structure, stability, and function of many proteins. Mass spectrometry is currently the preferred method for qualitative and quantitative characterization of glycosylation. However, the inherent heterogeneity of glycosylation makes its analysis difficult. Quantification of glycosylation occupancy, or macroheterogeneity, has proven to be especially challenging. Here, we used a variation of high-resolution multiple reaction monitoring (MRM^HR) or pseudo-MRM for targeted data-independent acquisition that we term SWAT (sequential window acquisition of targeted fragment ions). We compared the analytical performance of SWATH (sequential window acquisition of all theoretical fragment ions), SWAT, and SRM (selected reaction monitoring) using a suite of synthetic peptides spiked at various concentrations into a complex yeast tryptic digest sample. SWAT provided superior analytical performance to SWATH in a targeted approach. We then used SWAT to measure site-specific N-glycosylation occupancy in cell wall glycoproteins from yeast with defects in the glycosylation biosynthetic machinery. SWAT provided robust measurement of occupancy at more N-glycosylation sites and with higher precision than SWATH, allowing identification of novel glycosylation sites dependent on the Ost3p and Ost6p regulatory subunits of oligosaccharyltransferase.