Ascites syndrome is the most severe manifestation of pulmonary hypertension in fast-growing broilers. The disease can be attributed to increased body weights of birds, where the higher metabolic load ...is not matched by sufficient oxygen supply to the cells and tissues. Although there are environmental components, the disease exhibits moderate to high heritability. The current study uses high throughput whole genome resequencing (WGR) to identify genes and chromosomal regions associated with ascites.
The WGR data identified the CPQ gene on chromosome 2. The association was confirmed by genotyping a large collection of DNAs from phenotyped birds from three distinct broiler lines using SNPs in intron 6 and exon 8 of the CPQ gene. By combining the genotype data for these two SNP loci, we identified three different alleles segregating in the three broiler lines. Particular genotypes could be associated with resistance to ascites. We further determined that particular genotypes most associated with resistance overexpress CPQ mRNA in three tissues which might explain the role of these alleles in contributing to resistance.
Our findings indicate CPQ is an important determinant of pulmonary hypertension syndrome leading to ascites in broilers. We identified particular SNPs that can be used for marker-assisted selection of broilers for resistance to the disease. Our findings validate WGR as a highly efficient approach to map determinants contributing to complex phenotypic or disease-related traits. The CPQ gene has been associated with pulmonary hypertension in genome-wide association studies in humans. Therefore, ascites investigations in broilers are likely to provide insights into some forms of hypertension in humans.
Chemical biology has long sought to build protein switches for use in molecular diagnostics, imaging, and synthetic biology. The overarching challenge for any type of engineered protein switch is the ...ability to respond in a selective and predictable manner that caters to the specific environments and time scales needed for the application at hand. We previously described a general method to design switchable proteins, called “chemical rescue of structure”, that builds de novo allosteric control sites directly into a protein’s functional domain. This approach entails first carving out a buried cavity in a protein via mutation, such that the protein’s structure is disrupted and activity is lost. An exogenous ligand is subsequently added to substitute for the atoms that were removed by mutation, restoring the protein’s structure and thus its activity. Here, we begin to ask what principles dictate such switches’ response to different activating ligands. Using a redesigned β-glycosidase enzyme as our model system, we find that the designed effector site is quite malleable and can accommodate both larger and smaller ligands, but that optimal rescue comes only from a ligand that perfectly replaces the deleted atoms. Guided by these principles, we then altered the shape of this cavity by using different cavity-forming mutations, and predicted different ligands that would better complement these new cavities. These findings demonstrate how the protein switch’s response can be tuned via small changes to the ligand with respect to the binding cavity, and ultimately enabled us to design an improved switch. We anticipate that these insights will help enable the design of future systems that tune other aspects of protein activity, whereby, like evolved protein receptors, remolding the effector site can also adjust additional outputs such as substrate selectivity and activation of downstream signaling pathways.
The ciliate genus
Paramecium
served as one of the first model systems in microbial eukaryotic genetics, contributing much to the early understanding of phenomena as diverse as genome rearrangement, ...cryptic speciation, cytoplasmic inheritance, and endosymbiosis, as well as more recently to the evolution of mating types, introns, and roles of small RNAs in DNA processing. Substantial progress has recently been made in the area of comparative and population genomics.
Paramecium
species combine some of the lowest known mutation rates with some of the largest known effective populations, along with likely very high recombination rates, thereby harboring a population-genetic environment that promotes an exceptionally efficient capacity for selection. As a consequence, the genomes are extraordinarily streamlined, with very small intergenic regions combined with small numbers of tiny introns. The subject of the bulk of
Paramecium
research, the ancient
Paramecium aurelia
species complex, is descended from two whole-genome duplication events that retain high degrees of synteny, thereby providing an exceptional platform for studying the fates of duplicate genes. Despite having a common ancestor dating to several hundred million years ago, the known descendant species are morphologically indistinguishable, raising significant questions about the common view that gene duplications lead to the origins of evolutionary novelties.
I studied the evolution and cell biology of Paramecium tetraurelia—a model ciliate with over 40,000 distinct protein-coding genes resulting from as many as three ancient whole-genome duplication ...events. I was interested in the functional diversification of these gene duplicates at the level of protein localization, but the commonly used tools to study this were tedious. I instead applied a protein-correlation profiling approach to this system by way of generating a dozen sub-cellular fractions with different protein constituents due to the density of their resident organelle and then assayed these fractions using quantitative mass spectrometry. Each protein’s unique abundance profile provided evidence for its subcellular localization, and I used both supervised and unsupervised classification algorithms to cluster proteins together based on the similarity of these profiles to several hundred “marker proteins” which I manually curated. After expanding the protein inventory for numerous organelles by as many as a thousand proteins, I determined many features not previously understood or appreciated such as mosaic biochemical pathways, evidence for differential sorting mechanisms, and the abnormal evolutionary patterns of the mitochondrial proteome of ciliates. I developed a simple bioinformatic tool to probe spatial proteomics datasets more easily for proteins of interest. I demonstrate its applicability using a handful of well-characterized proteins in the budding yeast Saccharomyces cerevisiae as well as interesting proteins in less well-studied model systems like P. tetraurelia and the apicomplexan Toxoplasma gondii to both recapitulate known interactions and discover new ones. Finally, I look for large-scale evidence of gene duplicates relocalizing to new cellular compartments in P. tetraurelia and S. cerevisiae using this new dataset and a previously generated one, respectively. I find thousands of pairs of duplicates which are differentially identified and display evidence for subcellular divergence, and this seems to be largely decoupled from large changes in protein sequence but are instead associated with indels in their N-terminal peptide. These findings support the use of high-throughput proteomic techniques to determine evidence of functional divergence of gene duplicates. Taken together, this works provides a deep characterization of one of the largest unicellular proteomes in nature.
While coverage to the total HCP mixture in culture harvest stream has been traditionally requested regulatory agencies, the HCPs that purification process that are the most important with respect to ...and stability. major challenge for LC-MS-based methods for identification of HCP in DS is that there can be a more than 5 orders of magnitude difference in the concentration between HCPs and DS, for example a therapeutic antibody, in solution, which precludes the effective identification of low abundance HCPs. To overcome this challenge, efforts have been made to optimize the sample preparation to improve the dynamic range, such as online or offline fractionation 2–9, removal of mAbs by affinity depletion 10–13 or molecular weight cut-off 14, and HCP enrichment 15–17. DISCUSSION Residual HCPs in biopharmaceutics are undesired process-related impurities that need to be well controlled. Besides monitoring the total HCP levels, understanding their physicochemical and biochemical properties is important for achieving maximal HCP removal. The major challenge of LC-MS based methods for HCP characterization in drug substances is the limited dynamic range (3–4 orders of magnitude) that most highresolution mass spectrometers can achieve 22 compared wide dynamic ranges (>5 of magnitude) required to the low-level HCPs (<10 ppm).
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