Biochemical Characterization of Uracil Phosphoribosyltransferase from Mycobacterium tuberculosis

Uracil phosphoribosyltransferase (UPRT) catalyzes the conversion of uracil and 5-phosphoribosyl-α-1-pyrophosphate (PRPP) to uridine 5′-monophosphate (UMP) and pyrophosphate (PP_i). UPRT plays an important role in the pyrimidine salvage pathway since UMP is a common precursor of all pyrimidine nucleotides. Here we describe cloning, expression and purification to homogeneity of upp-encoded UPRT from Mycobacterium tuberculosis (MtUPRT). Mass spectrometry and N-terminal amino acid sequencing unambiguously identified the homogeneous protein as MtUPRT. Analytical ultracentrifugation showed that native MtUPRT follows a monomer-tetramer association model. MtUPRT is specific for uracil. GTP is not a modulator of MtUPRT ativity. MtUPRT was not significantly activated or inhibited by ATP, UTP, and CTP. Initial velocity and isothermal titration calorimetry studies suggest that catalysis follows a sequential ordered mechanism, in which PRPP binding is followed by uracil, and PP_i product is released first followed by UMP. The pH-rate profiles indicated that groups with pK values of 5.7 and 8.1 are important for catalysis, and a group with a pK value of 9.5 is involved in PRPP binding. The results here described provide a solid foundation on which to base upp gene knockout aiming at the development of strategies to prevent tuberculosis.     

Human proximity and habitat fragmentation are key drivers of the rangewide bonobo distribution

Habitat loss and hunting threaten bonobos (Pan paniscus), Endangered (IUCN) great apes endemic to lowland rainforests of the Democratic Republic of Congo. Conservation planning requires a current, data-driven, rangewide map of probable bonobo distribution and an understanding of key attributes of areas used by bonobos. We present a rangewide suitability model for bonobos based on a maximum entropy algorithm in which data associated with locations of bonobo nests helped predict suitable conditions across the species’ entire range. We systematically evaluated available biotic and abiotic factors, including a bonobo-specific forest fragmentation layer (forest edge density), and produced a final model revealing the importance of simple threat-based factors in a data poor environment. We confronted the issue of survey bias in presence-only models and devised a novel evaluation approach applicable to other taxa by comparing models built with data from geographically distinct sub-regions that had higher survey effort. The model’s classification accuracy was high (AUC = 0.82). Distance from agriculture and forest edge density best predicted bonobo occurrence with bonobo nests more likely to occur farther from agriculture and in areas of lower edge density. These results suggest that bonobos either avoid areas of higher human activity, fragmented forests, or both, and that humans reduce the effective habitat of bonobos. The model results contribute to an increased understanding of threats to bonobo populations, as well as help identify priority areas for future surveys and determine core bonobo protection areas.     

Partial genotyping at polymorphic markers can improve heritability estimates in sibling groups

Accurate estimates of heritability () are necessary to assess adaptive responses of populations and evolution of fitness‐related traits in changing environments. For plants, estimates generally rely on maternal progeny designs, assuming that offspring are either half‐sibs or unrelated. However, plant mating systems often depart from half‐sib assumptions, this can bias estimates. Here, we investigate how to accurately estimate in nonmodel species through the analysis of sibling designs with a moderate genotyping effort. We performed simulations to investigate how microsatellite marker information available for only a subset of offspring can improve estimates based on maternal progeny designs in the presence of nonrandom mating, inbreeding in the parental population or maternal effects. We compared the basic family method, considering or not adjustments based on average relatedness coefficients, and methods based on the animal model. The animal model was used with average relatedness information, or with hybrid relatedness information: associating one‐generation pedigree and family assumptions, or associating one‐generation pedigree and average relatedness coefficients. Our results highlighted that methods using marker‐based relatedness coefficients performed as well as pedigree‐based methods in the presence of nonrandom mating (i.e. unequal male reproductive contributions, selfing), offering promising prospects to investigate in situ heritabilities in natural populations. In the presence of maternal effects, only the use of pairwise relatednesses through pedigree information improved the accuracy of estimates. In that case, the amount of father‐related offspring in the sibling design is the most critical. Overall, we showed that the method using both one‐generation pedigree and average relatedness coefficients was the most robust to various ecological scenarios.     

Proteomics of Breast Muscle Tissue Associated with the Phenotypic Expression of Feed Efficiency within a Pedigree Male Broiler Line: I. Highlight on Mitochondria

As feed represents 60 to 70% of the cost of raising an animal to market weight, feed efficiency (the amount of dry weight intake to amount of wet weight gain) remains an important genetic trait in animal agriculture. To gain greater understanding of cellular mechanisms of feed efficiency (FE), shotgun proteomics was conducted using in-gel trypsin digestion and tandem mass spectrometry on breast muscle samples obtained from pedigree male (PedM) broilers exhibiting high feed efficiency (FE) or low FE phenotypes (n = 4 per group). The high FE group had greater body weight gain (P = 0.004) but consumed the same amount of feed (P = 0.30) from 6 to 7 wk resulting in higher FE (P < 0.001). Over 1800 proteins were identified, of which 152 were different (P < 0.05) by at least 1.3 fold and ≤ 15 fold between the high and low FE phenotypes. Data were analyzed for a modified differential expression (DE) metric (Phenotypic Impact Factors or PIF) and interpretation of protein expression data facilitated using the Ingenuity Pathway Analysis (IPA) program. In the entire data set, 228 mitochondrial proteins were identified whose collective expression indicates a higher mitochondrial expression in the high FE phenotype (binomial probability P < 0.00001). Within the top up and down 5% PIF molecules in the dataset, there were 15 mitoproteome proteins up-regulated and only 5 down-regulated in the high FE phenotype. Pathway enrichment analysis also identified mitochondrial dysfunction and oxidative phosphorylation as the number 1 and 5 differentially expressed canonical pathways (up-regulated in high FE) in the proteomic dataset. Upstream analysis (based on DE of downstream molecules) predicted that insulin receptor, insulin like growth receptor 1, nuclear factor, erythroid 2-like 2, AMP activated protein kinase (α subunit), progesterone and triiodothyronine would be activated in the high FE phenotype whereas rapamycin independent companion of target of rapamycin, mitogen activated protein kinase 4, and serum response factor would be inhibited in the high FE phenotype. The results provide additional insight into the fundamental molecular landscape of feed efficiency in breast muscle of broilers as well as further support for a role of mitochondria in the phenotypic expression of FE.Funding provided by USDA-NIFA (#2013–01953), Arkansas Biosciences Institute (Little Rock, AR), McMaster Fellowship (AUS to WB) and the Agricultural Experiment Station (Univ. of Arkansas, Fayetteville).     

Comprehensive manipulation of glycosylation profiles across development scales

The extent and pattern of glycosylation on therapeutic antibodies can influence their circulatory half-life, engagement of effector functions, and immunogenicity, with direct consequences to efficacy and patient safety. Hence, controlling glycosylation patterns is central to any drug development program, yet poses a formidable challenge to the bio-manufacturing industry. Process changes, which can affect glycosylation patterns, range from manufacturing at different scales or sites, to switching production process mode, all the way to using alternative host cell lines. In the emerging space of biosimilars development, often times all of these aspects apply. Gaining a deep understanding of the direction and extent to which glycosylation quality attributes can be modulated is key for efficient fine-tuning of glycan profiles in a stage appropriate manner, but establishment of such platform knowledge is time consuming and resource intensive. Here we report an inexpensive and highly adaptable screening system for comprehensive modulation of glycans on antibodies expressed in CHO cells. We characterize 10 media additives in univariable studies and in combination, using a design of experiments approach to map the design space for tuning glycosylation profile attributes. We introduce a robust workflow that does not require automation, yet enables rapid process optimization. We demonstrate scalability across deep wells, shake flasks, AMBR-15 cell culture system, and 2 L single-use bioreactors. Further, we show that it is broadly applicable to different molecules and host cell lineages. This universal approach permits fine-tuned modulation of glycan product quality, reduces development costs, and enables agile implementation of process changes throughout the product lifecycle.     

Role of the Spatzle Pro-domain in the generation of an active toll receptor ligand

The cytokine Sp?tzle is the ligand for Drosophila Toll, the prototype of an important family of membrane receptors that function in embryonic patterning and innate immunity. A dimeric precursor of Sp?tzle is processed by an endoprotease to produce a form (C-106) that cross-links Toll receptor ectodomains and establishes signaling. Here we show that before processing the pro-domain of Sp?tzle is required for correct biosynthesis and secretion. We mapped two loss-of-function mutations of Sp?tzle to a discrete site in the pro-domain and showed that the phenotype arises because of a defect in biosynthesis rather than signaling. We also report that the pro-domain and C-106 remain associated after cleavage and that this processed complex signals with the same characteristics as the C-terminal fragment. These results suggest that before activation the determinants on C-106 that bind specifically to Toll are sequestered by the pro-domain and that proteolytic processing causes conformational rearrangements that expose these determinants and enables binding to Toll. Furthermore, we show that the pro-domain is released when the Toll extracellular domain binds to the complex, a finding that has implications for the generation of a signaling-competent Toll dimer.     

Drosophila embryos lacking N-myristoyltransferase have multiple developmental defects

Lipid modification of proteins by the addition of myristic acid to the N-terminal is important in a number of critical cellular processes, for example, signal transduction and the modulation of membrane association by myristoyl switches. Myristic acid is added to proteins by the enzyme N-myristoyltransferase (NMT) and in this paper we detail the effects on embryonic development of a null mutation in the Drosophila NMT gene. Mutant embryos display a range of phenotypes, including failures of head involution, dorsal closure, and germ-band retraction, morphogenetic processes that require cellular movements. Embryos with milder phenotypes have more specific defects in the central nervous system, including thinning of the ventral nerve chord and, in some embryos, specific scission at parasegment 10. Staining of mutant embryos with phalloidin shows that the mutant embryos have a disrupted actin cytoskeleton and abnormal cell morphology. These phenotypes are strikingly similar to those caused by genes involved in dynamic rearrangement of the actin cytoskeleton. For example the myristoylated nonreceptor tyrosine kinases Dsrc42A and Dsrc64B were shown recently to be key regulators of dorsal closure. In addition, analysis of cell death reveals widespread ectopic apoptosis. Our findings are consistent with the hypothesis that the myristoyl switches and signaling pathways characterized at the biochemical level have important functions in fundamental morphogenetic processes.