16 research outputs found
A quick guide for student-driven community genome annotation
High quality gene models are necessary to expand the molecular and genetic
tools available for a target organism, but these are available for only a
handful of model organisms that have undergone extensive curation and
experimental validation over the course of many years. The majority of gene
models present in biological databases today have been identified in draft
genome assemblies using automated annotation pipelines that are frequently
based on orthologs from distantly related model organisms. Manual curation is
time consuming and often requires substantial expertise, but is instrumental in
improving gene model structure and identification. Manual annotation may seem
to be a daunting and cost-prohibitive task for small research communities but
involving undergraduates in community genome annotation consortiums can be
mutually beneficial for both education and improved genomic resources. We
outline a workflow for efficient manual annotation driven by a team of
primarily undergraduate annotators. This model can be scaled to large teams and
includes quality control processes through incremental evaluation. Moreover, it
gives students an opportunity to increase their understanding of genome biology
and to participate in scientific research in collaboration with peers and
senior researchers at multiple institutions
High-Resolution Analysis of Zn^2+ Coordination in the Alkaline Phosphatase Superfamily by EXAFS and X-ray Crystallography
Comparisons among evolutionarily related enzymes offer opportunities to reveal how structural differences produce different catalytic activities. Two structurally related enzymes, Escherichia coli alkaline phosphatase (AP) and
Xanthomonas axonopodis nucleotide pyrophosphatase/phosphodiesterase (NPP), have nearly identical binuclear Zn^2+ catalytic centers but show tremendous differential specificity for hydrolysis of phosphate monoesters or phosphate diesters. To determine if there are differences in Zn^2+ coordination in the two enzymes that might contribute to catalytic specificity, we analyzed both x-ray absorption spectroscopic and x-ray
crystallographic data. We report a 1.29-Ã… crystal structure of AP with bound phosphate, allowing evaluation of interactions at the AP metal site with high resolution. To make systematic comparisons between AP and NPP, we measured zinc extended x-ray absorption fine structure for AP and
NPP in the free-enzyme forms, with AMP and inorganic phosphate groundstate analogs and with vanadate transition-state analogs. These studies yielded average zinc–ligand distances in AP and NPP free-enzyme forms
and ground-state analog forms that were identical within error, suggesting little difference in metal ion coordination among these forms. Upon binding of vanadate to both enzymes, small increases in average metal–ligand distances were observed, consistent with an increased coordination number. Slightly longer increases were observed in NPP relative to AP, which could arise from subtle rearrangements of the active site or differences in the geometry of the bound vanadyl species. Overall, the
results suggest that the binuclear Zn^2+ catalytic site remains very similar between AP and NPP during the course of a reaction cycle
Sex difference and intra-operative tidal volume: Insights from the LAS VEGAS study
BACKGROUND: One key element of lung-protective ventilation is the use of a low tidal volume (VT). A sex difference in use of low tidal volume ventilation (LTVV) has been described in critically ill ICU patients.OBJECTIVES: The aim of this study was to determine whether a sex difference in use of LTVV also exists in operating room patients, and if present what factors drive this difference.DESIGN, PATIENTS AND SETTING: This is a posthoc analysis of LAS VEGAS, a 1-week worldwide observational study in adults requiring intra-operative ventilation during general anaesthesia for surgery in 146 hospitals in 29 countries.MAIN OUTCOME MEASURES: Women and men were compared with respect to use of LTVV, defined as VT of 8 ml kg-1 or less predicted bodyweight (PBW). A VT was deemed 'default' if the set VT was a round number. A mediation analysis assessed which factors may explain the sex difference in use of LTVV during intra-operative ventilation.RESULTS: This analysis includes 9864 patients, of whom 5425 (55%) were women. A default VT was often set, both in women and men; mode VT was 500 ml. Median [IQR] VT was higher in women than in men (8.6 [7.7 to 9.6] vs. 7.6 [6.8 to 8.4] ml kg-1 PBW, P < 0.001). Compared with men, women were twice as likely not to receive LTVV [68.8 vs. 36.0%; relative risk ratio 2.1 (95% CI 1.9 to 2.1), P < 0.001]. In the mediation analysis, patients' height and actual body weight (ABW) explained 81 and 18% of the sex difference in use of LTVV, respectively; it was not explained by the use of a default VT.CONCLUSION: In this worldwide cohort of patients receiving intra-operative ventilation during general anaesthesia for surgery, women received a higher VT than men during intra-operative ventilation. The risk for a female not to receive LTVV during surgery was double that of males. Height and ABW were the two mediators of the sex difference in use of LTVV.TRIAL REGISTRATION: The study was registered at Clinicaltrials.gov, NCT01601223
Improved annotation of the insect vector of citrus greening disease: Biocuration by a diverse genomics community
The Asian citrus psyllid (Diaphorina citri Kuwayama) is the insect vector of the bacterium Candidatus Liberibacter asiaticus (CLas), the pathogen associated with citrus Huanglongbing (HLB, citrus greening). HLB threatens citrus production worldwide. Suppression or reduction of the insect vector using chemical insecticides has been the primary method to inhibit the spread of citrus greening disease. Accurate structural and functional annotation of the Asian citrus psyllid genome, as well as a clear understanding of the interactions between the insect and CLas, are required for development of new molecular-based HLB control methods. A draft assembly of the D. citri genome has been generated and annotated with automated pipelines. However, knowledge transfer from well-curated reference genomes such as that of Drosophila melanogaster to newly sequenced ones is challenging due to the complexity and diversity of insect genomes. To identify and improve gene models as potential targets for pest control, we manually curated several gene families with a focus on genes that have key functional roles in D. citri biology and CLas interactions. This community effort produced 530 manually curated gene models across developmental, physiological, RNAi regulatory and immunity-related pathways. As previously shown in the pea aphid, RNAi machinery genes putatively involved in the microRNA pathway have been specifically duplicated. A comprehensive transcriptome enabled us to identify a number of gene families that are either missing or misassembled in the draft genome. In order to develop biocuration as a training experience, we included undergraduate and graduate students from multiple institutions, as well as experienced annotators from the insect genomics research community. The resulting gene set (OGS v1.0) combines both automatically predicted and manually curated gene models.Peer reviewedBiochemistry and Molecular BiologyEntomology and Plant Patholog
Site-Directed Mutagenesis Maps Interactions That Enhance Cognate and Limit Promiscuous Catalysis by an Alkaline Phosphatase Superfamily Phosphodiesterase
Catalytic promiscuity, an evolutionary
concept, also provides a
powerful tool for gaining mechanistic insights into enzymatic reactions.
Members of the alkaline phosphatase (AP) superfamily are highly amenable
to such investigation, with several members having been shown to exhibit
promiscuous activity for the cognate reactions of other superfamily
members. Previous work has shown that nucleotide pyrophosphatase/phosphodiesterase
(NPP) exhibits a >10<sup>6</sup>-fold preference for the hydrolysis
of phosphate diesters over phosphate monoesters, and that the reaction
specificity is reduced 10<sup>3</sup>-fold when the size of the substituent
on the transferred phosphoryl group of phosphate diester substrates
is reduced to a methyl group. Here we show additional specificity
contributions from the binding pocket for this substituent (herein
termed the R′ substituent) that account for an additional ∼250-fold
differential specificity with the minimal methyl substituent. Removal
of four hydrophobic side chains suggested on the basis of structural
inspection to interact favorably with R′ substituents decreases
phosphate diester reactivity 10<sup>4</sup>-fold with an optimal diester
substrate (R′ = 5′-deoxythymidine) and 50-fold with
a minimal diester substrate (R′ = CH<sub>3</sub>). These mutations
also enhance the enzyme’s promiscuous phosphate monoesterase
activity by nearly an order of magnitude, an effect that is traced
by mutation to the reduction of unfavorable interactions with the
two residues closest to the nonbridging phosphoryl oxygen atoms. The
quadruple R′ pocket mutant exhibits the same activity toward
phosphate diester and phosphate monoester substrates that have identical
leaving groups, with substantial rate enhancements of ∼10<sup>11</sup>-fold. This observation suggests that the Zn<sup>2+</sup> bimetallo core of AP superfamily enzymes, which is equipotent in
phosphate monoester and diester catalysis, has the potential to become
specialized for the hydrolysis of each class of phosphate esters via
addition of side chains that interact with the substrate atoms and
substituents that project away from the Zn<sup>2+</sup> bimetallo
core
Mechanistic and Evolutionary Insights from Comparative Enzymology of Phosphomonoesterases and Phosphodiesterases across the Alkaline Phosphatase Superfamily
Naively one might
have expected an early division between phosphate
monoesterases and diesterases of the alkaline phosphatase (AP) superfamily.
On the contrary, prior results and our structural and biochemical
analyses of phosphate monoesterase PafA, from <i>Chryseobacterium
meningosepticum</i>, indicate similarities to a superfamily phosphate
diesterase [<i>Xanthomonas citri</i> nucleotide pyrophosphatase/phosphodiesterase
(NPP)] and distinct differences from the three metal ion AP superfamily
monoesterase, from <i>Escherichia coli</i> AP (<i>Ec</i>AP). We carried out a series of experiments to map out and learn
from the differences and similarities between these enzymes. First,
we asked why there would be independent instances of monoesterases
in the AP superfamily? PafA has a much weaker product inhibition and
slightly higher activity relative to <i>Ec</i>AP, suggesting
that different metabolic evolutionary pressures favored distinct active-site
architectures. Next, we addressed the preferential phosphate monoester
and diester catalysis of PafA and NPP, respectively. We asked whether
the >80% sequence differences throughout these scaffolds provide
functional
specialization for each enzyme’s cognate reaction. In contrast
to expectations from this model, PafA and NPP mutants with the common
subset of active-site groups embedded in each native scaffold had
the same monoesterase:diesterase specificities; thus, the >10<sup>7</sup>-fold difference in native specificities appears to arise
from distinct interactions at a single phosphoryl substituent. We
also uncovered striking mechanistic similarities between the PafA
and <i>Ec</i>AP monoesterases, including evidence for ground-state
destabilization and functional active-site networks that involve different
active-site groups but may play analogous catalytic roles. Discovering
common network functions may reveal active-site architectural connections
that are critical for function, and identifying regions of functional
modularity may facilitate the design of new enzymes from existing
promiscuous templates. More generally, comparative enzymology and
analysis of catalytic promiscuity can provide mechanistic and evolutionary
insights