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Few studies have explored the impact of health insurance on patients with severe, chronic diseases. This retrospective study examined the association between health insurance and the risk of potentially avoidable rehospitalization in the 3 years following validated acute myocardial infarction (AMI) for a community-based probability sample of 683 patients admitted to 30 California hospitals in 1990-1991. In a multivariate analysis adjusted for measures of comorbidity burden, severity of illness, and AMI-related inpatient care, the risk of readmission was not significantly different among patients with no insurance, Medicare insurance, and non-Medicaid, non-Medicare ('private or other') insurance. However, compared to the latter group, patients with Medicaid were 2.6 times more likely to be readmitted for an AMI-related process (risk ratio. 2.61; 95% confidence interval, 1.33 to 5.11). Additional studies are needed to define the role of health insurance on clinical outcomes and health care access across a broader range of conditions and communities. [ABSTRACT FROM AUTHOR]

Introduction: Vibrio alginolyticus is a Gram-negative, rod-shaped bacterium belonging to the family of Vibrionaceae , a common pathogen in aquaculture animals, However, studies on its impact on Scylla serrata (mud crabs) are limited. In this study, we isolated V. alginolyticus SWS from dead mud crab during a disease outbreak in a Hong Kong aquaculture farm, which caused up to 70% mortality during summer., Methods: Experimental infection and histopathology were used to investigate the pathogenicity of V. alginolyticus SWS in S. serrata and validate Koch's postulates. Comprehensive whole-genome analysis and phylogenetic analysis antimicrobial susceptibility testing, and biochemical characterization were also performed., Results: Our findings showed that V. alginolyticus SWS caused high mortality (75%) in S. serrata with infected individuals exhibiting inactivity, loss of appetite, decolored and darkened hepatopancreas, gills, and opaque muscle in the claw. Histopathological analysis revealed tissue damage and degeneration in the hepatopancreas, gills, and claw muscle suggesting direct and indirect impacts of V. alginolyticus SWS infection., Conclusions: This study provides a comprehensive characterization of V. alginolyticus SWS as an emerging pathogen in S. serrata aquaculture. Our findings underscore the importance of ongoing surveillance, early detection, and the development of targeted disease management strategies to mitigate the economic impact of vibriosis outbreaks in mud crab aquaculture., Competing Interests: Author W-ML was employed by the company AI Farming Company Hong Kong Limited. The authors declare that this study received funding from AI Farming HK Ltd. The funder had the following involvement in the study: provided technical support and revised manuscript. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Kwok, Yu, Hau, Cheung, Ng, Fung, Wong, Yau, Liu, Kong, Siu, Chow and Seto.)

Objective: This study aimed to characterize the changing landscape of circulating SARS-CoV-2 lineages in the local community of Hong Kong throughout 2022. We examined how adjustments to quarantine arrangements influenced the transmission pattern of Omicron variants in a city with relatively rigorous social distancing measures at that time., Methods: In 2022, a total of 4684 local SARS-CoV-2 genomes were sequenced using the Oxford Nanopore GridION sequencer. SARS-CoV-2 consensus genomes were generated by MAFFT, and the maximum likelihood phylogeny of these genomes was determined using IQ-TREE. The dynamic changes in lineages were depicted in a time tree created by Nextstrain. Statistical analysis was conducted to assess the correlation between changes in the number of lineages and adjustments to quarantine arrangements., Results: By the end of 2022, a total of 83 SARS-CoV-2 lineages were identified in the community. The increase in the number of new lineages was significantly associated with the relaxation of quarantine arrangements (One-way ANOVA, F(5, 47) = 18.233, p < 0.001)). Over time, Omicron BA.5 sub-lineages replaced BA.2.2 and became the predominant Omicron variants in Hong Kong. The influx of new lineages reshaped the dynamics of Omicron variants in the community without fluctuating the death rate and hospitalization rate (One-way ANOVA, F(5, 47) = 2.037, p = 0.091)., Conclusion: This study revealed that even with an extended mandatory quarantine period for incoming travelers, it may not be feasible to completely prevent the introduction and subsequent community spread of highly contagious Omicron variants. Ongoing molecular surveillance of COVID-19 remains essential to monitor the emergence of new recombinant variants.

The prolonged incubation period of traditional culture methods leads to a delay in diagnosing invasive infections. Nanopore 16S rRNA gene sequencing (Nanopore 16S) offers a potential rapid diagnostic approach for directly identifying bacteria in infected body fluids. To evaluate the clinical utility of Nanopore 16S, we conducted a study involving the collection and sequencing of 128 monomicrobial samples, 65 polymicrobial samples, and 20 culture-negative body fluids. To minimize classification bias, taxonomic classification was performed using 3 analysis pipelines: Epi2me, Emu, and NanoCLUST. The result was compared to the culture references. The limit of detection of Nanopore 16S was also determined using simulated bacteremic blood samples. Among the three classifiers, Emu demonstrated the highest concordance with the culture results. It correctly identified the taxon of 125 (97.7%) of the 128 monomicrobial samples, compared to 109 (85.2%) for Epi2me and 102 (79.7%) for NanoCLUST. For the 230 cultured species in the 65 polymicrobial samples, Emu correctly identified 188 (81.7%) cultured species, compared to 174 (75.7%) for Epi2me and 125 (54.3%) for NanoCLUST. Through ROC analysis on the monomicrobial samples, we determined a threshold of relative abundance at 0.058 for distinguishing potential pathogens from background in Nanopore 16S. Applying this threshold resulted in the identification of 107 (83.6%), 117 (91.4%), and 114 (91.2%) correctly detected samples for Epi2me, Emu, and NanoCLUST, respectively, in the monomicrobial samples. Nanopore 16S coupled with Epi2me could provide preliminary results within 6 h. However, the ROC analysis of polymicrobial samples exhibited a random-like performance, making it difficult to establish a threshold. The overall limit of detection for Nanopore 16S was found to be about 90 CFU/ml., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Lao, Wong, Hui, Ng, Chan, Lo, Yau, Leung, Wong, Ho, Yip, Lam, Chow, Luk, Que, Chow and Siu.)

Background and Purpose: Despite the availability of a variety of treatment options, many asthma patients have poorly controlled disease with frequent exacerbations. Proteinase-activated receptor-2 (PAR2) has been identified in preclinical animal models as important to asthma initiation and progression following allergen exposure. Proteinase activation of PAR2 raises intracellular Ca 2+ , inducing MAPK and β-arrestin signalling in the airway, leading to inflammatory and protective effects. We have developed C391, a potent PAR2 antagonist effective in blocking peptidomimetic- and trypsin-induced PAR2 signalling in vitro as well as reducing inflammatory PAR2-associated pain in vivo. We hypothesized that PAR2 antagonism by C391 would attenuate allergen-induced acutely expressed asthma indicators in murine models., Experimental Approach: We evaluated the ability of C391 to alter Alternaria alternata-induced PAR2 signalling pathways in vitro using a human airway epithelial cell line that naturally expresses PAR2 (16HBE14o-) and a transfected embryonic cell line (HEK 293). We next evaluated the ability for C391 to reduce A. alternata-induced acutely expressed asthma indicators in vivo in two murine strains., Key Results: C391 blocked A. alternata-induced, PAR2-dependent Ca 2+ and MAPK signalling in 16HBE14o- cells, as well as β-arrestin recruitment in HEK 293 cells. C391 effectively attenuated A. alternata-induced inflammation, mucus production, mucus cell hyperplasia and airway hyperresponsiveness in acute allergen-challenged murine models., Conclusions and Implications: To our best knowledge, this is the first demonstration of pharmacological intervention of PAR2 to reduce allergen-induced asthma indicators in vivo. These data support further development of PAR2 antagonists as potential first-in-class allergic asthma drugs., (© 2021 The British Pharmacological Society.)

Bacterial pathogens that cannot be identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) are occasionally encountered in clinical laboratories. The 16S rRNA gene is often used for sequence-based analysis to identify these bacterial species. Nevertheless, traditional Sanger sequencing is laborious, time-consuming, and low throughput. Here, we compared two commercially available 16S rRNA gene sequencing tests that are based on Illumina and Nanopore sequencing technologies, respectively, in their ability to identify the species of 172 clinical isolates that failed to be identified by MALDI-TOF MS. Sequencing data were analyzed by the respective built-in programs (MiSeq Reporter software of Illumina and Epi2me of Nanopore) and BLAST+ (v2.11.0). Their agreement with Sanger sequencing on species-level identification was determined. Discrepancies were resolved by whole-genome sequencing. The diagnostic accuracy of each workflow was determined using the composite sequencing result as the reference standard. Despite the high base-calling accuracy of Illumina sequencing, we demonstrated that the Nanopore workflow had a higher taxonomic resolution at the species level. Using built-in analysis algorithms, the concordance of Sanger 16S with the Illumina and Nanopore workflows was 33.14% and 87.79%, respectively. The agreement was 65.70% and 83.14%, respectively, when BLAST+ was used for analysis. Compared with the reference standard, the diagnostic accuracy of Nanopore 16S was 96.36%, which was identical to that of Sanger 16S and better than that of Illumina 16S (69.07%). The turnaround time of the Illumina workflow and the Nanopore workflow was 78 h and 8.25 h, respectively. The per-sample cost of the Illumina and Nanopore workflows was US$28.5 and US$17.7, respectively.

The increasing prevalence of N501Y variants of SARS-CoV-2 has kindled global concern due to their enhanced transmissibility. Genome sequencing is the gold standard method to identify the emerging variants of concern. But it is time-consuming and expensive, limiting the widespread deployment of genome surveillance in some countries. Health authorities surge the development of alternative assay to expand screening capacity with reduced time and cost. In this study, we developed an in-house TaqMan minor groove binder (MGB) probe-based one-step RT-qPCR assay to detect the presence of N501Y mutation in SARS-CoV-2. A total of 168 SARS-CoV-2 positive respiratory specimens were collected to determine diagnostic accuracy of the RT-qPCR assay. As a reference standard, PANGO lineages and the mutation patterns of all samples were characterised by whole-genome sequencing. The analytical sensitivity and the ability of the assay to detect low frequency of N501Y variants were also evaluated. A total of 31 PANGO lineages were identified from 168 SARS-CoV-2 positive cases, in which 34 samples belonged to N501Y variants, including B.1.1.7 (n = 20), B.1.351 (n = 12) and P.3 (n = 2). The N501Y RT-qPCR correctly identified all 34 samples as N501Y-positive and the other 134 samples as wildtype. The limit-of-detection of the assay consistently achieved 1.5 copies/μL on four different qPCR platforms. N501Y mutation was successfully detected at an allele frequency as low as 10 % in a sample with mixed SARS-CoV-2 lineage. The N501Y RT-qPCR is simple and inexpensive (US$1.6 per sample). It enables robust high-throughput screening for surveillance of SARS-CoV-2 variants of concern harbouring N501Y mutation., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The integration of genomic data into health systems offers opportunities to identify genomic factors underlying the continuum of rare and common disease. We applied a population-scale haplotype association approach based on identity-by-descent (IBD) in a large multi-ethnic biobank to a spectrum of disease outcomes derived from electronic health records (EHRs) and uncovered a risk locus for liver disease. We used genome sequencing and in silico approaches to fine-map the signal to a non-coding variant (c.2784-12T>C) in the gene ABCB4. In vitro analysis confirmed the variant disrupted splicing of the ABCB4 pre-mRNA. Four of five homozygotes had evidence of advanced liver disease, and there was a significant association with liver disease among heterozygotes, suggesting the variant is linked to increased risk of liver disease in an allele dose-dependent manner. Population-level screening revealed the variant to be at a carrier rate of 1.95% in Puerto Rican individuals, likely as the result of a Puerto Rican founder effect. This work demonstrates that integrating EHR and genomic data at a population scale can facilitate strategies for understanding the continuum of genomic risk for common diseases, particularly in populations underrepresented in genomic medicine., Competing Interests: Declaration of interests N.S.A.-H. was previously employed by Regeneron Pharmaceuticals, has received an honorarium from Genentech, and serves on the Scientific Advisory Board for Allelica. E.E.K. has received speaker honoraria from Illumina and Regeneron Pharmaceuticals. C.R.G. owns stock in 23andMe, Inc. The remaining authors declare no competing interests., (Copyright © 2021. Published by Elsevier Inc.)

Pyoderma gangrenosum (PG) is an uncommon inflammatory neutrophilic disorder with a spectrum of clinical presentations with variable courses. Most cases are associated with an autoimmune disorder and manifest in middle-aged adults as a painful lesion that progresses to painful necrotizing ulcers of the lower extremity. Owing to its variability, clinical diagnosis remains difficult and many patients are often misdiagnosed, with resulting delay in treatment. While early immunosuppressant therapy is key to preventing progression of PG, surgical treatment has been met with criticism because of the risk of potentiating pathergy, an exaggerated skin reaction due to trauma. This article presents a case series in which 3 patients with PG lesions underwent different treatment methods, including surgical debridement and use of fetal bovine dermis (FBD). The use of FBD in conjunction with medical treatment provided pain relief and wound coverage as well as encouraged growth of granulation tissue and long-term stability. Commercial cellular and tissue-based products used to aid in accelerating PG wound closure are also reviewed.

The emergence of genomic data in biobanks and health systems offers new ways to derive medically important phenotypes, including acute phenotypes occurring during inpatient clinical care. Here we study the genetic underpinnings of the rapid response to phenylephrine, an α1-adrenergic receptor agonist commonly used to treat hypotension during anesthesia and surgery. We quantified this response by extracting blood pressure (BP) measurements 5 min before and after the administration of phenylephrine. Based on this derived phenotype, we show that systematic differences exist between self-reported ancestry groups: European-Americans (EA; n = 1387) have a significantly higher systolic response to phenylephrine than African-Americans (AA; n = 1217) and Hispanic/Latinos (HA; n = 1713) (31.3% increase, p value < 6e-08 and 22.9% increase, p value < 5e-05 respectively), after adjusting for genetic ancestry, demographics, and relevant clinical covariates. We performed a genome-wide association study to investigate genetic factors underlying individual differences in this derived phenotype. We discovered genome-wide significant association signals in loci and genes previously associated with BP measured in ambulatory settings, and a general enrichment of association in these genes. Finally, we discovered two low frequency variants, present at ~1% in EAs and AAs, respectively, where patients carrying one copy of these variants show no phenylephrine response. This work demonstrates our ability to derive a quantitative phenotype suited for comparative statistics and genome-wide association studies from dense clinical and physiological measures captured for managing patients during surgery. We identify genetic variants underlying non response to phenylephrine, with implications for preemptive pharmacogenomic screening to improve safety during surgery.

Initial cases of coronavirus disease in Hong Kong were imported from mainland China. A dramatic increase in case numbers was seen in February 2020. Most case-patients had no recent travel history, suggesting the presence of transmission chains in the local community. We collected demographic, clinical, and epidemiologic data from 50 patients, who accounted for 53.8% of total reported case-patients as of February 28, 2020. We performed whole-genome sequencing to determine phylogenetic relationship and transmission dynamics of severe acute respiratory syndrome coronavirus 2 infections. By using phylogenetic analysis, we attributed the community outbreak to 2 lineages; 1 harbored a common mutation, Orf3a-G251V, and accounted for 88.0% of the cases in our study. The estimated time to the most recent common ancestor of local coronavirus disease outbreak was December 24, 2019, with an evolutionary rate of 3.04 × 10 -3 substitutions/site/year. The reproduction number was 1.84, indicating ongoing community spread.

Alternaria alternata is a fungal allergen associated with severe asthma and asthma exacerbations. Similarly to other asthma-associated allergens, Alternaria secretes a serine-like trypsin protease(s) that is thought to act through the G protein-coupled receptor protease-activated receptor-2 (PAR 2 ) to induce asthma symptoms. However, specific mechanisms underlying Alternaria-induced PAR 2 activation and signaling remain ill-defined. We sought to determine whether Alternaria-induced PAR 2 signaling contributed to asthma symptoms via a PAR 2 /β-arrestin signaling axis, identify the protease activity responsible for PAR 2 signaling, and determine whether protease activity was sufficient for Alternaria-induced asthma symptoms in animal models. We initially used in vitro models to demonstrate Alternaria-induced PAR 2 /β-arrestin-2 signaling. Alternaria filtrates were then used to sensitize and challenge wild-type, PAR 2 -/- and β-arrestin-2 -/- mice in vivo. Intranasal administration of Alternaria filtrate resulted in a protease-dependent increase of airway inflammation and mucin production in wild-type but not PAR 2 -/- or β-arrestin-2 -/- mice. Protease was isolated from Alternaria preparations, and select in vitro and in vivo experiments were repeated to evaluate sufficiency of the isolated Alternaria protease to induce asthma phenotype. Administration of a single isolated serine protease from Alternaria, Alternaria alkaline serine protease (AASP), was sufficient to fully activate PAR 2 signaling and induce β-arrestin-2 -/- -dependent eosinophil and lymphocyte recruitment in vivo. In conclusion, Alternaria filtrates induce airway inflammation and mucus hyperplasia largely via AASP using the PAR 2 /β-arrestin signaling axis. Thus, β-arrestin-biased PAR 2 antagonists represent novel therapeutic targets for treating aeroallergen-induced asthma.

The colonization of novel environments often involves changes in gene expression, protein coding sequence, or both. Studies of how populations adapt to novel conditions, however, often focus on only one of these two processes, potentially missing out on the relative importance of different parts of the evolutionary process. In this study, our objectives were 1) to better understand the qualitative concordance between conclusions drawn from analyses of differential expression and changes in genic sequence and 2) to quantitatively test whether differentially expressed genes were enriched for sites putatively under positive selection within gene regions. To achieve this, we compared populations of fish (Poecilia mexicana) that have independently adapted to hydrogen-sulfide-rich environments in southern Mexico to adjacent populations residing in nonsulfidic waters. Specifically, we used RNA-sequencing data to compare both gene expression and DNA sequence differences between populations. Analyzing these two different data types led to similar conclusions about which biochemical pathways (sulfide detoxification and cellular respiration) were involved in adaptation to sulfidic environments. Additionally, we found a greater overlap between genes putatively under selection and differentially expressed genes than expected by chance. We conclude that considering both differential expression and changes in DNA sequence led to a more comprehensive understanding of how these populations adapted to extreme environmental conditions. Our results imply that changes in both gene expression and DNA sequence-sometimes at the same loci-may be involved in adaptation.

Domestication has had a strong impact on the development of modern societies. We sequenced 200 genomes of the chocolate plant Theobroma cacao L. to show for the first time to our knowledge that a single population, the Criollo population, underwent strong domestication ~3600 years ago (95% CI: 2481-13,806 years ago). We also show that during the process of domestication, there was strong selection for genes involved in the metabolism of the colored protectants anthocyanins and the stimulant theobromine, as well as disease resistance genes. Our analyses show that domesticated populations of T. cacao (Criollo) maintain a higher proportion of high-frequency deleterious mutations. We also show for the first time the negative consequences of the increased accumulation of deleterious mutations during domestication on the fitness of individuals (significant reduction in kilograms of beans per hectare per year as Criollo ancestry increases, as estimated from a GLM, P  = 0.000425)., Competing Interests: Co-authors Donald Livingstone III, Conrad Stack, Alberto Romero, Stefan Royaert, Osman Gutierrez, and Juan C. Motamayor are or have been employees of the funding agency MARS Inc. The authors declare no other competing financial or non-financial interests.

We analyzed 391 samples from 12 Argentinian populations from the Center-West, East and North-West regions with the Illumina Human Exome Beadchip v1.0 (HumanExome-12v1-A). We did Principal Components analysis to infer patterns of populational divergence and migrations. We identified proportions and patterns of European, African and Native American ancestry and found a correlation between distance to Buenos Aires and proportion of Native American ancestry, where the highest proportion corresponds to the Northernmost populations, which is also the furthest from the Argentinian capital. Most of the European sources are from a South European origin, matching historical records, and we see two different Native American components, one that spreads all over Argentina and another specifically Andean. The highest percentages of African ancestry were in the Center West of Argentina, where the old trade routes took the slaves from Buenos Aires to Chile and Peru. Subcontinentaly, sources of this African component are represented by both West Africa and groups influenced by the Bantu expansion, the second slightly higher than the first, unlike North America and the Caribbean, where the main source is West Africa. This is reasonable, considering that a large proportion of the ships arriving at the Southern Hemisphere came from Mozambique, Loango and Angola.

The mangrove rivulus, Kryptolebias marmoratus, is one of only two self-fertilizing hermaphroditic fish species and inhabits mangrove forests. While selfing can be advantageous, it reduces heterozygosity and decreases genetic diversity. Studies using microsatellites found that there are variable levels of selfing among populations of K. marmoratus, but overall, there is a low rate of outcrossing and, therefore, low heterozygosity. In this study, we used whole-genome data to assess the levels of heterozygosity in different lineages of the mangrove rivulus and infer the phylogenetic relationships among those lineages. We sequenced whole genomes from 15 lineages that were completely homozygous at microsatellite loci and used single nucleotide polymorphisms (SNPs) to determine heterozygosity levels. More variation was uncovered than in studies using microsatellite data because of the resolution of full genome sequencing data. Moreover, missense polymorphisms were found most often in genes associated with immune function and reproduction. Inferred phylogenetic relationships suggest that lineages largely group by their geographic distribution. The use of whole-genome data provided further insight into genetic diversity in this unique species. Although this study was limited by the number of lineages that were available, these data suggest that there is previously undescribed variation within lineages of K. marmoratus that could have functional consequences and (or) inform us about the limits to selfing (e.g., genetic load, accumulation of deleterious mutations) and selection that might favor the maintenance of heterozygosity. These results highlight the need to sequence additional individuals within and among lineages.

Achieving confidence in the causality of a disease locus is a complex task that often requires supporting data from both statistical genetics and clinical genomics. Here we describe a combined approach to identify and characterize a genetic disorder that leverages distantly related patients in a health system and population-scale mapping. We utilize genomic data to uncover components of distant pedigrees, in the absence of recorded pedigree information, in the multi-ethnic Bio Me biobank in New York City. By linking to medical records, we discover a locus associated with both elevated genetic relatedness and extreme short stature. We link the gene, COL27A1 , with a little-known genetic disease, previously thought to be rare and recessive. We demonstrate that disease manifests in both heterozygotes and homozygotes, indicating a common collagen disorder impacting up to 2% of individuals of Puerto Rican ancestry, leading to a better understanding of the continuum of complex and Mendelian disease.

Variation in gene expression can provide insights into organismal responses to environmental stress and physiological mechanisms mediating adaptation to habitats with contrasting environmental conditions. We performed an RNA-sequencing experiment to quantify gene expression patterns in fish adapted to habitats with different combinations of environmental stressors, including the presence of toxic hydrogen sulphide (H 2 S) and the absence of light in caves. We specifically asked how gene expression varies among populations living in different habitats, whether population differences were consistent among organs, and whether there is evidence for shared expression responses in populations exposed to the same stressors. We analysed organ-specific transcriptome-wide data from four ecotypes of Poecilia mexicana (nonsulphidic surface, sulphidic surface, nonsulphidic cave and sulphidic cave). The majority of variation in gene expression was correlated with organ type, and the presence of specific environmental stressors elicited unique expression differences among organs. Shared patterns of gene expression between populations exposed to the same environmental stressors increased with levels of organismal organization (from transcript to gene to physiological pathway). In addition, shared patterns of gene expression were more common between populations from sulphidic than populations from cave habitats, potentially indicating that physiochemical stressors with clear biochemical consequences can constrain the diversity of adaptive solutions that mitigate their adverse effects. Overall, our analyses provided insights into transcriptional variation in a unique system, in which adaptation to H 2 S and darkness coincide. Functional annotations of differentially expressed genes provide a springboard for investigating physiological mechanisms putatively underlying adaptation to extreme environments., (© 2017 John Wiley & Sons Ltd.)

Vertical growth of plants is a dynamic process that is influenced by genetic and environmental factors and has a pronounced effect on overall plant architecture and biomass composition. We have performed six controlled growth trials of an interspecific Setaria italica x Setaria viridis recombinant inbred line population to assess how the genetic architecture of plant height is influenced by developmental queues, water availability and planting density. The non-destructive nature of plant height measurements has enabled us to monitor height throughout the plant life cycle in both field and controlled environments. We find that plant height is reduced under water limitation and high density planting and affected by growth environment (field vs. growth chamber). The results support a model where plant height is a heritable, polygenic trait and that the major genetic loci that influence plant height function independent of growth environment. The identity and contribution of loci that influence height changes dynamically throughout development and the reduction of growth observed in water limited environments is a consequence of delayed progression through the genetic program which establishes plant height in Setaria. In this population, alleles inherited from the weedy S. viridis parent act to increase plant height early, whereas a larger number of small effect alleles inherited from the domesticated S. italica parent collectively act to increase plant height later in development.

The mangrove rivulus (Kryptolebias marmoratus) is one of two preferentially self-fertilizing hermaphroditic vertebrates. This mode of reproduction makes mangrove rivulus an important model for evolutionary and biomedical studies because long periods of self-fertilization result in naturally homozygous genotypes that can produce isogenic lineages without significant limitations associated with inbreeding depression. Over 400 isogenic lineages currently held in laboratories across the globe show considerable among-lineage variation in physiology, behavior, and life history traits that is maintained under common garden conditions. Temperature mediates the development of primary males and also sex change between hermaphrodites and secondary males, which makes the system ideal for the study of sex determination and sexual plasticity. Mangrove rivulus also exhibit remarkable adaptations to living in extreme environments, and the system has great promise to shed light on the evolution of terrestrial locomotion, aerial respiration, and broad tolerances to hypoxia, salinity, temperature, and environmental pollutants. Genome assembly of the mangrove rivulus allows the study of genes and gene families associated with the traits described above. Here we present a de novo assembled reference genome for the mangrove rivulus, with an approximately 900 Mb genome, including 27,328 annotated, predicted, protein-coding genes. Moreover, we are able to place more than 50% of the assembled genome onto a recently published linkage map. The genome provides an important addition to the linkage map and transcriptomic tools recently developed for this species that together provide critical resources for epigenetic, transcriptomic, and proteomic analyses. Moreover, the genome will serve as the foundation for addressing key questions in behavior, physiology, toxicology, and evolutionary biology., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Many important crops are members of the Poaceae family, which develop root systems characterized by a high degree of root initiation from the belowground basal nodes of the shoot, termed the crown. Although this postembryonic shoot-borne root system represents the major conduit for water uptake, little is known about the effect of water availability on its development. Here we demonstrate that in the model C4 grass Setaria viridis, the crown locally senses water availability and suppresses postemergence crown root growth under a water deficit. This response was observed in field and growth room environments and in all grass species tested. Luminescence-based imaging of root systems grown in soil-like media revealed a shift in root growth from crown-derived to primary root-derived branches, suggesting that primary root-dominated architecture can be induced in S. viridis under certain stress conditions. Crown roots of Zea mays and Setaria italica, domesticated relatives of teosinte and S. viridis, respectively, show reduced sensitivity to water deficit, suggesting that this response might have been influenced by human selection. Enhanced water status of maize mutants lacking crown roots suggests that under a water deficit, stronger suppression of crown roots actually may benefit crop productivity.

Hydrogen sulfide (H2S) is a potent toxicant interfering with oxidative phosphorylation in mitochondria and creating extreme environmental conditions in aquatic ecosystems. The mechanistic basis of adaptation to perpetual exposure to H2S remains poorly understood. We investigated evolutionarily independent lineages of livebearing fishes that have colonized and adapted to springs rich in H2S and compared their genome-wide gene expression patterns with closely related lineages from adjacent, nonsulfidic streams. Significant differences in gene expression were uncovered between all sulfidic and nonsulfidic population pairs. Variation in the number of differentially expressed genes among population pairs corresponded to differences in divergence times and rates of gene flow, which is consistent with neutral drift driving a substantial portion of gene expression variation among populations. Accordingly, there was little evidence for convergent evolution shaping large-scale gene expression patterns among independent sulfide spring populations. Nonetheless, we identified a small number of genes that was consistently differentially expressed in the same direction in all sulfidic and nonsulfidic population pairs. Functional annotation of shared differentially expressed genes indicated upregulation of genes associated with enzymatic H2S detoxification and transport of oxidized sulfur species, oxidative phosphorylation, energy metabolism, and pathways involved in responses to oxidative stress. Overall, our results suggest that modification of processes associated with H2S detoxification and toxicity likely complement each other to mediate elevated H2S tolerance in sulfide spring fishes. Our analyses allow for the development of novel hypotheses about biochemical and physiological mechanisms of adaptation to extreme environments., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Reduced representation sequencing methods such as genotyping-by-sequencing (GBS) enable low-cost measurement of genetic variation without the need for a reference genome assembly. These methods are widely used in genetic mapping and population genetics studies, especially with non-model organisms. Variant calling error rates, however, are higher in GBS than in standard sequencing, in particular due to restriction site polymorphisms, and few computational tools exist that specifically model and correct these errors. We developed a statistical method to remove errors caused by restriction site polymorphisms, implemented in the software package GBStools. We evaluated it in several simulated data sets, varying in number of samples, mean coverage and population mutation rate, and in two empirical human data sets (N = 8 and N = 63 samples). In our simulations, GBStools improved genotype accuracy more than commonly used filters such as Hardy-Weinberg equilibrium p-values. GBStools is most effective at removing genotype errors in data sets over 100 samples when coverage is 40X or higher, and the improvement is most pronounced in species with high genomic diversity. We also demonstrate the utility of GBS and GBStools for human population genetic inference in Argentine populations and reveal widely varying individual ancestry proportions and an excess of singletons, consistent with recent population growth.

Lifespan is a remarkably diverse trait ranging from a few days to several hundred years in nature, but the mechanisms underlying the evolution of lifespan differences remain elusive. Here we de novo assemble a reference genome for the naturally short-lived African turquoise killifish, providing a unique resource for comparative and experimental genomics. The identification of genes under positive selection in this fish reveals potential candidates to explain its compressed lifespan. Several aging genes are under positive selection in this short-lived fish and long-lived species, raising the intriguing possibility that the same gene could underlie evolution of both compressed and extended lifespans. Comparative genomics and linkage analysis identify candidate genes associated with lifespan differences between various turquoise killifish strains. Remarkably, these genes are clustered on the sex chromosome, suggesting that short lifespan might have co-evolved with sex determination. Our study provides insights into the evolutionary forces that shape lifespan in nature., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Although a variety of genetic alterations have been found across cancer types, the identification and functional characterization of candidate driver genetic lesions in an individual patient and their translation into clinically actionable strategies remain major hurdles. Here, we use whole genome sequencing of a prostate cancer tumor, computational analyses, and experimental validation to identify and predict novel oncogenic activity arising from a point mutation in the phosphatase and tensin homolog (PTEN) tumor suppressor protein. We demonstrate that this mutation (p.A126G) produces an enzymatic gain-of-function in PTEN, shifting its function from a phosphoinositide (PI) 3-phosphatase to a phosphoinositide (PI) 5-phosphatase. Using cellular assays, we demonstrate that this gain-of-function activity shifts cellular phosphoinositide levels, hyperactivates the PI3K/Akt cell proliferation pathway, and exhibits increased cell migration beyond canonical PTEN loss-of-function mutants. These findings suggest that mutationally modified PTEN can actively contribute to well-defined hallmarks of cancer. Lastly, we demonstrate that these effects can be substantially mitigated through chemical PI3K inhibitors. These results demonstrate a new dysfunction paradigm for PTEN cancer biology and suggest a potential framework for the translation of genomic data into actionable clinical strategies for targeted patient therapy.

Root systems develop different root types that individually sense cues from their local environment and integrate this information with systemic signals. This complex multi-dimensional amalgam of inputs enables continuous adjustment of root growth rates, direction, and metabolic activity that define a dynamic physical network. Current methods for analyzing root biology balance physiological relevance with imaging capability. To bridge this divide, we developed an integrated-imaging system called Growth and Luminescence Observatory for Roots (GLO-Roots) that uses luminescence-based reporters to enable studies of root architecture and gene expression patterns in soil-grown, light-shielded roots. We have developed image analysis algorithms that allow the spatial integration of soil properties, gene expression, and root system architecture traits. We propose GLO-Roots as a system that has great utility in presenting environmental stimuli to roots in ways that evoke natural adaptive responses and in providing tools for studying the multi-dimensional nature of such processes.

Large-scale sequencing efforts have documented extensive genetic variation within the human genome. However, our understanding of the origins, global distribution, and functional consequences of this variation is far from complete. While regulatory variation influencing gene expression has been studied within a handful of populations, the breadth of transcriptome differences across diverse human populations has not been systematically analyzed. To better understand the spectrum of gene expression variation, alternative splicing, and the population genetics of regulatory variation in humans, we have sequenced the genomes, exomes, and transcriptomes of EBV transformed lymphoblastoid cell lines derived from 45 individuals in the Human Genome Diversity Panel (HGDP). The populations sampled span the geographic breadth of human migration history and include Namibian San, Mbuti Pygmies of the Democratic Republic of Congo, Algerian Mozabites, Pathan of Pakistan, Cambodians of East Asia, Yakut of Siberia, and Mayans of Mexico. We discover that approximately 25.0% of the variation in gene expression found amongst individuals can be attributed to population differences. However, we find few genes that are systematically differentially expressed among populations. Of this population-specific variation, 75.5% is due to expression rather than splicing variability, and we find few genes with strong evidence for differential splicing across populations. Allelic expression analyses indicate that previously mapped common regulatory variants identified in eight populations from the International Haplotype Map Phase 3 project have similar effects in our seven sampled HGDP populations, suggesting that the cellular effects of common variants are shared across diverse populations. Together, these results provide a resource for studies analyzing functional differences across populations by estimating the degree of shared gene expression, alternative splicing, and regulatory genetics across populations from the broadest points of human migration history yet sampled.

The midge, Belgica antarctica, is the only insect endemic to Antarctica, and thus it offers a powerful model for probing responses to extreme temperatures, freeze tolerance, dehydration, osmotic stress, ultraviolet radiation and other forms of environmental stress. Here we present the first genome assembly of an extremophile, the first dipteran in the family Chironomidae, and the first Antarctic eukaryote to be sequenced. At 99 megabases, B. antarctica has the smallest insect genome sequenced thus far. Although it has a similar number of genes as other Diptera, the midge genome has very low repeat density and a reduction in intron length. Environmental extremes appear to constrain genome architecture, not gene content. The few transposable elements present are mainly ancient, inactive retroelements. An abundance of genes associated with development, regulation of metabolism and responses to external stimuli may reflect adaptations for surviving in this harsh environment.

An unexpectedly large fraction of genes in metazoans (human, mouse, zebrafish, worm, fruit fly) express high levels of circularized RNAs containing canonical exons. Here we report that circular RNA isoforms are found in diverse species whose most recent common ancestor existed more than one billion years ago: fungi (Schizosaccharomyces pombe and Saccharomyces cerevisiae), a plant (Arabidopsis thaliana), and protists (Plasmodium falciparum and Dictyostelium discoideum). For all species studied to date, including those in this report, only a small fraction of the theoretically possible circular RNA isoforms from a given gene are actually observed. Unlike metazoans, Arabidopsis, D. discoideum, P. falciparum, S. cerevisiae, and S. pombe have very short introns (∼ 100 nucleotides or shorter), yet they still produce circular RNAs. A minority of genes in S. pombe and P. falciparum have documented examples of canonical alternative splicing, making it unlikely that all circular RNAs are by-products of alternative splicing or 'piggyback' on signals used in alternative RNA processing. In S. pombe, the relative abundance of circular to linear transcript isoforms changed in a gene-specific pattern during nitrogen starvation. Circular RNA may be an ancient, conserved feature of eukaryotic gene expression programs.

Tools that allow for rapid, accurate and inexpensive assembly of multi-component combinatorial libraries of DNA for transformation into plants will accelerate the progress of synthetic biology research. Recent innovations in molecular cloning methods has vastly expanded the repertoire with which plant biologists can engineer a transgene. Here we describe a new set of binary vectors for use in Agrobacterium-mediated plant transformation that utilizes the Golden-Gate Cloning approach. Our optimized protocol facilitates the rapid and inexpensive generation of multi-component transgenes for later introduction into plants.

The Y chromosome and the mitochondrial genome have been used to estimate when the common patrilineal and matrilineal ancestors of humans lived. We sequenced the genomes of 69 males from nine populations, including two in which we find basal branches of the Y-chromosome tree. We identify ancient phylogenetic structure within African haplogroups and resolve a long-standing ambiguity deep within the tree. Applying equivalent methodologies to the Y chromosome and the mitochondrial genome, we estimate the time to the most recent common ancestor (T(MRCA)) of the Y chromosome to be 120 to 156 thousand years and the mitochondrial genome T(MRCA) to be 99 to 148 thousand years. Our findings suggest that, contrary to previous claims, male lineages do not coalesce significantly more recently than female lineages.

Methicillin-resistant Staphylococcus aureus (MRSA) has been endemic in Hong Kong for three decades. This study evaluated the practical use of high-resolution melting (HRM) real-time PCR analysis on MRSA staphylococcal Protein A (spa) typing on local MRSA isolates. Among 55 clinical MRSA isolates collected in 2011, 12 different spa types were observed by the conventional PCR-sequencing method including the locally predominant spa type t1081 and two locally predominant community acquired MRSA spa types t019 and t437. By using the HRM method, it could differentiate all 12 spa genotypes by distinct melting curves and HRM difference plot analysis. These two methods demonstrated 100% concordance whereas the HRM method required only 3h of turnaround time and one-fifth of reagent cost compared to the conventional method. Our study confirmed that the cost effective and rapid HRM typing approach is practically useful for MRSA community transmission monitoring and nosocomial outbreak control in Hong Kong., (Copyright © 2012 Elsevier B.V. All rights reserved.)

How organisms adapt to the range of environments they encounter is a fundamental question in biology. Elucidating the genetic basis of adaptation is a difficult task, especially when the targets of selection are not known. Emerging sequencing technologies and assembly algorithms facilitate the genomic dissection of adaptation and population differentiation in a vast array of organisms. Here we describe the attributes of Kryptolebias marmoratus, one of two known self-fertilizing hermaphroditic vertebrates that make this fish an attractive genetic system and a model for understanding the genomics of adaptation. Long periods of selfing have resulted in populations composed of many distinct naturally homozygous strains with a variety of identifiable, and apparently heritable, phenotypes. There also is strong population genetic structure across a diverse range of mangrove habitats, making this a tractable system in which to study differentiation both within and among populations. The ability to rear K. marmoratus in the laboratory contributes further to its value as a model for understanding the genetic drivers for adaptation. To date, microsatellite markers distinguish wild isogenic strains but the naturally high homozygosity improves the quality of de novo assembly of the genome and facilitates the identification of genetic variants associated with phenotypes. Gene annotation can be accomplished with RNA-sequencing data in combination with de novo genome assembly. By combining genomic information with extensive laboratory-based phenotyping, it becomes possible to map genetic variants underlying differences in behavioral, life-history, and other potentially adaptive traits. Emerging genomic technologies provide the required resources for establishing K. marmoratus as a new model organism for behavioral genetics and evolutionary genetics research.

Background: Elucidating the genomic basis of adaptation and speciation is a major challenge in natural systems with large quantities of environmental and phenotypic data, mostly because of the scarcity of genomic resources for non-model organisms. The Atlantic molly (Poecilia mexicana, Poeciliidae) is a small livebearing fish that has been extensively studied for evolutionary ecology research, particularly because this species has repeatedly colonized extreme environments in the form of caves and toxic hydrogen sulfide containing springs. In such extreme environments, populations show strong patterns of adaptive trait divergence and the emergence of reproductive isolation. Here, we used RNA-sequencing to assemble and annotate the first transcriptome of P. mexicana to facilitate ecological genomics studies in the future and aid the identification of genes underlying adaptation and speciation in the system., Description: We provide the first annotated reference transcriptome of P. mexicana. Our transcriptome shows high congruence with other published fish transcriptomes, including that of the guppy, medaka, zebrafish, and stickleback. Transcriptome annotation uncovered the presence of candidate genes relevant in the study of adaptation to extreme environments. We describe general and oxidative stress response genes as well as genes involved in pathways induced by hypoxia or involved in sulfide metabolism. To facilitate future comparative analyses, we also conducted quantitative comparisons between P. mexicana from different river drainages. 106,524 single nucleotide polymorphisms were detected in our dataset, including potential markers that are putatively fixed across drainages. Furthermore, specimens from different drainages exhibited some consistent differences in gene regulation., Conclusions: Our study provides a valuable genomic resource to study the molecular underpinnings of adaptation to extreme environments in replicated sulfide spring and cave environments. In addition, this study adds to the increasing number of genomic resources in the family Poeciliidae, which are widely used in comparative analyses of behavior, ecology, evolution, and medical genetics.

Naturally blond hair is rare in humans and found almost exclusively in Europe and Oceania. Here, we identify an arginine-to-cysteine change at a highly conserved residue in tyrosinase-related protein 1 (TYRP1) as a major determinant of blond hair in Solomon Islanders. This missense mutation is predicted to affect catalytic activity of TYRP1 and causes blond hair through a recessive mode of inheritance. The mutation is at a frequency of 26% in the Solomon Islands, is absent outside of Oceania, represents a strong common genetic effect on a complex human phenotype, and highlights the importance of examining genetic associations worldwide.

Postreplication repair (PRR) pathways play important roles in restarting stalled replication forks and regulating mutagenesis. In yeast, Rad5-mediated damage avoidance and Rad18-mediated translesion synthesis (TLS) are two forms of PRR. Two Rad5-related proteins, SHPRH and HLTF, have been identified in mammalian cells, but their specific roles in PRR are unclear. Here, we show that HLTF and SHPRH suppress mutagenesis in a damage-specific manner, preventing mutations induced by UV and MMS, respectively. Following UV, HLTF enhances PCNA monoubiquitination and recruitment of TLS polymerase η, while also inhibiting SHPRH function. In contrast, MMS promotes the degradation of HLTF and the interactions of SHPRH with Rad18 and polymerase κ. Our data suggest not only that cells differentially utilize HLTF and SHPRH for different forms of DNA damage, but also, surprisingly, that HLTF and SHPRH may coordinate the two main branches of PRR to choose the proper bypass mechanism for minimizing mutagenesis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Signaling pathways that respond to DNA damage are essential for the maintenance of genome stability and are linked to many diseases, including cancer. Here, a genome-wide siRNA screen was employed to identify additional genes involved in genome stabilization by monitoring phosphorylation of the histone variant H2AX, an early mark of DNA damage. We identified hundreds of genes whose downregulation led to elevated levels of H2AX phosphorylation (gammaH2AX) and revealed links to cellular complexes and to genes with unclassified functions. We demonstrate a widespread role for mRNA-processing factors in preventing DNA damage, which in some cases is caused by aberrant RNA-DNA structures. Furthermore, we connect increased gammaH2AX levels to the neurological disorder Charcot-Marie-Tooth (CMT) syndrome, and we find a role for several CMT proteins in the DNA-damage response. These data indicate that preservation of genome stability is mediated by a larger network of biological processes than previously appreciated.

Here, we demonstrate that primed, single-stranded DNA (ssDNA) is sufficient for activation of the ATR-dependent checkpoint pathway in Xenopus egg extracts. Using this structure, we define the contribution of the 5'- and 3'-primer ends to Chk1 activation when replication is blocked and ongoing. In addition, we show that although ssDNA is not sufficient for checkpoint activation, the amount of ssDNA adjacent to the primer influences the level of Chk1 phosphorylation. These observations define the minimal DNA requirements for checkpoint activation and suggest that primed ssDNA represents a common checkpoint activating-structure formed following many types of damage.

An essential component of the ATR (ataxia telangiectasia-mutated and Rad3-related)-activating structure is single-stranded DNA. It has been suggested that nucleotide excision repair (NER) can lead to activation of ATR by generating such a signal, and in yeast, DNA damage processing through the NER pathway is necessary for checkpoint activation during G1. We show here that ultraviolet (UV) radiation-induced ATR signaling is compromised in XPA-deficient human cells during S phase, as shown by defects in ATRIP (ATR-interacting protein) translocation to sites of UV damage, UV-induced phosphorylation of Chk1 and UV-induced replication protein A phosphorylation and chromatin binding. However, ATR signaling was not compromised in XPC-, CSB-, XPF- and XPG-deficient cells. These results indicate that damage processing is not necessary for ATR-mediated S-phase checkpoint activation and that the lesion recognition function of XPA may be sufficient. In contrast, XP-V cells deficient in the UV bypass polymerase eta exhibited enhanced ATR signaling. Taken together, these results suggest that lesion bypass and not lesion repair may raise the level of UV damage that can be tolerated before checkpoint activation, and that XPA plays a critical role in this activation.

Protein and lipid kinases are two important classes of biomedically relevant enzymes. The expression and activity of many kinases are known to be dysregulated in a variety of diseases, and proteomic tools that can assess the presence and activity of these enzymes are likely to be useful for their evaluation. Because many of the mechanisms by which protein kinases can become unregulated involve post-translational modifications or changes in protein localization, they can only be detected by examining protein activity, sometimes within the context of the living cell. Wortmannin is a steroid-derived fungal metabolite that covalently inhibits both protein and lipid kinases. Here we describe the synthesis of three wortmannin derivatives, biotin-wortmannin, BODIPY-wortmannin, and tetramethylrhodamine-wortmannin. We demonstrate that these reagents exhibit reactivity similarly as wortmannin and react with members of the phosphatidylinositol 3-kinase and PI3-kinase related kinase families in cellular lysates. Moreover, in some cases these reagents can differentiate between the active and inactive forms of the enzyme, indicating that they are activity-based probes. The reagents also exhibit complementary properties. The biotin-wortmannin reagent is effective in the isolation of labeled proteins; all three can be used for protein labeling, and BODIPY-wortmannin is cell-permeable and can be used to label proteins within cells.

The ATR-dependent DNA damage response pathway can respond to a diverse group of lesions as well as inhibitors of DNA replication. Using the Xenopus egg extract system, we show that lesions induced by UV irradiation and cis-platinum cause the functional uncoupling of MCM helicase and DNA polymerase activities, an event previously shown for aphidicolin. Inhibition of uncoupling during elongation with inhibitors of MCM7 or Cdc45, a putative helicase cofactor, results in abrogation of Chk1 phosphorylation, indicating that uncoupling is necessary for activation of the checkpoint. However, uncoupling is not sufficient for checkpoint activation, and DNA synthesis by Polalpha is also required. Finally, using plasmids of varying size, we demonstrate that all of the unwound DNA generated at a stalled replication fork can contribute to the level of Chk1 phosphorylation, suggesting that uncoupling amplifies checkpoint signaling at each individual replication fork. Taken together, these observations indicate that functional uncoupling of MCM helicase and DNA polymerase activities occurs in response to multiple forms of DNA damage and that there is a general mechanism for generation of the checkpoint-activating signal following DNA damage.

The function of the ATR (ataxia-telangiectasia mutated and Rad3-related)-ATRIP (ATR-interacting protein) protein kinase complex is central to the cellular response to replication stress and DNA damage. In order to better understand the function of this complex, we have studied its interaction with DNA. We find that both ATR and ATRIP associate with chromatin in vivo, and they exist as a large molecular weight complex that can bind single-stranded (ss)DNA cellulose in vitro. Although replication protein A (RPA) is sufficient for the recruitment of ATRIP to ssDNA, we show that a distinct ATR-ATRIP complex is able to bind to DNA with lower affinity in the absence of RPA. In this latter complex, we show that neither ATR nor ATRIP are able to bind DNA individually, nor do they bind DNA in a cooperative manner. However, the addition of HeLa nuclear extract is able to reconstitute the DNA binding of both ATR and ATRIP, suggesting the requirement for an additional protein activity. We also show that ATR is necessary for ATRIP to bind DNA in this low affinity mode and to form a large DNA binding complex. These observations suggest that there are at least two in vitro ATR-ATRIP DNA binding complexes, one which binds DNA with high affinity in an RPA-dependent manner and a second, which binds DNA with lower affinity in an RPA-independent manner but which requires an as of yet unidentified protein.

Using the Xenopus egg extract system, we investigated the involvement of DNA replication in activation of the DNA damage checkpoint. We show here that DNA damage slows replication in a checkpoint-independent manner and is accompanied by replication-dependent recruitment of ATR and Rad1 to chromatin. We also find that the replication proteins RPA and Polalpha accumulate on chromatin following DNA damage. Finally, damage-induced Chk1 phosphorylation and checkpoint arrest are abrogated when replication is inhibited. These data indicate that replication is required for activation of the DNA damage checkpoint and suggest a unifying model for ATR activation by diverse lesions during S phase.

The structure of anthranilate phosphoribosyltransferase from the enterobacterium Pectobacterium carotovorum has been solved at 2.4 A in complex with Mn(2+)-pyrophosphate, and at 1.9 A without ligands. The enzyme structure has a novel phosphoribosyltransferase (PRT) fold and displays close homology to the structures of pyrimidine nucleoside phosphorylases. The enzyme is a homodimer with a monomer of 345 residues. Each monomer consists of two subdomains, alpha and alpha/beta, which form a cleft containing the active site. The nature of the active site is inferred from the trapped MnPPi complex and detailed knowledge of the active sites of nucleoside phosphorylases. With the anthranilate (An)PRT structure solved, the structures of all the enzymes required for tryptophan biosynthesis are now known.

The crystal structure of anthranilate synthase (AS) from Serratia marcescens, a mesophilic bacterium, has been solved in the presence of its substrates, chorismate and glutamine, and one product, glutamate, at 1.95 A, and with its bound feedback inhibitor, tryptophan, at 2.4 A. In comparison with the AS structure from the hyperthermophile Sulfolobus solfataricus, the S. marcescens structure shows similar subunit structures but a markedly different oligomeric organization. One crystal form of the S. marcescens enzyme displays a bound pyruvate as well as a putative anthranilate (the nitrogen group is ambiguous) in the TrpE subunit. It also confirms the presence of a covalently bound glutamyl thioester intermediate in the TrpG subunit. The tryptophan-bound form reveals that the inhibitor binds at a site distinct from that of the substrate, chorismate. Bound tryptophan appears to prevent chorismate binding by a demonstrable conformational effect, and the structure reveals how occupancy of only one of the two feedback inhibition sites can immobilize the catalytic activity of both TrpE subunits. The presence of effectors in the structure provides a view of the locations of some of the amino acid residues in the active sites. Our findings are discussed in terms of the previously described AS structure of S. solfataricus, mutational data obtained from enteric bacteria, and the enzyme's mechanism of action.

Background: The DNA replication checkpoint ensures that mitosis is not initiated before DNA synthesis is completed. Recent studies using Xenopus extracts have demonstrated that activation of the replication checkpoint and phosphorylation of the Chk1 kinase are dependent on RNA primer synthesis by DNA polymerase alpha, and it has been suggested that the ATR kinase-so-called because it is related to the product of the gene that is mutated in ataxia telangiectasia (ATM) and to Rad3 kinase-may be an upstream component of this response. It has been difficult to test this hypothesis as an ATR-deficient system suitable for biochemical studies has not been available., Results: We have cloned the Xenopus laevis homolog of ATR (XATR) and studied the function of the protein in Xenopus egg extracts. Using a chromatin-binding assay, we found that ATR associates with chromatin after initiation of replication, dissociates from chromatin upon completion of replication, and accumulates in the presence of aphidicolin, an inhibitor of DNA replication. Its association with chromatin was inhibited by treatment with actinomycin D, an inhibitor of RNA primase. There was an early rise in the activity of Cdc2-cyclin B in egg extracts depleted of ATR both in the presence or absence of aphidicolin. In addition, the premature mitosis observed upon depletion of ATR was accompanied by the loss of Chk1 phosphorylation., Conclusions: ATR is a replication-dependent chromatin-binding protein, and its association with chromatin is dependent on RNA synthesis by DNA polymerase alpha. Depletion of ATR leads to premature mitosis in the presence and absence of aphidicolin, indicating that ATR is required for the DNA replication checkpoint.

Enzymes from thermophilic organisms often are barely active at low temperatures. To obtain a better understanding of this sluggishness, we used DNA shuffling to mutagenize the trpC gene, which encodes indoleglycerol phosphate synthase, from the hyperthermophile Sulfolobus solfataricus. Mutants producing more active protein variants were selected by genetic complementation of an Escherichia coli mutant bearing a trpC deletion. Single amino acid changes and combinations of these changes improved growth appreciably. Five singly and doubly altered protein variants with changes at the N- and C-termini, or at the phosphate binding site, were purified and characterized with regard to their kinetics of enzymatic catalysis, product binding, cleavage by trypsin, and inactivation by heat. Turnover numbers of the purified variant proteins correlated with the corresponding growth rates, showing that the turnover number was the selected trait. Although the affinities for both the substrate and the product decreased appreciably in most protein variants, these defects were offset by the accumulation of high levels of the enzyme's substrate. Rapid mixing of the product indoleglycerol phosphate with the parental enzyme revealed that the enzyme's turnover number at low temperatures is limited by the dissociation of the enzyme-product complex. In contrast, representative protein variants bind and release the product far more rapidly, shifting the bottleneck to the preceding chemical step. The turnover number of the parental enzyme increases with temperature, suggesting that its structural rigidity is responsible for its poor catalytic activity at low temperatures. In support of this interpretation, the rate of trypsinolysis or of thermal denaturation is accelerated significantly in the activated protein variants.

The role of helix 0 of the alpha chain (TrpA) of the tryptophan synthetase alpha2beta2 multi-functional enzyme complex of Escherichia coli was examined by deleting amino-terminal residues 2-6, 2-11, or 2-19 of TrpA. Selected substitutions were also introduced at TrpA positions 2-6. The altered genes encoding these polypeptides were overexpressed from a foreign promoter on a multicopy plasmid and following insertion at their normal chromosomal location. Each deletion polypeptide was functional in vivo. However all appeared to be somewhat more labile and insoluble and less active enzymatically than wild type TrpA. The deletion polypeptides were overproduced and solubilized from cell debris by denaturation and refolding. Several were partially purified and assayed in various reactions in the presence of tryptophan synthetase beta2 (TrpB). The purified TrpADelta2-6 and TrpADelta2-11 deletion polypeptides had low activity in both the indole + serine --> tryptophan reaction and the indoleglycerol phosphate + serine --> tryptophan reaction. Poor activity in each reaction was partly due to reduced association of TrpA with TrpB. The addition of the TrpA ligands, alpha-glycerophosphate or indoleglycerol phosphate, during catalysis of the indole + serine --> tryptophan reaction increased association and activity. These findings suggest that removal of helix 0 of TrpA decreases TrpA-TrpB association as well as the activity of the TrpA active site. Alignment of the TrpA sequences from different species indicates that several lack part or all of helix 0. In some of these polypeptides, extra residues at the carboxyl end may substitute for helix 0.