Polo-like kinase 4 maintains centriolar satellite integrity by phosphorylation of centrosomal protein 131 (CEP131).

The centrosome, consisting of two centrioles surrounded by a dense network of proteins, is the microtubule-organizing center of animal cells. Polo-like kinase 4 (PLK4) is a Ser/Thr protein kinase and the master regulator of centriole duplication, but it may play additional roles in centrosome function. To identify additional proteins regulated by PLK4, we generated an RPE-1 human cell line with a genetically engineered "analog-sensitive" PLK4AS, which genetically encodes chemical sensitivity to competitive inhibition via a bulky ATP analog. We used this transgenic line in an unbiased multiplex phosphoproteomic screen. Several hits were identified and validated as direct PLK4 substrates by in vitro kinase assays. Among them, we confirmed Ser-78 in centrosomal protein 131 (CEP131, also known as AZI1) as a direct substrate of PLK4. Using immunofluorescence microscopy, we observed that although PLK4-mediated phosphorylation of Ser-78 is dispensable for CEP131 localization, ciliogenesis, and centriole duplication, it is essential for maintaining the integrity of centriolar satellites. We also found that PLK4 inhibition or use of a nonphosphorylatable CEP131 variant results in dispersed centriolar satellites. Moreover, replacement of endogenous WT CEP131 with an S78D phosphomimetic variant promoted aggregation of centriolar satellites. We conclude that PLK4 phosphorylates CEP131 at Ser-78 to maintain centriolar satellite integrity.

Anillin Phosphorylation Controls Timely Membrane Association and Successful Cytokinesis.

During cytokinesis, a contractile ring generates the constricting force to divide a cell into two daughters. This ring is composed of filamentous actin and the motor protein myosin, along with additional structural and regulatory proteins, including anillin. Anillin is a required scaffold protein that links the actomyosin ring to membrane and its organizer, RhoA. However, the molecular basis for timely action of anillin at cytokinesis remains obscure. Here, we find that phosphorylation regulates efficient recruitment of human anillin to the equatorial membrane. Anillin is highly phosphorylated in mitosis, and is a substrate for mitotic kinases. We surveyed function of 46 residues on anillin previously found to be phosphorylated in human cells to identify those required for cytokinesis. Among these sites, we identified S635 as a key site mediating cytokinesis. Preventing S635 phosphorylation adjacent to the AH domain disrupts anillin concentration at the equatorial cortex at anaphase, whereas a phosphomimetic mutant, S635D, partially restores this localization. Time-lapse videomicroscopy reveals impaired recruitment of S635A anillin to equatorial membrane and a transient unstable furrow followed by ultimate failure in cytokinesis. A phosphospecific antibody confirms phosphorylation at S635 in late cytokinesis, although it does not detect phosphorylation in early cytokinesis, possibly due to adjacent Y634 phosphorylation. Together, these findings reveal that anillin recruitment to the equatorial cortex at anaphase onset is enhanced by phosphorylation and promotes successful cytokinesis.

Decoding Polo-like kinase 1 signaling along the kinetochore-centromere axis.

Protein kinase signaling along the kinetochore-centromere axis is crucial to assure mitotic fidelity, yet the details of its spatial coordination are obscure. Here, we examined how pools of human Polo-like kinase 1 (Plk1) within this axis control signaling events to elicit mitotic functions. To do this, we restricted active Plk1 to discrete subcompartments within the kinetochore-centromere axis using chemical genetics and decoded functional and phosphoproteomic signatures of each. We observe distinct phosphoproteomic and functional roles, suggesting that Plk1 exists and functions in discrete pools along this axis. Deep within the centromere, Plk1 operates to assure proper chromosome alignment and segregation. Thus, Plk1 at the kinetochore is a conglomerate of an observable bulk pool coupled with additional functional pools below the threshold of microscopic detection or resolution. Although complex, this multiplicity of locales provides an opportunity to decouple functional and phosphoproteomic signatures for a comprehensive understanding of Plk1's kinetochore functions.

Insights into the maize pan-genome and pan-transcriptome.

Genomes at the species level are dynamic, with genes present in every individual (core) and genes in a subset of individuals (dispensable) that collectively constitute the pan-genome. Using transcriptome sequencing of seedling RNA from 503 maize (Zea mays) inbred lines to characterize the maize pan-genome, we identified 8681 representative transcript assemblies (RTAs) with 16.4% expressed in all lines and 82.7% expressed in subsets of the lines. Interestingly, with linkage disequilibrium mapping, 76.7% of the RTAs with at least one single nucleotide polymorphism (SNP) could be mapped to a single genetic position, distributed primarily throughout the nonpericentromeric portion of the genome. Stepwise iterative clustering of RTAs suggests, within the context of the genotypes used in this study, that the maize genome is restricted and further sampling of seedling RNA within this germplasm base will result in minimal discovery. Genome-wide association studies based on SNPs and transcript abundance in the pan-genome revealed loci associated with the timing of the juvenile-to-adult vegetative and vegetative-to-reproductive developmental transitions, two traits important for fitness and adaptation. This study revealed the dynamic nature of the maize pan-genome and demonstrated that a substantial portion of variation may lie outside the single reference genome for a species.

Strictly conserved lysine of prolyl-tRNA Synthetase editing domain facilitates binding and positioning of misacylated tRNA(Pro.).

To ensure high fidelity in translation, many aminoacyl-tRNA synthetases, enzymes responsible for attaching specific amino acids to cognate tRNAs, require proof-reading mechanisms. Most bacterial prolyl-tRNA synthetases (ProRSs) misactivate alanine and employ a post-transfer editing mechanism to hydrolyze Ala-tRNA(Pro). This reaction occurs in a second catalytic site (INS) that is distinct from the synthetic active site. The 2'-OH of misacylated tRNA(Pro) and several conserved residues in the Escherichia coli ProRS INS domain are directly involved in Ala-tRNA(Pro) deacylation. Although mutation of the strictly conserved lysine 279 (K279) results in nearly complete loss of post-transfer editing activity, this residue does not directly participate in Ala-tRNA(Pro) hydrolysis. We hypothesized that the role of K279 is to bind the phosphate backbone of the acceptor stem of misacylated tRNA(Pro) and position it in the editing active site. To test this hypothesis, we carried out pKa, charge neutralization, and free-energy of binding calculations. Site-directed mutagenesis and kinetic studies were performed to verify the computational results. The calculations revealed a considerably higher pKa of K279 compared to an isolated lysine and showed that the protonated state of K279 is stabilized by the neighboring acidic residue. However, substitution of this acidic residue with a positively charged residue leads to a significant increase in Ala-tRNA(Pro) hydrolysis, suggesting that enhancement in positive charge density in the vicinity of K279 favors tRNA binding. A charge-swapping experiment and free energy of binding calculations support the conclusion that the positive charge at position 279 is absolutely necessary for tRNA binding in the editing active site.

QTL mapping and phenotypic variation for root architectural traits in maize (Zea mays L.).

KEY MESSAGE: QTL were identified for root architectural traits in maize. Root architectural traits, including the number, length, orientation, and branching of the principal root classes, influence plant function by determining the spatial and temporal domains of soil exploration. To characterize phenotypic patterns and their genetic control, three recombinant inbred populations of maize were grown for 28 days in solid media in a greenhouse and evaluated for 21 root architectural traits, including length, number, diameter, and branching of seminal, primary and nodal roots, dry weight of embryonic and nodal systems, and diameter of the nodal root system. Significant phenotypic variation was observed for all traits. Strong correlations were observed among traits in the same root class, particularly for the length of the main root axis and the length of lateral roots. In a principal component analysis, relationships among traits differed slightly for the three families, though vectors grouped together for traits within a given root class, indicating opportunities for more efficient phenotyping. Allometric analysis showed that trajectories of growth for specific traits differ in the three populations. In total, 15 quantitative trait loci (QTL) were identified. QTL are reported for length in multiple root classes, diameter and number of seminal roots, and dry weight of the embryonic and nodal root systems. Phenotypic variation explained by individual QTL ranged from 0.44% (number of seminal roots, NyH population) to 13.5% (shoot dry weight, OhW population). Identification of QTL for root architectural traits may be useful for developing genotypes that are better suited to specific soil environments.

Multiple pathways promote dynamical coupling between catalytic domains in Escherichia coli prolyl-tRNA synthetase.

Aminoacyl-tRNA synthetases are multidomain enzymes that catalyze covalent attachment of amino acids to their cognate tRNA. Cross-talk between functional domains is a prerequisite for this process. In this study, we investigate the molecular mechanism of site-to-site communication in Escherichia coli prolyl-tRNA synthetase (Ec ProRS). Earlier studies have demonstrated that evolutionarily conserved and/or co-evolved residues that are engaged in correlated motion are critical for the propagation of functional conformational changes from one site to another in modular proteins. Here, molecular simulation and bioinformatics-based analysis were performed to identify dynamically coupled and evolutionarily constrained residues that form contiguous pathways of residue-residue interactions between the aminoacylation and editing domains of Ec ProRS. The results of this study suggest that multiple pathways exist between these two domains to maintain the dynamic coupling essential for enzyme function. Moreover, residues in these interaction networks are generally highly conserved. Site-directed changes of on-pathway residues have a significant impact on enzyme function and dynamics, suggesting that any perturbation along these pathways disrupts the native residue-residue interactions that are required for effective communication between the two functional domains. Free energy analysis revealed that communication between residues within a pathway and cross-talk between pathways are important for coordinating functions of different domains of Ec ProRS for efficient catalysis.

Why Your Doctor Didn't Go to Class: Student Culture, High-Stakes Testing, and Novel Coupling Configurations in an Allopathic Medical School.

A clear pattern has emerged in allopathic medical schools across the United States: Most medical students have stopped going to class. While this trend among students is well known in medical education, few studies to date have examined the underlying sociological mechanisms driving this collective behavior or how these dynamics are related to institutional change in medical education. Drawing on 33 in-depth interviews with medical students in an allopathic medical school, we examine medical student culture and its role in shaping how medical students make sense of the institutionalized licensing requirement of the United States Medical Licensing Exam. We find that medical students learn to rely on digital recordings of their course content and third-party digital resources for Step 1 prep and stop attending their academic courses in person altogether. We argue that medical students create novel coupling configurations between local interaction and institutionalized licensure rules via their student cultures.

Role of coupled dynamics in the catalytic activity of prokaryotic-like prolyl-tRNA synthetases.

Prolyl-tRNA synthetases (ProRSs) have been shown to activate both cognate and some noncognate amino acids and attach them to specific tRNA(Pro) substrates. For example, alanine, which is smaller than cognate proline, is misactivated by Escherichia coli ProRS. Mischarged Ala-tRNA(Pro) is hydrolyzed by an editing domain (INS) that is distinct from the activation domain. It was previously shown that deletion of the INS greatly reduced cognate proline activation efficiency. In this study, experimental and computational approaches were used to test the hypothesis that deletion of the INS alters the internal protein dynamics leading to reduced catalytic function. Kinetic studies with two ProRS variants, G217A and E218A, revealed decreased amino acid activation efficiency. Molecular dynamics studies showed motional coupling between the INS and protein segments containing the catalytically important proline-binding loop (PBL, residues 199-206). In particular, the complete deletion of INS, as well as mutation of G217 or E218 to alanine, exhibited significant effects on the motion of the PBL. The presence of coupled dynamics between neighboring protein segments was also observed through in silico mutations and essential dynamics analysis. Altogether, this study demonstrates that structural elements at the editing domain-activation domain interface participate in coupled motions that facilitate amino acid binding and catalysis by bacterial ProRSs, which may explain why truncated or defunct editing domains have been maintained in some systems, despite the lack of catalytic activity.

A Genetic Toggle for Chemical Control of Individual Plk1 Substrates.

Polo-like kinase 1 has hundreds of substrates and multiple functions that operate within the ∼60 min of mitosis. Herein, we describe a chemical-genetic system that allows particular substrates to be "toggled" into or out of chemical control using engineered phosphoacceptor selectivity. Biochemical assays and phosphoproteomic analysis of mitotic cell extracts showed that Plk1S (L197F) and Plk1T (L197S/L211A) selectively phosphorylate Ser and Thr, respectively. Plk1S but not Plk1T sustains mitotic progression to anaphase, affording the opportunity to toggle substrate residues between Ser and Thr to place them under chemical control. Using this system, we evaluated Kif2b, a known substrate of Plk1 that regulates chromosome alignment. Toggling Ser to Thr on Kif2b places these phosphorylation sites under reversible chemical control, as indicated by a sharp increase in the frequency of misaligned chromosomes and prometaphase arrest. Thus, we demonstrate the ability to chemically control a single substrate by a genetic Ser/Thr toggle.

Marker density and read depth for genotyping populations using genotyping-by-sequencing.

Genotyping-by-sequencing (GBS) approaches provide low-cost, high-density genotype information. However, GBS has unique technical considerations, including a substantial amount of missing data and a nonuniform distribution of sequence reads. The goal of this study was to characterize technical variation using this method and to develop methods to optimize read depth to obtain desired marker coverage. To empirically assess the distribution of fragments produced using GBS, ∼8.69 Gb of GBS data were generated on the Zea mays reference inbred B73, utilizing ApeKI for genome reduction and single-end reads between 75 and 81 bp in length. We observed wide variation in sequence coverage across sites. Approximately 76% of potentially observable cut site-adjacent sequence fragments had no sequencing reads whereas a portion had substantially greater read depth than expected, up to 2369 times the expected mean. The methods described in this article facilitate determination of sequencing depth in the context of empirically defined read depth to achieve desired marker density for genetic mapping studies.

The costs and adequacy of safety net access for the uninsured in Genesee County, Michigan.

This commentary examines Genesee County (Flint) Michigan to explore whether a well-structured safety net system is able to provide low-income uninsured people adequate access to care at a reasonable cost. Genessee County is one of the more economically challenged communities in the country. This commentary explores the cost and adequacy of safety net care in Genessee County under the Genessee Health Plan (GHP). The analysis compares the cost of services under GHP to the cost of the same services offered by local private insurers and Medicaid. An analysis found that GHP, Flint-area physicians, hospitals, and foundations have succeeded in providing basic medical care access to a substantial majority of their low-income uninsured citizens. The costs of care, both paid by GHP and donated by local providers, are substantially less than the estimated costs if this population were covered by full Medicaid or private insurance.

A high-throughput core sampling device for the evaluation of maize stalk composition.

BACKGROUND: A major challenge in the identification and development of superior feedstocks for the production of second generation biofuels is the rapid assessment of biomass composition in a large number of samples. Currently, highly accurate and precise robotic analysis systems are available for the evaluation of biomass composition, on a large number of samples, with a variety of pretreatments. However, the lack of an inexpensive and high-throughput process for large scale sampling of biomass resources is still an important limiting factor. Our goal was to develop a simple mechanical maize stalk core sampling device that can be utilized to collect uniform samples of a dimension compatible with robotic processing and analysis, while allowing the collection of hundreds to thousands of samples per day. RESULTS: We have developed a core sampling device (CSD) to collect maize stalk samples compatible with robotic processing and analysis. The CSD facilitates the collection of thousands of uniform tissue cores consistent with high-throughput analysis required for breeding, genetics, and production studies. With a single CSD operated by one person with minimal training, more than 1,000 biomass samples were obtained in an eight-hour period. One of the main advantages of using cores is the high level of homogeneity of the samples obtained and the minimal opportunity for sample contamination. In addition, the samples obtained with the CSD can be placed directly into a bath of ice, dry ice, or liquid nitrogen maintaining the composition of the biomass sample for relatively long periods of time. CONCLUSIONS: The CSD has been demonstrated to successfully produce homogeneous stalk core samples in a repeatable manner with a throughput substantially superior to the currently available sampling methods. Given the variety of maize developmental stages and the diversity of stalk diameter evaluated, it is expected that the CSD will have utility for other bioenergy crops as well.

Spectral and copper binding properties of methanobactin from the facultative methanotroph Methylocystis strain SB2.

Methanobactin (mb) is the first characterized example of a chalkophore, a class of copper-binding chromopeptides similar to iron-binding siderophores. Structural, redox, themodynamic, and spectral studies on chalkophores have focused almost exclusively on the mb from Methylosinus trichosporium OB3b (mb-OB3b). The structural characterization of a second mb from Methylocystis strain SB2 (mb-SB2) provides a means to examine the core structural features and metal binding properties of this group of chromopeptides. With the exception of the 5-membered rings (either oxazolone or imidazolone), enethiol groups, and the N-terminus oxo group, the structure of mb-SB2 differs markedly from mb-OB3b. In particular the amino acids commonly associated with metal coordination and redox activity found in mb-OB3b, Cys, Met, and Try, are replaced by Ala or are missing in mb-SB2. In this report the spectral and thermodynamic properties of mb-SB2 are presented and compared to mb-OB3b. The results demonstrate that the spectral and basic copper binding properties of both methanobactins are similar and the unique copper binding capacity of both methanobactins lies primarily in the pair of five-membered rings and associated enethiol groups. The remaining portions of the methanobactin appear to provide the scaffolding that brings together of the two ring systems to produce the tetrahedral binding site for copper binding.

Tandem optimization of target activity and elimination of mutagenic potential in a potent series of N-aryl bicyclic hydantoin-based selective androgen receptor modulators.

Pharmacokinetic studies in cynomolgus monkeys with a novel prototype selective androgen receptor modulator revealed trace amounts of an aniline fragment released through hydrolytic metabolism. This aniline fragment was determined to be mutagenic in an Ames assay. Subsequent concurrent optimization for target activity and avoidance of mutagenicity led to the identification of a pharmacologically superior clinical candidate without mutagenic potential.

Is placenta previa more common in multiple gestations?

OBJECTIVE: This study was undertaken to compare the occurrence of placenta previa at delivery between singleton and multiple gestations. STUDY DESIGN: A historical cohort study was performed of placenta previa cases at Good Samaritan Regional Medical Center, Phoenix, Ariz, from January 1, 1997, to December 31, 2000. RESULTS: During the study, 29,268 deliveries (28,372 singleton and 896 multiple gestations) occurred at Good Samaritan Regional Medical Center. Placenta previa complicated 51 (0.18%) singleton gestations and 4 (0.46%) multiple gestations (P =.09). Singleton and multiple gestations with placenta previa were similar with respect to maternal demographic variables, prior uterine surgery (excluding cesarean section), and gestational age at delivery. Multiple gestations with placenta previa were more likely to have had a prior cesarean section (P <.001). There were no differences between the singleton and multiple gestations with placenta previa and need for cesarean hysterectomy and blood transfusion. CONCLUSION: The occurrence and complications of placenta previa do not differ between singleton and multiple gestations.