OptiFit: an Improved Method for Fitting Amplicon Sequences to Existing OTUs.

Assigning amplicon sequences to operational taxonomic units (OTUs) is an important step in characterizing microbial communities across large data sets. A notable difference between de novo clustering and database-dependent reference clustering methods is that OTU assignments from de novo methods may change when new sequences are added. However, one may wish to incorporate new samples to previously clustered data sets without clustering all sequences again, such as when comparing across data sets or deploying machine learning models. Existing reference-based methods produce consistent OTUs but only consider the similarity of each query sequence to a single reference sequence in an OTU, resulting in assignments that are worse than those generated by de novo methods. To provide an efficient method to fit sequences to existing OTUs, we developed the OptiFit algorithm. Inspired by the de novo OptiClust algorithm, OptiFit considers the similarity of all pairs of reference and query sequences to produce OTUs of the best possible quality. We tested OptiFit using four data sets with two strategies: (i) clustering to a reference database and (ii) splitting the data set into a reference and query set, clustering the references using OptiClust, and then clustering the queries to the references. The result is an improved implementation of reference-based clustering. OptiFit produces OTUs of a quality similar to that of OptiClust at faster speeds when using the split data set strategy. OptiFit provides a suitable option for users requiring consistent OTU assignments at the same quality as afforded by de novo clustering methods. IMPORTANCE Advancements in DNA sequencing technology have allowed researchers to affordably generate millions of sequence reads from microorganisms in diverse environments. Efficient and robust software tools are needed to assign microbial sequences into taxonomic groups for characterization and comparison of communities. The OptiClust algorithm produces high-quality groups by comparing sequences to each other, but the assignments can change when new sequences are added to a data set, making it difficult to compare different studies. Other approaches assign sequences to groups by comparing them to sequences in a reference database to produce consistent assignments, but the quality of the groups produced is reduced compared to that with OptiClust. We developed OptiFit, a new reference-based algorithm that produces consistent yet high-quality assignments like OptiClust. OptiFit allows researchers to compare microbial communities across different studies or add new data to existing studies without sacrificing the quality of the group assignments.

"Cool Knees" as a Measure of Systemic Vascular Resistance in Cardiac Patients.

INTRODUCTION: Clinical assessment of cardiac output (CO) and systemic vascular resistance (SVR) in cardiac patients is often inaccurate. Since the genicular arteries form a watershed zone accessible to physical examination, we hypothesized that "cool knees" would reflect abnormalities in central hemodynamics. METHODS: Nineteen patients with cardiac diagnoses, but without distributive shock, had a measurement of skin temperature over the thigh, knee, and foot in parallel with central hemodynamics derived from invasive monitoring. RESULTS: The temperature gradient from thigh to knee (DTK) reflected increased SVR, and was significantly correlated with SVR, cardiac index (CI), and CO. Cool feet (DTF) were significantly correlated only with systemic hypotension, but not central hemodynamics. CONCLUSION: Cool knees reflect increased SVR in cardiac patients and may be an important physical exam finding in their assessment and management.

A novel clock-face method for characterizing peridevice leaks after left atrial appendage occlusion.

OBJECTIVES: To propose a novel method for mapping leak location and frequency to a clock-face representation of the left atrial appendage (LAA) ostium. BACKGROUND: LAA occlusion with the Watchman device (WD) is an established therapy to reduce thromboembolic events in patients with atrial fibrillation (AF) and intolerance to long-term oral anticoagulation. Postimplantation leaks are known sequelae, but leak locations and characteristics are poorly described. METHODS: We retrospectively reviewed 101 consecutive WD implants from April 2015 to February 2018. Leak locations from 6-week post-implant transesophageal echocardiograms were mapped to a clock-face representation of the LAA ostium: 12:00 as cranial near the limbus, 3:00 as anterior toward the pulmonary artery, 6:00 as caudal near the mitral annulus, and 9:00 as posterior. Patient demographics, LAA dimensions, and procedural characteristics were also collected. RESULTS: Thirty-four patients had ≥1 leak totaling 45 leaks at 6-week follow-up. Baseline patient demographics showed a mean age 77, CHA2 DS2 VASc 4.69, and 64% of patients with permanent AF. No patient had a detectable leak at the time of implant. At 6 weeks, mean leak size was 2.67 ± 0.89 mm with no leak over 5 mm (largest 4.60 mm). Most leaks occurred along the posterior 6:00-12:00 segment (39/45) and the 6:00-9:00 quadrant (16/45). CONCLUSION: Six-week post-WD implant leaks localize to the posterior LAA ostium. This could result from the elliptical LAA orifice, differential LAA tissue composition, or implantation technique. This study provides a novel method for describing the location of post-implant leaks and serves as the basis for further investigations.

Watchman Occlusion in Long-Standing Persistent Atrial Fibrillation: Larger Left Atrial Appendages With Greater Residual Leak.

OBJECTIVES: This study sought to compare patients with and without long-standing persistent atrial fibrillation (LSPAF) undergoing Watchman left atrial appendage (LAA) occlusion. BACKGROUND: An increased burden of atrial fibrillation is associated with progressive left atrial remodeling and enlargement. METHODS: Transesophageal echocardiography (TEE) measures of LAA ostial diameter and depth, device compression, and residual leak were evaluated in 101 consecutive Watchman cases. The patients were categorized into LSPAF (n = 48) or non-LSPAF (n = 53) groups and compared. RESULTS: The average LAA ostial diameter for LSPAF versus non-LSPAF by TEE omniplane at 0° was 21.1 ± 4.1 mm versus 18.2 ± 3.6 mm (p = 0.0002); at 45° was 18.7 ± 3.4 mm versus 16.3 ± 3.1 mm (p = 0.0004); at 90° was 19.6 ± 3.8 mm versus 16.2 ± 3.4 mm (p = 0.00001); and at 135° was 21.0 ± 4.1 mm versus 18.0 ± 4.1 mm (p = 0.0005). The average LAA depth for LSPAF versus non-LSPAF by TEE at 0° was 28.1 ± 6.4 mm versus 25.2 ± 4.9 mm (p = 0.02); at 45° was 27.9 ± 5.8 mm versus 25.1 ± 4.3 mm (p = 0.007); at 90° was 27.2 ± 5.2 mm versus 22.8 ± 3.7 mm (p = 0.0001); and at 135° was 25.6 ± 5.4 mm versus 21.5 ± 3.8 mm (p = 0.0001). In successfully treated patients, 77% of the LSPAF group received larger device (27, 30, or 33 mm) implants versus only 46% in the non-LSPAF group (p = 0.003). While both groups had similar rates of moderate (3 to 5 mm) leaks at implant (2% vs. 0%; p = 0.14), 27% of the LSPAF vs. 4% of the non-LSPAF group had moderate leaks (p = 0.04) on 6-week follow-up TEE. CONCLUSIONS: Patients with LSPAF have significantly larger LAA sizes, require larger devices, and have more residual leak on follow-up TEE. LSPAF may represent a higher risk group that warrants more stringent long-term follow-up.

Relationship Between Psychosocial Stressors and Atrial Fibrillation in Women >45 Years of Age.

Negative emotions have been linked to the development of atrial fibrillation (AF), and positive effect may be protective. However, there are few large-scale studies examining the association between psychosocial stressors that may provoke these emotions and the occurrence of AF. We examined the cross-sectional relation between psychosocial stress and AF in 24,809 women participating in the Women's Health Study. Participants answered questions about work stress (e.g., excessive work, conflicting demands), work-family spillover stress (e.g., too stressed after work to participate in activities with family), financial stress (e.g., difficulty paying monthly bills), traumatic life events (e.g., death of a child), everyday discrimination (e.g., less respect, poor service), intimate partner stress (e.g., how judgmental is your spouse/partner), neighborhood stress (e.g., neighborhood safety, trust), negative life events within 5 years (e.g., life threatening illness, legal problems), and cumulative stress (a weighted measure of the stress domains). The prevalence of confirmed AF was 3.84% (N = 953) and risk factor profiles differed by AF status. Women with AF reported significantly higher financial stress, traumatic life events, and neighborhood stress (peach < 0.05). Only traumatic life events (odds ratio 1.37, 95% confidence interval 1.19 to 1.59) was significantly associated with AF after adjustment for cardiovascular risk factors, socioeconomic and psychosocial status. These large-scale cross-sectional data thus indicate a potential relationship between traumatic life events and AF in older women.

Prospective comparison of long-term pain relief rates after first-time microvascular decompression and stereotactic radiosurgery for trigeminal neuralgia.

OBJECTIVE Common surgical treatments for trigeminal neuralgia (TN) include microvascular decompression (MVD), stereotactic radiosurgery (SRS), and radiofrequency ablation (RFA). Although the efficacy of each procedure has been described, few studies have directly compared these treatment modalities on pain control for TN. Using a large prospective longitudinal database, the authors aimed to 1) directly compare long-term pain control rates for first-time surgical treatments for idiopathic TN, and 2) identify predictors of pain control. METHODS The authors reviewed a prospectively collected database for all patients who underwent treatment for TN between 1997 and 2014 at the University of California, San Francisco. Standardized collection of data on preoperative clinical characteristics, surgical procedure, and postoperative outcomes was performed. Data analyses were limited to those patients who received a first-time procedure for treatment of idiopathic TN with > 1 year of follow-up. RESULTS Of 764 surgical procedures performed at the University of California, San Francisco, for TN (364 SRS, 316 MVD, and 84 RFA), 340 patients underwent first-time treatment for idiopathic TN (164 MVD, 168 SRS, and 8 RFA) and had > 1 year of follow-up. The analysis was restricted to patients who underwent MVD or SRS. Patients who received MVD were younger than those who underwent SRS (median age 63 vs 72 years, respectively; p < 0.001). The mean follow-up was 59 ± 35 months for MVD and 59 ± 45 months for SRS. Approximately 38% of patients who underwent MVD or SRS had > 5 years of follow-up (60 of 164 and 64 of 168 patients, respectively). Immediate or short-term (< 3 months) postoperative pain-free rates (Barrow Neurological Institute Pain Intensity score of I) were 96% for MVD and 75% for SRS. Percentages of patients with Barrow Neurological Institute Pain Intensity score of I at 1, 5, and 10 years after MVD were 83%, 61%, and 44%, and the corresponding percentages after SRS were 71%, 47%, and 27%, respectively. The median time to pain recurrence was 94 months (25th-75th quartiles: 57-131 months) for MVD and 53 months (25th-75th quartiles: 37-69 months) for SRS (p = 0.006). A subset of patients who had MVD also underwent partial sensory rhizotomy, usually in the setting of insignificant vascular compression. Compared with MVD alone, those who underwent MVD plus partial sensory rhizotomy had shorter pain-free intervals (median 45 months vs no median reached; p = 0.022). Multivariable regression demonstrated that shorter preoperative symptom duration (HR 1.005, 95% CI 1.001-1.008; p = 0.006) was associated with favorable outcome for MVD and that post-SRS sensory changes (HR 0.392, 95% CI 0.213-0.723; p = 0.003) were associated with favorable outcome for SRS. CONCLUSIONS In this longitudinal study, patients who received MVD had longer pain-free intervals compared with those who underwent SRS. For patients who received SRS, postoperative sensory change was predictive of favorable outcome. However, surgical decision making depends upon many factors. This information can help physicians counsel patients with idiopathic TN on treatment selection.

OptiClust, an Improved Method for Assigning Amplicon-Based Sequence Data to Operational Taxonomic Units.

Assignment of 16S rRNA gene sequences to operational taxonomic units (OTUs) is a computational bottleneck in the process of analyzing microbial communities. Although this has been an active area of research, it has been difficult to overcome the time and memory demands while improving the quality of the OTU assignments. Here, we developed a new OTU assignment algorithm that iteratively reassigns sequences to new OTUs to optimize the Matthews correlation coefficient (MCC), a measure of the quality of OTU assignments. To assess the new algorithm, OptiClust, we compared it to 10 other algorithms using 16S rRNA gene sequences from two simulated and four natural communities. Using the OptiClust algorithm, the MCC values averaged 15.2 and 16.5% higher than the OTUs generated when we used the average neighbor and distance-based greedy clustering with VSEARCH, respectively. Furthermore, on average, OptiClust was 94.6 times faster than the average neighbor algorithm and just as fast as distance-based greedy clustering with VSEARCH. An empirical analysis of the efficiency of the algorithms showed that the time and memory required to perform the algorithm scaled quadratically with the number of unique sequences in the data set. The significant improvement in the quality of the OTU assignments over previously existing methods will significantly enhance downstream analysis by limiting the splitting of similar sequences into separate OTUs and merging of dissimilar sequences into the same OTU. The development of the OptiClust algorithm represents a significant advance that is likely to have numerous other applications. IMPORTANCE The analysis of microbial communities from diverse environments using 16S rRNA gene sequencing has expanded our knowledge of the biogeography of microorganisms. An important step in this analysis is the assignment of sequences into taxonomic groups based on their similarity to sequences in a database or based on their similarity to each other, irrespective of a database. In this study, we present a new algorithm for the latter approach. The algorithm, OptiClust, seeks to optimize a metric of assignment quality by shuffling sequences between taxonomic groups. We found that OptiClust produces more robust assignments and does so in a rapid and memory-efficient manner. This advance will allow for a more robust analysis of microbial communities and the factors that shape them.

Sequencing 16S rRNA gene fragments using the PacBio SMRT DNA sequencing system.

Over the past 10 years, microbial ecologists have largely abandoned sequencing 16S rRNA genes by the Sanger sequencing method and have instead adopted highly parallelized sequencing platforms. These new platforms, such as 454 and Illumina's MiSeq, have allowed researchers to obtain millions of high quality but short sequences. The result of the added sequencing depth has been significant improvements in experimental design. The tradeoff has been the decline in the number of full-length reference sequences that are deposited into databases. To overcome this problem, we tested the ability of the PacBio Single Molecule, Real-Time (SMRT) DNA sequencing platform to generate sequence reads from the 16S rRNA gene. We generated sequencing data from the V4, V3-V5, V1-V3, V1-V5, V1-V6, and V1-V9 variable regions from within the 16S rRNA gene using DNA from a synthetic mock community and natural samples collected from human feces, mouse feces, and soil. The mock community allowed us to assess the actual sequencing error rate and how that error rate changed when different curation methods were applied. We developed a simple method based on sequence characteristics and quality scores to reduce the observed error rate for the V1-V9 region from 0.69 to 0.027%. This error rate is comparable to what has been observed for the shorter reads generated by 454 and Illumina's MiSeq sequencing platforms. Although the per base sequencing cost is still significantly more than that of MiSeq, the prospect of supplementing reference databases with full-length sequences from organisms below the limit of detection from the Sanger approach is exciting.

De novo clustering methods outperform reference-based methods for assigning 16S rRNA gene sequences to operational taxonomic units.

Background. 16S rRNA gene sequences are routinely assigned to operational taxonomic units (OTUs) that are then used to analyze complex microbial communities. A number of methods have been employed to carry out the assignment of 16S rRNA gene sequences to OTUs leading to confusion over which method is optimal. A recent study suggested that a clustering method should be selected based on its ability to generate stable OTU assignments that do not change as additional sequences are added to the dataset. In contrast, we contend that the quality of the OTU assignments, the ability of the method to properly represent the distances between the sequences, is more important. Methods. Our analysis implemented six de novo clustering algorithms including the single linkage, complete linkage, average linkage, abundance-based greedy clustering, distance-based greedy clustering, and Swarm and the open and closed-reference methods. Using two previously published datasets we used the Matthew's Correlation Coefficient (MCC) to assess the stability and quality of OTU assignments. Results. The stability of OTU assignments did not reflect the quality of the assignments. Depending on the dataset being analyzed, the average linkage and the distance and abundance-based greedy clustering methods generated OTUs that were more likely to represent the actual distances between sequences than the open and closed-reference methods. We also demonstrated that for the greedy algorithms VSEARCH produced assignments that were comparable to those produced by USEARCH making VSEARCH a viable free and open source alternative to USEARCH. Further interrogation of the reference-based methods indicated that when USEARCH or VSEARCH were used to identify the closest reference, the OTU assignments were sensitive to the order of the reference sequences because the reference sequences can be identical over the region being considered. More troubling was the observation that while both USEARCH and VSEARCH have a high level of sensitivity to detect reference sequences, the specificity of those matches was poor relative to the true best match. Discussion. Our analysis calls into question the quality and stability of OTU assignments generated by the open and closed-reference methods as implemented in current version of QIIME. This study demonstrates that de novo methods are the optimal method of assigning sequences into OTUs and that the quality of these assignments needs to be assessed for multiple methods to identify the optimal clustering method for a particular dataset.

Interval From Imaging to Treatment Delivery in the Radiation Surgery Age: How Long Is Too Long?

PURPOSE: The purpose of this study was to evaluate workflow and patient outcomes related to frameless stereotactic radiation surgery (SRS) for brain metastases. METHODS AND MATERIALS: We reviewed all treatment demographics, clinical outcomes, and workflow timing, including time from magnetic resonance imaging (MRI), computed tomography (CT) simulation, insurance authorization, and consultation to the start of SRS for brain metastases. RESULTS: A total of 82 patients with 151 brain metastases treated with SRS were evaluated. The median times from consultation, insurance authorization, CT simulation, and MRI for treatment planning were 15, 7, 6, and 11 days to SRS. Local freedom from progression (LFFP) was lower in metastases with MRI ≥ 14 days before treatment (P = .0003, log rank). The 6- and 12-month LFFP rate were 95% and 75% for metastasis with interval of <14 days from MRI to treatment compared to 56% and 34% for metastases with MRI ≥ 14 days before treatment. On multivariate analysis, LFFP remained significantly lower for lesions with MRI ≥ 14 days at SRS (P = .002, Cox proportional hazards; hazard ratio: 3.4, 95% confidence interval: 1.6-7.3). CONCLUSIONS: Delay from MRI to SRS treatment delivery for brain metastases appears to reduce local control. Future studies should monitor the timing from imaging acquisition to treatment delivery. Our experience suggests that the time from MRI to treatment should be <14 days.

Diagnosis dialog for pediatric physical therapists: hypotonia, developmental coordination disorder, and pediatric obesity as examples.

PURPOSE: To clarify what diagnosis means for pediatric physical therapists, to provide several examples of human movement dysfunction syndromes, and to offer guidance for how pediatric physical therapists may continue this work in any clinical setting. KEY POINTS: The importance of diagnosis in pediatric physical therapy is presented along with examples of 3 different processes used to develop diagnostic labels. These processes included surveys to identify consensus opinion of clinicians, a literature review, and a combination of these 2. Hypotonia, developmental coordination disorder, and pediatric obesity are presented as examples. SUMMARY: The 3 diagnoses serve as a basis for ongoing dialogue, discussion, and development of diagnostic labels for human movement syndromes identified by pediatric physical therapists.

Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform.

Rapid advances in sequencing technology have changed the experimental landscape of microbial ecology. In the last 10 years, the field has moved from sequencing hundreds of 16S rRNA gene fragments per study using clone libraries to the sequencing of millions of fragments per study using next-generation sequencing technologies from 454 and Illumina. As these technologies advance, it is critical to assess the strengths, weaknesses, and overall suitability of these platforms for the interrogation of microbial communities. Here, we present an improved method for sequencing variable regions within the 16S rRNA gene using Illumina's MiSeq platform, which is currently capable of producing paired 250-nucleotide reads. We evaluated three overlapping regions of the 16S rRNA gene that vary in length (i.e., V34, V4, and V45) by resequencing a mock community and natural samples from human feces, mouse feces, and soil. By titrating the concentration of 16S rRNA gene amplicons applied to the flow cell and using a quality score-based approach to correct discrepancies between reads used to construct contigs, we were able to reduce error rates by as much as two orders of magnitude. Finally, we reprocessed samples from a previous study to demonstrate that large numbers of samples could be multiplexed and sequenced in parallel with shotgun metagenomes. These analyses demonstrate that our approach can provide data that are at least as good as that generated by the 454 platform while providing considerably higher sequencing coverage for a fraction of the cost.

Reducing the effects of PCR amplification and sequencing artifacts on 16S rRNA-based studies.

The advent of next generation sequencing has coincided with a growth in interest in using these approaches to better understand the role of the structure and function of the microbial communities in human, animal, and environmental health. Yet, use of next generation sequencing to perform 16S rRNA gene sequence surveys has resulted in considerable controversy surrounding the effects of sequencing errors on downstream analyses. We analyzed 2.7×10(6) reads distributed among 90 identical mock community samples, which were collections of genomic DNA from 21 different species with known 16S rRNA gene sequences; we observed an average error rate of 0.0060. To improve this error rate, we evaluated numerous methods of identifying bad sequence reads, identifying regions within reads of poor quality, and correcting base calls and were able to reduce the overall error rate to 0.0002. Implementation of the PyroNoise algorithm provided the best combination of error rate, sequence length, and number of sequences. Perhaps more problematic than sequencing errors was the presence of chimeras generated during PCR. Because we knew the true sequences within the mock community and the chimeras they could form, we identified 8% of the raw sequence reads as chimeric. After quality filtering the raw sequences and using the Uchime chimera detection program, the overall chimera rate decreased to 1%. The chimeras that could not be detected were largely responsible for the identification of spurious operational taxonomic units (OTUs) and genus-level phylotypes. The number of spurious OTUs and phylotypes increased with sequencing effort indicating that comparison of communities should be made using an equal number of sequences. Finally, we applied our improved quality-filtering pipeline to several benchmarking studies and observed that even with our stringent data curation pipeline, biases in the data generation pipeline and batch effects were observed that could potentially confound the interpretation of microbial community data.

Assessing and improving methods used in operational taxonomic unit-based approaches for 16S rRNA gene sequence analysis.

In spite of technical advances that have provided increases in orders of magnitude in sequencing coverage, microbial ecologists still grapple with how to interpret the genetic diversity represented by the 16S rRNA gene. Two widely used approaches put sequences into bins based on either their similarity to reference sequences (i.e., phylotyping) or their similarity to other sequences in the community (i.e., operational taxonomic units [OTUs]). In the present study, we investigate three issues related to the interpretation and implementation of OTU-based methods. First, we confirm the conventional wisdom that it is impossible to create an accurate distance-based threshold for defining taxonomic levels and instead advocate for a consensus-based method of classifying OTUs. Second, using a taxonomic-independent approach, we show that the average neighbor clustering algorithm produces more robust OTUs than other hierarchical and heuristic clustering algorithms. Third, we demonstrate several steps to reduce the computational burden of forming OTUs without sacrificing the robustness of the OTU assignment. Finally, by blending these solutions, we propose a new heuristic that has a minimal effect on the robustness of OTUs and significantly reduces the necessary time and memory requirements. The ability to quickly and accurately assign sequences to OTUs and then obtain taxonomic information for those OTUs will greatly improve OTU-based analyses and overcome many of the challenges encountered with phylotype-based methods.

Structural and functional analysis of phosphothreonine-dependent FHA domain interactions.

FHA domains are well established as phospho-dependent binding modules mediating signal transduction in Ser/Thr kinase signaling networks in both eukaryotic and prokaryotic species. Although they are unique in binding exclusively to phosphothreonine, the basis for this discrimination over phosphoserine has remained elusive. Here, we attempt to dissect overall binding specificity at the molecular level. We first determined the optimal peptide sequence for Rv0020c FHA domain binding by oriented peptide library screening. This served as a basis for systematic mutagenic and binding analyses, allowing us to derive relative thermodynamic contributions of conserved protein and peptide residues to binding and specificity. Structures of phosphopeptide-bound and uncomplexed Rv0020c FHA domain then directed molecular dynamics simulations which show how the extraordinary discrimination in favor of phosphothreonine occurs through formation of additional hydrogen-bonding networks that are ultimately stabilized by van der Waals interactions of the phosphothreonine γ-methyl group with a conserved pocket on the FHA domain surface.

Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities.

mothur aims to be a comprehensive software package that allows users to use a single piece of software to analyze community sequence data. It builds upon previous tools to provide a flexible and powerful software package for analyzing sequencing data. As a case study, we used mothur to trim, screen, and align sequences; calculate distances; assign sequences to operational taxonomic units; and describe the alpha and beta diversity of eight marine samples previously characterized by pyrosequencing of 16S rRNA gene fragments. This analysis of more than 222,000 sequences was completed in less than 2 h with a laptop computer.

An intramolecular switch regulates phosphoindependent FHA domain interactions in Mycobacterium tuberculosis.

Forkhead-associated (FHA) domains have gained considerable prominence as ubiquitous phosphothreonine-dependent binding modules; however, their precise roles in serine and threonine kinase (STK) pathways and mechanisms of regulation remain unclear. From experiments with Rv1827, an FHA domain-containing protein from Mycobacterium tuberculosis, we derived a complete molecular description of an FHA-mediated STK signaling process. First, binding of the FHA domain to each of three metabolic enzyme complexes regulated their catalytic activities but did not require priming phosphorylation. However, phosphorylation of a threonine residue within a conserved amino-terminal motif of Rv1827 triggered its intramolecular association with the FHA domain of Rv1827, thus blocking its interactions with each of the three enzymes. The solution structure of this inactivated form and further mutagenic studies showed how a previously unidentified intramolecular phosphoswitch blocked the access of the target enzymes to a common FHA interaction surface and how this shared surface accommodated three functionally related, but structurally diverse, binding partners. Thus, our data reveal an unsuspected versatility in the FHA domain that allows for the transformation of multiple kinase inputs into various downstream regulatory signals.

Broadband optical absorbance spectroscopy using a whispering gallery mode microsphere resonator.

We demonstrate the ability to excite and monitor many whispering gallery modes (WGMs) of a microsphere resonator simultaneously in order to make broadband optical absorbance measurements. The 340 microm diameter microsphere is placed in a microfluidic channel. A hemispherical prism is used for coupling the WGMs into and out of the microsphere. The flat surface of the prism seals the microfluidic channel. The slight nonsphericity in the microsphere results in coupling to precessed modes whose emission is spatially separated from the reflected excitation light. The evanescent fields of the light trapped in WGMs interact with the surrounding environment. The change in transmission observed in the precessed modes is used to determine the absorbance of the surrounding environment. In contrast to our broadband optical absorbance measurements, previous WGM sensors have used only a single narrow mode to measure properties such as refractive index. With the microfluidic cell, we have measured the absorbance of solutions of dyes (lissamine green B, sunset yellow, orange G, and methylene blue), aromatic molecules (benzylamine and benzoic acid), and biological molecules (tryptophan, phenylalanine, tyrosine, and o-phospho-L-tyrosine) at visible and ultraviolet wavelengths. The microsphere surface was reacted with organosilane molecules to attach octadecyl groups, amino groups, and fluorogroups to the surface. Both electrostatic and hydrophobic interactions were observed between the analytes and the microsphere surface, as indicated by changes in the measured effective pathlength with different organosilanes. For a given analyte and coated microsphere, the pathlength measurement was repeatable within a few percent. Methylene blue dye had a very strong interaction with the surface and pathlengths of several centimeters were measured. Choosing an appropriate surface coating to interact with a specific analyte should result in the highest sensitivity detection.

Structural basis of DNA replication origin recognition by an ORC protein.

DNA replication in archaea and in eukaryotes share many similarities. We report the structure of an archaeal origin recognition complex protein, ORC1, bound to an origin recognition box, a DNA sequence that is found in multiple copies at replication origins. DNA binding is mediated principally by a C-terminal winged helix domain that inserts deeply into the major and minor grooves, widening them both. However, additional DNA contacts are made with the N-terminal AAA+ domain, which inserts into the minor groove at a characteristic G-rich sequence, inducing a 35 degrees bend in the duplex and providing directionality to the binding site. Both contact regions also induce substantial unwinding of the DNA. The structure provides insight into the initial step in assembly of a replication origin and recruitment of minichromosome maintenance (MCM) helicase to that origin.