Reaction monitoring via benchtop nuclear magnetic resonance spectroscopy: A practical comparison of on-line stopped-flow and continuous-flow sampling methods.

The ability for nuclear magnetic resonance (NMR) spectroscopy to provide quantitative, structurally rich information makes this spectroscopic technique an attractive reaction monitoring tool. The practicality of NMR for this type of analysis has only increased in the recent years with the influx of commercially available benchtop NMR instruments and compatible flow systems. In this study, we aim to compare 19F NMR reaction profiles acquired under both on-line continuous-flow and stopped-flow sampling methods, with modern benchtop NMR instrumentation, and two reaction systems: a homogeneous imination reaction and a biphasic activation of a carboxylic acid to acyl fluoride. Reaction trends with higher data density can be acquired with on-line continuous-flow analyses, and this work highlights that representative reaction trends can be acquired without any correction when monitoring resonances with a shorter spin-lattice relaxation time (T1), and with the used flow conditions. On-line stopped-flow analyses resulted in representative reaction trends in all cases, including the monitoring of resonances with a long T1, without the need of any correction factors. The benefit of easier data analysis, however, comes with the cost of time, as the fresh reaction solution must be flowed into the NMR system, halted, and time must be provided for spins to become polarized in the instrument's external magnetic field prior to spectral measurement. Results for one of the reactions were additionally compared with the use of a high-field NMR.

The effect of tube quality on externally calibrated quantitative nuclear magnetic resonance analysis: How bad can it be?

Externally calibrated quantitative nuclear magnetic resonance (NMR) approaches offer practical means to simultaneously evaluate chemical identity and content without the addition of calibrants to the test sample. Despite continuous advances in external calibration over the last few decades, adoption of these approaches has been slower than expected. Variations in NMR tube geometry are a commonly overlooked factor that can have a substantial effect on externally calibrated quantitation methods. In this report, we investigate the extent to which tube-to-tube volume variability can affect quantitative NMR outcomes. The results highlight the importance of considering tube quality during the development stages of externally calibrated quantitative methods. In addition, we propose a simple, yet effective volume correction strategy using the residual protonated solvent signal that, based on experiments with mixed NMR tubes of varying quality, alleviates the effect of tube-to-tube variability.

A reliable external calibration method for reaction monitoring with benchtop NMR.

Nuclear magnetic resonance (NMR) spectroscopy is a powerful analytical technique with the ability to acquire both quantitative and structurally insightful data for multiple components in a test sample. This makes NMR spectroscopy a desirable tool to understand, monitor, and optimize chemical transformations. While quantitative NMR (qNMR) approaches relying on internal standards are well-established, using an absolute external calibration scheme is beneficial for reaction monitoring as resonance overlap complications from an added reference material to the sample can be avoided. Particularly, this type of qNMR technique is of interest with benchtop NMR spectrometers as the likelihood of resonance overlap is only enhanced with the lower magnetic field strengths of the used permanent magnets. The included study describes a simple yet robust methodology to determine concentration conversion factors for NMR systems using single- and multi-analyte linear regression models. This approach is leveraged to investigate a pharmaceutically relevant amide coupling batch reaction. An on-line stopped-flow (i.e., interrupted-flow or paused-flow) benchtop NMR system was used to monitor both the 1,1'-carbonyldiimidazole (CDI) promoted acid activation and the amide coupling. The results highlight how quantitative measurements in benchtop NMR systems can provide valuable information and enable analysts to make decisions in real time.

Application of CRAFT in two-dimensional NMR data processing.

Two-dimensional (2D) data are typically truncated in both dimensions, but invariably and severely so in the indirect dimension. These truncated FIDs and/or interferograms are extensively zero filled, and Fourier transformation of such zero-filled data is always preceded by a rapidly decaying apodization function. Hence, the frequency line width in the spectrum (at least parallel to the evolution dimension) is almost always dominated by the apodization function. Such apodization-driven line broadening in the indirect (t1 ) dimension leads to the lack of clear resolution of cross peaks in the 2D spectrum. Time-domain analysis (i.e. extraction of frequency, amplitudes, line width, and phase parameters directly from the FID, in this case via Bayesian modeling into a tabular format) of NMR data is another approach for spectral resonance characterization and quantification. The recently published complete reduction to amplitude frequency table (CRAFT) technique converts the raw FID data (i.e. time-domain data) into a table of frequencies, amplitudes, decay rate constants, and phases. CRAFT analyses of time-domain data require minimal or no apodization prior to extraction of the four parameters. We used the CRAFT processing approach for the decimation of the interferograms and compared the results from a variety of 2D spectra against conventional processing with and without linear prediction. The results show that use of the CRAFT technique to decimate the t1 interferograms yields much narrower spectral line width of the resonances, circumventing the loss of resolution due to apodization. Copyright © 2016 John Wiley & Sons, Ltd.

Orbital Plasmacytoma Mimicking an Orbital Abscess.

An 83-year-old male with a 15-month history of multiple myeloma presented with acute onset of swelling, redness, and pain around his right eye. CT scan was consistent with an orbital abscess. The patient was taken to the operating room for drainage of the orbital abscess. Abnormal tissue was encountered intraoperatively so biopsies were taken. His cultures grew only one colony of coagulase-negative Staphylococcus aureus. The histopathology from the biopsies showed a CD-138 positive plasma cell neoplasia consistent with a plasmacytoma. Plasmacytomas have been reported to present as orbital cellulitis and as abscesses in other locations in the body, but to our knowledge, this is the first case of a plasmacytoma presenting as an orbital abscess.

Long-Term Results for Entropion Repair by Tarsal Margin Rotation With Posterior Lamella Superadvancement.

PURPOSE: To report the long-term stability of tarsal margin rotation and posterior lamellar superadvancement (TMR PLS) for the repair of upper eyelid cicatricial entropion. METHODS: A retrospective chart review was performed from January 2000 through December 2014 to identify all patients who had TMR PLS at the authors' institution. Charts were reviewed for demographic information, recurrence of entropion or trichiasis, and surgical complications. Failure was defined as return of entropion. Patients with greater than 24 months of follow up were included. RESULTS: A total of 30 TMR PLS procedures were performed during the review period. Nineteen cases from 14 patients were included in the final analysis. None of the 19 cases demonstrated recurrence of entropion over an average follow-up period of 78.3 months. Eight cases demonstrated trichiasis after TMR PLS, 5 of which required treatment. CONCLUSION: This case series suggests that TMR PLS for the treatment of upper eyelid cicatricial entropion has excellent long-term stability.

High Resolution Ion Mobility Enables the Structural Characterization of Atropisomers of GDC-6036, a KRAS G12C Covalent Inhibitor.

GDC-6036 is a covalent KRAS G12C inhibitor that demonstrates high potency and selectivity. Structurally, GDC-6036 consists of several motifs that make the analytical characterization of this molecule challenging, including a highly basic pyrrolidine motif bonded to a quinazoline ring via an ether bond and an atropisomeric carbon-carbon bond between functionalized pyridine and quinazoline groups. Structurally, the desired atropisomer was synthesized via an atroposelective Negishi coupling with very high yield. However, having a direct way to analyze and confirm the presence of the atropisomeric species remained challenging in routine analytical workflows. In this study, both variable temperature nuclear magnetic resonance (VT-NMR) and two different approaches of in-line ion mobility coupled to liquid chromatography mass spectrometry (LC-MS) workflows were evaluated for the characterization of GDC-6036 and its undesired atropisomer (Compound B) to support synthetic route development. Briefly, both VT-NMR and traveling wave ion mobility spectrometry (TWIMS) enabled by structures for lossless ion manipulation (SLIM) technology coupled to high resolution MS (HRMS) are able to elucidate the structures of the atropisomers in a complex mixture. Drift tube IMS (DTIMS) was also evaluated, but lacked the resolving power to demonstrate separation between the two species in a mixture, but did show slight differences in their arrival times when multiplexed and injected separately. The determined resolving power (Rp) by multiplexing the ions via DTIMS was 67.3 and 60.5 for GDC-6036 and Compound B, respectively, while the two peak resolving power (Rpp) was determined to be 0.41, indicating inadequate resolution between the two species. Alternatively, the SLIM-IM studies showed Rp of 103.8 and 99.4, with a Rpp of 2.64, indicating good separation between the atropisomers. Furthermore, the CCS/z for GDC-6036 and Compound B was determined to be 231.2 Å2/z and 235.0 Å2/z, respectively. Quantitative experiments demonstrate linearity (R2 >0.99) for both GDC-6036 and Compound B while maintaining separation via SLIM-IM. Spike recoveries of one atropisomer relative to the other yielded strong recoveries (98.7% to 102.5%) while maintaining reproducibility (<7% RSD). The study herein describes the analytical process for evaluating new technologies and strategies for implementation in routine biopharmaceutical characterization workflows.

Evaluation and management of 2 ferocactus spines in the orbit.

A 49-year-old woman, who had fallen face first in a cactus 1 week earlier, presented with a small, mobile, noninflamed subcutaneous nodule at the rim of her right lateral orbit with no other functional deficits. A CT scan was obtained, which revealed a 4-cm intraorbital tubular-shaped foreign body resembling a large cactus spine. A second preoperative CT scan, obtained for an intraoperative guidance system, demonstrated a second cactus spine, which was initially not seen on the first CT scan. Both spines were removed surgically without complication. The authors discuss factors that can cause diagnosis delay, review the radiographic features of cactus spines, and discuss the often times benign clinical course of retained cactus spine foreign bodies. To the authors' knowledge, this is the first case report of cactus spines in the orbit. Health-care professionals should have a low threshold for imaging in cases of traumatic injuries involving cactus spines.

(1) H-NMR quantification of major saccharides in açaí raw materials: a comparison of the internal standard methodology with the absolute intensity qNMR method.

INTRODUCTION: While the use of internal standard methodology for qNMR is a proven and reliable form of quantification, simplified alternative approaches are needed. Agilent's absolute intensity qNMR utility software is a valuable alternative that has not yet been subjected to validation in the peer-reviewed literature. OBJECTIVE: To provide validation of Agilent's absolute intensity qNMR method with a specific application to natural product quantification by measuring saccharide content in açaí materials. METHODS: In order to validate the method, calibration test samples of ibuprofen were prepared in DMSO-d6 at nine different concentrations and measured with (1) H-NMR. A minimum of 40 spectra were collected for each sample, and the absolute intensity utility was used for quantification. The same methodology was then applied to the açaí materials, creating triplicates for each of the materials and using 3-(trimethylsilyl)-1-propanesulphonic acid sodium salt in water-d2 as both the solvent and internal standard. (1) H-NMR spectra were collected, and the amounts of glucose, sucrose and fructose were determined using both the internal standard approach and the absolute intensity qNMR method. RESULTS: Applying the absolute intensity utility to the ibuprofen samples demonstrated a linear response (R(2) = 0.99943). For the açaí investigations, results obtained from the absolute intensity method were comparable to those obtained from the internal standard approach, with percentage differences ranging from 0.5-6.2%. CONCLUSION: This study demonstrates the accuracy, precision and reliability of Agilent's absolute intensity qNMR method. In addition, practical information is provided for assessing the saccharide contents of açaí materials.

Effect of radiation therapy on intraocular pressure in patients with Graves' orbitopathy.

PURPOSE: To describe the effect of radiation therapy on intraocular pressure in patients who have had orbital radiation for Graves' orbitopathy, and to provide a descriptive analysis of these patients. DESIGN: Retrospective case series. PARTICIPANTS: 24 consecutive patients referred for orbital radiation therapy for Graves' orbitopathy from December 1st, 2001 through July 31st, 2009. INTERVENTION: Patients received a total of 2000 cGy fractionated over 10 days. MAIN OUTCOME MEASURES: Medical records were reviewed for: demographics, tobacco history, ocular history, history related to Graves' disease, medications, visual field tests, retinopathy, and physical exam findings. The two-tailed Student's t-test was used to determine statistically significant differences in intraocular pressure before radiation therapy and 0-3 (T1), 4-6 (T2), 7-12 (T3), and 13-18 (T4) months following radiation therapy. RESULTS: 34 eyes were available for analysis. There was no correlation between intraocular pressure and SPECS scores. Mean intraocular pressure prior to radiation therapy was 18.15 ± 3.83 mm Hg. Patients who had orbital decompression, eye muscle surgery, or glaucoma were excluded from the final analysis. There were 7, 11, 14, and 11 eyes with data at times T1, T2, T3, and T4 following radiation therapy, respectively. There was a significant decrease in mean intraocular pressure at T2, T3 and T4 of 26.00% ± 9.25%, 11.75% ± 27.58%, and 16.72% ± 13.94%, respectively. CONCLUSIONS: There was a significant drop in mean intraocular pressure between 4 and 18 months after radiation therapy in our patient population. The mechanism by which this decrease in intraocular pressure occurs is not understood.

Globally invariant metabolism but density-diversity mismatch in springtails.

Soil life supports the functioning and biodiversity of terrestrial ecosystems. Springtails (Collembola) are among the most abundant soil arthropods regulating soil fertility and flow of energy through above- and belowground food webs. However, the global distribution of springtail diversity and density, and how these relate to energy fluxes remains unknown. Here, using a global dataset representing 2470 sites, we estimate the total soil springtail biomass at 27.5 megatons carbon, which is threefold higher than wild terrestrial vertebrates, and record peak densities up to 2 million individuals per square meter in the tundra. Despite a 20-fold biomass difference between the tundra and the tropics, springtail energy use (community metabolism) remains similar across the latitudinal gradient, owing to the changes in temperature with latitude. Neither springtail density nor community metabolism is predicted by local species richness, which is high in the tropics, but comparably high in some temperate forests and even tundra. Changes in springtail activity may emerge from latitudinal gradients in temperature, predation and resource limitation in soil communities. Contrasting relationships of biomass, diversity and activity of springtail communities with temperature suggest that climate warming will alter fundamental soil biodiversity metrics in different directions, potentially restructuring terrestrial food webs and affecting soil functioning.

Challenges in the structure determination of a dimeric impurity found during development of GDC-0326.

While developing a synthetic route for GDC-0326, a PI3Kα selective inhibitor, a side product was identified which was adversely impacting process chemistry development. To aid in optimization of a viable synthetic pathway for the drug, it was decided to characterize this impurity. Initial efforts using typical high-resolution mass spectrometry data coupled with NMR analysis were unable to unambiguously identify the structure. The NMR analysis was hampered by a severe lack of protons in the core of the structure. While efforts were being made to produce suitable crystals for definitive x-ray analysis, Raman analysis was undertaken. The vibrational data were compared to DFT calculations for the two most likely structures. This data, along with chemical reasoning, eventually led to successful prediction of structure 2, which was ultimately confirmed by single crystal x-ray diffractometry data.

Bilateral spontaneous subperiosteal orbital hemorrhages following endoscopic retrograde cholangiopancreatography.

Spontaneous subperiosteal orbital hemorrhage is rare. The authors describe a case of acute bilateral eyelid swelling, eyelid ptosis, and double vision in a patient immediately following endoscopic retrograde cholangiopancreatography. CT revealed bilateral subperiosteal orbital hemorrhages. The patient demonstrated no signs of optic nerve compromise, and the hemorrhages resolved spontaneously without sequela. Previous reports in the literature have documented subperiosteal hemorrhages following increased intracranial venous pressure, coagulopathies, and prone positioning during procedures. To the authors' knowledge, subperiosteal orbital hemorrhages have not been previously described following endoscopic retrograde cholangiopancreatography. Ophthalmologists and gastroenterologists should be aware of this potential complication associated with this procedure.

Survival in hospice patients with dementia: the effect of home hospice and nurse visits.

BACKGROUND: Hospice patients with dementia are at increased risk for live discharge and long lengths of stay (>180 days), causing patient and family caregiver stress and burden. The location and timing of clinician visits are important factors influencing whether someone dies as expected, in hospice, or experiences a live discharge or long length of stay. OBJECTIVE: Examine how home hospice and nurse visit frequency relate to dying in hospice within the Medicare-intended 6-month period. DESIGN: Retrospective cohort study. SETTING: Non-profit hospice agency. PARTICIPANTS: Three thousand eight hundred and thirty seven patients with dementia who received hospice services from 2013 to 2017. METHODS: Multivariable survival analyses examined the effects of receiving home hospice (vs. nursing home) and timing of nurse visits on death within 6 months of hospice enrollment, compared to live discharge or long length of stay. Models adjust for relevant demographic and clinical factors. RESULTS: Thirty-nine percent (39%) of patients experienced live discharge or long length of stay. Home hospice patients were more likely to experience live discharge or long length of stays (HR for death: 0.77, 95%CI: 0.69-0.86, p < 0.001). Frequency of nurse visits was inversely associated with live discharge and long lengths of stay (HR for death: 2.87, 95%CI: 2.47-3.33, p < 0.001). CONCLUSION: Nearly 40% of patients with dementia in our study experienced live discharge or a long length of stay. Additional research is needed to understand why home hospice may result in live discharge or a long length of stay for patients with dementia. Nurse visits were associated with death, suggesting their responsiveness to deteriorating patient health. Hospice guidelines may need to permit longer stays so community-dwelling patients with dementia, a growing segment of hospice patients, can remain continuously enrolled in hospice and avoid burden and costs associated with live discharge.

Global data on earthworm abundance, biomass, diversity and corresponding environmental properties.

Earthworms are an important soil taxon as ecosystem engineers, providing a variety of crucial ecosystem functions and services. Little is known about their diversity and distribution at large spatial scales, despite the availability of considerable amounts of local-scale data. Earthworm diversity data, obtained from the primary literature or provided directly by authors, were collated with information on site locations, including coordinates, habitat cover, and soil properties. Datasets were required, at a minimum, to include abundance or biomass of earthworms at a site. Where possible, site-level species lists were included, as well as the abundance and biomass of individual species and ecological groups. This global dataset contains 10,840 sites, with 184 species, from 60 countries and all continents except Antarctica. The data were obtained from 182 published articles, published between 1973 and 2017, and 17 unpublished datasets. Amalgamating data into a single global database will assist researchers in investigating and answering a wide variety of pressing questions, for example, jointly assessing aboveground and belowground biodiversity distributions and drivers of biodiversity change.

A grammar-based distance metric enables fast and accurate clustering of large sets of 16S sequences.

BACKGROUND: We propose a sequence clustering algorithm and compare the partition quality and execution time of the proposed algorithm with those of a popular existing algorithm. The proposed clustering algorithm uses a grammar-based distance metric to determine partitioning for a set of biological sequences. The algorithm performs clustering in which new sequences are compared with cluster-representative sequences to determine membership. If comparison fails to identify a suitable cluster, a new cluster is created. RESULTS: The performance of the proposed algorithm is validated via comparison to the popular DNA/RNA sequence clustering approach, CD-HIT-EST, and to the recently developed algorithm, UCLUST, using two different sets of 16S rDNA sequences from 2,255 genera. The proposed algorithm maintains a comparable CPU execution time with that of CD-HIT-EST which is much slower than UCLUST, and has successfully generated clusters with higher statistical accuracy than both CD-HIT-EST and UCLUST. The validation results are especially striking for large datasets. CONCLUSIONS: We introduce a fast and accurate clustering algorithm that relies on a grammar-based sequence distance. Its statistical clustering quality is validated by clustering large datasets containing 16S rDNA sequences.

Race, Ethnicity, and Other Risks for Live Discharge Among Hospice Patients with Dementia.

OBJECTIVES: The end-of-life trajectory for persons with dementia is often protracted and difficult to predict, placing these individuals at heightened risk of live discharge from hospice. Risks for live discharge due to condition stabilization or failure to decline among patients with dementia are not well established. Our aim was to identify demographic, health, and hospice service factors associated with live discharge due to condition stabilization or failure to decline among hospice patients with dementia. DESIGN: Retrospective cohort study. SETTING: A large not-for-profit agency in New York City. PARTICIPANTS: A total of 2629 hospice patients with dementia age 65 years and older. MEASUREMENTS: Primary outcome was live discharge from hospice due to condition stabilization or failure to decline (vs death). Measures include demographic factors (race/ethnicity, Medicaid, sex, age, marital status, parental status), health characteristics (primary dementia diagnosis, comorbidities, functional status, prior hospitalization), and hospice service (location, length of service, number and timing of nurse visits). RESULTS: Logistic regression models indicated that compared with white hospice patients with dementia, African American and Hispanic hospice patients with dementia experienced increased risk of live discharge (African American: adjusted odds ratio [aOR] = 2.42; 95% confidence interval [CI] = 1.34-4.38; Hispanic: aOR = 2.99; 95% CI = 1.81-4.94). Home hospice (aOR = 7.57; 95% CI = 4.04-14.18), longer length of service (aOR = 1.04; 95% CI = 1.04-1.05), and more days between nurse visits and discharge (aOR = 1.86; 95% CI = 1.56-2.21) were also associated with live discharge. CONCLUSION: To avoid burdensome and disruptive transitions out of hospice in patients with dementia, interventions to reduce live discharge due to condition stabilization or failure to decline should be tailored to meet the needs of African American, Hispanic, and home hospice patients. Policies regarding sustained hospice eligibility should account for the variable and protracted end-of-life trajectory of patients with dementia. J Am Geriatr Soc 68:551-558, 2020.

Towards an integrative understanding of soil biodiversity.

Soil is one of the most biodiverse terrestrial habitats. Yet, we lack an integrative conceptual framework for understanding the patterns and mechanisms driving soil biodiversity. One of the underlying reasons for our poor understanding of soil biodiversity patterns relates to whether key biodiversity theories (historically developed for aboveground and aquatic organisms) are applicable to patterns of soil biodiversity. Here, we present a systematic literature review to investigate whether and how key biodiversity theories (species-energy relationship, theory of island biogeography, metacommunity theory, niche theory and neutral theory) can explain observed patterns of soil biodiversity. We then discuss two spatial compartments nested within soil at which biodiversity theories can be applied to acknowledge the scale-dependent nature of soil biodiversity.