Bayesian Optimization of Computer-Proposed Multistep Synthetic Routes on an Automated Robotic Flow Platform.

Computer-aided synthesis planning (CASP) tools can propose retrosynthetic pathways and forward reaction conditions for the synthesis of organic compounds, but the limited availability of context-specific data currently necessitates experimental development to fully specify process details. We plan and optimize a CASP-proposed and human-refined multistep synthesis route toward an exemplary small molecule, sonidegib, on a modular, robotic flow synthesis platform with integrated process analytical technology (PAT) for data-rich experimentation. Human insights address catalyst deactivation and improve yield by strategic choices of order of addition. Multi-objective Bayesian optimization identifies optimal values for categorical and continuous process variables in the multistep route involving 3 reactions (including heterogeneous hydrogenation) and 1 separation. The platform's modularity, robotic reconfigurability, and flexibility for convergent synthesis are shown to be essential for allowing variation of downstream residence time in multistep flow processes and controlling the order of addition to minimize undesired reactivity. Overall, the work demonstrates how automation, machine learning, and robotics enhance manual experimentation through assistance with idea generation, experimental design, execution, and optimization.

Extranodal Head and Neck Mantle Cell Lymphoma: Characteristics, Treatment, and Survival.

OBJECTIVES: To describe disease characteristics and treatment and to analyze survival and mortality for extranodal mantle cell lymphoma (MCL) of the head and neck. METHODS: Patients with extranodal MCL-excluding primary sites in the salivary glands, eye, and adnexa-were identified from the Surveillance, Epidemiology, and End Results (SEER) 18 Registries (2000-2015). Overall survival (OS) and cumulative incidence of MCL and non-MCL mortality were calculated. Factors associated with MCL and non-MCL mortality were analyzed with cause-specific hazard models. RESULTS: Five hundred nine patients met criteria for descriptive analysis and 294 patients met criteria for survival analysis, with a median follow-up of 58 months. The most common sites for MCL were the oropharynx (66.0%), nasopharynx (19.1%), and oral cavity (8.4%). The most common treatment received was chemotherapy alone (48.9%), followed by chemoradiation therapy (16.9%), and radiation therapy alone (10.4%). The proportion of cases diagnosed as early-stage disease ranged from 31% of sinonasal MCLs to 83% of laryngeal MCLs. At 5 years, OS was 63% (95% CI: 57%-69%). There was no significant difference in OS (P = .79), cumulative incidence of MCL mortality (P = .76), or cumulative incidence of non-MCL mortality (P = .98) by anatomic site. Comparing early-stage to late-stage disease, there was no significant difference in OS (P = .38), cumulative incidence of MCL mortality (P = .07), or cumulative incidence of non-MCL mortality (P = .14). Multivariate analysis showed increased hazard of MCL mortality for patients that were older or that presented with stage III or stage IV disease. CONCLUSION: The oropharynx is the most common subsite of head and neck MCLs, followed by the nasopharynx. Primary head and neck MCLs appear to present at an earlier stage than MCLs of other regions. In particular, laryngeal and hypopharyngeal MCLs may present as stage I or II disease.

Diversity and composition of macroinvertebrate communities in a rare inland salt marsh.

Inland salt marshes are rare habitats in the Great Lakes region of North America, formed on salt deposits from the Silurian period. These patchy habitats are abiotically stressful for the freshwater invertebrates that live there, and provide an opportunity to study the relationship between stress and diversity. We used morphological and COI metabarcoding data to assess changes in diversity and composition across both space (a transect from the salt seep to an adjacent freshwater area) and time (three sampling seasons). Richness was significantly lower at the seep site with both datatypes, while metabarcoding data additionally showed reduced richness at the freshwater transect end, consistent with a pattern where intermediate levels of stress show higher diversity. We found complementary, rather than redundant, patterns of community composition using the two datatypes: not all taxa were equally sequenced with the metabarcoding protocol. We identified taxa that are abundant at the salt seep of the marsh, including biting midges (Culicoides) and ostracods (Heterocypris). We conclude that (as found in other studies) molecular and morphological work should be used in tandem to identify the biodiversity in this rare habitat. Additionally, salinity may be a driver of community membership in this system, though further ecological research is needed to rule out alternate hypotheses.

Population growth of microcrustaceans in water from habitats with differing salinities.

Inland salt marshes are a rare habitat in North America. Little is known about the invertebrates in these habitats and their ability to cope with the brackish conditions of the marsh. We studied the population growth of ostracods found in an inland salt marsh (Maple River salt marsh) and of copepods found in the wetland habitat immediately adjacent to the freshwater Kalamazoo River. By studying these species in water from both habitats, we aimed to find out if they performed differently in the two habitats. We also tested Daphnia pulex in water from the two habitats due to the history of Daphnia spp. as model organisms. We found that copepods performed better in water taken from the Maple River salt marsh, and the ostracods and D. pulex performed equally well in either water. This was unexpected, since ostracods are found in the salt marsh and copepods in the freshwater area. As a second experiment, we tested the invertebrates in pairwise interactions. In water from the Kalamazoo River, ostracods outperformed the other two species, but there was no difference between D. pulex and copepods. No species outperformed the other in salt marsh water. Our results show no local adaptation to salinity, suggesting that ostracods and copepods may be limited in their respective distributions by dispersal limitation or habitat suitability.

An Analysis of Otolaryngology Medical Malpractice Payments From the National Practitioner Data Bank.

OBJECTIVE: To describe malpractice payments made on behalf of otolaryngologists, analyze trends over time, and test the association of payment amount with severity of alleged malpractice and patient age. STUDY DESIGN: Retrospective cross-sectional analysis. SETTING: National Practitioner Data Bank. SUBJECTS AND METHODS: This study comprised all payments made on behalf of otolaryngologists from 1991 to 2018 that were reported to the National Practitioner Data Bank. Descriptive statistics were calculated within and across years. Trends in payments were analyzed with the Mann-Kendall test. Generalized linear regression was utilized to test for association of payment amount with severity of the alleged injury and patient age. RESULTS: From 1991 to 2018, there was a significant decrease in the number of payments (272 to 81) and number of otolaryngologists on whose behalf payments were made (250 to 77). Mean and median payments increased significantly from $248,848 to $420,386 and from $96,813 to $275,000, respectively. By severity of alleged injury, mean payments ranged from $39,755 (95% CI, $20,957-$75,412) for insignificant injury to $754,349 (95% CI, $624,847-$910,692) for patients who were left quadriplegic, sustained brain damage, or required lifelong care. By patient age, mean payments for patients ≥60 years old were $191,465 (95% CI, $159,880-$229,292) versus $247,878 (95% CI, $209,416-$293,402) for patients aged 20 to 39 years and $232,225 (95% CI, $197,691-$272,793) for patients aged 40 to 59 years. CONCLUSION: The annual number and total value of malpractice payments decreased, while the annual mean and median payments increased. Payment amount was associated with severity of alleged malpractice and patient age.

Changes in Population-Level and Institutional-Level Prescribing Habits of Radioiodine Therapy for Papillary Thyroid Cancer.

Background: In the past two decades, new evidence and guidelines have emerged to refine recommendations for the use of radioactive iodine (RAI) therapy after thyroidectomy for cancer. We aim to describe national trends in RAI utilization, assess the impact of individual hospitals on RAI utilization, and examine whether variation in prescribing habits has declined over time. Methods: The National Cancer Database (NCDB) was queried from 2004 to 2016 for patients with papillary thyroid cancer (PTC) who received total thyroidectomy. Trends were analyzed using Joinpoint analysis. Hospital-specific effects and variation in prescribing habits were assessed through a hierarchical, mixed regression model. Results: RAI utilization declined from 61.0% in 2004 to 43.9% in 2016. RAI use declined most profoundly in patients with T1a, N0/X, M0 PTC without extrathyroidal extension (34.8% in 2004 to 9.5% in 2015), but continues to be used commonly in patients with advanced disease for whom it is routinely recommended (73.4% in 2004 to 72.0% in 2015). Furthermore, ∼80% of hospitals in 2016 utilized at or below the median utilization rate in 2006. Variation in RAI utilization across hospitals decreased by ∼50% from 2004 to 2016 (Levene's test p < 0.001), with a significant decline (p = 0.002) in the variation after 2012 (confidence interval: 2010 to 2014). Conclusions: Recommendations for whom to prescribe RAI appear to have impacted both the number of patients receiving RAI and the variation in prescribing habits across hospitals. Hospital selection has contributed less to the probability of receiving RAI over time.

Imaging Neurochemistry and Brain Structure Tracks Clinical Decline and Mechanisms of ALS in Patients.

Background: Oxidative stress and protein aggregation are key mechanisms in amyotrophic lateral sclerosis (ALS) disease. Reduced glutathione (GSH) is the most important intracellular antioxidant that protects neurons from reactive oxygen species. We hypothesized that levels of GSH measured by MR spectroscopic imaging (MRSI) in the motor cortex and corticospinal tract are linked to clinical trajectory of ALS patients. Objectives: Investigate the value of GSH imaging to probe clinical decline of ALS patients in combination with other neurochemical and structural parameters. Methods: Twenty-four ALS patients were imaged at 3 T with an advanced MR protocol. Mapping GSH levels in the brain is challenging, and for this purpose, we used an optimized spectral-edited 3D MRSI sequence with real-time motion and field correction to image glutathione and other brain metabolites. In addition, our imaging protocol included (i) an adiabatic T1ρ sequence to image macromolecular fraction of brain parenchyma, (ii) diffusion tensor imaging (DTI) for white matter tractography, and (iii) high-resolution anatomical imaging. Results: We found GSH in motor cortex (r = -0.431, p = 0.04) and corticospinal tract (r = -0.497, p = 0.016) inversely correlated with time between diagnosis and imaging. N-Acetyl-aspartate (NAA) in motor cortex inversely correlated (r = -0.416, p = 0.049), while mean water diffusivity (r = 0.437, p = 0.033) and T1ρ (r = 0.482, p = 0.019) positively correlated with disease progression measured by imputed change in revised ALS Functional Rating Scale. There is more decrease in the motor cortex than in the white matter for GSH compared to NAA, glutamate, and glutamine. Conclusions: Our study suggests that a panel of biochemical and structural imaging biomarkers defines a brain endophenotype, which can be used to time biological events and clinical progression in ALS patients. Such a quantitative brain endophenotype may stratify ALS patients into more homogeneous groups for therapeutic interventions compared to clinical criteria.

Loss of appetite in amyotrophic lateral sclerosis is associated with weight loss and decreased calorie consumption independent of dysphagia.

BACKGROUND: Loss of appetite has been reported to affect up to half of people with amyotrophic lateral sclerosis (ALS) and to be associated with weight loss. We wished to test whether loss of appetite correlates with reduced dietary intake independent of dysphagia. METHODS: Appetite was measured repeatedly using the Council on Nutrition Appetite Questionnaire (CNAQ) in participants in the Electronic health Application To Measure Outcomes REmotely study. Dietary intake and weight were compared to appetite, ALS Functional Rating Scale-Revised total and bulbar scores (dysphagia). RESULTS: The average baseline CNAQ score was 30.4 (n = 61; SD = 3.9) with 18.0% scoring <28 points (severe loss of appetite). Lower CNAQ scores correlated with greater weight loss since diagnosis (Pearson correlation coefficient, r = -0.34; P = 0.009) and lower baseline energy intake (P = 0.007), independent of dysphagia. CONCLUSIONS: Our results support an association between loss of appetite and decreased calorie intake and weight in ALS which is independent of dysphagia.

Continuous Flow Synthesis of ACE Inhibitors From N-Substituted l-Alanine Derivatives.

A strategy for the continuous flow synthesis of angiotensin converting enzyme (ACE) inhibitors is described. An optimization effort guided by in situ IR analysis resulted in a general amide coupling approach facilitated by N-carboxyanhydride (NCA) activation that was further characterized by reaction kinetics analysis in batch. The three-step continuous process was demonstrated by synthesizing 8 different ACE inhibitors in up to 88 % yield with throughputs in the range of ≈0.5 g h-1 , all while avoiding both isolation of reactive intermediates and process intensive reaction conditions. The process was further developed by preparing enalapril, a World Health Organization (WHO) essential medicine, in an industrially relevant flow platform that scaled throughput to ≈1 g h-1 .

A robotic platform for flow synthesis of organic compounds informed by AI planning.

The synthesis of complex organic molecules requires several stages, from ideation to execution, that require time and effort investment from expert chemists. Here, we report a step toward a paradigm of chemical synthesis that relieves chemists from routine tasks, combining artificial intelligence-driven synthesis planning and a robotically controlled experimental platform. Synthetic routes are proposed through generalization of millions of published chemical reactions and validated in silico to maximize their likelihood of success. Additional implementation details are determined by expert chemists and recorded in reusable recipe files, which are executed by a modular continuous-flow platform that is automatically reconfigured by a robotic arm to set up the required unit operations and carry out the reaction. This strategy for computer-augmented chemical synthesis is demonstrated for 15 drug or drug-like substances.

Reducing Phlebotomy Utilization With Education and Changes to Computerized Provider Order Entry.

INTRODUCTION: Daily phlebotomy is often a standard procedure in hospitalized patients. Recently, this practice has begun receiving attention as a potential target for efforts focused on eliminating overuse. Several organizations have published their efforts in this arena. Interventions have included education, feedback, and changes to computerized provider order entry (CPOE) but have yielded mixed results. METHODS: A quality improvement initiative to reduce the utilization of daily phlebotomy was conducted at a 505-bed Academic Medical Center. This project involved a combination of educational interventions and changes to CPOE. The primary end point evaluated was the daily performance of complete blood counts (CBCs) and basic metabolic profiles (BMPs) on medical and surgery units relative to the corresponding hospital census. RESULTS: Over the course of this project from August 1, 2013, to September 23, 2016, there was a 15.2% reduction in CBCs (p < .001 for linear trend) and 13.1% reduction in BMPs. DISCUSSION: Our results suggest that layering multimodal interventions that involve both "hard-wired" changes to CPOE and education and performance feedback can result in decreased utilization of phlebotomy.

Nutritional counseling with or without mobile health technology: a randomized open-label standard-of-care-controlled trial in ALS.

BACKGROUND: Nutritional status is an important prognostic factor in Amyotrophic Lateral Sclerosis (ALS). We wished to study the safety, tolerability and efficacy of nutritional counseling with or without an mHealth application to maintain or increase body weight in ALS, compared to standard care. METHODS: In this randomized open-label, standard-of-care-controlled, single-center clinical trial, we randomly assigned adults with ALS to one of three nutritional interventions: counseling by their physician or nurse ("standard care"), counseling by a registered dietitian (RD) ("in-person"), or counseling supported by a mHealth app ("mHealth"). Both intervention arms received tailored nutrition recommendations and recorded dietary intake and weight biweekly (mHealth) or monthly (in-person). The primary outcome of weight and secondary and tertiary outcomes of calorie intake, ALSFRS-R, and quality of life (QOL) were recorded at each clinic visit and analyzed in an ITT mixed model analysis. RESULTS: A total of 88 participants were enrolled of whom 78 were included in this analysis. The three arms were well-balanced except for excess males in the mHealth arm and greater weight lost at baseline in the in-person arm. Participants in the mHealth arm increased their calorie intake at month 3 to an average of 94% (95% CI: 85, 103) of recommended calories, compared to 81% (95% CI: 72, 91, p = 0.06 vs. mHealth) in the standard care arm. After 6 months, calorie intake was not different among the three arms. Overall weight was stable across all three groups. QOL scores in the mHealth arm were stable over 3 months (0.3 points, 95% CI: - 1.7, 2.2) compared to worsening in standard care (- 2.1 points, 95% CI: - 4.0, - 0.2, p = 0.09 vs. mHealth), but all scores declined by 6 months. ALSFRS-R total scores declined by an average of - 2.6 points (95% CI: - 5.1, - 0.1) over 6 months in the mHealth arm (p = 0.13 vs. standard care) compared to - 5.8 points (95% CI: - 8.2, - 3.4, p = 0.74 vs. standard care) in the in-person and - 5.2 points (95% CI: - 7.6, - 2.9) in the standard care arm. CONCLUSIONS: Nutritional counseling by a registered dietitian (with or without support by an mHealth app) is safe but did not maintain weight significantly better than standard care in ALS patients. TRIAL REGISTRATION: https://clinicaltrials.gov/ identifier NCT02418546. Registered April 16, 2015.

Pilot trial of inosine to elevate urate levels in amyotrophic lateral sclerosis.

OBJECTIVE: To test the safety, tolerability, and urate-elevating capability of the urate precursor inosine taken orally or by feeding tube in people with amyotrophic lateral sclerosis (ALS). METHODS: This was a pilot, open-label trial in 25 participants with ALS. Treatment duration was 12 weeks. The dose of inosine was titrated at pre-specified time points to elevate serum urate levels to 7-8 mg/dL. Primary outcomes were safety (as assessed by the occurrence of adverse events [AEs]) and tolerability (defined as the ability to complete the 12-week study on study drug). Secondary outcomes included biomarkers of oxidative stress and damage. As an exploratory analysis, observed outcomes were compared with a virtual control arm built using prediction algorithms to estimate ALSFRS-R scores. RESULTS: Twenty-four out of 25 participants (96%) completed 12 weeks of study drug treatment. One participant was unable to comply with study visits and was lost to follow-up. Serum urate rose to target levels in 6 weeks. No serious AEs attributed to study drug and no AEs of special concern, such as urolithiasis and gout, occurred. Selected biomarkers of oxidative stress and damage had significant changes during the study period. Observed changes in ALSFRS-R did not differ from baseline predictions. INTERPRETATION: Inosine appeared safe, well tolerated, and effective in raising serum urate levels in people with ALS. These findings, together with epidemiological observations and preclinical data supporting a neuroprotective role of urate in ALS models, provide the rationale for larger clinical trials testing inosine as a potential disease-modifying therapy for ALS.

Buprenorphine Supply, Access, and Quality: Where We Have Come and the Path Forward.

Buprenorphine is a form of opioid agonist treatment that has been demonstrated to be an effective medication for opioid addiction. It is available in different formulations and marketed under various trade names, including commonly as a buprenorphine/naloxone combination. This paper provides an overview of existing literature on the supply of buprenorphine treatment, the ability of people to access treatment with buprenorphine, and the quality of treatment received. We argue that better data for each of these aspects of treatment could inform policy to expand effective treatment with buprenorphine, and we suggest steps to obtain and act on such data.

Ni-Catalyzed Electrochemical Decarboxylative C-C Couplings in Batch and Continuous Flow.

An electrochemically driven, nickel-catalyzed reductive coupling of N-hydroxyphthalimide esters with aryl halides is reported. The reaction proceeds under mild conditions in a divided electrochemical cell and employs a tertiary amine as the reductant. This decarboxylative C(sp3)-C(sp2) bond-forming transformation exhibits excellent substrate generality and functional group compatibility. An operationally simple continuous-flow version of this transformation using a commercial electrochemical flow reactor represents a robust and scalable synthesis of value added coupling process.

Industry Perspective on Contemporary Protein-Binding Methodologies: Considerations for Regulatory Drug-Drug Interaction and Related Guidelines on Highly Bound Drugs.

Regulatory agencies have recently issued drug-drug interaction guidelines, which require determination of plasma protein binding (PPB). To err on the conservative side, the agencies recommend that a 0.01 lower limit of fraction unbound (fu) be used for highly bound compounds (>99%), irrespective of the actual measured values. While this may avoid false negatives, the recommendation would likely result in a high rate of false positive predictions, resulting in unnecessary clinical studies and more stringent inclusion/exclusion criteria, which may add cost and time in delivery of new medicines to patients. In this perspective, we provide a review of current approaches to measure PPB, and important determinants in enabling the accuracy and precision in these measurements. The ability to measure fu is further illustrated by a cross-company data comparison of PPB for warfarin and itraconazole, demonstrating good concordance of the measured fu values. The data indicate that fu values of ≤0.01 may be determined accurately across laboratories when appropriate methods are used. These data, along with numerous other examples presented in the literature, support the use of experimentally measured fu values for drug-drug interaction predictions, rather than using the arbitrary cutoff value of 0.01 as recommended in current regulatory guidelines.

Evaluation of Drug-Drug Interaction Potential Between Sacubitril/Valsartan (LCZ696) and Statins Using a Physiologically Based Pharmacokinetic Model.

Sacubitril/valsartan (LCZ696) has been approved for the treatment of heart failure. Sacubitril is an in vitro inhibitor of organic anion-transporting polypeptides (OATPs). In clinical studies, LCZ696 increased atorvastatin Cmax by 1.7-fold and area under the plasma concentration-time curve by 1.3-fold, but had little or no effect on simvastatin or simvastatin acid exposure. A physiologically based pharmacokinetics modeling approach was applied to explore the underlying mechanisms behind the statin-specific LCZ696 drug interaction observations. The model incorporated OATP-mediated clearance (CLint,T) for simvastatin and simvastatin acid to successfully describe the pharmacokinetic profiles of either analyte in the absence or presence of LCZ696. Moreover, the model successfully described the clinically observed drug effect with atorvastatin. The simulations clarified the critical parameters responsible for the observation of a low, yet clinically relevant, drug-drug interaction DDI between sacubitril and atorvastatin and the lack of effect with simvastatin acid. Atorvastatin is administered in its active form and rapidly achieves Cmax that coincide with the low Cmax of sacubitril. In contrast, simvastatin requires a hydrolysis step to the acid form and therefore is not present at the site of interactions at sacubitril concentrations that are inhibitory. Similar models were used to evaluate the drug-drug interaction risk for additional OATP-transported statins which predicted to maximally result in a 1.5-fold exposure increase.

Evaluation of plasma microsampling for dried plasma spots (DPS) in quantitative LC-MS/MS bioanalysis using ritonavir as a model compound.

Quantitative bioanalysis of dried plasma spots (DPS) is not subject to the impact of hematocrit and sample non-homogeneity that are often encountered in dried blood spot (DBS) assay. In the present report, an evaluation of plasma microsampling for DPS has been conducted for the first time using ritonavir as a model compound orally administered to dogs. For this evaluation, an LC-MS/MS method was developed and validated according to the current health authorities' guidance and industry practice for the analysis of ritonavir in DPS samples. The measured ritonavir concentrations in the DPS samples prepared using SAFE-TEC devices and directly from the conventional wet plasma using standard pipette were compared with each other and against those of conventional wet plasma. Both DPS results correlated well with each other and were comparable to those of the wet plasma. Good incurred sample reanalysis results were obtained for the two sets of DPS samples and wet plasma as well. The current plasma microsampling for DPS can serve as an alternative to DPS sampling via standard pipetting and wet plasma in in vivo studies.

Role of microRNAs 99b, 181a, and 181b in the differentiation of human embryonic stem cells to vascular endothelial cells.

MicroRNAs (miRNAs) are short noncoding RNAs, which post-transcriptionally regulate gene expression. miRNAs are transcribed as precursors and matured to active forms by a series of enzymes, including Dicer. miRNAs are important in governing cell differentiation, development, and disease. We have recently developed a feeder- and serum-free protocol for direct derivation of endothelial cells (ECs) from human embryonic stem cells (hESCs) and provided evidence of increases in angiogenesis-associated miRNAs (miR-126 and -210) during the process. However, the functional role of miRNAs in hESC differentiation to vascular EC remains to be fully interrogated. Here, we show that the reduction of miRNA maturation induced by Dicer knockdown suppressed hES-EC differentiation. A miRNA microarray was performed to quantify hES-EC miRNA profiles during defined stages of endothelial differentiation. miR-99b, -181a, and -181b were identified as increasing in a time- and differentiation-dependent manner to peak in mature hESC-ECs and adult ECs. Augmentation of miR-99b, -181a, and -181b levels by lentiviral-mediated transfer potentiated the mRNA and protein expression of EC-specific markers, Pecam1 and VE Cadherin, increased nitric oxide production, and improved hES-EC-induced therapeutic neovascularization in vivo. Conversely, knockdown did not impact endothelial differentiation. Our results suggest that miR-99b, -181a, and -181b comprise a component of an endothelial-miRNA signature and are capable of potentiating EC differentiation from pluripotent hESCs.