Boosting the Sensitivity of Quantitative Single-Cell Proteomics with Infrared-Tandem Mass Tags.

Single-cell proteomics is a powerful approach to precisely profile protein landscapes within individual cells toward a comprehensive understanding of proteomic functions and tissue and cellular states. The inherent challenges associated with limited starting material demand heightened analytical sensitivity. Just as advances in sample preparation maximize the amount of material that makes it from the cell to the mass spectrometer, we strive to maximize the number of ions that make it from ion source to the detector. In isobaric tagging experiments, limited reporter ion generation limits quantitative accuracy and precision. The combination of infrared photoactivation and ion parking circumvents the m/z dependence inherent in HCD, maximizing reporter generation and avoiding unintended degradation of TMT reporter molecules in infrared-tandem mass tags (IR-TMT). The method was applied to single-cell human proteomes using 18-plex TMTpro, resulting in 4-5-fold increases in reporter signal compared to conventional SPS-MS3 approaches. IR-TMT enables faster duty cycles, higher throughput, and increased peptide identification and quantification. Comparative experiments showcase 4-5-fold lower injection times for IR-TMT, providing superior sensitivity without compromising accuracy. In all, IR-TMT enhances the dynamic range of proteomic experiments and is compatible with gas-phase fractionation and real-time searching, promising increased gains in the study of cellular heterogeneity.

Improving Biological Joint Moment Estimation During Real-World Tasks with EMG and Instrumented Insoles.

OBJECTIVE: Real-time measurement of biological joint moment could enhance clinical assessments and generalize exoskeleton control. Accessing joint moments outside clinical and laboratory settings requires harnessing non-invasive wearable sensor data for indirect estimation. Previous approaches have been primarily validated during cyclic tasks, such as walking, but these methods are likely limited when translating to non-cyclic tasks where the mapping from kinematics to moments is not unique. METHODS: We trained deep learning models to estimate hip and knee joint moments from kinematic sensors, electromyography (EMG), and simulated pressure insoles from a dataset including 10 cyclic and 18 non-cyclic activities. We assessed estimation error on combinations of sensor modalities during both activity types. RESULTS: Compared to the kinematics-only baseline, adding EMG reduced RMSE by 16.9% at the hip and 30.4% at the knee (p<0.05) and adding insoles reduced RMSE by 21.7% at the hip and 33.9% at the knee (p<0.05). Adding both modalities reduced RMSE by 32.5% at the hip and 41.2% at the knee (p<0.05) which was significantly higher than either modality individually (p<0.05). All sensor additions improved model performance on non-cyclic tasks more than cyclic tasks (p<0.05). CONCLUSION: These results demonstrate that adding kinetic sensor information through EMG or insoles improves joint moment estimation both individually and jointly. These additional modalities are most important during non-cyclic tasks, tasks that reflect the variable and sporadic nature of the real-world. SIGNIFICANCE: Improved joint moment estimation and task generalization is pivotal to developing wearable robotic systems capable of enhancing mobility in everyday life.

Barriers to Implementing the Kidney Disease Outcomes Quality Initiative End-Stage Kidney Disease Life Plan Guideline.

OBJECTIVE: The updated 2019 National Kidney Foundation Kidney Disease Outcomes Quality Initiative vascular access guidelines recommend patient-centered, multi-disciplinary construction and regular update of an individualized end-stage kidney disease (ESKD) Life-Plan (LP) for each patient, a dramatic shift from previous recommendations and policy. The objective of this study was to examine barriers and facilitators to implementing the LP among key stakeholders. METHODS: Semi-structured individual interviews were analyzed using inductive and deductive coding. Codes were mapped to relevant domains in the Consolidated Framework for Implementation Research (CFIR). RESULTS: We interviewed 34 participants: 11 patients with end-stage kidney disease, 2 care partners, and 21 clinicians who care for patients with end-stage kidney disease. In both the clinician and the patient/care partner categories, saturation (where no new themes were identified) was reached at 8 participants. We identified significant barriers and facilitators to implementation of the ESKD LP across three CFIR domains: Innovation, Outer setting, and Inner setting. Regarding the Innovation domain, patients and care partners valued the concept of shared decision-making with their care team (CFIR construct: innovation design). However, both clinicians and patients had significant concerns about the complexity of decision-making around kidney substitutes and the ability of patients to digest the overwhelming amount of information needed to effectively participate in creating the LP (innovation complexity). Clinicians expressed concerns regarding the lack of existing evidence base which limits their ability to effectively counsel patients (innovation evidence base) and the implementation costs (innovation cost). Within the Outer Setting, both clinicians and patients were concerned about performance measurement pressure under the existing "Fistula First" policies and had concerns about reimbursement (financing). In the Inner Setting, clinicians and patients stressed the lack of available resources and access to knowledge and information. CONCLUSION: Given the complexity of decision-making around kidney substitutes and vascular access, our findings point to the need for implementation strategies, infrastructure development, and policy change to facilitate ESKD LP development.

Correction: Identifying Patients Who Meet Criteria for Genetic Testing of Hereditary Cancers Based on Structured and Unstructured Family Health History Data in the Electronic Health Record: Natural Language Processing Approach.

[This corrects the article DOI: 10.2196/37842.].

Identifying Patients Who Meet Criteria for Genetic Testing of Hereditary Cancers Based on Structured and Unstructured Family Health History Data in the Electronic Health Record: Natural Language Processing Approach.

BACKGROUND: Family health history has been recognized as an essential factor for cancer risk assessment and is an integral part of many cancer screening guidelines, including genetic testing for personalized clinical management strategies. However, manually identifying eligible candidates for genetic testing is labor intensive. OBJECTIVE: The aim of this study was to develop a natural language processing (NLP) pipeline and assess its contribution to identifying patients who meet genetic testing criteria for hereditary cancers based on family health history data in the electronic health record (EHR). We compared an algorithm that uses structured data alone with structured data augmented using NLP. METHODS: Algorithms were developed based on the National Comprehensive Cancer Network (NCCN) guidelines for genetic testing for hereditary breast, ovarian, pancreatic, and colorectal cancers. The NLP-augmented algorithm uses both structured family health history data and the associated unstructured free-text comments. The algorithms were compared with a reference standard of 100 patients with a family health history in the EHR. RESULTS: Regarding identifying the reference standard patients meeting the NCCN criteria, the NLP-augmented algorithm compared with the structured data algorithm yielded a significantly higher recall of 0.95 (95% CI 0.9-0.99) versus 0.29 (95% CI 0.19-0.40) and a precision of 0.99 (95% CI 0.96-1.00) versus 0.81 (95% CI 0.65-0.95). On the whole data set, the NLP-augmented algorithm extracted 33.6% more entities, resulting in 53.8% more patients meeting the NCCN criteria. CONCLUSIONS: Compared with the structured data algorithm, the NLP-augmented algorithm based on both structured and unstructured family health history data in the EHR increased the number of patients identified as meeting the NCCN criteria for genetic testing for hereditary breast or ovarian and colorectal cancers.

Using CDS Hooks to increase SMART on FHIR app utilization: a cluster-randomized trial.

OBJECTIVE: HL7 SMART on FHIR apps have the potential to improve healthcare delivery and EHR usability, but providers must be aware of the apps and use them for these potential benefits to be realized. The HL7 CDS Hooks standard was developed in part for this purpose. The objective of this study was to determine if contextually relevant CDS Hooks prompts can increase utilization of a SMART on FHIR medical reference app (MDCalc for EHR). MATERIALS AND METHODS: We conducted a 7-month, provider-randomized trial with 70 providers in a single emergency department. The intervention was a collection of CDS Hooks prompts suggesting the use of 6 medical calculators in a SMART on FHIR medical reference app. The primary outcome was the percentage of provider-patient interactions in which the app was used to view a recommended calculator. Secondary outcomes were app usage stratified by individual calculators. RESULTS: Intervention group providers viewed a study calculator in the app in 6.0% of interactions compared to 2.6% in the control group (odds ratio = 2.45, 95% CI, 1.2-5.2, P value .02), an increase of 130%. App use was significantly greater for 2 of 6 calculators. DISCUSSION AND CONCLUSION: Contextually relevant CDS Hooks prompts led to a significant increase in SMART on FHIR app utilization. This demonstrates the potential of using CDS Hooks to guide appropriate use of SMART on FHIR apps and was a primary motivation for the development of the standard. Future research may evaluate potential impacts on clinical care decisions and outcomes.

Infrared Photoactivation Boosts Reporter Ion Yield in Isobaric Tagging.

Isobaric tagging facilitates multiplexed experiments that can determine sequences and relative amounts of peptides in biological samples using tandem mass spectrometry (MSn). Limited reporter ion generation limits quantitative accuracy and precision. As reporter ions are susceptible to unintended fragmentation and scattering by high-energy collisions, we activated peptides with IR photons and prevented successive dissociation of generated reporter ions with ion parking, which altogether boosted reporter ion yield by up to 55%. Even so, unintended co-isolation of contaminating peaks in MS2 experiments distorts reporter ion intensities and can distort quantitative information. MS3 experiments address contamination by generating reporter ions via collisional activation (HCD) of one or more peptide product ions rather than the isolated peptide precursor ion. Because HCD performance is related to m/z, activation of multiple synchronously isolated product ions generates less than optimal reporter ion intensities. In this work, we show that using infrared multiphoton dissociation, which is not dependent on m/z, to generate reporter ions from 10 synchronously isolated peptide product ions results in a 2.4-fold increase in reporter ion intensities, significantly enhancing the sensitivity and dynamic range of quantitation via isobaric tagging.

Evaluation in Life Cycle of Information Technology (ELICIT) framework: Supporting the innovation life cycle from business case assessment to summative evaluation.

OBJECTIVE: Our objective was to develop an evaluation framework for electronic health record (EHR)-integrated innovations to support evaluation activities at each of four information technology (IT) life cycle phases: planning, development, implementation, and operation. METHODS: The evaluation framework was developed based on a review of existing evaluation frameworks from health informatics and other domains (human factors engineering, software engineering, and social sciences); expert consensus; and real-world testing in multiple EHR-integrated innovation studies. RESULTS: The resulting Evaluation in Life Cycle of IT (ELICIT) framework covers four IT life cycle phases and three measure levels (society, user, and IT). The ELICIT framework recommends 12 evaluation steps: (1) business case assessment; (2) stakeholder requirements gathering; (3) technical requirements gathering; (4) technical acceptability assessment; (5) user acceptability assessment; (6) social acceptability assessment; (7) social implementation assessment; (8) initial user satisfaction assessment; (9) technical implementation assessment; (10) technical portability assessment; (11) long-term user satisfaction assessment; and (12) social outcomes assessment. DISCUSSION: Effective evaluation requires a shared understanding and collaboration across disciplines throughout the entire IT life cycle. In contrast with previous evaluation frameworks, the ELICIT framework focuses on all phases of the IT life cycle across the society, user, and IT levels. Institutions seeking to establish evaluation programs for EHR-integrated innovations could use our framework to create such shared understanding and justify the need to invest in evaluation. CONCLUSION: As health care undergoes a digital transformation, it will be critical for EHR-integrated innovations to be systematically evaluated. The ELICIT framework can facilitate these evaluations.

Establishing a multidisciplinary initiative for interoperable electronic health record innovations at an academic medical center.

OBJECTIVE: To establish an enterprise initiative for improving health and health care through interoperable electronic health record (EHR) innovations. MATERIALS AND METHODS: We developed a unifying mission and vision, established multidisciplinary governance, and formulated a strategic plan. Key elements of our strategy include establishing a world-class team; creating shared infrastructure to support individual innovations; developing and implementing innovations with high anticipated impact and a clear path to adoption; incorporating best practices such as the use of Fast Healthcare Interoperability Resources (FHIR) and related interoperability standards; and maximizing synergies across research and operations and with partner organizations. RESULTS: University of Utah Health launched the ReImagine EHR initiative in 2016. Supportive infrastructure developed by the initiative include various FHIR-related tooling and a systematic evaluation framework. More than 10 EHR-integrated digital innovations have been implemented to support preventive care, shared decision-making, chronic disease management, and acute clinical care. Initial evaluations of these innovations have demonstrated positive impact on user satisfaction, provider efficiency, and compliance with evidence-based guidelines. Return on investment has included improvements in care; over $35 million in external grant funding; commercial opportunities; and increased ability to adapt to a changing healthcare landscape. DISCUSSION: Key lessons learned include the value of investing in digital innovation initiatives leveraging FHIR; the importance of supportive infrastructure for accelerating innovation; and the critical role of user-centered design, implementation science, and evaluation. CONCLUSION: EHR-integrated digital innovation initiatives can be key assets for enhancing the EHR user experience, improving patient care, and reducing provider burnout.

Estimating Knee Joint Load Using Acoustic Emissions During Ambulation.

Quantifying joint load in activities of daily life could lead to improvements in mobility for numerous people; however, current methods for assessing joint load are unsuitable for ubiquitous settings. The aim of this study is to demonstrate that joint acoustic emissions contain information to estimate this internal joint load in a potentially wearable implementation. Eleven healthy, able-bodied individuals performed ambulation tasks under varying speed, incline, and loading conditions while joint acoustic emissions and essential gait measures-electromyography, ground reaction forces, and motion capture trajectories-were collected. The gait measures were synthesized using a neuromuscular model to estimate internal joint contact force which was the target variable for subject-specific machine learning models (XGBoost) trained based on spectral, temporal, cepstral, and amplitude-based features of the joint acoustic emissions. The model using joint acoustic emissions significantly outperformed (p < 0.05) the best estimate without the sounds, the subject-specific average load (MAE = 0.31 ± 0.12 BW), for both seen (MAE = 0.08 ± 0.01 BW) and unseen (MAE = 0.21 ± 0.05 BW) conditions. This demonstrates that joint acoustic emissions contain information that correlates to internal joint contact force and that information is consistent such that unique cases can be estimated.

Low-profile elastic exosuit reduces back muscle fatigue.

We investigated the extent to which an un-motorized, low-profile, elastic exosuit reduced the rate of fatigue for six lumbar extensor muscles during leaning. Six healthy subjects participated in an A-B-A (withdrawal design) study protocol, which involved leaning at 45º for up to 90 s without exosuit assistance (A1), then with assistance (B), then again without assistance (A2). The exosuit provided approximately 12-16 Nm of lumbar extension torque. We measured lumbar muscle activity (via surface electromyography) and assessed fatigue rate via median frequency slope. We found that five of the six subjects showed consistent reductions in fatigue rate (ranging from 26% to 87%) for a subset of lumbar muscles (ranging from one to all six lumbar muscles measured). These findings objectively demonstrate the ability of a low-profile elastic exosuit to reduce back muscle fatigue during leaning, which may improve endurance for various occupations.

Troponin Testing and Coronary Syndrome in Geriatric Patients With Nonspecific Complaints: Are We Overtesting?

BACKGROUND: Elderly patients presenting to the emergency department (ED) with nonspecific complaints (NSCs) often undergo troponin testing to assess for atypical acute coronary syndrome (ACS). However, the rate of ACS and utility of troponin testing in this population is unknown. We sought to determine the rate of ACS and diagnostic yield of troponin testing in elderly patients with NSCs. METHODS: We retrospectively identified all patients aged ≥ 65 years triaged in the ED with NSCs from January 1, 2017, to June 30, 2017. NSCs were defined a priori and included complaints such as weakness, dizziness, or fatigue. NSCs were verified in ED provider notes by trained abstractors blind to testing results. Exclusions were focal chief complaint in provider notes, fever, and no troponin ordered. ACS was strictly defined and independently adjudicated by two trained physician researchers blind to the study hypothesis. We calculated the proportion of patients with ACS within 30 days and the test characteristics of troponin to diagnose ACS. RESULTS: Screening identified 1,146 encounters, and 552 were excluded for fever or focal chief complaints in the provider notes. Of the remaining 594 patients, troponin was ordered in 412 (69%), comprising the study cohort. The mean (±SD) age was 78.7 (±8.3) years, with 58% female and 75% admitted. Troponin elevation occurred in 81 patients (20%). ACS occurred in 5 of 412 (1.2%). Troponin was 100% sensitive (95% confidence interval [CI] = 48% to 100%) and 81% specific (95% CI = 77% to 85%) for ACS. Of patients with elevated troponin, 93.8% were false positives (no ACS). All patients with troponin elevation were admitted, but only one underwent angiography and no patients received reperfusion therapy. CONCLUSIONS: While consideration for ACS is prudent in selected elderly patients with NSCs, ACS was rare and no patients received reperfusion therapy. Given the false-positive rate in our study, our results may not support routine troponin testing for ACS in this population.

Evidence of polyclonality in neurofibromatosis type 2-associated multilobulated vestibular schwannomas.

BACKGROUND: Neurofibromatosis type 2 (NF2) is a tumor syndrome that results from mutation of the NF2 tumor suppressor gene. The hallmark of NF2 is the presence of bilateral vestibular schwannoma (VS). Though NF2-associated and sporadic VS share identical histopathologic findings and cytogenetic alterations, NF2-associated VS often appears multilobulated, is less responsive to radiosurgery, and has worse surgical outcomes. Temporal bone autopsy specimens and MRI of the inner ear performed on NF2 patients suggest that multiple discrete tumors may be present within the labyrinth and cerebellopontine angle. METHODS: Treatment-naïve ears in patients enrolled in a prospective NF2 natural history study (NIH#08-N-0044) were included for MRI analysis. T2-weighted and postcontrast T1-weighted MRIs were evaluated for the presence of multiple discrete tumors or a multilobulated mass. Peripheral blood (germline) and regional samples of tumor tissue were procured from consecutive patients enrolled in this study undergoing resection of a multilobulated VS (MVS). Histopathologic evaluation and genetic analysis (single nucleotide polymorphism array analysis, NF2 sequencing) were performed on each specimen. RESULTS: Over half of NF2 ears harbored either an MVS (60/139 ears) or multiple discrete masses (19/139 ears). For 4 successive MVSs, genetic analysis revealed an admixture of cell populations, each with its own somatic NF2 mutation or deletion. CONCLUSIONS: These findings suggest that the majority of NF2-associated VSs are polyclonal, such that the tumor mass represents a collision of multiple, distinct tumor clones. This explains the characteristic lobulated gross appearance of NF2-associated VS, and may also explain the substantially different treatment outcomes compared with sporadic VS.

Mechanism of superoxide generation by neuronal nitric-oxide synthase.

Neuronal nitric-oxide synthase (NOS I) in the absence of L-arginine has previously been shown to generate superoxide (O-2) (Pou, S., Pou, W. S., Bredt, D. S., Snyder, S. H., and Rosen, G. M. (1992) J. Biol. Chem. 267, 24173-24176). In the presence of L-arginine, NOS I produces nitric oxide (NO.). Yet the competition between O2 and L-arginine for electrons, and by implication formation of O-2, has until recently remained undefined. Herein, we investigated this relationship, observing O-2 generation even at saturating levels of L-arginine. Of interest was the finding that the frequently used NOS inhibitor NG-monomethyl L-arginine enhanced O-2 production in the presence of L-arginine because this antagonist attenuated NO. formation. Whereas diphenyliodonium chloride inhibited O-2, blockers of heme such as NaCN, 1-phenylimidazole, and imidazole likewise prevented the formation of O-2 at concentrations that inhibited NO. formation from L-arginine. Taken together these data demonstrate that NOS I generates O-2 and the formation of this free radical occurs at the heme domain.

Clostridial vaccination efficacy on stimulating and maintaining an immune response in beef cows and calves.

Experiments were conducted to determine the efficacy in stimulating and maintaining an immune response in the presence of maternal antibodies, compare the extent of the anamnestic responses to revaccination, and compare the maternal antibody response of 2- or 5-mL clostridial vaccination. In Exp. 1, 118 nursing calves were randomly assigned to receive a 2-mL (Alpha-7, A7) or a 5-mL clostridial vaccine (Ultrabac 7; UB7) at 50.4 +/- 15.30 (X +/- SD) d of age (d 0 = date of calving). Calves were revaccinated with the same treatment on d 170. Blood samples were collected from 10 calves of each treatment group on d 50, 170, and 191 to determine antitoxin units for Clostridium perfringens type C (PC) and D (PD) and agglutination titers for Cl. chauvoei (CC). The A7-treated calves tended (P < .10) to have higher PC units on d 170, an increased rate of change in PD units from d 170 to d 191 (P < .06), and a tendency (P < .10) for enhanced CC titers on d 191 compared with UB7-treated calves. In Exp. 2, 109 pregnant cows and 83 pregnant heifers were randomly assigned within a 2 x 2 x 2 factorial design. The main effects were dam age (cow or heifer), dam treatment (A7 or UB7), and calf treatment (A7 or UB7). Dams were vaccinated with A7 or UB7 d 124 prepartum (d -124) and d 53 after birth. At d 53.4 +/- 12.88, calves were vaccinated with A7 or UB7 (d 53). Calves were revaccinated with the same treatment on d 173. Blood samples were collected from 10 dams per treatment group on d -124, 53, and 173 and from their calves on d 53, 173, and 194. Cows had higher antitoxin levels for PC (P < .01) and PD (P < .05) than heifers. The A7-treated dams had higher (P < .01) PD units on d 53 and d 173 and CC on d 173 than did UB7-treated dams. Calves from A7-treated cows had higher (P < .03) PD units on d 53 than calves from UB7-treated cows. The A7-treated calves had higher titers for CC (P < .01) on d 173 than did UB7-treated calves, and this enhanced level seemed to continue to d 194 (P < .08). In conclusion, titers for clostridial diseases in 50- to 53-d-old calves can be enhanced if dams are vaccinated approximately 4 mo before calving, and 120 d between clostridial vaccinations seems to be too long for adequate protection.

Biological studies of a nitroso compound that releases nitric oxide upon illumination.

2-Methyl-2-nitrosopropane (MNP) has long been known to undergo photochemical and thermal decomposition, generating di-tert-butyl nitroxide, in organic solvent. The present study was undertaken to demonstrate that MNP can be used as a caged-nitric oxide (NO), which can liberate NO upon illumination. Photolysis of MNP leads to the generation of tert-butyl radical and NO, as detected by spin-trapping/ESR spectroscopy and by oxyhemoglobin/visible spectroscopy, respectively. Using soluble guanylate cyclase in neuroblastoma N1E-115 cells as an NO target, we found that MNP in the presence of light caused a dose- and time-dependent increase in cGMP. Finally, illumination of a solution of MNP was also found to induce relaxation of preconstricted isolated rat pulmonary artery rings. These studies demonstrated that MNP can be useful biochemical research tool for delivering NO in a controlled manner, by using light.

Can nitric oxide be spin trapped by nitrone and nitroso compounds?

Increasing interest in the study of nitric oxide (NO.) in many facets of biological research necessitates a search for accurate techniques to directly identify the free radical. One recently employed strategy for NO. detection is the method of electron spin resonance (ESR) used in combination with nitrone and nitroso spin traps. Applying this technique to our studies with nitric oxide synthase (NOS), we found that NO. generated directly from the enzyme system could not be detected. Further investigation revealed that 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS) inhibited NO. generation by NOS at concentrations used for spin trapping. Reexamining the ability of various nitrones and DBNBS to spin trap authentic NO. dissolved in buffer, we obtained ESR spectra similar to those previously reported for the spin trap DBNBS. However, continuing our studies with 15NO. and N-hydroxylamine, we found these spectra to be artifactual. Our results emphasize the need to synthesize new spin traps, since currently available compounds are not capable of spin trapping NO. generated by NOS.

Unfolding and release of heme from human hemoglobins A, S and F.

1. Analysis of the Soret spectra of hemoglobins A, S and F has been used to determine the extent of heme exposure and release from these hemoglobins in the presence of several solvent perturbants. 2. Oxyhemoglobin S unfolding in the presence of either urea or propyl urea resulted in greater heme exposure and release than either oxyhemoglobins A or F. 3. Methemoglobin formation resulted in lower denaturation midpoints for each hemoglobin compared to the reduced oxyhemoglobin state; methemoglobin F had the lowest denaturation midpoint under isothermal denaturing conditions. 4. Rate of heme exposure was greater for oxyhemoglobin S than oxyhemoglobin A in the presence of 200 microM the anionic detergent sodium dodecyl sulfate. 5. Evidence for increased levels of heme release in hemoglobin S may be related to the greater tendency of sickled red cell membranes to undergo lipid oxidation.