A Proteomics Pipeline for Generating Clinical Grade Biomarker Candidates from Data-Independent Acquisition Mass Spectrometry (DIA-MS) Discovery.

Clinical biomarker development has been stymied by inaccurate protein quantification from mass spectrometry (MS) discovery data and a prolonged validation process. To mitigate these issues, we created the Targeted Extraction Assessment of Quantification (TEAQ) software package that uses data-independent acquisition analysis from a discovery cohort to select precursors, peptides, and proteins that adhere to analytical criteria required for established targeted assays. TEAQ was applied to DIA-MS data from plasma samples acquired on a new high resolution accurate mass (HRAM) mass spectrometry platform where precursors were evaluated for linearity, specificity, repeatability, reproducibility, and intra-protein correlation based on 8- or 11-point loading curves at three throughputs. This data can be used as a general resource for developing other targeted assays. TEAQ analysis of data from a case and control cohort for inflammatory bowel disease (n=492) identified 1110 signature peptides for 326 quantifiable proteins from the 1179 identified proteins. Applying TEAQ analysis to discovery data will streamline targeted assay development and the transition to validation and clinical studies.

Fast and deep phosphoproteome analysis with the Orbitrap Astral mass spectrometer.

Owing to its roles in cellular signal transduction, protein phosphorylation plays critical roles in myriad cell processes. That said, detecting and quantifying protein phosphorylation has remained a challenge. We describe the use of a novel mass spectrometer (Orbitrap Astral) coupled with data-independent acquisition (DIA) to achieve rapid and deep analysis of human and mouse phosphoproteomes. With this method, we map approximately 30,000 unique human phosphorylation sites within a half-hour of data collection. The technology is benchmarked to other state-of-the-art MS platforms using both synthetic peptide standards and with EGF-stimulated HeLa cells. We apply this approach to generate a phosphoproteome multi-tissue atlas of the mouse. Altogether, we detect 81,120 unique phosphorylation sites within 12 hours of measurement. With this unique dataset, we examine the sequence, structural, and kinase specificity context of protein phosphorylation. Finally, we highlight the discovery potential of this resource with multiple examples of phosphorylation events relevant to mitochondrial and brain biology.

Paradigm shift in biomarker translation: a pipeline to generate clinical grade biomarker candidates from DIA-MS discovery.

Clinical biomarker development has been stymied by inaccurate protein quantification from mass spectrometry (MS) discovery data and a prolonged validation process. To mitigate these issues, we created the Targeted Extraction Assessment of Quantification (TEAQ) software package. This innovative tool uses the discovery cohort analysis to select precursors, peptides, and proteins that adhere to established targeted assay criteria. TEAQ was applied to Data-Independent Acquisition MS data from plasma samples acquired on an Orbitrap™ Astral™ MS. Identified precursors were evaluated for linearity, specificity, repeatability, reproducibility, and intra-protein correlation from 11-point loading curves under three throughputs, to develop a resource for clinical-grade targeted assays. From a clinical cohort of individuals with inflammatory bowel disease (n=492), TEAQ successfully identified 1116 signature peptides for 327 quantifiable proteins from 1180 identified proteins. Embedding stringent selection criteria adaptable to targeted assay development into the analysis of discovery data will streamline the transition to validation and clinical studies.

The One Hour Human Proteome.

We describe deep analysis of the human proteome in less than 1 h. We achieve this expedited proteome characterization by leveraging state-of-the-art sample preparation, chromatographic separations, and data analysis tools, and by using the new Orbitrap Astral mass spectrometer equipped with a quadrupole mass filter, a high-field Orbitrap mass analyzer, and an asymmetric track lossless (Astral) mass analyzer. The system offers high tandem mass spectrometry acquisition speed of 200 Hz and detects hundreds of peptide sequences per second within data-independent acquisition or data-dependent acquisition modes of operation. The fast-switching capabilities of the new quadrupole complement the sensitivity and fast ion scanning of the Astral analyzer to enable narrow-bin data-independent analysis methods. Over a 30-min active chromatographic method consuming a total analysis time of 56 min, the Q-Orbitrap-Astral hybrid MS collects an average of 4319 MS1 scans and 438,062 tandem mass spectrometry scans per run, producing 235,916 peptide sequences (1% false discovery rate). On average, each 30-min analysis achieved detection of 10,411 protein groups (1% false discovery rate). We conclude, with these results and alongside other recent reports, that the 1-h human proteome is within reach.

Ultra-fast label-free quantification and comprehensive proteome coverage with narrow-window data-independent acquisition.

Mass spectrometry (MS)-based proteomics aims to characterize comprehensive proteomes in a fast and reproducible manner. Here we present the narrow-window data-independent acquisition (nDIA) strategy consisting of high-resolution MS1 scans with parallel tandem MS (MS/MS) scans of ~200 Hz using 2-Th isolation windows, dissolving the differences between data-dependent and -independent methods. This is achieved by pairing a quadrupole Orbitrap mass spectrometer with the asymmetric track lossless (Astral) analyzer which provides >200-Hz MS/MS scanning speed, high resolving power and sensitivity, and low-ppm mass accuracy. The nDIA strategy enables profiling of >100 full yeast proteomes per day, or 48 human proteomes per day at the depth of ~10,000 human protein groups in half-an-hour or ~7,000 proteins in 5 min, representing 3× higher coverage compared with current state-of-the-art MS. Multi-shot acquisition of offline fractionated samples provides comprehensive coverage of human proteomes in ~3 h. High quantitative precision and accuracy are demonstrated in a three-species proteome mixture, quantifying 14,000+ protein groups in a single half-an-hour run.

Fast and Deep Phosphoproteome Analysis with the Orbitrap Astral Mass Spectrometer.

Owing to its roles in cellular signal transduction, protein phosphorylation plays critical roles in myriad cell processes. That said, detecting and quantifying protein phosphorylation has remained a challenge. We describe the use of a novel mass spectrometer (Orbitrap Astral) coupled with data-independent acquisition (DIA) to achieve rapid and deep analysis of human and mouse phosphoproteomes. With this method we map approximately 30,000 unique human phosphorylation sites within a half-hour of data collection. We applied this approach to generate a phosphoproteome multi-tissue atlas of the mouse. Altogether, we detected 81,120 unique phosphorylation sites within 12 hours of measurement. With this unique dataset, we examine the sequence and structural context of protein phosphorylation. Finally, we highlight the discovery potential of this resource with multiple examples of novel phosphorylation events relevant to mitochondrial and brain biology.

Parallelized Acquisition of Orbitrap and Astral Analyzers Enables High-Throughput Quantitative Analysis.

The growing trend toward high-throughput proteomics demands rapid liquid chromatography-mass spectrometry (LC-MS) cycles that limit the available time to gather the large numbers of MS/MS fragmentation spectra required for identification. Orbitrap analyzers scale performance with acquisition time and necessarily sacrifice sensitivity and resolving power to deliver higher acquisition rates. We developed a new mass spectrometer that combines a mass-resolving quadrupole, the Orbitrap, and the novel Asymmetric Track Lossless (Astral) analyzer. The new hybrid instrument enables faster acquisition of high-resolution accurate mass (HRAM) MS/MS spectra compared with state-of-the-art mass spectrometers. Accordingly, new proteomics methods were developed that leverage the strengths of each HRAM analyzer, whereby the Orbitrap analyzer performs full scans with a high dynamic range and resolution, synchronized with the Astral analyzer's acquisition of fast and sensitive HRAM MS/MS scans. Substantial improvements are demonstrated over previous methods using current state-of-the-art mass spectrometers.

Evaluating the Performance of the Astral Mass Analyzer for Quantitative Proteomics Using Data-Independent Acquisition.

We evaluate the quantitative performance of the newly released Asymmetric Track Lossless (Astral) analyzer. Using data-independent acquisition, the Thermo Scientific Orbitrap Astral mass spectrometer quantifies 5 times more peptides per unit time than state-of-the-art Thermo Scientific Orbitrap mass spectrometers, which have long been the gold standard for high-resolution quantitative proteomics. Our results demonstrate that the Orbitrap Astral mass spectrometer can produce high-quality quantitative measurements across a wide dynamic range. We also use a newly developed extracellular vesicle enrichment protocol to reach new depths of coverage in the plasma proteome, quantifying over 5000 plasma proteins in a 60 min gradient with the Orbitrap Astral mass spectrometer.

Evaluating the Performance of the Astral Mass Analyzer for Quantitative Proteomics Using Data Independent Acquisition.

We evaluate the quantitative performance of the newly released Asymmetric Track Lossless (Astral) analyzer. Using data independent acquisition, the Thermo Scientific™ Orbitrap™ Astral™ mass spectrometer quantifies 5 times more peptides per unit time than state-of-the-art Thermo Scientific™ Orbitrap™ mass spectrometers, which have long been the gold standard for high resolution quantitative proteomics. Our results demonstrate that the Orbitrap Astral mass spectrometer can produce high quality quantitative measurements across a wide dynamic range. We also use a newly developed extra-cellular vesicle enrichment protocol to reach new depths of coverage in the plasma proteome, quantifying over 5,000 plasma proteins in a 60-minute gradient with the Orbitrap Astral mass spectrometer.

Biodiversity of Phototrophs and Culturable Fungi in Gobustan Caves.

Unique natural objects, such as the caves of the Gobustan National Historical and Artistic Preserve, are also of great cultural and historical value due to rock art and sites of ancient people. A favorable microclimate makes these habitats convenient for colonization by microbiota, including phototrophs. In arid regions with intense seasonal fluctuations of microclimatic parameters, the conditions for survival are the least favorable; therefore, it becomes especially important to determine the composition of communities that are the most adapted to specific conditions. This work aimed to identify the biodiversity of communities of caves and grottoes of the Gobustan Reserve. The studies were carried out in July 2019. Samples were analyzed for cyanobacteria and algae by microscopy and cultivation methods, microfungi were isolated by soil dilution, and the fouling glass method was also used. In total, 29 taxa of cyanobacteria and algae, 18 taxa of fungi, and 3 species of mosses were identified. The studied habitats were dominated by the algae Chlorella vulgaris, Aphanocapsa sp., and Stichococcus bacillaris; the subdominants were Jaaginema subtilissimum, Leptolyngbya tenuis, Chlorococcum minutum, and Humidophila contenta. Microfungi had the highest occurrence of Aspergillus niger, Aureobasidium pullulans, Alternaria alternata, and Talaromyces ruber. It was noted that cyanobacteria dominated in morphologically differentiated biofilms and green algae on the rocks. The greatest number of microfungi was found in the aphotic zone and bryophyte tufts. The dominance of green algae is atypical for most caves of other regions and may be associated with intense lighting of habitats. The absence of protonema is a consequence of the aridity and low moisture content of the substrates.

The State of Dark Coniferous Forests on the East European Plain Due to Climate Change.

As a result of global climate changes, negative processes have been recorded in the coniferous forests of the Northern Hemisphere. Similar processes are observed in the Urals, including in Udmurtia. In the course of this research, archival analysis methods were used, as well as field research methods. In the process of analyzing archival materials in the Urals, a reduction of spruce forests was observed. If in the 20th century the share of spruce forests in the region was 50%, then in the 21th century it decreased to 35%. As a result of this research, it was revealed that the most unfavorable sanitary condition was recorded in the boreal-subboreal zone of Udmurtia, with a sanitary condition index of 3.2 (from 2.62 to 3.73). The main reason for the unfavorable sanitary condition of spruce forests was the vital activity of Ips typographus L. According to our research, in 11 sample plots out of 18, a high score for sanitary condition was associated with the vital activity of bark beetles. The correlation coefficient of the index of the sanitary condition of plantings and the number of individuals of Picea obovata Ledeb. affected by Ips typographus L. was0.93. Bark beetle activity has increased in the 21th century, which is associated with changing climatic factors. Unstable precipitation over recent years (differences of more than 100 mm) and an average temperature increase of 1.2 °C were observed in the region. The most significant increase in temperature over the past 10 years was observed in winter, which in turn affected the high survival rate of insect pests.

Protecting the Airway and the Physician: Lessons from 214 Cases of Endotracheal Intubation Litigation.

Objective: Medicolegal examination of an intervention as common as endotracheal intubation may be valuable to physicians in many specialties. Our objectives were to comprehensively detail the factors raised in litigation to better educate physicians on strategies for minimizing liability and augmenting patient safety. Methods: Publicly available court records were searched for pertinent litigation. Ultimately, 214 jury verdict and settlement reports were examined for various factors, including outcome, award, geographic location, defendant specialty, setting in which an injury occurred, patient demographics, and other causes of malpractice. Results: Ninety-two cases (43.0%) were resolved in the defendant's favor, with the remaining cases resulting in out-of-court settlement or a plaintiff's verdict. Payments from these cases were considerable, averaging $2.5 M. The most frequent physician defendants were anesthesiologists (59.8%) and emergency-physicians (19.2%), although other specialties were well represented. The most common setting of injury was the operating room (45.3%). Common factors included sustaining permanent deficits (89.2%), death (50.5%), and anoxic brain injury (37.4%). Injuries occurring in labor and delivery mostly involved newborns and had among the highest awards. Conclusions: Litigation involves injuries sustained in numerous settings. The most common factors present included sustaining permanent deficits, including anoxic brain injury. The presence of this latter injury increased the likelihood of a case being resolved with payment. Finally, deficits in informed consent were noted in numerous cases, stressing the importance of a clear process in which the physician explains specific risks (such as those detailed in this analysis), benefits, and alternatives.

Assessing the Readability of Anesthesia-Related Patient Education Materials from Major Anesthesiology Organizations.

Introduction: The National Institutes of Health (NIH), American Medical Association (AMA), and the US Department of Health and Human Services (USDHHS) recommend that patient education materials (PEMs) be written between the 4th to 6th grade reading level to ensure readability by the average American. In this study, we examine the reading levels of online patient education materials from major anesthesiology organizations. Methods: Readability analysis of PEMs found on the websites of anesthesiology organizations was performed using the Flesch Reading Ease score, Flesch-Kincaid Grade Level, Simple Measure of Gobbledygook, Gunning Frequency of Gobbledygook, New Dale-Chall test, Coleman-Liau Index, New Fog Count, Raygor Readability Estimate, the FORCAST test, and the Fry Score. Results: Most patient educational materials from the websites of the anesthesiology organizations evaluated were written at or above the 10th grade reading level. Conclusions: Online patient education materials from the major anesthesiology societies are written at levels higher than an average American adult reading skill level and higher than recommended by National Institute of Health, American Medical Association, and US Department of Health and Human Services. Online resources should be revised to improve readability. Simplifying text, using shorter sentences and terms are strategies online resources can implement to improve readability. Future studies should incorporate comprehensibility, user-friendliness, and linguistic ease to further understand the implications on overall healthcare.

Interscholastic Athletics and Bone Strength: The Iowa Bone Development Study.

ABSTRACT: Pashkova, A, Hartman, JM, Letuchy, EM, and Janz, KF. Interscholastic athletics and bone strength: the Iowa bone development study. J Strength Cond Res 36(5): 1271-1276, 2022-The objective of this study was to determine the relationship between adolescents' participation in various interscholastic sports and differences in bone strength outcomes. Subjects (N = 380) were recruited from the Iowa Bone Development Study and categorized based on sport participation into 3 power groups: no-power, low-power, and high-power. Sports such as basketball, cheerleading/poms, gymnastics, volleyball, track, football, tennis, and soccer were considered high-power. Peripheral quantitative computed tomography (pQCT) was used to determine bone measures of polar stress-strain index (measure of torsion strength), cortical content (measure of cortical bone size and area at the 66% tibia site), and bone strength index (measure of compression strength based on total bone density and area at the 4% tibia site). Adjusted pairwise comparison for group least squares means high-power sport participation compared with no-power sport participation showed significant differences in all bone strength outcomes for both men and women (p value < 0.01). There was a significant difference in all bone strength measures between low-power and no-power groups for men (p value < 0.05), but not women. Because of decreasing levels of physical activity in late adolescence, the promotion of high-power sports may be particularly important for optimal bone development in the final years before peak bone mass.

Controlled Substance Agreements and Other Best Opioid Prescription Practices.

Controlled substance agreements between providers and patients represent important strategies for setting expectations for chronic opioid therapy. These agreements generally summarize best opioid prescription practices and destigmatize practice policies such as regular toxicology screenings. These controlled substance agreements also set expectations for discontinuation of therapy if appropriate.

Preoperative Optimization for Perioperative Analgesia.

The perioperative analgesic plan begins with preoperative planning. The surgeon should be versed in practical approaches for managing analgesia in patients with chronic pain. The first step includes evaluating the patient and conducting a focused pain history. Confirming, documenting, and understanding current outpatient prescriptions is critical. Patients should be screened for medical conditions that preclude the use of certain analgesics, or place them at higher risk of respiratory depression. Providers should coordinate with the patient's outpatient prescribers and pain specialists to ensure a safe and effective analgesic plan. Multimodal analgesia should be implemented to optimize analgesia and decrease opioid requirements.

Pain Management for the Otolaryngologist: Overview of Perioperative Analgesia and Introduction to Opioids.

Nearly 50,000 US adults experience opioid-overdose deaths annually and 1.7 million experience a substance use disorder from prescription opioids. Hence, understanding analgesia strategies is of utmost importance. A pre-operative analgesic plan can consist of a brief conversation between the surgeon, patient, and anesthesiologist in an uncomplicated case or range all the way to an involved, multidisciplinary plan for a chronic pain patient. Over the past several decades, there have been myriad studies examining perioperative analgesic regimens for otolaryngologic procedures, many of which have demonstrated the efficacy of nonopioid analgesics.

Perioperative Analgesia for Patients Undergoing Thyroidectomy and Parathyroidectomy: An Evidence-Based Review.

OBJECTIVE: To perform an evidence-based systematic review evaluating perioperative analgesia, including opioid alternatives, used for patients undergoing thyroidectomy and parathyroidectomy. METHODS: A comprehensive literature search from 1997 to January 2018 of Pubmed, Cochrane, and EmBase libraries was performed for studies reporting analgesic administration following thyroid or parathyroid surgery. This systematic review was based on the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. Studies were evaluated for level of evidence and given a Jadad score to assess for risk of bias. Outcomes gathered included postoperative pain scores, time to rescue analgesia, rescue analgesic consumption, and adverse events. RESULTS: Thirty-eight randomized controlled trials met inclusion criteria. The GRADE criteria determined the overall evidence to be moderate-high. Studies utilizing NSAIDs reported reduced requirements for rescue analgesics. Acetaminophen studies presented with conflicting data on effectiveness. Gabapentinoid studies demonstrated lower pain scores and an increased time to rescue analgesic. Local anesthetics were effective at decreasing Visual Analogue Scale (VAS) and Numeric Rating Scale (NRS) pain scores while also reducing rescue analgesic consumption. Ketamine was shown to increased postoperative nausea and vomiting. NSAIDs and local anesthetic studies had an aggregate grade of evidence A, while all others had grade B evidence. CONCLUSION: There is significant evidence supporting the use of NSAIDs and local anesthetics in the perioperative period for pain management for thyroid and parathyroid surgeries. Acetaminophen, gabapentinoid and ketamine have some supporting evidence and may serve as adequate alternatives. Further multi-institutional RCTs are warranted to delineate optimal analgesic regimens. LEVEL OF EVIDENCE: NA.

Perioperative analgesia for patients undergoing otologic surgery: An evidence-based review.

OBJECTIVES/HYPOTHESIS: Opioid misuse and diversion is a major concern, with a negative impact on both the individual and society. The objective of this study was to perform an evidence-based systematic review of the efficacy of perioperative analgesic regimens following otologic surgery. METHODS: Embase, Cochrane Library, and PubMed/MEDLINE databases (January 1, 1947 to June 30, 2018) were searched for studies investigating pain management in otologic surgeries. All studies were assessed for quality and bias using the Cochrane bias tool. Patient demographics, type of surgery, medication class, dose, administration characteristics, pain scores, and adverse events were reported. RESULTS: Twenty-three studies encompassing 1,842 patients met inclusion criteria. In 21.4% of studies, an overall reduction in pain scores was reported when the treatment group included more than one analgesic. Nausea and vomiting were the most common adverse events across all medication types (10.2%), with local anesthetic patients experiencing these side effects most frequently (38.0%). Perioperative acetaminophen was reported to have the fewest adverse drug reactions overall (6.1%), but did not reduce pain scores as much as other modalities, such as nonsteroidal anti-inflammatory drugs (NSAIDs) or combination analgesics. CONCLUSIONS: There is evidence that combination analgesics, such as acetaminophen plus codeine, provide superior pain relief to monotherapy analgesics in the perioperative pain management of otologic surgeries. NSAIDs, α-agonists, and nerve blocks may also be viable single-therapy options. Further prospective randomized controlled trials into perioperative analgesia for patients undergoing otologic surgery may be helpful in establishing a definitive consensus. Laryngoscope, 130:190-199, 2020.