Confident Identification Research Articles

Exploring Factors Shaping Primary Health Care Readiness to Respond to Family Violence: Findings from a Rapid Evidence Assessment

Abstract Purpose Family violence (FV) is defined as any situation where an individual employs abusive behaviour to control and/or harm a former or current spouse, non-marital partner, or a member of their family. The health consequences of FV are vast, including a wide range of physical and mental health conditions for individuals experiencing violence or survivors, perpetrators, and their children. Primary health care (PHC) is recognized as a setting uniquely positioned to identify the risk and protective factors for FV, being an entry point into the health care system and a first, or only, point of contact for families with professionals who can facilitate access to specialist care and support. Methods A rapid evidence assessment of empirical studies on FV interventions in PHC was conducted to examine outcomes of effective FV interventions that promote identification, assessment, and care delivery within diverse PHC settings, factors shaping PHC provider and system readiness, and key intervention components that are important for sustaining PHC responses to FV. After completing data extraction, quality appraisal, and a hand search, a total of 49 articles were included in data synthesis and analysis. Results Several FV interventions that include multiple components such as, screening and identification of FV, training of PHC providers, advocacy, and referrals to supports, have been rigorously tested and evaluated in diverse PHC settings in rural and urban areas including primary care/family medicine practice clinics and community PHC centers. These interventions have demonstrated to be effective in identifying and responding to violence primarily experienced by women. There is a dearth of FV interventions or programs from empirical studies focused on men, children, and perpetrators. Additionally, provider and system readiness measurement tools and models have been implemented and evaluated in PHC specifically to assess physician or the health care team’s readiness to manage FV in terms of knowledge and awareness of FV. The findings highlight that there is no clear or standardized definition of provider or system “readiness” in the literature related to FV responses in PHC. Further, the findings revealed four key intervention components to facilitate PHC provider and organization readiness to address FV: (1) multidisciplinary teamwork and collaboration, (2) improving provider knowledge on the social and cultural determinants impacting FV, and (3) embedding system-level supports within PHC. Conclusions FV is a serious public health concern and PHC providers have a vital role in early detection of FV and the poor health outcomes associated with violence A focus on comprehensive or multi-component FV interventions are more likely to change provider behavior, and would allow for safe, confident, and professional identification and assessment of FV within PHC.

Sulfoproteomics Workflow with Precursor Ion Accurate Mass Shift Analysis Reveals Novel Tyrosine Sulfoproteins in the Golgi.

Tyrosine sulfation in the Golgi of secreted and membrane proteins is an important post-translational modification (PTM). However, its labile nature has limited analysis by mass spectrometry (MS), a major reason why no sulfoproteome studies have been previously reported. Here, we show that a phosphoproteomics experimental workflow, which includes serial enrichment followed by high resolution, high mass accuracy MS, and tandem MS (MS/MS) analysis, enables sulfopeptide coenrichment and identification via accurate precursor ion mass shift open MSFragger database search. This approach, supported by manual validation, allows the confident identification of sulfotyrosine-containing peptides in the presence of high levels of phosphorylated peptides, thus enabling these two sterically and ionically similar isobaric PTMs to be distinguished and annotated in a single proteomic analysis. We applied this approach to isolated interphase and mitotic rat liver Golgi membranes and identified 67 tyrosine sulfopeptides, corresponding to 26 different proteins. This work discovered 23 new sulfoproteins with functions related to, for example, Ca2+-binding, glycan biosynthesis, and exocytosis. In addition, we report the first preliminary evidence for crosstalk between sulfation and phosphorylation in the Golgi, with implications for functional control.

Equids (Equus sp.) in southern Spain from the Palaeolithic to the Bronze Age

ABSTRACTThe genus Equus was represented on the Iberian Peninsula by four species during the late Quaternary: the wild, now extinct, E. ferus (wild horse) and E. hydruntinus (European wild ass) and the extant, domestic E. caballus (horse) and E. asinus (donkey). The distribution and timing of the extinctions of the wild species and arrival of the domestic species is important to understand the changing environment and cultures through this dynamic period in one of the three southern Pleistocene glacial refugia in Europe. Here we collected data from all zooarchaeological studies that meet basic completeness criteria from the Palaeolithic to the Bronze Age (45–3.2 ka bp) and analysed the equine data in light of other large mammals at the same sites in order to document the species turnover and distributions through this culturally and ecologically dynamic period. The vast majority of Palaeolithic Equus were confidently identified as E. ferus, and by the Bronze age as E. caballus, with much uncertainty in between. Over time the larger equids (horses) were much more common than the smaller equids (asses). Equids were not common, but they were distributed across the southern Iberian peninsula through the Chalcolithic, and then appear to have become restricted to the drier eastern region in the Bronze Age. These analyses indicate that both E. ferus and E. hydruntinus went extinct by the end of the Pleistocene/Palaeolithic in Andalucia. Not all communities maintained equal numbers of equids, and their distribution changed with the changing climate through time, most notably between the Chalcolithic and the Bronze Age when confidence in species identification and local density of horses increase, but the distribution is restricted to the drier eastern region.

HILIC/MS quantitation of low-abundant phospholipids and sphingolipids in human plasma and serum: Dysregulation in pancreatic cancer

A new hydrophilic interaction liquid chromatography - mass spectrometry method is developed for low-abundant phospholipids and sphingolipids in human plasma and serum. The optimized method involves the Cogent Silica type C hydride column, the simple sample preparation by protein precipitation, and the removal of highly abundant lipid classes using the postcolumn valve directed to waste during two elution windows. The method allows a highly confident and sensitive identification of low-abundant lipid classes in human plasma (246 lipid species from 24 lipid subclasses) based on mass accuracy and retention dependencies in both polarity modes. The method is validated for quantitation using two internal standards (if available) for each lipid class and applied to human plasma and serum samples obtained from patients with pancreatic ductal adenocarcinoma (PDAC), healthy controls, and NIST SRM 1950. Multivariate data analysis followed by various statistical projection methods is used to determine the most dysregulated lipids. Significant downregulation is observed for lysophospholipids with fatty acyl composition 16:0, 18:0, 18:1, and 18:2. Distinct trends are observed for phosphatidylethanolamines (PE) in relation to the bonding type of fatty acyls, where most PE with acyl bonds are upregulated, while ether/plasmenyl PE are downregulated. For the sphingolipid category, sphingolipids with very long N-acyl chains are downregulated, while sphingolipids with shorter N-acyl chains were upregulated in PDAC. These changes are consistently observed for various classes of sphingolipids, ranging from ceramides to glycosphingolipids, indicating a possible metabolic disorder in ceramide biosynthesis caused by PDAC.

Identification of proteoforms of short open reading frame-encoded peptides in Blautia producta under different cultivation conditions.

Short open reading frames (sORF) have the potential to encode small proteins, sORF-encoded peptides (SEP), which are less than or equal to 100 amino acids in length. These SEP have been associated with various physiological processes and are known for their potential roles in modulating immune responses, signaling pathways, and cellular functions. Using bottom-up and top-down proteomics, we identified 45 novel and formerly reported SEP under different growth conditions and stress exposure in single cultures of Blautia producta that plays a central role in mediating colonization resistance and dampening inflammation in the human gut microbiome. Top-down analysis improved identification confidence and allowed the detection of a number of full-length and N- or C-terminal truncated proteoforms of the SEP and of proteoforms carrying post-translational modifications such as disulfides, which provides hints for a wide functional potential of these molecules. The identification of the SEP in single cultures of this bacterium shows that, in contrast to previous results, the biosynthesis of specific SEP is not restricted to direct bacterial interactions within the microbiome but is dependent on growth and environmental conditions during cultivation. IMPORTANCE The identification of short open reading frame-encoded peptides (SEP) and different proteoforms in single cultures of gut microbes offers new insights into a largely neglected part of the microbial proteome landscape. This is of particular importance as SEP provide various predicted functions, such as acting as antimicrobial peptides, maintaining cell homeostasis under stress conditions, or even contributing to the virulence pattern. They are, thus, taking a poorly understood role in structure and function of microbial networks in the human body. A better understanding of SEP in the context of human health requires a precise understanding of the abundance of SEP both in commensal microbes as well as pathogens. For the gut beneficial B. producta, we demonstrate the importance of specific environmental conditions for biosynthesis of SEP expanding previous findings about their role in microbial interactions.

Corneal Specialists’ Confidence in Identifying Causal Organisms of Microbial Keratitis

Purpose Microbial keratitis (MK) is a potentially blinding corneal disease caused by an array of microbial etiologies. However, the lack of early organism identification is a barrier to optimal care. We investigated clinician confidence in their diagnosis of organism type on initial presentation and the relationship between confidence and presenting features. Methods This research presents secondary data analysis of 72 patients from the Automated Quantitative Ulcer Analysis (AQUA) study. Cornea specialists reported their confidence in organism identification. Presenting sample characteristics were recorded including patient demographics, health history, infection morphology, symptoms, and circumstances of infection. The association between confidence and presenting characteristics was investigated with 2-sample t-tests, Wilcoxon tests, and Chi-square or Fisher’s exact tests. Results Clinicians reported being “confident or very confident” in their diagnosis of the causal organism in MK infections for 39 patients (54%) and “not confident” for 33 patients (46%). Confidence was not significantly associated with patient demographics, morphologic features, or symptoms related to MK. MK cases where clinicians reported they were confident, versus not confident in their diagnosis, showed significantly smaller percentages of previous corneal disease (0% versus 15%, p = 0.017), were not seen by an outside provider first (69% versus 94%, p = 0.015), or had no prior labs drawn (8% versus 33%, p = 0.046), and a significantly larger percentage of cases wore contact lenses (54% versus 28%, p = 0.029). Conclusion In almost half of MK cases, cornea specialists reported lack of confidence in identifying the infection type. Confidence was related to ocular history and circumstances of infection but not by observable signs and symptoms or patient demographics. Tools are needed to assist clinicians with early diagnosis of MK infection type to expedite care and healing.

Mass spectrometry imaging of natural carbonyl products directly from agar-based microbial interactions using 4-APEBA derivatization.

Aliphatic carboxylic acids, aldehydes, and ketones play diverse roles in microbial adaptation to their microenvironment, from excretion as toxins to adaptive metabolites for membrane fluidity. However, the spatial distribution of these molecules throughout biofilms and how microbes in these environments exchange these molecules remain elusive for many of these bioactive species due to inefficient molecular imaging strategies. Herein, we apply on-tissue chemical derivatization (OTCD) using 4-(2-((4-bromophenethyl)dimethylammonio)ethoxy)benzenaminium dibromide (4-APEBA) on a co-culture of a soil bacterium (Bacillus subtilis NCIB 3610) and fungus (Fusarium sp. DS 682) grown on agar as our model system. Using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), we spatially resolved more than 300 different metabolites containing carbonyl groups within this model system. Various spatial patterns are observable in these species, which indicate possible extracellular or intercellular processes of the metabolites and their up- or downregulation during microbial interaction. The unique chemistry of our approach allowed us to bring additional confidence in accurate carbonyl identification, especially when multiple isomeric candidates were possible, and this provided the ability to generate hypotheses about the potential role of some aliphatic carbonyls in this B. subtilis/Fusarium sp. interaction. The results shown here demonstrate the utility of 4-ABEBA-based OTCD MALDI-MSI in probing interkingdom interactions directly from microbial co-cultures, and these methods will enable future microbial interaction studies with expanded metabolic coverage.IMPORTANCEThe metabolic profiles within microbial biofilms and interkingdom interactions are extremely complex and serve a variety of functions, which include promoting colonization, growth, and survival within competitive and symbiotic environments. However, measuring and differentiating many of these molecules, especially in an in situ fashion, remains a significant analytical challenge. We demonstrate a chemical derivatization strategy that enabled highly sensitive, multiplexed mass spectrometry imaging of over 300 metabolites from a model microbial co-culture. Notably, this approach afforded us to visualize over two dozen classes of ketone-, aldehyde-, and carboxyl-containing molecules, which were previously undetectable from colonies grown on agar. We also demonstrate that this chemical derivatization strategy can enable the discrimination of isobaric and isomeric metabolites without the need for orthogonal separation (e.g., online chromatography or ion mobility). We anticipate that this approach will further enhance our knowledge of metabolic regulation within microbiomes and microbial systems used in bioengineering applications.

Measurement of very low-molecular weight metabolites by traveling wave ion mobility and its use in human urine samples

The collision cross-sections (CCS) measurement using ion mobility spectrometry (IMS) in combination with mass spectrometry (MS) offers a great opportunity to increase confidence in metabolite identification. However, owing to the lack of sensitivity and resolution, IMS has an analytical challenge in studying the CCS values of very low-molecular-weight metabolites (VLMs ≤ 250 Da). Here, we describe an analytical method using ultrahigh-performance liquid chromatography (UPLC) coupled to a traveling wave ion mobility-quadrupole-time-of-flight mass spectrometer optimized for the measurement of VLMs in human urine samples. The experimental CCS values, along with mass spectral properties, were reported for the 174 metabolites. The experimental data included the mass-to-charge ratio (m/z), retention time (RT), tandem MS (MS/MS) spectra, and CCS values. Among the studied metabolites, 263 traveling wave ion mobility spectrometry (TWIMS)-derived CCS values (TWCCSN2) were reported for the first time, and more than 70% of these were CCS values of VLMs. The TWCCSN2 values were highly repeatable, with inter-day variations of <1% relative standard deviation (RSD). The developed method revealed excellent TWCCSN2 accuracy with a CCS difference (ΔCCS) within ±2% of the reported drift tube IMS (DTIMS) and TWIMS CCS values. The complexity of the urine matrix did not affect the precision of the method, as evidenced by ΔCCS within ±1.92%. According to the Metabolomics Standards Initiative, 55 urinary metabolites were identified with a confidence level of 1. Among these 55 metabolites, 53 (96%) were VLMs. The larger number of confirmed compounds found in this study was a result of the addition of TWCCSN2 values, which clearly increased metabolite identification confidence.

Optimization of plasma extracellular vesicle enrichment for in‐depth lipidomic profiling in Alzheimer’s disease

AbstractBackgroundLipid dyshomeostasis is associated with various disorders including Alzheimer’s disease (AD). The work from our laboratory (Su et al, JEV 2021), and others, suggests that extracellular vesicles (EVs) contain lipid biomarkers that could aid in the diagnosis of AD and diseases associated with impaired lipid metabolism.Knowing the lipid content of EVs is a key first step to discovering EV‐based lipid biomarkers in blood. Some studies have reported the lipid content of ‘’EVs’’ in blood. However, used EV isolation techniques that are known to co‐isolate free lipid and lipoproteins.Here we: 1) demonstrate the importance of size exclusion resin size to isolating highly enriched EVs for lipidomic analysis, 2) have developed a reference EV plasma lipidome for the research community and 3) determining if plasma EVs report on lipid dyshomeostasis in Alzheimer’s disease.MethodHuman plasma EVs were isolated using an assortment of commercially available kits or by density fractionation and size exclusion chromatography and characterised by western blot, transmission electron microscopy, and quantitative mass spectrometry based proteomic and lipidome analysis.ResultOf the high‐throughput EV isolation kits tested, the majority were unable to separate lipoproteins from EVs for the purposes of EV lipidomics. We then relied on comparing labor intensive methods (density gradient and size exclusion) to identify a combination of techniques that isolate the analytes of interest, EV proteins and lipids. Quantitative proteome and lipidome analysis strategies are being applied to highly enriched EVs with the goal of providing the reference lipidome of EVs in plasma and in Alzheimer’s disease.ConclusionTo enhance the confidence of EV lipid identification and reveal the lipidome of EVs in plasma, our study compared and identified techniques to enrich EVs and deplete non‐EV associated lipid and protein from human plasma. These findings serve as a reference resource and the foundation for research investigating plasma EV lipids as Alzheimer’s disease biomarkers.

Secreted HLA-Fc fusion profiles immunopeptidome in hypoxic PDAC and cellular senescence.

Human leukocyte antigens (HLA) present peptides largely from intracellular proteins on cell surfaces. As these complexes can serve as biomarkers in disease, proper identification of peptides derived from disease-associated antigens and the corresponding presenting HLA is important for the design and execution of therapeutic strategies. Yet, current mass spectrometry methods for immunopeptidomic profiling require large and complex sample inputs, hindering the study of certain disease phenotypes and lowering confidence in peptide and allele identification. Here, we describe a secreted HLA (sHLA)-Fc fusion construct for simple single HLA allele profiling in hypoxic pancreatic ductal adenocarcinoma (PDAC) and cellular senescence. This method streamlines sample preparation, enables temporal control, and provides allele-restricted target identification. Over 30,000 unique HLA-associated peptides were identified across 2 different HLA alleles and 7 cell lines, with ∼9,300 peptides newly discovered. The sHLA-Fc fusion capture technology holds the potential to expedite immunopeptidomics and advance therapeutic interest in HLA-peptide complexes.

High-Throughput Identification of Graft-Vs-Leukemia TCRs in AML Patient Samples after Allogeneic Stem Cell Transplantation

For patients with intermediate and high-risk AML/MDS, allogeneic stem cell transplantation (aSCT) is a potentially curative therapy. Efficacy is driven by the graft-versus-leukemia (GvL) response, in which donor-derived T cells recognize peptide:MHC antigens presented by leukemia cells, and subsequently kill them. The antigens that drive GvL responses are attractive therapeutic targets for engineered cell therapies and/or biologics. However, only a small number of GvL antigens have been identified, limiting the therapeutic potential of targeted immunotherapies. Deep interrogation of the T cell repertoires of patients who have responded favorably to aSCT presents an opportunity to identify bona fide GvL antigens, but high-throughput methods need to be developed to do this effectively. A popular method to identify antigen-specific T cells is fluorescent peptide:MHC tetramer staining, whereby T cells whose T cell receptors (TCR) bind a particular peptide:MHC antigen become fluorescent and identifiable by flow cytometry. However, a major challenge to identifying GvL T cells using tetramers is that the prediction of immunogenic antigens - that is, those that induce a T cell response in vivo - is poor; studies of solid tumors suggest that only ~1% of predicted MHC-binding neoantigens are immunogenic in patients. In AML/MDS aSCT patients, there are thousands of peptides that are predicted to bind MHCI and represent potential GvL antigens, but due to practical limitations of patient blood and bone marrow sample quantities, only a small subset can be screened by flow cytometry. Other higher throughput methods using heavy-metal tagged peptide:MHC tetramers followed by mass cytometry, or DNA-barcoded tetramers followed by bulk sequencing have been employed to increase antigen screening scale, but these methods are limited in sensitivity and destroy the T cell without providing TCR sequences. To overcome these limitations and rapidly identify GvL T cells in patient samples, we developed a single cell sequencing based method that enables the screening of thousands of peptide:MHC antigens simultaneously, using a single patient sample. We generated peptide: MHCI monomers using UV-mediated peptide exchange and generated large panels of combinatorial DNA-barcoded peptide:MHCI tetramers at microscale. We integrated our methodology into the 10X Genomics Single Cell Immune Profiling workflow to enable simultaneous recovery of TCR antigen-specificity, paired TCR alpha beta sequences, and single cell RNA-Seq data. These methods were used to screen post-transplant AML patient blood and bone marrow, and we identified expanded GvL T cell clonotypes that target novel minor histocompatibility antigens, overexpressed “aberrant self” proteins, and other leukemia associated antigens. We generated primary CD8 T cells that retrovirally express these TCRs and demonstrate that they are potent and have the expected antigen specificities. The methods described are highly sensitive, specific, efficient, and scalable, and enable high confidence identification of antigen specific clonotypes in patient samples. They markedly expand the throughput compared to existing methods, and simultaneously yield paired alpha beta TCR sequences, facilitating a full characterization of the antigen-TCR drivers of GvL. Furthermore, these methods have broad utility beyond SCT, as they are applicable to studying T cell responses to other immune settings, including anti-cancer, autoimmune, and anti-microbial responses.

THE TADPOLES OF FROGS AND TOADS FOUND IN THE LOWLANDS OF NORTHERN COLOMBIA

Fifty-nine native species and two introduced species of frogs and toads occur in the lowlands of the Caribbean Region of Colombia. Of these 61 species, 54 have life cycles that include an aquatic larval stage. In order to promote ecological studies on these species, a key with illustrations (of 29 species) are provided to allow confident identification for most tadpoles encountered in these lowlands.

Historical sound changes in Central Chadic (Afroasiatic)

Abstract In its entirety as much as with regard to its four branches, the Chadic language family poses challenges to the application of the Neogrammarian-school comparative method, not the least because of the immense time-depth involved and aggravated by certain typological peculiarities of Chadic phonology and morphology as inherited from its Afroasiatic ancestry. This is particularly true for Central Chadic, which—with 80 languages—is the most numerous and most diverse branch of Chadic, which in total counts almost 200 languages and thereby more than half of all known Afroasiatic languages. Both more or less ‘regular’ and ‘sporadic’ sound changes criss-cross the territories currently occupied by speakers of Central Chadic languages in Nigeria, Cameroon, and Chad, which allow confident identification of cognates. Some cognates have survived many millennia of language history practically unchanged with regard to their phonetic realisations, while others differ remarkably from the reconstructed proto-language forms beyond ad hoc recognisability. Recent historical comparative research by Richard Gravina (2014, 2015) and the present author (2022, 2023) involving 66 spoken languages and some 230 cognate lexical items have unearthed much of the linguistic histories behind the massive synchronic diversity of the modern Central Chadic languages. The recent research by the author has added to our understanding of historical Central Chadic phonology by unravelling the phonological processes by which the languages have developed sets of new phonemes, in addition to tracing more or less ‘regular’ and ‘sporadic’ sound changes that the PCC segmental inventories of vowels and consonants underwent. Modern Central Chadic languages show sets of innovative vowels and consonant whose emergence can largely be attributed, besides occasional instances of segmental fusion, to the ‘colouring’ effects of so-called prosodies. This makes the analysis of prosodic features such as palatalisation, labialisation, prenasalisation and glottalisation essential in order to understand the evolution of modern Central Chadic languages.

Influence of extraction windows for data-independent acquisition on feature annotation during suspect screening

Non-target analysis (NTA) using high-resolution mass spectrometry is becoming a useful approach to screen for suspect and unknown chemicals. For comprehensive analyses, data-independent acquisition (DIA), like Sequential Windowed Acquisition of all THeoretical Mass Spectra (SWATH-MS) on Sciex instruments, is necessary, usually followed by library matching for feature annotation. The choice of parameters, such as acquisition window number and size, may influence the comprehensiveness of the suspect features detected. The goal of this study was to assess how mass spectrometric DIA settings may influence the ability to obtain confident annotations and identifications of features in environmental (river water, passive sample extract (PSE)), wastewater (unpreserved and acidified) and biological (urine) sample matrices. Each matrix was analysed using 11 different MS methods, with 5–15 variable size acquisition windows. True positive (TP) annotation (i.e., matching experimental and library spectra) rates were constant for PSE (40%) and highest for urine (18%), wastewater (34% and 36%, unpreserved and acidified, respectively) and river water (8%) when using higher numbers of windows (15). The number of annotated features was highest for PSE (12%) and urine (8.5%) when using more acquisition windows (9 and 14, respectively). Less complex matrices (based on average total ion chromatogram intensities) like river water, unpreserved and acidified wastewater have higher annotation rates (7.5%, 8% and 13.2%, respectively) when using less acquisition windows (5–6), indicating matrix dependency of optimum settings. Library scores varied widely for correct (scores between 6 and 100) as well as incorrect annotations (scores between 2 and 100), making it hard to define specific ideal cut-off values. Results highlight the need for properly curated libraries and careful optimization of SWATH-MS and other DIA methods for each individual matrix, finding the best ratio of total annotations to true positive, (i.e., correct) annotations to achieve best NTA results.

Revision of the Quaternary calcareous nannofossil biochronology of Arctic Ocean sediments

Despite extensive chronological studies, the relationship between the age and sub-seafloor depth of Arctic Ocean sediments remains ambiguous. This prevents confident identification of paleoceanographic changes in the Arctic during the Quaternary. Currently, age-depth models derived from uranium-series decay in Arctic sediments diverge by hundreds of thousands of years compared to those built on known evolutionary appearances and extinctions of calcareous nannoplankton, a group of globally valuable age-markers. Here we report on high-resolution biostratigraphic analysis of late Quaternary sediments in six cores from the central Arctic Ocean (CAO). We applied paired light microscope (LM) and scanning electron microscope (SEM) imaging to improve nannofossil diagnosis. We argue that low abundances and poor preservation have led to misidentification of the true stratigraphic depth of the critical Pleistocene nannofossil bio-events that have underpinned age models for many Arctic sedimentary records for decades. The revised calcareous nannofossil biochronology provides a radically different geochronological framework for CAO sediments – indicating that what had previously been identified as Marine Isotope Stage (MIS) 7 (191–243 ka) in many sedimentary records is older than MIS 12 (424–478 ka). Furthermore, it suggests that previously inferred sub-stages of MIS 5 could represent full interglacial periods rather than interstadials. The results help reconcile the different dating approaches and provide a transformative step towards resolving the disparity in Quaternary Arctic age-depth models, bringing us one step closer to accurate paleoceanographic reconstructions based on sediment cores.

Custom Workflow for the Confident Identification of Sulfotyrosine-Containing Peptides and Their Discrimination from Phosphopeptides.

Protein tyrosine sulfation (sY) is a post-translational modification (PTM) catalyzed by Golgi-resident tyrosyl protein sulfo transferases (TPSTs). Information on sY in humans is currently limited to ∼50 proteins, with only a handful having verified sites of sulfation. As such, the contribution of sulfation to the regulation of biological processes remains poorly defined. Mass spectrometry (MS)-based proteomics is the method of choice for PTM analysis but has yet to be applied for systematic investigation of the "sulfome", primarily due to issues associated with discrimination of sY-containing from phosphotyrosine (pY)-containing peptides. In this study, we developed an MS-based workflow for sY-peptide characterization, incorporating optimized Zr4+ immobilized metal-ion affinity chromatography (IMAC) and TiO2 enrichment strategies. Extensive characterization of a panel of sY- and pY-peptides using an array of fragmentation regimes (CID, HCD, EThcD, ETciD, UVPD) highlighted differences in the generation of site-determining product ions and allowed us to develop a strategy for differentiating sulfated peptides from nominally isobaric phosphopeptides based on low collision energy-induced neutral loss. Application of our "sulfomics" workflow to a HEK-293 cell extracellular secretome facilitated identification of 21 new sulfotyrosine-containing proteins, several of which we validate enzymatically, and reveals new interplay between enzymes relevant to both protein and glycan sulfation.

Evaluation of a Remote Curriculum to Improve Internal Medicine Resident Knowledge and Confidence in Oncologic Emergency Identification and Management

Background Oncologic emergencies (OEs) are life-threatening complications of malignancy that can present in multiple clinical settings and necessitate early recognition and intervention. Most medical resident training is experiential, but variability in exposure can yield gaps in medical knowledge that could result in delayed patient care. Remote curricula are commonly used to fill these gaps and reduce classroom burden on trainees. Given their time-sensitive nature, OEs are a prime target to augment clinical experience with a supplementary curriculum to prepare residents for new scenarios. This study evaluates whether an original, remote curriculum improves internal medicine resident knowledge of and confidence in recognition and management of OEs. Methods With expert faculty input, we developed a remote curriculum for first-year internal medicine residents at a tertiary care academic medical center. The curriculum includes 6 OEs: tumor lysis syndrome (TLS), hypercalcemia (hyperCa), neutropenic fever (NF), hyperviscosity syndrome and leukostasis (HVS/LS), superior vena cava (SVC) syndrome, and malignant spinal cord compression (CC). OEs were selected based upon prominence in the literature, morbidity and mortality, need for urgent management, and association with malignancy. We created an online lesson in Qualtrics for each OE. Each lesson was designed to be completed in 15 minutes via computer or smartphone. Lessons were delivered by email every 10 days, with reminders every 5 days. Learning objectives for each OE lesson were to 1) identify concerning signs and symptoms, 2) order appropriate workup, and 3) provide prompt management in the appropriate clinical setting. Lessons included clinical questions and evidence-based information delivery contextualized by case presentations. We evaluated the curriculum's efficacy with a randomized, controlled study. First-year residents were randomly assigned to two groups (“Intern A” and “Intern B”). We built assessments of knowledge and confidence with expert faculty input. Confidence in identifying and managing each OE was assessed by Likert scale (“1 - completely confident,” “5 - not at all confident”). Knowledge was evaluated with 3 multiple choice questions per OE, for a maximum knowledge score of 18. After both groups completed the pre-test, Intern A received the curriculum. After curriculum completion, both groups completed the post-test. All interns were required to complete the curriculum, but research participation was optional. Residents consented to participation prior to each test administration. All lessons and research activities were anonymous. Analysis was completed in GraphPad Prism 9 using the unpaired t-test or one-way ANOVA with post-hoc testing, as appropriate. Results Thirty-nine internal medicine residents completed the curriculum. Twenty were assigned to Intern A and 19 to Intern B. Intern A had 10 pre-test respondents (50.0%) with a mean knowledge score of 9.13 (50.7% correct), and Intern B had 13 pre-test respondents (68.4%) with a mean knowledge score of 9.35 (51.9% correct). There were no differences by OE-specific score. Baseline confidence was similar between groups (Table 1). Comparison of mean knowledge scores is shown in Figure 1. Intern A and B had 9 (45.0%) and 10 (52.6%) post-test respondents, respectively. Both Intern A (mean 14.2; 95% CI, 12.8 to 15.7; 78.9% correct) and Intern B (mean 12.2; 95% CI, 10.5 to 13.9; 67.8% correct) significantly improved their knowledge scores compared to prior, though Intern A had a more marked effect size (Intern A mean difference 5.02; 95% CI, 2.84 to 7.21; P&amp;lt;0.0001; Intern B mean difference 2.66; 95% CI, 0.660 to 4.66; P=0.006). Intern A confidence significantly improved from the pre-test in both the identification and management of HVS/LS and SVC syndrome. Intern A confidence was significantly better than Intern B confidence in management of SVC syndrome at the time of the post-test (Table 1). Conclusions Baseline intern OE knowledge scores were sub-optimal. Intern participation in a remote OE curriculum improved knowledge acquisition to a greater degree than experiential learning alone. Confidence in identification and management of OEs with low pre-existing confidence, such as HVS/LS and SVC syndrome, improved in the remote curriculum. Further study should evaluate knowledge retention over time, resident clinical performance, and patient outcomes.

Multi-Attribute Method (MAM) Analytical Workflow for Biotherapeutic Protein Characterization from Process Development to QC.

The multi-attribute method (MAM) has emerged significantly in recent years to support biotherapeutic protein characterization from process development to the QC environment. MAM is a liquid chromatography mass spectrometry (LC-MS) based peptide mapping approach, which combines the benefits from liquid chromatography coupled to high resolution accurate mass mass spectrometry (LC-HRAM MS), enabling direct assessment of protein sequence and product quality attributes with site specificity. These product quality attributes may impact efficacy, safety, stability, and process robustness. MAM is intended to replace conventional analytical approaches as it offers a more streamlined strategy for parallel monitoring of multiple attributes in a single analysis with high sensitivity and confidence, and ultimately supports more robust Quality by Design (QbD) approaches and faster decision cycles for biotherapeutic development. MAM consists of three main stages. The first stage is sample digestion, which typically entails proteolytic digestion of the protein. The second stage is reversed-phase chromatographic separation of the generated peptides and detection by HRAM MS in two phases. During MAM Phase I (discovery phase), data-dependent acquisition (DDA) MS/MS is performed to enable confident identification of peaks and development of a peptide workbook. During MAM Phase II (monitoring phase), full MS acquisition is only carried out for the monitoring of predefined product quality attributes (PQAs). The third stage is data processing, which entails analysis and reporting for each of the two phases including evaluation of sequence coverage, assessment of PQAs and peptide workbook creation during phase I, and targeted monitoring of predefined product attributes and new peak detection (NPD) during phase II. The latter is a comparative analysis that uses a base peak alignment algorithm to determine any non-monitored differences between the LC-MS chromatograms of a test sample and a reference standard. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: In-solution sample digestion Alternate Protocol: Automated sample digestion Basic Protocol 2: Reversed-phase chromatographic separation and detection by HRAM-MS (RPLC-HRAM MS) Basic Protocol 3: Data processing and reporting.

Electron activated dissociation - a complementary fragmentation technique to collision-induced dissociation for metabolite identification of synthetic cathinone positional isomers

Over the last decade, a remarkable number of new psychoactive substances (NPS) have emerged onto the drug market, resulting in serious threats to both public health and society. Despite their abundance and potential toxicity, there is little information available on their metabolism, a crucial piece of information for clinical and forensic purposes. NPS metabolism can be studied using in vitro models, such as liver microsomes, cytosol, hepatocytes, etc. The tentative structural elucidation of metabolites of NPS formed using in vitro models is typically carried out using liquid chromatography combined with high-resolution tandem mass spectrometry (LC-HRMS2) with collision-induced dissociation (CID) as a fragmentation method. However, the thermally-excited ions produced with CID may not be sufficient for unambiguous identification of metabolites or their complete characterization. Electron-activated dissociation (EAD), a relatively new fragmentation approach that can be used to fragment singly-charged ions, may provide complementary structural information that can be used to further improve the confidence in metabolite identification. The aim of this study was to compare CID and EAD as fragmentation methods for the characterization and identification of synthetic cathinone positional isomers and their metabolites. The in vitro metabolism of 2-methylethcathinone (2-MEC), 3-methylethcathinone (3-MEC) and 4-methylethcathinone (4-MEC) was investigated with both CID and EAD methods using LC-HRMS2. Four, seven and six metabolites were tentatively identified for the metabolism of 2-MEC, 3-MEC and 4-MEC, respectively. Here, the metabolism of 3-MEC and 2-MEC is reported for the first time. The EAD product ion mass spectra showed different fragmentation patterns compared to CID, where unique and abundant product ions were observed in EAD but not in CID. More importantly, certain EAD exclusive product ions play a significant role in structural elucidation of some metabolites. These results highlight the important role that EAD fragmentation can play in metabolite identification workflows, by providing additional fragmentation data compared with CID and, thus, enhancing the confidence in structural elucidation of drug metabolites.

O055 NIV titration: How to Match Ventilator Settings to the Patient?

Abstract Introduction Non-invasive ventilation (NIV) is a treatment that improves mortality and quality of life in chronic respiratory failure. The monitoring used to titrate NIV varies substantially; however, treatment is directed towards improving pulmonary gas exchange and sleep quality. The aim was to evaluate whether different montages of signals, influenced identification of respiratory and patient ventilator asynchrony (PVA) events and ventilation titration. Method 50 laboratory NIV polysomnography (PSG) studies were reviewed under 3 different signal conditions: NIV PSG, ventilator derived signals (V) and polygraphy (PG): ventilator signals, respiratory bands, transcutaneous carbon dioxide and oximetry. Two respiratory and sleep physicians reviewed and scored respiratory and PVA events for each individual study with agreement quantified with Cohen’s kappa statistic. Physicians’ confidence and satisfaction of ventilator settings and titration recommendations were ranked on a 5-point Likert scale and compared with ordinal logistic regression. Results There were marked differences in the identification of respiratory events including upper airway obstruction with reduced respiratory drive; PSG vs V kappa 0.1 (CI -0.12, 0.32) and PSG vs V kappa 0.43(CI 0.2, 0.67). Ineffective effort, a measure of PVA, PSG vs V studies demonstrated kappa 0.02 (CI 0, 0.05) compared with PSG vs PG kappa 0.34 (CI 0.16, 0.53). Physician confidence for ventilation prescription was lower in PG and V studies at -1.3 and -3.5 points respectively when compared with PSG. Conclusion Ventilator only derived signals resulted in key differences in respiratory event, PVA identification and physician confidence. This may help inform future models of NIV care.