Patient Participation in Multidisciplinary High-Risk Surgery Discussions: A Pilot Study.

Objective: Our medical center implemented a multidisciplinary team to improve surgical decision making for high-risk older adults. To make this a patient-centric process, a pilot program included the patient and their family/caregiver(s) in these conversations. Our hypothesis is that multidisciplinary team discussions can improve difficult surgical decision making. Methods: From January to June 2022, we offered patients and their family participation in multidisciplinary discussions at a Veterans Affairs medical center. Semistructured interviews were conducted 1-6 days after the meeting. Interview transcripts were analyzed with qualitative mixed-methods approach. Results: Six patients and caregivers participated in the interviews. They found the discussion helpful for improving their understanding of the surgical decision. Out of these, 50% (3 of 6) of the patients changed their decision regarding the planned operation based on the discussion. Conclusion: Including patients and caregiver(s) in multidisciplinary surgical decision-making discussions resulted in half of the patients changing their surgical plans. This pilot study demonstrated both acceptance and feasibility for all participants.

MEM-based pangenome indexing for k-mer queries.

Pangenomes are growing in number and size, thanks to the prevalence of high-quality long-read assemblies. However, current methods for studying sequence composition and conservation within pangenomes have limitations. Methods based on graph pangenomes require a computationally expensive multiple-alignment step, which can leave out some variation. Indexes based on k-mers and de Bruijn graphs are limited to answering questions at a specific substring length k. We present Maximal Exact Match Ordered (MEMO), a pangenome indexing method based on maximal exact matches (MEMs) between sequences. A single MEMO index can handle arbitrary-length queries over pangenomic windows. MEMO enables both queries that test k-mer presence/absence (membership queries) and that count the number of genomes containing k-mers in a window (conservation queries). MEMO's index for a pangenome of 89 human autosomal haplotypes fits in 2.04 GB, 8.8× smaller than a comparable KMC3 index and 11.4× smaller than a PanKmer index. MEMO indexes can be made smaller by sacrificing some counting resolution, with our decile-resolution HPRC index reaching 0.67 GB. MEMO can conduct a conservation query for 31-mers over the human leukocyte antigen locus in 13.89 seconds, 2.5x faster than other approaches. MEMO's small index size, lack of k-mer length dependence, and efficient queries make it a flexible tool for studying and visualizing substring conservation in pangenomes.

Heterogeneous distribution of kinesin-streptavidin complexes revealed by mass photometry.

Kinesin-streptavidin complexes are widely used in microtubule-based active-matter studies. The stoichiometry of the complexes is empirically tuned but experimentally challenging to determine. Here, mass photometry measurements reveal heterogenous distributions of kinesin-streptavidin complexes. Our binding model indicates that heterogeneity arises from both the kinesin-streptavidin mixing ratio and the kinesin-biotinylation efficiency.

Heterogeneous distribution of kinesin-streptavidin complexes revealed by Mass Photometry.

Kinesin-streptavidin complexes are widely used in microtubule-based active-matter studies. The stoichiometry of the complexes is empirically tuned but experimentally challenging to determine. Here, mass photometry measurements reveal heterogenous distributions of kinesin-streptavidin complexes. Our binding model indicates that heterogeneity arises from both the kinesin-streptavidin mixing ratio and the kinesin-biotinylation efficiency.

Movi: a fast and cache-efficient full-text pangenome index.

Efficient pangenome indexes are promising tools for many applications, including rapid classification of nanopore sequencing reads. Recently, a compressed-index data structure called the "move structure" was proposed as an alternative to other BWT-based indexes like the FM index and r-index. The move structure uniquely achieves both O(r) space and O(1)-time queries, where r is the number of runs in the pangenome BWT. We implemented Movi, an efficient tool for building and querying move-structure pangenome indexes. While the size of the Movi's index is larger than the r-index, it scales at a smaller rate for pangenome references, as its size is exactly proportional to r, the number of runs in the BWT of the reference. Movi can compute sophisticated matching queries needed for classification - such as pseudo-matching lengths and backward search - at least ten times faster than the fastest available methods, and in some cases more than 30-fold faster. Movi achieves this speed by leveraging the move structure's strong locality of reference, incurring close to the minimum possible number of cache misses for queries against large pangenomes. We achieve still further speed improvements by using memory prefetching to attain a degree of latency hiding that would be difficult with other index structures like the r-index. Movi's fast constant-time query loop makes it well suited to real-time applications like adaptive sampling for nanopore sequencing, where decisions must be made in a small and predictable time interval.

Chemoselective Labeling and Immobilization of Phosphopeptides with Phosphorimidazolide Reagents.

Protein phosphorylation is one of the most ubiquitous post-translational modifications, regulating numerous essential processes in cells. Accordingly, the large-scale annotation of phosphorylation sites continues to provide central insight into the regulation of signaling networks. The global analysis of the phosphoproteome typically relies on mass spectrometry analysis of phosphopeptides, with an enrichment step necessary due to the sub-stoichiometric nature of phosphorylation. Several affinity-based methods and chemical modification strategies have been developed to date, but the choice of enrichment method can have a considerable impact on the results. Here, we show that a biotinylated, photo-cleavable phosphorimidazolide reagent permits the immobilization and subsequent cleavage of phosphopeptides. The method is capable of the capture and release of phosphopeptides of varying characteristics, and this mild and selective strategy expands the current repertoire for phosphopeptide chemical modification with the potential to enrich and identify new phosphorylation sites in the future.

Augmented Thresholds for MONI.

MONI (Rossi et al., 2022) can store a pangenomic dataset T in small space and later, given a pattern P, quickly find the maximal exact matches (MEMs) of P with respect to T. In this paper we consider its one-pass version (Boucher et al., 2021), whose query times are dominated in our experiments by longest common extension (LCE) queries. We show how a small modification lets us avoid most of these queries which significantly speeds up MONI in practice while only slightly increasing its size.

Combined Structural Analysis and Molecular Dynamics Reveal Penicillin-Binding Protein Inhibition Mode with ß-Lactones.

ß-Lactam antibiotics comprise one of the most widely used therapeutic classes to combat bacterial infections. This general scaffold has long been known to inhibit bacterial cell wall biosynthesis by inactivating penicillin-binding proteins (PBPs); however, bacterial resistance to ß-lactams is now widespread, and new strategies are urgently needed to target PBPs and other proteins involved in bacterial cell wall formation. A key requirement in the identification of strategies to overcome resistance is a deeper understanding of the roles of the PBPs and their associated proteins during cell growth and division, such as can be obtained with the use of selective chemical probes. Probe development has typically depended upon known PBP inhibitors, which have historically been thought to require a negatively charged moiety that mimics the C-terminus of the PBP natural peptidoglycan substrate, d-Ala-d-Ala. However, we have identified a new class of ß-lactone-containing molecules that interact with PBPs, often in an isoform-specific manner, and do not incorporate this C-terminal mimetic. Here, we report a series of structural biology experiments and molecular dynamics simulations that we utilized to evaluate specific binding modes of this novel PBP inhibitor class. In this work, we obtained <2 Å resolution X-ray structures of four ß-lactone probes bound to PBP1b from Streptococcus pneumoniae. Despite their diverging recognition modes beyond the site of covalent modification, these four probes all efficiently labeled PBP1b, as well as other PBPs from S. pneumoniae. From these structures, we analyzed protein-ligand interactions and characterized the ß-lactone-bound active sites using in silico mutagenesis and molecular dynamics. Our approach has clarified the dynamic interaction profile in this series of ligands, expanding the understanding of PBP inhibitor binding.

Psychometric Evaluation of the Creighton Competency Evaluation Instrument in a Population of Working Nurses.

BACKGROUND AND PURPOSE: This study explored the psychometric properties of the Creighton Competency Evaluation Instrument (C-CEI), previously validated for use with nursing students, to assess simulation performance among registered nurses working 12-hour shifts. Valid and reliable measurements are needed to test clinical and simulation competencies and characterize the effects of fatigue on nursing performance. METHODS: Trained raters scored nurses' patient care performance in simulation scenarios using the C-CEI. We analyzed the instrument's principal components, internal reliability, and construct validity. RESULTS: Internal reliability of the C-CEI aggregate score and the Clinical Decision-making component were high (>.70). The latter robustly correlated with predicted cognitive effectiveness, a measure of fatigue. CONCLUSIONS: The C-CEI is a reliable measure for use among registered nurses and its further development will be important for testing performance of working nurses and fatigue-mitigation innovations.

Perioperative Clerkship Design for Students with Physical Disabilities: A Model for Implementation.

OBJECTIVE: Disability inclusion is an important and growing area of focus for medical education that may be stymied by stereotypes about disabilities, lack of knowledge about accommodations for students with physical disabilities, or outdated technical standards that preclude participation of people with mobility disabilities. To support the inclusion of students with physical disability in surgical clerkships, we describe a proactive, progressive approach to the accommodations process for a student with a thoracic spinal cord injury entering a surgical clerkship. DESIGN: Working proactively, medical school leadership, disability professionals and the clerkship team collaborated on the development of reasonable accommodations for a student with a thoracic spinal cord injury entering a surgical clerkship. SETTING: University of Colorado, Department of Surgery and Department of Medical Education, Aurora, CO. PARTICIPANTS: A third-year medical student and faculty from the medical school and surgical clerkship leaders. RESULTS: An M3 student with a thoracic spinal cord injury successfully completed an 8-week surgical clerkship completing all required procedural and clinical skills utilizing reasonable accommodation. The student achieved a grade of honors for the rotation. CONCLUSIONS: Early communication and planning for disability-related adjustments are critical to ensure an accessible experience for students with physical disabilities. The addition of a student with a disability adds to a better understanding of inclusive practices for surgical education and adds to the diversity of thought and experience for the medical education community.

The effects of three consecutive 12-hour shifts on cognition, sleepiness, and domains of nursing performance in day and night shift nurses: A quasi-experimental study.

BACKGROUND: Healthcare systems have widely adopted consecutive 12 h day and night shifts for nurses, but the effects of these shifts on cognition, sleepiness, and nursing performance remains understudied. OBJECTIVE: To determine the extent of changes in cognition and sleepiness in nurses working three consecutive 12 h shifts, quantify the respective impacts of these changes on different aspects of nursing performance, and investigate individual differences in all measures. DESIGN: A quasi-experimental, between-within design collected data from nurses between November 2018 and March 2020. The between-groups component was comprised of day shift nurses vs. night shift nurses, while the within-groups component was comprised of two separate test sessions for each nurse: one immediately following a third consecutive shift (fatigued) and one after three consecutive days off work (rested). SETTING: Participants were tested in a northwestern US university's nursing simulation laboratory. PARTICIPANTS: A volunteer sample of 94 registered nurses involved in direct patient care working 12 h shifts were recruited from two local hospitals. METHODS: Simulated nursing performance was measured in seven separate domains and an aggregate score from the Creighton Competence and Evaluation Inventory, covering both lower- and higher-level constructs like procedural skills, assessment, decision-making, etc. Cognition and sleepiness were assessed through measures of sustained attention, predicted cognitive effectiveness, and subjective sleepiness. RESULTS: In our 94 nurses, individual differences in all our measures varied from trivial to extensive. For six domains of performance and the aggregate score there were no significant differences in means across groups or conditions. For the seventh, Communication skills were lower for night nurses than day nurses, but this effect was small. After three consecutive shifts, sustained attention and predicted cognitive effectiveness decreased, and subjective sleepiness increased. Predicted cognitive effectiveness was particularly low for fatigued night nurses relative to other conditions and was positively correlated with Communication while controlling for other predictors. CONCLUSIONS AND RELEVANCE: Nurses maintained their levels of performance for all domains after three consecutive shifts. Individual differences in predicted cognitive effectiveness could account for variation in performance by shift type for Communication skills but for no other domain of performance. Communication skills and predicted cognitive effectiveness may interest researchers in the development of fatigue-mitigation strategies for night nurses, but our findings also suggest that more sensitive measures of performance may be necessary to capture other meaningful effects of long, consecutive shifts-if any-on patient care. Tweetable abstract: The effects of three consecutive 12 h shifts on cognition, sleepiness, and domains of nursing performance in day and night shift nurses: A quasi-experimental study.

Affinity-based proteomics reveals novel targets of inositol pyrophosphate (5-IP7 )-dependent phosphorylation and binding in Trypanosoma cruzi replicative stages.

Diphosphoinositol-5-pentakisphosphate (5-PP-IP5 ), also known as inositol heptakisphosphate (5-IP7 ), has been described as a high-energy phosphate metabolite that participates in the regulation of multiple cellular processes through protein binding or serine pyrophosphorylation, a posttranslational modification involving a ß-phosphoryl transfer. In this study, utilizing an immobilized 5-IP7 affinity reagent, we performed pull-down experiments coupled with mass spectrometry identification, and bioinformatic analysis, to reveal 5-IP7 -regulated processes in the two proliferative stages of the unicellular parasite Trypanosoma cruzi. Our protein screen clearly defined two cohorts of putative targets either in the presence of magnesium ions or in metal-free conditions. We endogenously tagged four protein candidates and immunopurified them to assess whether 5-IP7 -driven phosphorylation is conserved in T. cruzi. Among the most interesting targets, we identified a choline/o-acetyltransferase domain-containing phosphoprotein that undergoes 5-IP7 -mediated phosphorylation events at a polyserine tract (Ser578-580 ). We also identified a novel SPX domain-containing phosphoribosyltransferase [EC 2.7.6.1] herein termed as TcPRPPS4. Our data revealed new possible functional roles of 5-IP7 in this divergent eukaryote, and provided potential new targets for chemotherapy.

Comparison of Bioorthogonal ß-Lactone Activity-Based Probes for Selective Labeling of Penicillin-Binding Proteins.

Penicillin-binding proteins (PBPs) are a family of bacterial enzymes that are key components of cell-wall biosynthesis and the target of ß-lactam antibiotics. Most microbial pathogens contain multiple structurally homologous PBP isoforms, making it difficult to target individual PBPs. To study the roles and regulation of specific PBP isoforms, a panel of bioorthogonal ß-lactone probes was synthesized and compared. Fluorescent labeling confirmed selectivity, and PBPs were selectively enriched from Streptococcus pneumoniae lysates. Comparisons between fluorescent labeling of probes revealed that the accessibility of bioorthogonal reporter molecules to the bound probe in the native protein environment exerts a more significant effect on labeling intensity than the bioorthogonal reaction used, observations that are likely applicable beyond this class of probes or proteins. Selective, bioorthogonal activity-based probes for PBPs will facilitate the activity-based determination of the roles and regulation of specific PBP isoforms, a key gap in knowledge that has yet to be filled.

Sleep health and predicted cognitive effectiveness of nurses working 12-hour shifts: an observational study.

BACKGROUND: Due to the 24hr nature of society, shift work has become an integral part of many industries. Within the literature there exists an abundance of evidence linking shift work-related sleep restriction and fatigue with errors, accidents, and adverse long-term health outcomes. OBJECTIVE: The study goal was to physiologically measure sleep patterns and predicted cognitive decline of nurses working both 12hr day and night shifts to address the growing concern about sleep restriction among healthcare workers. DESIGN: This study presents the results of a quasi-experimental, mixed between-within design where the sleep of 12hr day and night shift nurses was measured using ReadiBand wrist actigraphs. The between groups component was comprised of day v. night shift nurses. The within groups component was comprised of two separate measurement periods for each nurse-once for three consecutive days while they were working shifts (on duty) and once for three consecutive days off work (off duty). SETTING: Participants wore the wrist actigraph at home and in the hospital, and were instructed to adhere to their regular sleep schedule. PARTICIPANTS: Participants were recruited from two hospitals in Washington State (n=90). Participants were 48 night- and 42 day-shift nurses. All participants worked 12-hour shifts. METHODS: Sleep was measured using ReadiBand wrist actigraphs, which are licensed with the Sleep, Activity, Fatigue, and Task Effectiveness (SAFTEtm) Alertness Score model, a biomathematical model that predicts cognitive effectiveness based on sleep/wake schedule. ReadiBands also calculate sleep quantity, sleep efficiency, and sleep latency. Results were analysed in SPSS (v26) through multilevel modelling. RESULTS: Differences were observed in sleep quantity, efficiency, and latency based on shift type (day vs. night) and shift duty (on vs. off). The most extreme differences, however, were noted in cognitive effectiveness (SAFTEtm), whereby night shift nurses experienced substantial decline-frequently into the "high risk" zone-throughout their shifts compared to day shift nurses. CONCLUSIONS: The present study identifies sleep characteristics that differ between day and night nurses working 12-hour shifts using objective measurements of sleep. Biomathematical modelling can offer a novel method to estimate hours of greatest cognitive decline, and have implications for policy around shift duration, timing, and overtime allocation.

On-chip electrochemical detection of glucose towards the miniaturised quality control of carbohydrate-based radiotracers.

The miniaturisation of positron emission tomography (PET) radiotracer production is facilitating a move towards a dose-on-demand strategy that would enable a stratified approach to patient diagnostics, but while the on-chip synthesis steps have been demonstrated, the subsequent quality control (QC) testing steps have received much less attention. As part of the development of an integrated QC platform for PET tracers, we have developed two microfluidic electrochemical detectors for the pulsed amperometric detection (PAD) of carbohydrate-based radiotracers, with a particular view to the QC testing of the most important tracer, [18F]2-fluoro-2-deoxy-d-glucose ([18F]FDG). The first device employed a commercial screen-printed electrode (SPE) to enable a single-use format, while the second device incorporated wire electrodes for use as a more permanent fixture in a QC instrument. A flow-injection analysis (FIA)-style setup was used to inject boluses of d-glucose into the chips in a proxy for intended chromatographic separations prior to PAD. In proof-of-concept testing of the devices, the chips featuring the SPE and the wire electrodes yielded limits of detection of 0.1 ppm and 9 ppm, respectively, each below the required limits for [18F]FDG, and thus making both methodologies viable for the QC testing of PET radiotracers in a dose-on-demand format.

Chemical tools for selective activity profiling of bacterial penicillin-binding proteins.

Penicillin-binding proteins (PBPs) are membrane-associated proteins involved in the biosynthesis of peptidoglycan (PG), the main component of bacterial cell walls. These proteins were discovered and named for their affinity to bind the ß-lactam antibiotic penicillin. The importance of the PBPs has long been appreciated; however, specific roles of individual family members in each bacterial strain, as well as their protein-protein interactions, are yet to be understood. The apparent functional redundancy of the 4-18 PBPs that most eubacteria possess makes determination of their individual roles difficult. Existing techniques to study PBPs are not ideal because they do not directly visualize protein activity and can suffer from artifacts and perturbations of native PBP function. Therefore, development of new methods for studying the roles of individual PBPs in cell wall synthesis is required. We recently generated a library of fluorescent chemical probes containing a ß-lactone scaffold that specifically targets the PBPs, enabling the visualization of their catalytic activity. Herein, we describe a general protocol to label and detect the activity of individual PBPs in Streptococcus pneumoniae using our fluorescent ß-lactone probes.