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Background Neural networks are increasingly used to assess physiological processes or pathologies, as well as to predict the increased likelihood of an impending medical crisis, such as hypotension. Method We compared the capabilities of a single hidden layer neural network of 12 nodes to those of a discrete-feature discrimination approach with the goal being to predict the likelihood of a given patient developing significant hypotension under spinal anesthesia when undergoing a Cesarean section (C/S). Physiological input information was derived from a non-invasive blood pressure device (Caretaker [CT]) that utilizes a finger cuff to measure blood pressure and other hemodynamic parameters via pulse contour analysis. Receiver-operator-curve/area-under-curve analyses were used to compare performance. Results The results presented here suggest that a neural network approach (Area Under Curve [AUC] = 0.89 [p p Conclusion This pilot study has demonstrated that increased coherence in Arterial Stiffness (AS) variability obtained from the pulse wave analysis of a continuous non-invasive blood pressure device appears to be an effective predictor of hypotension after spinal anesthesia in the obstetrics population undergoing C/S. This allowed us to predict specific dosing thresholds of phenylephrine required to maintain systolic blood pressure above 90 mmHg.

Introduction The reliable detection and, ultimately, prediction of hypotensive events in post-operative settings remains an unsolved problem, as patients are currently only monitored intermittently because of the lack of validated, non-invasive/non-intrusive and continuous physiological monitoring technologies. With this goal in mind, the aim of this study was to validate a next-generation platform version of the currently FDA-cleared non-invasive Caretaker (CT) physiological monitor in the hemodynamically challenging environment of abdominal surgeries in comparison with blood pressures obtained from arterial catheters, evaluated against ANSI/AAMI/ISO 81060–2:2019 standards as well as against current non-invasive standard of care measurements provided by clinical-grade automatic oscillometric cuffs. Methods Comparison data from 41 major abdominal surgery patients at Cooper Hospital (Camden NJ) were analyzed in this IRB approved study. Each patient was monitored with a radial arterial catheter and CT using a finger cuff applied to the contralateral middle finger. Systolic and diastolic blood pressures continuously collected from the arterial catheter and CT were compared using Pearson correlation coefficients and Bland-Altman analysis. In addition, a trend analysis using 4Q plots was performed. Both the CT’s continuous BP tracking and the CT’s self-calibration capability were analyzed. Results The continuous data comparisons were performed with and without taking the CT recalibrations into account. With the recalibrations the mean differences and standard deviations (STDs) for systole and diastole were, respectively, -1.14 mmHg (13.82 mmHg) and -2.49 mmHg (9.42 mmHg), while the correlations were 0.80 and 0.78. Mean differences and STDs for an initial calibration and no subsequent recalibrations were, respectively for systole and diastole, -0.42 mmHg (16.73 mmHg) and -2.57 mmHg (10.36 mmHg), while the correlations were 0.64 and 0.67. For the CT’s self-calibrations alone, correlations for systole and diastole were, respectively, 0.83 and 0.75, while corresponding mean differences (STD) were -3.19 mmHg (10.86 mmHg) and -2.41 mmHg (8.18 mmHg). For 41% of total surgery time, both systole and diastole were within 8 mmHg of the arterial catheter Gold Standard. The concordances for systolic and diastolic blood pressure changes on a 30-second time scale were 0.87 and 0.86. The same comparison analysis for the automatic cuff and the arterial catheter data yielded: correlations for systole and diastole: 0.69 and 0.61, mean differences and STDs: 2.48 mmHg (15.82 mmHg) and 0.65 mmHg (10.68 mmHg). Conclusions The results of this study are significant in that they validate the future use of the CT physiological monitor, which utilizes Pulse Decomposition Analysis (PDA), in the post-operative monitoring scenario both as a monitor to detect hypotensive events to facilitate clinical intervention as well as provide signal inputs that could enable anticipatory measures.

Caspases are a family of proteases found in all metazoans, including a dozen in humans, that drive the terminal stages of apoptosis as well as other cellular remodeling and inflammatory events. Caspases are named because they are cysteine class enzymes shown to cleave after aspartate residues. In the past decade, we and others have developed unbiased proteomic methods that collectively identified ~2000 native proteins cleaved during apoptosis after the signature aspartate residues. Here, we explore non-aspartate cleavage events and identify 100s of substrates cleaved after glutamate in both human and murine apoptotic samples. The extended consensus sequence patterns are virtually identical for the aspartate and glutamate cleavage sites suggesting they are cleaved by the same caspases. Detailed kinetic analyses of the dominant apoptotic executioner caspases-3 and -7 show that synthetic substrates containing DEVD↓ are cleaved only twofold faster than DEVE↓, which is well within the 500-fold range of rates that natural proteins are cut. X-ray crystallography studies confirm that the two acidic substrates bind in virtually the same way to either caspases-3 or -7 with minimal adjustments to accommodate the larger glutamate. Lastly, during apoptosis we found 121 proteins cleaved after serine residues that have been previously annotated to be phosphorylation sites. We found that caspase-3, but not caspase-7, can cleave peptides containing DEVpS↓ at only threefold slower rate than DEVD↓, but does not cleave the unphosphorylated serine peptide. There are only a handful of previously reported examples of proteins cleaved after glutamate and none after phosphorserine. Our studies reveal a much greater promiscuity for cleaving after acidic residues and the name 'cacidase' could aptly reflect this broader specificity.

BackgroundDespite increased interest in non-invasive arterial pressure monitoring, the majority of commercially available technologies have failed to satisfy the limits established for the validation of automatic arterial pressure monitoring by the Association for the Advancement of Medical Instrumentation (AAMI). According to the ANSI/AAMI/ISO 81060-2:2013 standards, the group-average accuracy and precision are defined as acceptable if bias is not greater than 5mmHg and standard deviation is not greater than 8mmHg. In this study, these standards are used to evaluate the CareTaker® (CT) device, a device measuring continuous non-invasive blood pressure via a pulse contour algorithm called Pulse Decomposition Analysis.MethodsA convenience sample of 24 patients scheduled for major abdominal surgery were consented to participate in this IRB approved pilot study. Each patient was monitored with a radial arterial catheter and CT using a finger cuff applied to the contralateral thumb. Hemodynamic variables were measured and analyzed from both devices for the first thirty minutes of the surgical procedure including the induction of anesthesia. The mean arterial pressure (MAP), systolic and diastolic blood pressures continuously collected from the arterial catheter and CT were compared. Pearson correlation coefficients were calculated between arterial catheter and CT blood pressure measurements, a Bland-Altman analysis, and polar and 4Q plots were created.ResultsThe correlation of systolic, diastolic, and mean arterial pressures were 0.92, 0.86, 0.91, respectively (p

Background Captive cetacean attractions are growing in number globally, their operators citing entertainment, education, and conservation as benefits. Those for and against developing such attractions claim public support. Previous public opinion research, however, shows little consensus, partly due to the introduction of biases in study design that influence participants’ responses. Those involved in, or concerned with, developing and licensing these attractions need to better understand what drives the lack of consensus to take socially-acceptable decisions. Methods We reviewed previous research on public opinion of cetacean captivity, noting possible sources of bias. Survey question wording can be a major source of introduced bias, so we used an open-ended photo elicitation approach. We showed tourists in the Turks Caicos Islands (N = 292) images of a marine mammal park (MMP) killer whale show and a swim-with-the-dolphins (SWTD) attraction and asked for their qualitative comments on the potential development of each. They also indicated how likely they would be to visit each on a Likert scale. Results Respondents were generally against visiting MMP killer whale shows, with 60.9% not likely to visit. SWTD attractions were more popular; 60.3% were likely to visit. For SWTD, USA residents were more likely to visit; older respondents and those staying in all-inclusive resorts were less likely. Those staying in all-inclusive resorts were also less likely to visit MMP killer whale shows. The great majority of qualitative comments centred on either entertainment value or animal welfare concerns. There were very few, if any, comments on the education or conservation value of these attractions. Discussion Our findings contradict several previous studies on public opinion of captive cetaceans that did not use photo elicitation. The support shown for MMP killer whale shows in this survey was well below that claimed by studies conducted on behalf of captive cetacean attraction operators. Opposition to SWTD was also noticeably lower than that found in surveys conducted with wild cetacean tourism participants. This difference can likely be attributed to the different survey populations and settings, but this variation is also very likely attributable to researcher-introduced bias. While photo selection can introduce bias, photo elicitation reduces reliance on pre-scripted questions and responses, and seems to effectively reduce other forms of bias. Allowing open-ended responses, where participants responded to an image, seems to have given a more representative understanding of what is at the forefront of the public’s mind than closed questioning. These conclusions, among others made in this study, suggest that development decisions for captive cetacean attractions are being made on imprecise data. Going forward, data collected via responder-led, open-ended, bias-minimising approaches should at least be considered when informing such decisions.

Proteolysis is a critical modification leading to alteration of protein function with important outcomes in many biological processes. However, for the majority of proteases, we have an incomplete understanding of both cellular substrates and downstream effects. Here, we describe detailed protocols and applications for using the rationally engineered peptide ligase, subtiligase, to specifically label and capture protein N-termini generated by proteases either induced or added to complex biological samples. This method allows identification of the protein targets as well as their precise cleavage locations. This approach has revealed >8000 proteolytic sites in healthy and apoptotic cells including >1700 caspase cleavages. One can further determine substrate preferences through rate analysis with quantitative mass spectrometry, physiological substrate specificities, and even infer the identity of proteases operating in the cell. In this chapter, we also describe how this experimental method can be generalized to investigate proteolysis in any biological sample.

Proteolysis is a critical post-translational modification for regulation of cellular processes. Our lab has previously developed a technique for specifically labeling unmodified protein N termini, the α-aminome, using the engineered enzyme, subtiligase. Here we present a database, called the DegraBase (http://wellslab.ucsf.edu/degrabase/), which compiles 8090 unique N termini from 3206 proteins directly identified in subtiligase-based positive enrichment mass spectrometry experiments in healthy and apoptotic human cell lines. We include both previously published and unpublished data in our analysis, resulting in a total of 2144 unique α-amines identified in healthy cells, and 6990 in cells undergoing apoptosis. The N termini derive from three general categories of proteolysis with respect to cleavage location and functional role: translational N-terminal methionine processing (∼10% of total proteolysis), sites close to the translational N terminus that likely represent removal of transit or signal peptides (∼25% of total), and finally, other endoproteolytic cuts (∼65% of total). Induction of apoptosis causes relatively little change in the first two proteolytic categories, but dramatic changes are seen in endoproteolysis. For example, we observed 1706 putative apoptotic caspase cuts, more than double the total annotated sites in the CASBAH and MEROPS databases. In the endoproteolysis category, there are a total of nearly 3000 noncaspase nontryptic cleavages that are not currently reported in the MEROPS database. These studies significantly increase the annotation for all categories of proteolysis in human cells and allow public access for investigators to explore interesting proteolytic events in healthy and apoptotic human cells.

Caspases, cysteine proteases with aspartate specificity, are key players in programmed cell death across the metazoan lineage. Hundreds of apoptotic caspase substrates have been identified in human cells. Some have been extensively characterized, revealing key functional nodes for apoptosis signaling and important drug targets in cancer. But the functional significance of most cuts remains mysterious. We set out to better understand the importance of caspase cleavage specificity in apoptosis by asking which cleavage events are conserved across metazoan model species. Using N-terminal labeling followed by mass spectrometry, we identified 257 caspase cleavage sites in mouse, 130 in Drosophila, and 50 in Caenorhabditis elegans. The large majority of the caspase cut sites identified in mouse proteins were found conserved in human orthologs. However, while many of the same proteins targeted in the more distantly related species were cleaved in human orthologs, the exact sites were often different. Furthermore, similar functional pathways are targeted by caspases in all four species. Our data suggest a model for the evolution of apoptotic caspase specificity that highlights the hierarchical importance of functional pathways over specific proteins, and proteins over their specific cleavage site motifs.