Dynamics of Single-Cell Protein Covariation during Epithelial-Mesenchymal Transition.

Physiological processes, such as the epithelial-mesenchymal transition (EMT), are mediated by changes in protein interactions. These changes may be better reflected in protein covariation within a cellular cluster than in the temporal dynamics of cluster-average protein abundance. To explore this possibility, we quantified proteins in single human cells undergoing EMT. Covariation analysis of the data revealed that functionally coherent protein clusters dynamically changed their protein-protein correlations without concomitant changes in the cluster-average protein abundance. These dynamics of protein-protein correlations were monotonic in time and delineated protein modules functioning in actin cytoskeleton organization, energy metabolism, and protein transport. These protein modules are defined by protein covariation within the same time point and cluster and, thus, reflect biological regulation masked by the cluster-average protein dynamics. Thus, protein correlation dynamics across single cells offers a window into protein regulation during physiological transitions.

Harm-to-benefit ratio of fecal immunochemical test-based screening for colorectal cancer given prior fecal hemoglobin concentrations.

BACKGROUND AND AIMS: This study aims to provide evidence on the harm-to-benefit ratio of Fecal Immunochemical Test (FIT)-based colorectal cancer (CRC) screening by previous fecal Hemoglobine (f-Hb) concentrations, as reflected in Number Needed to Screen (NNS) and Number Needed to Scope (NNSc). METHODS: Participants in up to four FIT screening rounds of the Dutch CRC screening program were included. The main outcomes of this study were the NNS and NNSc to detect one CRC and/or AN in screening round two, three or four, conditional on previous f-Hb concentrations. Outcomes were compared between participants using chi-squared tests, and logistic regression. RESULTS: In total, 2,428,883 study participants completed at least two consecutive FITs, 1,308,684 completed three FITs, and 150,958 completed four FITs. There were 31,400, 16,060, and 2,007 AN detected by round, respectively. The NNS for individuals with vs without a history of detectable f-Hb differed significantly irrespective of screening round. Individuals without detectable f-Hb in previous negative FITs had almost nine times the NNS to detect one AN compared to those with detectable f-Hb (OR 8.71, 95%CI 8.51-8.92). A similar directional pattern was observed for NNSc, although the differences were smaller (OR 2.7, 95%CI 2.7-2.8). CONCLUSIONS: The harm-to-benefit ratio of FIT-based screening is substantially greater in individuals without vs. with prior detectable f-Hb. Less intensive screening should be considered for this lower-risk group.

Nonclassical Spin-Multiplexing Metasurfaces Enabled Multifunctional Meta-Scope.

Dielectric metasurfaces have emerged as attractive devices for advanced imaging systems because of their high efficiency, ability of wavefront manipulation, and lightweight. The classical spin-multiplexing metasurfaces can only provide two orthogonal circular polarization channels and require high phase contrast which limits their applications. Here, metasurfaces with arbitrary three independent channels are demonstrated by proposing a nonclassical spin-multiplexing approach exploring the low refractive index meta-atoms. A zoom microscope with on-axis tri-foci and a synchronous achiral-chiral microscope with in-plane tri-foci based on silicon nitride metasurfaces are experimentally demonstrated. Based on the on-axis tri-foci metasurface, singlet zoom imaging with three magnifications and a broadband response (blue to red) based on a single metasurface is first demonstrated. A compact microscope (meta-scope) consisting of two metasurfaces with three magnifications of 9.5, 10, and 29X with diffraction-limited resolutions is further constructed, respectively. Utilizing the in-plane tri-foci metasurface, a singlet microscope with three achiral-chiral channels is demonstrated. It offers a magnification of 53X and a diffraction-limited resolution, enabling simultaneous imaging of an object's achiral and chiral properties. Our multifunctional metasurfaces and meta-scope approaches could boost the applications in biological imaging and machine vision.

Mechanisms of enhanced cardiorespiratory performance under hyperoxia differ with exposure duration in yellowtail kingfish.

Hyperoxia has been shown to expand the aerobic capacity of some fishes, although there have been very few studies examining the underlying mechanisms and how they vary across different exposure durations. Here, we investigated the cardiorespiratory function of yellowtail kingfish (Seriola lalandi) acutely (~20 h) and chronically (3-5 weeks) acclimated to hyperoxia (~200% air saturation). Our results show that the aerobic performance of kingfish is limited in normoxia and increases with environmental hyperoxia. The aerobic scope was elevated in both hyperoxia treatments driven by a ~33% increase in maximum O2 uptake (MO2max), although the mechanisms differed across treatments. Fish acutely transferred to hyperoxia primarily elevated tissue O2 extraction, while increased stroke volume-mediated maximum cardiac output was the main driving factor in chronically acclimated fish. Still, an improved O2 delivery to the heart in chronic hyperoxia was not the only explanatory factor as such. Here, maximum cardiac output only increased in chronic hyperoxia compared with normoxia when plastic ventricular growth occurred, as increased stroke volume was partly enabled by an ~8%-12% larger relative ventricular mass. Our findings suggest that hyperoxia may be used long term to boost cardiorespiratory function potentially rendering fish more resilient to metabolically challenging events and stages in their life cycle.

Engineering of a Baeyer-Villiger monooxygenase to Improve Substrate Scope, Stereoselectivity and Regioselectivity.

Baeyer-Villiger monooxygenases belong to a family of flavin-binding proteins that catalyze the Baeyer-Villiger (BV) oxidation of ketones to produce lactones or esters, which are important intermediates in pharmaceuticals or sustainable materials. Phenylacetone monooxygenase (PAMO) from Thermobifida fusca with moderate thermostability catalyzes the oxidation of aryl ketone substrates, but is limited by high specificity and narrow substrate scope. In the present study, we applied loop optimization by loop swapping followed by focused saturation mutagenesis in order to evolve PAMO mutants capable of catalyzing the regioselective BV oxidation of cyclohexanone and cyclobutanone derivatives with formation of either normal or abnormal esters or lactones. We further modulated PAMO to increase enantioselectivity. Crystal structure studies indicate that rotation occurs in the NADP-binding domain and that the high B-factor region is predominantly distributed in the catalytic pocket residues. Computational analyses further revealed dynamic character in the catalytic pocket and reshaped hydrogen bond interaction networks, which is more favorable for substrate binding. Our study provides useful insights for studying enzyme-substrate adaptations.

The Biocatalytic Potential of Aromatic Ammonia-Lyase from Loktanella atrilutea.

Characterization of the aromatic ammonia-lyase from Loktanella atrilutea (LaAAL) revealed reduced activity towards canonical AAL substrates: l-Phe, l-Tyr, and l-His, contrasted by its pronounced efficiency towards 3,4-dimethoxy-l-phenylalanine. Assessing the optimal conditions, LaAAL exhibited maximal activity at pH 9.5 in the ammonia elimination reaction route, distinct from the typical pH ranges of most PALs and TALs. Within the exploration of the ammonia source for the opposite, synthetically valuable ammonia addition reaction, the stability of LaAAL exhibited a positive correlation with the ammonia concentration, with the highest stability in 4 M ammonium carbamate of unadjusted pH of ~9.5. While the enzyme activity increased with rising temperatures yet, the highest operational stability and highest stationary conversions of LaAAL were observed at 30 °C. The substrate scope analysis highlighted the catalytic adaptability of LaAAL in the hydroamination of diverse cinnamic acids, especially of meta-substituted and di-/multi-substituted analogues, with structural modelling exposing steric clashes between the substrates' ortho-substituents and catalytic site residues. LaAAL showed a predilection for ammonia elimination, while classifying as a tyrosine ammonia-lyase (TAL) among the natural AAL classes. However, its distinctive attributes, such as genomic context, unique substrate specificity and catalytic fingerprint, suggest a potential natural role beyond those of known AAL classes.

Substrate-Multiplexed Assessment of Aromatic Prenyltransferase Activity.

An increasingly effective strategy to identify synthetically useful enzymes is to sample the diversity already present in Nature. Here, we construct and assay a panel of phylogenetically diverse aromatic prenyltransferases (PTs). These enzymes catalyze a variety of C-C bond forming reactions in natural product biosynthesis and are emerging as tools for synthetic chemistry and biology. Homolog screening was further empowered through substrate-multiplexed screening, which provides direct information on enzyme specificity. We perform a head-to-head assessment of the model members of the PT family and further identify homologs with divergent sequences that rival these superb enzymes. This effort revealed the first bacterial O-Tyr PT and, together, provide valuable benchmarking for future synthetic applications of PTs.

Asymmetric Synthesis of Axially Chiral N, N-1,2-Pentatomic Heterobiaryl Diamines by an Organocatalytic Arylation Reaction.

The utilization of axially chiral biaryl diamines has been widely acknowledged as highly advantageous structures for the advancement of chiral catalysts and ligands. This highlights their extensive range of applications in asymmetric catalysis and synthesis. Herein, we devised a direct arylation reactions of 5-aminopyrazoles with azonaphthalenes, utilizing chiral phosphoric acid as the catalyst. This method delivers structurally novel atroposelective N, N-1,2-azole heteroaryl diamines with high yields (up to >98%) and good to excellent enantiomeric ratios while exhibiting a wide range of substrate compatibility.

Thermal constraints on exercise and metabolic performance do not explain the use of dormancy as an overwintering strategy in the cunner (Tautogolabrus adspersus).

Winter cold slows ectotherm physiology, potentially constraining activities and ecological opportunities at poleward latitudes. Yet, many fishes are winter-active, facilitated by thermal compensation that improves cold performance. Conversely, winter-dormant fishes (e.g. cunner, Tautogolabrus adspersus) become inactive and non-feeding overwinter. Why are certain fishes winter-dormant? We hypothesized that winter dormancy is an adaptive behavioural response arising in poleward species that tolerate severe, uncompensated constraints of cold on their physiological performance. We predicted that below their dormancy threshold of 7--8°C, exercise and metabolic performance of cunner are greatly decreased, even after acclimation (i.e. shows above-normal, uncompensated thermal sensitivity, Q10>1-3). We measured multiple key performance metrics (e.g. C-start maximum velocity, chase swimming speed, aerobic scope) in cunner after acute exposure to 26-2°C (3°C intervals using 14°C-acclimated fish) or acclimation (5-8 weeks) to 14-2°C (3°C intervals bracketing the dormancy threshold). Performance declined with cooling, and the acute Q10 of all six performance rate metrics was significantly greater below the dormancy threshold temperature (Q10,acute8-2°C=1.5-4.9, mean=3.3) than above (Q10,acute14-8°C=1.1-1.9, mean=1.5), inferring a cold constraint. However, 2°C acclimation (temporally more relevant to seasonal cooling) improved performance, abolishing the acute constraint (Q10,acclimated8-2°C=1.4-3.0, mean=2.0; also cf. Q10,acclimated14-8°C=1.2-2.9, mean=1.7). Thus, dormant cunner show partial cold-compensation of exercise and metabolic performance, similar to winter-active species. However, responsiveness to C-start stimuli was greatly cold-constrained even following acclimation, suggesting dormancy involves sensory limitation. Thermal constraints on metabolic and exercise physiology are not significant drivers of winter dormancy in cunner. In fact, compensatory plasticity at frigid temperatures is retained even in a dormant fish.

Coronary circulation enhances the aerobic performance of wild Pacific salmon.

Female Pacific salmon often experience higher mortality than males during their once-in-a-lifetime up-river spawning migration, particularly when exposed to secondary stressors (e.g. high temperatures). However, the underlying mechanisms remain unknown. One hypothesis is that female Pacific salmon hearts are more oxygen-limited than those of males and are less able to supply oxygen to the body's tissues during this demanding migration. Notably, female hearts have higher coronary blood flow, which could indicate a greater reliance on this oxygen source. Oxygen limitations can develop from naturally occurring coronary blockages (i.e. coronary arteriosclerosis) found in mature salmon hearts. If female hearts rely more heavily on coronary blood flow but experience similar arteriosclerosis levels as males, they will have disproportionately impaired aerobic performance. To test this hypothesis, we measured resting (RMR) and maximum metabolic rate (MMR), aerobic scope (AS) and acute upper thermal tolerance in coho salmon (Oncorhynchus kisutch) with an intact or artificially blocked coronary oxygen supply. We also assessed venous blood oxygen and chemistry (cortisol, ions and metabolite concentrations) at different time intervals during recovery from exhaustive exercise. We found that coronary blockage impaired MMR, AS and the partial pressure of oxygen in venous blood (PvO2) during exercise recovery but did not differ between sexes. Coronary ligation lowered acute upper thermal tolerance by 1.1°C. Although we did not find evidence of enhanced female reliance on coronary supply, our findings highlight the importance of coronary blood supply for mature wild salmon, where migration success may be linked to cardiac performance, particularly during warm water conditions.

Hyperoxia disproportionally benefits the aerobic performance of large fish at elevated temperature.

Increasing evidence shows that larger fish are more vulnerable to acute warming than smaller individuals of the same species. This size-dependency of thermal tolerance has been ascribed to differences in aerobic performance, largely owing to a decline in oxygen supply relative to demand. To shed light on these ideas, we examined metabolic allometry in 130 rainbow trout ranging from 12 to 358 g under control conditions (17°C) and in response to acute heating (to 25°C), with and without supplemental oxygen (100% versus 150% air saturation). Under normoxia, high temperature caused an average 17% reduction in aerobic scope compared with 17°C. Aerobic performance disproportionally deteriorated in bigger fish as the scaling exponent (b) for aerobic scope declined from b=0.87 at 17°C to b=0.74 at 25°C. Hyperoxia increased maximum metabolic rate and aerobic scope at both temperatures and disproportionally benefited larger fish at 25°C as the scaling exponent for aerobic scope was reestablished to the same level as at 17°C (b=0.86). This suggests that hyperoxia may provide metabolic refuge for larger individuals, allowing them to sustain aerobic activities when facing acute warming. Notably, the elevated aerobic capacity afforded by hyperoxia did not appear to improve thermal resilience, as mortality in 25°C hyperoxia (13.8%, n=4) was similar to that in normoxia (12.1%, n=4), although we caution that this topic warrants more targeted research. We highlight the need for mechanistic investigations of the oxygen transport system to determine the consequences of differential metabolic scaling across temperature in a climate warming context.

Hyperoxia Does Not Improve the Acute Upper Thermal Tolerance of a Tropical Marine Fish (Lutjanus apodus).

Fish can experience hyperoxia in shallow environments due to photosynthetic activity, and this has been suggested to provide them with a metabolic refuge during acute warming. However, this hypothesis has never been tested on a tropical marine species. Thus, we fitted 29°C-acclimated wild schoolmaster snapper (Lutjanus apodus; a species known to experience diel hyperoxia in mangrove creeks and coastal waters) with Transonic® flow probes, and exposed them to an acute increase in temperature (at 1°C h-1) in respirometers under normoxia and hyperoxia (150% air saturation), until their critical thermal maximum (CTmax). The CTmax of both groups was ∼39°C, and no differences in maximum cardiac function were recorded as the fish were warmed. However, temperature-induced factorial aerobic scope was significantly greater in fish tested under hyperoxia. These data suggest that hyperoxia will not protect coastal tropical fish species during marine heat waves, despite its effects on metabolic scope / capacity.

Thermal performance curves for aerobic scope and specific dynamic action in a sexually dimorphic piscivore: implications for a warming climate.

Digestion can make up a substantial proportion of animal energy budgets, yet our understanding of how it varies with sex, body mass and ration size is limited. A warming climate may have consequences for animal growth and feeding dynamics that will differentially impact individuals in their ability to efficiently acquire and assimilate meals. Many species, such as walleye (Sander vitreus), exhibit sexual size dimorphism (SSD), whereby one sex is larger than the other, suggesting sex differences in energy acquisition and/or expenditure. Here, we present the first thorough estimates of specific dynamic action (SDA) in adult walleye using intermittent-flow respirometry. We fed male (n=14) and female (n=9) walleye two ration sizes, 2% and 4% of individual body mass, over a range of temperatures from 2 to 20°C. SDA was shorter in duration and reached higher peak rates of oxygen consumption with increasing temperature. Peak SDA increased with ration size and decreased with body mass. The proportion of digestible energy lost to SDA (i.e. the SDA coefficient) was consistent at 6% and was unrelated to temperature, body mass, sex or ration size. Our findings suggest that sex has a negligible role in shaping SDA, nor is SDA a contributor to SSD for this species. Standard and maximum metabolic rates were similar between sexes but maximum metabolic rate decreased drastically with body mass. Large fish, which are important for population growth because of reproductive hyperallometry, may therefore face a bioenergetic disadvantage and struggle most to perform optimally in future, warmer waters.

Direct in-scope suction: an in vitro evaluation of a single use flexible ureteroscope with integrated suction capability.

PURPOSE: To evaluate the stone clearance rate and limitations of a novel integrated suction capability within a single-use flexible ureteroscope according to stone particle size. METHODS: Varying sized stone particles were created using a stone phantom (Begostone Plus, Bego ©, Lincoln, RI, USA). Particle size ranged as follows: 63-125 µm, 125-250 µm, 250-500 µm, 500 µm- 1 mm and 1-2 mm. These were mixed with Normal saline (0.9%) to mimic in vivo conditions. The suction enabled single-use flexible ureteroscope (Pusen, Zhuhai, China) was used to aspirate stone fragments in three trials. Firstly, aspiration of 5 g of each dust range was attempted. Secondly, a direct comparison of the integrated suction to a manual syringe technique was applied to 1 g of each size range. Finally, aspiration was applied to 5 g of a heterogenous 1:1:1:1:1 mixture. Endoscopic clearance rate (g/min) and number of blockages were recorded. Each challenge was repeated three times. RESULTS: The integrated suction cleared 100% of dust < 250 µm. Endoscopic clearance rates were significantly faster than manual aspiration (3.01 g/min versus 0.41 g/min) for dust between 125 and 250 µm (p = 0.008). Complete endoscopic clearance by 180 s (without encountering test limiting blockages) was unsuccessful for particles > 250 µm. Clearance rates were greatly limited by stone particle size heterogeneity above 250 µm, to 0.09 g/min. CONCLUSION: This technology works better in vitro than previously DISS evaluated methods when challenged by stone dust < 250 µm. However, this adaptation is significantly challenged in the presence of stone dust particles > 250 µm.

Pharmacist Prescribing Models for HIV Pre-exposure and Post-exposure Prophylaxis.

State strategies for pharmacist prescribing exist on a continuum from most restrictive to least restrictive. Using human immunodeficiency virus (HIV) pre-exposure prophylaxis and post-exposure prophylaxis as a case study, there are 3 viable pharmacist prescribing models: (1) population-based collaborative practice agreements; (2) government protocols; and (3) standard of care prescribing. The advantages and disadvantages of these 3 models are reviewed.

Prognostic performance of a two-step method using the Fibro-Scope system for metabolic dysfunction-associated steatotic liver disease.

AIM: Fibro-Scope is an artificial intelligence/neural network system for the noninvasive diagnosis of hepatic fibrosis in patients with metabolic dysfunction-associated steatotic liver disease. We aimed to examine the diagnostic performance of a two-step method that used the Fibrosis-4 (FIB-4) index and Fibro-Scope system for the assessment of Japanese patients with metabolic dysfunction-associated steatotic liver disease. METHODS: We analyzed a longitudinal study cohort of 796 Japanese patients with biopsy-proven metabolic dysfunction-associated steatotic liver disease during a follow-up period of 6.4 years. The predictive performance of the two-step method of FIB-4 index and Fibro-Scope for liver-related events and prognostic performance of that were assessed in the patients. RESULTS: In the 796 patients, by classifying the intermediate zone, defined by FIB-4 index 1.30-2.67, using the Fibro-Scope, the final classification was 69.6% low risk, 28.3% high risk, and 2.1% in the middle-risk group. The sensitivity and specificity for predicting advanced fibrosis (≥F3) was 84.0% and 84.0%. During the follow-up period, 52 (6.5%) patients developed liver-related events and 35 died. Multivariate analysis revealed that high-risk patients derived from the two-step method had hazard ratios of 30.1 or the development of liver-related events and 7.8 for outcome. CONCLUSIONS: The two-step method using the FIB-4 index and Fibro-Scope contributed to improving the diagnostic performance by picking up high-risk patients from those classified as intermediate risk with the FIB-4 index. This noninvasive method, which uses a blood sample is a cost-effective screening method, is suitable for clinical practice in Japan.

Scope of practice and opioid prescribing behavior of nurse practitioners serving Medicare beneficiaries.

Policymakers aiming to increase access to health care while simultaneously keeping costs low and quality high are considering expanding the practice authority and prescriptive authority of nurse practitioners in order to address primary care shortages. While we know this increases access, some researchers argue that the expansion of job autonomy of nurse practitioners can compromise the quality and safety of rendered medical services. This paper investigates quality and safety outcomes in prescribing behaviors of nurse practitioners who have prescribed opioids for Medicare Part D beneficiaries using a unique source of policy variation, nurse practitioners with the ability to prescribe medication who move to either states with or without physician supervision. We find that scope of practice expansions do not compromise quality and safety in terms of potential abuse or misuse of prescriptive authority.

The effects of dental hygienist autonomy on dental care utilization.

We investigate the effects of regulations governing the practice autonomy of dental hygienists on dental care use with the 2001-2014 Medical Expenditure Panel Survey. We measure the strength of autonomy regulations by extending the Dental Hygiene Professional Practice Index to the years 2001-2014, allowing us to capture changes in regulations within states over time. Using a difference-in-differences framework applied to selected states, we find that relaxing supervision requirements to provide dental hygienists moderate autonomy results in an increase in total dental visits due to greater use of preventive dental care. However, the use of dental treatment decreases when states adopt the highest level of autonomy. Both sets of estimates increase in magnitude when we subset the sample to dental care provider shortage areas. In support of these findings, we show that dental visits shift to dental hygienists in shortage areas when states expand the scope of practice of hygienists, and that there is an increase in tasks performed by hygienists, such as cleanings and dental exams.

How do dental practices respond to changes in scope of practice regulations?

Regulations that restrict the tasks that credentialed workers are allowed to perform may affect a firm's input choices, output, and which part of the market the firm serves. Using dental practice survey data from 1989 to 2014 and a stacked difference-in-differences design, this paper examines the effects of state-level scope of practice regulations on the behavior of dental practices. Results suggest that scope of practice deregulation in regards to dental hygienists' ability to administer nitrous oxide or local anesthesia is associated with fewer dentist visits per week in the short-term, lower patient wait times, and an increased likelihood of treating lower revenue generating publicly insured patients. There is weak evidence that scope of practice deregulation alters a practice's labor inputs.

Paediatric emergency front-of-neck airway: issues of ethics, law, and philosophy.

Practitioners can face significant challenges when managing the airways of infants and neonates because of their unique anatomical and physiological features. The requirement for emergency airway management in this age group is rare. Details of emergency airway techniques in paediatric practice guidelines are missing or lack consensus, and it is known that outcomes for affected children can be poor. Ideally, these children should be managed by experienced paediatric airway practitioners working in a team. However, situations can arise where practitioners, unfamiliar and inexperienced with infants, find themselves in charge. So, what happens when such a practitioner encounters this life-or-death scenario and feels ill-equipped to act? The ethical and legal issues surrounding the management of this emergency are clearly defined, but they can be unknown or misunderstood by doctors. Compounding the extreme stress of the scenario is the moral and ethical dilemma of whether to act or not. The following discussion explores these issues and examines the philosophical and psychological perspectives.