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Background: Autofluorescence imaging of the coenzyme, reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H), provides a label-free technique to assess cellular metabolism. Because NAD(P)H is localized in the cytosol and mitochondria, instance segmentation of cell cytoplasms from NAD(P)H images allows quantification of metabolism with cellular resolution. However, accurate cytoplasmic segmentation of autofluorescence images is difficult due to irregular cell shapes and cell clusters., Method: Here, a cytoplasm segmentation method is presented and tested. First, autofluorescence images are segmented into cells via either hand-segmentation or Cellpose, a deep learning-based segmentation method. Then, a cytoplasmic post-processing algorithm (CPPA) is applied for cytoplasmic segmentation. CPPA uses a binarized segmentation image to remove non-segmented pixels from the NAD(P)H image and then applies an intensity-based threshold to identify nuclei regions. Errors at cell edges are removed using a distance transform algorithm. The nucleus mask is then subtracted from the cell segmented image to yield the cytoplasm mask image. CPPA was tested on five NAD(P)H images of three different cell samples, quiescent T cells, activated T cells, and MCF7 cells., Results: Using POSEA, an evaluation method tailored for instance segmentation, the CPPA yielded F-measure values of 0.89, 0.87, and 0.94 for quiescent T cells, activated T cells, and MCF7 cells, respectively, for cytoplasm identification of hand-segmented cells. CPPA achieved F-measure values of 0.84, 0.74, and 0.72 for Cellpose segmented cells., Conclusion: These results exceed the F-measure value of a comparative cell segmentation method (CellProfiler, ∼0.50-0.60) and support the use of artificial intelligence and post-processing techniques for accurate segmentation of autofluorescence images for single-cell metabolic analyses., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Significance: The ability to observe and monitor cell density and morphology has been imperative for assessing the health of a cell culture and for producing high quality, high yield cell cultures for decades. Microcarrier-based cultures, used for large-scale cellular expansion processes, are not compatible with traditional visualization-based methods, such as widefield microscopy, due to their thickness and material composition., Aim: Here, we assess the optical imaging compatibilities of commercial polystyrene microcarriers versus custom-fabricated gelatin methacryloyl (gelMA) microcarriers for non-destructive and non-invasive visualization of the entire microcarrier surface, direct cell enumeration, and sub-cellular visualization of mesenchymal stem/stromal cells., Approach: Mie scattering and wavefront error simulations of the polystyrene and gelMA microcarriers were performed to assess the potential for elastic scattering-based imaging of adherent cells. A Zeiss Z.1 light-sheet microscope was adapted to perform light-sheet tomography using label-free elastic scattering contrast from planar side illumination to achieve optical sectioning and permit non-invasive and non-destructive, in toto , three-dimensional, high-resolution visualization of cells cultured on microcarriers., Results: The polystyrene microcarrier prevents visualization of cells on the distal half of the microcarrier using either fluorescence or elastic scattering contrast, whereas the gelMA microcarrier allows for high fidelity visualization of cell morphology and quantification of cell density using light-sheet fluorescence microscopy and tomography., Conclusions: The combination of optical-quality gelMA microcarriers and label-free light-sheet tomography will facilitate enhanced control of bioreactor-microcarrier cell culture processes., (© 2024 The Authors.)

Neural stem cells (NSCs) must exit quiescence to produce neurons; however, our understanding of this process remains constrained by the technical limitations of current technologies. Fluorescence lifetime imaging (FLIM) of autofluorescent metabolic cofactors has been used in other cell types to study shifts in cell states driven by metabolic remodeling that change the optical properties of these endogenous fluorophores. Using this non-destructive, live-cell, and label-free strategy, we found that quiescent NSCs (qNSCs) and activated NSCs (aNSCs) have unique autofluorescence profiles. Specifically, qNSCs display an enrichment of autofluorescence localizing to a subset of lysosomes, which can be used as a graded marker of NSC quiescence to predict cell behavior at single-cell resolution. Coupling autofluorescence imaging with single-cell RNA sequencing, we provide resources revealing transcriptional features linked to deep quiescence and rapid NSC activation. Together, we describe an approach for tracking mouse NSC activation state and expand our understanding of adult neurogenesis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Migratory species trade-off long-distance movement with survival and reproduction, but the spatio-temporal scales at which these decisions occur are relatively unknown. Technological and statistical advances allow fine-scale study of animal decision-making, improving our understanding of possible causes and therefore conservation management. We quantified effects of reproductive preparation during spring migration on subsequent breeding outcomes, breeding outcomes on autumn migration characteristics and autumn migration characteristics on subsequent parental survival in Greenland white-fronted geese ( Anser albifrons flavirostris ). These are long-distance migratory birds with an approximately 50% population decline from 1999 to 2022. We deployed GPS-acceleration devices on adult females to quantify up to 5 years of individual decision-making throughout the annual cycle. Weather and habitat-use affected time spent feeding and overall dynamic body acceleration (i.e. energy expenditure) during spring and autumn. Geese that expended less energy and fed longer during spring were more likely to successfully reproduce. Geese with offspring expended more energy and fed for less time during autumn, potentially representing adverse fitness consequences of breeding. These behavioural comparisons among Greenland white-fronted geese improve our understanding of fitness trade-offs underlying abundance. We provide a reproducible framework for full annual cycle modelling using location and behaviour data, applicable to similarly studied migratory animals.

Fluorescence lifetime imaging of the co-enzyme reduced nicotinamide adenine dinucleotide (NADH) offers a label-free approach for detecting cellular metabolic perturbations. However, the relationships between variations in NADH lifetime and metabolic pathway changes are complex, preventing robust interpretation of NADH lifetime data relative to metabolic phenotypes. Here, a three-dimensional convolutional neural network (3D CNN) trained at the cell level with 3D NAD(P)H lifetime decay images (two spatial dimensions and one time dimension) was developed to identify metabolic pathway usage by cancer cells. NADH fluorescence lifetime images of MCF7 breast cancer cells with three isolated metabolic pathways, glycolysis, oxidative phosphorylation, and glutaminolysis were obtained by a multiphoton fluorescence lifetime microscope and then segmented into individual cells as the input data for the classification models. The 3D CNN models achieved over 90% accuracy in identifying cancer cells reliant on glycolysis, oxidative phosphorylation, or glutaminolysis. Furthermore, the model trained with human breast cancer cell data successfully predicted the differences in metabolic phenotypes of macrophages from control and POLG-mutated mice. These results suggest that the integration of autofluorescence lifetime imaging with 3D CNNs enables intracellular spatial patterns of NADH intensity and temporal dynamics of the lifetime decay to discriminate multiple metabolic phenotypes. Furthermore, the use of 3D CNNs to identify metabolic phenotypes from NADH fluorescence lifetime decay images eliminates the need for time- and expertise-demanding exponential decay fitting procedures. In summary, metabolic-prediction CNNs will enable live-cell and in vivo metabolic measurements with single-cell resolution, filling a current gap in metabolic measurement technologies., Competing Interests: The authors have no conflicts to disclose., (© 2024 Author(s).)

Metabolic reprogramming at a cellular level contributes to many diseases including cancer, yet few assays are capable of measuring metabolic pathway usage by individual cells within living samples. Here, autofluorescence lifetime imaging is combined with single-cell segmentation and machine-learning models to predict the metabolic pathway usage of cancer cells. The metabolic activities of MCF7 breast cancer cells and HepG2 liver cancer cells were controlled by growing the cells in culture media with specific substrates and metabolic inhibitors. Fluorescence lifetime images of two endogenous metabolic coenzymes, reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavin adenine dinucleotide (FAD), were acquired by a multi-photon fluorescence lifetime microscope and analyzed at the cellular level. Quantitative changes of NADH and FAD lifetime components were observed for cells using glycolysis, oxidative phosphorylation, and glutaminolysis. Conventional machine learning models trained with the autofluorescence features classified cells as dependent on glycolytic or oxidative metabolism with 90%-92% accuracy. Furthermore, adapting convolutional neural networks to predict cancer cell metabolic perturbations from the autofluorescence lifetime images provided improved performance, 95% accuracy, over traditional models trained via extracted features. Additionally, the model trained with the lifetime features of cancer cells could be transferred to autofluorescence lifetime images of T cells, with a prediction that 80% of activated T cells were glycolytic, and 97% of quiescent T cells were oxidative. In summary, autofluorescence lifetime imaging combined with machine learning models can detect metabolic perturbations between glycolysis and oxidative metabolism of living samples at a cellular level, providing a label-free technology to study cellular metabolism and metabolic heterogeneity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Hu, Wang, Bryant, Liu, Xie, West and Walsh.)

Monitoring marine fauna is essential for mitigating the effects of disturbances in the marine environment, as well as reducing the risk of negative interactions between humans and marine life. Drone-based aerial surveys have become popular for detecting and estimating the abundance of large marine fauna. However, sightability errors, which affect detection reliability, are still apparent. This study tested the utility of spectral filtering for improving the reliability of marine fauna detections from drone-based monitoring. A series of drone-based survey flights were conducted using three identical RGB (red-green-blue channel) cameras with treatments: (i) control (RGB), (ii) spectrally filtered with a narrow 'green' bandpass filter (transmission between 525 and 550 nm), and, (iii) spectrally filtered with a polarising filter. Video data from nine flights comprising dolphin groups were analysed using a machine learning approach, whereby ground-truth detections were manually created and compared to AI-generated detections. The results showed that spectral filtering decreased the reliability of detecting submerged fauna compared to standard unfiltered RGB cameras. Although the majority of visible contrast between a submerged marine animal and surrounding seawater (in our study, sites along coastal beaches in eastern Australia) is known to occur between 515-554 nm, isolating the colour input to an RGB sensor does not improve detection reliability due to a decrease in the signal to noise ratio, which affects the reliability of detections.

Vascularization is a key pre-requisite to engineered anatomical scale three dimensional (3-D) constructs to ensure their nutrient and oxygen supply upon implantation. Presently, engineered pre-vascularized 3-D tissues are limited to only micro-scale hydrogels, which meet neither the anatomical scale needs nor the complexity of natural extracellular matrix (ECM) environments. Anatomical scale perfusable constructs are critically needed for translational applications. To overcome this challenge, we previously developed pre-vascularized ECM sheets with long and oriented dense microvascular networks. The present study further evaluated the patency, perfusability and innate immune response toward these pre-vascularized constructs. Macrophage-co-cultured pre-vascularized constructs were evaluated in vitro to confirm micro-vessel patency and perturbations in macrophage metabolism. Subcutaneously implanted pre-vascularized constructs remained viable and formed a functional anastomosis with host vasculature within 3 days of implantation. This completely biological pre-vascularized construct holds great potential as a building block to engineer perfusable anatomical scale tissues., (© 2023 The Authors.)

Researchers generally ascribe demographic drivers in a single sub-population and presume they are representative. With this information, practitioners implement blanket conservation measures across metapopulations to reverse declines. However, such approaches may not be appropriate in circumstances where sub-populations are spatiotemporally segregated and exposed to different environmental variation. The Greenland White-fronted Goose, Anser albifrons flavirostris , is an Arctic-nesting migrant that largely comprises two sub-populations (delineated by northerly and southerly breeding areas in west Greenland). The metapopulation has declined since 1999 but this trend is only mirrored in one sub-population and the causes of this disparity are unclear. Here we compare the drivers and trends of productivity in both sub-populations using population- and individual-level analysis. We examined how temperature and precipitation influenced population-level reproductive success over 37 years and whether there was a change in the relationship when metapopulation decline commenced. In addition, we used biologging devices to remotely classify incubation events for 86 bird-years and modelled how phenology and environmental conditions influenced individual-level nest survival. Correlations between reproductive success and temperature/precipitation on the breeding grounds have weakened for both sub-populations. This has resulted in lower reproductive success for the northerly, but not southerly breeding sub-population, which at the individual-level appears to be driven by lower nest survival. Earlier breeding ground arrival and less precipitation during incubation increased nest survival in the northerly breeding population, while no factors examined were important for the southerly breeding sub-population. This suggests reproductive success is driven by different factor(s) in the two sub-populations. Demographic rates and their environmental drivers differ between the sub-populations examined here and consequently we encourage further decomposition of demography within metapopulations. This is important for conservation practitioners to consider as bespoke conservation strategies, targeting different limiting factors, may be required for different sub-populations., Competing Interests: None to declare., (© 2023 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Introduction: Autofluorescence imaging of the coenzymes reduced nicotinamide (phosphate) dinucleotide (NAD(P)H) and oxidized flavin adenine dinucleotide (FAD) provides a label-free method to detect cellular metabolism and phenotypes. Time-domain fluorescence lifetime data can be analyzed by exponential decay fitting to extract fluorescence lifetimes or by a fit-free phasor transformation to compute phasor coordinates. Methods: Here, fluorescence lifetime data analysis by biexponential decay curve fitting is compared with phasor coordinate analysis as input data to machine learning models to predict cell phenotypes. Glycolysis and oxidative phosphorylation of MCF7 breast cancer cells were chemically inhibited with 2-deoxy-d-glucose and sodium cyanide, respectively; and fluorescence lifetime images of NAD(P)H and FAD were obtained using a multiphoton microscope. Results: Machine learning algorithms built from either the extracted lifetime values or phasor coordinates predict MCF7 metabolism with a high accuracy (∼88%). Similarly, fluorescence lifetime images of M0, M1, and M2 macrophages were acquired and analyzed by decay fitting and phasor analysis. Machine learning models trained with features from curve fitting discriminate different macrophage phenotypes with improved performance over models trained using only phasor coordinates. Discussion: Altogether, the results demonstrate that both curve fitting and phasor analysis of autofluorescence lifetime images can be used in machine learning models for classification of cell phenotype from the lifetime data., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Hu, Ter Hofstede, Sharma, Zhao and Walsh.)

Background: Cisplatin (CDDP) is a mainstay treatment for advanced head and neck squamous cell carcinomas (HNSCC) despite a high frequency of innate and acquired resistance. We hypothesised that tumours acquire CDDP resistance through an enhanced reductive state dependent on metabolic rewiring., Methods: To validate this model and understand how an adaptive metabolic programme might be imprinted, we performed an integrated analysis of CDDP-resistant HNSCC clones from multiple genomic backgrounds by whole-exome sequencing, RNA-seq, mass spectrometry, steady state and flux metabolomics., Results: Inactivating KEAP1 mutations or reductions in KEAP1 RNA correlated with Nrf2 activation in CDDP-resistant cells, which functionally contributed to resistance. Proteomics identified elevation of downstream Nrf2 targets and the enrichment of enzymes involved in generation of biomass and reducing equivalents, metabolism of glucose, glutathione, NAD(P), and oxoacids. This was accompanied by biochemical and metabolic evidence of an enhanced reductive state dependent on coordinated glucose and glutamine catabolism, associated with reduced energy production and proliferation, despite normal mitochondrial structure and function., Conclusions: Our analysis identified coordinated metabolic changes associated with CDDP resistance that may provide new therapeutic avenues through targeting of these convergent pathways., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Many techniques and software packages have been developed to segment individual cells within microscopy images, necessitating a robust method to evaluate images segmented into a large number of unique objects. Currently, segmented images are often compared with ground-truth images at a pixel level; however, this standard pixel-level approach fails to compute errors due to pixels incorrectly assigned to adjacent objects. Here, we define a per-object segmentation evaluation algorithm (POSEA) that calculates segmentation accuracy metrics for each segmented object relative to a ground truth segmented image. To demonstrate the performance of POSEA, precision, recall, and f-measure metrics are computed and compared with the standard pixel-level evaluation for simulated images and segmented fluorescence microscopy images of three different cell samples. POSEA yields lower accuracy metrics than the standard pixel-level evaluation due to correct accounting of misclassified pixels of adjacent objects. Therefore, POSEA provides accurate evaluation metrics for objects with pixels incorrectly assigned to adjacent objects and is robust for use across a variety of applications that require evaluation of the segmentation of unique adjacent objects., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The adoption of cell-based therapies into the clinic will require tremendous large-scale expansion to satisfy future demand, and bioreactor-microcarrier cultures are best suited to meet this challenge. The use of spherical microcarriers, however, precludes in-process visualization and monitoring of cell number, morphology, and culture health. The development of novel expansion methods also motivates the advancement of analytical methods used to characterize these microcarrier cultures. A robust optical imaging and image-analysis assay to non-destructively quantify cell number and cell volume was developed. This method preserves 3D cell morphology and does not require membrane lysing, cellular detachment, or exogenous labeling. Complex cellular networks formed in microcarrier aggregates were imaged and analyzed in toto. Direct cell enumeration of large aggregates was performed in toto for the first time. This assay was successfully applied to monitor cellular growth of mesenchymal stem cells attached to spherical hydrogel microcarriers over time. Elastic scattering and fluorescence lightsheet microscopy were used to quantify cell volume and cell number at varying spatial scales. The presented study motivates the development of on-line optical imaging and image analysis systems for robust, automated, and non-destructive monitoring of bioreactor-microcarrier cell cultures., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Benavides et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Cell-based therapies harness functional cells or tissues to mediate healing and treat disease. Assessment of cellular therapeutics requires methods that are non-destructive to ensure therapies remain viable and uncontaminated for use in patients. Optical imaging of endogenous collagen, by second-harmonic generation, and the metabolic coenzymes NADH and FAD, by autofluorescence microscopy, provides tissue structure and cellular information. Here, we review applications of label-free nonlinear optical imaging of cellular metabolism and collagen second-harmonic generation for assessing cell-based therapies. Additionally, we discuss the potential of label-free imaging for quality control of cell-based therapies, as well as the current limitations and potential future directions of label-free imaging technologies.

Arctic-nesting geese face energetic challenges during spring migration, including ecological barriers and weather conditions (e.g., precipitation and temperature), which in long-lived species can lead to a trade-off to defer reproduction in favor of greater survival. We used GPS location and acceleration data collected from 35 greater white-fronted geese of the North American midcontinent and Greenland populations at spring migration stopovers, and novel applications of Bayesian dynamic linear models to test daily effects of minimum temperature and precipitation on energy expenditure (i.e., overall dynamic body acceleration, ODBA) and proportion of time spent feeding (PTF), then examined the daily and additive importance of ODBA and PTF on probability of breeding deferral using stochastic antecedent models. We expected distinct responses in behavior and probability of breeding deferral between and within populations due to differences in stopover area availability. Time-varying coefficients of weather conditions were variable between ODBA and PTF, and often did not show consistent patterns among birds, indicating plasticity in how individuals respond to conditions. An increase in antecedent ODBA was associated with a slightly increased probability of deferral in midcontinent geese but not Greenland geese. Probability of deferral decreased with increased PTF in both populations. We did not detect any differentially important time periods. These results suggest either that movements and behavior throughout spring migration do not explain breeding deferral or that ecological linkages between bird decisions during spring and subsequent breeding deferral were different between populations and across migration but occurred at different time scales than those we examined., (© 2022. The Author(s).)

Gallbladder polyps (also known as polypoid lesions of the gallbladder) are a common incidental finding. The vast majority of gallbladder polyps smaller than 10 mm are not true neoplastic polyps but are benign cholesterol polyps with no inherent risk of malignancy. In addition, recent studies have shown that the overall risk of gallbladder cancer is not increased in patients with small gallbladder polyps, calling into question the rationale for frequent and prolonged follow-up of these common lesions. In 2021, a Society of Radiologists in Ultrasound, or SRU, consensus conference was convened to provide recommendations for the management of incidentally detected gallbladder polyps at US. See also the editorial by Sidhu and Rafailidis in this issue., (© RSNA, 2022.)

Metastatic cancers are chemoresistant, involving complex interplay between disseminated cancer cell aggregates and the distant organ microenvironment (extracellular matrix and stromal cells). Conventional metastasis surrogates (scratch/wound healing, Transwell migration assays) lack 3D architecture and ECM presence. Metastasis studies can therefore significantly benefit from biomimetic 3D in vitro models recapitulating the complex cascade of distant organ invasion and colonization by collective clusters of cells. We aimed to engineer reproducible and quantifiable 3D models of highly therapy-resistant cancer processes: (i) colorectal cancer liver metastasis; and (ii) breast cancer lung metastasis. Metastatic seeds are engineered using 3D tumor spheroids to recapitulate the 3D aggregation of cancer cells both in the tumor and in circulation throughout the metastatic cascade of many cancers. Metastatic soil was engineered by decellularizing porcine livers and lungs to generate biomatrix scaffolds, followed by extensive materials characterization. HCT116 colorectal and MDA-MB-231 breast cancer spheroids were generated on hanging drop arrays to initiate clustered metastatic seeding into liver and lung biomatrix scaffolds, respectively. Between days 3-7, biomatrix cellular colonization was apparent with increased metabolic activity and the presence of cellular nests evaluated via multiphoton microscopy. HCT116 and MDA-MB-231 cells colonized liver and lung biomatrices, and at least 15% of the cells invaded more than 20 μm from the surface. Engineered metastases also expressed increased signatures of genes associated with the metastatic epithelial to mesenchymal transition (EMT). Importantly, inhibition of matrix metalloproteinase-9 inhibited metastatic invasion into the biomatrix. Furthermore, metastatic nests were significantly more chemoresistant (>3 times) to the anti-cancer drug oxaliplatin, compared to 3D spheroids. Together, our data indicated that HCT116 and MDA-MB-231 spheroids invade, colonize, and proliferate in livers and lungs establishing metastatic nests in 3D settings in vitro . The metastatic nature of these cells was confirmed with functional readouts regarding EMT and chemoresistance. Modeling the dynamic metastatic cascade in vitro has potential to identify therapeutic targets to treat or prevent metastatic progression in chemoresistant metastatic cancers.

Background: Anterior glenohumeral instability occurs most commonly in those aged 15-29, with 72% of individuals younger than 22 years suffering recurrent episodes; collision athletes are at particular risk. In the setting of subcritical glenoid bone loss, arthroscopic Bankart repair is widely used despite concerns of recurrent dislocations when compared with open techniques. Furthermore, indications for bone-block procedures are evolving with the Latarjet procedure being favored amongst recent authors as a primary stabilization method in elite and contact athletes., Objective: To determine the efficacy of open modified Bankart stabilization in treating anterior glenohumeral instability in young collision athletes., Methods: This was a retrospective review of outcomes of consecutive patients aged 15-20 years who underwent unilateral or bilateral open stabilization for recurrent anterior glenohumeral instability over a 7-year period (2007-2015). The cohort was selected as recent literature suggests that this is the group with the highest redislocation rate and poorest outcomes. Outcome assessments included redislocation rate, return to sport, pain score, patient-related satisfaction scores, and the Western Ontario Shoulder Instability Index., Results: A total of 60 patients (55 male: 5 female) of mean age 18 years (range: 15-20 years) were available for follow-up at 7 years. All but 3 were competitive athletes with 18 competing regionally and 9 internationally; rugby union and rugby league represent the most common sports. Fifty-five of 60 (92%) have returned to their desired level of sport with 62% of athletes returning to their previous level of competition sport and 1 retuning at a higher level. The mean postoperative pain score was 2.5/10. The mean time to return to play was 14 months (range: 5-48 months). Eight of 60 shoulders reported redislocation (13%), 7 of these being traumatic dislocations after return to high-impact sporting activities. At 7 years of follow-up, 4 of 60 shoulders (7%) had undergone revision surgery., Conclusion: Young collision athletes represent a challenging cohort of instability patients. This study of open modified Bankart in young collision sport athletes revealed excellent outcomes with 92% return to sport and a low revision rate. The results from this cohort rival those of arthroscopic repair. The open procedure described here in this 7-year series represents a robust, reliable technique that could be considered as an alternative to arthroscopic Bankart due to concern for recurrence, while avoiding potential morbidity and complication of bone-block procedures. There is still a role for the open modified Bankart procedure in treating traumatic anterior instability., (Copyright © 2022 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Elsevier Inc. All rights reserved.)

Abstract: Gallbladder polyps (GPs) are a common incidental finding on ultrasound; however, important differences in recommended management exist among professional society guidelines.An electronic survey was sent to 189 fellows of the Society of Radiologists in Ultrasound. Main outcomes included preferences and current practice patterns for evaluation, management, and surveillance of GPs as well as personal lifetime experience with gallbladder sonography and GPs.A total of 64 subjects (34%) with experience in gallbladder sonography completed the study. The estimated combined total number of gallbladder scans seen by the responders was 3,071,880. None of fellows had ever seen a pedunculated GP <1 cm detected on ultrasound that was proven to be malignant at the time of detection or during subsequent follow-up. All of the fellows used size as a feature to stratify recommendations. The median size threshold currently used by Society of Radiologists in Ultrasound fellows for recommending ultrasound follow-up was 6 mm, and their preferred threshold was 7 mm. The median size threshold for recommending surgical consultation was 10 mm, and the preferred threshold was 10 mm. Wall thickening and shape were considered important factors by 76% and 67% of respondents, respectively.Society of Radiologists in Ultrasound fellows tend to provide recommendations most similar to the American College of Radiology and Canadian Association of Radiology guidelines for management of GPs. Many would prefer guidelines that result in fewer recommendations for follow-up and surgical consultation. Despite a substantial combined experience, this survey did not uncover any case of a small GP that was malignant., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Drug-resistant cells and anti-inflammatory immune cells within tumor masses contribute to tumor aggression, invasion, and worse patient outcomes. These cells can be a small proportion (<10%) of the total cell population of the tumor. Due to their small quantity, the identification of rare cells is challenging with traditional assays. Single cell analysis of autofluorescence images provides a live-cell assay to quantify cellular heterogeneity. Fluorescence intensities and lifetimes of the metabolic coenzymes reduced nicotinamide adenine dinucleotide and oxidized flavin adenine dinucleotide allow quantification of cellular metabolism and provide features for classification of cells with different metabolic phenotypes. In this study, Gaussian distribution modeling and machine learning classification algorithms are used for the identification of rare cells within simulated autofluorescence lifetime image data of a large tumor comprised of tumor cells and T cells. A Random Forest machine learning algorithm achieved an overall accuracy of 95% for the identification of cell type from the simulated optical metabolic imaging data of a heterogeneous tumor of 20,000 cells consisting of 70% drug responsive breast cancer cells, 5% drug resistant breast cancer cells, 20% quiescent T cells and 5% activated T cells. High resolution imaging methods combined with single-cell quantitative analyses allows identification and quantification of rare populations of cells within heterogeneous cultures., (© 2022 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.)

BACKGROUND. Previous European multisociety guidelines recommend routine follow-up imaging of gallbladder polyps (including polyps < 6 mm in patients without risk factors) and cholecystectomy for polyp size changes of 2 mm or more. OBJECTIVE. The purpose of this study was to assess longitudinal changes in the number and size of gallbladder polyps on serial ultrasound examinations. METHODS. This retrospective study included patients who underwent at least one ultrasound examination between January 1, 2010, and December 31, 2020 (as part of a hepatocellular carcinoma screening and surveillance program) that showed a gallbladder polyp. Number of polyps and size of largest polyp were recorded based primarily on review of examination reports. Longitudinal changes on serial examinations were summarized. Pathologic findings from cholecystectomy were reviewed. RESULTS. Among 9683 patients, 759 (8%) had at least one ultrasound examination showing a polyp. Of these, 434 patients (248 men, 186 women; mean age, 50.6 years) had multiple examinations (range, 2-19 examinations; mean, 4.8 examinations per patient; mean interval between first and last examinations, 3.6 ± 3.1 [SD] years; maximum interval, 11.0 years). Among these 434 patients, 257 had one polyp, 40 had two polyps, and 137 had more than two polyps. Polyp size was 6 mm or less in 368 patients, 7-9 mm in 52 patients, and 10 mm or more in 14 patients. Number of polyps increased in 9% of patients, decreased in 14%, both increased and decreased on serial examinations in 22%, and showed no change in 55%. Polyp size increased in 10% of patients, decreased in 16%, both increased and decreased on serial examinations in 18%, and showed no change in 56%. In 9% of patients, gallbladder polyps were not detected on follow-up imaging; in 6% of patients, gallbladder polyps were not detected on a follow-up examination but were then detected on later studies. No gallbladder carcinoma was identified in 19 patients who underwent cholecystectomy. CONCLUSION. Gallbladder polyps fluctuate in size, number, and visibility over serial examinations. Using a 2-mm threshold for growth, 10% increased in size. No carcinoma was identified. CLINICAL IMPACT. European multisociety guidelines that propose surveillance of essentially all polyps and a 2-mm size change as the basis for cholecystectomy are likely too conservative for clinical application.

The function of macrophages in vitro is linked to their metabolic rewiring. However, macrophage metabolism remains poorly characterized in situ. Here, we used two-photon intensity and lifetime imaging of autofluorescent metabolic coenzymes, nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD), to assess the metabolism of macrophages in the wound microenvironment. Inhibiting glycolysis reduced NAD(P)H mean lifetime and made the intracellular redox state of macrophages more oxidized, as indicated by reduced optical redox ratio. We found that TNFα+ macrophages had lower NAD(P)H mean lifetime and were more oxidized compared to TNFα- macrophages. Both infection and thermal injury induced a macrophage population with a more oxidized redox state in wounded tissues. Kinetic analysis detected temporal changes in the optical redox ratio during tissue repair, revealing a shift toward a more reduced redox state over time. Metformin reduced TNFα+ wound macrophages, made intracellular redox state more reduced and improved tissue repair. By contrast, depletion of STAT6 increased TNFα+ wound macrophages, made redox state more oxidized and impaired regeneration. Our findings suggest that autofluorescence of NAD(P)H and FAD is sensitive to dynamic changes in intracellular metabolism in tissues and can be used to probe the temporal and spatial regulation of macrophage metabolism during tissue damage and repair., Competing Interests: VM, KT, ML, EB, AW, LZ, CM, MC, JF, JS, MS, AH No competing interests declared, (© 2022, Miskolci et al.)

Cellular metabolism is the process by which cells generate energy, and many diseases, including cancer, are characterized by abnormal metabolism. Reduced nicotinamide adenine (phosphate) dinucleotide (NAD(P)H) and oxidized flavin adenine dinucleotide (FAD) are coenzymes of metabolic reactions. NAD(P)H and FAD exhibit autofluorescence and can be spectrally isolated by excitation and emission wavelengths. Both coenzymes, NAD(P)H and FAD, can exist in either a free or protein-bound configuration, each of which has a distinct fluorescence lifetime-the time for which the fluorophore remains in the excited state. Fluorescence lifetime imaging (FLIM) allows quantification of the fluorescence intensity and lifetimes of NAD(P)H and FAD for label-free analysis of cellular metabolism. Fluorescence intensity and lifetime microscopes can be optimized for imaging NAD(P)H and FAD by selecting the appropriate excitation and emission wavelengths. Metabolic perturbations by cyanide verify autofluorescence imaging protocols to detect metabolic changes within cells. This article will demonstrate the technique of autofluorescence imaging of NAD(P)H and FAD for measuring cellular metabolism.

Significance: Autofluorescence measurements of the metabolic cofactors NADH and flavin adenine dinucleotide (FAD) provide a label-free method to quantify cellular metabolism. However, the effect of extracellular pH on flavin lifetimes is currently unknown., Aim: To quantify the relationship between extracellular pH and the fluorescence lifetimes of FAD, flavin mononucleotide (FMN), and reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]., Approach: Human breast cancer (BT474) and HeLa cells were placed in pH-adjusted media. Images of an intracellular pH indicator or endogenous fluorescence were acquired using two-photon fluorescence lifetime imaging. Fluorescence lifetimes of FAD and FMN in solutions were quantified over the same pH range., Results: The relationship between intracellular and extracellular pH was linear in both cell lines. Between extracellular pH 4 to 9, FAD mean lifetimes increased with increasing pH. NAD(P)H mean lifetimes decreased with increasing pH between extracellular pH 5 to 9. The relationship between NAD(P)H lifetime and extracellular pH differed between the two cell lines. Fluorescence lifetimes of FAD, FAD-cholesterol oxidase, and FMN solutions decreased, showed no trend, and showed no trend, respectively, with increasing pH., Conclusions: Changes in endogenous fluorescence lifetimes with extracellular pH are mostly due to indirect changes within the cell rather than direct pH quenching of the endogenous molecules.

There is a critical need for adjuvants that can safely elicit potent and durable T cell-based immunity to intracellular pathogens. Here, we report that parenteral vaccination with a carbomer-based adjuvant, Adjuplex (ADJ), stimulated robust CD8 T-cell responses to subunit antigens and afforded effective immunity against respiratory challenge with a virus and a systemic intracellular bacterial infection. Studies to understand the metabolic and molecular basis for ADJ's effect on antigen cross-presentation by dendritic cells (DCs) revealed several unique and distinctive mechanisms. ADJ-stimulated DCs produced IL-1β and IL-18, suggestive of inflammasome activation, but in vivo activation of CD8 T cells was unaffected in caspase 1-deficient mice. Cross-presentation induced by TLR agonists requires a critical switch to anabolic metabolism, but ADJ enhanced cross presentation without this metabolic switch in DCs. Instead, ADJ induced in DCs, an unique metabolic state, typified by dampened oxidative phosphorylation and basal levels of glycolysis. In the absence of increased glycolytic flux, ADJ modulated multiple steps in the cytosolic pathway of cross-presentation by enabling accumulation of degraded antigen, reducing endosomal acidity and promoting antigen localization to early endosomes. Further, by increasing ROS production and lipid peroxidation, ADJ promoted antigen escape from endosomes to the cytosol for degradation by proteasomes into peptides for MHC I loading by TAP-dependent pathways. Furthermore, we found that induction of lipid bodies (LBs) and alterations in LB composition mediated by ADJ were also critical for DC cross-presentation. Collectively, our model challenges the prevailing metabolic paradigm by suggesting that DCs can perform effective DC cross-presentation, independent of glycolysis to induce robust T cell-dependent protective immunity to intracellular pathogens. These findings have strong implications in the rational development of safe and effective immune adjuvants to potentiate robust T-cell based immunity., Competing Interests: The authors have declared that no competing interests exist.

The function of a T cell depends on its subtype and activation state. Here, we show that imaging of the autofluorescence lifetime signals of quiescent and activated T cells can be used to classify the cells. T cells isolated from human peripheral blood and activated in culture using tetrameric antibodies against the surface ligands CD2, CD3 and CD28 showed specific activation-state-dependent patterns of autofluorescence lifetime. Logistic regression models and random forest models classified T cells according to activation state with 97-99% accuracy, and according to activation state (quiescent or activated) and subtype (CD3 + CD8 + or CD3 + CD4 + ) with 97% accuracy. Autofluorescence lifetime imaging can be used to non-destructively determine T-cell function.

The auto-fluorescent coenzymes reduced nicotinamide dinucleotide (NADH) and oxidized flavin adenine dinucleotide (FAD) allow label-free detection of cellular metabolism. The optical redox ratio, which is traditionally computed as the ratio of NADH and FAD intensities, allows quantification of cell redox state. In addition to multiple formulations of the optical redox ratio from NADH and FAD intensity measurements, a fluorescence lifetime redox ratio (FLIRR) based on the fractions of protein-bound NADH and FAD was developed to overcome the limitations of experimental factors that influence fluorescence intensity measurements. In this paper, we compare fluorescence-intensity computations of the optical redox ratio with the fluorescence lifetime redox ratio for quiescent and activated T cells. Fluorescence lifetime images of NAD(P)H and FAD of T cells were acquired with a two-photon fluorescence lifetime microscope. Metabolic perturbation experiments, including inhibition of glycolysis, oxidative phosphorylation, glutaminolysis, and fatty acid synthesis revealed differences between the intensity and lifetime redox ratios. Statistical analysis reveals that the FLIRR has a lower standard deviation and skewness (two-tail T-test, P value = 0.05) than the intensity redox ratio. Correlation analysis revealed a weak relationship between FLIRR and intensity redox ratio for individual cells, with a stronger correlation identified for activated T cells (Linear regression, R-value = 0.450) than quiescent T cells (R-value = 0.172). Altogether, the results demonstrate that while both the fluorescence lifetime and intensity redox ratios resolve metabolic perturbations in T cells, the endpoints are influenced by different metabolic processes., Competing Interests: The authors declare no conflicts of interest., (© 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement.)

The importance of T cells in immunotherapy has motivated developing technologies to improve therapeutic efficacy. One objective is assessing antigen-induced T cell activation because only functionally active T cells are capable of killing the desired targets. Autofluorescence imaging can distinguish T cell activity states in a non-destructive manner by detecting endogenous changes in metabolic co-enzymes such as NAD(P)H. However, recognizing robust activity patterns is computationally challenging in the absence of exogenous labels. We demonstrate machine learning methods that can accurately classify T cell activity across human donors from NAD(P)H intensity images. Using 8260 cropped single-cell images from six donors, we evaluate classifiers ranging from traditional models that use previously-extracted image features to convolutional neural networks (CNNs) pre-trained on general non-biological images. Adapting pre-trained CNNs for the T cell activity classification task provides substantially better performance than traditional models or a simple CNN trained with the autofluorescence images alone. Visualizing the images with dimension reduction provides intuition into why the CNNs achieve higher accuracy than other approaches. Our image processing and classifier training software is available at https://github.com/gitter-lab/t-cell-classification., (© 2019 The Authors. Journal of Biophotonics published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Aims: To inform a discussion for the applicability of using the Nursing Role Effectiveness Model (NREM) in the primary health care setting through a synthesis of the literature that has used the model in all health care sectors., Design: Scoping Review., Methods: Articles were considered for inclusion if they discussed any aspect of the NREM in health care research that presented information related to any nursing regulatory designation, such as nurse practitioner (NP), registered nurse (RN), licensed/registered practical nurse (LPN/RPN) and considered both quantitative and qualitative study designs, including expert opinions and reports., Results: A total of 22 articles that cited and/or used the NREM were identified in this review. Only two studies were focused in the primary health care setting. There is precedence for the use of the NREM to guide research in primary health care. The NREM should be modified to incorporate the unique characteristics of the primary health care setting., Competing Interests: No conflict of interest has been declared by the authors.