Perspectives on label-free microscopy of heterogeneous and dynamic biological systems.

Significance: Advancements in label-free microscopy could provide real-time, non-invasive imaging with unique sources of contrast and automated standardized analysis to characterize heterogeneous and dynamic biological processes. These tools would overcome challenges with widely used methods that are destructive (e.g., histology, flow cytometry) or lack cellular resolution (e.g., plate-based assays, whole animal bioluminescence imaging). Aim: This perspective aims to (1) justify the need for label-free microscopy to track heterogeneous cellular functions over time and space within unperturbed systems and (2) recommend improvements regarding instrumentation, image analysis, and image interpretation to address these needs. Approach: Three key research areas (cancer research, autoimmune disease, and tissue and cell engineering) are considered to support the need for label-free microscopy to characterize heterogeneity and dynamics within biological systems. Based on the strengths (e.g., multiple sources of molecular contrast, non-invasive monitoring) and weaknesses (e.g., imaging depth, image interpretation) of several label-free microscopy modalities, improvements for future imaging systems are recommended. Conclusion: Improvements in instrumentation including strategies that increase resolution and imaging speed, standardization and centralization of image analysis tools, and robust data validation and interpretation will expand the applications of label-free microscopy to study heterogeneous and dynamic biological systems.

Assessment of the services provided at the family medicine staff clinics in King Saud Medical City, Riyadh.

Background: Patient satisfaction is the subjective evaluation of a patient's cognitive and emotional responses. This reflects their expectations regarding the ideal healthcare to be provided. This study aims to assess the satisfaction level of the attendees to the family medicine staff clinics at King Saud Medical City, Riyadh. Materials and Methods: A descriptive cross-sectional study recruited 378 participants. An adapted and piloted questionnaire was used to gather the data. The questionnaire enquires about the communication skills of the receptionist, the triage nurse, and the assigned physician. It included questions regarding the structure of the clinics. Results: Females represent the majority of about 255 (67.5%). About 2/3 of the respondents were within the age group of 18-34 years, 245 (64.8%). A high level of satisfaction was reported by the participants regarding the professionalism, kindness, interest of the staff member, and waiting time. Gender, marital status, and age group have no significant effect on the satisfaction level; the P value was uniformly more than 0.05. Conclusion: The result of this study identified high satisfaction responses regarding the communication skills of the receptionist, triage nurse, and physicians. The overall evaluation of the experience during staff clinic visits was satisfactory. Periodic evaluation of these attributes and other indicators that promote patient-centered care should be undertaken to improve the overall quality of care.

Leveraging electrocardiography signals for deep learning-driven cardiovascular disease classification model.

Electrocardiography (ECG) is the most non-invasive diagnostic tool for cardiovascular diseases (CVDs). Automatic analysis of ECG signals assists in accurately and rapidly detecting life-threatening arrhythmias like atrioventricular blockage, atrial fibrillation, ventricular tachycardia, etc. The ECG recognition models need to utilize algorithms to detect various kinds of waveforms in the ECG and identify complicated relationships over time. However, the high variability of wave morphology among patients and noise are challenging issues. Physicians frequently utilize automated ECG abnormality recognition models to classify long-term ECG signals. Recently, deep learning (DL) models can be used to achieve enhanced ECG recognition accuracy in the healthcare decision making system. In this aspect, this study introduces an automated DL enabled ECG signal recognition (ADL-ECGSR) technique for CVD detection and classification. The ADL-ECGSR technique employs three most important subprocesses: pre-processed, feature extraction, parameter tuning, and classification. Besides, the ADL-ECGSR technique involves the design of a bidirectional long short-term memory (BiLSTM) based feature extractor, and the Adamax optimizer is utilized to optimize the trained method of the BiLSTM model. Finally, the dragonfly algorithm (DFA) with a stacked sparse autoencoder (SSAE) module is applied to recognize and classify EEG signals. An extensive range of simulations occur on benchmark PTB-XL datasets to validate the enhanced ECG recognition efficiency. The comparative analysis of the ADL-ECGSR methodology showed a remarkable performance of 91.24 % on the existing methods.

Impact of Tranexamic Acid on Bleeding Outcomes and Complication Rates in Facelift: A Systematic Review and Meta-analysis.

BACKGROUND: Rhytidectomy poses a significant risk of bleeding. Several studies have reported the safety profile and efficacy of tranexamic acid (TXA), an antifibrinolytic agent, in minimizing perioperative sequelae, particularly hematoma and bleeding. OBJECTIVES: The aim of this systematic review was to analyze the effect of TXA administration in facelift surgery, its effect with different routes of administration, and to compare different administration routes in reducing intraoperative blood loss, postoperative edema, and ecchymosis in rhytidectomy. METHODS: A systematic literature search was conducted to identify studies that reported on TXA in facelift surgery. The primary outcomes of interest were intraoperative blood loss, time to achieve hemostasis, operation duration, and postoperative hematoma, edema, ecchymosis, drain output, and major and minor complications. Meta-analyses of hematoma, operation duration, drain output, and major and minor complications were performed, and the risk of bias was assessed with ROBINS-I for nonrandomized studies, and Cochrane's RoB 2.0, a tool for randomized controlled trials. RESULTS: In total, 104 articles were included in the initial screening. Out of 388 participants 170 patients were administered TXA, predominantly female (over 91%), with ages from the late 50s to mid-60s. TXA administration varied, with subcutaneous injection being the most common method. The meta-analysis revealed that the pooled prevalence of minor and major hematoma in TXA recipients was remarkably low, with a significant reduction in the risk of minor hematoma (odds ratio [OR] = 0.18, 95% CI 0.05-0.62, P < .001) and no significant difference in major hematoma risk. Interestingly, TXA significantly reduced postoperative drainage compared to the controls (mean difference = -25.59, 95% CI, -30.4--20.77, P < .01). Additionally, neither minor nor major complications were significantly different between the TXA recipients and controls. Specifically, the pooled odds for the incidence of major complications were not significantly different (OR = 1.47, 95% CI, 0.23-9.19, P = .68), and similar results were found for minor complications (OR = 0.59, 95% CI, 0.23-1.48, P = .26). CONCLUSIONS: TXA significantly reduces postoperative drain output and minor hematomas in facelift surgery without increasing major complications. It also reduces edema, ecchymosis, and intraoperative blood loss. However, further studies are required to explore the efficacy of TXA with different dosages and administration routes.

Distribution and Antimicrobial Resistance of Complicated Intraabdominal Infection Pathogens in Two Tertiary Hospitals in Egypt.

Background: Management of complicated intraabdominal infections (cIAIs) requires containment of the source and appropriate initial antimicrobial therapy. Identifying the local data is important to guide the empirical selection of antimicrobial therapy. In this study, we aimed to describe the pathogen distribution and antimicrobial resistance of cIAI. Methods: In two major tertiary care hospitals in Egypt, we enrolled patients who met the case definition of cIAI from October 2022 to September 2023. Blood cultures were performed using the BACTAlert system (BioMerieux, Marcy l'Etoile, France). A culture of aspirated fluid, resected material, or debridement of the infection site was performed. Identification of pathogens and antimicrobial susceptibility testing were conducted by the VITEK-2 system (BioMerieux, Marcy l'Etoile, France). Gram-negative resistance genes were identified by PCR and confirmed by whole bacterial genome sequencing using the Nextera XT DNA Library Preparation Kit and sequencing with the MiSeq Reagent Kit 600 v3 (Illumina, USA) on the Illumina MiSeq. Results: We enrolled 423 patients, 275 (65.01%) males. The median age was 61.35 (range 25-72 years). We studied 452 recovered bacterial isolates. Gram-negative bacteria were the vast majority, dominated by E. coli, followed by Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, and Proteus mirabilis (33.6%, 30.5%, 13.7%, 13%, and 5.4%, respectively). High rates of resistance were detected to third- and fourth-generation cephalosporins and fluoroquinolones. No resistance was detected to colistin. Resistance to amikacin and tigecycline was low among all isolates. Resistance to meropenem and ceftazidime/avibactam was moderate. ESBL genes were common in E. coli and K. pneumoniae. CTX-M15 gene was the most frequent. Among Enterobacterales, blaOXA-48 and blaNDM were the most prevalent carbapenemase genes. Pseudomonas aeruginosa isolates harbored a wide variety of carbapenemase genes (OXA, NDM, VIM, SIM, GIM, SPM, IMP, AIM), dominated by metallo-beta-lactamases. In 20.6% of isolates, we identified two or more resistance genes. Conclusion: High resistance rates were detected to third- and fourth-generation cephalosporins and fluoroquinolones. Amikacin and tigecyclines were the most active antimicrobials. Our data call for urgent implementation of antimicrobial stewardship programs and reinforcement of infection control.

Receptors Involved in COVID-19-Related Anosmia: An Update on the Pathophysiology and the Mechanistic Aspects.

Olfactory perception is an important physiological function for human well-being and health. Loss of olfaction, or anosmia, caused by viral infections such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has received considerable attention, especially in persistent cases that take a long time to recover. This review discusses the integration of different components of the olfactory epithelium to serve as a structural and functional unit and explores how they are affected during viral infections, leading to the development of olfactory dysfunction. The review mainly focused on the role of receptors mediating the disruption of olfactory signal transduction pathways such as angiotensin converting enzyme 2 (ACE2), transmembrane protease serine type 2 (TMPRSS2), neuropilin 1 (NRP1), basigin (CD147), olfactory, transient receptor potential vanilloid 1 (TRPV1), purinergic, and interferon gamma receptors. Furthermore, the compromised function of the epithelial sodium channel (ENaC) induced by SARS-CoV-2 infection and its contribution to olfactory dysfunction are also discussed. Collectively, this review provides fundamental information about the many types of receptors that may modulate olfaction and participate in olfactory dysfunction. It will help to understand the underlying pathophysiology of virus-induced anosmia, which may help in finding and designing effective therapies targeting molecules involved in viral invasion and olfaction. To the best of our knowledge, this is the only review that covered all the receptors potentially involved in, or mediating, the disruption of olfactory signal transduction pathways during COVID-19 infection. This wide and complex spectrum of receptors that mediates the pathophysiology of olfactory dysfunction reflects the many ways in which anosmia can be therapeutically managed.

Oral Care and Positioning to Prevent Ventilator-Associated Pneumonia: A Systematic Review.

Introduction: Ventilator-associated pneumonia (VAP) is one of the most common nosocomial infections in critical patients. The negative impacts of VAP on patient outcomes emphasize the importance of effective preventive measures such as oral care and patient positioning. The aim of this review was to investigate the impact of oral care and positioning on the prevention of VAP among patients in the intensive care unit. Methods: This review was reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. A comprehensive search was performed. The methodological quality of included studies was appraised using Joanna Briggs Institute checklists. Results: In total 13 studies were included, all of them were randomized controlled trials. Six out of nine studies about oral care have significant results on VAP incidence. Regarding the position, Patients positioned at a 45-degree were less likely to develop VAP than those positioned at a 30-degree and those in a supine position. Conclusion: Although patients positioned at 45-degree angle were less likely to develop VAP than those at 30-degree, it is necessary to individualize this practice before recommending it, once there are some contraindications, such as neurocritical patients. Regarding oral care to prevent VAP, considering the current guidelines' recommendation not to use oral chlorexidine, further studies evaluating alternatives are needed.

Breast self-examination knowledge, attitude, and practice among Jazan women, Kingdom of Saudi Arabia

Breast cancer is the most common cancer among women worldwide. It is a major cause of cancer death, and its incidence rate has been gradually increasing in the Arab world, Saudi Arabia has a higher proportion of young females getting breast cancer than other countries. This study was conducted to investigate the knowledge, attitude, and practice regarding breast self-examination among females from 20 to 60 years old in Jazan Region, Saudi Arabia. Using A Community-based descriptive cross-sectional approach, data were gathered from 567 Saudi and non-Saudi women using structured interviews and then analyzed using the Statistical Package for Social Sciences (IBM) version 21.0 software program. Where the study indicated that around half of the participants (55.7%) had good total knowledge scores regarding breast self-examination, and most had positive attitudes. However, only 205 (36.2%) respondents practice it regularly. Moreover, the study reveals a significant association between respondents' knowledge and attitudes toward breast self-examination (P = 0.05). These findings indicate moderate knowledge, poor breast self-examination practices, and a significant association between knowledge and attitudes. Therefore, conducting health education programs is necessary to raise awareness about breast self-examination (BSE) among Jazan women.

Le cancer du sein est le cancer le plus répandu chez les femmes dans le monde. Il s'agit d'une cause majeure de décès par cancer et son taux d'incidence augmente progressivement dans le monde arabe. L'Arabie saoudite compte une proportion plus élevée de jeunes femmes atteintes d'un cancer du sein que les autres pays. Cette étude a été menée pour étudier les connaissances, l'attitude et la pratique concernant l'auto-examen des seins chez les femmes de 20 à 60 ans dans la région de Jazan, en Arabie Saoudite. À l'aide d'une approche transversale descriptive communautaire, les données ont été recueillies auprès de 567 femmes saoudiennes et non saoudiennes à l'aide d'entretiens structurés, puis analysées à l'aide du logiciel Statistical Package for Social Sciences (IBM) version 21.0. L'étude a indiqué qu'environ la moitié des participantes (55,7 %) avaient de bons scores totaux de connaissances concernant l'auto-examen des seins et que la plupart avaient des attitudes positives. Cependant, seuls 205 (36,2%) répondants le pratiquent régulièrement. De plus, l'étude révèle une association significative entre les connaissances et les attitudes des répondants à l'égard de l'auto-examen des seins (P = 0,05). Ces résultats indiquent des connaissances modérées, de mauvaises pratiques d'auto-examen des seins et une association significative entre les connaissances et les attitudes. Par conséquent, il est nécessaire de mener des programmes d'éducation sanitaire pour sensibiliser les femmes de Jazan à l'auto-examen des seins (ESB).

RELIABILITY OF CLINICAL GRADING OF DIABETIC RETINOPATHY COMPARED WITH GRADING OF ULTRA-WIDEFIELD IMAGES.

PURPOSE: To evaluate the reliability of clinical grading of diabetic retinopathy (DR) severity compared with grading on ultra-widefield pseudocolor fundus (UWF-CF) and ultra-widefield fluorescein angiography (UWF-FA) images and their relative detection of sight-threatening DR and referable DR. METHODS: A total of 184 diabetic eyes were analyzed. UWF-CF and UWF-FA images were graded based on the International Clinical Diabetic Retinopathy severity scale. Agreement between clinical and UWF-based severity grading was evaluated using Cohen's kappa coefficient. The rate of sight-threatening DR and referable DR was evaluated for each grading method. RESULTS: Moderate agreement was found between clinical grading and UWF-CF (k = 0.456, P < 0.001) and between UWF-CF and UWF-FA (k = 0.443, P < 0.001). The agreement between clinical grading and UWF-FA was fair (k = 0.397, P < 0.001). UWF-based grading identified a higher DR grade in 56 eyes (30%) on UWF-CF and 85 eyes (46.2%) on UWF-FA. Compared with clinical grading, UWF-FA detected a higher rate of sight-threatening DR (44%; 81/184 vs. 22.3%; 41/184), while UWF-CF detected more referable eyes (58.1%; 107/184 vs. 45.65%; 84/184). CONCLUSION: Ultra-widefield pseudocolor fundus is a valuable tool for identifying referable eyes and can be a useful, noninvasive adjunct to clinical grading. The results suggest that UWF-FA is particularly useful for detecting unsuspected sight-threatening DR in eyes with clinically referable DR.

Factors Affecting Patients' Atttendance for Periodontal Follow-up Visits after Crown Lengthening Surgery.

PURPOSE: To assess adherence to follow-up maintenance visits among patients who had previously undergone crown-lengthening surgery and investigate the different factors impacting their compliance. MATERIALS AND METHODS: A total of 314 patients were identified for follow-up appointments. Based on their responses, participants were categorised into four groups: attendees, non-attendees, refusals, and unreachable. Furthermore, data on sociodemographic factors (age, sex, nationality, marital status, education, occupation, and residential area), medical history, dental history (including missing teeth, implants, or orthodontic treatment history), and past appointment attendance (average yearly appointments, missed appointment percentage, and last appointment date) were collected and analysed to understand their influence on patient compliance. RESULTS: In a sample of 314 patients, 102 (32.5%) attended the appointments successfully. Improved attendance rates were significantly associated with being female, Saudi Arabian, married, and employed (p < 0.05). Moreover, patients with a high frequency of annual appointments and a recent history of appointments exhibited better compliance. None of the analysed dental factors affected the attendance rates. CONCLUSION: About one-third of patients who had undergone crown lengthening surgery were compliant with the follow-up visits. Different factors influenced this compliance pattern to varying extents, with more efforts needed to enhance patients' commitment to these visits.

Knowledge and Attitudes Toward Pressure Ulcer Prevention Evidence-based Guidelines among Nursing Students at Jazan University, Saudi Arabia.

BACKGROUND: Pressure ulcers (PUs) are a common global problem. They affect health-care costs and patient health because of increased morbidity and mortality rates. Critically ill patients are more prone to acquiring PUs than other patients. As the prevalence of PUs is high in Saudi Arabia, more attention is required for PU prevention. Nurses' knowledge and attitudes toward PU prevention are crucial. AIMS: This study aimed to assess the knowledge and attitudes of nursing students regarding PU prevention evidence-based guidelines. SETTING AND DESIGN: This descriptive, cross-sectional study was conducted among nursing students at Jazan University. MATERIALS AND METHODS: Data were collected using an electronic questionnaire. STATISTICAL ANALYSIS USED: Data were entered, coded, and analyzed using the SPSS version 23. RESULTS: A total of 378 participants were included. The mean age was 22.4 ± 3.4 years, and the mean knowledge score was 7.6 ± 2.64 (range = 0-16). Of the participants, 365 (96.6%) and 13 (3.4%) were considered to have poor and good knowledge about PUs, respectively. Only 72 (19%) correctly identified a lack of oxygen as the cause of PUs; other participants answered malnutrition and moisture. Conversely, 200 (52.9%) participants indicated that recent weight loss below patients' ideal weight increases the risk for PUs. Concerning knowledge about preventive measures, 64 (16.9%) participants correctly answered that a backward sitting position, with both legs resting on a footrest, can reduce the amount of pressure. Meanwhile, 169 (44.7%) participants correctly selected thick air cushion as a means to reduce the magnitude of pressure when patients are sliding down on a chair. Some participants answered that PUs can be reduced if patients are mobilized. Seventy-two (19%) participants indicated that patients laying on a visco-elastic foam mattress should be repositioned every 2 h to reduce the duration of pressure. Age, year of education, training experience, and department were not significantly associated with knowledge about PUs (P = 0.333, P = 0.370, P = 0.700, and P = 0.810, respectively). CONCLUSIONS: The general knowledge and attitudes toward PUs of the nursing students at Jazan University were poor. Nevertheless, they had an average knowledge level regarding PU prevention. Age and training experience were not significantly associated with knowledge about PUs.

Résumé Contexte:Les ulcères de pression (UP) sont un problème mondial courant. Ils affectent les coûts de santé et la santé des patients en raison de l'augmentation des taux de morbidité et de mortalité. Les patients gravement malades sont plus susceptibles de développer des UP que d'autres patients. Comme la prévalence des UP est élevée en Arabie saoudite, une attention accrue est nécessaire pour la prévention des UP. Les connaissances et attitudes des infirmiers en matière de prévention des UP sont cruciales.Objectifs:Cette étude visait à évaluer les connaissances et attitudes des étudiants en soins infirmiers concernant les directives de prévention des UP basées sur des preuves. Cadre et conception : Cette étude descriptive et transversale a été menée auprès des étudiants en soins infirmiers de l'Université de Jazan.Matériels et méthodes:Les données ont été collectées à l'aide d'un questionnaire électronique. Analyse statistique utilisée: Les données ont été saisies, codées et analysées à l'aide de la version 23 du logiciel SPSS.Résultats:Au total, 378 participants ont été inclus. L'âge moyen était de 22,4 ± 3,4 ans et le score moyen de connaissance était de 7,6 ± 2,64 (plage = 0-16). Parmi les participants, 365 (96,6 %) avaient une connaissance médiocre et 13 (3,4 %) une bonne connaissance des UP. Seuls 72 (19 %) ont correctement identifié un manque d'oxygène comme étant la cause des UP; les autres participants ont répondu la malnutrition et l'humidité. En revanche, 200 (52,9 %) participants ont indiqué qu'une perte de poids récente en dessous du poids idéal des patients augmente le risque de UP. En ce qui concerne les connaissances sur les mesures préventives, 64 (16,9 %) participants ont répondu correctement qu'une position assise inclinée vers l'arrière, avec les deux jambes reposant sur un repose-pieds, peut réduire la pression. Pendant ce temps, 169 (44,7 %) participants ont correctement sélectionné un coussin d'air épais comme moyen de réduire l'ampleur de la pression lorsque les patients glissent sur une chaise. Certains participants ont répondu que les UP peuvent être réduits si les patients sont mobilisés. Soixante-douze (19 %) participants ont indiqué que les patients allongés sur un matelas en mousse viscoélastique devraient être repositionnés toutes les 2 heures pour réduire la durée de la pression. L'âge, l'année d'éducation, l'expérience de formation et le département n'étaient pas significativement associés aux connaissances sur les UP (P = 0,333, P = 0,370, P = 0,700 et P = 0,810, respectivement).Conclusions:Les connaissances générales et les attitudes à l'égard des UP des étudiants en soins infirmiers de l'Université de Jazan étaient médiocres. Néanmoins, ils avaient un niveau de connaissance moyen concernant la prévention des UP. L'âge et l'expérience de formation n'étaient pas significativement associés aux connaissances sur les UP.

Effect of mesenchymal stem cells and melatonin on experimentally induced peripheral nerve injury in rats.

Injury of a peripheral nerve (PNI) leads to both ischemic and inflammatory alterations. Sciatic nerve injury (SNI) represents the most widely used model for PNI. Mesenchymal stem cell-based therapy (MSCs) has convenient properties on PNI by stimulating the nerve regeneration. Melatonin has cytoprotective activity. The neuroprotective characteristics of MSCs and melatonin separately or in combination remain a knowledge need. In the rats-challenged SNI, therapeutic roles of intralesional MSCs and intraperitoneal melatonin injections were evaluated by functional assessment of peripheral nerve regeneration by walking track analysis involving sciatic function index (SFI) and two electrophysiological tests, electromyography and nerve conduction velocity, as well as measurement of antioxidant markers in serum, total antioxidant capacity (TAC) and malondialdehyde, and mRNA expression of brain derived neurotrophic factor (BDNF) in nerve tissues in addition to the histopathological evaluation of nerve tissue. Both individual and combination therapy with MSCs and melatonin therapies could effectively ameliorate this SNI and promote its regeneration as evidenced by improving the SFI and two electrophysiological tests and remarkable elevation of TAC with decline in lipid peroxidation and upregulation of BDNF levels. All of these led to functional improvement of the damaged nerve tissues and good recovery of the histopathological sections of sciatic nerve tissues suggesting multifactorial synergistic approach of the concurrent usage of melatonin and MSCs in PNI. The combination regimen has the most synergistic neuro-beneficial effects in PNI that should be used as therapeutic option in patients with PNI to boost their quality of life.

Autofluorescence lifetime flow cytometry with time-correlated single photon counting.

Autofluorescence lifetime imaging microscopy (FLIM) is sensitive to metabolic changes in single cells based on changes in the protein-binding activities of the metabolic co-enzymes NAD(P)H. However, FLIM typically relies on time-correlated single-photon counting (TCSPC) detection electronics on laser-scanning microscopes, which are expensive, low-throughput, and require substantial post-processing time for cell segmentation and analysis. Here, we present a fluorescence lifetime-sensitive flow cytometer that offers the same TCSPC temporal resolution in a flow geometry, with low-cost single-photon excitation sources, a throughput of tens of cells per second, and real-time single-cell analysis. The system uses a 375 nm picosecond-pulsed diode laser operating at 50 MHz, alkali photomultiplier tubes, an FPGA-based time tagger, and can provide real-time phasor-based classification (i.e., gating) of flowing cells. A CMOS camera produces simultaneous brightfield images using far-red illumination. A second PMT provides two-color analysis. Cells are injected into the microfluidic channel using a syringe pump at 2-5 mm/s with nearly 5 ms integration time per cell, resulting in a light dose of 2.65 J/cm2 that is well below damage thresholds (25 J/cm2 at 375 nm). Our results show that cells remain viable after measurement, and the system is sensitive to autofluorescence lifetime changes in Jurkat T cells with metabolic perturbation (sodium cyanide), quiescent versus activated (CD3/CD28/CD2) primary human T cells, and quiescent versus activated primary adult mouse neural stem cells, consistent with prior studies using multiphoton FLIM. This TCSPC-based autofluorescence lifetime flow cytometer provides a valuable label-free method for real-time analysis of single-cell function and metabolism with higher throughput than laser-scanning microscopy systems.

Cellpose as a reliable method for single-cell segmentation of autofluorescence microscopy images.

Autofluorescence microscopy uses intrinsic sources of molecular contrast to provide cellular-level information without extrinsic labels. However, traditional cell segmentation tools are often optimized for high signal-to-noise ratio (SNR) images, such as fluorescently labeled cells, and unsurprisingly perform poorly on low SNR autofluorescence images. Therefore, new cell segmentation tools are needed for autofluorescence microscopy. Cellpose is a deep learning network that is generalizable across diverse cell microscopy images and automatically segments single cells to improve throughput and reduce inter-human biases. This study aims to validate Cellpose for autofluorescence imaging, specifically from multiphoton intensity images of NAD(P)H. Manually segmented nuclear masks of NAD(P)H images were used to train new Cellpose models. These models were applied to PANC-1 cells treated with metabolic inhibitors and patient-derived cancer organoids (across 9 patients) treated with chemotherapies. These datasets include co-registered fluorescence lifetime imaging microscopy (FLIM) of NAD(P)H and FAD, so fluorescence decay parameters and the optical redox ratio (ORR) were compared between masks generated by the new Cellpose model and manual segmentation. The Dice score between repeated manually segmented masks was significantly lower than that of repeated Cellpose masks (p<0.0001) indicating greater reproducibility between Cellpose masks. There was also a high correlation (R2>0.9) between Cellpose and manually segmented masks for the ORR, mean NAD(P)H lifetime, and mean FAD lifetime across 2D and 3D cell culture treatment conditions. Masks generated from Cellpose and manual segmentation also maintain similar means, variances, and effect sizes between treatments for the ORR and FLIM parameters. Overall, Cellpose provides a fast, reliable, reproducible, and accurate method to segment single cells in autofluorescence microscopy images such that functional changes in cells are accurately captured in both 2D and 3D culture.

Hypertension Likely Drives Arteriolar Wall Thickening in Preclinical Diabetic Retinopathy While Diabetes Drives Wall Thickness in Clinical Retinopathy.

Purpose: Both hypertension and diabetes are known to increase the wall-to-lumen ratio (WLR) of retinal arterioles, but the differential effects are unknown. Here, we study the timing and relative impact of hypertension versus diabetes on the WLR in diabetic retinopathy (DR) to address this unresolved question. Methods: This prospective cross-sectional study compared the retinal arteriolar WLR in 17 healthy eyes, 15 with diabetes but no apparent DR (DM no DR), and 8 with diabetic macular edema (DME) and either nonproliferative or proliferative DR. We imaged each arteriole using adaptive optics scanning laser ophthalmoscopy and measured the WLR using ImageJ. Multiple linear regression (MLR) was performed to estimate the effects of hypertension, diabetes, and age on the WLR. Results: Both subjects with DM no DR and subjects with DME had significantly higher WLR than healthy subjects (0.36 ± 0.08 and 0.42 ± 0.08 vs. 0.29 ± 0.07, 1-way ANOVA P = 0.0009). MLR in healthy subjects and subjects with DM no DR showed hypertension had the strongest effect (regression coefficient = 0.08, P = 0.009), whereas age and diabetes were not significantly correlated with WLR. MLR in all three groups together (healthy, DM no DR, and DME) showed diabetes had the strongest effect (regression coefficient = 0.05, P = 0.02), whereas age and hypertension were not significantly correlated with WLR. Conclusions: Hypertension may be an early driver of retinal arteriolar wall thickening in preclinical DR, independent of age or diabetes, whereas changes specific to DR may drive wall thickening in DME and later DR stages. Translational Relevance: We offer a framework for understanding the relative contributions of hypertension and diabetes on the vascular wall, and emphasize the importance of hypertension control early in diabetes even before DR onset.

Bilateral Exudative Retinal Detachment in Preeclampsia: A Case Report and Literature Review.

Preeclampsia, a complex multisystem disorder predominantly impacting the kidneys and liver, manifests through hypertension and organ dysfunction in expectant mothers. Preeclampsia can also cause ocular signs, but they are uncommon. Exudative retinal detachment (ERD) is one such unusual but dangerous consequence. A thorough clinical description and therapy of a patient who experienced exudative retinal detachment while experiencing preeclampsia are provided in this study. A 28-year-old Saudi female, with no medical or surgical history, underwent an emergency cesarean section (CS) due to severe preeclampsia and failed induction of labor. The patient complained of painless blurry vision, with central dark spot and decreased vision starting from labor daytime. The patient was admitted to the hospital for blood pressure monitoring and further investigations. The patient was started on hydralazine intravenous (IV) and labetalol PO to control BP. The patient was delivered by cesarean section for preeclampsia with severe features after the failure of labor induction, and she had improved her vision by four weeks postpartum. Retinal detachment as a consequence of preeclampsia is conservatively managed, with a generally favorable prognosis. Previous studies have consistently emphasized the critical importance of a multidisciplinary approach that fosters collaboration between obstetricians and ophthalmologists. This collaborative strategy not only ensures comprehensive care but also facilitates early detection, timely intervention, and improved management outcomes for conditions affecting both maternal health and ophthalmic well-being during pregnancy.

FOXC1 regulates endothelial CD98 (LAT1/4F2hc) expression in retinal angiogenesis and blood-retina barrier formation.

Angiogenesis, the growth of new blood vessels from pre-existing vasculature, is essential for the development of new organ systems, but transcriptional control of angiogenesis remains incompletely understood. Here we show that FOXC1 is essential for retinal angiogenesis. Endothelial cell (EC)-specific loss of Foxc1 impairs retinal vascular growth and expression of Slc3a2 and Slc7a5, which encode the heterodimeric CD98 (LAT1/4F2hc) amino acid transporter and regulate the intracellular transport of essential amino acids and activation of the mammalian target of rapamycin (mTOR). EC-Foxc1 deficiency diminishes mTOR activity, while administration of the mTOR agonist MHY-1485 rescues perturbed retinal angiogenesis. EC-Foxc1 expression is required for retinal revascularization and resolution of neovascular tufts in a model of oxygen-induced retinopathy. Foxc1 is also indispensable for pericytes, a critical component of the blood-retina barrier during retinal angiogenesis. Our findings establish FOXC1 as a crucial regulator of retinal vessels and identify therapeutic targets for treating retinal vascular disease.

Perfusion Deficits in Diabetes Without Retinopathy Localize to the Perivenular Deep Capillaries Near the Fovea on OCT Angiography.

Purpose: To localize early capillary perfusion deficits in patients with diabetes mellitus (DM) without clinical diabetic retinopathy (DR) using averaged OCT angiography (OCTA). Design: Retrospective cross-sectional study. Participants: Patients with DM without DR and healthy controls. Methods: We measured perfusion deficits in the full retina, superficial capillary plexus (SCP), and deep capillary plexus (DCP) on averaged 3 × 3-mm OCTA images. Perfusion deficits were defined as the percentage of retinal tissue located >30 µm from blood vessels, excluding the foveal avascular zone (FAZ). One eye from each patient was selected based on image quality. We measured deficits in the parafoveal region, the 300 µm surrounding the FAZ, and 300 to 1000 µm surrounding the FAZ. If a capillary layer within one of these regions was significantly different in DM without DR compared with controls, we further characterized the location of perfusion deficit as periarteriolar, perivenular, or the capillaries between these 2 zones. Main Outcome Measures: Location of increased perfusion deficits in patients with DM without DR compared with controls. Results: Sixteen eyes from 16 healthy controls were compared with 16 eyes from 16 patients with DM without DR (age 45.1 ± 10.7 and 47.4 ± 15.2 years respectively, P = 0.64). Foveal avascular zone area and perfusion deficits in the entire parafovea and the 300 to 1000-µm ring around the FAZ were not significantly different between groups (P > 0.05 for all). Perfusion deficits in 300 µm around the FAZ were significantly increased in patients with DM without DR in full retinal thickness, SCP, and DCP (P < 0.05 for all). When analyzing the perivenular, periarteriolar, and capillary zones, only the perivenular DCP perfusion deficits were significantly increased (5.03 ± 2.92% in DM without DR and 2.73 ± 1.97% in controls, P = 0.014). Conclusions: Macular perfusion deficits in patients with DM without DR were significantly increased in the region nearest the FAZ, mainly at the perivenular deep capillaries. Further research on these early changes may improve our understanding of the capillaries most susceptible to vascular injury and disruption during diabetes. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Autofluorescence lifetime flow cytometry with time-correlated single photon counting.

Autofluorescence lifetime imaging microscopy (FLIM) is sensitive to metabolic changes in single cells based on changes in the protein-binding activities of the metabolic co-enzymes NAD(P)H. However, FLIM typically relies on time-correlated single-photon counting (TCSPC) detection electronics on laser-scanning microscopes, which are expensive, low-throughput, and require substantial post-processing time for cell segmentation and analysis. Here, we present a fluorescence lifetime-sensitive flow cytometer that offers the same TCSPC temporal resolution in a flow geometry, with low-cost single-photon excitation sources, a throughput of tens of cells per second, and real-time single-cell analysis. The system uses a 375nm picosecond-pulsed diode laser operating at 50MHz, alkali photomultiplier tubes, an FPGA-based time tagger, and can provide real-time phasor-based classification ( i.e ., gating) of flowing cells. A CMOS camera produces simultaneous brightfield images using far-red illumination. A second PMT provides two-color analysis. Cells are injected into the microfluidic channel using a syringe pump at 2-5 mm/s with nearly 5ms integration time per cell, resulting in a light dose of 2.65 J/cm 2 that is well below damage thresholds (25 J/cm 2 at 375 nm). Our results show that cells remain viable after measurement, and the system is sensitive to autofluorescence lifetime changes in Jurkat T cells with metabolic perturbation (sodium cyanide), quiescent vs. activated (CD3/CD28/CD2) primary human T cells, and quiescent vs. activated primary adult mouse neural stem cells, consistent with prior studies using multiphoton FLIM. This TCSPC-based autofluorescence lifetime flow cytometer provides a valuable label-free method for real-time analysis of single-cell function and metabolism with higher throughput than laser-scanning microscopy systems.