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Diagnostics (Basel, Switzerland) Aug 2023The aim of this clinical study was to compare the diagnostic performance of dual short wavelength infrared (SWIR) occlusal transillumination and reflectance...
The aim of this clinical study was to compare the diagnostic performance of dual short wavelength infrared (SWIR) occlusal transillumination and reflectance multispectral imaging with conventional visual assessment and radiography for caries detection on premolars scheduled for extraction for orthodontics reasons. Polarized light microscopy (PLM) and micro-computed tomography (microCT) performed after tooth extraction were used as gold standards. The custom-fabricated imaging probe was 3D-printed and the imaging system employed a SWIR camera and fiber-optic light sources emitting light at 1300 nm for occlusal transillumination and 1600 nm for reflectance measurements. Teeth (n = 135) on 40 test subjects were imaged in vivo using the SWIR imaging prototype in the study and teeth were extracted after imaging. Our study demonstrates for the first time that near-simultaneous real-time transillumination and reflectance video can be successfully acquired for caries detection. Both SWIR imaging modalities had markedly higher sensitivity for lesions on proximal and occlusal surfaces compared to conventional methods (visual and radiographic). Reflectance imaging at 1600 nm had higher sensitivity and specificity than transillumination at 1300 nm. The combined SWIR methods yielded higher specificity but the combined sensitivity was lower than for each individual method.
PubMed: 37685362
DOI: 10.3390/diagnostics13172824 -
Archives of Dermatological Research May 2024Skin cancer treatment is a core aspect of dermatology that relies on accurate diagnosis and timely interventions. Teledermatology has emerged as a valuable asset across... (Review)
Review
Skin cancer treatment is a core aspect of dermatology that relies on accurate diagnosis and timely interventions. Teledermatology has emerged as a valuable asset across various stages of skin cancer care including triage, diagnosis, management, and surgical consultation. With the integration of traditional dermoscopy and store-and-forward technology, teledermatology facilitates the swift sharing of high-resolution images of suspicious skin lesions with consulting dermatologists all-over. Both live video conference and store-and-forward formats have played a pivotal role in bridging the care access gap between geographically isolated patients and dermatology providers. Notably, teledermatology demonstrates diagnostic accuracy rates that are often comparable to those achieved through traditional face-to-face consultations, underscoring its robust clinical utility. Technological advancements like artificial intelligence and reflectance confocal microscopy continue to enhance image quality and hold potential for increasing the diagnostic accuracy of virtual dermatologic care. While teledermatology serves as a valuable clinical tool for all patient populations including pediatric patients, it is not intended to fully replace in-person procedures like Mohs surgery and other necessary interventions. Nevertheless, its role in facilitating the evaluation of skin malignancies is gaining recognition within the dermatologic community and fostering high approval rates from patients due to its practicality and ability to provide timely access to specialized care.
Topics: Humans; Artificial Intelligence; Dermatology; Dermoscopy; Remote Consultation; Skin Neoplasms; Telemedicine
PubMed: 38696032
DOI: 10.1007/s00403-024-02884-7 -
Cell Communication and Signaling : CCS Oct 2023Specific interactions between G protein-coupled receptors (GPCRs) and G proteins play a key role in mediating signaling events. While there is little doubt regarding...
BACKGROUND
Specific interactions between G protein-coupled receptors (GPCRs) and G proteins play a key role in mediating signaling events. While there is little doubt regarding receptor preference for Gα subunits, the preferences for specific Gβ and Gγ subunits and the effects of different Gβγ dimer compositions on GPCR signaling are poorly understood. In this study, we aimed to investigate the subcellular localization and functional response of Gαi-based heterotrimers with different combinations of Gβ and Gγ subunits.
METHODS
Live-cell imaging microscopy and colocalization analysis were used to investigate the subcellular localization of Gαi in combination with Gβ or Gβ heterotrimers, along with representative Gγ subunits. Furthermore, fluorescence lifetime imaging microscopy (FLIM-FRET) was used to investigate the nanoscale distribution of Gαi-based heterotrimers in the plasma membrane, specifically with the dopamine D receptor (DR). In addition, the functional response of the system was assessed by monitoring intracellular cAMP levels and conducting bioinformatics analysis to further characterize the heterotrimer complexes.
RESULTS
Our results show that Gαi heterotrimers mainly localize to the plasma membrane, although the degree of colocalization is influenced by the accompanying Gβ and Gγ subunits. Heterotrimers containing Gβ showed slightly lower membrane localization compared to those containing Gβ, but certain combinations, such as Gαiβγ and Gαiβγ, deviated from this trend. Examination of the spatial arrangement of Gαi in relation to DR and of changes in intracellular cAMP level showed that the strongest functional response is observed for those trimers for which the distance between the receptor and the Gα subunit is smallest, i.e. complexes containing Gβ and Gγ or Gγ subunit. Deprivation of Gαi lipid modifications resulted in a significant decrease in the amount of protein present in the cell membrane, but did not always affect intracellular cAMP levels.
CONCLUSION
Our studies show that the composition of G protein heterotrimers has a significant impact on the strength and specificity of GPCR-mediated signaling. Different heterotrimers may exhibit different conformations, which further affects the interactions of heterotrimers and GPCRs, as well as their interactions with membrane lipids. This study contributes to the understanding of the complex signaling mechanisms underlying GPCR-G-protein interactions and highlights the importance of the diversity of Gβ and Gγ subunits in G-protein signaling pathways. Video Abstract.
Topics: GTP-Binding Protein alpha Subunits; GTP-Binding Protein gamma Subunits; GTP-Binding Proteins; Signal Transduction; Carrier Proteins; Receptors, G-Protein-Coupled
PubMed: 37817242
DOI: 10.1186/s12964-023-01307-w -
Cell Communication and Signaling : CCS Jan 2024The incidence of melanoma is increasing worldwide. Since metastatic melanoma is highly aggressive, it is important to decipher all the biological aspects of melanoma...
BACKGROUND
The incidence of melanoma is increasing worldwide. Since metastatic melanoma is highly aggressive, it is important to decipher all the biological aspects of melanoma cells. In this context, we have previously shown that metastatic FEMX-I melanoma cells release small (< 150 nm) extracellular vesicles (EVs) known as exosomes and ectosomes containing the stem (and cancer stem) cell antigenic marker CD133. EVs play an important role in intercellular communication, which could have a micro-environmental impact on surrounding tissues.
RESULTS
We report here a new type of large CD133 EVs released by FEMX-I cells. Their sizes range from 2 to 6 µm and they contain lipid droplets and mitochondria. Real-time video microscopy revealed that these EVs originate from the lipid droplet-enriched cell extremities that did not completely retract during the cell division process. Once released, they can be taken up by other cells. Silencing CD133 significantly affected the cellular distribution of lipid droplets, with a re-localization around the nuclear compartment. As a result, the formation of large EVs containing lipid droplets was severely compromised.
CONCLUSION
Given the biochemical effect of lipid droplets and mitochondria and/or their complexes on cell metabolism, the release and uptake of these new large CD133 EVs from dividing aggressive melanoma cells can influence both donor and recipient cells, and therefore impact melanoma growth and dissemination.
Topics: Humans; Melanoma; Lipid Droplets; Extracellular Vesicles; Cell Division; Mitochondria
PubMed: 38243233
DOI: 10.1186/s12964-024-01471-7 -
Chemical & Biomedical Imaging Dec 2023Vomocytosis is a process that occurs when internalized fungal pathogens escape from phagocytes without compromising the viability of the pathogen and the host cell....
Vomocytosis is a process that occurs when internalized fungal pathogens escape from phagocytes without compromising the viability of the pathogen and the host cell. Manual quantification of time-lapse microscopy videos is currently used as the standard to study pathogen behavior and vomocytosis incidence. However, human-driven quantification of vomocytosis (and the closely related phenomenon, exocytosis) is incredibly burdensome, especially when a large volume of cells and interactions needs to be analyzed. In this study, we designed a MATLAB algorithm that measures the extent of colocalization between the phagocyte and fungal cell (; CN) and rapidly reports the occurrence of vomocytosis in a high throughput manner. Our code processes multichannel, time-lapse microscopy videos of cocultured CN and immune cells that have each been fluorescently stained with unique dyes and provides quantitative readouts of the spatiotemporally dynamic process that is vomocytosis. This study also explored metrics, such as the rate of change of pathogen colocalization with the host cell, that could potentially be used to predict vomocytosis occurrence based on the quantitative data collected. Ultimately, the algorithm quantifies vomocytosis events and reduces the time for video analysis from over 1 h to just 10 min, a reduction in labor of 83%, while simultaneously minimizing human error. This tool significantly minimizes the vomocytosis analysis pipeline, accelerates our ability to elucidate unstudied aspects of this phenomenon, and expedites our ability to characterize CN strains for the study of their epidemiology and virulence.
PubMed: 38155727
DOI: 10.1021/cbmi.3c00102 -
Bioinformatics (Oxford, England) Oct 2023Reliable label-free methods are needed for detecting and profiling apoptotic events in time-lapse cell-cell interaction assays. Prior studies relied on fluorescent...
MOTIVATION
Reliable label-free methods are needed for detecting and profiling apoptotic events in time-lapse cell-cell interaction assays. Prior studies relied on fluorescent markers of apoptosis, e.g. Annexin-V, that provide an inconsistent and late indication of apoptotic onset for human melanoma cells. Our motivation is to improve the detection of apoptosis by directly detecting apoptotic bodies in a label-free manner.
RESULTS
Our trained ResNet50 network identified nanowells containing apoptotic bodies with 92% accuracy and predicted the onset of apoptosis with an error of one frame (5 min/frame). Our apoptotic body segmentation yielded an IoU accuracy of 75%, allowing associative identification of apoptotic cells. Our method detected apoptosis events, 70% of which were not detected by Annexin-V staining.
AVAILABILITY AND IMPLEMENTATION
Open-source code and sample data provided at https://github.com/kwu14victor/ApoBDproject.
Topics: Humans; Microscopy, Video; Time-Lapse Imaging; Neural Networks, Computer; Extracellular Vesicles; Annexins
PubMed: 37773981
DOI: 10.1093/bioinformatics/btad584 -
BioRxiv : the Preprint Server For... Oct 2023HIV-1 budding as well as many other cellular processes require the Endosomal Sorting Complex Required for Transport (ESCRT) machinery. Understanding the architecture of...
HIV-1 budding as well as many other cellular processes require the Endosomal Sorting Complex Required for Transport (ESCRT) machinery. Understanding the architecture of the native ESCRT-III complex at HIV-1 budding sites is limited due to spatial resolution and transient ESCRT-III recruitment. Here, we developed a drug-inducible transient HIV-1 budding inhibitory tool to enhance the ESCRT-III lifetime at budding sites. We generated auto-cleavable CHMP2A, CHMP3, and CHMP4B fusion proteins with the hepatitis C virus NS3 protease. We characterized the CHMP-NS3 fusion proteins in the absence and presence of protease inhibitor Glecaprevir with regard to expression, stability, localization and HIV-1 Gag VLP budding. Immunoblotting experiments revealed rapid and stable accumulation of CHMP-NS3 fusion proteins with variable modification of Gag VLP budding upon drug administration. Notably, CHMP2A-NS3 and CHMP4B-NS3 fusion proteins substantially decrease VLP release while CHMP3-NS3 exerted a minor effect and synergized with CHMP2A-NS3. Localization studies demonstrated the re-localization of CHMP-NS3 fusion proteins to the plasma membrane, endosomes, and Gag VLP budding sites. Through the combined use of transmission electron microscopy and video-microscopy, we unveiled drug-dependent accumulation of CHMP2A-NS3 and CHMP4B-NS3, causing a delay in HIV-1 Gag-VLP release. Our findings provide novel insight into the functional consequences of inhibiting ESCRT-III during HIV-1 budding and establish new tools to decipher the role of ESCRT-III at HIV-1 budding sites and other ESCRT-catalyzed cellular processes.
PubMed: 37905063
DOI: 10.1101/2023.10.16.562494 -
MBio Feb 2024The infection cycle of phage λ terminates in lysis mediated by three types of lysis proteins, each disrupting a layer in the bacterial envelope: the S105 holin, the R...
The infection cycle of phage λ terminates in lysis mediated by three types of lysis proteins, each disrupting a layer in the bacterial envelope: the S105 holin, the R endolysin, and the Rz/Rz1 spanin complex targeting the inner membrane, cell wall or peptidoglycan, and the outer membrane, respectively. Video microscopy has shown that in most infections, lysis occurs as a sudden, explosive event at a cell pole, such that the initial product is a less refractile ghost that retains rod-shaped morphology. Here, we investigate the molecular basis of polar lysis using time-lapse fluorescence microscopy. The results indicate that the holin determines the morphology of lysis by suddenly forming two-dimensional rafts at the poles about 100 s prior to lysis. Given the physiological and biochemical similarities between the lambda holin and other class I holins, dynamic redistribution and sudden concentration may be common features of holins, probably reflecting the fitness advantage of all-or-nothing lysis regulation.IMPORTANCEIn this study, we use fluorescent video microscopy to track -green fluorescent protein (GFP)-labeled holin in the minutes prior to phage lysis. Our work contextualizes prior genetic and biochemical data, showing when hole formation starts and where holin oligomers form in relation to the site of lytic rupture. Furthermore, prior work showed that the morphology of lambda-infected cells is characterized by an explosive event starting at the cell pole; however, the basis for this was not clear. This study shows that holin most often oligomerizes at cell poles and that the site of the oligomerization is spatially correlated with the site of lytic blowout. Therefore, the holin is the key contributor to polar lysis morphology for phage lambda.
Topics: Viral Proteins; Bacteriophage lambda; Cell Death; Cell Wall; Bacteriolysis
PubMed: 38126784
DOI: 10.1128/mbio.01290-23 -
Biophysical Journal Apr 2024The binding of calcium/calmodulin (CAM) to calcium/calmodulin-dependent protein kinase II (CaMKII) initiates an ATP-driven cascade that triggers CaMKII...
The binding of calcium/calmodulin (CAM) to calcium/calmodulin-dependent protein kinase II (CaMKII) initiates an ATP-driven cascade that triggers CaMKII autophosphorylation. The autophosphorylation in turn increases the CaMKII affinity for CAM. Here, we studied the ATP dependence of CAM association with the actin-binding CaMKIIβ isoform using single-molecule total internal reflection fluorescence microscopy. Rhodamine-CAM associations/dissociations to surface-immobilized Venus-CaMKIIβ were resolved with 0.5 s resolution from video records, batch-processed with a custom algorithm. CAM occupancy was determined simultaneously with spot-photobleaching measurement of CaMKII holoenzyme stoichiometry. We show the ATP-dependent increase of the CAM association requires dimer formation for both the α and β isoforms. The study of mutant β holoenzymes revealed that the ATP-dependent increase in CAM affinity results in two distinct states. The phosphorylation-defective (T287.306-307A) holoenzyme resides only in the low-affinity state. CAM association is further reduced in the T287A holoenzyme relative to T287.306-307A. In the absence of ATP, the affinity of CAM for the T287.306-307A mutant and the wild-type monomer are comparable. The affinity of the ATP-binding impaired (K43R) mutant is even weaker. In ATP, the K43R holoenzyme resides in the low-affinity state. The phosphomimetic mutant (T287D) resides only in a 1000-fold higher-affinity state, with mean CAM occupancy of more than half of the 14-mer holoenzyme stoichiometry in picomolar CAM. ATP promotes T287D holoenzyme disassembly but does not elevate CAM occupancy. Single Poisson distributions characterized the ATP-dependent CAM occupancy of mutant holoenzymes. In contrast, the CAM occupancy of the wild-type population had a two-state distribution with both low- and high-affinity states represented. The low-affinity state was the dominant state, a result different from published in vitro assays. Differences in assay conditions can alter the balance between activating and inhibitory autophosphorylation. Bound ATP could be sufficient for CaMKII structural function, while antagonistic autophosphorylations may tune CaMKII kinase-regulated action-potential frequency decoding in vivo.
Topics: Calmodulin; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium; Single Molecule Imaging; Adenosine Triphosphate; Holoenzymes; Phosphorylation
PubMed: 38414237
DOI: 10.1016/j.bpj.2024.02.021 -
Diagnostics (Basel, Switzerland) Mar 2024Artificial intelligence (AI) has seen significant progress in medical diagnostics, particularly in image and video analysis. This review focuses on the application of AI... (Review)
Review
Artificial intelligence (AI) has seen significant progress in medical diagnostics, particularly in image and video analysis. This review focuses on the application of AI in analyzing in vivo confocal microscopy (IVCM) images for corneal diseases. The cornea, as an exposed and delicate part of the body, necessitates the precise diagnoses of various conditions. Convolutional neural networks (CNNs), a key component of deep learning, are a powerful tool for image data analysis. This review highlights AI applications in diagnosing keratitis, dry eye disease, and diabetic corneal neuropathy. It discusses the potential of AI in detecting infectious agents, analyzing corneal nerve morphology, and identifying the subtle changes in nerve fiber characteristics in diabetic corneal neuropathy. However, challenges still remain, including limited datasets, overfitting, low-quality images, and unrepresentative training datasets. This review explores augmentation techniques and the importance of feature engineering to address these challenges. Despite the progress made, challenges are still present, such as the "black-box" nature of AI models and the need for explainable AI (XAI). Expanding datasets, fostering collaborative efforts, and developing user-friendly AI tools are crucial for enhancing the acceptance and integration of AI into clinical practice.
PubMed: 38611606
DOI: 10.3390/diagnostics14070694