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Cell Jun 2024Gamete formation and subsequent offspring development often involve extended phases of suspended cellular development or even dormancy. How cells adapt to recover and...
Gamete formation and subsequent offspring development often involve extended phases of suspended cellular development or even dormancy. How cells adapt to recover and resume growth remains poorly understood. Here, we visualized budding yeast cells undergoing meiosis by cryo-electron tomography (cryoET) and discovered elaborate filamentous assemblies decorating the nucleus, cytoplasm, and mitochondria. To determine filament composition, we developed a "filament identification" (FilamentID) workflow that combines multiscale cryoET/cryo-electron microscopy (cryoEM) analyses of partially lysed cells or organelles. FilamentID identified the mitochondrial filaments as being composed of the conserved aldehyde dehydrogenase Ald4 and the nucleoplasmic/cytoplasmic filaments as consisting of acetyl-coenzyme A (CoA) synthetase Acs1. Structural characterization further revealed the mechanism underlying polymerization and enabled us to genetically perturb filament formation. Acs1 polymerization facilitates the recovery of chronologically aged spores and, more generally, the cell cycle re-entry of starved cells. FilamentID is broadly applicable to characterize filaments of unknown identity in diverse cellular contexts.
Topics: Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Cryoelectron Microscopy; Mitochondria; Gametogenesis; Meiosis; Aldehyde Dehydrogenase; Electron Microscope Tomography; Coenzyme A Ligases; Spores, Fungal; Cytoplasm; Cell Nucleus
PubMed: 38906101
DOI: 10.1016/j.cell.2024.04.026 -
Biomaterials Jun 2024Biohybrid tissue-engineered vascular grafts (TEVGs) promise long-term durability due to their ability to adapt to hosts' needs. However, the latter calls for sensitive...
Biohybrid tissue-engineered vascular grafts (TEVGs) promise long-term durability due to their ability to adapt to hosts' needs. However, the latter calls for sensitive non-invasive imaging approaches to longitudinally monitor their functionality, integrity, and positioning. Here, we present an imaging approach comprising the labeling of non-degradable and degradable TEVGs' components for their in vitro and in vivo monitoring by hybrid H/F MRI. TEVGs (inner diameter 1.5 mm) consisted of biodegradable poly(lactic-co-glycolic acid) (PLGA) fibers passively incorporating superparamagnetic iron oxide nanoparticles (SPIONs), non-degradable polyvinylidene fluoride scaffolds labeled with highly fluorinated thermoplastic polyurethane (F-TPU) fibers, a smooth muscle cells containing fibrin blend, and endothelial cells. H/F MRI of TEVGs in bioreactors, and after subcutaneous and infrarenal implantation in rats, revealed that PLGA degradation could be faithfully monitored by the decreasing SPIONs signal. The F signal of F-TPU remained constant over weeks. PLGA degradation was compensated by cells' collagen and α-smooth-muscle-actin deposition. Interestingly, only TEVGs implanted on the abdominal aorta contained elastin. XTT and histology proved that our imaging markers did not influence extracellular matrix deposition and host immune reaction. This concept of non-invasive longitudinal assessment of cardiovascular implants using H/F MRI might be applicable to various biohybrid tissue-engineered implants, facilitating their clinical translation.
PubMed: 38906013
DOI: 10.1016/j.biomaterials.2024.122669 -
Science Advances Jun 2024Integral membrane proteins (IMPs) constitute a large fraction of organismal proteomes, playing fundamental roles in physiology and disease. Despite their importance, the...
Integral membrane proteins (IMPs) constitute a large fraction of organismal proteomes, playing fundamental roles in physiology and disease. Despite their importance, the mechanisms underlying dynamic features of IMPs, such as anomalous diffusion, protein-protein interactions, and protein clustering, remain largely unknown due to the high complexity of cell membrane environments. Available methods for in vitro studies are insufficient to study IMP dynamics systematically. This publication introduces the freestanding bilayer microscope (FBM), which combines the advantages of freestanding bilayers with single-particle tracking. The FBM, based on planar lipid bilayers, enables the study of IMP dynamics with single-molecule resolution and unconstrained diffusion. This paper benchmarks the FBM against total internal reflection fluorescence imaging on supported bilayers and is used here to estimate ion channel open probability and to examine the diffusion behavior of an ion channel in phase-separated bilayers. The FBM emerges as a powerful tool to examine membrane protein/lipid organization and dynamics to understand cell membrane processes.
Topics: Lipid Bilayers; Single Molecule Imaging; Membrane Proteins; Ion Channels; Diffusion; Cell Membrane
PubMed: 38905330
DOI: 10.1126/sciadv.ado4722 -
PloS One 2024In a clinical context, conventional optical microscopy is commonly used for the visualization of biological samples for diagnosis. However, the availability of molecular...
In a clinical context, conventional optical microscopy is commonly used for the visualization of biological samples for diagnosis. However, the availability of molecular techniques and rapid diagnostic tests are reducing the use of conventional microscopy, and consequently the number of experienced professionals starts to decrease. Moreover, the continuous visualization during long periods of time through an optical microscope could affect the final diagnosis results due to induced human errors and fatigue. Therefore, microscopy automation is a challenge to be achieved and address this problem. The aim of the study is to develop a low-cost automated system for the visualization of microbiological/parasitological samples by using a conventional optical microscope, and specially designed for its implementation in resource-poor settings laboratories. A 3D-prototype to automate the majority of conventional optical microscopes was designed. Pieces were built with 3D-printing technology and polylactic acid biodegradable material with Tinkercad/Ultimaker Cura 5.1 slicing softwares. The system's components were divided into three subgroups: microscope stage pieces, storage/autofocus-pieces, and smartphone pieces. The prototype is based on servo motors, controlled by Arduino open-source electronic platform, to emulate the X-Y and auto-focus (Z) movements of the microscope. An average time of 27.00 ± 2.58 seconds is required to auto-focus a single FoV. Auto-focus evaluation demonstrates a mean average maximum Laplacian value of 11.83 with tested images. The whole automation process is controlled by a smartphone device, which is responsible for acquiring images for further diagnosis via convolutional neural networks. The prototype is specially designed for resource-poor settings, where microscopy diagnosis is still a routine process. The coalescence between convolutional neural network predictive models and the automation of the movements of a conventional optical microscope confer the system a wide range of image-based diagnosis applications. The accessibility of the system could help improve diagnostics and provide new tools to laboratories worldwide.
Topics: Microscopy; Humans; Printing, Three-Dimensional; Software; Robotics; Smartphone; Automation; Imaging, Three-Dimensional
PubMed: 38905190
DOI: 10.1371/journal.pone.0304085 -
PLoS Biology Jun 2024Throughout human life, the brain undergoes intricate structural changes that support cognition. A study in PLOS Biology introduces new avenues for depicting the...
Throughout human life, the brain undergoes intricate structural changes that support cognition. A study in PLOS Biology introduces new avenues for depicting the trajectory of the brain morphometric connectome and its underlying genetic and molecular mechanisms.
Topics: Brain; Connectome; Humans; Longevity; Magnetic Resonance Imaging
PubMed: 38905164
DOI: 10.1371/journal.pbio.3002669 -
EJNMMI Radiopharmacy and Chemistry Jun 2024In the last decade the development of new PSMA-ligand based radiopharmaceuticals for the imaging and therapy of prostate cancer has been a highly active and important...
BACKGROUND
In the last decade the development of new PSMA-ligand based radiopharmaceuticals for the imaging and therapy of prostate cancer has been a highly active and important area of research. The most promising derivative in terms of interaction with the antigen and clinical properties has been found to be "PSMA-617", and its lutetium-177 radiolabelled version has recently been approved by EU and USA regulatory agencies for therapeutic purposes. For the above reasons, the development of new derivatives of PSMA-617 radiolabelled with fluorine-18 may still be of great interest. This paper proposes the comparison of two different PSMA-617 derivatives functionalized with NODA and RESCA chelators, respectively, radiolabelled via [F]AlF complexation.
RESULTS
The organic synthesis of two PSMA-617 derivatives and their radiolabelling via [F]AlF complexation resulted to proceed efficiently and successfully. Moreover, stability in solution and in plasma has been evaluated. The whole radiosynthesis procedure has been fully automated, and the final products have been obtained with radiochemical yield and purity potentially suitable for clinical studies. The biodistribution of the two derivatives was performed both in prostate cancer and glioma tumour models. Compared with the reference [F]F-PSMA-1007 and [F]F-PSMA-617-RESCA, [F]F-PSMA-617-NODA derivative showed a higher uptake in both tumors, faster clearance in non-target organs, and lower uptake in salivary glands.
CONCLUSION
PSMA-617 NODA and RESCA derivatives were radiolabelled successfully via [F]AlF chelation, the former being more stable in solution and human plasma. Moreover, preclinical biodistribution studies showed that [F]F-PSMA-617-NODA might be of potential interest for clinical applications.
PubMed: 38904859
DOI: 10.1186/s41181-024-00280-0 -
Investigative Ophthalmology & Visual... Jun 2024Interphotoreceptor retinoid-binding protein's (IRBP) role in eye growth and its involvement in cell homeostasis remain poorly understood. One hypothesis proposes early...
PURPOSE
Interphotoreceptor retinoid-binding protein's (IRBP) role in eye growth and its involvement in cell homeostasis remain poorly understood. One hypothesis proposes early conditional deletion of the IRBP gene could lead to a myopic response with retinal degeneration, whereas late conditional deletion (after eye size is determined) could cause retinal degeneration without myopia. Here, we sought to understand if prior myopia was required for subsequent retinal degeneration in the absence of IRBP. This study investigates if any cell type or developmental stage is more important in myopia or retinal degeneration.
METHODS
IBRPfl/fl mice were bred with 5 Cre-driver lines: HRGP-Cre, Chx10-Cre, Rho-iCre75, HRGP-Cre Rho-iCre75, and Rx-Cre. Mice were analyzed for IRBP gene expression through digital droplet PCR (ddPCR). Young adult (P30) mice were tested for retinal degeneration and morphology using spectral-domain optical coherence tomography (SD-OCT) and hematoxylin and eosin (H&E) staining. Function was analyzed using electroretinograms (ERGs). Eye sizes and axial lengths were compared through external eye measurements and whole eye biometry.
RESULTS
Across all outcome measures, when bred to IRBPfl/fl, HRGP-Cre and Chx10-Cre lines showed no differences from IRBPfl/fl alone. With the Rho-iCre75 line, small but significant reductions were seen in retinal thickness with SD-OCT imaging and postmortem H&E staining without increased axial length. Both the HRGP-Cre+Rho-iCre75 and the Rx-Cre lines showed significant decreases in retinal thickness and outer nuclear layer cell counts. Using external eye measurements and SD-OCT imaging, both lines showed an increase in eye size. Finally, function in both lines was roughly halved across scotopic, photopic, and flicker ERGs.
CONCLUSIONS
Our studies support hypotheses that for both eye size determination and retinal homeostasis, there are two critical timing windows when IRBP must be expressed in rods or cones to prevent myopia (P7-P12) and degeneration (P21 and later). The rod-specific IRBP knockout (Rho-iCre75) showed significant retinal functional losses without myopia, indicating that the two phenotypes are independent. IRBP is needed for early development of photoreceptors and eye size, whereas Rho-iCre75 IRBPfl/fl knockout results in retinal degeneration without myopia.
Topics: Animals; Mice; Retinal Degeneration; Eye Proteins; Tomography, Optical Coherence; Mice, Knockout; Retinol-Binding Proteins; Electroretinography; Disease Models, Animal; Myopia; Mice, Inbred C57BL; Retina
PubMed: 38904640
DOI: 10.1167/iovs.65.6.32 -
Investigative Ophthalmology & Visual... Jun 2024Recent studies have shown that the retinal pigment epithelium (RPE) relies on fatty acid oxidation (FAO) for energy, however, its role in overall retinal health is...
PURPOSE
Recent studies have shown that the retinal pigment epithelium (RPE) relies on fatty acid oxidation (FAO) for energy, however, its role in overall retinal health is unknown. The only FAO disorder that presents with chorioretinopathy is long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD). Studying the molecular mechanisms can lead to new treatments for patients and elucidate the role of FAO in the RPE. This paper characterizes the chorioretinopathy progression in a recently reported LCHADD mouse model.
METHODS
Visual assessments, such as optokinetic tracking and fundus imaging, were performed in wildtype (WT) and LCHADD mice at 3, 6, 10, and 12 months of age. Retinal morphology was analyzed in 12-month retinal cross-sections using hematoxylin and eosin (H&E), RPE65, CD68, and TUNEL staining, whereas RPE structure was assessed using transmission electron microscopy (TEM). Acylcarnitine profiles were measured in isolated RPE/sclera samples to determine if FAO was blocked. Bulk RNA-sequencing of 12 month old male WT mice and LCHADD RPE/sclera samples assessed gene expression changes.
RESULTS
LCHADD RPE/sclera samples had a 5- to 7-fold increase in long-chain hydroxyacylcarnitines compared to WT, suggesting an impaired LCHAD step in long-chain FAO. LCHADD mice have progressively decreased visual performance and increased RPE degeneration starting at 6 months. LCHADD RPE have an altered structure and a two-fold increase in macrophages in the subretinal space. Finally, LCHADD RPE/sclera have differentially expressed genes compared to WT, including downregulation of genes important for RPE function and angiogenesis.
CONCLUSIONS
Overall, this LCHADD mouse model recapitulates early-stage chorioretinopathy seen in patients with LCHADD and is a useful model for studying LCHADD chorioretinopathy.
Topics: Animals; Disease Models, Animal; Mice; Retinal Pigment Epithelium; Mice, Inbred C57BL; Long-Chain-3-Hydroxyacyl-CoA Dehydrogenase; Choroid Diseases; Male; Retinal Diseases; Microscopy, Electron, Transmission
PubMed: 38904639
DOI: 10.1167/iovs.65.6.33 -
Frontiers in Endocrinology 2024Thyroid cancer rarely occurs in children and adolescents. Molecular markers such as , , and have been widely used in adult PTC. It is currently unclear whether these...
OBJECTIVES
Thyroid cancer rarely occurs in children and adolescents. Molecular markers such as , , and have been widely used in adult PTC. It is currently unclear whether these molecular markers have equivalent potential for application in pediatric patients. This study aims to explore the potential utility of a multi-gene conjoint analysis based on next-generation targeted sequencing for pediatric papillary thyroid carcinoma (PTC).
MATERIALS AND METHODS
The patients diagnosed with PTC (aged 18 years or younger) in the pediatrics department of Lishui District Hospital of Traditional Chinese Medicine were retrospectively screened. A targeted enrichment and sequencing analysis of 116 genes associated with thyroid cancer was performed on paraffin-embedded tumor tissues and paired paracancerous tissue of fifteen children (average age 14.60) and nine adults (average age 49.33) PTC patients. Demographic information, clinical indicators, ultrasonic imaging information and pathological data were collected. The Kendall correlation test was used to establish a correlation between molecular variations and clinical characteristics in pediatric patients.
RESULTS
A sample of 15 pediatric PTCs revealed a detection rate of 73.33% (11/15) for driver gene mutations and fusion. Compared to adult PTCs, the genetic mutation landscape of pediatric PTCs was more complex. Six mutant genes overlap between the two groups, and an additional seventeen unique mutant genes were identified only in pediatric PTCs. There was only one unique mutant gene in adult PTCs. The tumor diameter of pediatric PTCs tended to be less than 4cm (p<0.001), and the number of lymph node metastases was more than five (p<0.001). Mutations in specific genes unique to pediatric PTCs may contribute to the onset and progression of the disease by adversely affecting hormone synthesis, secretion, and action mechanisms, as well as the functioning of thyroid hormone signaling pathways. But, additional experiments are required to validate this hypothesis.
CONCLUSION
mutation and fusion are involved in the occurrence and development of adolescent PTC. For pediatric thyroid nodules that cannot be determined as benign or malignant by fine needle aspiration biopsy, multiple gene combination testing can provide a reference for personalized diagnosis and treatment by clinical physicians.
Topics: Humans; Female; Adolescent; Thyroid Cancer, Papillary; Male; Child; Thyroid Neoplasms; Mutation; Retrospective Studies; Proto-Oncogene Proteins B-raf; Adult; Middle Aged; Biomarkers, Tumor; Proto-Oncogene Proteins c-ret; High-Throughput Nucleotide Sequencing; DNA Mutational Analysis
PubMed: 38904052
DOI: 10.3389/fendo.2024.1405142 -
Frontiers in Pharmacology 2024Systemic chemotherapy is typically administered following radical gastrectomy for advanced stage. To attenuate systemic side effects, we evaluated the effectiveness of...
Systemic chemotherapy is typically administered following radical gastrectomy for advanced stage. To attenuate systemic side effects, we evaluated the effectiveness of regional chemotherapy using paclitaxel, albumin-paclitaxel, and liposome-encapsulated albumin-paclitaxel via subserosal injection in rat models employing nuclear medicine and molecular imaging technology. Nine Sprague Dawley rats were divided into three groups: paclitaxel ( = 3), albumin-paclitaxel nano-particles (APNs; = 3), and liposome-encapsulated APNs ( = 3). [I]Iodo-paclitaxel ([I]I-paclitaxel) was synthesized by conventional electrophilic radioiodination using -butylstannyl substituted paclitaxel as the precursor. Albumin-[I]iodo-paclitaxel nanoparticles ([I]APNs) were prepared using a desolvation technique. Liposome-encapsulated APNs (L-[I]APNs) were prepared by thin-film hydration using DSPE-PEG2000, HSPC, and cholesterol. The rats in each group were injected with each test drug into the subserosa of the stomach antrum. After predetermined times (30 min, 2, 4, 8 h, and 24 h), molecular images of nuclear medicine were acquired using single-photon emission computed tomography/computed tomography. Paclitaxel, APNs, and L-APNs showed a high cumulative distribution in the stomach, with L-APNs showing the largest area under the curve. Most drugs administered via the gastric subserosal route are distributed in the stomach and intestines, with a low uptake of less than 1% in other major organs. The time to reach the maximum concentration in the intestine for L-APNs, paclitaxel, and APNs was 6.67, 5.33, and 4.00 h, respectively. These preliminary results imply that L-APNs have the potential to serve as a novel paclitaxel preparation method for the regional treatment of gastric cancer.
PubMed: 38904000
DOI: 10.3389/fphar.2024.1381406