-
Microbiome Sep 2023In the last few years, considerable attention has been focused on the plastic-degrading capability of insects and their gut microbiota in order to develop novel,...
BACKGROUND
In the last few years, considerable attention has been focused on the plastic-degrading capability of insects and their gut microbiota in order to develop novel, effective, and green strategies for plastic waste management. Although many analyses based on 16S rRNA gene sequencing are available, an in-depth analysis of the insect gut microbiome to identify genes with plastic-degrading potential is still lacking.
RESULTS
In the present work, we aim to fill this gap using Black Soldier Fly (BSF) as insect model. BSF larvae have proven capability to efficiently bioconvert a wide variety of organic wastes but, surprisingly, have never been considered for plastic degradation. BSF larvae were reared on two widely used plastic polymers and shotgun metagenomics was exploited to evaluate if and how plastic-containing diets affect composition and functions of the gut microbial community. The high-definition picture of the BSF gut microbiome gave access for the first time to the genomes of culturable and unculturable microorganisms in the gut of insects reared on plastics and revealed that (i) plastics significantly shaped bacterial composition at species and strain level, and (ii) functions that trigger the degradation of the polymer chains, i.e., DyP-type peroxidases, multicopper oxidases, and alkane monooxygenases, were highly enriched in the metagenomes upon exposure to plastics, consistently with the evidences obtained by scanning electron microscopy and H nuclear magnetic resonance analyses on plastics.
CONCLUSIONS
In addition to highlighting that the astonishing plasticity of the microbiota composition of BSF larvae is associated with functional shifts in the insect microbiome, the present work sets the stage for exploiting BSF larvae as "bioincubators" to isolate microbial strains and enzymes for the development of innovative plastic biodegradation strategies. However, most importantly, the larvae constitute a source of enzymes to be evolved and valorized by pioneering synthetic biology approaches. Video Abstract.
Topics: Animals; Larva; Gastrointestinal Microbiome; Plastics; RNA, Ribosomal, 16S; Diptera
PubMed: 37705113
DOI: 10.1186/s40168-023-01649-0 -
International Journal of Molecular... Sep 2023Polyploidy and metastasis are associated with a low probability of disease-free survival in cancer patients. Polyploid cells are known to facilitate tumorigenesis....
Polyploidy and metastasis are associated with a low probability of disease-free survival in cancer patients. Polyploid cells are known to facilitate tumorigenesis. However, few data associate polyploidization with metastasis. Here, by generating and using diploid (2n) and tetraploid (4n) clones from malignant fibrous histiocytoma (MFH) and colon carcinoma (RKO), we demonstrate the migration and invasion advantage of tetraploid cells in vitro using several assays, including the wound healing, the OrisTM two-dimensional cell migration, single-cell migration tracking by video microscopy, the Boyden chamber, and the xCELLigence RTCA real-time cell migration. Motility advantage was observed despite tetraploid cell proliferation weakness. We could also demonstrate preferential metastatic potential in vivo for the tetraploid clone using the tail vein injection in mice and tracking metastatic tumors in the lung. Using the Mitelman Database of Chromosome Aberrations in Cancer, we found an accumulation of polyploid karyotypes in metastatic tumors compared to primary ones. This work reveals the clinical relevance of the polyploid subpopulation and the strategic need to highlight polyploidy in preclinical studies as a therapeutic target for metastasis.
Topics: Humans; Animals; Mice; Tetraploidy; Polyploidy; Chromosome Aberrations; Colonic Neoplasms
PubMed: 37762227
DOI: 10.3390/ijms241813926 -
Scientific Reports Nov 2023Human induced pluripotent stem cell (hiPSC)-derived cardiomyocyte (CM) models have become an attractive tool for in vitro cardiac disease modeling and drug studies....
Human induced pluripotent stem cell (hiPSC)-derived cardiomyocyte (CM) models have become an attractive tool for in vitro cardiac disease modeling and drug studies. These models are moving towards more complex three-dimensional microphysiological organ-on-chip systems. Label-free imaging-based techniques capable of quantifying contractility in 3D are needed, as traditional two-dimensional methods are ill-suited for 3D applications. Here, we developed multifocal (MF) optical projection microscopy (OPM) by integrating an electrically tunable lens to our in-house built optical projection tomography setup for extended depth of field brightfield imaging in CM clusters. We quantified cluster biomechanics by implementing our previously developed optical flow-based CM video analysis for MF-OPM. To demonstrate, we acquired and analyzed multiangle and multifocal projection videos of beating hiPSC-CM clusters in 3D hydrogel. We further quantified cluster contractility response to temperature and adrenaline and observed changes to beating rate and relaxation. Challenges emerge from light penetration and overlaying textures in larger clusters. However, our findings indicate that MF-OPM is suitable for contractility studies of 3D clusters. Thus, for the first time, MF-OPM is used in CM studies and hiPSC-CM 3D cluster contraction is quantified in multiple orientations and imaging planes.
Topics: Humans; Myocytes, Cardiac; Microscopy; Induced Pluripotent Stem Cells
PubMed: 37957157
DOI: 10.1038/s41598-023-46510-4 -
Scientific Data Oct 2023Detecting and tracking multiple moving objects in a video is a challenging task. For living cells, the task becomes even more arduous as cells change their morphology...
Detecting and tracking multiple moving objects in a video is a challenging task. For living cells, the task becomes even more arduous as cells change their morphology over time, can partially overlap, and mitosis leads to new cells. Differently from fluorescence microscopy, label-free techniques can be easily applied to almost all cell lines, reducing sample preparation complexity and phototoxicity. In this study, we present ALFI, a dataset of images and annotations for label-free microscopy, made publicly available to the scientific community, that notably extends the current panorama of expertly labeled data for detection and tracking of cultured living nontransformed and cancer human cells. It consists of 29 time-lapse image sequences from HeLa, U2OS, and hTERT RPE-1 cells under different experimental conditions, acquired by differential interference contrast microscopy, for a total of 237.9 hours. It contains various annotations (pixel-wise segmentation masks, object-wise bounding boxes, tracking information). The dataset is useful for testing and comparing methods for identifying interphase and mitotic events and reconstructing their lineage, and for discriminating different cellular phenotypes.
Topics: Humans; Cell Cycle; Cell Tracking; HeLa Cells; Image Processing, Computer-Assisted; Microscopy, Fluorescence; Time-Lapse Imaging
PubMed: 37794110
DOI: 10.1038/s41597-023-02540-1 -
Journal of Indian Prosthodontic Society 2023The intraoral scanners are digital devices used to digitise the oral tissues. The accuracy of the intraoral scanners has been studied under different environmental... (Clinical Trial)
Clinical Trial
Comparative analysis on efficiency and accuracy of parallel confocal microscopy and three-dimensional in motion video with triangulation technology-based intraoral scanner under influence of moisture and mouth opening - A crossover clinical trial.
AIM
The intraoral scanners are digital devices used to digitise the oral tissues. The accuracy of the intraoral scanners has been studied under different environmental conditions, but there might be differences that occur in the actual oral environment, which is still in question. The aim of the study was to evaluate the accuracy and efficiency of Parallel Confocal Microscopy and 3D in motion video with triangulation technology-based intraoral scanners under the influence of moisture and mouth opening.
SETTINGS AND DESIGN
This was an Cross over clinical controlled study.
MATERIALS AND METHODS
The controlled in vivo study included healthy subjects who were in need of CBCT for the purpose of locating the position of unerupted third molars before going abroad for a job. The subjects were exposed to scans in the upper and lower jaws with two intraoral scanners based on 3D motion video technology with triangulation (Medit) and parallel confocal microscopy (Trios) under the influence of two oral conditions, which were moisture (presence and absence of moisture) and mouth opening (30 mm and 50 mm, respectively). A total of 96 scans were obtained and superimposed individually over the reference CBCT scans to find the deviations in the Geomagic Rapidform (version 2020, USA) software. The efficiency of the scanners was calculated by recording the time taken and the number of images obtained after each scan.
STATISTICAL ANALYSIS USED
The significance was calculated by using the independent and paired sample t test in SPSS software (IBM, version 23).
RESULTS
Based on the surface analysis, the trueness of the intra-oral scanners had statistically significant differences when compared between 3D in motion video technology with Triangulation and Parallel Confocal Microscopy (P < 0.05) whereas no statistical significance was observed in precision. There was a significant difference observed in the efficiency of the intra-oral scanners (P < 0.05).
CONCLUSION
There is a significant difference in the accuracy and efficiency of the intraoral scanners under the influence of oral conditions, such as different moisture levels and mouth opening conditions. 3D in motion video technology with Triangulation showed better results with the least deviation than Parallel Confocal Microscopy.
Topics: Humans; Computer-Aided Design; Dental Impression Technique; Imaging, Three-Dimensional; Microscopy, Confocal; Technology; Cross-Over Studies
PubMed: 37929362
DOI: 10.4103/jips.jips_65_23 -
Stem Cell Research & Therapy Sep 2023Polymorphic ventricular tachycardia (PMVT) is a rare genetic disease associated with structurally normal hearts which in 8% of cases can lead to sudden cardiac death,...
BACKGROUND
Polymorphic ventricular tachycardia (PMVT) is a rare genetic disease associated with structurally normal hearts which in 8% of cases can lead to sudden cardiac death, typically exercise-induced. We previously showed a link between the RyR2-H29D mutation and a clinical phenotype of short-coupled PMVT at rest using patient-specific hiPSC-derived cardiomyocytes (hiPSC-CMs). In the present study, we evaluated the effects of clinical and experimental anti-arrhythmic drugs on the intracellular Ca handling, contractile and molecular properties in PMVT hiPSC-CMs in order to model a personalized medicine approach in vitro.
METHODS
Previously, a blood sample from a patient carrying the RyR2-H29D mutation was collected and reprogrammed into several clones of RyR2-H29D hiPSCs, and in addition we generated an isogenic control by reverting the RyR2-H29D mutation using CRIPSR/Cas9 technology. Here, we tested 4 drugs with anti-arrhythmic properties: propranolol, verapamil, flecainide, and the Rycal S107. We performed fluorescence confocal microscopy, video-image-based analyses and biochemical analyses to investigate the impact of these drugs on the functional and molecular features of the PMVT RyR2-H29D hiPSC-CMs.
RESULTS
The voltage-dependent Ca channel inhibitor verapamil did not prevent the aberrant release of sarcoplasmic reticulum (SR) Ca in the RyR2-H29D hiPSC-CMs, whereas it was prevented by S107, flecainide or propranolol. Cardiac tissue comprised of RyR2-H29D hiPSC-CMs exhibited aberrant contractile properties that were largely prevented by S107, flecainide and propranolol. These 3 drugs also recovered synchronous contraction in RyR2-H29D cardiac tissue, while verapamil did not. At the biochemical level, S107 was the only drug able to restore calstabin2 binding to RyR2 as observed in the isogenic control.
CONCLUSIONS
By testing 4 drugs on patient-specific PMVT hiPSC-CMs, we concluded that S107 and flecainide are the most potent molecules in terms of preventing the abnormal SR Ca release and contractile properties in RyR2-H29D hiPSC-CMs, whereas the effect of propranolol is partial, and verapamil appears ineffective. In contrast with the 3 other drugs, S107 was able to prevent a major post-translational modification of RyR2-H29D mutant channels, the loss of calstabin2 binding to RyR2. Using patient-specific hiPSC and CRISPR/Cas9 technologies, we showed that S107 is the most efficient in vitro candidate for treating the short-coupled PMVT at rest.
Topics: Humans; Calcium; Myocytes, Cardiac; Flecainide; Propranolol; Anti-Arrhythmia Agents; Precision Medicine; Ryanodine Receptor Calcium Release Channel; Tachycardia, Ventricular; Verapamil
PubMed: 37740238
DOI: 10.1186/s13287-023-03502-5 -
Scientific Reports May 2024Endothelial glycocalyx (eGC) covers the inner surface of the vessels and plays a role in vascular homeostasis. Syndecan is considered the "backbone" of this structure....
Endothelial glycocalyx (eGC) covers the inner surface of the vessels and plays a role in vascular homeostasis. Syndecan is considered the "backbone" of this structure. Several studies have shown eGC shedding in sepsis and its involvement in organ dysfunction. Matrix metalloproteinases (MMP) contribute to eGC shedding through their ability for syndecan-1 cleavage. This study aimed to investigate if doxycycline, a potent MMP inhibitor, could protect against eGC shedding in lipopolysaccharide (LPS)-induced sepsis and if it could interrupt the vascular hyperpermeability, neutrophil transmigration, and microvascular impairment. Rats that received pretreatment with doxycycline before LPS displayed ultrastructural preservation of the eGC observed using transmission electronic microscopy of the lung and heart. In addition, these animals exhibited lower serum syndecan-1 levels, a biomarker of eGC injury, and lower perfused boundary region (PBR) in the mesenteric video capillaroscopy, which is inversely related to the eGC thickness compared with rats that only received LPS. Furthermore, this study revealed that doxycycline decreased sepsis-related vascular hyperpermeability in the lung and heart, reduced neutrophil transmigration in the peritoneal lavage and inside the lungs, and improved some microvascular parameters. These findings suggest that doxycycline protects against LPS-induced eGC shedding, and it could reduce vascular hyperpermeability, neutrophils transmigration, and microvascular impairment.
Topics: Glycocalyx; Animals; Sepsis; Doxycycline; Rats; Male; Lipopolysaccharides; Capillary Permeability; Lung; Syndecan-1; Rats, Wistar; Endothelium, Vascular; Endothelial Cells; Neutrophils; Matrix Metalloproteinase Inhibitors
PubMed: 38714743
DOI: 10.1038/s41598-024-60919-5 -
Scientific Reports Aug 2023Automatic mitosis detection from video is an essential step in analyzing proliferative behaviour of cells. In existing studies, a conventional object detector such as...
Automatic mitosis detection from video is an essential step in analyzing proliferative behaviour of cells. In existing studies, a conventional object detector such as Unet is combined with a link prediction algorithm to find correspondences between parent and daughter cells. However, they do not take into account the biological constraint that a cell in a frame can correspond to up to two cells in the next frame. Our model called GNN-DOL enables mitosis detection by complementing a graph neural network (GNN) with a differentiable optimization layer (DOL) that implements the constraint. In time-lapse microscopy sequences cultured under four different conditions, we observed that the layer substantially improved detection performance in comparison with GNN-based link prediction. Our results illustrate the importance of incorporating biological knowledge explicitly into deep learning models.
Topics: Mitosis; Cell Nucleus Division; Neural Networks, Computer; Algorithms; Knowledge
PubMed: 37653108
DOI: 10.1038/s41598-023-41562-y -
Indian Journal of Ophthalmology Sep 2023Ophthalmic dirofilariasis is an uncommon zoonotic parasitic infection caused by species of Dirofilaria, a dog tapeworm that is transmitted to human by mosquitoes. Man is...
BACKGROUND
Ophthalmic dirofilariasis is an uncommon zoonotic parasitic infection caused by species of Dirofilaria, a dog tapeworm that is transmitted to human by mosquitoes. Man is a dead-end host for the parasite. Ophthalmic involvement is rare and includes periorbital, subconjunctival, subtenon, and intra-ocular involvement. We report the removal of a subconjunctival worm and identification by light microscopy (LM) and scanning electron microscopy (SEM).
PURPOSE
: A 62-year-old female presented with complaints of redness, discharge, and foreign body sensation with difficulty in opening eyes in the left eye for the last 3 days. The patient is a non-vegetarian. On examination, her best corrected visual acuity in both eyes was 20/20. On slit lamp examination, there was a long, thin, round, coiled white subconjunctival live worm in the left eye superiorly. The rest of anterior segment evaluation, intra-ocular pressure, and fundus was normal in both eyes. The parasite was removed under local anesthesia from subconjunctival space [Video]. External surface morphology under LM revealed fine transverse cuticular striations with tapered cephalic and caudal ends. Uterus was long and coiled with indistinguishable masses inside. The finding was also confirmed by SEM.
SYNOPSIS
A subconjuctival parasite was removed and identified as Dirofilaria repens by characteristic LM and SEM findings.
HIGHLIGHT
Dirofilaria species may lodge in many tissues of human bodies including eye and adnexa. Dirofilaria is a natural parasite of carnivorous animals, mostly dogs, cats, and foxes. The most common mode of transmission to human is usually by bite of mosquitoes like Culex and Aedes, which are considered as vectors, and it is often thought that parasitemia is because of accidental conduction. Simple surgical removal of the worm is curative. After removal, the worm should be visualized directly under LM. All the internal structures of the transparent worm could be seen and compared with those under SEM.
Topics: Humans; Female; Male; Animals; Dogs; Middle Aged; Microscopy, Electron, Scanning; Parasites; Mosquito Vectors; Eye; Face
PubMed: 37602631
DOI: 10.4103/IJO.IJO_830_23 -
Pathogens (Basel, Switzerland) Jul 2023Bacterial adhesion is the first step in the formation of surface biofilms. The number of bacteria that bind to a surface from the solution depends on how many bacteria...
Bacterial adhesion is the first step in the formation of surface biofilms. The number of bacteria that bind to a surface from the solution depends on how many bacteria can reach the surface (bacterial transport) and the strength of interactions between bacterial adhesins and surface receptors (adhesivity). By using microfluidic channels and video microscopy as well as computational simulations, we investigated how the interplay between bacterial transport and adhesivity affects the number of the common human pathogen that bind to heterogeneous surfaces with different receptor densities. We determined that gravitational sedimentation causes bacteria to concentrate at the lower surface over time as fluid moves over a non-adhesive region, so bacteria preferentially adhere to adhesive regions on the lower, inflow-proximal areas that are downstream of non-adhesive regions within the entered compartments. Also, initial bacterial attachment to an adhesive region of a heterogeneous lower surface may be inhibited by shear due to mass transport effects alone rather than shear forces per se, because higher shear washes out the sedimented bacteria. We also provide a conceptual framework and theory that predict the impact of sedimentation on adhesion between and within adhesive regions in flow, where bacteria would likely bind both in vitro and in vivo, and how to normalize the bacterial binding level under experimental set-ups based on the flow compartment configuration.
PubMed: 37513788
DOI: 10.3390/pathogens12070941