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The British Journal of Ophthalmology Jan 1969
Topics: Adult; Contusions; Eye Injuries; Female; Humans; Male; Ophthalmoscopy; Retinal Detachment
PubMed: 5775574
DOI: 10.1136/bjo.53.1.59 -
Frontiers in Cellular Neuroscience 2022Retinal detachment is a sight-threatening disorder, which occurs when the photoreceptors are separated from their vascular supply. The aim of the present study was to...
Retinal detachment is a sight-threatening disorder, which occurs when the photoreceptors are separated from their vascular supply. The aim of the present study was to shed light on photoreceptor energy metabolism during experimental detachment in rats. Retinal detachment was induced in the eyes of rats subretinal injection of sodium hyaluronate. Initially, we investigated whether detachment caused hypoxia within photoreceptors, as evaluated by the exogenous and endogenous biomarkers pimonidazole and HIF-1α, as well as by qPCR analysis of HIF target genes. The results showed no unequivocal staining for pimonidazole or HIF-1α within any detached retina, nor upregulation of HIF target genes, suggesting that any reduction in pO is of insufficient magnitude to produce hypoxia-induced covalent protein adducts or HIF-1α stabilisation. Subsequently, we analysed expression of cellular bioenergetic enzymes in photoreceptors during detachment. We documented loss of mitochondrial, and downregulation of glycolytic enzymes during detachment, indicating that photoreceptors have reduced energetic requirements and/or capacity. Given that detachment did not cause widespread hypoxia, but did result in downregulated expression of bioenergetic enzymes, we hypothesised that substrate insufficiency may be critical in terms of pathogenesis, and that boosting metabolic inputs may preserve photoreceptor bioenergetic production and, protect against their degeneration. Thus, we tested whether supplementation with the bioavailable energy substrate pyruvate mitigated rod and cone injury and degeneration. Despite protecting photoreceptors in culture from nutrient deprivation, pyruvate failed to protect against apoptotic death of rods, loss of cone opsins, and loss of inner segment mitochondria, , when evaluated at 3 days after detachment. The regimen was also ineffective against cumulative photoreceptor deconstruction and degeneration when evaluated after 4 weeks. Retinal metabolism, particularly the bioenergetic profiles and pathological responses of the various cellular subtypes still presents a considerable knowledge gap that has important clinical consequences. While our data do not support the use of pyruvate supplementation as a means of protecting detached photoreceptors, they do provide a foundation and motivation for future research in this area.
PubMed: 36467607
DOI: 10.3389/fncel.2022.1036834 -
Scientific Reports Feb 2021During cancer, a major challenge faced by oncologists is the treatment of metastasis; a leading cause of cancer-related deaths around the world. Metastasis involves a...
During cancer, a major challenge faced by oncologists is the treatment of metastasis; a leading cause of cancer-related deaths around the world. Metastasis involves a highly ordered sequence of events starting with the detachment of tumor cells from the extracellular matrix (E.C.M.). In normal cells, detachment from E.C.M. triggers programmed cell death, termed anoikis. However, tumor cells dodge their way to anoikis and spread to distant sites for initiating the metastatic program. In this work, we explored the impact of E.C.M. detachment on the expression of some major oncogenic histone methyltransferases. Results showed both EZH2 expression and its enzymatic activity were significantly increased in E.C.M. detached cancer cells when compared to the attached cells. Inhibition of EZH2 results in a significant reduction in cell proliferation, spheroids size, and induction in apoptosis in E.C.M. detached cells. Furthermore, we observed a reduction in EZH2 expression levels in single cells when compared to clusters of E.C.M. detached cells. Finally, we combined the EZH2 inhibition with AMPK, known to be highly expressed in E.C.M. detached cancer cells and observed antagonistic effects between the two pathways. The observed results clearly showed that E.C.M. detached cancer cells require oncogenic EZH2 and can be targeted by EZH2 inhibitors.
Topics: Cell Adhesion; Cell Movement; Enhancer of Zeste Homolog 2 Protein; Extracellular Matrix; HCT116 Cells; HeLa Cells; Humans; Neoplasms
PubMed: 33531586
DOI: 10.1038/s41598-021-82431-w -
Frontiers in Immunology 2022The nature of the culture dish surface and the technique used to detach adherent cells could very likely influence the cell viability and cell membrane protein integrity...
The nature of the culture dish surface and the technique used to detach adherent cells could very likely influence the cell viability and cell membrane protein integrity of harvested macrophages. Several previous studies assessed the detachment efficacies of enzymatic and non-enzymatic methods for harvesting the single cell suspensions of macrophages, but a comprehensive study assessing different dissociation methods and culture conditions for detaching functionally different macrophage populations has not yet been reported. In this study, the well-established GM-CSF and M-CSF differentiated bone marrow derived macrophage models (GM-BMDMs and M-BMDMs), we compared four commonly used enzymatic (trypsin and accutase) and non-enzymatic (PBS and EDTA) dissociation methods along with necessary mechanical detaching steps (scraping and pipetting) to evaluate the viable cell recovery and cell surface marker integrality of GM-BMDMs and M-BMDMs cultured on standard cell culture dish (TC dish), or on culture dish (noTC dish) that was not conditioned to enhance adherence. The data showed that accutase yielded a better recovery of viable cells comparing with PBS and EDTA, especially for tightly adherent GM-BMDMs on TC dishes, with a relatively higher level of detected cell membrane marker F4/80 than trypsin. An additional gradient centrifugation-based dead cell removal approach could increase the proportion of viable cells for TC cultured GM-BMDMs after accutase dissociation. Furthermore, transcriptome analysis was performed to evaluate the putative influence of culture dishes. At steady state, BMDMs cultured on noTC dishes exhibited more proinflammatory gene expression signatures (e.g. IL6, CXCL2 and ILlβ) and functions (e.g. TNF and IL17 signaling pathways). Similar inflammatory responses were observed upon LPS challenge regardless of culture conditions and differentiation factors. However, in LPS treated samples, the difference of gene expression patterns, signaling pathways and molecular functions between TC and noTC cultured BMDMs were largely dependent on the types of growth factors (M-CSF and GM-CSF). This observation might provide valuable information for macrophage studies.
Topics: Animals; Cell Culture Techniques; Edetic Acid; Granulocyte-Macrophage Colony-Stimulating Factor; Lipopolysaccharides; Macrophage Colony-Stimulating Factor; Macrophages; Mice; Phenotype; Trypsin
PubMed: 35874686
DOI: 10.3389/fimmu.2022.920232 -
Journal of Ophthalmic & Vision Research 2018To determine 1) which components of retinal function are impaired after rhegmatogenous retinal detachment, 2) which outer retinal pathways (rod- or cone-driven) are more...
PURPOSE
To determine 1) which components of retinal function are impaired after rhegmatogenous retinal detachment, 2) which outer retinal pathways (rod- or cone-driven) are more severely affected, and 3) whether there is concomitant inner retinal dysfunction.
METHODS
We conducted a prospective observational study in a large academic institution. We performed preoperative electroretinography on eight patients to assess outer and inner retinal function. In all cases, a comparison between the eye with the detached retina and the control fellow eye was made.
RESULTS
Eyes with a detached retina had significantly lower a-wave and b-wave amplitudes with respect to both rod- and cone-dominated testing parameters ( < 0.05) and reduced 30 Hz flicker responses compared to fellow eyes ( < 0.05); the effect size was similar for all significantly reduced parameters (r~0.6). There were no significant differences between eyes with detached retinas and control fellow eyes with respect to b/a-wave ratios, a-wave latencies, or b-wave latencies.
CONCLUSION
Patients with rhegmatogenous retinal detachment have preoperative outer retinal dysfunction equally affecting both rod- and cone-driven pathways, and they have minimal inner retinal dysfunction.
PubMed: 30090179
DOI: 10.4103/jovr.jovr_161_17 -
Journal of Molecular Biology Jan 2019Meters of DNA wrap around histone proteins to form nucleosomes and fit inside the micron-diameter nucleus. For the genetic information encoded in the DNA to become...
Meters of DNA wrap around histone proteins to form nucleosomes and fit inside the micron-diameter nucleus. For the genetic information encoded in the DNA to become available for transcription, replication, and repair, the DNA-histone assembly must be disrupted. Experiment has indicated that the outer stretches of nucleosomal DNA "breathe" by spontaneously detaching from and reattaching to the histone core. Here, we report direct observation of spontaneous DNA breathing in atomistic molecular dynamics simulations, detailing a microscopic mechanism of the DNA breathing process. According to our simulations, the outer stretches of nucleosomal DNA detach in discrete steps involving 5 or 10 base pairs, with the detachment process being orchestrated by the motion of several conserved histone residues. The inner stretches of nucleosomal DNA are found to be more stably associated with the histone core by more abundant nonspecific DNA-protein contacts, providing a microscopic interpretation of nucleosome unraveling experiments. The CG content of nucleosomal DNA is found to anticorrelate with the extent of unwrapping, supporting the possibility that AT-rich segments may signal the start of transcription by forming less stable nucleosomes.
Topics: DNA; Histones; Humans; Molecular Dynamics Simulation; Nucleic Acid Conformation; Nucleosomes
PubMed: 30468737
DOI: 10.1016/j.jmb.2018.11.013 -
Scientific Reports Dec 2022Adherent cell cultures are often dissociated from their culture vessel (and each other) through enzymatic harvesting, where the detachment response is monitored by an...
Adherent cell cultures are often dissociated from their culture vessel (and each other) through enzymatic harvesting, where the detachment response is monitored by an operator. However, this approach is lacking standardisation and reproducibility, and prolonged exposure or too high concentrations can affect the cell's viability and differentiation potential. Quantitative monitoring systems are required to characterise the cell detachment response and objectively determine the optimal time-point to inhibit the enzymatic reaction. State-of-the-art methodologies rely on bulky imaging systems and/or features (e.g. circularity) that lack robustness. In this study, lens-free imaging (LFI) technology was used to develop a novel cell detachment feature. Seven different donors were cultured and subsequently harvested with a (diluted) enzymatic harvesting solution after 3, 5 and 7 days of culture. Cell detachment was captured with the LFI set-up over a period of 20 min (every 20 s) and by optimising the reconstruction of the LFI intensity images, a new feature could be identified. Bright regions in the intensity image were identified as detaching cells and using image analysis, a method was developed to automatically extract this feature, defined as the percentage of detached cell regions. Next, the method was quantitatively and qualitatively validated on a diverse set of images. Average absolute error values of 1.49%, 1.34% and 1.97% were obtained for medium to high density and overconfluent cultures, respectively. The detachment response was quantified for all conditions and the optimal time for enzyme inhibition was reached when approximately 92.5% of the cells were detached. On average, inhibition times of 9.6-11.1 and 16.2-17.2 min were obtained for medium to high density and overconfluent cultures, respectively. In general, overconfluent cultures detached much slower, while their detachment rate was also decreased by the diluted harvesting solution. Moreover, several donors exhibited similar trends in cell detachment behaviour, with two clear outliers. Using the novel feature, measurements can be performed with an increased robustness, while the compact LFI design could pave the way for in situ monitoring in a variety of culture vessels, including bioreactors.
Topics: Reproducibility of Results; Cell Culture Techniques; Lens, Crystalline; Lenses; Diagnostic Imaging
PubMed: 36564377
DOI: 10.1038/s41598-022-22561-x -
Cornea Nov 2023The aim of this study was to explore types of Descemet membrane detachment (DMD) after ocular surface burns by anterior segment optical coherence tomography. (Observational Study)
Observational Study
PURPOSE
The aim of this study was to explore types of Descemet membrane detachment (DMD) after ocular surface burns by anterior segment optical coherence tomography.
METHODS
This is a pilot, case series, observational study. Patients with DMD after ocular surface burns were enrolled. Ophthalmologic examinations were performed in all patients including slit-lamp photography and anterior segment optical coherence tomography.
RESULTS
Three types of DMDs in 9 eyes of 9 patients with ocular surface burns were identified depending on the detachment components involved with the pre-Descemet layer (PDL). Type A was referred as a taut chord that the PDL and Descemet membrane (DM) detached simultaneously but were remained attached to each other, while type B was identified as a wavy line separated from the stroma by a dark slit that demonstrated the detachment of DM from the PDL and stroma. Type C was defined as the DM detached with or without PDL but they were separated from each other. We found that DM and PDL were detached simultaneously in most condition, with type A in 4 cases, type C in 5 cases, and type B in only 1 case.
CONCLUSIONS
Our study demonstrated 3 types of DMDs after ocular surface burns and revealed that the limbal involvement and retrocorneal exudations may give clues to DMD in the corresponding areas. DMDs may be neglected for long in patients with extensive limbal involvement in early stages and also play an important role in unstable ocular surface condition until the late stages of conjunctivalization after ocular surface burns.
Topics: Humans; Burns; Corneal Diseases; Descemet Membrane; Face; Tomography, Optical Coherence; Pilot Projects
PubMed: 36729715
DOI: 10.1097/ICO.0000000000003210 -
MSphere May 2024Biofilm formation is an important virulence factor for methicillin-resistant (MRSA). The extracellular matrix of MRSA biofilms contains significant amounts of...
Biofilm formation is an important virulence factor for methicillin-resistant (MRSA). The extracellular matrix of MRSA biofilms contains significant amounts of double-stranded DNA that hold the biofilm together. MRSA cells secrete micrococcal nuclease (Nuc1), which degrades double-stranded DNA. In this study, we used standard methodologies to investigate the role of Nuc1 in MRSA biofilm formation and dispersal. We quantified biofilm formation and extracellular DNA (eDNA) levels in broth and agar cultures. In some experiments, cultures were supplemented with sub-MIC amoxicillin to induce biofilm formation. Biofilm erosion was quantitated by culturing biofilms on rods and enumerating detached colony-forming units (CFUs), and biofilm sloughing was investigated by perfusing biofilms cultured in glass tubes with fresh broth and measuring the sizes of the detached cell aggregates. We found that an MRSA mutant strain produced significantly more biofilm and more eDNA than a wild-type strain, both in the absence and presence of sub-MIC amoxicillin. mutant biofilms grown on rods detached significantly less than wild-type biofilms. Detachment was restored by exogenous DNase or complementing the mutant. In the sloughing assay, mutant biofilms released cell aggregates that were significantly larger than those released by wild-type biofilms. Our results suggest that Nuc1 modulates biofilm formation, biofilm detachment, and the sizes of detached cell aggregates. These processes may play a role in the spread and subsequent survival of MRSA biofilms during biofilm-related infections.IMPORTANCEInfections caused by antibiotic-resistant bacteria known as methicillin-resistant (MRSA) are a significant problem in hospitals. MRSA forms adherent biofilms on implanted medical devices such as catheters and breathing tubes. Bacteria can detach from biofilms on these devices and spread to other parts of the body such as the blood or lungs, where they can cause life-threatening infections. In this article, researchers show that MRSA secretes an enzyme known as thermonuclease that causes bacteria to detach from the biofilm. This is important because understanding the mechanism by which MRSA detaches from biofilms could lead to the development of procedures to mitigate the problem.
Topics: Biofilms; Methicillin-Resistant Staphylococcus aureus; Micrococcal Nuclease; Anti-Bacterial Agents; Bacterial Proteins; DNA, Bacterial; Virulence Factors; Microbial Sensitivity Tests; Amoxicillin
PubMed: 38695568
DOI: 10.1128/msphere.00126-24 -
Microbiology Spectrum Feb 2022Patients with chronic respiratory diseases use home nebulizers that are often contaminated with pathogenic microbes to deliver aerosolized medications. The conditions...
Patients with chronic respiratory diseases use home nebulizers that are often contaminated with pathogenic microbes to deliver aerosolized medications. The conditions under which these microbes leave the surface as bioaerosols during nebulization are not well characterized. The objectives of this study were to (i) determine whether different pathogens detach and disperse from the nebulizer surface during aerosolization and (ii) measure the effects of relative humidity and drying times on bacterial surface detachment and aerosolization. Bacteria were cultured from bioaerosols after Pari LC Plus albuterol nebulization using two different sources, as follows: (i) previously used nebulizers donated by anonymous patients with cystic fibrosis (CF) and (ii) nebulizers inoculated with bacteria isolated from the lungs of CF patients. Fractionated bioaerosols were collected with a Next-Generation Impactor. For a subset of bacteria, surface adherence during rewetting was measured with fluorescence microscopy. Bacteria dispersed from the surface of used CF patient nebulizers during albuterol nebulization. Eighty percent (16/20) of clinical isolates inoculated on the nebulizer in the laboratory formed bioaerosols. Detachment from the plastic surface into the chamber solution predicted bioaerosol production. Increased relative humidity and decreased drying times after inoculation favored bacterial dispersion on aerosols during nebulized therapy. Pathogenic bacteria contaminating nebulizer surfaces detached from the surface as bioaerosols during nebulized therapies, especially under environmental conditions when contaminated nebulizers were dried or stored at high relative humidity. This finding emphasizes the need for appropriate nebulizer cleaning, disinfection, and complete drying during storage and informs environmental conditions that favor bacterial surface detachment during nebulization. Studies from around the world have demonstrated that many patients use contaminated nebulizers to deliver medication into their lungs. While it is known that using contaminated medications in a nebulizer can lead to a lung infection, whether bacteria on the surface of a contaminated nebulizer detach as bioaerosols capable of reaching the lung has not been studied. This work demonstrates that a subset of clinical bacteria enter solution from the surface during nebulization and are aerosolized. Environmental conditions of high relative humidity during storage favor dispersion from the surface. We also provide results of an assay conducted to monitor bacterial surface detachment during multiple cycles of rewetting that correlate with the results of nebulizer/bacterial surface interactions. These studies demonstrate for the first time that pathogenic bacteria on the nebulizer surface pose a risk of bacterial inhalation to patients who use contaminated nebulizers.
Topics: Aerosols; Bacteria; Bacterial Adhesion; Cystic Fibrosis; Equipment Contamination; Humans; Nebulizers and Vaporizers
PubMed: 35107362
DOI: 10.1128/spectrum.02535-21