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Cell Communication and Signaling : CCS Jan 2024Retinal ganglion cell (RGC) degeneration and death cause vision loss in patients with glaucoma. Regulated cell death, once initiated, is generally considered to be an...
BACKGROUND
Retinal ganglion cell (RGC) degeneration and death cause vision loss in patients with glaucoma. Regulated cell death, once initiated, is generally considered to be an irreversible process. Recently, we showed that, by timely removing the cell death stimulus, stressed neuronal PC12 cells can recover from phosphatidylserine (PS) exposure, nuclear shrinkage, DNA damage, mitochondrial fragmentation, mitochondrial membrane potential loss, and retraction of neurites, all hallmarks of an activated cell death program. Whether the cell death process can be reversed in neurons of the central nervous system, like RGCs, is still unknown. Here, we studied reversibility of the activated cell death program in primary rat RGCs (prRGCs).
METHODS
prRGCs were exposed to ethanol (5%, vol/vol) to induce cell death. At different stages of the cell death process, ethanol was removed by washing and injured prRGCs were further cultured in fresh medium to see whether they recovered. The dynamics of single cells were monitored by high-resolution live-cell spinning disk microscopy. PS exposure, mitochondrial structure, membrane potential, and intracellular Ca were revealed by annexin A5-FITC, Mito-tracker, TMRM, and Fluo 8-AM staining, respectively. The distribution of cytochrome c was investigated by immunofluorescence. The ultrastructure of mitochondria was studied by electron microscopy.
RESULTS
Analysis of temporal relationships between mitochondrial changes and PS exposure showed that fragmentation of the mitochondrial network and loss of mitochondrial membrane potential occurred before PS exposure. Mitochondrial changes proceeded caspase-independently, while PS exposure was caspase dependent. Interestingly, prRGCs recovered quickly from these mitochondrial changes but not from PS exposure at the plasma membrane. Correlative light and electron microscopy showed that stress-induced decrease in mitochondrial area, length and cristae number was reversible. Intracellular Ca was elevated during this stage of reversible mitochondrial injury, but there was no sign of mitochondrial cytochrome c release.
CONCLUSIONS
Our study demonstrates that RGCs with impaired mitochondrial structure and function can fully recover if there is no mitochondrial cytochrome c release yet, and no PS is exposed at the plasma membrane. This finding indicates that there is a time window for rescuing dying or injured RGCs, by simply removing the cell death stimulus. Video Abstract.
Topics: Animals; Rats; Apoptosis; Caspases; Cytochromes c; Ethanol; Retinal Ganglion Cells
PubMed: 38297331
DOI: 10.1186/s12964-023-01427-3 -
Light, Science & Applications Jul 2023Advancement in mid-infrared (MIR) technology has led to promising biomedical applications of MIR spectroscopy, such as liquid biopsy or breath diagnosis. On the...
Advancement in mid-infrared (MIR) technology has led to promising biomedical applications of MIR spectroscopy, such as liquid biopsy or breath diagnosis. On the contrary, MIR microscopy has been rarely used for live biological samples in an aqueous environment due to the lack of spatial resolution and the large water absorption background. Recently, mid-infrared photothermal (MIP) imaging has proven to be applicable to 2D and 3D single-cell imaging with high spatial resolution inherited from visible light. However, the maximum measurement rate has been limited to several frames s, limiting its range of use. Here, we develop a significantly improved wide-field MIP quantitative phase microscope with two orders-of-magnitude higher signal-to-noise ratio than previous MIP imaging techniques and demonstrate live-cell imaging beyond video rate. We first derive optimal system design by numerically simulating thermal conduction following the photothermal effect. Then, we develop the designed system with a homemade nanosecond MIR optical parametric oscillator and a high full-well-capacity image sensor. Our high-speed and high-spatial-resolution MIR microscope has great potential to become a new tool for life science, in particular for live-cell analysis.
PubMed: 37463888
DOI: 10.1038/s41377-023-01214-2 -
Microbiology Spectrum Aug 2023Lysins (peptidoglycan hydrolases) are promising new protein-based antimicrobial candidates under development to address rising antibiotic resistance encountered among...
Lysins (peptidoglycan hydrolases) are promising new protein-based antimicrobial candidates under development to address rising antibiotic resistance encountered among pathogenic bacteria. Exebacase is an antistaphylococcal lysin and the first member of the lysin class to have entered clinical trials in the United States. In this study, the bacteriolytic activity of exebacase was characterized with time-kill assays, turbidity reduction assays, and microscopy. Three methicillin-susceptible and three methicillin-resistant isolates were tested in time-kill assays over a range of concentrations from 0.25 to 8 × MIC. Exebacase demonstrated a concentration-dependent killing and showed bactericidal activity (≥3 log kill achieved relative to the starting inoculum) within 3 h at 1 × MIC against all strains tested. Dose-dependent lysis by exebacase was, furthermore, observed in the turbidity reduction assay, wherein decreases in initial OD of 50% were observed within ~15 min at concentrations as low as 4 µg/mL. Membrane dissolution, loss of cytoplasmic material, and lysis were confirmed by video and electron microscopy. The demonstrated rapid bacteriolytic effect of exebacase is an important distinguishing feature of this novel modality. IMPORTANCE To guide the development of an investigational new antibacterial entity, microbiological data are required to evaluate the killing kinetics against target organism(s). Exebacase is a lysin (peptidoglycan hydrolase) that represents a novel antimicrobial modality based on degradation of the cell wall of . Killing by exebacase was determined in multiple assay formats including time-kill assays, wherein reductions of viability of ≥3 log colony-forming units/mL were observed within 3 h for multiple different isolates tested, consistent with very rapid bactericidal activity. Rapid reductions in optical density were likewise observed in exebacase-treated cultures, which were visually consistent with microscopic observations of rapid lysis. Overall, exebacase provides a novel antimicrobial modality against , characterized by a rapid cidal and lytic activity.
PubMed: 37561151
DOI: 10.1128/spectrum.01906-23 -
In Vivo (Athens, Greece) 2023Silicone implants or tissue expanders placed under the pectoralis major (PM) muscle are often used for breast reconstruction. However, the disruption of PM insertions,... (Review)
Review
Silicone implants or tissue expanders placed under the pectoralis major (PM) muscle are often used for breast reconstruction. However, the disruption of PM insertions, which is often an inevitable part of the surgical procedure, is known to cause PM morbidity and, subsequently, problems with the use of the ipsilateral arm. In this systematic review, we present current knowledge regarding the effect of submuscular silicone-based breast reconstruction on the function of PM and the ipsilateral arm. A search of the relevant English literature was performed through PubMed and ten eligible studies were identified. Articles reporting breast augmentation were accepted as the techniques of implant insertion are similar to reconstruction. Questionnaires reporting the status of the arm, analysis of the range of motion of the shoulder with 3-D video, isometric or isokinetic dynamometry, ultrasound shear-wave elastography, volumetric MRI, electromyography and light and electron microscopy of the PM fibers were used for the assessment of PM and arm status. The insertion of implants under the PM, especially when combined with dissection of some of its insertions, seems to be associated with measurable abnormal microscopic, imaging, and dynamometric findings. However, the intact part of the muscle and possibly other nearby muscular structures are able to compensate for the lost part of PM. Thus, the insertion of implants fully or partially under the PM seems to have no or little effect on the function of the ipsilateral upper limb in daily life.
Topics: Pectoralis Muscles; Silicones; Mammaplasty; Prostheses and Implants; Magnetic Resonance Imaging; Breast Implants
PubMed: 37652471
DOI: 10.21873/invivo.13289 -
Bioengineering (Basel, Switzerland) Sep 2023Current auricular cartilage replacements for pediatric microtia fail to address the need for long-term integration and neocartilage formation. While collagen hydrogels...
Current auricular cartilage replacements for pediatric microtia fail to address the need for long-term integration and neocartilage formation. While collagen hydrogels have been successful in fostering neocartilage formation, the toughness and extensibility of these materials do not match that of native tissue. This study used the N-terminal functionalization of collagen with alginate oligomers to improve toughness and extensibility through metal-ion complexation. Alginate conjugation was confirmed via FTIR spectroscopy. The retention of native collagen fibrillar structure, thermal gelation, and helical conformation in functionalized gels was confirmed via scanning electron microscopy, oscillatory shear rheology, and circular dichroism spectroscopy, respectively. Alginate-calcium complexation enabled a more than two-fold increase in modulus and work density in functionalized collagen with the addition of 50 mM CaCl, whereas unmodified collagen decreased in both modulus and work density with increasing calcium concentration. Additionally, the extensibility of alginate-functionalized collagen was increased at 25 and 50 mM CaCl. Following 2-week culture with auricular chondrocytes, alginate-functionalization had no effect on the cytocompatibility of collagen gels, with no effects on cell density, and increased glycosaminoglycan deposition. Custom MATLAB video analysis was then used to quantify fracture toughness, which was more than 5-fold higher following culture in functionalized collagen and almost three-fold higher in unmodified collagen.
PubMed: 37760139
DOI: 10.3390/bioengineering10091037 -
Scientific Reports Jul 2023The possibility to extract motion of a single organism from video recordings at a large-scale provides means for the quantitative study of its behavior, both individual...
The possibility to extract motion of a single organism from video recordings at a large-scale provides means for the quantitative study of its behavior, both individual and collective. This task is particularly difficult for organisms that interact with one another, overlap, and occlude parts of their bodies in the recording. Here we propose WormSwin-an approach to extract single animal postures of Caenorhabditis elegans (C. elegans) from recordings of many organisms in a single microscope well. Based on transformer neural network architecture our method segments individual worms across a range of videos and images generated in different labs. Our solutions offers accuracy of 0.990 average precision ([Formula: see text]) and comparable results on the benchmark image dataset BBBC010. Finally, it allows to segment challenging overlapping postures of mating worms with an accuracy sufficient to track the organisms with a simple tracking heuristic. An accurate and efficient method for C. elegans segmentation opens up new opportunities for studying of its behaviors previously inaccessible due to the difficulty in the worm extraction from the video frames.
Topics: Animals; Caenorhabditis elegans; Neural Networks, Computer; Microscopy; Video Recording; Behavior, Animal; Image Processing, Computer-Assisted
PubMed: 37419938
DOI: 10.1038/s41598-023-38213-7 -
National Science Review Jul 2023Precursor liquid is a nanoscale liquid creeping ahead of the macroscopic edge of spreading liquids, whose behaviors tightly correlate with the three-phase reaction...
Precursor liquid is a nanoscale liquid creeping ahead of the macroscopic edge of spreading liquids, whose behaviors tightly correlate with the three-phase reaction efficiency and patterning accuracy. However, the important spatial-temporal characteristic of the precursor liquid still remains obscure because its real-time spreading process has not been directly observed. Here, we report that the spreading ionic liquid precursors in a silicon corner can be directly captured on video using scanning electron microscopy. spreading videos show that the precursor liquid spreads linearly over time ([Formula: see text]) rather than obeying the classic Lucas-Washburn law ([Formula: see text]) and possesses a characteristic width of ∼250-310 nm. Theoretical analyses and molecular dynamics simulations demonstrate that the unique behaviors of precursor liquids originate from the competing effect of van der Waals force and surface energy. These findings provide avenues for directly observing liquid/solid interfacial phenomena on a microscopic level.
PubMed: 37266559
DOI: 10.1093/nsr/nwad119 -
Diagnostics (Basel, Switzerland) Jun 2023Nailfolds of patients with systemic sclerosis (SSc) provide an opportunity to directly visualize microvascular remodeling in SSc. Nailfold video capillaroscopy (NVC)... (Review)
Review
INTRODUCTION
Nailfolds of patients with systemic sclerosis (SSc) provide an opportunity to directly visualize microvascular remodeling in SSc. Nailfold video capillaroscopy (NVC) remains the gold standard for assessing nailfold capillaroscopy (NFC). However, access to NVC is limited by expense and expertise. This review aims to synthesize current research on other NFC devices compared to NVC.
METHODS
The literature search included the primary research of adult patients with SSc as defined by the 2013 ACR/EULAR criteria. Methods of assessing NFC included stereomicroscopy/wide-field microscopy, ophthalmoscopy, dermatoscopy, smartphone devices, and digital USB microscopy. Primary outcomes included both qualitative (normal vs. abnormal nailfolds, overall pattern recognition, presence/absence of giant capillaries, hemorrhages, and abnormal morphology) and quantitative (capillary density and dimension) measures.
RESULTS
The search yielded 471 studies, of which 9 were included. Five studies compared NVC to dermatoscopy, two compared it to widefield/stereomicroscopy, one to smartphone attachments, and one to USB microscopy. In dermatoscopy studies, NVC had a higher percentage of images that were interpretable (63-77% vs. 100%), classifiable (70% vs. 84%), or gradable (70% vs. 79.3%) across three studies. Dermatoscopy had a lower sensitivity (60.2% vs. 81.6%) and higher specificity (92.5% vs. 84.6%) compared to NVC. One stereomicroscopy study found a significant difference between methods in capillary density in limited cutaneous SSc, while another found correlations in all parameters between stereomicroscopy and NVC. One smartphone lens had good agreement with NVC on abnormal capillary morphology and density. USB microscopy was able to differentiate between SSc and healthy controls using mean capillary width but not by capillary density.
DISCUSSION
A dermatoscope may serve as a more portable and affordable screening tool to identify a normal "scleroderma pattern", and images that need further corroboration by NVC. NFC parameters reported are heterogenous and the standardization of these parameters is important, especially in non-gold-standard devices.
PubMed: 37443598
DOI: 10.3390/diagnostics13132204 -
Neurosurgical Focus: Video Apr 2024Minimally invasive surgery (MIS) is increasingly being adopted for spinal intradural tumors. Through the use of conventional microscopy or exoscopy for large lobulated...
Minimally invasive surgery (MIS) is increasingly being adopted for spinal intradural tumors. Through the use of conventional microscopy or exoscopy for large lobulated nerve sheath tumors, the posterior root attachment is often visualized only after mobilizing the tumor. Here, the authors describe the utility of angled endoscopy with its panoramic view for a T10 nerve sheath tumor. Gross-total extracapsular excision was achieved utilizing a minimally invasive right paraspinous approach, fenestration, lateral durotomy, sliding delivery of the tumor, sharp dissection of radicular attachments under neuromonitoring, and dural closure with oblique clips. Angled endoscopes help visualize the attachments behind large multilobulated tumors and confirm the totality of excision. The video can be found here: https://stream.cadmore.media/r10.3171/2024.1.FOCVID23214.
PubMed: 38616904
DOI: 10.3171/2024.1.FOCVID23214 -
BMC Pediatrics Jan 2024In children with sepsis, circulatory shock and multi-organ failure remain major contributors to mortality. Prolonged capillary refill time (PCRT) is a clinical tool...
BACKGROUNDS
In children with sepsis, circulatory shock and multi-organ failure remain major contributors to mortality. Prolonged capillary refill time (PCRT) is a clinical tool associated with disease severity and tissue hypoperfusion. Microcirculation assessment with videomicroscopy represents a promising candidate for assessing and improving hemodynamic management strategies in children with sepsis. Particularly when there is loss of coherence between the macro and microcirculation (hemodynamic incoherence). We sought to evaluate the association between PCRT and microcirculation changes in sepsis.
METHODS
This was a prospective cohort study in children hospitalized with sepsis. Microcirculation was measured using sublingual video microscopy (capillary density and flow and perfused boundary region [PBR]-a parameter inversely proportional to vascular endothelial glycocalyx thickness), phalangeal tissue perfusion, and endothelial activation and glycocalyx injury biomarkers. The primary outcome was the association between PCRT and microcirculation changes.
RESULTS
A total of 132 children with sepsis were included, with a median age of two years (IQR 0.6-12.2). PCRT was associated with increased glycocalyx degradation (PBR 2.21 vs. 2.08 microns; aOR 2.65, 95% CI 1.09-6.34; p = 0.02) and fewer 4-6 micron capillaries recruited (p = 0.03), with no changes in the percentage of capillary blood volume (p = 0.13). Patients with hemodynamic incoherence had more PBR abnormalities (78.4% vs. 60.8%; aOR 2.58, 95% CI 1.06-6.29; p = 0.03) and the persistence of these abnormalities after six hours was associated with higher mortality (16.5% vs. 6.1%; p < 0.01). Children with an elevated arterio-venous CO difference (DCO) had an abnormal PBR (aOR 1.13, 95% CI 1.01-1.26; p = 0.03) and a lower density of small capillaries (p < 0.05). Prolonged capillary refill time predicted an abnormal PBR (AUROC 0.81, 95% CI 0.64-0.98; p = 0.03) and relative percentage of blood in the capillaries (AUROC 0.82, 95% CI 0.58-1.00; p = 0.03) on admission. A normal CRT at 24 h predicted a shorter hospital stay (aOR 0.96, 95% CI 0.94-0.99; p < 0.05).
CONCLUSIONS
We found an association between PCRT and microcirculation changes in children with sepsis. These patients had fewer small capillaries recruited and more endothelial glycocalyx degradation. This leads to nonperfused capillaries, affecting oxygen delivery to the tissues. These disorders were associated with hemodynamic incoherence and worse clinical outcomes when the CRT continued to be abnormal 24 h after admission.
Topics: Child; Humans; Infant; Child, Preschool; Microcirculation; Prospective Studies; Sepsis; Capillaries; Biomarkers
PubMed: 38245695
DOI: 10.1186/s12887-024-04524-5