Search Results (1,141)

Iron redistribution in the intestine after total body irradiation is an established phenomenon. However, in the literature, there are no reports about the use of X-ray fluorescence microscopy or equivalent techniques to generate semi-quantitative 2D maps of iron in sectioned intestine samples from irradiated mice. In this work, we used X-ray fluorescence microscopy (XFM) to map the elemental content of iron as well as phosphorus, sulfur, calcium, copper and zinc in tissue sections of the small intestine from eight-week-old BALB/c male mice that developed gastrointestinal acute radiation syndrome (GI-ARS) in response to exposure to 8 Gray of gamma rays. Seven days after irradiation, we found that the majority of the iron is localized as hot spots in the intercellular regions of the area surrounding crypts and stretching between the outer perimeter of the intestine and the surface cell layer of villi. In addition, this study represents our current efforts to develop elemental cell classifiers that could be used for the automated generation of regions of interest for analyses of X-ray fluorescence maps. Once developed, such a tool will be instrumental for studies of effects of radiation and other toxicants on the elemental content in cells and tissues. While XFM studies cannot be conducted on living organisms, it is possible to envision future scenarios where XFM imaging of single cells sloughed from the human (or rodent) intestine could be used to follow up on the progression of GI-ARS. Full article

Background/Objectives: Adeno-associated viruses (AAVs) are widely used as viral vectors for gene delivery in mammalian cells. We focused on the efficacy of the transduction of AAV2/5, 2/6, 2/8 and 2/9 expressing GFP in preadipocyte cells by live imaging microscopy using IncuCyte S3 and flow cytometry. Methods: Three transduction modes in 3T3-L1 preadipocyte cells assessed: AAV transduction in 3T3-L1 preadipocyte cells, transduction with further differentiation into mature adipocyte-like cells and the transduction of differentiated 3T3-L1 adipocytes. For the in vivo study, we injected AAV2/6, AAV2/8 and AAV2/9 in adipose tissue of C57BL6 mice, and the transduction capacity of AAV2/6, along with AAV2/8 and AAV2/9 was evaluated. Results: AAV2/6 demonstrated the highest transduction efficiency in 3T3-L1 preadipocytes, as it was 1.5–2-fold more effective than AAV2/5, and AAV2/8 in the range of viral concentrations from 2 × 10⁴ to 1.6 × 10⁵ VG/cell. AAV2/5 and AAV2/8 showed transduction efficiencies similar to each other. The expression of GFP under the CMV promoter remained stable for up to 20 days. The induction of 3T3-L1 differentiation in three days after AAV transduction did not alter the GFP expression level, and AAV2/6 showed the best transduction efficiency. AAV2/6 demonstrated the ability to transduce mature adipocytes. These results were confirmed by in vivo studies on C57BL6 mice. AAV2/6 had the highest transducing activity on both inguinal and interscapular adipose tissue. Conclusions: Thus, AAV2/6 has demonstrated higher transduction efficacy compared to AAV2/5, AAV2/8 and AAV2/9 both in 3T3-L1 adipocytes and adipose tissue in vivo, which proves its usability along with AAV2/8 and AAV2/9 for gene delivery to adipocytes. Full article

Lactate is a critical regulatory factor secreted by tumors, influencing tumor development, metastasis, and clinical prognosis. Precise analysis of tumor-cell-secreted lactate is pivotal for early cancer diagnosis. This study describes a paper-based microfluidic chip to enable the detection of lactate levels secreted externally by living cells. Under optimized conditions, the lactate biosensor can complete the assay in less than 30 min. In addition, the platform can be used to distinguish lactate secretion levels in different cell lines and can be applied to the screening of antitumor drugs. Through enzymatic chemical conversion, this platform generates fluorescent signals, enabling qualitative assessment under a handheld UV lamp and quantitative analysis via grayscale intensity measurements using ImageJ (Ver. 1.50i) software. The paper-based platform presented in this study is rapid and highly sensitive and does not necessitate other costly and intricate instruments, thus making it applicable in resource-constrained areas and serving as a valuable tool for investigating cell lactate secretion and screening various anti-cancer drugs. Full article

Intracellular biothiols, including cysteine (Cys), glutathione (GSH), and homocysteine (Hcy), play a critical role in many physiological and pathological processes. Among them, GSH is the most abundant non-protein mercaptan (1–10 mM) in cells, and the change in GSH concentration level is closely related to the occurrence of many diseases, such as Parkinson’s disease, Alzheimer’s disease, and neurological diseases. Fluorescent probes have attracted much attention due to their advantages of high specificity, high sensitivity, high selectivity, low cost, and high quantum yield. Methods that use optical probes for selective detection of GSH in vitro and in vivo are in high demand. In this paper, we reviewed the most recent five years of research on fluorescence probes for the detection of GSH, including the specific detection of GSH, dual-channel identification of GSH and other substances, and the detection of GSH and other biothiols. According to the type of fluorophore, we classified GSH fluorescent probes into eight classes, including BODIPY, 1,8-Naphthalimide, coumarin, xanthene, rhodamine, cyanine, benzothiazoles, and others. In addition, we roundly discuss the synthesis, detection mechanism, photophysical properties, and biological applications of fluorescent probes. We hope that this review will inspire the exploration of new fluorescent probes for GSH and other related analyses. Full article

Streptococcus suis is an important bacterial pathogen that affects the global pig industry. The immunosuppressive nature of S. suis infection is recognized, and our previous research has confirmed thymus atrophy with a large number of necrotic cells. In this current work, we aimed to uncover the role of pyroptosis in cellular necrosis in thymic cells of S. suis-infected mice. Confocal microscopy revealed that S. suis activated the M1 phenotype and primed pyroptosis in the macrophages of atrophied thymus. Live cell imaging further confirmed that S. suis could induce porcine alveolar macrophage (PAM) pyroptosis in vitro, displaying cell swelling and forming large bubbles on the plasma membrane. Meanwhile, the levels of p-p38, p-extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) were increased, which indicated the mitogen-activated protein kinase (MAPK) and AKT pathways were also involved in the inflammation of S. suis-infected PAMs. Furthermore, RT-PCR revealed significant mRNA expression of pro-inflammatory mediators, including interleukin (IL)-1β, IL-6, IL-18, tumor necrosis factor (TNF)-α and chemokine CXCL8. The data indicated that the inflammation induced by S. suis was in parallel with pro-inflammatory activities of M1 macrophages, pyroptosis and MAPK and AKT pathways. Pyroptosis contributes to necrotic cells and thymocyte reduction in the atrophied thymus of mice. Full article

Adeno-associated viral vectors (AAVs) are the predominant viral vectors used for gene therapy applications. A significant challenge in obtaining effective doses is removing non-therapeutic empty viral capsids lacking DNA cargo. Current methods for separating full (gene-containing) and empty capsids are challenging to scale, produce low product yields, are slow, and are difficult to operationalize for continuous biomanufacturing. This communication demonstrates the feasibility of separating full and empty capsids by ultrafiltration. Separation performance was quantified by measuring the sieving coefficients for full and empty capsids using ELISA, qPCR, and an infectivity assay based on the live cell imaging of green fluorescent protein expression. We demonstrated that polycarbonate track-etched membranes with a pore size of 30 nm selectively permeated empty capsids to full capsids, with a high recovery yield (89%) for full capsids. The average sieving coefficients of full and empty capsids obtained through ELISA/qPCR were calculated as 0.25 and 0.49, indicating that empty capsids were about twice as permeable as full capsids. Establishing ultrafiltration as a viable unit operation for separating full and empty AAV capsids has implications for developing the scale-free continuous purification of AAVs. Full article

The cytoskeleton mediates fundamental cellular processes by organizing inter-organelle interactions. Pathogenic variants of inverted formin 2 (INF2) CAAX isoform, an actin assembly factor that is predominantly expressed in the endoplasmic reticulum (ER), are linked to focal segmental glomerulosclerosis (FSGS) and Charcot–Marie–Tooth (CMT) neuropathy. To investigate how pathogenic INF2 variants alter ER integrity, we used high-resolution live imaging of HeLa cells. Cells expressing wild-type (WT) INF2 showed a predominant tubular ER with perinuclear clustering. Cells expressing INF2 FSGS variants that cause mild and intermediate disease induced more sheet-like ER, a pattern similar to that seen for cells expressing WT-INF2 that were treated with actin and microtubule (MT) inhibitors. Dual CMT-FSGS INF2 variants led to more severe ER dysmorphism, with a diffuse, fragmented ER and coarse INF2 aggregates. Proper organization of both F-actin and MT was needed to modulate the tubule vs. sheet conformation balance, while MT arrays regulated spatial expansion of tubular ER in the cell periphery. Pathogenic INF2 variants also induced mitochondria fragmentation and dysregulated mitochondria distribution. Such mitochondrial abnormalities were more prominent for cells expressing CMT-FSGS compared to those with FSGS variants, indicating that the severity of the dysfunction is linked to the degree of cytoskeletal disorganization. Our observations suggest that pathogenic INF2 variants disrupt ER continuity by altering interactions between the ER and the cytoskeleton that in turn impairs inter-organelle communication, especially at ER–mitochondria contact sites. ER continuity defects may be a common disease mechanism involved in both peripheral neuropathy and glomerulopathy. Full article

Long-term live cell imaging requires sophisticated and fully automated commercial-stage incubators equipped with specified inverted microscopes to regulate temperature, CO₂ content, and humidity. In this study, we present a CO₂-free on-stage incubator specifically designed for use across various cell culture platforms, enabling live cell imaging applications. A simple and transparent incubator was fabricated from acrylic sheets to be easily placed on the stages of most inverted microscopes. We successfully performed live-cell imaging of cholangiocarcinoma (CCA) cells and HeLa cell dynamics in both 2D and 3D microenvironments over three days. We also analyzed directed cell migration under high serum induction within a microfluidic device. Interesting phenomena such as “whole-colony migration”, “novel type of collective cell migration” and “colony formation during cell and colony migration” are reported here for the first time, to the best of our knowledge. These phenomena may improve our understanding of the nature of cell migration and cancer metastasis. Full article

Penetrating deep into the cells of the human body in real time has become increasingly possible with the implementation of modern technologies in medicine. Atomic force microscopy (AFM) enables the effective live imaging of cellular and molecular structures of biological samples (such as cells surfaces, components of biological membranes, cell nuclei, actin networks, proteins, and DNA) and provides three-dimensional surface visualization (in X-, Y-, and Z-planes). Furthermore, the AFM technique enables the study of the mechanical, electrical, and magnetic properties of cells and cell organelles and the measurements of interaction forces between biomolecules. The technique has found wide application in cancer research. With the use of AFM, it is not only possible to differentiate between healthy and cancerous cells, but also to distinguish between the stages of cancerous conditions. For many years, AFM has been an important tool for the study of neurodegenerative diseases associated with the deposition of peptide amyloid plaques. In recent years, a significant amount of research has been conducted on the application of AFM in the evaluation of connective tissue cell mechanics. This review aims to provide the spectrum of the most important applications of the AFM technique in medicine to date. Full article

Ketone bodies are considered alternative fuels for the brain when glucose availability is limited. To determine the neuroregenerative potential of D,L-sodium-beta-hydroxybutyrate (D/L-BHB), Sprague Dawley rat primary cortical neurons were exposed to simulated central nervous system injury using a scratch assay. The neuronal cell migration, cell density and degree of regeneration in the damaged areas (gaps) in the absence (control) and presence of BHB (2 mM) were documented with automated live-cell imaging by the CytoSMART system over 24 h, which was followed by immunocytochemistry, labeling synapsin-I and β3-tubulin. The cell density was significantly higher in the gaps with BHB treatment after 24 h compared to the control. In the control, only 1.5% of the measured gap areas became narrower over 24 h, while in the BHB-treated samples 49.23% of the measured gap areas became narrower over 24 h. In the control, the gap expanded by 63.81% post-injury, while the gap size decreased by 10.83% in response to BHB treatment, compared to the baseline. The cell density increased by 97.27% and the gap size was reduced by 74.64% in response to BHB, compared to the control. The distance travelled and velocity of migrating cells were significantly higher with BHB treatment, while more synapsin-I and β3-tubulin were found in the BHB-treated samples after 24 h, compared to the control. The results demonstrate that D/L-BHB enhanced neuronal migration and molecular processes associated with neural regeneration and axonogenesis. These results may have clinical therapeutic applications in the future for nervous system injuries, such as for stroke, concussion and TBI patients. Full article

To explore new compounds with antitumour activity, fifteen phenolic nor-tripterpenes isolated from Celastraceae species, Maytenus jelskii, Maytenus cuzcoina, and Celastrus vulcanicola, have been studied. Their chemical structures were elucidated through spectroscopic and spectrometric techniques, resulting in the identification of three novel chemical compounds. Evaluation on human tumour cell lines (A549 and SW1573, non-small cell lung; HBL-100 and T-47D, breast; HeLa, cervix, and WiDr, colon) revealed that three compounds, named 6-oxo-pristimerol, demethyl-zeylasteral, and zeylasteral, exhibited significant activity (GI₅₀ ranging from 0.45 to 8.6 µM) on at least five of the cell lines tested. Continuous live cell imaging identified apoptosis as the mode of action of selective cell killing in HeLa cells. Furthermore, their effect on a drug-sensitive Saccharomyces cerevisiae strain has been investigated to deepen on their mechanism of action. In dose-response growth curves, zeylasteral and 7α-hydroxy-blepharodol were markedly active. Additionally, halo assays were conducted to assess the involvement of oxidative stress and/or mitochondrial function in the anticancer profile, ruling out these modes of action for the active compounds. Finally, we also delve into the structure-activity relationship, providing insights into how the molecular structure of these compounds influences their biological activity. This comprehensive analysis enhances our understanding of the therapeutic potential of this triterpene type and underscores its relevance for further research in this field. Full article

DNA double strand breaks (DSBs) are critical for the efficacy of radiotherapy as they lead to cell death if not repaired. DSBs caused by ionizing radiation (IR) initiate histone modifications and accumulate DNA repair proteins, including 53BP1, which forms distinct foci at damage sites and serves as a marker for DSBs. DSB repair primarily occurs through Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR). NHEJ directly ligates DNA ends, employing proteins such as DNA-PK_cs, while HR, involving proteins such as Rad54, uses a sister chromatid template for accurate repair and functions in the S and G2 phases of the cell cycle. Both pathways are crucial, as illustrated by the IR sensitivity in cells lacking DNA-PK_cs or Rad54. We generated mouse embryonic stem (mES) cells which are knockout (KO) for DNA-PK_cs and Rad54 to explore the combined role of HR and NHEJ in DSB repair. We found that cells lacking both DNA-PK_cs and Rad54 are hypersensitive to X-ray radiation, coinciding with impaired 53BP1 focus resolution and a more persistent G2 phase cell cycle block. Additionally, mES cells deficient in DNA-PK_cs or both DNA-PK_cs and Rad54 exhibit an increased nuclear size approximately 18–24 h post-irradiation. To further explore the role of Rad54 in the absence of DNA-PK_cs, we generated DNA-PK_cs KO mES cells expressing GFP-tagged wild-type (WT) or ATPase-defective Rad54 to track the Rad54 foci over time post-irradiation. Cells lacking DNA-PK_cs and expressing ATPase-defective Rad54 exhibited a similar phenotypic response to IR as those lacking both DNA-PK_cs and Rad54. Despite a strong G2 phase arrest, live-cell imaging showed these cells eventually progress through mitosis, forming micronuclei. Additionally, mES cells lacking DNA-PK_cs showed increased Rad54 foci over time post-irradiation, indicating an enhanced reliance on HR for DSB repair without DNA-PK_cs. Our findings underscore the essential roles of HR and NHEJ in maintaining genomic stability post-IR in mES cells. The interplay between these pathways is crucial for effective DSB repair and cell cycle progression, highlighting potential targets for enhancing radiotherapy outcomes. Full article

In recent years, various technologies have emerged for the imaging of chromatin loci in living cells via catalytically inactive Cas9 (dCas9). These technologies facilitate a deeper understanding of the mechanisms behind the chromatin dynamics and provide valuable kinetic data that could not have previously been obtained via FISH applied to fixed cells. However, such technologies are relatively complicated, as they involve the expression of several chimeric proteins as well as sgRNAs targeting the visualized loci, a process that entails many technical subtleties. Therefore, the effectiveness in visualizing a specific target locus may be quite low. In this study, we directly compared two versions of a previously published CRISPR-Sirius method based on the use of sgRNAs containing eight MS2 or PP7 stem loops and the expression of MCP or PCP fused to fluorescent proteins. We assessed the visualization efficiency for several unique genomic loci by comparing the two approaches in delivering sgRNA genes (transient transfection and lentiviral transduction), as well as two CRISPR-Sirius versions (with PCP and with MCP). The efficiency of visualization varied among the loci, and not all loci could be visualized. However, the MCP-sfGFP version provided more efficient visualization in terms of the number of cells with signals than PCP-sfGFP for all tested loci. We also showed that lentiviral transduction was more efficient in locus imaging than transient transfection for both CRISPR-Sirius systems. Most of the target loci in our study were located at the borders of topologically associating domains, and we defined a set of TAD borders that could be effectively visualized using the MCP-sfGFP version of the CRISPR-Sirius system. Altogether, our study validates the use of the CRISPR-Sirius technology for live-cell visualization and highlights various technical details that should be considered when using this method. Full article

Cryofixation by ultra-rapid freezing is widely regarded as the gold standard for preserving cell structure without artefacts for electron microscopy. However, conventional cryofixation technologies are not compatible with live imaging, making it difficult to capture dynamic cellular processes at a precise time. To overcome this limitation, we recently introduced a new technology, called microfluidic cryofixation. The principle is based on micro-hotplates counter-cooled with liquid nitrogen. While the power is on, the sample inside a foil-embedded microchannel on top of the micro-hotplate is kept warm. When the heater is turned off, the thermal energy is drained rapidly and the sample freezes. While this principle has been demonstrated experimentally with small samples (<0.5 mm²), there is an important trade-off between the attainable cooling rate, sample size, and heater power. Here, we elucidate these connections by theoretical modeling and by measurements. Our findings show that cooling rates of 10⁶ K s⁻¹, which are required for the vitrification of pure water, can theoretically be attained in samples up to ∼1 mm wide and 5 μm thick by using diamond substrates. If a heat sink made of silicon or copper is used, the maximum thickness for the same cooling rate is reduced to ∼3 μm. Importantly, cooling rates of 10⁴ K s⁻¹ to 10⁵ K s⁻¹ can theoretically be attained for samples of arbitrary area. Such rates are sufficient for many real biological samples due to the natural cryoprotective effect of the cytosol. Thus, we expect that the vitrification of millimeter-scale specimens with thicknesses in the 10 μm range should be possible using micro-hotplate-based microfluidic cryofixation technology. Full article

Direct neuronal reprogramming is a promising approach to replace neurons lost due to disease via the conversion of endogenous glia reacting to brain injury into neurons. However, it is essential to demonstrate that the newly generated neurons originate from glial cells and/or show that they are not pre-existing endogenous neurons. Here, we use controls for both requirements while comparing two viral vector systems (Mo-MLVs and AAVs) for the expression of the same neurogenic factor, the phosphorylation-resistant form of Neurogenin2. Our results show that Mo-MLVs targeting proliferating glial cells after traumatic brain injury reliably convert astrocytes into neurons, as assessed by genetic fate mapping of astrocytes. Conversely, expressing the same neurogenic factor in a flexed AAV system results in artefactual labelling of endogenous neurons fatemapped by birthdating in development that are negative for the genetic fate mapping marker induced in astrocytes. These results are further corroborated by chronic live in vivo imaging. Taken together, the phosphorylation-resistant form of Neurogenin2 is more efficient in reprogramming reactive glia into neurons than its wildtype counterpart in vivo using retroviral vectors (Mo-MLVs) targeting proliferating glia. Conversely, AAV-mediated expression generates artefacts and is not sufficient to achieve fate conversion. Full article