Traditional biochemical methods for enzyme detection are mainly based on antibody-based immunoassays, which lack the ability to monitor the spatiotemporal distribution and, in particular, the in situ ...activity of enzymes in live cells and in vivo. In this review, we comprehensively summarize recent progress that has been made in the development of small-molecule as well as material-based fluorogenic probes for sensitive detection of the activities of enzymes that are related to a number of human diseases. The principles utilized to design these probes as well as their applications are reviewed. Specific attention is given to fluorogenic probes that have been developed for analysis of the activities of enzymes including oxidases and reductases, those that act on biomacromolecules including DNAs, proteins/peptides/amino acids, carbohydrates and lipids, and those that are responsible for translational modifications. We envision that this review will serve as an ideal reference for practitioners as well as beginners in relevant research fields.
To identify the effects of superoxide dismutase (SOD)3 on diabetes mellitus (DM)-induced retinal changes in a diabetic rat model.
Diabetic models were established by a single intraperitoneal ...injection of streptozotocin (STZ) in Sprague-Dawley rats. After purification of the recombinant SOD3, intravitreal injection of SOD3 was performed at the time of STZ injection, and 1 and 2 weeks following STZ injection. Scotopic and photopic electroretinography (ERG) were recorded. Immunofluorescence staining with ɑ-smooth muscle actin (SMA), glial fibrillary acidic protein (GFAP), pigment epithelium-derived factor (PEDF), Flt1, recoverin, parvalbumin, extracellular superoxide dismutase (SOD3), 8-Hydroxy-2'deoxyguanosine (8-OHdG) and tumor necrosis factor-ɑ (TNF-ɑ) were evaluated.
In the scotopic ERG, the diabetic group showed reduced a- and b-wave amplitudes compared with the control group. In the photopic ERG, b-wave amplitude showed significant (p < 0.0005) reduction at 8 weeks following DM induction. However, the trend of a- and b-wave reduction was not evident in the SOD3 treated group. GFAP, Flt1, 8-OHdG and TNF-ɑ immunoreactivity were increased, and ɑ-SMA, PEDF and SOD3 immunoreactivity were decreased in the diabetic retina. The immunoreactivity of these markers was partially recovered in the SOD3 treated group. Parvalbumin expression was not decreased in the SOD3 treated group. In the diabetic retinas, the immunoreactivity of recoverin was weakly detected in both of the inner nuclear layer and inner plexiform layer compared to the control group but not in the SOD3 treated group.
SOD3 treatment attenuated the loss of a/b-wave amplitudes in the diabetic rats, which was consistent with the immunohistochemical evaluation. We also suggest that in rod-dominant rodents, the use of blue on green photopic negative response (PhNR) is effective in measuring the inner retinal function in animal models of diabetic retinopathy. SOD3 treatment ameliorated the retinal Müller cell activation in diabetic rats and pericyte dysfunction. These results suggested that SOD3 exerted protective effects on the development of diabetic retinopathy.
We examined the functional morphology of loach Misgurnus anguillicaudatus skin by using synchrotron X‐ray micro‐computed tomography (SR‐μCT) and high‐contrast staining using osmium tetroxide or ...phosphotungstic acid (PTA), which enhances the image contrast of soft tissues. The captured high‐spatial resolution images revealed that the surface ornamentations were stuck in the basement membrane of the loach scales. The ornamentations consisting of grooves (radii) and ridges (circuli) that can move freely and bend flexibly. The cross‐sectional lateral microstructures of flat, concave and convex loach skins were observed from a live image of loach skin obtained through dark‐field optical coherence tomography (OCT) imaging. The thickness of loach skin was changed with varying empty space between the mucous‐cell layer and the scales by bending motion of loach. In addition, through direct measurement of drag reduction of loach skin, the mucous layer was found to have a strong influence on the reduction of skin friction. The present results enhance the understanding of the functional morphologies of mucous layer of loach to secrete mucus for skin friction reduction.
Urinary tract infection (UTI) is the most prevalent urological condition worldwide. Choosing appropriate antibiotics for patients who have fever before receiving a culture result is challenging. This ...retrospective study enrolled patients 394 patients hospitalized at Gangneung Asan Hospital for UTI from May 2017 to April 2021. Fever at 48 h of hospitalization was the analysis point, as this is when the response to antibiotic therapy manifest, although the results of antibiogram are not available. Multivariate analysis was performed to assess the correlation between ESBL producing bacteria (EPB) and fever at 48 h. Overall, 36.3% of patients had EPB and 27.9% had fever at 48 h. In multivariate analysis, a significant positive association was found between EPB and fever (odds ratio 1.17, 95% CI 1.05-1.30, P = 0.004) Female had negative association with multivariate model (OR 0.83, 95% CI 0.73-0.94, P = 0.004). Diabetes did not demonstrate a significant association with EPB. (OR 1.10, 95% CI 0.99-1.22, P = 0.072). Fever at 48 h is associated with EPB and could be considered a predictive factor for EPB infection in patients with UTI. Antibiotic escalation may be considered in patients with fever at 48 h.
Advancing the technologies for cellular reprogramming with high efficiency has significant impact on regenerative therapy, disease modeling, and drug discovery. Biophysical cues can tune the cell ...fate, yet the precise role of external physical forces during reprogramming remains elusive. Here the authors show that temporal cyclic-stretching of fibroblasts significantly enhances the efficiency of induced pluripotent stem cell (iPSC) production. Generated iPSCs are proven to express pluripotency markers and exhibit in vivo functionality. Bulk RNA-sequencing reveales that cyclic-stretching enhances biological characteristics required for pluripotency acquisition, including increased cell division and mesenchymal-epithelial transition. Of note, cyclic-stretching activates key mechanosensitive molecules (integrins, perinuclear actins, nesprin-2, and YAP), across the cytoskeletal-to-nuclear space. Furthermore, stretch-mediated cytoskeletal-nuclear mechano-coupling leads to altered epigenetic modifications, mainly downregulation in H3K9 methylation, and its global gene occupancy change, as revealed by genome-wide ChIP-sequencing and pharmacological inhibition tests. Single cell RNA-sequencing further identifies subcluster of mechano-responsive iPSCs and key epigenetic modifier in stretched cells. Collectively, cyclic-stretching activates iPSC reprogramming through mechanotransduction process and epigenetic changes accompanied by altered occupancy of mechanosensitive genes. This study highlights the strong link between external physical forces with subsequent mechanotransduction process and the epigenetic changes with expression of related genes in cellular reprogramming, holding substantial implications in the field of cell biology, tissue engineering, and regenerative medicine.
Bacterial infection raises serious concerns in tissue repair settings involved with implantable biomaterials, devastating the regenerative process and even life-threatening. When hard tissues are ...infected with bacteria (called ‘osteomyelitis’), often the cases in open fracture or chronic inflammation, a complete restoration of regenerative capacity is significantly challenging even with highly-dosed antibiotics or surgical intervention. The implantable biomaterials are thus needed to be armored to fight bacteria then to relay regenerative events. To this end, here we propose a nanoglass paste made of ~200-nm-sized silicate-glass (with Ca, Cu) particles that are hardened in contact with aqueous medium and multiple-therapeutic, i.e., anti-bacterial, pro-angiogenic and osteopromotive. The nanoglass paste self-hardened via networks of precipitated nano-islands from leached ions to exhibit ultrahigh surface area (~300 m2/g), amenable to fill tunable defects with active biomolecular interactions. Also, the nanoglass paste could release multiple ions (silicate, calcium, and copper) at therapeutically relevant doses and sustainably (for days to weeks), implying possible roles in surrounding cells/tissues as a therapeutic-ions reservoir. The osteopromotive effects of nanoglass paste were evidenced by the stimulated osteogenic differentiation of MSCs. Also, the nanoglass paste promoted angiogenesis of endothelial cells in vitro and vasculature formation in vivo. Furthermore, the significant bactericidal effect of nanoglass paste, as assessed with E. coli and S. aureus, highlighted the role of copper played in elevating ROS level and destroying homeostasis, which salvaged tissue cells from co-cultivated bacteria contamination. When administered topically to rat tibia osteomyelitis defects, the nanoglass paste enhanced in vivo bone healing and fracture resistance. The developed nanoglass paste, given its self-setting property and the coordinated therapeutic actions, is considered to be a promising drug-free inorganic biomaterial platform for the regenerative therapy of bacteria-infected hard tissues.
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Superoxide dismutase 3 (SOD3), also known as extracellular superoxide dismutase, is an enzyme that scavenges reactive oxygen species (ROS). It has been reported that SOD3 exerts anti-inflammatory ...abilities in several immune disorders. However, the effect of SOD3 and the underlying mechanism in inflammatory bowel disease (IBD) have not been uncovered. Therefore, in the present study, we investigated whether SOD3 can protect intestinal cells or organoids from inflammation-mediated epithelial damage. Cells or mice were treated with SOD3 protein or SOD3-transduced mesenchymal stem cells (MSCs). Caco-2 cells or intestinal organoids stimulated with pro-inflammatory cytokines were used to evaluate the protective effect of SOD3 on epithelial junctional integrity. Dextran sulfate sodium (DSS)-induced colitis mice received SOD3 or SOD3-transduced MSCs (SOD3-MSCs), and were assessed for severity of disease and junctional protein expression. The activation of the mitogen-activated protein kinase (MAPK) pathway and elevated expression of cytokine-encoding genes decreased in TNF-α-treated Caco-2 cells or DSS-induced colitis mice when treated with SOD3 or SOD3-MSCs. Moreover, the SOD3 supply preserved the expression of tight junction (ZO-1, occludin) or adherence junction (E-cadherin) proteins when inflammation was induced. SOD3 also exerted a protective effect against cytokine- or ROS-mediated damage to intestinal organoids. These results indicate that SOD3 can effectively alleviate enteritis symptoms by maintaining the integrity of epithelial junctions and regulating inflammatory- and oxidative stress.
Ulcerative colitis is an inflammatory bowel disease (IBD) with relapsing and remitting patterns, and it is caused by varied factors, such as the intestinal inflammation extent and duration. We ...examined the preventative effects of human milk oligosaccharides (HMOs) on epithelial barrier integrity and intestinal inflammation in an interleukin (IL)-6-induced cell model and dextran sodium sulfate (DSS)-induced acute mouse colitis model. HMOs including 2'-fucosyllactose (FL) and 3-FL and positive controls including fructooligosaccharide (FOS) and 5-acetylsalicylic acid (5-ASA) were orally administrated once per day to C57BL/6J mice with colitis induced by 5% DSS in the administered drinking water. 2'-FL and 3-FL did not affect the cell viability in Caco-2 cells. Meanwhile, these agents reversed IL-6-reduced intestinal barrier function in Caco-2 cells. Furthermore, 2'-FL and 3-FL reversed the body weight loss and the remarkably short colon lengths in DSS-induced acute colitis mice. Moreover, 2'-FL and 3-FL obviously protected the decreasing expression of zonula occluden-1 and occludin in colon tissue relative to the findings in the DSS-treated control group. 2'-FL and 3-FL significantly reduced IL-6 and tumor necrosis factor-α levels in serum relative to the control findings. The summary of these results shows that HMOs prevent colitis mainly by enhancing intestinal barrier function and advancing anti-inflammatory responses. Therefore, HMOs might suppress inflammatory responses and represent candidate treatments for IBD that protect intestinal integrity.
Repair of defective hard-tissues in osteoporotic patients faces significantly challenges with limited therapeutic options. Although biomedical cements are considered promising materials for healthy ...bone repair, their uses for healing osteoporotic fracture are clinically limited. Herein, strontium-releasing-nanoscale cement was introduced to provide dual therapeutic-actions (pro-osteogenesis and anti-osteoclastogenesis), eventually for the regeneration of osteoporotic bone defect. The Sr-nanocement hardened from the Sr-doped nanoscale-glass particles was shown to release multiple ions including silicate, calcium and strontium at doses therapeutically relevant over time. When the Sr-nanocement was treated to pre-osteoblastic cells, the osteogenic mRNA level (Runx2, Opn, Bsp, Ocn), alkaline phosphatase activity, calcium deposition, and target luciferase reporter were stimulated with respect to the case with Sr-free-nanocement. When treated to pre-osteoclastic cells, the Sr-nanocement substantially reduced the osteoclastogenesis, such as osteoclastic mRNA level (Casr, Nfatc1, c-fos, Acp, Ctsk, Mmp-9), tartrate-resistant acid trap activity, and bone resorption capacity. In particular, the osteoclastic inhibition resulted in part from the interactive effect of osteoblasts which were activated by the Sr-nanocement, i.e., blockage of RANKL (receptor activator of nuclear factor-κB ligand) binding by enhanced osteoprotegerin and the deactivated Nfatc1. The Sr-nanocement, administered to an ovariectomized tibia defect (osteoporotic model) in rats, exhibited profound bone regenerative potential in cortical and surrounding trabecular area, including increased bone volume and density, enhanced production of osteopromotive proteins, and more populated osteoblasts, together with reduced signs of osteoclastic bone resorption. These results demonstrate that Sr-nanocement, with its dual effects of osteoclastic inhibition and osteogenic-stimulation, can be considered an effective nanotherapeutic implantable biomaterial platform for the treatment of osteoporotic bone defects.
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Leuconostoc citreum is widely utilized across numerous industries due to its beneficial properties. Thus, a detection method for this target species is essential to track the presence of L. citreum ...in commercial products. We developed a rapid, on-site L. citreum detection method using the loop-mediated isothermal amplification (LAMP) technique in this study. The designed LAMP primer set exhibited high specificity, only targeting the intended species among the 76 tested. Moreover, this assay demonstrated a sensitivity of 2 pg of target DNA. To evaluate the LAMP assay's applicability, we selected 20 varied types of processed products. The results confirmed these products' successful and precise detection of L. citreum within 35 min. Additionally, we used samples spiked with L. citreum to determine the detection limit, revealing L. citreum detection up to 102 or 103 CFU/mL. Thus, the LAMP assay developed herein is a specific, sensitive, precise, and rapid on-site detection method for L. citreum in various processed products.
•We developed a rapid on-site detection method for Leuconostoc citreum.•Our novel assay exhibits high sensitivity, detecting as little as 2 pg of target DNA.•We detected L. citreum (up to 102 or 103 CFU/mL) in 20 products within 35 min.
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