Search Results (45)

Resveratrol, a bioactive phytoalexin, has been extensively studied as a pharmaceutical and nutraceutical candidate for the treatment of various diseases. Although its therapeutic effects have been largely attributed to its anti-oxidant properties, its underlying mechanisms and dose dependency are not well understood. Recent studies have shown that cell-free chromatin particles (cfChPs), which are released daily from billions of dying cells, can enter circulation and be internalized by healthy cells, wherein they trigger various damaging effects, including double-strand DNA breaks. Notably, deactivating cfChPs using a mixture of resveratrol and copper can neutralize their harmful effects. The addition of copper imparts a novel therapeutic property to resveratrol viz. the generation of reactive oxygen species (ROS), which are capable of deactivating cfChPs without damaging the genomic DNA. This perspective article discusses how the deactivation of cfChPs via the ROS generated by combining resveratrol with copper can have multiple therapeutic effects. Exploiting the damaging effects of ROS to deactivate cfChPs and ameliorate disease conditions may be a viable therapeutic approach. Full article

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, the world’s deadliest infectious disease. Mtb uses a variety of mechanisms to evade the human host’s defenses and survive intracellularly. Mtb’s oxidative stress response enables Mtb to survive within activated macrophages, an environment with reactive oxygen species and low pH. Dye-decolorizing peroxidase (DyP), an enzyme involved in Mtb’s oxidative stress response, is encapsulated in a nanocompartment, encapsulin (Enc), and is important for Mtb’s survival in macrophages. Encs are homologs of viral capsids and encapsulate cargo proteins of diverse function, including those involved in iron storage and stress responses. DyP contains a targeting peptide (TP) at its C-terminus that recognizes and binds to the interior of the Enc nanocompartment. Here, we present the crystal structure of the Mtb-Enc•DyP complex and compare it to cryogenic-electron microscopy (cryo-EM) Mtb-Enc structures. Investigation into the canonical pores formed at symmetrical interfaces reveals that the five-fold pore for the Mtb-Enc crystal structure is strikingly different from that observed in cryo-EM structures. We also observe DyP-TP electron density within the Mtb-Enc shell. Finally, investigation into crystallographic small-molecule binding sites gives insight into potential novel avenues by which substrates could enter Mtb-Enc to react with Mtb-DyP. Full article

Duchenne Muscular Dystrophy (DMD) is a genetic disorder characterized by disruptions in the dystrophin gene. This study aims to investigate potential a therapeutic approach using genetically modified human iPS-derived mesoangioblast-like cells (HIDEMs) in mdx mouse model. This study utilizes patient-specific myoblasts reprogrammed to human induced pluripotent stem cells (iPSCs) and then differentiated into HIDEMs. Lentiviral vectors carrying microdystrophin sequences have been employed to deliver the genetic construct to express a shortened, functional dystrophin protein in HIDEMs. The study indicated significant changes within redox potential between healthy and pathological HIDEM cells derived from DMD patients studied by catalase and superoxide dismutase activities. Microdystrophin expressing HIDEMs also improved expression of genes involved in STARS (striated muscle activator of Rho signaling) pathway albeit in selective DMD patients (with mild phenotype). Although in vivo observations did not bring progress in the mobility of mdx mice with HIDEMs, microdystrophin interventions this may argue against “treadmill test” as suitable for assessment of mdx mice recovery. Low-level signaling of the Rho pathway and inflammation-related factors in DMD myogenic cells can also contribute to the lack of success in a functional study. Overall, this research contributes to the understanding of DMD pathogenesis and provides insights into potential novel therapeutic strategy, highlighting the importance of personalized gene therapy. Full article

Coriolopsis gallica (Cga) is a white-rot fungus renowned for its ability to secrete ligninolytic enzymes that are capable of oxidizing phenolic compounds. This study aimed to investigate the biochemical characteristics of a dye-decolorizing peroxidase named CgaDyP1 and test its ability to biotransform antibiotics. CgaDyP1 was cloned and heterologously expressed in Escherichia coli. We fully characterized the biochemical properties of CgaDyP1 and evaluated its dye-decolorizing potential to confirm that it belongs to the DyP class of enzymes. We also tested its fluoroquinolone antibiotic biotransformation potential for possible biotechnological applications. Alignment of the primary amino acid sequence with DyP homolog sequences showed that CgaDyP1 has high similarity with other fungal DyPs. The recombinant CgaDyP1 exhibited activity on substrates such as ABTS and 2,6-dimethoxyphenol (DMP) with optimal performance at a pH of 3, although activity at pH 2.5, pH 4, and pH5 diminished over time. Thermostability tests indicated that the enzyme remains stable at temperatures between 30 °C and 50 °C and retains 70% of its initial activity after 180 min at 50 °C. Tests on the effect of hydrogen peroxide on CgaDyP1 activity found peak activity at 0.25 mM H₂O₂. CgaDyP1 decolorized five industrial dyes, and kinetics data confirmed that it belongs to the DyP class of enzymes. CgaDyP1 was shown to biotransform some of the 7 recalcitrant fluoroquinolone antibiotics tested here, including levofloxacin, moxifloxacin, and norfloxacin, and thus holds potential for biotechnological applications. Full article

Objectives: To identify the effect of alternating monocular instillation (AMI) of 0.125% atropine in Korean children with progressive myopia. Methods: This retrospective single-center study included 120 children with progressive myopia. A total of 60 children (mean age 9.2 ± 2.0 years) wearing glasses who received AMI of 0.125% atropine for one year were allocated to the treatment group. The remaining 60 children (mean age 9.2 ± 1.9 years) with the same refraction, SE, and axial length (AL) who did not receive any treatments except for wearing glasses were allocated to the control group. Ocular findings and the progression rate were compared between the groups pre- and post-treatment, and adverse events were investigated in the treatment group. Results: The mean spherical equivalent (SE) at baseline was −3.87 ± 1.55 D in the control group and −3.90 ± 1.56 D in the treatment group. Pre-treatment SE, age, and AL were similar between the groups; however, post-treatment SE and AL changes were smaller in the treatment group (−0.36 ± 0.46 D/y, 0.21 ± 0.20 mm/year in the treatment group vs. −1.02 ± 0.57 D/y, 0.51 ± 0.20 mm/year in the control group) (Ps < 0.001). The pre-treatment progression rate diminished in the treatment group compared to the control group after one year (p < 0.001), and the changes in pupil size under mesopic and photopic conditions in the treatment group increased by 0.03 ± 0.05 mm and 0.76 ± 0.90 mm, respectively. Regarding adverse events, a tingling sensation was noted in two patients (3.3%) in the treatment group. Conclusions: Alternating monocular 0.125% atropine eye drop instillation may be effective and suitable for progressive myopia in Korean children. Full article

The degeneration of axon terminals before the soma, referred to as “dying back”, is a feature of Parkinson’s disease (PD). Axonal assays are needed to model early PD pathogenesis as well as identify protective therapeutics. We hypothesized that defects in axon lysosomal trafficking as well as injury repair might be important contributing factors to “dying back” pathology in PD. Since primary human PD neurons are inaccessible, we developed assays to quantify axonal trafficking and injury repair using induced pluripotent stem cell (iPSC)-derived neurons with LRRK2 G2019S, which is one of the most common known PD mutations, and isogenic controls. We observed a subtle axonal trafficking phenotype that was partially rescued by a LRRK2 inhibitor. Mutant LRRK2 neurons showed increased phosphorylated Rab10-positive lysosomes, and lysosomal membrane damage increased LRRK2-dependent Rab10 phosphorylation. Neurons with mutant LRRK2 showed a transient increase in lysosomes at axotomy injury sites. This was a pilot study that used two patient-derived lines to develop its methodology; we observed subtle phenotypes that might correlate with heterogeneity in LRRK2-PD patients. Further analysis using additional iPSC lines is needed. Therefore, our axonal lysosomal assays can potentially be used to characterize early PD pathogenesis and test possible therapeutics. Full article

Lignocellulosic materials are composed of cellulose, hemicellulose and lignin and are one of the most abundant biopolymers in marine environments. The extent of the involvement of marine microorganisms in lignin degradation and their contribution to the oceanic carbon cycle remains elusive. In this study, a novel lignin-degrading bacterial strain, LCG003, was isolated from intertidal seawater in Lu Chao Harbor, East China Sea. Phylogenetically, strain LCG003 was affiliated with the genus Aliiglaciecola within the family Alteromonadaceae. Metabolically, strain LCG003 contains various extracellular (signal-fused) glycoside hydrolase genes and carbohydrate transporter genes and can grow with various carbohydrates as the sole carbon source, including glucose, fructose, sucrose, rhamnose, maltose, stachyose and cellulose. Moreover, strain LCG003 contains many genes of amino acid and oligopeptide transporters and extracellular peptidases and can grow with peptone as the sole carbon and nitrogen source, indicating a proteolytic lifestyle. Notably, strain LCG003 contains a gene of dyp-type peroxidase and strain-specific genes involved in the degradation of 4-hydroxy-benzoate and vanillate. We further confirmed that it can decolorize aniline blue and grow with lignin as the sole carbon source. Our results indicate that the Aliiglaciecola species can depolymerize and mineralize lignocellulosic materials and potentially play an important role in the marine carbon cycle. Full article

Dye-decolorizing peroxidases (DyPs) are heme proteins with distinct structural properties and substrate specificities compared to classical peroxidases. Here, we demonstrate that DyP from the extremely radiation-resistant bacterium Deinococcus radiodurans is, like some other homologues, inactive at physiological pH. Resonance Raman (RR) spectroscopy confirms that the heme is in a six-coordinated-low-spin (6cLS) state at pH 7.5 and is thus unable to bind hydrogen peroxide. At pH 4.0, the RR spectra of the enzyme reveal the co-existence of high-spin and low-spin heme states, which corroborates catalytic activity towards H₂O₂ detected at lower pH. A sequence alignment with other DyPs reveals that DrDyP possesses a Methionine residue in position five in the highly conserved GXXDG motif. To analyze whether the presence of the Methionine is responsible for the lack of activity at high pH, this residue is substituted with a Glycine. UV-vis and RR spectroscopies reveal that the resulting DrDyPM190G is also in a 6cLS spin state at pH 7.5, and thus the Methionine does not affect the activity of the protein. The crystal structures of DrDyP and DrDyPM190G, determined to 2.20 and 1.53 Å resolution, respectively, nevertheless reveal interesting insights. The high-resolution structure of DrDyPM190G, obtained at pH 8.5, shows that one hydroxyl group and one water molecule are within hydrogen bonding distance to the heme and the catalytic Asparagine and Arginine. This strong ligand most likely prevents the binding of the H₂O₂ substrate, reinforcing questions about physiological substrates of this and other DyPs, and about the possible events that can trigger the removal of the hydroxyl group conferring catalytic activity to DrDyP. Full article

Bioluminescence is a common phenomenon in nature, especially in the deep ocean. The physiological role of bacterial bioluminescence involves protection against oxidative and UV stresses. Yet, it remains unclear if bioluminescence contributes to deep-sea bacterial adaptation to high hydrostatic pressure (HHP). In this study, we constructed a non-luminescent mutant of ΔluxA and its complementary strain c-ΔluxA of Photobacterium phosphoreum ANT-2200, a deep-sea piezophilic bioluminescent bacterium. The wild-type strain, mutant and complementary strain were compared from aspects of pressure tolerance, intracellular reactive oxygen species (ROS) level and expression of ROS-scavenging enzymes. The results showed that, despite similar growth profiles, HHP induced the accumulation of intracellular ROS and up-regulated the expression of ROS-scavenging enzymes such as dyp, katE and katG, specifically in the non-luminescent mutant. Collectively, our results suggested that bioluminescence functions as the primary antioxidant system in strain ANT-2200, in addition to the well-known ROS-scavenging enzymes. Bioluminescence contributes to bacterial adaptation to the deep-sea environment by coping with oxidative stress generated from HHP. These results further expanded our understanding of the physiological significance of bioluminescence as well as a novel strategy for microbial adaptation to a deep-sea environment. Full article

Under physiological conditions, phosphatidylserine (PS) predominantly localizes to the cytosolic leaflet of the plasma membrane of cells. During apoptosis, PS is exposed on the cell surface and serves as an “eat-me” signal for macrophages to prevent releasing self-immunogenic cellular components from dying cells which could potentially lead to autoimmunity. However, increasing evidence indicates that viable cells can also expose PS on their surface. Interestingly, tumor cell-derived extracellular vesicles (EVs) externalize PS. Recent studies have proposed PS-exposing EVs as a potential biomarker for the early detection of cancer and other diseases. However, there are confounding results regarding subtypes of PS-positive EVs, and knowledge of PS exposure on the EV surface requires further elucidation. In this study, we enriched small EVs (sEVs) and medium/large EVs (m/lEVs) from conditioned media of breast cancer cells (MDA-MB-231, MDA-MB-468) and non-cancerous cells (keratinocytes, fibroblasts). Since several PS-binding molecules are available to date, we compared recombinant proteins of annexin A5 and the carboxylated glutamic acid domain of Protein S (GlaS), also specific for PS, to detect PS-exposing EVs. Firstly, PS externalization in each EV fraction was analyzed using a bead-based EV assay, which combines EV capture using microbeads and analysis of PS-exposing EVs by flow cytometry. The bulk EV assay showed higher PS externalization in m/lEVs derived from MDA-MB-468 cells but not from MDA-MB-231 cells, while higher binding of GlaS was also observed in m/lEVs from fibroblasts. Second, using single EV flow cytometry, PS externalization was also analyzed on individual sEVs and m/lEVs. Significantly higher PS externalization was detected in m/lEVs (annexin A1⁺) derived from cancer cells compared to m/lEVs (annexin A1⁺) from non-cancerous cells. These results emphasize the significance of PS-exposing m/lEVs (annexin A1⁺) as an undervalued EV subtype for early cancer detection and provide a better understanding of PS externalization in disease-associated EV subtypes. Full article

Lignin is Nature’s major source of aromatic chemistry and is by many seen as the green entry-point alternative to the fossil-based chemical industry. Due to its chemically recalcitrant structure, the utilization of lignin is challenging, wherein enzymes might be the key to overcome this challenge. Here, we focus on the characterization of dye-decolorizing peroxidases from Streptomyces coelicolor A3(2) (ScDyPs) in the context of enzymatic modification of organosolv lignins from aspen and Miscanthus × giganteus. In this study, we show that the ScDyPB can remodel organosolv lignins from grassy biomass, leading to higher molecular weight species, while ScDyPAs can deconstruct hardwood lignin, leading to an overall reduction in its molecular weight. Additionally, we show that ScDyPB is effective in polymerizing low-molecular-weight phenolics, leading to their removal from the solution. Full article

Barley bran has potential bioactivities due to its high content of polyphenols and dietary fiber, etc. Fermentation has been considered as an effective way to promote the functional activity of food raw materials. In this study, polysaccharides from barley bran extract fermented by Lactiplantibacillus plantarum dy-1 (FBBE-PS) were analyzed, and its effects on lipid accumulation and oxidative stress in high-fat HepG2 cells induced by sodium oleate were evaluated. The results showed that the molecular weight decreased and monosaccharide composition of polysaccharides changed significantly after fermentation. In addition, 50 μg/mL FBBE-PS could reduce the triglyceride (TG) content and reaction oxygen species (ROS) level in high-fat HepG2 cells by 21.62% and 30.01%, respectively, while increasing the activities of superoxide dismutase (SOD) and catalase (CAT) represented by 64.87% and 22.93%, respectively. RT-qPCR analysis revealed that FBBE-PS could up-regulate the lipid metabolism-related genes such as ppar-α, acox-1 and cpt-1α, and oxidation-related genes such as nrf2, ho-1, nqo-1, sod1, cat, etc. The metabolomics analysis indicated that FBBE-PS could alleviate lipid deposition by inhibiting the biosynthesis of unsaturated fatty acids, which is consistent with the downregulation of scd-1 expression. It is demonstrated that fermentation can alter the properties and physiological activities of polysaccharides in barley bran, and FBBE-PS exhibited an alleviating effect on lipid deposition and oxidative stress in high-fat cells. Full article

Mutations in presenilin 2 (PS2) have been causally linked to the development of inherited Alzheimer’s disease (AD). Besides its role as part of the γ-secretase complex, mammalian PS2 is also involved, as an individual protein, in a growing number of cell processes, which result altered in AD. To gain more insight into PS2 (dys)functions, we have generated a presenilin2 (psen2) knockout zebrafish line. We found that the absence of the protein does not markedly influence Notch signaling at early developmental stages, suggesting a Psen2 dispensable role in the γ-secretase-mediated Notch processing. Instead, loss of Psen2 induces an exaggerated locomotor response to stimulation in fish larvae, a reduced number of ER-mitochondria contacts in zebrafish neurons, and an increased basal autophagy. Moreover, the protein is involved in mitochondrial axonal transport, since its acute downregulation reduces in vivo organelle flux in zebrafish sensory neurons. Importantly, the expression of a human AD-linked mutant of the protein increases this vital process. Overall, our results confirm zebrafish as a good model organism for investigating PS2 functions in vivo, representing an alternative tool for the characterization of new AD-linked defective cell pathways and the testing of possible correcting drugs. Full article

Fungi are the main contaminants of books and archival documents. In addition to their degrading power, offered by various types of lignolytic and cellulolytic enzymes, they can also hue the surface of the paper through the production of pigments. The fungi on paper release various types of pigments belonging mostly to two chemical groups (polyketides and carotenoids), which cause unpleasant anaesthetic stains. The paper surface can also be hued with several synthetic colors, which are part, for example, of stamps and inks. These synthetic colors could be degraded by lignin-modifying enzymes (LMEs) and also by dye-decolorizing peroxidases (DyPs). Therefore, the mechanism of action of LEMs and DyPs is illustrated. Moreover, we have examined the potentiality of LEMs and DyPs to remove the synthetic stains and also their hypothetical application in order to clean the fungal hues from the paper surface. Our review article, using the enzymatic removal parallelism between fungal and synthetic pigments, would like to show prospective solutions to this arduous problem. Full article

iPSCs and their derivatives are the most promising cell sources for creating skin equivalents. However, their properties are not fully understood. In addition, new approaches and parameters are needed for studying cells in 3D models without destroying their organization. Thus, the aim of our work was to study and compare the metabolic status and pH of dermal spheroids created from dermal papilla cells differentiated from pluripotent stem cells (iDP) and native dermal papilla cells (hDP) using fluorescence microscopy and fluorescence lifetime imaging microscopy (FLIM). For this purpose, fluorescence intensities of NAD(P)H and FAD, fluorescence lifetimes, and the contributions of NAD(P)H, as well as the fluorescence intensities of SypHer-2 and BCECF were measured. iDP in spheroids were characterized by a more glycolytic phenotype and alkaline intra-cellular pH in comparison with hDP cells. Moreover, the metabolic activity of iDP in spheroids depends on the source of stem cells from which they were obtained. So, less differentiated and condensed spheroids from iDP-iPSDP and iDP-iPSKYOU are characterized by a more glycolytic phenotype compared to dense spheroids from iDP-DYP0730 and iDP-hES. FLIM and fluorescent microscopy in combination with the metabolism and pH are promising tools for minimally invasive and long-term analyses of 3D models based on stem cells. Full article