Korea Research Institute of Bioscience and Biotechnology
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CRISPR-mediated gene correction links the ATP7A M1311V mutations with amyotrophic lateral sclerosis pathogenesis in one individual
No full textAmyotrophic lateral sclerosis (ALS) is a severe disease causing motor neuron death, but a complete cure has not been developed and related genes have not been defined in more than 80% of cases. Here we compared whole genome sequencing results from a male ALS patient and his healthy parents to identify relevant variants, and chose one variant in the X-linked ATP7A gene, M1311V, as a strong disease-linked candidate after profound examination. Although this variant is not rare in the Ashkenazi Jewish population according to results in the genome aggregation database (gnomAD), CRISPR-mediated gene correction of this mutation in patient-derived and re-differentiated motor neurons drastically rescued neuronal activities and functions. These results suggest that the ATP7A M1311V mutation has a potential responsibility for ALS in this patient and might be a potential therapeutic target, revealed here by a personalized medicine strategy.
Wnt3a stimulation promotes primary ciliogenesis through beta-catenin phosphorylation-induced reorganization of centriolar satellites
No full textPrimary cilium is an antenna-like microtubule-based cellular sensing structure. Abnormal regulation of the dynamic assembly and disassembly cycle of primary cilia is closely related to ciliopathy and cancer. The Wnt signaling pathway plays a major role in embryonic development and tissue homeostasis, and defects in Wnt signaling are associated with a variety of human diseases, including cancer. In this study, we provide direct evidence of Wnt3a-induced primary ciliogenesis, which includes a continuous pathway showing that the stimulation of Wnt3a, a canonical Wnt ligand, promotes the generation of β-catenin p-S47 epitope by CK1δ, and these events lead to the reorganization of centriolar satellites resulting in primary ciliogenesis. We have also confirmed the application of our findings in MCF-7/ADR cells, a multidrug-resistant tumor cell model. Thus, our data provide a Wnt3a-induced primary ciliogenesis pathway and may provide a clue on how to overcome multidrug resistance in cancer treatment.
PyMPV17, the MPV17 homolog of Pyropia yezoensis (Rhodophyta), enhances osmotic stress tolerance in Chlamydomonas
No full textPyropia yezoensis is the most cultivated marine red algae in East Asia and is desiccation tolerant. PyMPV17, a desiccation stress response gene isolated from P. yezoensis, shares significant sequence similarity with mouse MPV17. A yeast knock-out mutant for SYM1, an MPV17 ortholog, could not grow on ethanol-containing medium at 38°C. Expression of PyMPV17 in yeast sym1 cells complemented the ethanol growth defect at 38°C, which suggests that PyMPV17 is a functional ortholog of SYM1. PyMPV17-green fluorescent protein (GFP) fluorescence was detected in mitochondria and PyMPV17 transcription was upregulated under desiccation stress in P. yezoensis gametophytes. Transcription of the PyMPV17 gene also increased in response to H2O2 and abscisic acid treatments. When PyMPV17 was introduced into the green alga Chlamydomonas, transgenic cells grew better than control cells on agar plates containing mannitol. Total malondialdehyde content in the transgenic cells was lower than that in the control cells under stress conditions. These results suggest that PyMPV17 reduces oxidative damage and contributes to the mechanism underlying tolerance to osmotic stress in red algae.
Using bioengineered fluorescence for selective in vivo and ex vivo tracking of intestinal organoids derived from human pluripotent stem cells
No full textFor current and future applications of human intestinal organoids (hIOs) to various aspects of in vivo research and their potential clinical use, an efficient noninvasive system is needed to directly visualize the stage of intestinal differentiation and graft-host interactions and for further safety monitoring and efficacy. Here, we describe a detailed method for monitoring and histologically identifying implanted hIO-expressing eGFP and mCherry fluorescence under the kidney capsule of immunodeficient mice with fluorescence imaging (FLI). We then describe the orthotropic transplantation method of hIOs and methods to confirm successful engraftment in the small intestines of immunodeficient mice. These methods provide an approach for tracking the location of intestinal cells in hIOs in vivo and ex vivo using a fluorescent reporter system from the beginning of engraftment to various subsequent experiments.
Evidence for binary Smc complexes lacking kite subunits in archaea
No full textSMC complexes play a central role in chromosome organization in all domains of life. The bacterial Smc-ScpAB complex is a three-subunit complex composed of Smc, ScpA and ScpB. ScpA bridges the two ATPase domains of the Smc homodimer, while ScpB, which belongs to the kite family of proteins, interacts with ScpA. The three subunits are known to be equally important for the function of Smc-ScpAB in bacteria. From crystallographic and biochemical studies, evidence is provided that six archaeal ScpA proteins are unable to interact with the only putative ScpB found in these species. Structure-based sequence alignment reveals that these archaeal ScpAs lack the ScpB-binding segment that is commonly present in the middle of bacterial ScpA sequences, which is thus responsible for their inability to interact with ScpB. ScpA proteins lacking the ScpB-binding segment are found to prevail in archaea. Moreover, two archaeal ScpA proteins with a longer middle region also failed to bind their putative ScpB partner. Furthermore, all or most species belonging to five out of 14 euryarchaeotal orders contain Smc and ScpA but not a detectable ScpB homologue. These data support the notion that archaeal Smc-based complexes generally function as a two-subunit complex composed of only Smc and ScpA.
IL-10 protects mice from the lung infection of Acinetobacter baumannii and contributes to bacterial clearance by regulating STAT3-mediated MARCO expression in macrophages
No full textInterleukin-10 plays important, yet contrasting, roles in host protection against bacterial infections and in the septic response. To determine the role of IL-10 in the host defense against Acinetobacter baumannii infection, wild-type (WT) and IL-10-deficient mice were infected intranasally with the bacteria. IL-10-deficient mice exhibited increased mortality, severe pathology, and excess production of proinflammatory cytokines and chemokines in the lungs, and increased bacterial burdens in bronchoalveolar lavage (BAL) fluids and lung homogenates after A. baumannii infection, compared to WT mice. Intranasal administration of recombinant IL-10 rescued mice from the lethality of the bacterial infection by promoting bacterial clearance and reducing production of cytokines and chemokines in the lungs. In vitro experiments revealed that IL-10 enhanced phagocytosis and bacterial killing by macrophages by upregulating the macrophage receptor with collagenous structure (MARCO). In addition, A. baumannii-induced activation of STAT3 was impaired in IL-10-deficient macrophages, which was essential for expression of MARCO. Intranasal adoptive transfer of WT macrophages resulted in significant increases in mice survival and bacterial clearance in IL-10-deficient mice infected with A. baumannii. Our results show that IL-10 played an important role in the host defense against pulmonary infection of A. baumannii by promoting the antibacterial function of macrophages by regulating MARCO expression through the STAT3-mediated pathway.
Inhibitory functions of maslinic acid, a natural triterpene, on HMGB1-mediated septic responses
No full textBACKGROUND:
Maslinic acid (MA), a natural triterpenoid from Olea europaea, prevents oxidative stress and pro-inflammatory cytokine generation. High mobility group box 1 (HMGB1) has been recognized as a late mediator of sepsis, and the inhibition of the release of HMGB1 and the recovery of vascular barrier integrity have emerged as attractive therapeutic strategies for the management of sepsis.
METHODS:
We tested the hypothesis that MA induces sirtuin 1 and heme oxygenase-1, which inhibit the release of HMGB1 in lipopolysaccharide (LPS)-stimulated cells, thus inhibiting HMGB1-induced hyperpermeability and increasing the survival of septic mice. MA was administered after LPS or HMGB1 challenge, and the antiseptic activity of MA was determined based on permeability, the activation of pro-inflammatory proteins, and the production of markers for tissue injury in HMGB1-activated human umbilical vein endothelial cells (HUVECs) and a cecal ligation and puncture (CLP)-induced sepsis mouse model.
RESULTS:
MA significantly reduced the release of HMGB1 in LPS-activated HUVECs and attenuated the CLP-induced release of HMGB1. Additionally, MA alleviated HMGB1-mediated vascular disruption and inhibited hyperpermeability in mice, and in vivo analysis revealed that MA reduced sepsis-related mortality and tissue injury.
CONCLUSION:
Taken together, the present results suggest that MA reduced HMGB1 release and septic mortality and thus may be useful in the treatment of sepsis.
Antibacterial AZT derivative regulates metastasis of breast cancer cells
No full textAntimicrobial peptides (AMP) with anticancer activity have drawn remarkable attention in modern treatments. However, long peptide length and protease instability are the most addressing factors, which hampers their further development as therapeutic agents. In view of this, herein, we designed and synthesized a series of AZT-based cationic small molecule incorporating a variety of hydrophobic groups and cationic charges, including amine and guanidine groups to mimic the amphipathic structure of AMPs. These compounds were evaluated for their antibacterial activity against Gram-positive and Gram-negative bacteria. Through an extensive structure activity relationship study (SAR), we identified ADG-2e as the most potent antibacterial agent, which exhibited remarkable potency against drug resistant bacterial strains such as MRSA and MDRPA. Further, ADG-2e was examined for their anti-metastatic ability by investigating the cancer cell migration and invasiveness through scratch wound-healing assay and transwell invasive assay, respectively. In addition, time-lapse cell tracking analysis also performed for analyzing the cell movement pattern. Treatment of ADG-2e against metastatic breast cancer cells (MDA-MB-231) suppressed tumor cell migration by multi-directional lamellipodium formation, indicating their anti-metastatic potential. Thus, our cationic AZT based small molecules may evolve as an appealing class of antibacterial agents with anti-metastasis potential.
Inhibition of skin inflammation by scytonemin, an ultraviolet sunscreen pigment
No full textScytonemin is a yellow-green ultraviolet sunscreen pigment present in different genera of aquatic and terrestrial blue-green algae, including marine cyanobacteria. In the present study, the anti-inflammatory activities of scytonemin were evaluated in vitro and in vivo. Topical application of scytonemin inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear swelling in BALB/c mice. The expression of tumor necrosis factor-a (TNF-a) and inducible nitric oxide synthase (iNOS) was also suppressed by scytonemin treatment in the TPA-treated ear of BALB/c mice. In addition, scytonemin inhibited lipopolysaccharide (LPS)-induced production of TNF-a and nitric oxide (NO) in RAW 264.7 cells, a murine macrophage-like cell line, and the mRNA expressions of TNF-a and iNOS were also suppressed by scytonemin in LPS-stimulated RAW 264.7 cells. Further study demonstrated that LPS-induced NF-kB activity was significantly suppressed by scytonemin treatment in RAW 264.7 cells. Our results also showed that the degradation of IkBa and nuclear translocation of the p65 subunit were blocked by scytonemin in LPS-stimulated RAW 264.7 cells. Collectively, these results suggest that scytonemin inhibits skin inflammation by blocking the expression of inflammatory mediators, and the anti-inflammatory effect of scytonemin is mediated, at least in part, by down-regulation of NF-kB activity. Our results also suggest that scytonemin might be used as a multi-function skin care ingredient for UV protection and anti-inflammation.
Evaluation of the wastewater generated during alkaline pretreatment of biomass for feasibility of recycling and reusing
No full textWe evaluated the wastewater generated in the alkali pretreatment process of Miscanthus sacchariflorus with sodium hydroxide for the feasibility of recycling and reusing. After thermal alkali pretreatment of the biomass, the separated alkaline solution and the biomass wash water were filtrated using several different types of membranes to recover sodium hydroxide, as well as to eliminate lignin-hemicellulose fractions from the wastewater. Ion concentration, total organic carbon, and acid-hydrolyzed carbohydrate contents were analyzed to evaluate the quality of the alkaline wastewater. The data indicated that a poly(ether)sulfone-based ultrafiltration (UF) membrane filter with a low molecular weight cut-off was able to recover > 98% sodium ions and to remove > 60% of solubilized organic carbon fractions from the alkaline wastewater. These results indicated that filtration using UF membrane could be an alternative strategy to reuse wastewater and to recover the alkali catalyst in the alkali pretreatment process.