Korea Research Institute of Bioscience and Biotechnology
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LIMK1/2 is required for actin filament and cell junction assembly in porcine embryos developing in vitro
No full textObjective
This study was conducted to investigate the roles of LIM kinases (LIMK1 and LIMK2) during porcine early embryo development. We checked the mRNA expression patterns and localization of LIMK1/2 to evaluate their characterization. We further explored the function of LIMK1/2 in developmental competence and their relationship between actin assembly and cell junction integrity, specifically during the first cleavage and compaction.
Methods
Pig ovaries were transferred from a local slaughterhouse within 1 h and cumulus oocyte complexes (COCs) were collected. COCs were matured in in vitro maturation medium in a CO2 incubator. Metaphase II oocytes were activated using an Electro Cell Manipulator 2001 and microinjected to insert LIMK1/2 dsRNA into the cytoplasm. To confirm the roles of LIMK1/2 during compaction and subsequent blastocyst formation, we employed a LIMK inhibitor (LIMKi3).
Results
LIMK1/2 was localized in cytoplasm in embryos and co-localized with actin in cell-to-cell boundaries after the morula stage. LIMK1/2 knockdown using LIMK1/2 dsRNA significantly decreased the cleavage rate, compared to the control group. Protein levels of E-cadherin and β-catenin, present in adherens junctions, were reduced at the cell-to-cell boundaries in the LIMK1/2 knockdown embryos. Embryos treated with LIMKi3 at the morula stage failed to undergo compaction and could not develop into blastocysts. Actin intensity at the cortical region was considerably reduced in LIMKi3-treated embryos. LIMKi3-induced decrease in cortical actin levels was attributed to the disruption of adherens junction and tight junction assembly. Phosphorylation of cofilin was also reduced in LIMKi3-treated embryos.
Conclusion
The above results suggest that LIMK1/2 is crucial for cleavage and compaction through regulation of actin organization and cell junction assembly.
Exome sequencing reveals novel candidate gene variants associated with clinical characteristics in alopecia areata patients
No full textUnbiased investigation of specificities of prime editing systems in human cells
No full textPrime editors (PEs) enable targeted precise editing, including the generation of substitutions, insertions and deletions, in eukaryotic genomes. However, their genome-wide specificity has not been explored. Here, we developed Nickase-based Digenome-seq (nDigenome-seq), an in vitro assay that uses whole-genome sequencing to identify single-strand breaks induced by CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) nickase. We used nDigenome-seq to screen for potential genome-wide off-target sites of Cas9 H840A nickase, a PE component, targeted to nine human genomic sites. Then, using targeted amplicon sequencing of off-target candidates identified by nDigenome-seq, we showed that only five off-target sites showed detectable PE-induced modifications in cells, at frequencies ranging from 0.1 to 1.9%, suggesting that PEs provide a highly specific method of precise genome editing. We also found that PE specificity in human cells could be further improved by incorporating mutations from engineered Cas9 variants, particularly eSpCas9 and Sniper Cas9, into PE.
Tracking Alexandrium catenella from seed-bed to bloom on the southern coast of Korea
No full textAlexandrium catenella was tracked from seed-bed to bloom at a hot spot of cyst deposition on the southern coast of Korea from June 2016 to Feb. 2020. Changes in cyst abundance and germinability from sediment, as well as the vegetative cell abundance and encystment in the water column were intensively monitored. Cyst germination of ca. 73% occurred synchronously in November of 2016 to 2019, when bottom water temperature was around 15 °C. After mass germination, vegetative cells formed a seed populations at low density (<10 cells L？1) during winter. Overwintering populations initiated growth in March and then proliferated into high density (ca. 4 × 104 cells L？1) spring blooms in mid-April 2017 when moderate temperature (15 °C) was recorded. There was no bloom in spring of 2018 and 2019, but small vegetative populations developed. Decline of the spring bloom was followed by massive encystment and an increase in Noctiluca abundance. An average spring encystment ratio of 0.002 was estimated for the study years. Newly formed cysts lay dormant during the warm season lasting about six months and then seeded the next population of vegetative cells. An average contribution ratio of cells recruited from the sediment was ca. 0.09 for seeding winter populations. The range in shift ratios for spring production of a daughter cyst population to prior cyst abundance of the mother population in fall was 0.1 to 0.6 for consecutive years, depending on annual variation of local environments. Tracking mass transformation of A. catenella cysts will contribute to more effective science based management of paralytic shellfish poisoning on the southern Korean coast.
FABP3-mediated membrane lipid saturation alters fluidity and induces ER stress in skeletal muscle with aging
No full textSarcopenia is characterized by decreased skeletal muscle mass and function with age. Aged muscles have altered lipid compositions; however, the role and regulation of lipids are unknown. Here we report that FABP3 is upregulated in aged skeletal muscles, disrupting homeostasis via lipid remodeling. Lipidomic analyses reveal that FABP3 overexpression in young muscles alters the membrane lipid composition to that of aged muscle by decreasing polyunsaturated phospholipid acyl chains, while increasing sphingomyelin and lysophosphatidylcholine. FABP3-dependent membrane lipid remodeling causes ER stress via the PERK-eIF2α pathway and inhibits protein synthesis, limiting muscle recovery after immobilization. FABP3 knockdown induces a young-like lipid composition in aged muscles, reduces ER stress, and improves protein synthesis and muscle recovery. Further, FABP3 reduces membrane fluidity and knockdown increases fluidity in vitro, potentially causing ER stress. Therefore, FABP3 drives membrane lipid composition-mediated ER stress to regulate muscle homeostasis during aging and is a valuable target for sarcopenia.
Ceria-Zirconia antioxidant nanoparticles attenuate hypoxia-induced acute kidney injury by restoring autophagy flux and alleviating mitochondrial damage
No full textOxidative stress is one of the principal causes of hypoxia-induced kidney injury. The ceria nanoparticle (CNP) is known to exhibit free radical scavenger and catalytic activities. When zirconia is attached to CNPs (CZNPs), the ceria atom tends to remain in a Ce3+ form and its efficacy as a free radical scavenger thus increases. We determined the effectiveness of CNP and CZNP antioxidant activities against hypoxia-induced acute kidney injury (AKI) and observed that these nanoparticles suppress the apoptosis of hypoxic HK-2 cells by restoring autophagy flux and alleviating mitochondrial damage. In vivo experiments revealed that CZNPs effectively attenuate hypoxia-induced AKI by preserving renal structures and glomerulus function. These nanoparticles can successfully diffuse into HK-2 cells and effectively counteract reactive oxygen species (ROS) to block hypoxia-induced AKI. This suggests that these particles represent a novel approach to controlling this condition.
Dynamic transcriptome analysis unveils key proresolving factors of chronic inflammatory arthritis
No full textDespite recent advances in understanding chronic inflammation remission, global analyses have not been explored to systematically discover genes or pathways underlying the resolution dynamics of chronic inflammatory diseases. Here, we performed time-course gene expression profiling of mouse synovial tissues along progression and resolution of collagen-induced arthritis (CIA) and identified genes associated with inflammation resolution. Through network analysis of these genes, we predicted 3 key secretory factors responsible for the resolution of CIA: Itgb1, Rps3, and Ywhaz. These factors were predominantly expressed by Tregs and antiinflammatory M2 macrophages, suppressing production of proinflammatory cytokines. In particular, Ywhaz was elevated in the sera of mice with arthritis resolution and in the urine of rheumatoid arthritis (RA) patients with good therapeutic responses. Moreover, adenovirus-mediated transfer of the Ywhaz gene to the affected joints substantially inhibited arthritis progression in mice with CIA and suppressed expression of proinflammatory cytokines in joint tissues, lymph nodes, and spleens, suggesting Ywhaz is an excellent target for RA therapy. Therefore, our comprehensive analysis of dynamic synovial transcriptomes provides previously unidentified antiarthritic genes, Itgb1, Rps3, and Ywhaz, which can serve as molecular markers to predict disease remission, as well as therapeutic targets for chronic inflammatory arthritis.
Peroxiredoxin 2 deficiency reduces white adipogenesis due to the excessive ROS generation
No full textReactive oxygen species (ROS) act as signaling molecules to regulate various cell functions. Numerous studies have demonstrated ROS to be essential for the differentiation of adipocytes. Peroxiredoxins (Prxs) are a ubiquitous family of antioxidant enzymes in mammalian cells. Prx2 is present in the cytoplasm and cell membranes and demonstrates ROS scavenging activity. We focused on Prx2 involvement in regulating adipogenesis and lipid accumulation and demonstrated that Prx2 expression was upregulated during adipocyte differentiation. In addition, the silencing of Prx2 (shPrx2) inhibited adipogenesis by modulating adipogenic gene expression, and cell death was enhanced via increased ROS production in shPrx2？3T3？L1 cells. These results demonstrate that shPrx2 triggers adipocyte cell death and weakens adipocyte function via ROS production. Taken together, our data suggest the participation of Prx2 in adipocyte function and differentiation. Our results also imply that the downregulation of Prx2 activity could help prevent obesity. Overall, findings support the development of ROS？based therapeutic solutions for the treatment of obesity and obesity？related metabolic disorders.
Long-term surveillance of bat coronaviruses in Korea: diversity and distribution pattern
No full textBats harbour diverse coronaviruses (CoVs), some of which are associated with zoonotic infections, as well as inter-species transmission. In this study, a total of 512 bat faecal samples from the bat habitats at different geographical locations in South Korea were investigated between 2016 and 2019. Seventy-eight samples were positive for coronaviruses (15.2%), comprising 68 alphacoronaviruses (13.3%) and 10 betacoronaviruses (2.0%). The positive rates tended to increase during the awakening (April) period. Notably, betacoronaviruses were only found in the site where Rhinolophus ferrumequinum was the major species of bats, and were related to SARS- and MERS-related CoVs identified in China and South Korea, respectively. No betacoronaviruses were closely related to SARS-CoV-2 in this study. Alphacoronaviruses were detected in the sites where Hypsugo alaschanicus, Miniopterus fuliginosus, Miniopterus schreibersii, Rhinolophus ferrumequinum, Myotis bombinus, Myotis macrodactylus and Myotis petax were found to be the major bat species. Furthermore, alphacoronaviruses had higher genetic diversity than betacoronaviruses and had a wider distribution in Korea. Considering that different bat species are co-roosting in crowded conditions in the same habitat, the diverse coronaviruses in Korean bats are likely to undergo cross-species transmission events due to the richness in host species. Therefore, continuous monitoring should be performed, especially at the awakening time of the hibernating bats in the habitats where diverse bat species co-roost, to better understand the evolution of coronaviruses in bats.
Sestrin2 attenuates cellular senescence by inhibiting NADPH oxidase 4 expression
No full textBackground: Sestrin2 (Sesn2) is involved in the maintenance of metabolic homeostasis and aging via modulation of the 5' AMP-activated protein kinase-mammalian target of rapamycin (AMPK-mTOR) pathway.
Methods: Wild-type and Sesn2 knockout (KO) mice of the 129/SvJ background were maintained in a pathogen-free authorized facility under a 12-hour dark/light cycle at 20°C？22°C and 50%？60% humidity. Mouse embryonic fibroblasts (MEFs) were prepared from 13.5-day-old embryos derived from Sesn2-KO mice mated with each other.
Results: The MEFs from Sesn2-KO mice showed enlarged and flattened morphologies and senescence-associated β-galactosidase activity, accompanied by an elevated level of reactive oxygen species. These senescence phenotypes recovered following treatment with N-acetyl-cysteine. Notably, the mRNA levels of NADPH oxidase 4 (NOX4) and transforming growth factor (TGF)-β were markedly increased in Sesn2-KO MEFs. Treatment of Sesn2-KO MEFs with the NOX inhibitor diphenyleneiodonium and the TGF-β inhibitor SB431542 restored cell growth inhibited by Sesn2-KO.
Conclusion: Sesn2 attenuates cellular senescence via suppression of TGF-β- and NOX4-induced reactive oxygen species generation and subsequent inhibition of AMPK.