Korea Research Institute of Bioscience and Biotechnology
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Exploration of alternative splicing events in mesenchymal stem cells from human induced pluripotent stem cells
No full textAlthough comparative genome-wide transcriptomic analysis has provided insight into the biology of human induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs), the distinct alternative splicing (AS) signatures of iMSCs remain elusive. Here, we performed Illumina RNA sequencing analysis to characterize AS events in iMSCs compared with tissue-derived MSCs. A total of 4586 differentially expressed genes (|FC| > 2) were identified between iMSCs and umbilical cord blood-derived MSCs (UCB-MSCs), including 2169 upregulated and 2417 downregulated genes. Of these, 164 differentially spliced events (BF > 20) in 112 genes were identified between iMSCs and UCB-MSCs. The predominant type of AS found in iMSCs was skipped exons (43.3%), followed by retained introns (19.5%), alternative 3′ (15.2%) and 5′ (12.8%) splice sites, and mutually exclusive exons (9.1%). Functional enrichment analysis showed that the differentially spliced genes (|FC| > 2 and BF > 20) were mainly enriched in functions associated with focal adhesion, extracellular exosomes, extracellular matrix organization, cell adhesion, and actin binding. Splice isoforms of selected genes including TRPT1, CNN2, and AP1G2, identified in sashimi plots, were further validated by RT-PCR analysis. This study provides valuable insight into the biology of iMSCs and the translation of mechanistic understanding of iMSCs into therapeutic applications.
Characterization of terminal-ileal and colonic Crohn's disease in treatment-naive paediatric patients based on transcriptomic profile using logistic regression
No full textBackground: Inflammatory bowel disease (IBD) is a chronic and idiopathic inflammatory disorder of the gastrointestinal tract and comprises ulcerative colitis (UC) and Crohn's disease (CD). Crohn's disease can affect any part of the gastrointestinal tract, but mainly the terminal ileum and colon. In the present study, we aimed to characterize terminal-ileal CD (ICD) and colonic CD (CCD) at the molecular level, which might enable a more optimized approach for the clinical care and scientific research of CD.
Methods: We analyzed differentially expressed genes in samples from 23 treatment-naive paediatric patients with CD and 25 non-IBD controls, and compared the data with previously published RNA-Seq data using multi-statistical tests and confidence intervals. We implemented functional profiling and proposed statistical methods for feature selection using a logistic regression model to identify genes that are highly associated in ICD or CCD. We also validated our final candidate genes in independent paediatric and adult cohorts.
Results: We identified 550 genes specifically expressed in patients with CD compared with those in healthy controls (p < 0.05). Among these DEGs, 240 from patients with CCD were mainly involved in mitochondrial dysfunction, whereas 310 from patients with ICD were enriched in the ileum functions such as digestion, absorption, and metabolism. To choose the most effective gene set, we selected the most powerful genes (p-value ≤ 0.05, accuracy ≥ 0.8, and AUC ≥ 0.8) using logistic regression. Consequently, 33 genes were identified as useful for discriminating CD location; the accuracy and AUC were 0.86 and 0.83, respectively. We then validated the 33 genes with data from another independent paediatric cohort (accuracy = 0.93, AUC = 0.92) and adult cohort (accuracy = 0.88, AUC = 0.72).
Conclusions: In summary, we identified DEGs that are specifically expressed in CCD and ICD compared with those in healthy controls and patients with UC. Based on the feature selection analysis, 33 genes were identified as useful for discriminating CCD and ICD with high accuracy and AUC, for not only paediatric patients but also independent cohorts. We propose that our approach and the final gene set are useful for the molecular classification of patients with CD, and it could be beneficial in treatments based on disease location.
First report of the marine benthic dinoflagellate Bysmatrum subsalsum from Korean tidal pools
No full textDense patches were observed in the tidal pools of the southern area of Korea. To clarify the causative organisms, the cells were collected and their morphological features were examined using light and scanning electron microscopy (SEM). In addition, after establishing strains for the cells the molecular phylogeny was inferred with concatenated small subunit (SSU) and large subunit (LSU) rRNA sequences. The cells were characterized by a nucleus in the hypotheca, strong reticulations in thecal plates, the separation of plates 2a and 3a, the tear-shaped apical pore complex, an elongated rectangular 1a plate and the absence of the right sulcal list. The thecal plate formula was Po, X, 4′, 3a, 7″, 6c, 4S, 5′′′, 2′′′′. Based on these morphological features, the cells were identified as Bysmatrum subsalsum. In the culture, the spherical cysts of B. subsalsum without thecal plates were observed. Molecular phylogeny revealed two ribotypes of B. subsalsum are identified; The Korean isolates were nested within the ribotype B consisting of the isolates from China, Malaysia and the French Atlantic, whereas the ribotype A includes only the isolates from the Mediterranean Sea. In the phylogeny, B. subsalsum and B. austrafrum were grouped. This can be supported by the morphological similarity between the two species, indicating that the two species may be conspecific, however B. subsalsum may distinguish from B. austrafrum, because of differences in the types of eyespots reported in previous studies. These findings support the idea that there is cryptic diversity within B. subsalsum.
Bifidobacterium longum subsp. infantis YB0411 inhibits adipogenesis in 3T3-L1 pre-adipocytes and reduces high-fat-diet-iInduced obesity in mice
No full textAlthough the health benefits of probiotics have been widely known for decades, there has still been limited use of probiotic bacteria in anti-obesity therapy. Herein, we demonstrated the role of Bifidobacterium longum subsp. infantis YB0411 (YB, which was selected by an in vitro adipogenesis assay) in adipogenic differentiation in 3T3-L1 pre-adipocytes. We observed that YB-treatment effectively reduced triglyceride accumulation and the expression of CCAAT/enhancer-binding protein α, β, and δ (C/EBPα, C/EBPβ, and C/EBPδ), peroxisome proliferator-activated receptor γ (PPARγ), fatty acid-binding protein 4 (aP2), and acetyl-CoA carboxylase (ACC). YB-treatment also reduced the levels of core autophagic markers (p62 and LC3B) in 3T3-L1 pre-adipocytes. Small-interfering-RNA-mediated knockdown and competitive-chemical-inhibition assays showed that AMP-activated protein kinase (AMPK) commenced the anti-adipogenic effect of YB. In addition, YB supplement markedly reduced body weight and fat accretion in mice with high-fat-diet-induced obesity. Our findings suggest that YB may be used as a potential probiotic candidate to ameliorate obesity.
Complete genome sequence of platycodon closterovirus 1, a novel putative member of the genus Closterovirus
No full textA new member of the genus Closterovirus was detected in Platycodon grandiflorus using high-throughput RNA sequencing analysis. The complete genome sequence of this new virus isolate, tentatively named “platycodon closterovirus 1” (PlaCV1), comprises 16,771 nucleotides with nine predicted open reading frames (ORFs) having the typical closterovirus genome organization. PlaCV1 shares 37%？50% nucleotide sequence identity with other known closterovirus genome sequences. The putative RNA-dependent RNA polymerase (RdRp), heat shock protein 70-like protein (HSP70h), viral heat shock protein 90-like protein (HSP90h), minor coat protein (CPm), and coat protein (CP) show 47？68%, 39？66%, 24？52%, 21？57%, and 16？35% amino acid sequence identity, respectively, to homologous proteins in previously identified closteroviruses, suggesting that it represents a distinct, new species in the genus. Phylogenetic analysis of HSP70h sequences places PlaCV1 alongside other members of the genus Closterovirus in the family Closteroviridae. To our knowledge, this study is the first report of the complete genome sequence of PlaCV1 infecting P. grandiflorus in the Republic of Korea.
Aptamer-linked in vitro expression assay for ultrasensitive detection of biomarkers
No full textSignal amplification is a key step that determines the sensitivity of molecular assays. Although studies on aptamers have mostly focused on their target-binding ability, taking advantage of the gene-coding function of nucleic acids, we demonstrate here that aptamers can be engineered into diagnostic reagents that can both recognize a target and generate highly amplified detection signals. We developed a strategy that employs a ‘readable’ aptamer that consists of a single-stranded aptamer and a double-stranded reporter gene. After binding to its target via the aptamer region, the reporter gene of the readable aptamer produces amplified number of signal-generating enzymes through a subsequent in vitro expression reaction. In contrast to conventional enzyme-conjugation methods, this method allows the generation of far more amplified detection signals, thereby markedly increasing the sensitivity of detection enough to analyze a target present in aM concentrations.
Development of CRISPR technology for precise single-base genome editing: a brief review
No full textThe clustered regularly interspaced short palindromic repeats (CRISPR) system is a family of DNA sequences originally discovered as a type of acquired immunity in prokaryotes such as bacteria and archaea. In many CRISPR systems, the functional ribonucleoproteins (RNPs) are composed of CRISPR protein and guide RNAs. They selectively bind and cleave specific target DNAs or RNAs, based on sequences complementary to the guide RNA. The specific targeted cleavage of the nucleic acids by CRISPR has been broadly utilized in genome editing methods. In the process of genome editing of eukaryotic cells, CRISPRmediated DNA double-strand breaks (DSB) at specific genomic loci activate the endogenous DNA repair systems and induce mutations at the target sites with high efficiencies. Two of the major endogenous DNA repair machineries are non-homologous end joining (NHEJ) and homology-directed repair (HDR). In case of DSB, the two repair pathways operate in competition, resulting in several possible outcomes including deletions, insertions, and substitutions. Due to the inherent stochasticity of DSB-based genome editing methods, it was difficult to achieve defined single-base changes without unanticipated random mutation patterns. In order to overcome the heterogeneity in DSB-mediated genome editing, novel methods have been developed to incorporate precise single-base level changes without inducing DSB. The approaches utilized catalytically compromised CRISPR in conjunction with base-modifying enzymes and DNA polymerases, to accomplish highly efficient and precise genome editing of single and multiple bases. In this review, we introduce some of the advances in single-base level CRISPR genome editing methods and their applications.
Mitigation of hematopoietic syndrome of acute radiation syndrome by 1-Palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) is associated with regulation of systemic inflammation in a murine model of total-body irradiation
No full textThe growing risk of accidental radiation exposure due to increased usage of ionizing radiation, such as in nuclear power, industries and medicine, has increased the necessity for the development of radiation countermeasures. Previously, we demonstrated the therapeutic potential of the acetylated diacylglycerol, 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG), as a radiation countermeasure by mitigating radiation-associated mortality and hematopoietic acute radiation syndrome (H-ARS) in BALB/c mice after a lethal dose (LD70/30) of gamma-ray total-body irradiation (TBI). In this study, we show that PLAG mitigates symptoms of H-ARS, as characterized by mature blood cell recovery and restoration of bone marrow cellularity, by regulating systemic inflammation. Log-rank test demonstrated that high levels of WBCs, lymphocytes and neutrophils on day 10 post-TBI resulted in significantly improved survival rate. PLAG significantly enhanced the nadir values of all major blood cell types as well as bone marrow cellularity. A single TBI at LD70/30 induced an immediate increase in the blood levels of CXCL1 (12.5 fold), CXCL2 (1.5 fold), IL-6 (86.9 fold), C-reactive protein (CRP; 1.3 fold) and G-CSF (15.7 fold) at 6 h post-TBI, but the cytokine levels returned to baseline level afterward. When the irradiated mice started to die around 15 days post-TBI, they exhibited a second surge in blood levels of CXCL1 (49.3 fold), CXCL2 (87.1 fold), IL-6 (208 fold), CRP (3.6 fold) and G-CSF (265.7 fold). However, PLAG-treated groups showed a significant decrease in these same blood levels (P < 0.001). Considering the inverse correlation between inflammatory cytokine levels and hematological nadirs, PLAG exerts its therapeutic effects on H-ARS by regulating inflammatory cytokine production. These data suggest that PLAG has high potential as a radiation countermeasure to mitigate H-ARS after accidental exposure to radiation.
Acoustofluidic separation of proteins using aptamer-functionalized microparticles
No full textWe propose an acoustofluidic method for the triseparation of proteins conjugated with aptamer-coated microparticles inside a microchannel. Traveling surface acoustic waves (TSAWs) produced from a slanted-finger interdigital transducer (SFIT) are used to separate the protein-loaded microparticles of different sizes via the TSAW-driven acoustic radiation force (ARF). The acoustofluidic device consists of an SFIT deposited onto a piezoelectric lithium niobate substrate and a polydimethylsiloxane (PDMS) microfluidic channel on top of the substrate. The TSAWs propagating on the substrate penetrate into the sample fluid flow, where the human protein-conjugated microparticles are suspended, inside the PDMS microchannel. The microparticles are subjected to the TSAW-driven ARF with varying magnitude depending on their size and thus flow along different streamlines, leading to triseparation of the proteins. In this work, we used two different-sized streptavidin-functionalized polystyrene (PS) microparticles to capture two kinds of aptamers (apt15 and aptD17.4), which were labeled with a respective biotin molecule at one end. The biotin ends of the aptamers were attached to the microparticles through streptavidin-biotin linkage, whereas the free ends of the aptamers were used to capture their target proteins of thrombin (th) and immunoglobulin E (IgE). The resultant PS-apt15-th and PS-aptD17.4-IgE complexes, as well as mCardinal2, were used for experimental demonstration of acoustofluidic triseparation of the human proteins. We achieved simultaneous separation of proteins of three kinds (th, IgE, and mCardinal2) for the first time via the TSAW-driven ARF in the proposed acoustofluidic device.