Establishment of TUBB3-mCherry knock-in human pluripotent stem cell line using CRISPR/Cas9 (SNUe003-A-4).

TUBB3 is a structural neuronal protein important for multiple neuronal functions including axonal guidance and maturation. This study aimed to generate a human pluripotent stem cell (hPSC) line with a TUBB3-mCherry reporter using CRISPR/SpCas9 nuclease. The stop codon in the last exon of TUBB3 was replaced with a T2A-mCherry cassette using CRISPR/SpCas9-mediated homologous recombination. The established TUBB3-mCherry knock-in cell line exhibited typical pluripotent characteristics. The mCherry reporter faithfully replicated the endogenous level of TUBB3 upon induction of neuronal differentiation. The reporter cell line could contribute to the investigation of neuronal differentiation, neuronal toxicity, and neuronal tracing.

Geometric analysis algorithm based on a neural network with localized simulation data for nano-grating structure using Mueller matrix spectroscopic ellipsometry.

Mueller matrix spectroscopic ellipsometry (MMSE) is a nondestructive tool for nanostructure analysis, and recently the enhanced computational power, combining neural networks and simulation data, enhance its analysis ability on more complex geometries. This study introduces a deep learning method to realize fast and accurate analysis; predicting nanostructure parameters by pairing Mueller matrices with relatively limited library data and then applying neural network algorithm. Thus, it was realized to predict the width and height of 1D grating structure with an accuracy of MAE below 0.1 nm through the proposed two-step prediction algorithm. Finally, experimental validation on SiO2 grating of 38 nm width and 100 nm height showed a good agreement in the dimensions with reasonable range compared to those measured by scanning electron microscopy.

Generation of αMHC-EGFP knock-in in human pluripotent stem cell line, SNUe003-A-3 using CRISPR/Cas9-based gene targeting.

The cardiac muscle-specific protein, α-myosin heavy chain (αMHC), is a major component of cardiac muscle filaments involved in cardiac muscle contraction. Here, we established an αMHC-enhanced fluorescent protein (EGFP) knock-in human pluripotent stem cell (hPSC) line by linking the EGFP gene to the C-terminal region of αMHC via a 2A non-joining peptide using CRISPR/Cas9 nuclease. The EGFP reporter precisely reflected the endogenous level of αMHC upon the induction of cardiac differentiation. This reporter cell line will be a valuable platform for cardiotoxicity tests, drug screening, and investigating the pathological mechanisms of cardiomyocytes.

Population genetic analysis reveals secondary contact between Eriocheir sinensis and E. japonica in South Korea.

BACKGROUND: The Chinese mitten crab (Eriocheir sinensis) and the Japanese mitten crab (E. japonica) of the family Varunidae, which are also critical fishery species in their native habitats, including China, Japan, and South Korea, exhibit a rare migration life history for the decapod life cycle. Eriocheir sinensis and E. japonica in South Korea may have originated from speciation or secondary contact in South Korea after speciation of these two species; however, the genetic relationship between these South Korean populations remains unclear, and need to be clarified. OBJECTIVE: This study examined the population genetic properties of E. sinensis and E. japonica in South Korea to reveal the origin of their co-existence. METHODS: Mitochondrial DNA cytochrome c oxidase I sequences of 120 E. sinensis and E. japonica from 6 sampling sites in South Korea were sequenced. Their genetic diversity and haplotype data were compared to previously reported data from the Chinese mainland, Taiwan, Japan, and Russia. RESULTS: The haplotype network, analysis of molecular variance, FST values, Mantel test, and Bayesian skyline plot results of South Korean E. sinensis and E. japonica demonstrated that E. sinensis and E. japonica were isolated from their central populations, Chinese and Japanese populations, by the distance due to the temperature, salinity, ocean current flow, or a geohistorical event. In addition, genetic analysis indicated that the South Korean populations of the two species were marginal populations, exhibiting low genetic diversity, significant negative neutrality values, and radial haplotype networks. The current results also suggest that the Geum River is an overlapping habitat for the natural populations of E. sinensis and E. japonica in South Korea. Further research is required to examine the geohistorical and evolutionary event between E. sinensis and E. japonica in South Korea. CONCLUSIONS: The co-existence of E. sinensis and E. japonica in South Korea may have originated from secondary contact after their speciation.

Enhancement of image sharpness and height measurement using a low-speckle light source based on a patterned quantum dot film in dual-wavelength digital holography.

A dual-wavelength single light source based on a patterned quantum dot (QD) film was developed with a 405nm LED and bandpass filters to increase color conversion efficiency as well as to decouple the two peaks of dual-wavelength emitted from the QD film. A QD film was patterned laterally with two different sizes of QDs and was combined with bandpass filters to produce a high efficiency and low-speckle dual-wavelength light source. The experimental results showed that the developed dual-wavelength light source can decrease speckle noise to improve the reconstructed image sharpness and the accuracy on height measurement in dual-wavelength digital holography.

Generation of Brachyury-mCherry knock-in reporter human pluripotent stem cell line (SNUe003-A-2) using CRISPR/CAS9 nuclease.

Brachyury is an embryonic nuclear transcription factor required for mesoderm formation and differentiation. Here, we introduced an mCherry reporter into the C-terminus of Brachyury in the human pluripotent stem cell line SNUhES3 using the CRISPR/Cas9 nuclease approach. Successful gene editing was verified by DNA sequencing. SNUhES3-Brachyury-mCherry cells expressed pluripotent stem cell markers, exhibited a normal karyotype, and could generate all three germ layers. This cell line expressed the red fluorescence protein mCherry upon the induction of mesoderm differentiation. This reporter cell line could be used to monitor mesodermal population enrichment during mesodermal differentiation.

Integrative lipidomic and transcriptomic analysis of X-linked adrenoleukodystrophy reveals distinct lipidome signatures between adrenomyeloneuropathy and childhood cerebral adrenoleukodystrophy.

Precise pathophysiology with respect to the phenotypic variations and severity of X-ALD, specifically between adrenomyeloneuropathy (AMN) and childhood cerebral adrenoleukodystrophy (CCALD), has not been fully discovered. Herein, a systematic analysis using multi-layered lipidomics and transcriptomics was conducted to elucidate distinctive metabolic biosignatures among healthy control, AMN, and CCALD. Significant alterations regarding the accumulation of very long chain fatty acids were found in various lipid species such as phospholipids, glycerolipids, and sphingolipids. Remarkably, TG and CER that are physiologically essential were markedly down-regulated in CCALD than AMN. Transcriptomic analysis further supported the robustness of our findings by providing valuable information on the gene expressions of the regulatory factors. For instance, regulators of sphingolipid catabolism (SMPD1, CERK, and SPHK1) and TG anabolism (GPAM, GPAT2, and MBOAT2) were more up-regulated in AMN than in CCALD. These observations, among others, were in line with the recognized alterations of the associated lipidomes. In conclusion, the homeostatic imbalance of the complex lipid networks may be pathogenically important in X-ALD and the particular dysregulations of TG and CER may further influence the severity of CCALD among X-ALD patients.

Clean-Up Human Embryonic Stem Cell Lines Using Humanized Culture Condition.

Human embryonic stem cell (hESC) culture system has been changing culture conditions from conventional to xeno-free for therapeutic cell applications, and N-glycolylneuraminic acid (Neu5Gc) could be a useful indicator of xenogeneic contaminations in hESCs because human cells can no longer produce it genetically. We set up the humanized culture condition using commercially available humanized materials and two different adaptation methods: sequential or direct. SNUhES4 and H1 hESC lines, previously established in conventional culture conditions, were maintained using the humanized culture condition and were examined for the presence of Neu5Gc. The hESCs showed the same morphology and character as those of the conventional culture condition. Moreover, they were negative for Neu5Gc within two passages without loss of pluripotency. This study suggested that this method can effectively cleanse previously established hESC lines, bringing them one step closer to being clinical-grade hESCs.

Propagation of Human Embryonic Stem Cells on Human Amniotic Fluid Cells as Feeder Cells in Xeno-Free Culture Conditions.

Human embryonic stem cells (hESCs) have been routinely cultured on mouse embryonic fibroblast feederlayers with a medium containing animal materials. For clinical application of hESCs, animal-derived products from the animal feeder cells, animal substrates such as gelatin or Matrigel and animal serum are strictly to be eliminated in the culture system. In this study, we performed that SNUhES32 and H1 were cultured on human amniotic fluid cells (hAFCs) with KOSR XenoFree and a humanized substrate. All of hESCs were relatively well propagated on hAFCs feeders with xeno-free conditions and they expressed pluripotent stem cell markers, alkaline phosphatase, SSEA-4, TRA1-60, TRA1-81, Oct-4, and Nanog like hESCs cultured on STO or human foreskin fibroblast feeders. In addition, we observed the expression of nonhuman N-glycolylneuraminic acid (Neu5GC) molecules by flow cytometry, which was xenotransplantation components of contamination in hESCs cultured on animal feeder conditions, was not detected in this xeno-free condition. In conclusion, SNUhES32 and H1 could be maintained on hAFCs for humanized culture conditions, therefore, we suggested that new xenofree conditions for clinical grade hESCs culture will be useful data in future clinical studies.

Clinical insights for early detection of acute transverse myelitis in the emergency department.

OBJECTIVE: Acute transverse myelitis (ATM) is characterized by motor weakness, sensory changes, and autonomic dysfunction. However, diagnosis of ATM is based on early-stage clinical features only (and clarification of the cause of disease), which are difficult for emergency department (ED) physicians owing to low incidence rates. We performed retrospective analysis of ATM in order to provide clinical insights for early detection. METHODS: Medical records of patients, who were finally diagnosed with ATM from January 2005 to February 2013, were investigated. Data, including demographics, clinical findings, and radiographic findings, were reviewed. RESULTS: Forty-six patients were included in the present study, with a mean age of 43.4 years. Sensory changes were identified in 45 patients (97.8%), motor weakness in 33 patients (71.7%), and autonomic dysfunction in 35 patients (76.1%). Thirty patients (65.2%) showed high signal intensity in T2-weighted magnetic resonance imaging (MRI), with lesions most frequently found in the thoracic level of the spinal cord (56.7%). There were discrepancies between sensory changes and levels of MRI lesions. Thirty-five patients (76.1%) were diagnosed with idiopathic ATM. Initial diagnostic impressions in the ED were herniated intervertebral disc (38.7%), stroke (19.4%), Guillain-Barré syndrome (12.9%), cauda equina syndrome (9.7%), ATM (9.7%), and others (9.7%). CONCLUSION: When a patient presents with motor weakness, sensory changes, or autonomic dysfunction, ATM should be initially considered as a differential diagnosis, unless the ED physician's impression after initial evaluation is clear.