Simple modeling of familial Alzheimer's disease using human pluripotent stem cell-derived cerebral organoid technology.

BACKGROUND: Cerebral organoids (COs) are the most advanced in vitro models that resemble the human brain. The use of COs as a model for Alzheimer's disease (AD), as well as other brain diseases, has recently gained attention. This study aimed to develop a human AD CO model using normal human pluripotent stem cells (hPSCs) that recapitulates the pathological phenotypes of AD and to determine the usefulness of this model for drug screening. METHODS: We established AD hPSC lines from normal hPSCs by introducing genes that harbor familial AD mutations, and the COs were generated using these hPSC lines. The pathological features of AD, including extensive amyloid-ß (Aß) accumulation, tauopathy, and neurodegeneration, were analyzed using enzyme-linked immunosorbent assay, Amylo-Glo staining, thioflavin-S staining, immunohistochemistry, Bielschowsky's staining, and western blot analysis. RESULTS: The AD COs exhibited extensive Aß accumulation. The levels of paired helical filament tau and neurofibrillary tangle-like silver deposits were highly increased in the AD COs. The number of cells immunoreactive for cleaved caspase-3 was significantly increased in the AD COs. In addition, treatment of AD COs with BACE1 inhibitor IV, a ß-secretase inhibitor, and compound E, a γ-secretase inhibitor, significantly attenuated the AD pathological features. CONCLUSION: Our model effectively recapitulates AD pathology. Hence, it is a valuable platform for understanding the mechanisms underlying AD pathogenesis and can be used to test the efficacy of anti-AD drugs.

Human embryonic stem cell-specific role of YAP in maintenance of self-renewal and survival.

Human embryonic stem cells (hESCs) have unique characteristics, such as self-renewal and pluripotency, which are distinct from those of other cell types. These characteristics of hESCs are tightly regulated by complex signaling mechanisms. In this study, we demonstrate that yes-associated protein (YAP) functions in an hESC-specific manner to maintain self-renewal and survival in hESCs. hESCs were highly sensitive to YAP downregulation to promote cell survival. Interestingly, hESCs displayed dynamic changes in YAP expression in response to YAP downregulation. YAP was critical for the maintenance of self-renewal. Additionally, the function of YAP in maintenance of self-renewal and cell survival was hESC-specific. Doxycycline upregulated YAP in hESCs and attenuated the decreased cell survival induced by YAP downregulation. However, decreased expression of self-renewal markers triggered by YAP downregulation and neural/cardiac differentiation were affected by doxycycline treatment. Collectively, the results reveal the mechanism underlying the role of YAP and the novel function of doxycycline in hESCs.

Seasonal characteristics of atmospheric water-soluble organic nitrogen in PM2.5 in Seoul, Korea: Source and atmospheric processes of free amino acids and aliphatic amines.

The seasonal characteristics of atmospheric water-soluble organic nitrogen (WSON) in particulate matter with a diameter of 2.5 µm or smaller (PM2.5) were analyzed focusing on sources and atmospheric processing. Daily collected samples over 23 h (10:00-9:00) from 7 August 2018 to 31 December 2019 on quartz filters with a high-volume sampler at the Korea Institute of Science and Technology (KIST) in Seoul were considered. The most common species in the Seoul atmosphere included Glycine (5.45 ± 9.81 ng/m3) among free amino acids (FAAs) and trimethylamine (TMA) (5.35 ± 3.80 ng/m3) among aliphatic amines (AAs). The top 10 WSON species (93.6% of all WSON species) were categorized into three groups based on correlation analysis considering meteorological data, (e.g., temperature, rainfall, relative humidity (RH), wind speed) gaseous pollutants (e.g., SO2, CO, NO2) and mass concentration of PM10 and PM2.5. Those three groups are G1 (Glycine, Alanine, and Threonine), G2 (Gln Glutamine, Lys Lysine, and Glutamic acid) and G3 (Trimethylamine (TMA), dimethylamine (DMA), and methylamine (MA)), where G1, G2 and G3 accounted for 31.1%, 8.8% and 51.1%, respectively, of the total species. Among these three groups, G1 and G3 are from combustion sources, and G2 shows secondary features generated by photochemical reactions involving ozone. Although both G1 and G3 exhibited features influenced by combustion sources, the AA species (TMA, DMA, and MA) in G3 demonstrated typical features enhanced under high-humidity conditions, suggesting not only primary sources but also secondary formation at the local scale influence to the AA in G3 group. Based on long-term measurements more than a year, our findings suggest that complex and diverse sources of atmospheric WSON are in Seoul, Korea both from primary and secondary, which may affect its environmental, climate and health.

A simple method to improve the quality and yield of human pluripotent stem cell-derived cerebral organoids.

The development of cerebral organoid technology has allowed the human neural tissue to be collected for studying human brain development and neurological diseases. Human pluripotent stem cell-derived cerebral organoids (hCOs) are a theoretically infinite source of fresh human brain tissue for various research purposes. However, hCOs have limitations, including core necrotic cell death. To solve this problem, we tested a simple method, which has been previously overlooked. In this study, we mechanically cut 70-day-old hCOs with a scalpel blade into 2 to 4 pieces, each depending on their original size. After culturing cut hCOs for additional 7 days, their size was less variable and smaller than uncut hCOs and there were no histological differences between uncut and cut hCOs. Note that hypoxia-inducible factor (HIF)-1α was expressed in the central area of uncut hCOs but not in cut hCOs. Uncut hCOs, therefore, showed broad core areas stained with terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL), whereas cut hCOs did not. In conclusion, this simple mechanical cutting method allowed us to acquire a larger number of hCOs without a necrotic core.

Trolox-induced cardiac differentiation is mediated by the inhibition of Wnt/ß-catenin signaling in human embryonic stem cells.

Cardiac differentiation of human pluripotent stem cells may be induced under chemically defined conditions, wherein the regulation of Wnt/ß-catenin pathway is often desirable. Here, we examined the effect of trolox, a vitamin E analog, on the cardiac differentiation of human embryonic stem cells (hESCs). 6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) significantly enhanced cardiac differentiation in a time- and dose-dependent manner after the mesodermal differentiation of hESCs. Trolox promoted hESC cardiac differentiation through its inhibitory activity against the Wnt/ß-catenin pathway. This study demonstrates an efficient cardiac differentiation method and reveals a novel Wnt/ß-catenin regulator.

Ingestion of Exopolymers from Aureobasidium pullulans Reduces the Duration of Cold and Flu Symptoms: A Randomized, Placebo-Controlled Intervention Study.

AIM: The objective of the study was to assess the efficacy of exopolymers from Aureobasidium pullulans (EAP) on the incidence of colds and flu in healthy adults. METHODS: We conducted a randomized, double-blind, placebo-controlled study at the onset of the influenza season. A total of 76 subjects (30-70 years of age) were recruited from the general population. The subjects were instructed to take one capsule per day of either EAP or a placebo for a period of 8 weeks. The duration of cold and flu symptoms, a primary variable in assessing effectiveness, and serum cytokine levels as well as WBC counts as secondary variables were also evaluated. RESULTS: EAP was associated with a statistically significant decrease in the duration of cold and flu symptoms, a primary variable in assessing effectiveness. Although cold and flu symptom levels were not significantly different at a significance level of 5%, the cold and flu symptom levels of the EAP group were less severe compared to the placebo group. No statistically significant changes of serum cytokine levels as well as WBC counts were observed. CONCLUSION: The results showed that EAP is a useful pharmaceutical and functional food material for preventing and treating colds and flu.

Effect of chongmyungtang, a traditional Korean polyherbal formula, on the Pharmacokinetic profiles of donepezil in rats.

Chongmyungtang (CMT) is a famous Korean herbal medicine for improving learning and memory, which has been reported to have anti-cholinergic and neuroprotective effects. Therefore, drug-drug interactions were examined between CMT and donepezil as a first screening of combination therapy for cognitive deficits. Rats received oral co-administration of donepezil with distilled water as a control or donepezil with CMT as a combination. The distilled water or CMT was co-administered at intervals within 5min after donepezil or 1.5h intervals. The plasma samples were analyzed for donepezil concentration and its pharmacokinetic parameters of Tmax, Cmax, AUC, t1/2 and MRTinf. In the single co-administration at intervals within 5min, donepezil was detected lower in the combination than control at 0.5h and 2h post-treatment (P<0.05). In addition, the combination showed significant increases in MRTinf compared to the control (P<0.05). This suggests drug-drug interactions between donepezil and CMT in the co-administration within 5 min. However, no meaningful differences were found in the pharmacokinetic profiles of donepezil by single dosing with CMT at 1.5h intervals and even by the repeated dosing for a week at 1.5h intervals potential combination therapy of donepezil with CMT.

Relative effectiveness of dominant versus non-dominant hand position for rescuer's side of approach during chest compressions between right-handed and left-handed novice rescuers.

INTRODUCTION: The major components affecting high quality cardiopulmonary resuscitation (CPR) have been defined as the ability of the rescuer, hand position, position of the rescuer and victim, depth and rate of chest compressions, and fatigue. Until now, there have been no studies on dominant versus non-dominant hand position and the rescuer's side of approach. This study was designed to evaluate the effectiveness of hand position and approach side on the quality of CPR between right-handed (RH) and left-handed (LH) novice rescuers. MATERIAL AND METHODS: 44 health science university students with no previous experience of basic life support (BLS) volunteered for the study. We divided volunteers into two groups by handedness. Adult BLS was performed on a manikin for 2 min in each session. The sequences were randomly performed on the manikin's left side of approach (Lap) with the rescuer's left hand in contact with the sternum (Lst), Lap/Rst, Rap/Lst and Rap/Rst. RESULTS: We compared the quality of chest compressions between the RH and LH groups according to predetermined positions. A significant decrease in mean compression depth between the two groups was only observed when rescuers performed in the Rap/Lst scenario, regardless of hand dominance. The frequency of correct hand placement also significantly decreased in the Lap/Rst position for the LH group. CONCLUSIONS: The performance of novice rescuers during chest compressions is influenced by the position of the dominant hand and the rescuer's side of approach. In CPR training and real world situations, a novice rescuer, regardless of handedness, should consider hand positions for contacting the sternum identical to the side of approach after approaching from the nearest and most accessible side, for optimal CPR performance.

Pharmacokinetic alteration of baclofen by multiple oral administration of herbal medicines in rats.

The potential pharmacokinetic (PK) interaction of conventional western drug, baclofen, and oriental medications Oyaksungisan (OY) and Achyranthes bidentata radix (AB) extract for the treatment of spasticity has been evaluated. Rats were pretreated with distilled water (DW), OY, or AB extract by oral administration every day for 7 days. After 10 min of the final dose of DW or each herbal medication, baclofen (1 mg/kg) was given by oral administration and plasma concentrations of baclofen were determined by LC/MS/MS. The plasma baclofen concentration-time profiles were then analyzed by noncompartmental analysis and a population PK model was developed. Baclofen was rapidly absorbed, showed biexponential decline with elimination half-life of 3.42-4.10 hr, and mostly excreted into urine. The PK of baclofen was not affected by AB extract pretreatment. However, significantly lower maximum plasma concentration (C max) and longer time to reach C max (T max) were observed in OY pretreated rats without changes in the area under the curve (AUC) and the fraction excreted into urine (F urine). The absorption rate (K a ) of baclofen was significantly decreased in OY pretreated rats. These data suggested that repeated doses of OY might delay the absorption of baclofen without changes in extent of absorption, which needs further evaluation for clinical significance.

Continuous hypoxia attenuates paraquat-induced cytotoxicity in the human A549 lung carcinoma cell line.

Paraquat (1,1'-dimethyl-4,4'-bipyridinium dichloride; PQ), an effective and widely used herbicide, was commercially introduced in 1962. It is reduced by the electron donor NADPH, and then reduced PQ transfers the electrons to molecular oxygen, resulting in the production of reactive oxygen species (ROS), which are related to cellular toxicity. However, the influence of continuous hypoxia on PQ-induced ROS production has not fully been investigated. We evaluated in vitro the protective effect of continuous hypoxia on PQ-induced cytotoxicity in the human carcinogenic alveolar basal epithelial cell line (A549 cells) by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and live and dead assay, and by measuring lactate dehydrogenase (LDH) release. To elucidate the mechanism underlying this effect, we monitored the immunofluorescence of intracellular ROS and measured malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities. Continuous hypoxia protected the A549 cells from PQ-induced cytotoxicity. Continuous hypoxia for a period of 24 h significantly reduced intracellular ROS, decreased MDA concentration in the supernatant, and normalized SOD and GPx activities. Continuous hypoxia attenuated PQ-induced cell toxicity in A549 cells. This protective effect might be attributable to the suppression of PQ-induced ROS generation.

Transforming growth factor-ß1 bioassay involving matrix metalloproteinase-2 induction.

The transforming growth factor-ß1 (TGF-ß1) bioassay developed in this study monitors increased luciferase activity in MCF10A cells containing the matrix metalloproteinase-2 (MMP-2) promoter with a luciferase reporter and treated with increasing TGF-ß1 concentrations. The response was linear in the concentration range from 75 to 2,500 pg/mL. The abilities of 3 types of TGF-ß in inducing MMP-2 were different. The luciferase activity induced by TGF-ß1 was about 2 times more than that by TGF-ß2 and TGF-ß3. The MMP-2 promoter bioassay showed greater reproducibility (coefficient of variation [CV] 10%) than the previously developed anticell proliferation assay of TF-1 cell (CV 16%) and the MMP-2 zymogram assay (CV 40%).

Validation of a HeLa Mx2/Luc reporter cell line for the quantification of human type I interferons.

Although antiviral assays have been the most widely available biological assays for interferons (IFNs), they are less sensitive and provide considerable interassay variation. In this study, we demonstrate a new reporter cell line, which is based on HeLa cells transfected with a plasmid containing a human Mx2 promoter driving a luciferase (Luc) cDNA. To characterize the specific gene expression profiles induced by interferon alpha, we analyzed the microarray results of interferon response gene expression induced by IFN-alpha2a or IFN-alpha2b treatment with HeLa cells. We found that the Mx2 gene increased the most by treatment with both IFN-alpha2a and IFN-alpha2b. Based on this result, we designed a reporter cell line, HeLa-Mx2, suitable for determination of IFN-alpha. HeLa cells were stably transfected with the luciferase gene under the control of Mx2 promoter. The expression of luciferase can be easily measured for luminescence using a 96-well luminometer and has been correlated with the concentration of added IFN and cell density. In the validation results, our reporter cell line had specificity for type I IFN, but the significant effects of a number of other cytokines such as tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-2, IL-5, IL-6 and GM-CSF, or type II interferon (IFN-gamma) were not observed. Moreover, the robustness of our cell line is demonstrated by the lack of an effect of the HeLa-Mx2 cell culture's age on the performance of the reporter gene assay. The reporter gene assay demonstrated reproducible dose-response curves for IFN-alpha2a in the range of 1-10,000 IU/ml. The 95% confidential limit and total coefficient of variation estimates ranged between 96 and 116 and 10.51% in the reducible range mentioned above, respectively. In conclusion, we established a stable IFN-responsible HeLa-Mx2 cell line, which has advantages with regard to simplicity, selectivity, and reliability over conventional cytopathic effect reduction assays used to quantify IFN-alpha activity.