Evaluation of NAD+ precursors for improved metabolism and productivity of antibody-producing CHO cell.

In the previous study, the culture medium was treated with nicotinamide adenine dinucleotide (NAD+) under the hypothesis that NAD+ regeneration is a major factor causing excessive lactate accumulation in Chinese hamster ovary (CHO) cells. The NAD+ treatment improved metabolism by not only reducing the Warburg effect but also enhancing oxidative phosphorylation, leading to enhanced antibody production. Building on this, four NAD+ precursors - nicotinamide mononucleotide (NMN), nicotinic acid (NA), nicotinamide riboside (NR), and nicotinamide (NAM) - were tested to elevate intracellular NAD+ levels more economically. First, the ability of CHO cells to utilize both the salvage and Preiss-Handler pathways for NAD+ biosynthesis was verified, and then the effect of NAD+ precursors on CHO cell cultures was evaluated. These precursors increased intracellular NAD+ levels by up to 70.6% compared to the non-treated group. Culture analysis confirmed that all the precursors induced metabolic changes and that NMN, NA, and NR improved productivity akin to NAD+ treatment, with comparable integral viable cell density. Despite the positive effects such as the increase in the specific productivity and changes in cellular glucose metabolism, none of the precursors surpassed direct NAD+ treatment in antibody titer, presumably due to the reduction in nucleoside availability, as evidenced by the decrease in ATP levels in the NAD+ precursor-treated groups. These results underscore the complexity of cellular metabolism as well as the necessity for further investigation to optimize NAD+ precursor treatment strategies, potentially with the supplementation of nucleoside precursors. Our findings suggest a feasible approach for improving CHO cell culture performances by using NAD+ precursors as medium and feed components for the biopharmaceutical production.

RepID represses megakaryocytic differentiation by recruiting CRL4A-JARID1A at DAB2 promoter.

BACKGROUND: Megakaryocytes (MKs) are platelet precursors, which arise from hematopoietic stem cells (HSCs). While MK lineage commitment and differentiation are accompanied by changes in gene expression, many factors that modulate megakaryopoiesis remain to be uncovered. Replication initiation determinant protein (RepID) which has multiple histone-code reader including bromodomain, cryptic Tudor domain and WD40 domains and Cullin 4-RING E3 ubiquitin ligase complex (CRL4) recruited to chromatin mediated by RepID have potential roles in gene expression changes via epigenetic regulations. We aimed to investigate whether RepID-CRL4 participates in transcriptional changes required for MK differentiation. METHODS: The PCR array was performed using cDNAs derived from RepID-proficient or RepID-deficient K562 erythroleukemia cell lines. Correlation between RepID and DAB2 expression was examined in the Cancer Cell Line Encyclopedia (CCLE) through the CellMinerCDB portal. The acceleration of MK differentiation in RepID-deficient K562 cells was determined by estimating cell sizes as well as counting multinucleated cells known as MK phenotypes, and by qRT-PCR analysis to validate transcripts of MK markers using phorbol 12-myristate 13-acetate (PMA)-mediated MK differentiation condition. Interaction between CRL4 and histone methylation modifying enzymes were investigated using BioGRID database, immunoprecipitation and proximity ligation assay. Alterations of expression and chromatin binding affinities of RepID, CRL4 and histone methylation modifying enzymes were investigated using subcellular fractionation followed by immunoblotting. RepID-CRL4-JARID1A-based epigenetic changes on DAB2 promoter were analyzed by chromatin-immunoprecipitation and qPCR analysis. RESULTS: RepID-deficient K562 cells highly expressing MK markers showed accelerated MKs differentiation exhibiting increases in cell size, lobulated nuclei together with reaching maximum levels of MK marker expression earlier than RepID-proficient K562 cells. Recovery of WD40 domain-containing RepID constructs in RepID-deficient background repressed DAB2 expression. CRL4A formed complex with histone H3K4 demethylase JARID1A in soluble nucleus and loaded to the DAB2 promoter in a RepID-dependent manner during proliferation condition. RepID, CRL4A, and JARID1A were dissociated from the chromatin during MK differentiation, leading to euchromatinization of the DAB2 promoter. CONCLUSION: This study uncovered a role for the RepID-CRL4A-JARID1A pathway in the regulation of gene expression for MK differentiation, which can form the basis for the new therapeutic approaches to induce platelet production. Video Abstract.

RepID represses megakaryocytic differentiation by recruiting CRL4A-JARID1A at DAB2 promoter.

Background Megakaryocytes (MKs) are platelet precursors, which arise from hematopoietic stem cells (HSCs). While MK lineage commitment and differentiation are accompanied by changes in gene expression, many factors that modulate megakaryopoiesis remain to be uncovered. Replication origin binding protein (RepID) which has multiple histone-code reader including bromodomain, cryptic Tudor domain and WD40 domains and Cullin 4-RING ubiquitin ligase complex (CRL4) recruited to chromatin mediated by RepID have potential roles in gene expression changes via epigenetic regulations. We aimed to investigate whether RepID-CRL4 participates in transcriptional changes required for MK differentiation. Methods The PCR array was performed using cDNAs derived from RepID-proficient or RepID-deficient K562 erythroleukemia cell lines. Correlation between RepID and DAB2 expression was examined in the Cancer Cell Line Encyclopedia (CCLE) through the CellMinerCDB portal. The acceleration of MK differentiation in RepID-deficient K562 cells was determined by estimating cell sizes as well as counting multinucleated cells known as MK phenotypes, and by qRT-PCR analysis to validate transcripts of MK markers using phorbol 12-myristate 13-acetate (PMA)-mediated MK differentiation condition. Interaction between CRL4 and histone methylation modifying enzymes were investigated using BioGRID database, immunoprecipitation and proximity ligation assay. Alterations of expression and chromatin binding affinities of RepID, CRL4 and histone methylation modifying enzymes were investigated using subcellular fractionation followed by immunoblotting. RepID-CRL4-JARID1A-based epigenetic changes on DAB2 promoter were analyzed by chromatin-immunoprecipitation and qPCR analysis. Results RepID-deficient K562 cells highly expressing MK markers showed accelerated MKs differentiation exhibiting increases in cell size, lobulated nuclei together with reaching maximum levels of MK marker expression earlier than RepID-proficient K562 cells. Recovery of WD40 domain-containing RepID constructs in RepID-deficient background repressed DAB2 expression. CRL4A formed complex with histone H3K4 demethylase JARID1A in soluble nucleus and loaded to the DAB2 promoter in a RepID-dependent manner during proliferation condition. RepID, CRL4A, and JARID1A were dissociated from the chromatin during MK differentiation, leading to euchromatinization of the DAB2 promoter. Conclusion This study uncovered a role for the RepID-CRL4A-JARID1A pathway in the regulation of gene expression for MK differentiation, which can form the basis for the new therapeutic approaches to induce platelet production.

The differentially expressed gene signatures of the Cullin 3-RING ubiquitin ligases in neuroendocrine cancer.

Cullin-RING ubiquitin E3 ligase (CRLs) composed of four components including cullin scaffolds, adaptors, substrate receptors, and RING proteins mediates the ubiquitination of approximately 20% of cellular proteins that are involved in numerous biological processes. While CRLs deregulation contributes to the pathogenesis of many diseases, including cancer, how CRLs deregulation occurs is yet to be fully investigated. Here, we demonstrate that components of CRL3 and its transcriptional regulators are possible prognosis marker of neuroendocrine (NE) cancer. Analysis of Cancer Cell Line Encyclopedia (CCLE) through the CellMinerCDB portal revealed that expression of CRL3 scaffold Cullin 3 (CUL3) highly correlates with NE signature, and CUL3 silencing inhibited NE cancer proliferation. Moreover, subset of 151 BTB (Bric-a-brac, Tramtrack, Broad complex) domain-containing proteins that have dual roles as substrate receptors and adaptor subunits in CRL3, as well as the expression of transcription factors (TFs) that control the transcription of BTB genes were upregulated in NE cancer. Analysis using published ChIP-sequencing data in small cell lung cancer (SCLC), including NE or non-NE SCLC verified that gene promoter of candidates which show high correlation with NE signature enriched H3K27Ac. These observations suggest that CRL3 is a master regulator of NE cancer and knowledge of specifically regulated CRL3 genes in NE cancer may accelerate new therapeutic approaches.

COVID-19 Depression and Infection Prevention Behavior among College Students: A Health Belief Perspective.

The COVID-19 pandemic has resulted in an increase in depression among college students due to anxiety and fear of infection. Nonetheless, COVID-19 infection prevention measures should be actively implemented. In this study, the mediating effect of health belief on the relationship between depression and infection prevention behavior was investigated. A survey of 220 South Korean college students was conducted. Depression was found to be the independent variable, health belief the mediating variable, and infection prevention behavior the dependent variable. The model fit index according to confirmatory factor analysis was found to be suitable. Depression among college students was not directly related to COVID-19 infection prevention behavior; however, depression was confirmed to be related to infection prevention behavior via the mediation of health belief. Arbitration measures, focusing on perceived severity and susceptibility during health belief, are required.

A Prediction Algorithm for Hypoglycemia Based on Support Vector Machine Using Glucose Level and Electrocardiogram.

A prediction algorithm for hypoglycemic events is proposed using glucose levels and electrocardiogram (ECG) with support vector machine (SVM). We extracted the corrected QT interval and five heart rate variability parameters from the ECG, along with glucose level from a continuous glucose monitoring system (CGMS). This feature set is used as input to the SVM, and hypoglycemic events are predicted every 5 min using the trained SVM model for up to 30 min in advance. The proposed algorithm was developed and evaluated for nine Type-1 diabetes patients in the D1NAMO dataset. The prediction sensitivity, specificity, and accuracy values for the test set were 91.1%, 87.0%, and 89.0% (10 min before); 88.0%, 84.3%, and 86.2% (20 min before); 80.1%, 83.3%, and 81.7% (30 min before), respectively. These results show higher performance of the proposed method compared to previous studies and suggest the possibility of predicting hypoglycemia in advance.

Deep Learning for Automatic Detection of Periodic Limb Movement Disorder Based on Electrocardiogram Signals.

In this study, a deep learning model (deepPLM) is shown to automatically detect periodic limb movement syndrome (PLMS) based on electrocardiogram (ECG) signals. The designed deepPLM model consists of four 1D convolutional layers, two long short-term memory units, and a fully connected layer. The Osteoporotic Fractures in Men sleep (MrOS) study dataset was used to construct the model, including training, validating, and testing the model. A single-lead ECG signal of the polysomnographic recording was used for each of the 52 subjects (26 controls and 26 patients) in the MrOS dataset. The ECG signal was normalized and segmented (10 s duration), and it was divided into a training set (66,560 episodes), a validation set (16,640 episodes), and a test set (20,800 episodes). The performance evaluation of the deepPLM model resulted in an F1-score of 92.0%, a precision score of 90.0%, and a recall score of 93.0% for the control set, and 92.0%, 93.0%, and 90.0%, respectively, for the patient set. The results demonstrate the possibility of automatic PLMS detection in patients by using the deepPLM model based on a single-lead ECG. This could be an alternative method for PLMS screening and a helpful tool for home healthcare services for the elderly population.

Subacute Oral Toxicity Evaluation of Expanded-Polystyrene-Fed Tenebrio molitor Larvae (Yellow Mealworm) Powder in Sprague-Dawley Rats.

Tenebrio molitor larvae, as known as edible insects, has advantages of being rich in protein, and has been recognized as a suitable alternate protein source for broiler and pig feed. Moreover, given their ability to biodegrade polystyrene, a major pollutant, Tenebrio molitor larvae has been proposed as an innovative solution to environmental problems. In the present study, we investigated the toxicity of Tenebrio molitor larvae powder (TMlp) ingested with expanded-polystyrene (W/ eps) through in vitro and in vivo experiments. The objective of this study was to determine whether TMlp W/ eps can be applied as livestock alternative protein source. For in vitro experiments, cytotoxicity test was performed to investigate the effects of TMlp-extract on the viability of estrogen-dependent MCF-7 cells. The possibility of estrogen response was investigated in two groups: Expanded-polystyrene-fed (W/ eps) TMlp group and without expanded-polystyrene-fed (W/o eps) TMlp group. For in vivo experiments, The male Sprague-Dawley rats were divided based on the dosage of TMlp administered and oral administration was performed to every day for 5 weeks. A toxicological assessments were performed, which included clinical signs, food consumption, body and organ weights, hematology, serum chemistry, and hematoxylin and eosin staining of liver and kidney. There were no specific adverse effect of TMlp W/ eps-related findings under the experimental conditions of this study, but further studies on both sexes and animal species differences should be investigated. In conclusion, TMlp W/ eps was considered non-toxic and observed to be applicable as an alternative protein source for livestock feed.

Pulse Broadening Effects on Ranging Performance of a Laser Altimeter with Return-to-Zero Pseudorandom Noise Code Modulation.

A laser altimeter using code modulation techniques receives a backscattered pulse wider than the transmitted rectangular pulse when scanning a rough or sloped target surface. This leads to degrading the ranging performance in terms of signal-to-noise ratio (SNR) and detection probability. Unlike the pulsed techniques, little work has focused on the pulse broadening effect of the code modulation techniques. In this study, mathematical models were derived to investigate the pulse broadening effect on the ranging performance of a return-to-zero pseudorandom noise (RZPN) laser altimeter. Considering that the impulse response can be approximated by a Gaussian function, the analytical waveform was derived using a new flat-topped multi-Gaussian beam (FMGB) model. The closed-form expressions were also analytically derived for a peak cross-correlation, SNR, and detection probability in terms of the pulse broadening effect. With the use of a three-dimensional model of asteroid Itokawa for practical surface profiles, the analytical expressions were validated by comparing to the results obtained from numerical simulations. It was also demonstrated that the pulse broadening effect dropped down the peak cross-correlation and then deteriorated the ranging performance. These analytical expressions will play an important role in not only designing a laser altimeter using the RZPN code modulation technique but also analyzing its ranging performance.

Deep Convolutional Recurrent Model for Automatic Scoring Sleep Stages Based on Single-Lead ECG Signal.

BACKGROUND: Sleep stage scoring, which is an essential step in the quantitative analysis of sleep monitoring, relies on human experts and is therefore subjective and time-consuming; thus, an easy and accurate method is needed for the automatic scoring of sleep stages. METHODS: In this study, we constructed a deep convolutional recurrent (DCR) model for the automatic scoring of sleep stages based on a raw single-lead electrocardiogram (ECG). The DCR model uses deep convolutional and recurrent neural networks to apply the complex and cyclic rhythms of human sleep. It consists of three convolutional and two recurrent layers and is optimized by dropout and batch normalization. The constructed DCR model was evaluated using multiclass classification, including five-class sleep stages (wake, N1, N2, N3, and rapid eye movement (REM)) and three-class sleep stages (wake, non-REM (NREM), and REM), using a raw single-lead ECG signal. The single-lead ECG signal was collected from 112 subjects in two groups: control (52 subjects) and sleep apnea (60 subjects). The single-lead ECG signal was preprocessed, segmented at a duration of 30 s, and divided into a training set of 89 subjects and test set of 23 subjects. RESULTS: We achieved an overall accuracy of 74.2% for five classes and 86.4% for three classes. CONCLUSIONS: These results show the DCR model's superior performance over those in the previous studies, highlighting that the model can be an alternative tool for sleep monitoring and sleep screening.