Nm-Nano: Predicting 2′-O-methylation (Nm) Sites in Nanopore RNA Sequencing Data

Nm (2′-O-methylation) is one of the most abundant modifications of mRNAs and non-coding RNAs occurring when a methyl group (–CH3) is added to the 2′ hydroxyl (–OH) of the ribose moiety. This modification can appear on any nucleotide (base) regardless of the type of nitrogenous base, because each ribose sugar has a hydroxyl group and so 2′-O-methyl ribose can occur on any base. Nm modification has a great contribution in many biological processes such as the normal functioning of tRNA, the protection of mRNA against degradation by DXO, and the biogenesis and specificity of rRNA. Recently, the single-molecule sequencing techniques for long reads of RNA sequences data offered by Oxford Nanopore technologies have enabled the direct detection of RNA modifications on the molecule that is being sequenced, but to our knowledge there was only one research attempt that applied this technology to predict the stoichiometry of Nm-modified sites in RNA sequence of yeast cells. To this end, in this paper, we extend this research direction by proposing a bio-computational framework, Nm-Nano for predicting Nm sites in Nanopore direct RNA sequencing reads of human cell lines. Nm-Nano framework integrates two supervised machine learning models for predicting Nm sites in Nanopore sequencing data, namely Xgboost and Random Forest (RF). Each model is trained with set of features that are extracted from the raw signal generated by the Oxford Nanopore MinION device, as well as the corresponding basecalled k-mer resulting from inferring the RNA sequence reads from the generated Nanopore signals. The results on two benchmark data sets generated from RNA Nanopore sequencing data of Hela and Hek293 cell lines show a great performance of Nm-Nano. In independent validation testing, Nm-Nano has been able to identify Nm sites with a high accuracy of 93% and 88% using Xgboost and RF models respectively by training each model with Hela benchmark dataset and testing it for identifying Nm sites on Hek293 benchmark dataset. Thus, Nm-Nano outperforms the Nm sites predictors existing in the literature (not relying on Nanopore technology) that were only limited to predict Nm sites on short reads of RNA sequences and unable to predict Nm sites on long RNA sequence reads. By deploying Nm-Nano to predict Nm sites in Hela cell line, it was revealed that a total of 196 genes was identified to have the most abundance of Nm modification among all other genes that have been modified by Nm in this cell line. Similarly, deploying Nm-Nano to predict Nm sites in Hek393 cell line revealed that a total of 196 genes line was identified to have the most abundance of Nm modification among all other genes that have been modified by Nm in this cell line. According to this, a significant enrichment of a wide range of functional processes like high confidences (adjusted p-val < 0.05) enriched ontologies that were more representative of Nm modification role in immune response and cellular homeostasis were revealed in Hela cell line, and "MHC class 1 protein complex", "mitotic spindle assembly", "response to glucocorticoid", and "nucleocytoplasmic transport" were revealed in Hek293 cell line. The source code of Nm-Nano can be freely accessed https://github.com/Janga-Lab/Nm-Nano.

The Cytotoxicity and Nephroprotective Activity of the Ethanol Extracts of Angelica keiskei Koidzumi Stems and Leaves against the NAPQI-Induced Human Embryonic Kidney (HEK293) Cell Line

Ethnopharmacological Relevance. In Indonesia, Angelica keiskei Koidzumi (ashitaba or Japanese celery) has been traditionally used to maintain health and to achieve longevity. Previously, the chlorophyll-rich extract of A. keiskei planted in Korea exhibited a strong antioxidant activity. The objective of the present study was to investigate the cytotoxicity and nephroprotective activity of the ethanol extract of A. keiskei Koidzumi on the N-acetyl-p-benzoquinone imine (NAPQI) induced human embryonic kidney (HEK293) cell line. Materials and Methods. A. keiskei Koidzumi plant was collected from Mount Rinjani, Lombok, Indonesia, and was identified at the School of Biology Sciences and Technology, Bandung Institute of Technology, Indonesia. Extraction of the stems (ASE) and leaves (ALE) was performed by employing ethanol 70% for 3 × 24 h at 26°C. The cytotoxicity study of the extracts was assessed using the water-soluble tetrazolium salt-8 (WST-8) reagent on the HEK293 cell line, while the nephroprotective activity assay was determined on the NAPQI-induced HEK293 cell line. Results. The WST-8 assay showed that the cytotoxicity IC50 of ASE = 2322 μg/mL and IC50 of ALE = 2283 μg/mL. The nephroprotective activity assay revealed that ASE possesses nephroprotective activity against the NAPQI-induced HEK293 cell line at 1161 μg/mL, while ALE does not show the nephroprotective activity. Conclusion. Taken together, lower concentrations of ASE and ALE (<2000 μg/mL) are not toxic to the HEK293 cell line, and only ASE indicates the activity to protect the HEK293 cell line against NAPQI damage. This Japanese celery could be further explored for its potential as a plant-based nephroprotective drug.

BK Polyomavirus Activates HSF1 Stimulating Human Kidney Hek293 Cell Proliferation

Objectives. Some DNA viruses, such as BKPyV, are capable of inducing neoplastic transformation in human tissues through still unclear mechanisms. The goal of this study is to investigate the carcinogenic potential of BK polyomavirus (BKPyV) in human embryonic kidney 293 (Hek293) cells, dissecting the molecular mechanism that determines the neoplastic transformation. Materials and Methods. BKPyV, isolated from urine samples of infected patients, was used to infect monolayers of Hek293 cells. Subsequently, intracellular redox changes, GSH/GSSH concentration by HPLC, and reactive oxygen/nitrogen species (ROS/RNS) production were monitored. Moreover, to understand the signaling pathway underlying the neoplastic transformation, the redox-sensitive HFS1-Hsp27 molecular axis was examined using the flavonoid quercetin and polishort hairpin RNA technologies. Results. The data obtained show that while BKPyV replication is closely linked to the transcription factor p53, the increase in Hek293 cell proliferation is due to the activation of the signaling pathway mediated by HSF1-Hsp27. In fact, its inhibition blocks viral replication and cell growth, respectively. Conclusions. The HSF1-Hsp27 signaling pathway is involved in BKPyV infection and cellular replication and its activation, which could be involved in cell transformation.

Comparison of different technologies for producing recombinant adeno-associated virus on a laboratory scale

Adeno-associated virus vectors are among the most promising ones for the delivery of transgenes to various organs and tissues. Recombinant adeno-associated virus (rAAV) is able to transduce both dividing and non-dividing cells, has low immunogenicity, and is able to provide long-term expression of transgenes. Modern technologies make it possible to obtain rAAV for in vivo use, but they are not without drawbacks associated with laboriousness, scalability difficulties, and high cost, therefore, improvement of technological schemes for obtaining rAAV is an urgent issue. The aim of the study was to compare different technological approaches to rAAV production based on different conditions of the transfected HEK293 cell line cultivation on a laboratory scale. Materials and methods: HEK293 cell culture, AAV-DJ Packaging System, PlasmidSelect Xtra Starter Kit were used in the study. The technologies were compared using a model rAAV vector with a single-domain antibody transgene fused to the Fc-fragment of IgG1 specific to botulinum toxin. HEK293 cells were transfected with supercoiled plasmid DNA isolated by three-step chromatographic purification. The identity of the rAAV preparation was determined by electrophoresis, immunoblotting, and real-time polymerase chain reaction. Results: the study demonstrated the efficiency of the chromatographic method for obtaining a supercoiled form of plasmid DNA that can be used for efficient transfection of cell culture in order to produce rAAV. The study compared the following processes of rAAV production: using transient transfection and cultivation of the transfected HEK293 cell suspension in Erlenmeyer flasks, adherent culture in T-flasks, and adherent culture in a BioBLU 5p bioreactor on a matrix of Fibra-Cel disks. Conclusions: the data obtained showed the possibility of using the described approaches to purification of plasmid DNA, cell transfection, and cultivation of the transfected cells under various conditions to obtain rAAV samples that expresses the antibody gene. The BioBLU 5p reactor with Fibra-Cel discs was used for the first time to produce preparative quantities of rAAV on a laboratory scale, which increased the adherent surface area during cell culture and transfection, and, as a result, increased the yield of the target product.

OaAEP1-mediated PNA-protein conjugation enables erasable imaging of membrane protein

Methods to efficiently and site-specifically conjugate proteins to nucleic acids could enable exciting application in bioanalytics and biotechnology. Here, we report the use of the strict protein ligase to covalently ligate a protein to a peptide nucleic acid (PNA). The rapid ligation requires only a short N-terminal GL dipeptide in target protein and a C-terminal NGL tripeptide in PNA. We demonstrate the versatility of this approach by conjugating three different types of proteins with a PNA strand. The biostable PNA strand then serves as a generic landing platform for nucleic acid hybridization. Lastly, we show the erasable imaging of EGFR on HEK293 cell membrane through toehold-mediated strand displacement. This work provides a controlled tool for precise conjugation of proteins with nucleic acids through an extremely small peptide linker and facilitates further study of membrane proteins.

Effect of Quercetin on Injury to Indomethacin-Treated Human Embryonic Kidney 293 Cells

Nonsteroidal anti-inflammatory drugs (NSAIDs) are used to treat inflammation and pain and even to prevent the progression of cardiovascular disease. They have become widely used because of their effectiveness, especially among athletes performing high-intensity training. Indomethacin is used for pain management in sports medicine and is highly effective and versatile. However, several clinical studies have reported that indomethacin induces acute renal damage. In the present study, we determined that indomethacin reduced human embryonic kidney 293 (HEK293) cell viability in a concentration-dependent manner by triggering apoptosis. In addition, we demonstrated the effect of quercetin on indomethacin-treated HEK293 cells by inactivating the caspase-3 and caspase-9 signals. Furthermore, quercetin reduced ROS production and increased mitochondrial membrane potential (ΔΨm) in indomethacin-treated HEK293 cells. Our results indicate that quercetin can interrupt the activated caspase and mitochondrial pathway induced by indomethacin in HEK293 cells and affect apoptotic mRNA expression. Quercetin can protect against indomethacin-induced HEK293 cell apoptosis by regulating abnormal ΔΨm and apoptotic mRNA expression.

Synthesis, Antioxidant activity and Structure-Activity Relationship of gallic hydrazones Analogues

A series of gallic acid hydrazones were designed and synthesized as new potential anti-oxidant agents. Most of these compounds are potent antioxidants. The strongest compounds are 11 and 15 (EC50: 6.42 μg·mL−1, 6.86 μg·mL−1, DPPH) and (EC50: 12.85μg·mL−1, 12.49μg·mL−1ABTS), more potent than the positive control Trolox. Furthermore, the promising compounds 11 and 15 exhibited very low cytotoxic activity against HEK293 cell (IC50 >56.4 µM). The SAR study revealed that the pattern of hydroxyl, methoxy and methyl substituents on the gallic hydrazones framework can increase the antioxidant properties of the prototype compounds. Moreover, the results also showed that the activity increased with the number of the groups and increased following hydroxyl > methoxy > methyl. Overall, the present study suggests that the designed compounds may serve as lead molecules for developing novel anti-oxidative agents in food industry.

The adenoviral E1B-55k protein present in HEK293 cells mediates abnormal accumulation of key WNT signaling proteins in large cytoplasmic aggregates

HEK293 cells are one of the most widely used cell lines in research and HEK293 cells are frequently used as an in vitro model for studying the WNT signaling pathway. The HEK293 cell line was originally established by transfection of human embryonic kidney cells with sheared adenovirus 5 DNA and it is known that that HEK293 cells stably express the adenoviral E1A and E1B-55k proteins. Here we show that HEK293 cells display an unexpected distribution of key components of the WNT/β-catenin signaling pathway where AXIN1, APC, DVL2 and tankyrase are all co-localized in large spherical cytoplasmic aggregates. The cytoplasmic aggregates are enclosed by a narrow layer of the adenoviral E1B-55k protein. Reduction of E1B-55k protein levels leads to disappearance of the cytoplasmic aggregates thus corroborating an essential role of the E1B-55k protein in mediating the formation of the aggregates. Furthermore, HEK293 cells with reduced E1B-55k protein levels display reduced levels of transcriptional activation of WNT/β-catenin signaling upon stimulation by the Wnt3A agonist. The demonstrated influence of the E1B-55k protein on the cellular localization of WNT/β-catenin signaling components and on transcriptional regulation of WNT/β-catenin signaling asks for caution in the interpretation of data derived from the HEK293 cell line.

Affecting HEK293 Cell Growth and Production Performance by Modifying the Expression of Specific Genes

The HEK293 cell line has earned its place as a producer of biotherapeutics. In addition to its ease of growth in serum-free suspension culture and its amenability to transfection, this cell line’s most important attribute is its human origin, which makes it suitable to produce biologics intended for human use. At the present time, the growth and production properties of the HEK293 cell line are inferior to those of non-human cell lines, such as the Chinese hamster ovary (CHO) and the murine myeloma NSO cell lines. However, the modification of genes involved in cellular processes, such as cell proliferation, apoptosis, metabolism, glycosylation, secretion, and protein folding, in addition to bioprocess, media, and vector optimization, have greatly improved the performance of this cell line. This review provides a comprehensive summary of important achievements in HEK293 cell line engineering and on the global engineering approaches and functional genomic tools that have been employed to identify relevant genes for targeted engineering.