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.

Methylation of 2-Aryl-2-(3-indolyl)acetohydroxamic Acids and Evaluation of Cytotoxic Activity of the Products

2-Aryl-2-(3-indolyl)acetohydroxamic acids demonstrate promising antitumor activity, but quickly metabolize in vivo via glucuronidation of hydroxamic acid residue. In an attempt to improve their pharmacokinetics, methyl esters were synthesized via a newly developed protocol for chemoselective mono-methylation of hydroxamic acids. The cytotoxicity of these derivatives against the HeLa cell line was evaluated and found to be inferior compared to the parent lead compounds.

Microencapsulation of an Extract of Sechium edule (Jacq.) Sw., with Antineoplastic Activity

Microencapsulation is a technique used in pharmaceuticals as an administration vehicle. Encapsulating secondary metabolites for therapeutic purposes has been promoted recently. Microencapsulation based on chitosan was developed for the methanol extract of cv. Perla negra (S. edule (Jacq.) Sw.) (Cucurbitaceae) fruits to evaluate its viability as an administration vehicle and to assess the possible negative interaction between the extract and chitosan. Microencapsulation was performed by coacervation, implementing a method with constant sonication. The microparticles obtained were registered by means of Scanning Electron Microscopy. The presence of the bioactive in aqueous medium was recorded for release tests, measuring with spectrophotometry its concentration as a function of time. The assessment of the biological effect of the microencapsulated extract was done on the HeLa cell line and control cells (lymphocytes). Microspheres with an average size of 20 µm and a loading capacity of 98% were obtained. The highest concentration of released extract was 24 µg mL−1 at 23 h. The mainly chitosan-based microspheres did not affect the antiproliferative activity of the extract of cv. Perla negra and proved to be a potential vehicle for its therapeutic administration. The empty microspheres made with chitosan also showed to have an antiproliferative effect, and those loaded with extract showed cellular inhibition (statistical IC50) of 8 µg mL−1 without affecting the lymphocytes. Chitosan does not interfere with the biological activity of the metabolites incorporated into the microspheres since they retain their inhibitory activity on proliferation in tumor cells, thus constituting a potential vehicle for the therapeutic administration of fruit extract.

A Comprehensive Analysis of the Thrombin Binding Aptamer Containing Functionalized Pyrrolo-2’-deoxycytidines

Aptamers constitute an answer for the growing need for targeted therapy development. One of the most well-known representatives of this group of compounds is thrombin binding aptamers (TBA) targeted towards thrombin. The TBA inhibitory activity is determined by its spatial arrangement, which consists of two G-tetrads linked by two shorter TT loops and one longer TGT loop and folds into a unimolecular, antiparallel G-quadruplex structure. Interesting properties of the aptamer can be further improved via the introduction of a number of chemical modifications. Herein, a comprehensive analysis of the influence of pyrrolo-2’-deoxycytidine (Py-dC) and its derivatives on TBA physicochemical and biological properties has been presented. The studies have shown that the presence of modified residues at the T7 position of the TGT loop has only minor effects on TBA thermodynamic stability without affecting its folding topology. All analyzed oligomers exhibit anticoagulant properties, but only aptamer modified with a decyl derivative of Py-dC was able to inhibit thrombin activity more efficiently than unmodified, parental compounds. Importantly, the same compound also possessed the potential to effectively restrain HeLa cell line growth.

A study on ER stress-induced apoptosis pathway in cervical cancer HeLa cells treated with biosynthesized gold nanoparticles

Background The expression of apoptotic family of protein plays a major role in induction of programmed cell death. There are six major apoptotic proteins such as Caspase 12, Bcl 2, BAX, Cytochrome c, PARP3 and Mcl1. All these proteins have crucial role in the regulation of apoptosis through mitochondrial degradation, DNA damage, nuclear condensation and eventually cell death of the cancerous cells. It was observed that the apoptotic pathway has been initiated in the cancer cells from the expression of the apoptotic proteins. The results emphasized that the apoptotic cell death has been induced by the nanomaterials against cervical cancer HeLa cell line. Methods Initially, the nanomaterials were individually checked for potential anticancer activities through MTT assay. The cervical cancer HeLa cell line was treated with nanoparticles, nanoconjugates, nano-dox conjugate and chitosan–nano-dox conjugates. The cell lysates were processed for SDS–PAGE followed by Western blotting. The apoptotic expression has been studied for six major apoptotic proteins such as Caspase 12, Bcl 2, BAX, Cytochrome c, PARP3 and Mcl 1. Results In the present study, the biosynthesized gold nanoparticles, nanoconjugates, nano-dox conjugate, chitosan–nano-dox conjugate were treated against cervical cancer HeLa cell line. The results demonstrated anticancer effects of the nanocompounds implying nanoparticles induced apoptotic pathway in the cancer cells. Further apoptotic expression was studied for six major apoptotic proteins such as Caspase 12, Bcl 2, BAX, Cytochrome c, PARP3 and Mcl 1. The present study was focussed on anticancer efficiency of biosynthesized nanomaterials. Conclusions The in vitro anticancer study showed that the nanomaterials induced cell death over the treated cervical cancer cells. In the process of apoptotic cell death, the caspase cascade pathway was activated. The gene expression was checked in line with some of the genes involved in apoptosis, cell death. The expression was checked for Caspase 12, BAX, Bcl2, cyt c, PARP3 and Mcl 1. The expression of apoptotic proteins suggested that the cancer cell death was mediated through ER stress-induced pathway involving the major apoptotic proteins.

L’héritage d’Henrietta Lacks

Many developments in biology and biotechnology have relied on the HeLa cell line, originally derived in 1951 from a Black cancer patient without her knowledge. This origin became generally known at the turn of the century, and the patient’s descendants have sought and obtained some recognition and some control but little compensation. They have now retained two famous attorneys to sue a biotech firm for very extensive damages, with more legal action planned against other companies. This may have important repercussions for the biotech industry, and raises complex issues regarding ownership of biological material and compensation to patients from whom these materials have been obtained.

Evaluating the in vitro Cytotoxicity of Thymus vulgaris Essential Oil on MCF-7 and HeLa Cancer Cell Lines

Thyme essential oil (TEO) was extracted from dried leaves of Thymus vulgaris. The air-dried aerial parts of the plant produced 1.0% yield of TEO. The detection of this essential oil’s compounds was performed by GC-MASS. The cytotoxic activity of TEO was evaluated against two human cancer cell lines, namely HeLa (human epithelial cervical cancer) and MCF-7 (human breast carcinoma). Cells grown in 96 multi-well plates were treated with six concentrations of EO (6.25, 12.5, 25, 50, 100, 200 ppm) and incubated at 37 °C for 72 hrs. Cancer cell lines elicited various degrees of sensitivity to the cytotoxic effect of essential oil. The TEO exhibited significant differences (p≤ 0.01) between the effects of all concentrations against these two human cell lines. The results showed the highest toxicity of TEO on HeLa cell line (78.67%) and MCF-7 cell line (83.60%) at 200 ppm concentration. Also the values of half-maximal inhibitory concentration (IC50) of TEO against HeLa and MCF-7 cell lines were 34.63 and 27.66 ppm, respectively. Cells treated with the IC50 of TEO showed a significant difference (p ≤ 0.01) in p53 fold expression between HeLa cell line (4.33±0.41 folds) and MCF-7 cell line (5.10±0.32 folds). In general, a dose-dependent decrease the survival of the two cell lines was observed. In addition, MCF-7 cell line revealed higher sensitivity against TEO than HeLa cell line.