microRNAs in viral oncogenesis

MicroRNAs are a recently discovered class of small noncoding functional RNAs. These molecules mediate post-transcriptional regulation of gene expression in a sequence specific manner. MicroRNAs are now known to be key players in a variety of biological processes and have been shown to be deregulated in a number of cancers. The discovery of viral encoded microRNAs, especially from a family of oncogenic viruses, has attracted immense attention towards the possibility of microRNAs as critical modulators of viral oncogenesis. The host-virus crosstalk mediated by microRNAs, messenger RNAs and proteins, is complex and involves the different cellular regulatory layers. In this commentary, we describe models of microRNA mediated viral oncogenesis

IoT Based Gas Leakage Detection and Alert Generation

Nowadays the use of the gas is increased the gas leakage has been a notable issue. The gas leakage causes the wastage of the gas and mainly as its combustible serious harm can be done to the living thing and other property. To overcome such an incident, we are developing a IoT based Gas Leakage Detection and Alert Generating system this will notify the user about the leakage and take the safety measures instantly. The proposed system can be use in the area where the gas is frequently used such as kitchens and industries and even in the gas operated vehicles too. The idea of the system is that the gas sensor is used which detects the presence on the gas in the surrounding atmosphere and if the value of the gas in atmosphere is increased then the threshold value alert is generated by the system and the system can be monitored and controlled remotel

Implementation and evaluation of a high-throughput siRNA screening system for suspension CHO cells

Chinese Hamster Ovary (CHO) cells are the most frequently used mammalian cell factory for the production of human-like recombinant proteins. Due to existing limitations in growth and protein production, genetic optimization of CHO cell lines may significantly enhance bioprocess productivities. Knockdown of genes by siRNAs is a standard method to identify genes involved in a desirable phenotype, either because their knockdown improves or degenerates the property. As at least 13000 different transcripts are present in a cell at any time, it is of interest to develop a method that is able to efficiently test the effect of gene knockdown at an appropriate throughput and scale. Here we describe the implementation of a high-throughput and small scale siRNA screening assay for suspension CHO cells that produce a secreted fluorescent protein. First, growth of CHO cells in 384 well plates was optimized. Second, a suitable method to deliver siRNAs into CHO cells was implemented and optimized. The optimization procedure was conducted by varying initial cell number, lipofection reagent concentration, media composition and incubation time with the help of several control siRNAs. Laser cytometry was used to detect the number of cells, the amount of fluorescent protein per cell and the total fluorescence per well. In addition, cell viability was determined afterwards by the CellTiter Glo® Luminescent Cell Viability Assay. The screening system was evaluated by a pilot screen, consisting of a set of kinome-targeting siRNAs (n=2112). For assessment of reproducibility, this entire screen was conducted twice. While the viability assay shows bad reproducibility, questioning its suitability, the cell number, amount of fluorescent protein per cell, and the total fluorescence per well show a good correlation between the two screens. Target genes, capable of enhancing the phenotype of CHO cells towards a higher growth and/or productivity upon their siRNA-induced knockdown were identified. This indicates the suitability of this high-throughput siRNA screening system to identify genes that are involved in the enhancement of growth and/or productivity in CHO cells

Generation of a Chinese Hamster Ovary cell genome-wide deletion library

Nowadays, around 70% of all industrially produced biopharmaceuticals are generated from Chinese Hamster Ovary (CHO) cells showing the high interest for further characterization and optimization of this cell line and its derivates. Despite their importance, the connection between the CHO cell genome sequence and function has not been explored in detail so far. Forward genetic screens are the state-of-the-art approach to investigate the link between genotype and phenotype using the CRISPR system as an efficient tool for this purpose. These screens are usually focusing on the ~ 28,000 protein coding genes, which cover only ~ 3 % of the genome. Our approach aims to correlate larger functional regions of the genome, including coding and non-coding sequences, with process relevant cell behavior, such as growth and productivity. To this end, we designed a deletion library approach that targets larger genomic regions of 100 – 150 kb using paired CRISPR gRNAs. So far, we demonstrated successful and efficient deletions up to 150 kb, resulting in proper loss-of-function mutations. These modifications were analyzed on genome and phenotype level, demonstrating that deletion efficiencies are size independent. Furthermore, to enable the presence of active gRNA pairs in each individual cell, we implemented bicistronic transcription of gRNAs separated by a tRNA sequence that unequivocally links each pair. Additionally, we determined CRISPR Cpf1 – an alternative CRISPR enzyme – activity in CHO with no cross-interaction to the CRISPR/Cas9 system, providing the possibility to use the two systems in parallel, one for targeted insertion of the gRNA pair into the genome for later identification of the deleted region, the other for deletion of the corresponding genomic region itself. Currently we are working on the generation of a first smallscale deletion library targeting lncRNAs in CHO for the implementation of the strategy before going genomewide. This will then open the opportunity both of generating large scale gene knockout libraries and of characterizing non-coding genomic regions, gene clusters or regulatory elements

Artificial Intelligence: Exploring the Attitude of Secondary Students

This paper aims to find the “attitude of secondary students towards artificial intelligence.” Intelligence is a blessing received to humankind through we can learn new things, experience surrounding, and solve complex problems by making our life at pace (Ewert, 2018). Likewise, artificial intelligence (AI) is one of such abilities given to machines by humans to perform all possible tasks which humans can perform (Kengam, 2020). Rapidly growing technology has continuously changed human existence, including robotics and automation leading to the magical transformation. However, this technological transformation has not left the education field untouched. Through the Digital survey, this paper analyzes the understanding of secondary students towards artificial intelligence and its possible effect in the field of education. The researcher found the high attitude in Pune city from the qualitative and quantitative data collected

CRISPR/Cas9 mediated knockout of microRNAs for precise cell engineering

Recent advances in the development of molecular tools available for cell line engineering has opened avenues for precise modulation of production cell lines to optimize cellular process relevant parameters. Among those, the CRISPR/Cas9 system represents a rapid and straightforward cell line engineering tool which allows for precise gene editing of host genomes to modify relevant signaling pathways. In addition, microRNAs (miRNAs) have proven to serve as versatile molecular tools to improve production cells regarding protein production and growth characteristics. These small non-coding RNA molecules are regulators of gene expression and regulate process relevant cellular pathways as growth, apoptosis, protein expression or stress. With an individual microRNA being able to regulate up to several hundred target genes, the modulation of miRNA expression may mimic simultaneous modification of several hundred target genes. Therefore, miRNAs may offer great potential to serve as targets for CRISPR/Cas9-mediated genome editing, as an individual microRNA can regulate up to several hundred target genes and therefore mimics multiplexing of hundreds of target genes in one single step. While the overexpression of miRNAs to improve the performance of biopharmaceutical production hosts has received major interest in the past years, effects of precise knockout of unfavorable miRNAs in Chinese hamster ovary (CHO) production cells have not been reported yet. Therefore, the current study aimed at applying a novel strategy to increase product yields by enhancing viability and culture longevity of CHO cells using CRISPR/Cas9-mediated deletion of a miRNA causing adverse effects. In a previous high content screen with more than 1000 miRNAs we were able to identify miR-744 as being involved in the regulation of apoptosis. After the apoptosis-inducing capacity of miR-744 in monoclonal antibody-producing CHO-mAb1 cells was revalidated, the genomic precursor sequence of miR-744 was deleted by two sgRNA-Cas9-mediated DNA double-strand breaks flanking the miR-744 gene in the CHO genome. A plasmid encoding both required sgRNAs in a tandem array, Cas9 and GFP was used to allow for simultaneous delivery of all required components and thereby reducing the screening effort for potential knockout (KO) cell lines. After fluorescent-activated cell sorting (FACS) seven putative, clonal miR-744-KO cell lines could be recovered. From these, three were identified and confirmed as miR-744-KOs by sequencing of their miR-744 locus respectively. In a subsequent growth experiment over a period of nine days considerable differences in the investigated parameters viable cell count, viability, apoptosis, necrosis and antibody titers of the miR-744-KO cell lines in comparison to untreated cells and non-targeting sgRNA transfected cells could be observed. Further experiments are conducted to analyze the observed effects. In summary, the present study elucidates the role of microRNAs, which bear adverse effects on production cells, as targets for the novel CRISPR/Cas9 genome editing technique in the context of cell line engineering. This approach contributes to the idea of a rational design of CHO production hosts with controlled modulation of signaling pathways or metabolic characteristics to enable the generation of optimized production cells

Variation in karyotype and chromsome numbers in CHO cell lines and subclones

Genomic rearrangements are a common phenomenon in rapidly growing cell lines such as Chinese hamster ovary cells, a feature that, while it provides the ability to adapt to different conditions and to select the rare variants with high productivity, in the final production clone may lead to batch irreproducibility and instability. Few methods exist to assess such genome wide instability. Here we use the population distribution of chromosome numbers per cell as well as chromosome painting to identify large scale chromosomal rearrangements for quantification of karyotypic variation in several CHO cell lines, including host and recombinant cell lines, both at the pool, minipool and subclone level. Apart from investigating differences between cell lines and subclones, we followed changes in chromosome number distribution and chromosome pattern over a period of 6 months for stability assessment. Please click Additional Files below to see the full abstract

Microlearning strategies for teacher professional development in the era of fourth industrial revolution in India

The Fourth Industrial Revolution which is characterized by rapid breakthroughs in digital technology, automation, and artificial intelligence, is revolutionizing both the classroom and workplace learning settings. This change in the classroom and workplace setting is characterized by changing skill requirements, accelerating knowledge obsolescence, need for personalized and adaptive learning experiences, a culture of continuous learning, and on-demand performance support. The educational landscape is changing with the intervention of technology into our lives and new and innovative ways of learning are emerging. The key stakeholders in the education system are teachers and their professional development has utmost significance in helping education pick pace with societal changes. The various strategies of microlearning are useful for teachers to enhance their skills and mitigate the impact fourth industrial revolution is having in terms of quicker knowledge obsolescence and new job creation. Microlearning can provide considerable benefits in the Indian classroom context since traditional educational systems struggle to keep up with these changes. According to a Deloitte University Press research report, microlearning can improve learning retention rates because it provides learners with bite-sized, focused content that is easier to digest and retain than lengthy classroom lectures or training sessions. The interactive and self-directed learning approaches, such as microlearning, can lead to higher learning outcomes compared to traditional classroom teaching (Sitzmann, 2011). The paper provides a look into effective microlearning strategies that can help in dealing with the problems of the traditional classrooms in the current digital society

Analysis of microRNA transcription and post-transcriptional processing by Dicer in the context of CHO cell proliferation

AbstractCHO cells are the mammalian cell line of choice for recombinant production of therapeutic proteins. However, their low rate of proliferation limits obtainable space-time yields due to inefficient biomass accumulation. We set out to correlate microRNA transcription to cell-specific growth-rate by microarray analysis of 5 CHO suspension cell lines with low to high specific growth rates. Global microRNA expression analysis and Pearson correlation studies showed that mature microRNA transcript levels are predominately up-regulated in a state of fast proliferation (46 positively correlated, 17 negatively correlated). To further validate this observation, the expression of three genes that are central to microRNA biogenesis (Dicer, Drosha and Dgcr8) was analyzed. The expression of Dicer, which mediates the final step in microRNA maturation, was found to be strongly correlated to growth rate. Accordingly, knockdown of Dicer impaired cell growth by reducing growth-correlating microRNA transcripts. Moderate ectopic overexpression of Dicer positively affected cell growth, while strong overexpression impaired growth, presumably due to the concomitant increase of microRNAs that inhibit cell growth. Our data therefore suggest that Dicer dependent microRNAs regulate CHO cell proliferation and that Dicer could serve as a potential surrogate marker for cellular proliferation

CHO microRNA engineering is growing up : recent successes and future challenges

microRNAs with their ability to regulate complex pathways that control cellular behavior and phenotype have been proposed as potential targets for cell engineering in the context of optimization of biopharmaceutical production cell lines, specifically of Chinese Hamster Ovary cells. However, until recently, research was limited by a lack of genomic sequence information on this industrially important cell line. With the publication of the genomic sequence and other relevant data sets for CHO cells since 2011, the doors have been opened for an improved understanding of CHO cell physiology and for the development of the necessary tools for novel engineering strategies. In the present review we discuss both knowledge on the regulatory mechanisms of microRNAs obtained from other biological models and proof of concepts already performed on CHO cells, thus providing an outlook of potential applications of microRNA engineering in production cell lines