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1.
Front Genet ; 14: 1273296, 2023.
Article in English | MEDLINE | ID: mdl-38146340

ABSTRACT

MicroRNAs (miRNAs) are small non-coding RNAs that participate as powerful genetic regulators. MiRNAs can interfere with cellular processes by interacting with a broad spectrum of target genes under physiological and pathological states, including cancer development and progression. Major histocompatibility complex major histocompatibility complex class I-related chain A (MICA) belongs to a family of proteins that bind the natural-killer group 2, member D (NKG2D) receptor on Natural Killer cells and other cytotoxic lymphocytes. MICA plays a crucial role in the host's innate immune response to several disease settings, including cancer. MICA harbors various single nucleotide polymorphisms (SNPs) located in its 3'-untranslated region (3'UTR), a characteristic that increases the complexity of MICA regulation, favoring its post-transcriptional modulation by miRNAs under physiological and pathological conditions. Here, we conducted an in-depth analysis of MICA 3'UTR sequences according to each MICA allele described to date using NCBI database. We also systematically evaluated interactions between miRNAs and their putative targets on MICA 3'UTR containing SNPs using in silico analysis. Our in silico results showed that MICA SNPs rs9266829, rs 1880, and rs9266825, located in the target sequence of miRNAs hsa-miR-106a-5p, hsa-miR-17-5p, hsa-miR-20a-5p, hsa-miR-20b-5p, hsa-miR-93, hsa-miR-1207.5p, and hsa-miR-711 could modify the binding free energy between -8.62 and -18.14 kcal/mol, which may affect the regulation of MICA expression. We believe that our results may provide a starting point for further exploration of miRNA regulatory effects depending on MICA allelic variability; they may also be a guide to conduct miRNA in silico analysis for other highly polymorphic genes.

2.
Sci Rep ; 13(1): 1482, 2023 01 27.
Article in English | MEDLINE | ID: mdl-36707606

ABSTRACT

Improving the cellular capacity of Chinese hamster ovary (CHO) cells to produce large amounts of therapeutic proteins remains a major challenge for the biopharmaceutical industry. In previous studies, we observed strong correlations between the performance of CHO cells and expression of two transcription factors (TFs), MYC and XBP1s. Here, we have evaluated the effective of overexpression of these two TFs on CHO cell productivity. To address this goal, we generated an EPO-producing cell line (CHOEPO) using a targeted integration approach, and subsequently engineered it to co-overexpress MYC and XBP1s (a cell line referred to as CHOCXEPO). Cells overexpressing MYC and XBP1s increased simultaneously viable cell densities and EPO production, leading to an enhanced overall performance in cultures. These improvements resulted from the individual effect of each TF in the cell behaviour (i.e., MYC-growth and XBP1s-productivity). An evaluation of the CHOCXEPO cells under different environmental conditions (temperature and media glucose concentration) indicated that CHOCXEPO cells increased cell productivity in high glucose concentration. This study showed the potential of combining TF-based cell engineering and process optimisation for increasing CHO cell productivity.


Subject(s)
Glucose , Animals , Cricetinae , Cell Proliferation , CHO Cells , Cricetulus , Recombinant Proteins/metabolism , Proto-Oncogene Proteins c-myc/metabolism , X-Box Binding Protein 1/metabolism
3.
J Ind Microbiol Biotechnol ; 47(3): 299-309, 2020 Mar.
Article in English | MEDLINE | ID: mdl-32086638

ABSTRACT

Piscirickettsia salmonis is a facultative Gram-negative intracellular bacterium that produces piscirickettsiosis, disease that causes a high negative impact in salmonid cultures. The so-far-unidentified nutritional requirements have hindered its axenic culture at laboratory and industrial scales for the formulation of vaccines. The present study describes the development of a defined culture medium for P. salmonis. The culture medium was formulated through rational design involving auxotrophy test and statistical designs of experiments, considering the genome-scale metabolic reconstruction of P. salmonis reported by our group. The whole optimization process allowed for a twofold increase in biomass and a reduction of about 50% of the amino acids added to the culture medium. The final culture medium contains twelve amino acids, where glutamic acid, threonine and arginine were the main carbon and energy sources, supporting 1.65 g/L of biomass using 6.5 g/L of amino acids in the formulation. These results will contribute significantly to the development of new operational strategies to culture this bacterium for the production of vaccines.


Subject(s)
Piscirickettsia/growth & development , Vaccines/immunology , Culture Media , Vaccines/metabolism
4.
Crit Rev Biotechnol ; 39(5): 665-679, 2019 Aug.
Article in English | MEDLINE | ID: mdl-31030575

ABSTRACT

The continuous increase of approved biopharmaceutical products drives the development of more efficient recombinant protein expression systems. Chinese hamster ovary (CHO) cells are the mainstay for this purpose but have some drawbacks, such as low levels of expression. Several strategies have been applied to increase the productivity of CHO cells with different outcomes. Transcription factor (TF) engineering has emerged as an interesting and successful approach, as these proteins can act as master regulators; the expression and function of a TF can be controlled by small molecules, and it is possible to design tailored TFs and promoters with desired features. To date, the majority of studies have focused on the use of TFs with growth, metabolic, cell cycle or endoplasmic reticulum functions, although there is a trend to develop new, synthetic TFs. Moreover, new synthetic biological approaches are showing promising advances for the development of specific TFs, even with tailored ligand sensitivity. In this article, we summarize the strategies to increase recombinant protein expression by modulating and designing TFs and with advancements in synthetic biology. We also illustrate how this class of proteins can be used to develop more robust expression systems.


Subject(s)
Transcription Factors/metabolism , Animals , Apoptosis , CHO Cells , Cell Cycle , Cricetulus , Endoplasmic Reticulum/metabolism , Humans , Promoter Regions, Genetic , Protein Engineering , Transcription Factors/genetics
5.
Bioresour Technol ; 223: 105-114, 2017 Jan.
Article in English | MEDLINE | ID: mdl-27788423

ABSTRACT

Piscirickettsia salmonis is a fish bacterium that causes the disease piscirickettsiosis in salmonids. This pathology is partially controlled by vaccines. The lack of knowledge has hindered its culture on laboratory and industrial scale. The study describes the metabolic phenotype of P. salmonis in culture. This study presents the first genome-scale model (iPF215) of the LF-89 strain of P. salmonis, describing the central metabolic pathway, biosynthesis and molecule degradation and transport mechanisms. The model was adjusted with experiment data, allowing the identification of the capacities that were not predicted by the automatic annotation of the genome sequences. The iPF215 model is comprised of 417 metabolites, 445 reactions and 215 genes, was used to reproduce the growth of P. salmonis (µmax 0.052±0.005h-1). The metabolic reconstruction of the P. salmonis LF-89 strain obtained in this research provides a baseline that describes the metabolic capacities of the bacterium and is the basis for developing improvements to its cultivation for vaccine formulation.


Subject(s)
Aquaculture , Fish Diseases/genetics , Models, Biological , Piscirickettsiaceae/genetics , Salmonidae/microbiology , Animals , Base Sequence , Piscirickettsiaceae Infections
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