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BioTech, Volume 10, Issue 4 (December 2021) – 9 articles

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Article
Public Perceptions Regarding Genomic Technologies Applied to Breeding Farm Animals: A Qualitative Study
by , , and
BioTech 2021, 10(4), 28; https://0-doi-org.brum.beds.ac.uk/10.3390/biotech10040028 (registering DOI) - 03 Dec 2021
Viewed by 131
Abstract
The societal acceptability of different applications of genomic technologies to animal production systems will determine whether their innovation trajectories will reach the commercialisation stage. Importantly, technological implementation and commercialisation trajectories, regulation, and policy development need to take account of public priorities and attitudes. [...] Read more.
The societal acceptability of different applications of genomic technologies to animal production systems will determine whether their innovation trajectories will reach the commercialisation stage. Importantly, technological implementation and commercialisation trajectories, regulation, and policy development need to take account of public priorities and attitudes. More effective co-production practices will ensure the application of genomic technologies to animals aligns with public priorities and are acceptable to society. Consumer rejection of, and limited demand for, animal products developed using novel genomic technologies will determine whether they are integration into the food system. However, little is known about whether genomic technologies that accelerate breeding but do not introduce cross-species genetic changes are more acceptable to consumers than those that do. Five focus groups, held in the north east of England, were used to explore the perceptions of, and attitudes towards, the use of genomic technologies in breeding farm animals for the human food supply chain. Overall, study participants were more positive towards genomic technologies applied to promote animal welfare (e.g., improved disease resistance), environmental sustainability, and human health. Animal “disenhancement” was viewed negatively and increased food production alone was not perceived as a potential benefit. In comparison to gene editing, research participants were most negative about genetic modification and the application of gene drives, independent of the benefits delivered. Full article
(This article belongs to the Special Issue Biotechnology and Bioethics)
Article
VirusLab: A Tool for Customized SARS-CoV-2 Data Analysis
BioTech 2021, 10(4), 27; https://0-doi-org.brum.beds.ac.uk/10.3390/biotech10040027 - 06 Nov 2021
Viewed by 584
Abstract
Since the beginning of 2020, the COVID-19 pandemic has posed unprecedented challenges to viral data analysis and connected host disease diagnostic methods. We propose VirusLab, a flexible system for analysing SARS-CoV-2 viral sequences and relating them to metadata or clinical information about the [...] Read more.
Since the beginning of 2020, the COVID-19 pandemic has posed unprecedented challenges to viral data analysis and connected host disease diagnostic methods. We propose VirusLab, a flexible system for analysing SARS-CoV-2 viral sequences and relating them to metadata or clinical information about the host. VirusLab capitalizes on two existing resources: ViruSurf, a database of public SARS-CoV-2 sequences supporting metadata-driven search, and VirusViz, a tool for visual analysis of search results. VirusLab is designed for taking advantage of these resources within a server-side architecture that: (i) covers pipelines based on approaches already in use (ARTIC, Galaxy) but entirely cutomizable upon user request; (ii) predigests analysis of raw sequencing data from different platforms (Oxford Nanopore and Illumina); (iii) gives access to public archives datasets; (iv) supplies user-friendly reporting – making it a tool that can also be integrated into a business environment. VirusLab can be installed and hosted within the premises of any organization where information about SARS-CoV-2 sequences can be safely integrated with information about hosts (e.g., clinical metadata). A system such as VirusLab is not currently available in the landscape of similar providers: our results show that VirusLab is a powerful tool to generate tabular/graphical and machine readable reports that can be integrated in more complex pipelines. We foresee that the proposed system can support many research-oriented and therapeutic scenarios within hospitals or the tracing of viral sequences and their mutational processes within organizations for viral surveillance. Full article
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Article
Drought Resistance Loci in Recombinant Lines of Iranian Oryza sativa L. in Germination Stage
BioTech 2021, 10(4), 26; https://0-doi-org.brum.beds.ac.uk/10.3390/biotech10040026 - 06 Nov 2021
Viewed by 353
Abstract
In order to locate control genes related to Oryza sativa L. traits at the germination stage under normal conditions and at drought stress levels (−4.5 and −9.0 bar), we evaluated 120 F8 generation offspring from the cross between two cultivars Neda × Ahlemitarum [...] Read more.
In order to locate control genes related to Oryza sativa L. traits at the germination stage under normal conditions and at drought stress levels (−4.5 and −9.0 bar), we evaluated 120 F8 generation offspring from the cross between two cultivars Neda × Ahlemitarum in a factorial experiment in a completely randomized block design with three replications in 2013 in the botanical laboratory of Gonbad Kavous University. A linkage map was prepared using 90 Simple Sequence Repeats (SSR) markers and 28 Inter Simple Sequence Repeats (ISSR), and 6 iPBS and 9 IRAP markers (265 polymorphic alleles). The results of the analysis of variance showed that all of the evaluated traits had a significant difference at the probability level of 1%. Hence, it can be noted that the desired genetic diversity can be found between genotypes. The results of the stepwise regression analysis for the germination percentage as a dependent variable and other traits as independent variables in the studied treatments showed that under normal conditions, there was variable coleoptile length, but under drought stress of −4.5 and −9.0 bar, the variable plumule dry weight entered the model. In this study, the markers included in RM1-RM490 and ISSR2-3-RM133 of chromosomes 1 and 6 of Oryza sativa were identified as the main regulators of traits associated with Oryza sativa drought resistance. In particular, they present the quantitative trait loci (QTL) that control the first stages of germination of Oryza sativa in water stress conditions. Full article
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Communication
In-Line Monitoring of Downstream Purification Processes for VSV Based SARS-CoV-2 Vaccine Using a Novel Technique
BioTech 2021, 10(4), 25; https://0-doi-org.brum.beds.ac.uk/10.3390/biotech10040025 - 03 Nov 2021
Viewed by 300
Abstract
The COVID-19 pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) increases the need for a rapid development of efficient vaccines. Among other vaccines in clinical trials, a recombinant VSV-∆G-spike vaccine was developed by the Israel Institute for Biological Research (IIBR) and [...] Read more.
The COVID-19 pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) increases the need for a rapid development of efficient vaccines. Among other vaccines in clinical trials, a recombinant VSV-∆G-spike vaccine was developed by the Israel Institute for Biological Research (IIBR) and is being evaluated. The development of an efficient downstream purification process (DSP) enables the vaccine to be advanced to clinical trials. The DSP must eliminate impurities, either process- or product-related, to yield a sufficient product with high purity, potency and quality. To acquire critical information on process restrictions and qualities, the application of in-line monitoring is vital and should significantly impact the process yield, product quality and economy of the entire process. Here, we describe an in-line monitoring technique that was applied in the DSP of the VSV-∆G-spike vaccine. The technique is based on determining the concentrations of metabolites, nutrients and a host cell protein using the automatic chemistry analyzer, Cobas Integra 400 Plus. The analysis revealed critical information on process parameters and significantly impacted purification processes. The technique is rapid, easy and efficient. Adopting this technique during the purification process improves the process yield and the product quality and enhances the economy of the entire downstream process for biotechnology and bio pharmaceutical products. Full article
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Article
Cell-Free Protein Synthesis by Diversifying Bacterial Transcription Machinery
BioTech 2021, 10(4), 24; https://0-doi-org.brum.beds.ac.uk/10.3390/biotech10040024 - 14 Oct 2021
Viewed by 387
Abstract
We have evaluated several approaches to increase protein synthesis in a cell-free coupled bacterial transcription and translation system. A strong pargC promoter, originally isolated from a moderate thermophilic bacterium Geobacillus stearothermophilus, was used to improve the performance of a cell-free system [...] Read more.
We have evaluated several approaches to increase protein synthesis in a cell-free coupled bacterial transcription and translation system. A strong pargC promoter, originally isolated from a moderate thermophilic bacterium Geobacillus stearothermophilus, was used to improve the performance of a cell-free system in extracts of Escherichia coli BL21 (DE3). A stimulating effect on protein synthesis was detected with extracts prepared from recombinant cells, in which the E. coli RNA polymerase subunits α, β, β’ and ω are simultaneously coexpressed. Appending a 3′ UTR genomic sequence and a T7 transcription terminator to the protein-coding region also improves the synthetic activity of some genes from linear DNA. The E. coli BL21 (DE3) rna::Tn10 mutant deficient in a periplasmic RNase I was constructed. The mutant cell-free extract increases by up to four-fold the expression of bacterial and human genes mediated from both bacterial pargC and phage pT7 promoters. By contrast, the RNase E deficiency does not affect the cell-free expression of the same genes. The regulatory proteins of the extremophilic bacterium Thermotoga, synthesized in a cell-free system, can provide the binding capacity to target DNA regions. The advantageous characteristics of cell-free systems described open attractive opportunities for high-throughput screening assays. Full article
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Article
Primer Binding Site (PBS) Profiling of Genetic Diversity of Natural Populations of Endemic Species Allium ledebourianum Schult.
BioTech 2021, 10(4), 23; https://0-doi-org.brum.beds.ac.uk/10.3390/biotech10040023 - 13 Oct 2021
Viewed by 430
Abstract
Endemic species are especially vulnerable to biodiversity loss caused by isolation or habitat specificity, small population size, and anthropogenic factors. Endemic species biodiversity analysis has a critically important global value for the development of conservation strategies. The rare onion Allium ledebourianum is a [...] Read more.
Endemic species are especially vulnerable to biodiversity loss caused by isolation or habitat specificity, small population size, and anthropogenic factors. Endemic species biodiversity analysis has a critically important global value for the development of conservation strategies. The rare onion Allium ledebourianum is a narrow-lined endemic species, with natural populations located in the extreme climatic conditions of the Kazakh Altai. A. ledebourianum populations are decreasing everywhere due to anthropogenic impact, and therefore, this species requires preservation and protection. Conservation of this rare species is associated with monitoring studies to investigate the genetic diversity of natural populations. Fundamental components of eukaryote genome include multiple classes of interspersed repeats. Various PCR-based DNA fingerprinting methods are used to detect chromosomal changes related to recombination processes of these interspersed elements. These methods are based on interspersed repeat sequences and are an effective approach for assessing the biological diversity of plants and their variability. We applied DNA profiling approaches based on conservative sequences of interspersed repeats to assess the genetic diversity of natural A. ledebourianum populations located in the territory of Kazakhstan Altai. The analysis of natural A. ledebourianum populations, carried out using the DNA profiling approach, allowed the effective differentiation of the populations and assessment of their genetic diversity. We used conservative sequences of tRNA primer binding sites (PBS) of the long-terminal repeat (LTR) retrotransposons as PCR primers. Amplification using the three most effective PBS primers generated 628 PCR amplicons, with an average of 209 amplicons. The average polymorphism level varied from 34% to 40% for all studied samples. Resolution analysis of the PBS primers showed all of them to have high or medium polymorphism levels, which varied from 0.763 to 0.965. Results of the molecular analysis of variance showed that the general biodiversity of A. ledebourianum populations is due to interpopulation (67%) and intrapopulation (33%) differences. The revealed genetic diversity was higher in the most distant population of A. ledebourianum LD64, located on the Sarymsakty ridge of Southern Altai. This is the first genetic diversity study of the endemic species A. ledebourianum using DNA profiling approaches. This work allowed us to collect new genetic data on the structure of A. ledebourianum populations in the Altai for subsequent development of preservation strategies to enhance the reproduction of this relict species. The results will be useful for the conservation and exploitation of this species, serving as the basis for further studies of its evolution and ecology. Full article
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Article
Highly Efficient Purification of Recombinant VSV-∆G-Spike Vaccine against SARS-CoV-2 by Flow-Through Chromatography
BioTech 2021, 10(4), 22; https://0-doi-org.brum.beds.ac.uk/10.3390/biotech10040022 - 12 Oct 2021
Cited by 2 | Viewed by 444
Abstract
This study reports a highly efficient, rapid one-step purification process for the production of the recombinant vesicular stomatitis virus-based vaccine, rVSV-∆G-spike (rVSV-S), recently developed by the Israel Institute for Biological Research (IIBR) for the prevention of COVID-19. Several purification strategies are evaluated using [...] Read more.
This study reports a highly efficient, rapid one-step purification process for the production of the recombinant vesicular stomatitis virus-based vaccine, rVSV-∆G-spike (rVSV-S), recently developed by the Israel Institute for Biological Research (IIBR) for the prevention of COVID-19. Several purification strategies are evaluated using a variety of chromatography methods, including membrane adsorbers and packed-bed ion-exchange chromatography. Cell harvest is initially treated with endonuclease, clarified, and further concentrated by ultrafiltration before chromatography purification. The use of anion-exchange chromatography in all forms results in strong binding of the virus to the media, necessitating a high salt concentration for elution. The large virus and spike protein binds very strongly to the high surface area of the membrane adsorbents, resulting in poor virus recovery (<15%), while the use of packed-bed chromatography, where the surface area is smaller, achieves better recovery (up to 33%). Finally, a highly efficient chromatography purification process with CaptoTM Core 700 resin, which does not require binding and the elution of the virus, is described. rVSV-S cannot enter the inner pores of the resin and is collected in the flow-through eluent. Purification of the rVSV-S virus with CaptoTM Core 700 resulted in viral infectivity above 85% for this step, with the efficient removal of host cell proteins, consistent with regulatory requirements. Similar results were obtained without an initial ultrafiltration step. Full article
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Review
Flow-FISH as a Tool for Studying Bacteria, Fungi and Viruses
BioTech 2021, 10(4), 21; https://0-doi-org.brum.beds.ac.uk/10.3390/biotech10040021 - 11 Oct 2021
Viewed by 408
Abstract
Many techniques are currently in use to study microbes. These can be aimed at detecting, identifying, and characterizing bacterial, fungal, and viral species. One technique that is suitable for high-throughput analysis is flow cytometry-based fluorescence in situ hybridization, or Flow-FISH. This technique employs [...] Read more.
Many techniques are currently in use to study microbes. These can be aimed at detecting, identifying, and characterizing bacterial, fungal, and viral species. One technique that is suitable for high-throughput analysis is flow cytometry-based fluorescence in situ hybridization, or Flow-FISH. This technique employs (fluorescently labeled) probes directed against DNA or (m)RNA, for instance targeting a gene or microorganism of interest and provides information on a single-cell level. Furthermore, by combining Flow-FISH with antibody-based protein detection, proteins of interest can be measured simultaneously with genetic material. Additionally, depending on the type of Flow-FISH assay, Flow-FISH can also be multiplexed, allowing for the simultaneous measurement of multiple gene targets and/or microorganisms. Together, this allows for, e.g., single-cell gene expression analysis or identification of (sub)strains in mixed cultures. Flow-FISH has been used in mammalian cells but has also been extensively employed to study diverse microbial species. Here, the use of Flow-FISH for studying microorganisms is reviewed. Specifically, the detection of (intracellular) pathogens, studying microorganism biology and disease pathogenesis, and identification of bacterial, fungal, and viral strains in mixed cultures is discussed, with a particular focus on the viruses EBV, HIV-1, and SARS-CoV-2. Full article
Communication
Searching for G-Quadruplex-Binding Proteins in Plants: New Insight into Possible G-Quadruplex Regulation
BioTech 2021, 10(4), 20; https://0-doi-org.brum.beds.ac.uk/10.3390/biotech10040020 - 22 Sep 2021
Viewed by 927
Abstract
G-quadruplexes are four-stranded nucleic acid structures occurring in the genomes of all living organisms and viruses. It is increasingly evident that these structures play important molecular roles; generally, by modulating gene expression and overall genome integrity. For a long period, G-quadruplexes have been [...] Read more.
G-quadruplexes are four-stranded nucleic acid structures occurring in the genomes of all living organisms and viruses. It is increasingly evident that these structures play important molecular roles; generally, by modulating gene expression and overall genome integrity. For a long period, G-quadruplexes have been studied specifically in the context of human promoters, telomeres, and associated diseases (cancers, neurological disorders). Several of the proteins for binding G-quadruplexes are known, providing promising targets for influencing G-quadruplex-related processes in organisms. Nonetheless, in plants, only a small number of G-quadruplex binding proteins have been described to date. Thus, we aimed to bioinformatically inspect the available protein sequences to find the best protein candidates with the potential to bind G-quadruplexes. Two similar glycine and arginine-rich G-quadruplex-binding motifs were described in humans. The first is the so-called “RGG motif”-RRGDGRRRGGGGRGQGGRGRGGGFKG, and the second (which has been recently described) is known as the “NIQI motif”-RGRGRGRGGGSGGSGGRGRG. Using this general knowledge, we searched for plant proteins containing the above mentioned motifs, using two independent approaches (BLASTp and FIMO scanning), and revealed many proteins containing the G4-binding motif(s). Our research also revealed the core proteins involved in G4 folding and resolving in green plants, algae, and the key plant model organism, Arabidopsis thaliana. The discovered protein candidates were annotated using STRINGdb and sorted by their molecular and physiological roles in simple schemes. Our results point to the significant role of G4-binding proteins in the regulation of gene expression in plants. Full article
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