protein variant
Recently Published Documents


TOTAL DOCUMENTS

261
(FIVE YEARS 125)

H-INDEX

36
(FIVE YEARS 6)

2022 ◽  
Vol 14 ◽  
Author(s):  
Despina Tsortouktzidis ◽  
Anna R. Tröscher ◽  
Herbert Schulz ◽  
Thoralf Opitz ◽  
Susanne Schoch ◽  
...  

Precise genome editing in combination with viral delivery systems provides a valuable tool for neuroscience research. Traditionally, the role of genes in neuronal circuits has been addressed by overexpression or knock-out/knock-down systems. However, those techniques do not manipulate the endogenous loci and therefore have limitations. Those constraints include that many genes exhibit extensive alternative splicing, which can be regulated by neuronal activity. This complexity cannot be easily reproduced by overexpression of one protein variant. The CRISPR activation and interference/inhibition systems (CRISPRa/i) directed to promoter sequences can modulate the expression of selected target genes in a highly specific manner. This strategy could be particularly useful for the overexpression of large proteins and for alternatively spliced genes, e.g., for studying large ion channels known to be affected in ion channelopathies in a variety of neurological diseases. Here, we demonstrate the feasibility of a newly developed CRISPRa/i toolbox to manipulate the promoter activity of the Cacna1h gene. Impaired, function of the low-voltage-activated T-Type calcium channel CaV3.2 is involved in genetic/mutational as well as acquired/transcriptional channelopathies that emerge with epileptic seizures. We show CRISPR-induced activation and inhibition of the Cacna1h locus in NS20Y cells and primary cortical neurons, as well as activation in mouse organotypic slice cultures. In future applications, the system offers the intriguing perspective to study functional effects of gain-of-function or loss-of-function variations in the Cacna1h gene in more detail. A better understanding of CaV3.2 channelopathies might result in a major advancement in the pharmacotherapy of CaV3.2 channelopathy diseases.


2022 ◽  
Vol 42 ◽  
pp. 01009
Author(s):  
E. Yu. Podlasova ◽  
S. V. Lebedev

It is impossible to organize proper feeding of farm animals without a detailed study of the composition of the nutritional content of feed. Legume-grass mixtures occupy one of main places in solving the problem of high-quality feed production. Since mixed crops of legumes and cereals retain high fodder quality of green mass due to high protein content in legumes. To meet the need for a high-quality feed base, it is necessary to look for ways to increase the yield of fodder crops. The use of innovative technologies is one of the components of modern crop production. The aim of the study was to study the methods of seed treatment in conjunction with the technology of mixed crops; it allows obtaining a green mass with the desired productive properties. Thus, the article presents the materials of a field study on the development of a highly productive green feed. The results of the study of combined and mixed crops on the nutritional value and productivity of annual forages of peas, millet, and barley using pre-sowing seed treatment with ultrafine particles of SiO2; MoO2; Fe3O4 and growth regulators AgroVerm and RibavExtra were obtained. As a result of the experiment, it was shown that the use of pre-sowing treatment of seeds with ultradispersed SiO2 particles in combined crops showed the maximum leaf surface area of 85.5 thousand m2/ha. By the time of harvesting, the safety of plants on variants with the use of SiO2; Fe3O4; MoO2 was 88.4%; 87.5%; 86.5% respectively. According to the collection of digestible protein, variant with the use of ultrafine particles SiO2 was 20% in both sowing methods. Low protein content was 16.1%; 16.4% in the control variants respectively. According to the content of feed units in 1 kg of dry matter, SiO2 variants prevail with 0.91. Thus, the research is the basis for possible studying of combined and mixed crops with the use of pre-sowing seed treatment with ultrafine particles. It allows obtaining high-quality feed.


Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2548
Author(s):  
Gabriel E. Wagner ◽  
Massimo G. Totaro ◽  
André Volland ◽  
Michaela Lipp ◽  
Sabine Saiger ◽  
...  

Rapid molecular surveillance of SARS-CoV-2 S-protein variants leading to immune escape and/or increased infectivity is of utmost importance. Among global bottlenecks for variant monitoring in diagnostic settings are sequencing and bioinformatics capacities. In this study, we aimed to establish a rapid and user-friendly protocol for high-throughput S-gene sequencing and subsequent automated identification of variants. We designed two new primer pairs to amplify only the immunodominant part of the S-gene for nanopore sequencing. Furthermore, we developed an automated “S-Protein-Typer” tool that analyzes and reports S-protein mutations on the amino acid level including a variant of concern indicator. Validation of our primer panel using SARS-CoV-2-positive respiratory specimens covering a broad Ct range showed successful amplification for 29/30 samples. Restriction to the region of interest freed sequencing capacity by a factor of 12–13, compared with whole-genome sequencing. Using either the MinION or Flongle flow cell, our sequencing strategy reduced the time required to identify SARS-CoV-2 variants accordingly. The S-Protein-Typer tool identified all mutations correctly when challenged with our sequenced samples and 50 deposited sequences covering all VOCs (December 2021). Our proposed S-protein variant screening offers a simple, more rapid, and low-cost entry into NGS-based SARS-CoV-2 analysis, compared with current whole-genome approaches.


PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0260957
Author(s):  
Edit Szabó ◽  
Anna Kulin ◽  
Orsolya Mózner ◽  
László Korányi ◽  
Botond Literáti-Nagy ◽  
...  

Type 2 diabetes mellitus (T2DM) is a complex metabolic disease and variations in multispecific membrane transporter functions may affect T2DM development, complications or treatment. In this work we have analyzed the potential effects of a major polymorphism, the Q141K variant of the ABCG2 transporter in T2DM. The ABCG2 protein is a multispecific xeno- and endobiotic transporter, affecting drug metabolism and playing a key role in uric acid extrusion. The ABCG2-Q141K variant, with reduced expression level and function, is present in 15–35% of individuals, depending on the genetic background of the population, and has been shown to significantly affect gout development. Several other diseases, including hypertension, chronic renal failure, and T2DM have also been reported to be associated with high serum uric acid levels, suggesting that ABCG2 may also play a role in these conditions. In this work we have compared relatively small cohorts (n = 203) of T2DM patients (n = 99) and healthy (n = 104) individuals regarding the major laboratory indicators of T2DM and determined the presence of the SNP rs2231142 (C421A), resulting the ABCG2-Q141K protein variant. We found significantly higher blood glucose and HbA1c levels in the T2DM patients carrying the ABCG2-Q141K variant. These findings may emphasize the potential metabolic role of ABCG2 in T2DM and indicate that further research should explore how prevention and treatment of this disease may be affected by the frequent polymorphism of ABCG2.


2021 ◽  
Author(s):  
J. Steen Hoyer ◽  
Olivia W. Wilkins ◽  
Aanandi Munshi ◽  
Emma Wiese ◽  
Divya Dubey ◽  
...  

Cabbage leaf curl virus (CabLCV) has a bipartite single-stranded DNA genome and infects the model plant Arabidopsis thaliana. CabLCV serves as a model for the genus Begomovirus, members of which cause tremendous crop losses worldwide. We have used CabLCV as a model for within-plant virus evolution by inoculating individual plants with infectious clones of both wild-type and mutagenized versions of the CabLCV genome. Consistent with previous reports, detrimental substitutions in the Replication-associated gene (Rep) were readily compensated for by direct reversion and/or alternative mutations. A surprising number of common mutations were detected elsewhere in both viral segments (DNA-A and DNA-B) indicating convergent evolution and suggesting that CabLCV may not be as well adapted to A. thaliana as commonly presumed. Consistent with this idea, a spontaneous coat protein variant consistently rose to higher allele frequency in a hypersusceptible A. thaliana accession (Sei-0) than in another susceptible accession (Col-0). Numerous high-frequency mutations were also detected in a candidate Rep binding site in DNA-B. Our results reinforce the fact that spontaneous mutation of this type of virus occurs rapidly and can change the majority consensus sequence of a within-plant virus population in weeks.


2021 ◽  
Author(s):  
Bruce J Wittmann ◽  
Kadina E Johnston ◽  
Patrick J Almhjell ◽  
Frances H Arnold

Widespread availability of protein sequence-fitness data would revolutionize both our biochemical understanding of proteins and our ability to engineer them. Unfortunately, even though thousands of protein variants are generated and evaluated for fitness during a typical protein engineering campaign, most are never sequenced, leaving a wealth of potential sequence-fitness information untapped. This largely stems from the fact that sequencing is unnecessary for many protein engineering strategies; the added cost and effort of sequencing is thus unjustified. Here, we present every variant sequencing (evSeq), an efficient protocol for sequencing a variable region within every variant gene produced during a protein engineering campaign at a cost of cents per variant. Execution of evSeq is simple, requires no sequencing experience to perform, relies only on resources and services typically available to biology labs, and slots neatly into existing protein engineering workflows. Analysis of evSeq data is likewise made simple by its accompanying software (found at github.com/fhalab/evSeq, documentation at fhalab.github.io/evSeq), which can be run on a personal laptop and was designed to be accessible to users with no computational experience. Low-cost and easy to use, evSeq makes collection of extensive protein variant sequence-fitness data practical.


2021 ◽  
Author(s):  
Alexandra M Blee ◽  
Bian Li ◽  
Turner Pecen ◽  
Jens Meiler ◽  
Zachary D Nagel ◽  
...  

For precision medicine to reach its full potential for treatment of cancer and other diseases, protein variant effect prediction tools are needed that characterize variants of unknown significance (VUS) in a patient's genome with respect to their likelihood to influence treatment response and outcomes. However, the performance of most variant prediction tools is limited by the difficulty of acquiring sufficient training and validation data. To overcome these limitations, we applied an iterative active learning approach starting from available biochemical, evolutionary, and functional annotations. The potential of active learning to improve variant interpretation was first demonstrated by applying it to synthetic and deep mutational scanning (DMS) datasets for four cancer-relevant proteins. We then probed its utility to guide interpretation and functional validation of tumor VUS in a potential biomarker for cancer therapy sensitivity, the nucleotide excision repair (NER) protein Xeroderma Pigmentosum Complementation Group A (XPA). A quantitative high-throughput cell-based NER activity assay, fluorescence-based multiplex flow-cytometric host cell reactivation (FM-HCR), was used to validate XPA VUS selected by the active learning strategy. In all cases, selecting VUS for validation by active learning yielded an improvement in performance over traditional learning. These analyses suggest that active learning is well-suited to significantly improve interpretation of VUS and cancer patient genomes.


2021 ◽  
Vol 8 ◽  
Author(s):  
Ana M. Esteves ◽  
Efthymia Papaevangelou ◽  
Dorota Smolarek ◽  
Prokar Dasgupta ◽  
Christine Galustian

Interleukin-15 (IL-15) is a cytokine previously suggested as a potential immunotherapy for cancer treatment. IL-15 can effectively reduce tumor growth in many preclinical tumor models including prostate cancer. This is due to its ability to expand and activate immune cells, such as CD8+ T cells and natural killer cells. To increase the potency of IL-15, we have engineered a protein variant that can be modified to localize and be retained in tissues where it is administered. However, the production of recombinant IL-15, the purity, and correct refolding of the final protein is not always ideal. In the current study, we aimed to optimize the methodology for production and purification of a modified recombinant human IL-15 and investigate the efficacy of the produced protein in the treatment of prostate tumors. Human IL-15 with its polypeptide sequence modified at the C-terminus to enable thiol conjugation with membrane localizing peptides, was produced in E. coli and purified using mild denaturing conditions (2M urea) from a washing step or from solubilization of inclusion bodies. The purified protein from the wash fraction was conjugated to a myristoylated peptide to form a membrane-localizing IL-15 (cyto-IL-15). The efficacy of cyto-IL-15 was investigated in subcutaneous TRAMP-C2 prostate tumors in mice and compared with cyto-IL-15 derived from protein purified from inclusion bodies (cyto-IL-15 Gen). When mild denaturing conditions were used for purification, the largest amount of IL-15 was collected from the wash fraction and a smaller amount from inclusion bodies. The protein from the wash fraction was mainly present as a monomer, whereas the one from inclusion bodies formed homodimers and higher complexes. After cytotopic modification, the purified IL-showed great efficacy in delaying prostate tumor growth (∼50%) and increased mice survival by ∼1.8-fold compared with vehicle. This study demonstrates an alternative, inexpensive and efficient method to produce and purify a modified version of IL-15 using mild denaturing conditions. This IL-15, when cytotopically modified, showed great efficacy as a monotherapy in prostate tumors in mice further highlighting the potential of IL-15 as a cancer immunotherapy.


2021 ◽  
Author(s):  
Juan A Perez-Bermejo ◽  
Luke M Judge ◽  
Christina L Jensen ◽  
Kenneth Wu ◽  
Annie Truong ◽  
...  

AbstractMultiple genetic association studies have correlated a common allelic block linked to the BAG3 gene with a decreased incidence of heart failure, but the molecular mechanism for such protection remains elusive. One of the variants in this allele block is coding, changing cysteine to arginine at position 151 of BAG3 (rs2234962-BAG3C151R). Here, we use induced pluripotent stem cells (iPSC) to test if the BAG3C151R variant alters protein and cellular function in human cardiac myocytes. Quantitative protein interaction network analysis identified specific changes in BAG3C151R protein interaction partners in cardiomyocytes but not in iPSCs or an immortalized cell line. Knockdown of BAG3 interacting factors in cardiomyocytes followed by myofibrillar analysis revealed that BAG3C151R associates more strongly with proteins involved in the maintenance of myofibrillar integrity. Finally, we demonstrate that cardiomyocytes expressing the BAG3C151R variant have improved response to proteotoxic stress in an allele dose-dependent manner. This study suggests that the BAG3C151R variant increases cardiomyocyte protection from stress by enhancing the recruitment of factors critical to the maintenance of myofibril integrity, hinting that this variant could be responsible for the cardioprotective effect of the haplotype block. By revealing specific changes in preferential binding partners of the BAG3C151R protein variant, we also identify potential targets for the development of novel cardioprotective therapies.


Biomedicines ◽  
2021 ◽  
Vol 9 (10) ◽  
pp. 1390
Author(s):  
Giovanna Cardiero ◽  
Gennaro Musollino ◽  
Romeo Prezioso ◽  
Giuseppina Lacerra

An insertion or deletion of a nucleotide (nt) in the penultimate or the last exon can result in a frameshift and premature termination codon (PTC), giving rise to an unstable protein variant, showing a dominant phenotype. We described two α-globin mutants created by the deletion of a nucleotide in the penultimate or the last exon of the α1-globin gene: the Hb Campania or α1 cod95 (−C), causing a frameshift resulting in a PTC at codon 102, and the Hb Sciacca or α1 cod109 (−C), causing a frameshift and formation of a PTC at codon 133. The carriers showed α-thalassemia alterations (mild microcytosis with normal Hb A2) and lacked hemoglobin variants. The 3D model indicated the α-chain variants’ instability, due to the severe structural alterations with impairment of the chaperone alpha-hemoglobin stabilizing protein (AHSP) interaction. The qualitative and semiquantitative analyses of the α1mRNA from the reticulocytes of carriers highlighted a reduction in the variant cDNAs that constituted 34% (Hb Campania) and 15% (Hb Sciacca) of the total α1-globin cDNA, respectively. We developed a workflow for the in silico analysis of mechanisms triggering no-go decay, and its results suggested that the reduction in the variant mRNA was likely due to no-go decay caused by the presence of a rare triplet, and, in the case of Hb Sciacca, also by the mRNA’s secondary structure variation. It would be interesting to correlate the phenotype with the quantity of other frameshift mRNA variants, but very few data concerning α- and β-globin variants are available.


Sign in / Sign up

Export Citation Format

Share Document