scholarly journals Novel diagnostic tool for prediction of variant spliceogenicity derived from a set of 395 combined in silico/in vitro studies: an international collaborative effort

2018 ◽  
Vol 46 (15) ◽  
pp. 7913-7923 ◽  
Author(s):  
Raphaël Leman ◽  
Pascaline Gaildrat ◽  
Gérald Le Gac ◽  
Chandran Ka ◽  
Yann Fichou ◽  
...  
Keyword(s):  
In Silico ◽  
Mrna Level ◽  
Optimal Decision ◽  
Diagnostic Laboratory ◽  
Consensus Sequences ◽  
Site Consensus ◽  
Brca1 Brca2 ◽  
The Impact ◽  
Generation Sequencing

AbstractVariant interpretation is the key issue in molecular diagnosis. Spliceogenic variants exemplify this issue as each nucleotide variant can be deleterious via disruption or creation of splice site consensus sequences. Consequently, reliable in silico prediction of variant spliceogenicity would be a major improvement. Thanks to an international effort, a set of 395 variants studied at the mRNA level and occurring in 5′ and 3′ consensus regions (defined as the 11 and 14 bases surrounding the exon/intron junction, respectively) was collected for 11 different genes, including BRCA1, BRCA2, CFTR and RHD, and used to train and validate a new prediction protocol named Splicing Prediction in Consensus Elements (SPiCE). SPiCE combines in silico predictions from SpliceSiteFinder-like and MaxEntScan and uses logistic regression to define optimal decision thresholds. It revealed an unprecedented sensitivity and specificity of 99.5 and 95.2%, respectively, and the impact on splicing was correctly predicted for 98.8% of variants. We therefore propose SPiCE as the new tool for predicting variant spliceogenicity. It could be easily implemented in any diagnostic laboratory as a routine decision making tool to help geneticists to face the deluge of variants in the next-generation sequencing era. SPiCE is accessible at (https://sourceforge.net/projects/spicev2-1/).

scholarly journals Phycobilins as Potent Food Bioactive Broad-Spectrum Inhibitors Against Proteases of SARS-CoV-2 and Other Coronaviruses: A Preliminary Study

2021 ◽  
Vol 12 ◽  
Author(s):  
Brahmaiah Pendyala ◽  
Ankit Patras ◽  
Chandravanu Dash
Keyword(s):  
Binding Affinity ◽  
Broad Spectrum ◽  
In Silico ◽  
Research Area ◽  
Therapeutic Drug ◽  
Inhibitor Activity ◽  
Current Vaccine ◽  
Mutant Strains ◽  
The Impact

In the 21st century, we have witnessed three coronavirus outbreaks: SARS in 2003, MERS in 2012, and the ongoing pandemic coronavirus disease 2019 (COVID-19). The search for efficient vaccines and development and repurposing of therapeutic drugs are the major approaches in the COVID-19 pandemic research area. There are concerns about the evolution of mutant strains (e.g., VUI – 202012/01, a mutant coronavirus in the United Kingdom), which can potentially reduce the impact of the current vaccine and therapeutic drug development trials. One promising approach to counter the mutant strains is the “development of effective broad-spectrum antiviral drugs” against coronaviruses. This study scientifically investigates potent food bioactive broad-spectrum antiviral compounds by targeting main protease (Mpro) and papain-like protease (PLpro) proteases of coronaviruses (CoVs) using in silico and in vitro approaches. The results reveal that phycocyanobilin (PCB) shows potential inhibitor activity against both proteases. PCB had the best binding affinity to Mpro and PLpro with IC50 values of 71 and 62 μm, respectively. Also, in silico studies with Mpro and PLpro enzymes of other human and animal CoVs indicate broad-spectrum inhibitor activity of the PCB. As with PCB, other phycobilins, such as phycourobilin (PUB), phycoerythrobilin (PEB), and phycoviolobilin (PVB) show similar binding affinity to SARS-CoV-2 Mpro and PLpro.

scholarly journals Tec1p-Independent Activation of a Hypha-Associated Candida albicans Virulence Gene during Infection

2004 ◽  
Vol 72 (4) ◽  
pp. 2386-2389 ◽  
Author(s):  
Peter Staib ◽  
Ayfer Binder ◽  
Marianne Kretschmar ◽  
Thomas Nichterlein ◽  
Klaus Schröppel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Candida Albicans ◽  
Animal Models ◽  
Environmental Conditions ◽  
Deletion Mutant ◽  
Virulence Gene ◽  
Consensus Sequences ◽  
Site Consensus

ABSTRACT The Tec1p transcription factor is involved in the expression of hypha-specific genes in Candida albicans. Although the induction of the hypha-associated SAP5 gene by serum in vitro depends on Tec1p, deletion of all Tec1p binding site consensus sequences from the SAP5 promoter did not affect its activation. In two different animal models of candidiasis, the SAP5 promoter was induced even in a Δtec1 deletion mutant, demonstrating that the requirement for Tec1p in gene expression in C. albicans depends on the environmental conditions within the host.

scholarly journals An in silico and in vitro approach to elucidate the impact of residues flanking the cleavage scissile bonds of FVIII

PLoS ONE ◽  
2017 ◽  
Vol 12 (7) ◽  
pp. e0180456 ◽  
Author(s):  
Behnaz Pezeshkpoor ◽  
Ursula Schreck ◽  
Arijit Biswas ◽  
Julia Driesen ◽  
Ann-Cristin Berkemeier ◽  
...  
Keyword(s):  
In Silico ◽  
The Impact

scholarly journals Regulation of translation by upstream translation initiation codons of surfactant protein A1 splice variants

2015 ◽  
Vol 308 (1) ◽  
pp. L58-L75 ◽  
Author(s):  
Nikolaos Tsotakos ◽  
Patricia Silveyra ◽  
Zhenwu Lin ◽  
Neal Thomas ◽  
Mudit Vaid ◽  
...  
Keyword(s):  
In Silico ◽  
Splice Variants ◽  
Surfactant Protein ◽  
In Vitro Translation ◽  
Start Codon ◽  
Translation Efficiency ◽  
Human Lung Tissue ◽  
In Vitro Techniques ◽  
The Impact

Surfactant protein A (SP-A), a molecule with roles in lung innate immunity and surfactant-related functions, is encoded by two genes in humans: SFTPA1 (SP-A1) and SFTPA2 (SP-A2). The mRNAs from these genes differ in their 5′-untranslated regions (5′-UTR) due to differential splicing. The 5′-UTR variant ACD′ is exclusively found in transcripts of SP-A1, but not in those of SP-A2. Its unique exon C contains two upstream AUG codons (uAUGs) that may affect SP-A1 translation efficiency. The first uAUG (u1) is in frame with the primary start codon (p), but the second one (u2) is not. The purpose of this study was to assess the impact of uAUGs on SP-A1 expression. We employed RT-qPCR to determine the presence of exon C-containing SP-A1 transcripts in human RNA samples. We also used in vitro techniques including mutagenesis, reporter assays, and toeprinting analysis, as well as in silico analyses to determine the role of uAUGs. Exon C-containing mRNA is present in most human lung tissue samples and its expression can, under certain conditions, be regulated by factors such as dexamethasone or endotoxin. Mutating uAUGs resulted in increased luciferase activity. The mature protein size was not affected by the uAUGs, as shown by a combination of toeprint and in silico analysis for Kozak sequence, secondary structure, and signal peptide and in vitro translation in the presence of microsomes. In conclusion, alternative splicing may introduce uAUGs in SP-A1 transcripts, which in turn negatively affect SP-A1 translation, possibly affecting SP-A1/SP-A2 ratio, with potential for clinical implication.

scholarly journals Andean Berry (Vaccinium meridionale Swartz) Juice in Combination With Aspirin Modulated Apoptotic Signaling in Colon Cancer In Vitro and In Vivo

2021 ◽  
Vol 5 (Supplement_2) ◽  
pp. 261-261
Author(s):  
Sandra Arango-Varela ◽  
Ivan Luzardo ◽  
Maria Maldonado-Celis
Keyword(s):  
Colorectal Cancer ◽  
Colon Cancer ◽  
In Silico ◽  
Aberrant Crypt Foci ◽  
Apoptotic Signaling ◽  
Polyphenolic Composition ◽  
Apoptotic Effects ◽  
The Impact

Abstract Objectives This research aimed to assess the impact of Andean Berry (Vaccinium meridionale Swartz) juice (ABJ) in combination with Aspirin in the apoptotic signaling in colon cancer in vitro and in vivo. We hypothesized that ABJ + Aspirin would produce the most effective anti-proliferative and pro-apoptotic effects in vitro and in vivo. Methods The polyphenolic composition of ABJ was carried out by HPLC-DAD. ABJ (0–30% v/v), Aspirin (0–20 mM), and their mixture were evaluated for their pro-apoptotic effects in human SW480 colorectal cancer cells, followed by human apoptosis proteomic and bioinformatic analysis and in silico docking potential between ABJ components and selected pro-apoptotic targets. For the in vivo assays, colorectal cancer was induced with two injections (separated 1 week each) of azoxymethane (AOM: 15 mg/kg body weight, BW), and treatments were evaluated for its chemopreventive and chemoprotective effects. Hence, 30 male and female Balb/c mice were randomly divided in 5 groups: negative control (basal diet, BD); and four AOM-induced groups: positive control (BD), Aspirin (25 mg/kg BW + BD), ABJ (30% v/v in drinking water ABJ + BD), and ABJ + Aspirin (30% v/v ABJ + 25 mg/kg BW Aspirin + BD). Macroscopic and histopathological parameters were evaluated in vivo. Results The mixture displayed the highest antiproliferative effects (+46%), arrested cell cycle at the G2/M phase, decreased cloning efficiency, but reduced Caspase 3/7 activity, suggesting an alternative apoptotic pathway, compared to untreated SW480 cells. Several pro-apoptotic (cytochrome C, TNFRSF1A, Bax, and Bad) and anti-apoptotic (Hsp70/Hsp32) proteins were decreased. ABJ flavonoids (rutin and kaempferol) exhibited the highest in silico affinity with proteins like TRAILR2 or Catalase. Both chemopreventive and chemoprotective approaches showed similar body/liver weight outcomes, but the mixture displayed the strongest aberrant crypt foci reduction in vivo. The chemopreventive approach was more effective in protecting the colon from AOM. Conclusions Results suggested the potential of ABJ to reduce Aspirin use in the alleviation of colorectal cancer markers in vitro and in vivo, modulating alternate pro-apoptotic signaling. Funding Sources The funding provided by COLCIENCIAS and DGAPA-CTIC-UNAM is appreciated.

scholarly journals Meta analysis of variant predictions in congenital adrenal hyperplasia caused by mutations in CYP21A2.

2021 ◽  
Author(s):  
Mayara Jorgens Prado ◽  
Rodrigo Ligabue-Braun ◽  
Arnaldo Zaha ◽  
Maria Lucia Rosa Rossetti ◽  
Amit V Pandey
Keyword(s):  
Congenital Adrenal Hyperplasia ◽  
In Silico ◽  
Meta Analysis ◽  
Genetic Disorders ◽  
In Silico Prediction ◽  
Adrenal Hyperplasia ◽  
Single Nucleotide Variants ◽  
Mild Phenotype ◽  
The Impact

Context: CYP21A2 deficiency represents 95% of congenital adrenal hyperplasia cases (CAH), a group of genetic disorders that affect steroid biosynthesis. The genetic and functional analysis provides critical tools to elucidate complex CAH cases. One of the most accessible tools to infer the pathogenicity of new variants is in silico prediction. Objective: Analyze the performance of in silico prediction tools to categorize missense single nucleotide variants (SNVs) of the CYP21A2. Methods: SNVs of the CYP21A2 characterized in vitro by functional assays were selected to assess the performance of online single and meta predictors. SNVs were tested separately or in combination with the related phenotype (severe or mild CAH form). In total, 103 SNVs of the CYP21A2 (90 pathogenic and 13 neutral) were used to test the performance of 13 single-predictors and four meta-predictors. Results: SNVs associated with the severe phenotypes were well categorized by all tools, with an accuracy between 0.69 (PredictSNP2) and 0.97 (CADD), and Matthews' correlation coefficient (MCC) between 0.49 (PoredicSNP2) and 0.90 (CADD). However, SNVs related to the mild phenotype had more variation, with the accuracy between 0.47 (S3Ds&GO and MAPP) and 0.88 (CADD), and MCC between 0.18 (MAPP) and 0.71 (CADD). Conclusion: From our analysis, we identified four predictors of CYP21A2 pathogenicity with good performance. These results can be used for future analysis to infer the impact of uncharacterized SNVs' in CYP21A2.

scholarly journals Optimization of the Transwell® System for Assessing the Dissolution Behavior of Orally Inhaled Drug Products through In Vitro and In Silico Approaches

Pharmaceutics ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1109
Author(s):  
Elham Amini ◽  
Abhinav Kurumaddali ◽  
Sharvari Bhagwat ◽  
Simon M. Berger ◽  
Günther Hochhaus
Keyword(s):  
In Silico ◽  
Mass Effect ◽  
Dissolution Behavior ◽  
Experimental Conditions ◽  
Drug Products ◽  
Starting Point ◽  
Orally Inhaled Drug Products ◽  
Model Drug ◽  
The Impact

The aim of this study was to further evaluate and optimize the Transwell® system for assessing the dissolution behavior of orally inhaled drug products (OIDPs), using fluticasone propionate as a model drug. Sample preparation involved the collection of a relevant inhalable dose fraction through an anatomical mouth/throat model, resulting in a more uniform presentation of drug particles during the subsequent dissolution test. The method differed from previously published procedures by (1) using a 0.4 µm polycarbonate (PC) membrane, (2) stirring the receptor compartment, and (3) placing the drug-containing side of the filter paper face downwards, towards the PC membrane. A model developed in silico, paired with the results of in vitro studies, suggested that a dissolution medium providing a solubility of about 5 µg/mL would be a good starting point for the method’s development, resulting in mean transfer times that were about 10 times longer than those of a solution. Furthermore, the model suggested that larger donor/receptor and sampling volumes (3, 3.3 and 2 mL, respectively) will significantly reduce the so-called “mass effect”. The outcomes of this study shed further light on the impact of experimental conditions on the complex interplay of dissolution and diffusion within a volume-limited system, under non-sink conditions.

scholarly journals In silico ADME in drug design – enhancing the impact

ADMET & DMPK ◽  
2018 ◽  
Vol 6 (1) ◽  
pp. 15 ◽  
Author(s):  
Susanne Winiwarter ◽  
Ernst Ahlberg ◽  
Edmund Watson ◽  
Ioana Oprisiu ◽  
Mickael Mogemark ◽  
...  
Keyword(s):  
In Silico ◽  
In Vitro Assays ◽  
Lead Compounds ◽  
Human Dose ◽  
Pharmacokinetic Properties ◽  
In Silico Predictions ◽  
In Silico Models ◽  
The Impact ◽  
Selection Of

<p>Each year the pharmaceutical industry makes thousands of compounds, many of which do not meet the desired efficacy or pharmacokinetic properties, describing the absorption, distribution, metabolism and excretion (ADME) behavior. Parameters such as lipophilicity, solubility and metabolic stability can be measured in high throughput in vitro assays. However, a compound needs to be synthesized in order to be tested. In silico models for these endpoints exist, although with varying quality. Such models can be used before synthesis and, together with a potency estimation, influence the decision to make a compound. In practice, it appears that often only one or two predicted properties are considered prior to synthesis, usually including a prediction of lipophilicity. While it is important to use all information when deciding which compound to make, it is somewhat challenging to combine multiple predictions unambiguously. This work investigates the possibility of combining in silico ADME predictions to define the minimum required potency for a specified human dose with sufficient confidence. Using a set of drug discovery compounds,in silico predictions were utilized to compare the relative ranking based on minimum potency calculation with the outcomes from the selection of lead compounds. The approach was also tested on a set of marketed drugs and the influence of the input parameters investigated.</p>

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