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1.
J Matern Fetal Neonatal Med ; 34(1): 99-104, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32429717

RESUMO

Background: Intrauterine adhesions are usually detected incidentally during routine obstetric ultrasound and remain one of the reasons for concern for both clinicians and patients.Objective: Our objective was to document ultrasonographic findings of intrauterine adhesions detected in obstetric ultrasound and to investigate their correlation with obstetric history.Study Design: Detailed scans were performed in 685 singleton pregnancies at 16-24 weeks' gestation. Intrauterine adhesion was referred to as "adhesion-membrane complex'' (AMC). Patients were divided into three groups: Group I consisted of patients with ≥1 therapeutic D&C associated with pregnancy but with neither vaginal delivery nor Cesarean section (CS). Group II consisted of patients with ≥1 CS but with neither vaginal delivery nor therapeutic D&C associated with pregnancy. Group III consisted of patients who were in their first pregnancy. Ultrasonographic properties of AMC and relationship between AMCs and obstetric history were investigated.Results: The incidence of AMC in Group I (n = 108), Group II (n = 189), and Group III (n = 388) was 11.1% (n = 12), 1.05% (n = 2) and 1.03% (n = 4), respectively. Positive history of D&C is associated with significantly increased risk of AMC (risk ratio:10.778; 95% confidence interval: 3.55-32.75). Also, previous history of CS is not associated with significantly increased risk of AMC (risk ratio: 1.026; 95% confidence interval: 0.19-5.55). The AMCs were located in the upper half in 7 (38,9%) and in the lower half of the uterus in 11 (61.1%) patients. The midpoint thickness of the AMC was between 0.75 and 5.10 mm (mean: 2.65 mm; SD ± 1.2). The width of the AMC was between 2 and 52 mm (mean: 20.98; SD ± 15.3), the heights of the AMCs were 5-60 mm (mean: 33.27 mm; SD ±17.0). In ten of the AMC positive patients (55.6%) a thick and bulbous free end and in eleven of them (61.1%) a "Y image" was detected. The mean gestational age at birth was 37.4 (SD ± 3.3) weeks in 18 patients with AMC. There were no intrauterine fetal or perinatal deaths. None of the neonates had congenital abnormalities.Conclusions: Intrauterine adhesions detected in obstetric ultrasonography were redefined and renamed in a more comprehensible manner. Our results pointed out that while the positive history of D&C is associated with significantly increased risk of AMC, previous history of CS is not associated with significantly increased risk of AMC.

2.
Acta Pharm ; 71(2): 163-174, 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-33151166

RESUMO

The current outbreak of novel coronavirus (COVID-19) infections urges the need to identify potential therapeutic agents. Therefore, the repurposing of FDA-approved drugs against today's diseases involves the use of de-risked compounds with potentially lower costs and shorter development timelines. In this study, the recently resolved X-ray crystallographic structure of COVID-19 main protease (Mpro) was used to generate a pharmacophore model and to conduct a docking study to capture antiviral drugs as new promising COVID-19 main protease inhibitors. The developed pharmacophore successfully captured five FDA-approved antiviral drugs (lopinavir, remdesivir, ritonavir, saquinavir and raltegravir). The five drugs were successfully docked into the binding site of COVID-19 Mpro and showed several specific binding interactions that were comparable to those tying the co-crystallized inhibitor X77 inside the binding site of COVID-19 Mpro. Three of the captured drugs namely, remdesivir, lopinavir and ritonavir, were reported to have promising results in COVID-19 treatment and therefore increases the confidence in our results. Our findings suggest an additional possible mechanism of action for remdesivir as an antiviral drug inhibiting COVID-19 Mpro. Additionally, a combination of structure-based pharmacophore modeling with a docking study is expected to facilitate the discovery of novel COVID-19 Mpro inhibitors.


Assuntos
Infecções por Coronavirus/enzimologia , Pneumonia Viral/enzimologia , Inibidores de Proteases/farmacologia , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/química , Monofosfato de Adenosina/farmacologia , Monofosfato de Adenosina/uso terapêutico , Alanina/análogos & derivados , Alanina/química , Alanina/farmacologia , Alanina/uso terapêutico , Antivirais/química , Antivirais/farmacologia , Infecções por Coronavirus/tratamento farmacológico , Cristalografia por Raios X , Descoberta de Drogas/métodos , Reposicionamento de Medicamentos , Humanos , Modelos Químicos , Simulação de Acoplamento Molecular , Estrutura Molecular , Pandemias , Pneumonia Viral/tratamento farmacológico , Inibidores de Proteases/química , Relação Estrutura-Atividade
3.
Acta Pharm ; 71(2): 175-184, 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-33151168

RESUMO

Recently, an outbreak of a fatal coronavirus, SARS-CoV-2, has emerged from China and is rapidly spreading worldwide. Possible interaction of SARS-CoV-2 with DPP4 peptidase may partly contribute to the viral pathogenesis. An integrative bioinformatics approach starting with mining the biomedical literature for high confidence DPP4-protein/gene associations followed by functional analysis using network analysis and pathway enrichment was adopted. The results indicate that the identified DPP4 networks are highly enriched in viral processes required for viral entry and infection, and as a result, we propose DPP4 as an important putative target for the treatment of COVID-19. Additionally, our protein-chemical interaction networks identified important interactions between DPP4 and sitagliptin. We conclude that sitagliptin may be beneficial for the treatment of COVID-19 disease, either as monotherapy or in combination with other therapies, especially for diabetic patients and patients with pre-existing cardiovascular conditions who are already at higher risk of COVID-19 mortality.


Assuntos
Infecções por Coronavirus/tratamento farmacológico , Dipeptidil Peptidase 4/efeitos dos fármacos , Inibidores da Dipeptidil Peptidase IV/farmacologia , Inibidores da Dipeptidil Peptidase IV/uso terapêutico , Pneumonia Viral/tratamento farmacológico , Fosfato de Sitagliptina/farmacologia , Fosfato de Sitagliptina/uso terapêutico , Doenças Cardiovasculares/complicações , Doenças Cardiovasculares/tratamento farmacológico , Biologia Computacional , Infecções por Coronavirus/complicações , Cristalografia por Raios X , Mineração de Dados , Complicações do Diabetes/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Reposicionamento de Medicamentos , Redes Reguladoras de Genes , Humanos , Estrutura Molecular , Pandemias , Pneumonia Viral/complicações
4.
Life Sci Alliance ; 4(1)2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33158978

RESUMO

Compared with the ubiquitous expression of type I (IFNα and IFNß) interferon receptors, type III (IFNλ) interferon receptors are mainly expressed in epithelial cells of mucosal barriers of the of the intestine and respiratory tract. Consequently, IFNλs are important for innate pathogen defense in the lung and intestine. IFNλs also determine the outcome of hepatitis C virus (HCV) infections, with IFNλ4 inhibiting spontaneous clearance of HCV. Because viral clearance is dependent on T cells, we explored if IFNλs can directly bind to and regulate human T cells. We found that human B cells and CD8+ T cells express the IFNλ receptor and respond to IFNλs, including IFNλ4. IFNλs were not inhibitors but weak stimulators of B- and T-cell responses. Furthermore, IFNλ4 showed neither synergistic nor antagonistic effects in co-stimulatory experiments with IFNλ1 or IFNα. Multidimensional flow cytometry of cells from liver biopsies of hepatitis patients from IFNλ4-producers showed accumulation of activated CD8+ T cells with a central memory-like phenotype. In contrast, CD8+ T cells with a senescent/exhausted phenotype were more abundant in IFNλ4-non-producers. It remains to be elucidated how IFNλ4 promotes CD8 T-cell responses and inhibits the host immunity to HCV infections.

5.
Talanta ; 222: 121444, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33167198

RESUMO

The development of simple detection methods aimed at widespread screening and testing is crucial for many infections and diseases, including prostate cancer where early diagnosis increases the chances of cure considerably. In this paper, we report on genosensors with different detection principles for a prostate cancer specific DNA sequence (PCA3). The genosensors were made with carbon printed electrodes or quartz coated with layer-by-layer (LbL) films containing gold nanoparticles and chondroitin sulfate and a layer of a complementary DNA sequence (PCA3 probe). The highest sensitivity was reached with electrochemical impedance spectroscopy with the detection limit of 83 pM in solutions of PCA3, while the limits of detection were 2000 pM and 900 pM for cyclic voltammetry and UV-vis spectroscopy, respectively. That detection could be performed with an optical method is encouraging, as one may envisage extending it to colorimetric tests. Since the morphology of sensing units is known to be affected in detection experiments, we applied machine learning algorithms to classify scanning electron microscopy images of the genosensors and managed to distinguish those exposed to PCA3-containing solutions from control measurements with an accuracy of 99.9%. The performance in distinguishing each individual PCA3 concentration in a multiclass task was lower, with an accuracy of 88.3%, which means that further developments in image analysis are required for this innovative approach.

6.
Talanta ; 222: 121533, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33167241

RESUMO

This study assesses the application of a handheld, near infrared spectroscopy (NIRS) device, namely the NeoSpectra Micro, for the determination of oregano authenticity. Utilising a large sample set of oregano (n = 295) and potential adulterants of oregano (n = 109), models were developed and validated using SIMCA 15 software. The models demonstrated excellent predictability for the determination of authentic oregano and adulterant samples. The optimal model resulted in a 93.0% and 97.5% correct prediction for oregano and adulterants, respectively. Different standardisation approaches were assessed to determine model transferability to a second NIRS device. In the case of the second device, the best predictions were achieved with data that had not undergone any spectral standardisation (raw). Subsequently, the optimal model was able to correctly predict 90% of authentic oregano samples and 100% of the adulterant samples on the second device. This study demonstrates the potential of the device to be used as a simple, cost effective, reliable and handheld screening tool for the determination of oregano authenticity, at various stages of the food supply chain. It is believed that such forms of monitoring could be highly beneficial in other areas of food authenticity analysis to help combat the negative economical and health implications of food fraud.

7.
Methods Mol Biol ; 2189: 1-17, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33180289

RESUMO

Hybrid promoter engineering takes advantage of the modular nature of eukaryotic promoters by combining discrete promoter motifs to confer novel regulatory function. By combinatorially screening sequence libraries for trans-acting transcriptional operators, activators, repressors and core promoter sequences, it is possible to derive constitutive or inducible promoter collections covering a broad range of expression strengths. However, combinatorial approaches to promoter design can result in highly complex, multidimensional design spaces, which can be experimentally costly to thoroughly explore in vivo. Here, we describe an in silico pipeline for the design of hybrid promoter libraries that employs a Design of Experiments (DoE) approach to reduce experimental burden and efficiently explore the promoter fitness landscape. We also describe a software pipeline to ensure that the designed promoter sequences are compatible with the YTK assembly standard.

8.
Methods Mol Biol ; 2189: 19-30, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33180290

RESUMO

The success of any oligonucleotide-based experiment strongly depends on the accurate design of the components. Oli2go is a user-friendly web tool that provides efficient multiplex oligonucleotide design including specificity and primer dimer checks. Its fully automated workflow involves important design steps that use specific parameters to produce high-quality oligonucleotides. This chapter describes how these steps are computationally implemented by oli2go.

9.
Methods Mol Biol ; 2189: 31-43, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33180291

RESUMO

Synthetic biology aims at engineering new biological systems and functions that can be used to provide new technological solutions to worldwide challenges. Detection and processing of multiple signals are crucial for many synthetic biology applications. A variety of logic circuits operating in living cells have been implemented. One particular class of logic circuits uses site-specific recombinases mediating specific DNA inversion or excision. Recombinase logic offers many interesting features, including single-layer architectures, memory, low metabolic footprint, and portability in many species. Here, we present two automated design strategies for recombinase-based logic circuits, one based on the distribution of computation within a multicellular consortia and the other one being a single-cell design. The two design strategies are complementary and are both adapted for none expert as a design web-interface exits for each strategy, the CALIN and RECOMBINATOR web-interface for respectively the multicellular and single-cell design strategy. In this book chapter, we are guiding the reader step by step through recombinase-logic circuit design from selecting the design strategy fitting to his/her final system of interest to obtaining the final design using one of our design web-interface.

10.
Methods Mol Biol ; 2189: 45-63, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33180292

RESUMO

The System Biology Markup Language (SBML) Level 2 has been used extensively to make models for biological systems of different complexity. However, the lack of modularity was a serious hurdle for its application to Synthetic Biology where genetic circuits are preferably modeled by putting together the models of their components. SBML Level 3 with the Hierarchical Composition Package overcame this limit. Here, we describe how to realize a modular model for a eukaryotic AND gate in SBML Level 3. Circuit modules, such as transcription units and pools of molecules, are modeled separately and connected, to close the circuit, via Python scripts that utilize the libSBML API. Circuit simulations with COPASI confirm the validity of this modeling approach.

11.
Methods Mol Biol ; 2189: 65-69, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33180293

RESUMO

The CRISPR/Cas9 system has been developed as a powerful technology for both targeted genome editing and gene regulation. However, the design of efficient single-guide RNAs (sgRNAs) remains challenging with the consideration of many criteria. In this section, we introduce how to design sgRNA sequences and build genome-wide sgRNA library using CRISPR-ERA, which is one of the state-of-the-art designer webserver tools for sgRNA design based on a set of sgRNA design rules summarized from published reports.

12.
Methods Mol Biol ; 2189: 71-80, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33180294

RESUMO

With the advent of genome editing technologies, scientists have recognized that these technologies can be prone to nonspecific or off-target activity. As many areas of the genome are sensitive and can give rise to abnormalities if mutated, it is imperative that scientists identify regions of off-target activity in order to utilize these new technologies for medical benefits. GUIDE-seq and iGUIDE both use an oligo-based marker method to identify regions of DNA double-strand breaks in an unbiased manner. The repeated observation of these double-strand breaks across the genome in comparison with target sequences (such as guide RNAs) has allowed researchers to identify on- and off-target sites related to their targeted-nuclease technologies.

13.
Methods Mol Biol ; 2189: 81-88, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33180295

RESUMO

The CRISPR-Cas system is broadly used for genome editing because of its convenience and relatively low cost. However, the use of CRISPR nucleases to induce specific nucleotide changes in target DNA requires complex procedures and additional donor DNAs. Furthermore, CRISPR nuclease-mediated DNA cleavage at target sites frequently causes large deletions or genomic rearrangements. In contrast, base editors that consist of catalytically dead Cas9 (dCas9) or Cas9 nickase (nCas9) connected to a cytidine or a guanine deaminase can correct point mutations in the absence of additional donor DNA and without generating double-strand breaks (DSBs) in the target region. To design target sites and assess mutation ratios for cytosine and adenine base editors (CBEs and ABEs), we have developed web tools, named BE-Designer and BE-Analyzer. These tools are easy to use (such that tasks are accomplished by clicking on relevant buttons) and do not require a deep knowledge of bioinformatics.

14.
Methods Mol Biol ; 2189: 89-103, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33180296

RESUMO

Synthetic biology aims at engineering synthetic circuits with pre-defined target functions. From a systems (model-based) perspective, the following problems are of central importance: (1) given the model of a biomolecular circuit, elucidate whether it is capable of a certain behavior/functionality; and (2) starting from a pre-defined required functionality and a library of biological parts, find the biomolecular circuit that, built as a combination of such parts, achieves the desired behavior. These two problems, framed, respectively, as nonlinear analysis and automated design problems, are tackled here by efficient optimization methods. We illustrate these methods with case studies considering the analysis and design of biocircuits capable of bistability (bistable switches). Bistability is of particular interest in the context of systems and synthetic biology because it endows cells with the capacity to make decisions.

15.
Methods Mol Biol ; 2189: 105-118, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33180297

RESUMO

This chapter describes an electrode-integrated microfluidic system with multiple functions of manipulating and monitoring single S. cerevisiae cells. In this system, hydrodynamic trapping and negative dielectrophoretic (nDEP) releasing of S. cerevisiae cells are implemented, providing a flexible method for single-cell manipulation. The multiplexing microelectrodes also enable sensitive electrical impedance spectroscopy (EIS) to discern the number of immobilized cells, classify different orientations of captured cells, as well as detect potential movements of immobilized single yeast cells during the overall recording duration by using principal component analysis (PCA) in data mining. The multifrequency EIS measurements can, therefore, obtain sufficient information of S. cerevisiae cells at single-cell level.

16.
Methods Mol Biol ; 2189: 119-132, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33180298

RESUMO

In this post-genomic era, protein network can be used as a complementary way to shed light on the growing amount of data generated from current high-throughput technologies. Protein network is a powerful approach to describe the molecular mechanisms of the biological events through protein-protein interactions. Here, we describe the computational methods used to construct the protein network using expression data. We provide a list of available tools and databases that can be used in constructing the network.

17.
Methods Mol Biol ; 2189: 133-155, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33180299

RESUMO

The deduction of design principles for complex biological functionalities has been a source of constant interest in the fields of systems and synthetic biology. A number of approaches have been adopted, to identify the space of network structures or topologies that can demonstrate a specific desired functionality, ranging from brute force to systems theory-based methodologies. The former approach involves performing a search among all possible combinations of network structures, as well as the parameters underlying the rate kinetics for a given form of network. In contrast to the search-oriented approach in brute force studies, the present chapter introduces a generic approach inspired by systems theory to deduce the network structures for a particular biological functionality. As a first step, depending on the functionality and the type of network in consideration, a measure of goodness of attainment is deduced by defining performance parameters. These parameters are computed for the most ideal case to obtain the necessary condition for the given functionality. The necessary conditions are then mapped as specific requirements on the parameters of the dynamical system underlying the network. Following this, admissible minimal structures are deduced. The proposed methodology does not assume any particular rate kinetics in this case for deducing the admissible network structures notwithstanding a minimum set of assumptions on the rate kinetics. The problem of computing the ideal set of parameter/s or rate constants, unlike the problem of topology identification, depends on the particular rate kinetics assumed for the given network. In this case, instead of a computationally exhaustive brute force search of the parameter space, a topology-functionality specific optimization problem can be solved. The objective function along with the feasible region bounded by the motif specific constraints amounts to solving a non-convex optimization program leading to non-unique parameter sets. To exemplify our approach, we adopt the functionality of adaptation, and demonstrate how network topologies that can achieve adaptation can be identified using such a systems-theoretic approach. The outcomes, in this case, i.e., minimum network structures for adaptation, are in agreement with the brute force results and other studies in literature.

18.
Methods Mol Biol ; 2189: 157-167, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33180300

RESUMO

Mathematical models for the spread of diseases help us understand the mechanisms on how diseases spread, evaluate the possible effects of interventions, predict outcomes of epidemics, and forecast the course of outbreaks. Compartmental models are widely used in synthetic biology since they can represent a biological system as an assembly of various parts or compartments with different functions. Here we present a framework for the analysis of a compartmental model for the transmission of diseases using ordinary differential equations. We apply this method on a study about the spread of tuberculosis.

19.
Methods Mol Biol ; 2189: 169-181, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33180301

RESUMO

Synthetic biology aims at engineering biological systems, ranging from genes to entire genomes. The emerging field of synthetic genomics provides new tools to address questions and tackle challenges in biology and biotechnology impossible to address with current methods. Chromosome scale engineering requires computational tools and workflows to streamline designing, building and verifying long DNA molecules. While a systematic and generic genome design workflow does not exist, here we outline chromosome assembly and verification operations that are at the foundation of every genome scale engineering efforts.

20.
Methods Mol Biol ; 2189: 183-198, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33180302

RESUMO

Synthetic biologists engineer cells and cellular functions using design-build-test cycles; when the task is to extensively engineer entire genomes, the lack of appropriate design tools and biological knowledge about each gene in a cell can lengthen the process, requiring time-consuming and expensive experimental iterations.Whole-cell models represent a new avenue for genome design; the bacteria Mycoplasma genitalium has the first (and currently only published) whole-cell model which combines 28 cellular submodels and represents the integrated functions of every gene and molecule in a cell.We created two minimal genome design algorithms, GAMA and Minesweeper, that produced 1000s of in silico minimal genomes by running simulations on multiple supercomputers. Here we describe the steps to produce in silico cells with reduced genomes, combining minimisation algorithms with whole-cell model simulations.We foresee that the combination of similar algorithms and whole-cell models could later be used for a broad spectrum of genome design applications across cellular species when appropriate models become available.

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