scholarly journals Quality control and statistical evaluation of combinatorial DNA libraries using nanopore sequencing

BioTechniques ◽  
2020 ◽  
Vol 69 (5) ◽  
pp. 379-383
Author(s):  
Cédric Lood ◽  
Hans Gerstmans ◽  
Yves Briers ◽  
Vera van Noort ◽  
Rob Lavigne
Keyword(s):  
Ad Hoc ◽  
Comprehensive Evaluation ◽  
Combinatorial Libraries ◽  
Initial Step ◽  
Building Blocks ◽  
Small Subset ◽  
Sequencing Analysis ◽  
Nanopore Sequencing ◽  
Long Reads

Protein engineering and synthetic biology applications increasingly rely on the assembly of modular libraries composed of thousands of different combinations of DNA building blocks. At present, the validation of such libraries is performed by Sanger sequencing analysis on a small subset of clones on an ad hoc basis. Here, we implement a systematic procedure for the comprehensive evaluation of combinatorial libraries, immediately after their creation in vitro, using long reads sequencing technology. After an initial step of nanopore sequencing, we use straightforward bioinformatics tools to tabulate the composition and synteny of the building blocks in each read. We subsequently use exploratory statistics to assess the library and validate its diversity before carrying downstream cloning and screening assays.

scholarly journals Biosynthetic Strategies for Macrocyclic Peptides

Molecules ◽  
2021 ◽  
Vol 26 (11) ◽  
pp. 3338
Author(s):  
Wei Wang ◽  
S. Cyrus Khojasteh ◽  
Dian Su
Keyword(s):  
Cyclic Peptide ◽  
Chemical Space ◽  
Combinatorial Libraries ◽  
Building Blocks ◽  
Peptide Library ◽  
In Vitro Translation ◽  
Split Intein ◽  
Macrocyclic Peptides

Macrocyclic peptides are predominantly peptide structures bearing one or more rings and spanning multiple amino acid residues. Macrocyclization has become a common approach for improving the pharmacological properties and bioactivity of peptides. A variety of ribosomal-derived and non-ribosomal synthesized cyclization approaches have been established. The biosynthesis of backbone macrocyclic peptides using seven new emerging methodologies will be discussed with regard to the features and strengths of each platform rather than medicinal chemistry tools. The mRNA display variant, known as the random nonstandard peptide integrated discovery (RaPID) platform, utilizes flexible in vitro translation (FIT) to access macrocyclic peptides containing nonproteinogenic amino acids (NAAs). As a new discovery approach, the ribosomally synthesized and post-translationally modified peptides (RiPPs) method involves the combination of ribosomal synthesis and the phage screening platform together with macrocyclization chemistries to generate libraries of macrocyclic peptides. Meanwhile, the split-intein circular ligation of peptides and proteins (SICLOPPS) approach relies on the in vivo production of macrocyclic peptides. In vitro and in vivo peptide library screening is discussed as an advanced strategy for cyclic peptide selection. Specifically, biosynthetic bicyclic peptides are highlighted as versatile and attractive modalities. Bicyclic peptides represent another type of promising therapeutics that allow for building blocks with a heterotrimeric conjugate to address intractable challenges and enable multimer complexes via linkers. Additionally, we discuss the cell-free chemoenzymatic synthesis of macrocyclic peptides with a non-ribosomal catalase known as the non-ribosomal synthetase (NRPS) and chemo-enzymatic approach, with recombinant thioesterase (TE) domains. Novel insights into the use of peptide library tools, activity-based two-hybrid screening, structure diversification, inclusion of NAAs, combinatorial libraries, expanding the toolbox for macrocyclic peptides, bicyclic peptides, chemoenzymatic strategies, and future perspectives are presented. This review highlights the broad spectrum of strategy classes, novel platforms, structure diversity, chemical space, and functionalities of macrocyclic peptides enabled by emerging biosynthetic platforms to achieve bioactivity and for therapeutic purposes.

scholarly journals Nanopore sequencing enables high-resolution analysis of resistance determinants and mobile elements in the human gut microbiome

2018 ◽  
Author(s):  
Denis Bertrand ◽  
Jim Shaw ◽  
Manesh Kalathiappan ◽  
Amanda Hui Qi Ng ◽  
Senthil Muthiah ◽  
...  
Keyword(s):  
Complex Dynamics ◽  
Human Microbiome ◽  
Abundant Species ◽  
Error Rates ◽  
Nanopore Sequencing ◽  
High Quality ◽  
Short Read ◽  
Long Reads ◽  
Long Read

AbstractThe analysis of information rich whole-metagenome datasets acquired from complex microbial communities is often restricted by the fragmented nature of assembly from short-read sequencing. The availability of long-reads from third-generation sequencing technologies (e.g. PacBio or Oxford Nanopore) can help improve assembly quality in principle, but high error rates and low throughput have limited their application in metagenomics. In this work, we describe the first hybrid metagenomic assembler which combines the advantages of short and long-read technologies, providing an order of magnitude improvement in contiguity compared to short read assemblies, and high base-pair level accuracy. The proposed approach (OPERA-MS) integrates a novel assembly-based metagenome clustering technique with an exact scaffolding algorithm that can efficiently assemble repeat rich sequences. Based on evaluations with defined in vitro communities and virtual gut microbiomes, we show that it is possible to assemble near complete genomes from metagenomes with as little as 9× long read coverage, thus enabling high quality assembly of lowly abundant species (<1%). Furthermore, OPERA-MS’s fine-grained clustering is able to deconvolute and assemble multiple genomes of the same species in a single sample, allowing us to study the complex dynamics of the human microbiome at the sub-species level. Applying nanopore sequencing to gut metagenomes of patients undergoing antibiotic treatment, we show that long reads can be obtained from stool samples in clinical studies to produce more meaningful metagenomic assemblies (up to 200× improvement over short-read assemblies), including the closed assembly of >80 putative plasmid/phage sequences and a 263kbp jumbo phage. Our results highlight that high-quality hybrid assemblies provide an unprecedented view of the gut resistome in these patients, including strain dynamics and identification of novel plasmid sequences.

Language, procedures, and the non-perceptual origin of natural number concepts

2016 ◽  
Author(s):  
David Barner
Keyword(s):  
General Framework ◽  
Ad Hoc ◽  
Building Blocks ◽  
Number Words ◽  
Linguistic Markers ◽  
Logical Foundations ◽  
Large Numbers ◽  
Perceptual Representations ◽  
Positive Integers ◽  
Perceptual Systems

Perceptual representations – e.g., of objects or approximate magnitudes –are often invoked as building blocks that children combine with linguisticsymbols when they acquire the positive integers. Systems of numericalperception are either assumed to contain the logical foundations ofarithmetic innately, or to supply the basis for their induction. Here Ipropose an alternative to this general framework, and argue that theintegers are not learned from perceptual systems, but instead arise toexplain perception as part of language acquisition. Drawing oncross-linguistic data and developmental data, I show that small numbers(1-4) and large numbers (~5+) arise both historically and in individualchildren via entirely distinct mechanisms, constituting independentlearning problems, neither of which begins with perceptual building blocks.Specifically, I propose that children begin by learning small numbers(i.e., *one, two, three*) using the same logical resources that supportother linguistic markers of number (e.g., singular, plural). Several yearslater, children discover the logic of counting by inferring the logicalrelations between larger number words from their roles in blind countingprocedures, and only incidentally associate number words with perception ofapproximate magnitudes, in an *ad hoc* and highly malleable fashion.Counting provides a form of explanation for perception but is not causallyderived from perceptual systems.

Design, Synthesis and Bioactivity of Core 1 O-glycan and its Derivative on Human Gut Microbiota

2019 ◽  
Vol 16 (12) ◽  
pp. 1348-1353
Author(s):  
Huanhuan Qu ◽  
Baixue Li ◽  
Jingyi Yang ◽  
Huaiwen Liang ◽  
Meixia Li ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Initial Step ◽  
Building Blocks ◽  
Glycan Structure ◽  
Human Gut ◽  
Design Synthesis ◽  
The Core ◽  
Human Gut Microbiota ◽  
Biochemical Studies ◽  
Gut Symbionts

Background: Disaccharide core 1 (Galβ1-3GalNAc) is a common O-glycan structure in nature. Biochemical studies have confirmed that the formation of the core 1 structure is an important initial step in O-glycan biosynthesis and it is of great importance for human body. Objective: Our study will provide meaningful and useful sights for O-glycan synthesis and their bioassay. And all the synthetic glycosides would be used as intermediate building blocks in the scheme developed for oligosaccharide construction. Methods: In this article, we firstly used chemical procedures to prepare core 1 and its derivative, and a novel disaccharide was efficiently synthesized. The structures of the synthesized compounds were elucidated and confirmed by 1H NMR, 13C NMR and MS. Then we employed three human gut symbionts belonging to Bacteroidetes, a predominantphyla in the distal gut, as models to study the bioactivity of core 1 and its derivative on human gut microbiota. Results: According to our results, both core 1 and derivative could support the growth of B. fragilis, especially the core 1 derivative, while failed to support the growth of B. thetaiotaomicron and B. ovatus. Conclusion: This suggested that the B. fragilis might have the specificity glycohydrolase to cut the glycosidic bond for acquiring monosaccharide.

Synthesis and antioxidant activity of new selenium-containing quinolines

2020 ◽  
Vol 16 ◽  
Author(s):  
Benedetta Bocchini ◽  
Bruna Goldani ◽  
Fernanda S.S. Sousa ◽  
Paloma T. Birmann ◽  
Cesar A. Brüning ◽  
...  
Keyword(s):  
Antioxidant Activity ◽  
Superoxide Dismutase ◽  
Radical Scavenging ◽  
Building Blocks ◽  
Antioxidant Potential ◽  
In Vitro Assays ◽  
Pharmacological Activities ◽  
Antioxidant Power ◽  
Antioxidant Agents

Background: Quinoline derivatives have been attracted much attention in drug discovery and synthetic derivatives of these scaffolds present a range of pharmacological activities. Therefore, organoselenium compounds are valuable scaffolds in organic synthesis because their pharmacological activities and their use as versatile building blocks for regio-, chemio-and stereoselective reactions. Thus, the synthesis of selenium-containing quinolines has great significance, and their applicability range from simple antioxidant agents, to selective DNA-binding and photocleaving agents. Objective: In the present study we describe the synthesis and antioxidant activity in vitro of new 7-chloroN(arylselanyl)quinolin-4-amines 5 by the reaction of 4,7-dichloroquinoline 4 with (arylselanyl)-amines 3. Methods: For the synthesis of 7-chloro-N(arylselanyl)quinolin-4-amines 5, we performed the reaction of (arylselanyl)- amines 3 with 4,7-dichloroquinoline 4 in the presence of Et3N at 120 °C in a sealed tube. The antioxidant activities of the compounds 5 were evaluated by the following in vitro assays: 2,2- diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, 2,2-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), ferric ion reducing antioxidant power (FRAP), nitric oxide (NO) scavenging and superoxide dismutase-like activity (SOD-Like). Results: 7-Chloro-N(arylselanyl)quinolin-4-amines 5a-d has been synthesized in yields ranging from 68% to 82% by the reaction of 4,7-dichloroquinoline 4 with arylselanyl-amines 3a-d using Et3N as base, at 120 °C, in a sealed tube for 24 hours and tolerates different substituents, such as -OMe and -Cl, in the arylselanyl moiety. The obtained compounds 5a-d presented significant results with respect to the antioxidant potential, which had effect in the tests of inhibition of radical’s DPPH, ABTS+ and NO, as well as in the test that evaluates the capacity (FRAP) and in the superoxide dismutase-like activity assay (SOD-Like). It is worth mentioning that 7-chloro-N(arylselanyl)quinolin-4-amine 5b presented excellent results, demonstrating a better antioxidant capacity when compared to the others. Conclusion: According to the obtained results 7-chloro-N(arylselanyl)quinolin-4-amines 5 were synthesized in good yields by the reaction of 4,7-dichloroquinoline with arylselanyl-amines and tolerates different substituents in the arylselanyl moiety. The tested compounds presented significant antioxidant potential in the tests of inhibition of DPPH, ABTS+ and NO radicals, as well as in the FRAP and superoxide dismutase-like activity assays (SOD-Like).

Kinase Targets for Mycolic Acid Biosynthesis in Mycobacterium tuberculosis

2019 ◽  
Vol 12 (1) ◽  
pp. 27-49 ◽  
Author(s):  
Shahinda S.R. Alsayed ◽  
Chau C. Beh ◽  
Neil R. Foster ◽  
Alan D. Payne ◽  
Yu Yu ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Enzymatic Activity ◽  
Bacterial Pathogen ◽  
Building Blocks ◽  
Mycolic Acid ◽  
Adaptive Responses ◽  
Mycolic Acids ◽  
Hydroxylated Fatty Acids

Background:Mycolic acids (MAs) are the characteristic, integral building blocks for the mycomembrane belonging to the insidious bacterial pathogen Mycobacterium tuberculosis (M.tb). These C60-C90 long α-alkyl-β-hydroxylated fatty acids provide protection to the tubercle bacilli against the outside threats, thus allowing its survival, virulence and resistance to the current antibacterial agents. In the post-genomic era, progress has been made towards understanding the crucial enzymatic machineries involved in the biosynthesis of MAs in M.tb. However, gaps still remain in the exact role of the phosphorylation and dephosphorylation of regulatory mechanisms within these systems. To date, a total of 11 serine-threonine protein kinases (STPKs) are found in M.tb. Most enzymes implicated in the MAs synthesis were found to be phosphorylated in vitro and/or in vivo. For instance, phosphorylation of KasA, KasB, mtFabH, InhA, MabA, and FadD32 downregulated their enzymatic activity, while phosphorylation of VirS increased its enzymatic activity. These observations suggest that the kinases and phosphatases system could play a role in M.tb adaptive responses and survival mechanisms in the human host. As the mycobacterial STPKs do not share a high sequence homology to the human’s, there have been some early drug discovery efforts towards developing potent and selective inhibitors.Objective:Recent updates to the kinases and phosphatases involved in the regulation of MAs biosynthesis will be presented in this mini-review, including their known small molecule inhibitors.Conclusion:Mycobacterial kinases and phosphatases involved in the MAs regulation may serve as a useful avenue for antitubercular therapy.

Performance and Scalability Assessment for Non-Certificate-Based Public Key Management in VANETs

2012 ◽  
Vol 6 (1) ◽  
pp. 33-56 ◽  
Author(s):  
Pei-Yuan Shen ◽  
Maolin Tang ◽  
Vicky Liu ◽  
William Caelli
Keyword(s):  
Key Management ◽  
Vehicular Ad Hoc Networks ◽  
Ad Hoc ◽  
Comprehensive Evaluation ◽  
Public Key ◽  
Secure Messaging ◽  
Evaluation Of Performance ◽  
Management Regime ◽  
Verification Process ◽  
Hoc Networks

Current research in secure messaging for Vehicular Ad hoc Networks (VANETs) focuses on employing a digital certificate-based Public Key Cryptosystem (PKC) to support security. However, the security overhead of such a scheme creates a transmission delay and introduces a time-consuming verification process to VANET communications. This paper proposes a non-certificate-based public key management for VANETs. A comprehensive evaluation of performance and scalability of the proposed public key management regime is presented, which is compared with a certificate-based PKC by employing a number of quantified analyses and simulations. In this paper, the authors demonstrate that the proposal can maintain security and assert that it can improve overall performance and scalability at a lower cost, compared with certificate-based PKC. The proposed scheme adds a new dimension to key management and verification services for VANETs.

scholarly journals Beyond Oncological Hyperthermia: Physically Drivable Magnetic Nanobubbles as Novel Multipurpose Theranostic Carriers in the Central Nervous System

Molecules ◽  
2020 ◽  
Vol 25 (9) ◽  
pp. 2104 ◽  
Author(s):  
Eleonora Ficiarà ◽  
Shoeb Anwar Ansari ◽  
Monica Argenziano ◽  
Luigi Cangemi ◽  
Chiara Monge ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Brain Tumors ◽  
Ad Hoc ◽  
Superparamagnetic Iron Oxide ◽  
Conventional Radiotherapy ◽  
The Central Nervous System ◽  
Magnetic Resonance Imaging Mri ◽  
The Brain

Magnetic Oxygen-Loaded Nanobubbles (MOLNBs), manufactured by adding Superparamagnetic Iron Oxide Nanoparticles (SPIONs) on the surface of polymeric nanobubbles, are investigated as theranostic carriers for delivering oxygen and chemotherapy to brain tumors. Physicochemical and cyto-toxicological properties and in vitro internalization by human brain microvascular endothelial cells as well as the motion of MOLNBs in a static magnetic field were investigated. MOLNBs are safe oxygen-loaded vectors able to overcome the brain membranes and drivable through the Central Nervous System (CNS) to deliver their cargoes to specific sites of interest. In addition, MOLNBs are monitorable either via Magnetic Resonance Imaging (MRI) or Ultrasound (US) sonography. MOLNBs can find application in targeting brain tumors since they can enhance conventional radiotherapy and deliver chemotherapy being driven by ad hoc tailored magnetic fields under MRI and/or US monitoring.

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