scholarly journals Cas9-Assisted Targeting of CHromosome segments CATCH enables one-step targeted cloning of large gene clusters

2015 ◽  
Vol 6 (1) ◽  
Author(s):  
Wenjun Jiang ◽  
Xuejin Zhao ◽  
Tslil Gabrieli ◽  
Chunbo Lou ◽  
Yuval Ebenstein ◽  
...  
Keyword(s):  
Gene Clusters ◽  
Single Step ◽  
Gene Synthesis ◽  
Gibson Assembly ◽  
Large Gene ◽  
Cas9 Nuclease ◽  
Bacterial Chromosomes ◽  
One Step ◽  
Chromosome Segments

Abstract The cloning of long DNA segments, especially those containing large gene clusters, is of particular importance to synthetic and chemical biology efforts for engineering organisms. While cloning has been a defining tool in molecular biology, the cloning of long genome segments has been challenging. Here we describe a technique that allows the targeted cloning of near-arbitrary, long bacterial genomic sequences of up to 100 kb to be accomplished in a single step. The target genome segment is excised from bacterial chromosomes in vitro by the RNA-guided Cas9 nuclease at two designated loci, and ligated to the cloning vector by Gibson assembly. This technique can be an effective molecular tool for the targeted cloning of large gene clusters that are often expensive to synthesize by gene synthesis or difficult to obtain directly by traditional PCR and restriction-enzyme-based methods.

scholarly journals Customized one-step preparation of sgRNA transcription templates via overlapping PCR Using short primers and its application in vitro and in vivo gene editing

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Zheng Hu ◽  
Li Wang ◽  
Zhaoying Shi ◽  
Jing Jiang ◽  
Xiangning Li ◽  
...  
Keyword(s):  
Dna Assembly ◽  
Dna Templates ◽  
Core Fragment ◽  
Cas9 Nuclease ◽  
Overlap Extension ◽  
One Step ◽  
Dna Fragment ◽  
Polymerase Chain

Abstract Overlap extension polymerase chain reaction (PCR) is a powerful technology for DNA assembly. Based on this technology, we synthesized DNA templates, which were transcribed into sgRNA in vitro, and further detected their efficiency of purified sgRNAs with Cas9 nuclease. The sgRNAs synthesized by this approach can effectively cleave the DNA fragments of interest in vitro and in vivo. Compared with the conventional method for generating sgRNA, it does not require construction of recombinant plasmids and design of primers to amplify sgRNA core fragment. Only several short primers with overlapped sequences are needed to assemble a DNA fragment as the template of sgRNA. This modified and simplified method is highly applicable and less time-consuming.

One-Step Extraction and Cleanup Procedure for Determination of p,p'-DDT, p,p'-DDD, and p,p'-DDE in Fish

1986 ◽  
Vol 69 (4) ◽  
pp. 581-586 ◽  
Author(s):  
Nazir Ahmad ◽  
Robert Slobodan Marolt
Keyword(s):  
Residue Analysis ◽  
Trisodium Citrate ◽  
Single Step ◽  
Chromatographic Column ◽  
Fish Samples ◽  
Average Coefficient ◽  
Cleanup Procedure ◽  
One Step

Abstract A simplified method that combines extraction, partitioning, and cleanup in a single step for measuring p,p'-DDT and its metabolites in fish is described. Minced fish samples are emulsified with disodium hydrogen orthophosphate and trisodium citrate, ground with sodium sulfate, and eluted from a chromatographic column prepacked with alumina and silicic acid. The fats and fatty acids are solubilized and easily extracted from the tissues and retained by the column, while p,p'-DDT and its metabolites are quantitatively eluted with 40 mL n-hexane. The eluate is directly applied to a gas chromatographic column. Average recoveries of p,p'-DDT and its metabolites added to fish in vitro are 81%. The average coefficient of variation for recoveries of p,p'-DDT and its metabolites is less than 6.5% and the detection limit is 0.001 Hg/g for p,p'-DDE, thus making this method very suitable for residue analysis.

scholarly journals O-P05 Gemcitabine and paclitaxel loaded microbubbles for the treatment of pancreatic cancer

2021 ◽  
Vol 108 (Supplement_9) ◽  
Author(s):  
Keiran Logan ◽  
John Callan
Keyword(s):  
Pancreatic Cancer ◽  
Drug Loading ◽  
Tumour Volume ◽  
Single Step ◽  
Promising Alternative ◽  
One Step ◽  
Transphosphatidylation Reaction ◽  
Subsequent Incorporation

Abstract Background Ultrasound targeted microbubble destruction (UTMD) has emerged as an effective strategy for the delivery of drug payloads to solid tumours. However, loading a single microbubble (MB) formulation with two drug payloads is challenging and often involves several manipulations post-MB preparation to enable attachment of drug payloads which can be cumbersome and generally results in low / inconsistent drug loading. Here we report a one-step synthesis of a gemcitabine-functionalised phospholipid and its subsequent incorporation into a stable MB formulation co-loaded with paclitaxel (PTX). The efficacy of the MB conjugate was determined in a Panc-1 spheroid model and ectopic BxPC-3 tumour model of pancreatic cancer. Methods Gemcitabine-modified phospholipid (Lipid-Gem MB) was prepared from 1,2-dibehenoyl-sn-glycero-3-phosphocholine (DBPC) though a transphosphatidylation reaction using gemcitabine (Gem) as the acceptor alcohol. Lipid-Gem MB and Lipid-Gem-PTX MB were prepared from Lipid-Gem MB and/or PTX using a standard thin-film hydration technique followed by sonication in the presence of PFB gas. In vitro efficacy of Lipid-Gem MB and Lipid-Gem-PTX MB were determined in Panc-1 spheroids using an MTT assay. The in vivo effectiveness was determined in BxPC-3 tumour bearing mice following IV administration of either Lipid-Gem MB or Lipid-Gem-PTX MB plus ultrasound (US). Free Gem, free Gem + PTX and untreated mice were used for comparative purposes. Results Spheroids treated with Lipid-Gem MB +US or Lipid-Gem-PTX MB +US were significantly reduced relative to spheroids treated with US alone (p = 0.033 and p = 0.0031 respectively) or with the respective MB formulation alone (i.e. no US) (p = 0.0336 and p = 0.0037 respectively). Furthermore, cell viability for spheroids treated with Lipid-Gem-PTX MB +US was significantly reduced compared with spheroids treated with Lipid-Gem MB +US (p = 0.0077) (Figure a). Mice treated with Lipid-Gem MB +US or Lipid-Gem-PTX MB +US showed an average change in tumour volume of + 7 ± 7% and -10 ± 10 % respectively compared with +45 ±10% and +30 ± 10% for free gem and free gem + PTX respectively (Figure b). Conclusions A Gem-modified lipid was succesfully synthesised using a single step reaction and was subsequently incorporated into MBs containging PTX, eliminating the need for cumbersome drug conjgation methods. UTMD mediated treatment of Panc-1 spheroids and BxPC-3 tumours demonstrated the efficacy and tolerability of the formulations. Given that all components of this formulation are already clinicaly apporved, UTMD using Lipid-Gem-PTX MB offers a promising alternative to existing treatments

Synthesis of Polycycles and Oxacycles by Tandem Metathesis of endo–norbornene Derivatives

Synthesis ◽  
2021 ◽  
Author(s):  
Sambasivarao Kotha ◽  
Sunil Pulletikurti ◽  
Ambareen Fatma ◽  
gopal dhangar ◽  
gonna somu Naidu
Keyword(s):  
Natural Products ◽  
Metal Complex ◽  
Carbonyl Group ◽  
Single Step ◽  
Time Dependent ◽  
Computational Studies ◽  
Nmr Studies ◽  
Tricyclic Compounds ◽  
One Step ◽  
Norbornene Derivatives

Here, we have demonstrated that the presence of a carbonyl group at C7 position is preventing the olefin metathesis of endo-norbornene derivatives due to the complexation of the metal alkylidene. Time-dependent NMR studies showed the presence of new proton signals in the metal alkylidene region, which indicate the formation of metal complex with the carbonyl group of the substrate. These observations were further proved by ESI-MS analysis. Whereas, computational studies provided that the catalyst was interacting with the C7 carbonyl group and aligned perpendicular to that of norbornene olefin. Later, these endo-keto norbornene derivatives were reduced to hydroxyl derivatives diastereoselectively. Ring-rearrangement metathesis (RRM) of these hydroxyl derivatives, produced the [6/5/6], and [5/6/5] carbo-tricyclic cores of the natural products in one step. Whereas the RRM of O-allyl derivatives, delivered the oxa-tricyclic compounds in a single step with excellent yields.

scholarly journals A Nitronaphthalimide Probe for Fluorescence Imaging of Hypoxia in Cancer Cells

2021 ◽  
Author(s):  
Rashmi Kumari ◽  
Vasumathy R ◽  
Dhanya Sunil ◽  
Raghumani Singh Ningthoujam ◽  
Badri Narain Pandey ◽  
...  
Keyword(s):  
Fluorescence Imaging ◽  
Tumor Hypoxia ◽  
Binding Pocket ◽  
Docking Studies ◽  
Single Step ◽  
Naphthalic Anhydride ◽  
Hypoxic Conditions ◽  
Fluorescence Measurements ◽  
Enzymatic Reduction

AbstractThe bioreductive enzymes typically upregulated in hypoxic tumor cells can be targeted for developing diagnostic and drug delivery applications. In this study, a new fluorescent probe 4−(6−nitro−1,3−dioxo−1H−benzo[de]isoquinolin−2(3H)−yl)benzaldehyde (NIB) based on a nitronaphthalimide skeleton that could respond to nitroreductase (NTR) overexpressed in hypoxic tumors is designed and its application in imaging tumor hypoxia is demonstrated. The docking studies revealed favourable interactions of NIB with the binding pocket of NTR-Escherichia coli. NIB, which is synthesized through a simple and single step imidation of 4−nitro−1,8−naphthalic anhydride displayed excellent reducible capacity under hypoxic conditions as evidenced from cyclic voltammetry investigations. The fluorescence measurements confirmed the formation of identical products (NIB-red) during chemical as well as NTR−aided enzymatic reduction in the presence of NADH. The potential fluorescence imaging of hypoxia based on NTR-mediated reduction of NIB is confirmed using in-vitro cell culture experiments using human breast cancer (MCF−7) cells, which displayed a significant change in the fluorescence colour and intensity at low NIB concentration within a short incubation period in hypoxic conditions. Graphical abstract

scholarly journals Tetracalcium Phosphate/Monetite/Calcium Sulfate Hemihdrate Biocement Powder Mixtures Prepared by the One-Step Synthesis for Preparation of Nanocrystalline Hydroxyapatite Biocement-Properties and In Vitro Evaluation

Materials ◽  
2021 ◽  
Vol 14 (9) ◽  
pp. 2137
Author(s):  
Lubomir Medvecky ◽  
Maria Giretova ◽  
Radoslava Stulajterova ◽  
Lenka Luptakova ◽  
Tibor Sopcak
Keyword(s):  
Simulated Body Fluid ◽  
Body Fluid ◽  
Calcium Sulfate ◽  
Powder Mixtures ◽  
Liquid Component ◽  
Tetracalcium Phosphate ◽  
Nanocrystalline Hydroxyapatite ◽  
One Step ◽  
Calcium Sulfate Hemihydrate

A modified one-step process was used to prepare tetracalcium phosphate/monetite/calcium sulfate hemihydrate powder cement mixtures (CAS). The procedure allowed the formation of monetite and calcium sulfate hemihydrate (CSH) in the form of nanoparticles. It was hypothesized that the presence of nanoCSH in small amounts enhances the in vitro bioactivity of CAS cement in relation to osteogenic gene markers in mesenchymal stem cells (MSCs). The CAS powder mixtures with 15 and 5 wt.% CSH were prepared by milling powder tetracalcium phosphate in an ethanolic solution of both orthophosphoric and sulfuric acids. The CAS cements had short setting times (around 5 min). The fast setting of the cement samples after the addition of the liquid component (water solution of NaH2PO4) was due to the partial formation of calcium sulfate dihydrate and hydroxyapatite before soaking in SBF with a small change in the original phase composition in cement powder samples after milling. Nanocrystalline hydroxyapatite biocement was produced by soaking of cement samples after setting in simulated body fluid (SBF). The fast release of calcium ions from CAS5 cement, as well as a small rise in the pH of SBF during soaking, were demonstrated. After soaking in SBF for 7 days, the final product of the cement transformation was nanocrystalline hydroxyapatite. The compressive strength of the cement samples (up to 30 MPa) after soaking in simulated body fluid (SBF) was comparable to that of bone. Real time polymerase chain reaction (RT-PCR) analysis revealed statistically significant higher gene expressions of alkaline phosphatase (ALP), osteonectin (ON) and osteopontin (OP) in cells cultured for 14 days in CAS5 extract compared to CSH-free cement. The addition of a small amount of nanoCSH (5 wt.%) to the tetracalcium phosphate (TTCP)/monetite cement mixture significantly promoted the over expression of osteogenic markers in MSCs. The prepared CAS powder mixture with its enhanced bioactivity can be used for bone defect treatment and has good potential for bone healing.

scholarly journals Efficient Cryopreservation of Populus tremula by In Vitro-Grown Axillary Buds and Genetic Stability of Recovered Plants

Plants ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 77
Author(s):  
Elena O. Vidyagina ◽  
Nikolay N. Kharchenko ◽  
Konstantin A. Shestibratov
Keyword(s):  
Survival Rate ◽  
Axillary Buds ◽  
Long Term Storage ◽  
Average Survival ◽  
Transgenic Lines ◽  
Ssr Loci ◽  
One Step ◽  
The One

Axillary buds of in vitro microshoots were successfully frozen at –196 °C by the one-step freezing method using the protective vitrification solution 2 (PVS2). Microshoots were taken from 11 transgenic lines and three wild type lines. Influence of different explant pretreatments were analyzed from the point of their influence towards recovery after cryopreservation. It was found out that the use of axillary buds as explants after removal of the apical one increases recovery on average by 8%. The cultivation on growth medium of higher density insignificantly raises the regenerants survival rate. Pretreatment of the osmotic fluid (OF) shows the greatest influence on the survival rate. It leads to the increase in survival rate by 20%. The cryopreservation technology providing regenerants average survival rate of 83% was developed. It was based on the experimental results obtained with explant pretreatment. Incubation time in liquid nitrogen did not affect the explants survival rate after thawing. After six months cryostorage of samples their genetic variability was analyzed. Six variable simple sequence repeat (SSR) loci were used to analyze genotype variability after the freezing-thawing procedure. The microsatellite analysis showed the genetic status identity of plants after cryopreservation and of the original genotypes. The presence of the recombinant gene in the transgenic lines after cryostorage were confirmed so as the interclonal variation in the growth rate under greenhouse conditions. The developed technique is recommended for long-term storage of various breeding and genetically modified lines of aspen plants, as it provides a high percentage of explants survival with no changes in genotype.

scholarly journals Structural comparison of Acinetobacter baumannii β-ketoacyl-acyl carrier protein reductases in fatty acid and aryl polyene biosynthesis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Woo Cheol Lee ◽  
Sungjae Choi ◽  
Ahjin Jang ◽  
Kkabi Son ◽  
Yangmee Kim
Keyword(s):  
Fatty Acid ◽  
Acinetobacter Baumannii ◽  
Gene Cluster ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Gene Clusters ◽  
Carrier Protein ◽  
Aryl Group ◽  
Visible Absorption

AbstractSome Gram-negative bacteria harbor lipids with aryl polyene (APE) moieties. Biosynthesis gene clusters (BGCs) for APE biosynthesis exhibit striking similarities with fatty acid synthase (FAS) genes. Despite their broad distribution among pathogenic and symbiotic bacteria, the detailed roles of the metabolic products of APE gene clusters are unclear. Here, we determined the crystal structures of the β-ketoacyl-acyl carrier protein (ACP) reductase ApeQ produced by an APE gene cluster from clinically isolated virulent Acinetobacter baumannii in two states (bound and unbound to NADPH). An in vitro visible absorption spectrum assay of the APE polyene moiety revealed that the β-ketoacyl-ACP reductase FabG from the A. baumannii FAS gene cluster cannot be substituted for ApeQ in APE biosynthesis. Comparison with the FabG structure exhibited distinct surface electrostatic potential profiles for ApeQ, suggesting a positively charged arginine patch as the cognate ACP-binding site. Binding modeling for the aryl group predicted that Leu185 (Phe183 in FabG) in ApeQ is responsible for 4-benzoyl moiety recognition. Isothermal titration and arginine patch mutagenesis experiments corroborated these results. These structure–function insights of a unique reductase in the APE BGC in comparison with FAS provide new directions for elucidating host–pathogen interaction mechanisms and novel antibiotics discovery.

scholarly journals Single Step 2-Port Device De-Embedding Algorithm for Fixture-DUT-Fixture Network Assembly

Electronics ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1275
Author(s):  
Simone Scafati ◽  
Enza Pellegrino ◽  
Francesco de Paulis ◽  
Carlo Olivieri ◽  
James Drewniak ◽  
...  
Keyword(s):  
Standard Technique ◽  
Single Step ◽  
Scattering Parameters ◽  
Linear Set ◽  
S Parameter ◽  
Parameter Conversion ◽  
Before And After ◽  
One Step ◽  
Typical Measurement

The de-embedding of measurement fixtures is relevant for an accurate experimental characterization of radio frequency and digital electronic devices. The standard technique consists in removing the effects of the measurement fixtures by the calculation of the transfer scattering parameters (T-parameters) from the available measured (or simulated) global scattering parameters (S-parameters). The standard de-embedding is achieved by a multiple steps process, involving the S-to-T and subsequent T-to-S parameter conversion. In a typical measurement setup, two fixtures are usually placed before and after the device under test (DUT) allowing the connection of the device to the calibrated vector network analyzer coaxial ports. An alternative method is proposed in this paper: it is based on the newly developed multi-network cascading algorithm. The matrices involved in the fixture-DUT-fixture cascading gives rise to a non-linear set of equations that is in one step analytically solved in closed form, obtaining a unique solution. The method is shown to be effective and at least as accurate as the standard multi-step de-embedding one.

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