scholarly journals Erratum for: Large-scale in Vitro Transcription, RNA Purification and Chemical Probing Analysis

2019 ◽  
Vol 52 (5) ◽  
pp. 1253-1253
Keyword(s):  
Large Scale ◽  
In Vitro Transcription ◽  
Rna Purification ◽  
Chemical Probing

scholarly journals Large-Scale in Vitro Transcription, RNA Purification and Chemical Probing Analysis

2018 ◽  
Vol 48 (5) ◽  
pp. 1915-1927 ◽  
Author(s):  
Fariha Kanwal ◽  
Ting Chen ◽  
Yunlon Zhang ◽  
Altaf Simair ◽  
Cai Rujie ◽  
...  
Keyword(s):  
Structure Analysis ◽  
Rna Structure ◽  
In Vitro Transcription ◽  
Size Exclusion ◽  
Nucleoside Triphosphate ◽  
Separation Procedure ◽  
Rna Purification ◽  
Chemical Probing ◽  
Rna Interaction

Background/Aims: RNA elements such as catalytic RNA, riboswitch, microRNA, and long non coding RNA (lncRNA) play central roles in many cellular processes. Studying diverse RNA functions require large quantities of RNA for precise structure analysis. Current RNA structure and function studies can benefit from improved RNA quantity and quality, simpler separation procedure and enhanced accuracy of structural analysis. Methods: Here we present an optimized protocol for analyzing the structure of any RNA, including in vitro transcription, size-exclusion chromatography (SEC) based denaturing purification and improved secondary structure analysis by chemical probing. Results: We observed that higher Mg2+, nucleoside triphosphate (NTP) concentrations and longer reaction duration can improve the RNA yield from in vitro transcription, specifically for longer and more complicated constructs. Our improved SEC-based denaturing RNA purification effectively halved the experiment duration and labor without introducing any contaminant. Finally, this study increased the accuracy and signal-to-noise ratio (SNR) of selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) chemical probing for analyzing RNA structure. Conclusion: Part or all of our modified method can improve almost any RNA-related study from protein-RNA interaction analysis to crystallography.

scholarly journals Production and Purification of Artificial Circular RNA Sponges for Application in Molecular Biology and Medicine

2020 ◽  
Vol 3 (2) ◽  
pp. 42 ◽  
Author(s):  
Janina Breuer ◽  
Oliver Rossbach
Keyword(s):  
Molecular Biology ◽  
Large Scale ◽  
Circular Rna ◽  
In Vitro Transcription ◽  
Circular Rnas ◽  
Small Scale ◽  
Limiting Factor ◽  
Scale Production ◽  
Naturally Occurring

Characterized by their covalently closed structure and thus an elevated stability compared to linear RNA molecules, circular RNAs (circRNAs) form a novel class of mainly non-coding RNAs. Although the biological functions of naturally occurring circRNAs are largely unknown, they were reported to act as molecular sponges, sequestering microRNAs (miRNAs), resulting in a de-repression of target mRNAs. Taking these characteristics of naturally occurring circRNAs into account, artificial circRNAs could be a potential tool in molecular biology and medicine. Using the Hepatitis C virus (HCV) as a model system, this application of artificial circular RNAs was demonstrated. The virus requires cellular miRNA miR-122 for its life cycle, and circRNAs specifically engineered to efficiently sequester this miRNA impacted viral propagation. Since in this context the production of engineered circRNA remains the limiting factor, we present a method to produce and efficiently purify artificial circRNA sponges (ciRS) in vitro. In this protocol we provide insights into a small-scale and large-scale production technique of artificial circular RNA sponges relying on in vitro transcription and RNA ligation.

Purification of the Antihemophilic Factor by Gel Filtration on Agarose

1969 ◽  
Vol 22 (03) ◽  
pp. 577-583 ◽  
Author(s):  
M.M.P Paulssen ◽  
A.C.M.G.B Wouterlood ◽  
H.L.M.A Scheffers
Keyword(s):  
Factor Viii ◽  
Plasma Proteins ◽  
Large Scale ◽  
Gel Filtration ◽  
Large Scale Preparation ◽  
Scale Preparation ◽  
Antihemophilic Factor

SummaryFactor VIII can be isolated from plasma proteins, including fibrinogen by chromatography on agarose. The best results were obtained with Sepharose 6B. Large scale preparation is also possible when cryoprecipitate is separated by chromatography. In most fractions containing factor VIII a turbidity is observed which may be due to the presence of chylomicrons.The purified factor VIII was active in vivo as well as in vitro.

Naphthoquinones and derivatives for chemotherapy: perspectives and limitations of their anti-trypanosomatids activities

2020 ◽  
Vol 26 ◽  
Author(s):  
Luíza Dantas-Pereira ◽  
Edézio F. Cunha-Junior ◽  
Valter V. Andrade-Neto ◽  
John F. Bower ◽  
Guilherme A. M. Jardim ◽  
...  
Keyword(s):  
Large Scale ◽  
Neglected Tropical Diseases ◽  
Folk Medicine ◽  
Leishmania Species ◽  
Parasitic Infections ◽  
Mechanistic Analysis ◽  
Leishmania Spp ◽  
Nanomolar Range

: Chagas disease, Sleeping sickness and Leishmaniasis, caused by trypanosomatids Trypanosoma cruzi, Trypanosoma brucei and Leishmania spp., respectively, are considered neglected tropical diseases, and they especially affect impoverished populations in the developing world. The available chemotherapies are very limited and a search for alternatives is still necessary. In folk medicine, natural naphthoquinones have been employed for the treatment of a great variety of illnesses, including parasitic infections. This review is focused on the anti-trypanosomatid activity and mechanistic analysis of naphthoquinones and derivatives. Among all the series of derivatives tested in vitro, naphthoquinone-derived 1,2,3-triazoles were very active on T. cruzi infective forms in blood bank conditions, as well as in amastigotes of Leishmania spp. naphthoquinones containing a CF3 on a phenyl amine ring inhibited T. brucei proliferation in the nanomolar range, and naphthopterocarpanquinones stood out for their activity on a range of Leishmania species. Some of these compounds showed a promising selectivity index (SI) (30 to 1900), supporting further analysis in animal models. Indeed, high toxicity to the host and inactivation by blood components are crucial obstacles to be overcome to use naphthoquinones and/or their derivatives for chemotherapy. Multidisciplinary initiatives embracing medicinal chemistry, bioinformatics, biochemistry, and molecular and cellular biology need to be encouraged to allow the optimization of these compounds. Large scale automated tests are pivotal for the efficiency of the screening step, and subsequent evaluation of both the mechanism of action in vitro and pharmacokinetics in vivo are essential for the development of a novel, specific and safe derivative, minimizing adverse effects.

Dehydroepiandrosterone (DHEA) and its Sulphate (DHEAS) in Alzheimer’s Disease

2020 ◽  
Vol 17 (2) ◽  
pp. 141-157 ◽  
Author(s):  
Dubravka S. Strac ◽  
Marcela Konjevod ◽  
Matea N. Perkovic ◽  
Lucija Tudor ◽  
Gordana N. Erjavec ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Large Scale ◽  
Therapeutic Potential ◽  
Relevant Literature ◽  
Comprehensive Overview ◽  
Brain Functions ◽  
Specific Effects ◽  
And Behavior

Background: Neurosteroids Dehydroepiandrosterone (DHEA) and Dehydroepiandrosterone Sulphate (DHEAS) are involved in many important brain functions, including neuronal plasticity and survival, cognition and behavior, demonstrating preventive and therapeutic potential in different neuropsychiatric and neurodegenerative disorders, including Alzheimer’s disease. Objective: The aim of the article was to provide a comprehensive overview of the literature on the involvement of DHEA and DHEAS in Alzheimer’s disease. Method: PubMed and MEDLINE databases were searched for relevant literature. The articles were selected considering their titles and abstracts. In the selected full texts, lists of references were searched manually for additional articles. Results: We performed a systematic review of the studies investigating the role of DHEA and DHEAS in various in vitro and animal models, as well as in patients with Alzheimer’s disease, and provided a comprehensive discussion on their potential preventive and therapeutic applications. Conclusion: Despite mixed results, the findings of various preclinical studies are generally supportive of the involvement of DHEA and DHEAS in the pathophysiology of Alzheimer’s disease, showing some promise for potential benefits of these neurosteroids in the prevention and treatment. However, so far small clinical trials brought little evidence to support their therapy in AD. Therefore, large-scale human studies are needed to elucidate the specific effects of DHEA and DHEAS and their mechanisms of action, prior to their applications in clinical practice.

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