Interconversion of Anthozoa GFP-like fluorescent and non-fluorescent proteins by mutagenesis

BACKGROUND: Within the family of green fluorescent protein (GFP) homologs, one can mark two main groups, specifically, fluorescent proteins (FPs) and non-fluorescent or chromoproteins (CPs). Structural background of differences between FPs and CPs are poorly understood to date. RESULTS: Here, we applied site-directed and random mutagenesis in order to to transform CP into FP and vice versa. A purple chromoprotein asCP (asFP595) from Anemonia sulcata and a red fluorescent protein DsRed from Discosoma sp. were selected as representatives of CPs and FPs, respectively. For asCP, some substitutions at positions 148 and 165 (numbering in accordance to GFP) were found to dramatically increase quantum yield of red fluorescence. For DsRed, substitutions at positions 148, 165, 167, and 203 significantly decreased fluorescence intensity, so that the spectral characteristics of these mutants became more close to those of CPs. Finally, a practically non-fluorescent mutant DsRed-NF was generated. This mutant carried four amino acid substitutions, specifically, S148C, I165N, K167M, and S203A. DsRed-NF possessed a high extinction coefficient and an extremely low quantum yield (< 0.001). These spectral characteristics allow one to regard DsRed-NF as a true chromoprotein. CONCLUSIONS: We located a novel point in asCP sequence (position 165) mutations at which can result in red fluorescence appearance. Probably, this finding could be applied onto other CPs to generate red and far-red fluorescent mutants. A possibility to transform an FP into CP was demonstrated. Key role of residues adjacent to chromophore's phenolic ring in fluorescent/non-fluorescent states determination was revealed

Isolation, characterization and molecular cloning of Duplex-Specific Nuclease from the hepatopancreas of the Kamchatka crab

<p>Abstract</p> <p>Background</p> <p>Nucleases, which are key components of biologically diverse processes such as DNA replication, repair and recombination, antiviral defense, apoptosis and digestion, have revolutionized the field of molecular biology. Indeed many standard molecular strategies, including molecular cloning, studies of DNA-protein interactions, and analysis of nucleic acid structures, would be virtually impossible without these versatile enzymes. The discovery of nucleases with unique properties has often served as the basis for the development of modern molecular biology methods. Thus, the search for novel nucleases with potentially exploitable functions remains an important scientific undertaking.</p> <p>Results</p> <p>Using degenerative primers and the rapid amplification of cDNA ends (RACE) procedure, we cloned the Duplex-Specific Nuclease (DSN) gene from the hepatopancreas of the Kamchatka crab and determined its full primary structure. We also developed an effective method for purifying functional DSN from the crab hepatopancreas. The isolated enzyme was highly thermostable, exhibited a broad pH optimum (5.5 – 7.5) and required divalent cations for activity, with manganese and cobalt being especially effective. The enzyme was highly specific, cleaving double-stranded DNA or DNA in DNA-RNA hybrids, but not single-stranded DNA or single- or double-stranded RNA. Moreover, only DNA duplexes containing at least 9 base pairs were effectively cleaved by DSN; shorter DNA duplexes were left intact.</p> <p>Conclusion</p> <p>We describe a new DSN from Kamchatka crab hepatopancreas, determining its primary structure and developing a preparative method for its purification. We found that DSN had unique substrate specificity, cleaving only DNA duplexes longer than 8 base pairs, or DNA in DNA-RNA hybrids. Interestingly, the DSN primary structure is homologous to well-known Serratia-like non-specific nucleases structures, but the properties of DSN are distinct. The unique substrate specificity of DSN should prove valuable in certain molecular biology applications.</p

Mechanistic Insights into the Desorption of Methanol and Dimethyl Ether Over ZSM-5 Catalysts

Acknowledgements Financial support from the Petroleum Technology Development Fund of Nigeria (PTDF/ED/PHD/OO/766/15) and from the European Commission in the scope of the 7th Framework program BIOGO project (Grant Number: 604296) https://www.biogo. eu/ is acknowledged.Peer reviewedPublisher PD

Practical and reliable FRET/FLIM pair of fluorescent proteins

Background: In spite of a great number of monomeric fluorescent proteins developed in the recent years, the reported fluorescent protein-based FRET pairs are still characterized by a number of disadvantageous features, complicating their use as reporters in cell biology and for high-throughput cell-based screenings. Results: Here we screened some of the recently developed monomeric protein pairs to find the optimal combination, which would provide high dynamic range FRET changes, along with high pH- and photo-stability, fast maturation and bright fluorescence, and reliable detection in any fluorescent imaging system. Among generated FRET pairs, we have selected TagGFP-TagRFP, combining all the mentioned desirable characteristics. On the basis of this highly efficient FRET pair, we have generated a bright, high contrast, pH- and photo-stable apoptosis reporter, named CaspeR3 (Caspase 3 Reporter). Conclusion: The combined advantages suggest that the TagGFP-TagRFP is one of the most efficient green/red couples available to date for FRET/FLIM analyses to monitor interaction of proteins of interest in living cells and to generate FRET-based sensors for various applications. CaspeR3 provides reliable detection of apoptosis, and should become a popular tool both for cell biology studies and high throughput screening assays

Analysis of alternative splicing of cassette exons at single-cell level using two fluorescent proteins

Alternative splicing plays a major role in increasing proteome complexity and regulating gene expression. Here, we developed a new fluorescent protein-based approach to quantitatively analyze the alternative splicing of a target cassette exon (skipping or inclusion), which results in an open-reading frame shift. A fragment of a gene of interest is cloned between red and green fluorescent protein (RFP and GFP)-encoding sequences in such a way that translation of the normally spliced full-length transcript results in expression of both RFP and GFP. In contrast, alternative exon skipping results in the synthesis of RFP only. Green and red fluorescence intensities can be used to estimate the proportions of normal and alternative transcripts in each cell. The new method was successfully tested for human PIG3 (p53-inducible gene 3) cassette exon 4. Expected pattern of alternative splicing of PIG3 minigene was observed, including previously characterized effects of UV light irradiation and specific mutations. Interestingly, we observed a broad distribution of normal to alternative transcript ratio in individual cells with at least two distinct populations with ∼45% and >95% alternative transcript. We believe that this method is useful for fluorescence-based quantitative analysis of alternative splicing of target genes in a variety of biological models

Микроакселерометр на поверхностных акустических волнах с кольцевым резонатором на анизотропном материале

Introduction. Diagnostic systems are designed to monitor the condition of operational components (for example, on the railway). It is imperative that micro-electromechanical systems (MEMS) equipped with acceleration sensors (accelerometers) be used as part of measuring diagnostic systems. It is known that accelerometers are operated under increased vibration and repeated shock loads. This imposes a limitation both on the accelerometer design and the properties of materials from which these devices are produced.Aim. To develop a micromechanical accelerometer (MMA) for surface acoustic waves (SAW), capable of measuring shock effects.Materials and methods. The theoretical part of the study was carried out using the mathematical theory of differential equations, theoretical mechanics, finite element analysis and elements of SAW theory. In the course of the work, the following methods of mathematical processing were applied: MATLAB, Mathcad, Maple, COMSOL Multiphysics, OOFELIE: Multiphysics, Bluehill3 software, CorelDRAW. Experimental studies were also conducted using the INSTRON 5985 floor automated test system.Results. An original design of MMA on a SAW capable of measuring shock effects in hundreds of g was proposed. A sensing element (SE) of the sensor was developed. An analysis of the plate materials for their use as part of the SAW-based MMA design showed that SE from the quartz ST-cut material has a wider range of measured accelerations and a higher sensitivity threshold than SE from the YX-128˚ cut-off lithium niobate material. Requirements were developed to increase the SE sensitivity threshold. Design requirements were developed, and an interdigital transducer (IDT) topology in the form of a ring resonator was proposed. The following output characteristics were assessed: sensitivity threshold, dynamic range and scale factor. In addition, a procedure was developed for calculating MMA on a SAW with a ring resonator on an anisotropic material. It was found that the developed SE is characterized by a high sensitivity threshold, a wide dynamic range and a low transverse sensitivity.Conclusion. The technique proposed for designing a sensing element for use in solid-state linear acceleration sensors facilitates, depending on technical requirements, selection of construction materials and sensor design. Due to the originality of the design and engineering solutions, the proposed accelerometer allows measurements to be carried out across a wide range of impact loads.Введение. Состояние объектов эксплуатации (например, на железной дороге) контролируется системами диагностики. В их составе используются микроэлектромеханические системы, комплектуемые датчиками ускорения (акселерометрами). В процессе эксплуатации акселерометры подвергаются значительным вибрациям и многократно повторяющимся ударным воздействиям. Это накладывает ограничения на конструкцию и материалы, из которых изготавливаются акселерометры.Цель работы. Разработка микромеханического акселерометра (ММА) на поверхностных акустических волнах (ПАВ), способного измерять ударные воздействия.Материалы и методы. Теоретическая часть работы выполнялась с применением математической теории дифференциальных уравнений, теоретической механики, конечно-элементарного анализа и элементов теории ПАВ. В ходе работы применялась математическая обработка в программах MATLAB, Mathcad, Maple, COMSOL Multiphysics, OOFELIE::Multiphysics, ПО Bluehill3, CorelDRAW. Экспериментальные исследования проведены с привлечением напольной автоматизированной испытательной системы INSTRON 5985.Результаты. Разработана концепция построения и предложена оригинальная конструкция ММА на ПАВ, способного измерять ударные воздействия в сотни g. Разработан чувствительный элемент (ЧЭ) сенсора. Анализ материалов для пластин в составе конструкции ММА на ПАВ показал, что ЧЭ из кварца ST-среза отличается более широким диапазоном измеряемых ускорений и более высоким порогом чувствительности, чем ЧЭ из ниобата лития среза YX-128°. Выработаны требования и исследована возможность повышения порога чувствительности датчика. Сформулированы требования к проектированию и предложена топология встречно-штыревого преобразователя (ВШП) в виде кольцевого резонатора. Предложена оригинальная топология резонатора с неэквидистантным ВШП для учета анизотропии материала чувствительного элемента. Оценены выходные характеристики: порог чувствительности, динамический диапазон, масштабный коэффициент. Предложена методика расчета ММА на ПАВ с кольцевым резонатором на анизотропном материале. ЧЭ ММА такой конструкции имеет высокий порог чувствительности, широкий динамический диапазон и малую поперечную чувствительность.Заключение. Предложенная методика проектирования ЧЭ твердотельного датчика линейных ускорений позволяет выбрать материал и систему съема измерительной информации в зависимости от технических требований. Благодаря оригинальности конструкторско-технологического решения предложенный акселерометр позволяет проводить измерения в широком диапазоне ударных воздействий

Circular Permutation of Red Fluorescent Proteins

Circular permutation of fluorescent proteins provides a substrate for the design of molecular sensors. Here we describe a systematic exploration of permutation sites for mCherry and mKate using a tandem fusion template approach. Circular permutants retaining more than 60% (mCherry) and 90% (mKate) brightness of the parent molecules are reported, as well as a quantitative evaluation of the fluorescence from neighboring mutations. Truncations of circular permutants indicated essential N- and C- terminal segments and substantial flexibility in the use of these molecules. Structural evaluation of two cp-mKate variants indicated no major conformational changes from the previously reported wild-type structure, and cis conformation of the chromophores. Four cp-mKates were identified with over 80% of native fluorescence, providing important new building blocks for sensor and complementation experiments

Omecamtiv mecarbil in chronic heart failure with reduced ejection fraction, GALACTIC‐HF: baseline characteristics and comparison with contemporary clinical trials

Aims: The safety and efficacy of the novel selective cardiac myosin activator, omecamtiv mecarbil, in patients with heart failure with reduced ejection fraction (HFrEF) is tested in the Global Approach to Lowering Adverse Cardiac outcomes Through Improving Contractility in Heart Failure (GALACTIC‐HF) trial. Here we describe the baseline characteristics of participants in GALACTIC‐HF and how these compare with other contemporary trials. Methods and Results: Adults with established HFrEF, New York Heart Association functional class (NYHA) ≥ II, EF ≤35%, elevated natriuretic peptides and either current hospitalization for HF or history of hospitalization/ emergency department visit for HF within a year were randomized to either placebo or omecamtiv mecarbil (pharmacokinetic‐guided dosing: 25, 37.5 or 50 mg bid). 8256 patients [male (79%), non‐white (22%), mean age 65 years] were enrolled with a mean EF 27%, ischemic etiology in 54%, NYHA II 53% and III/IV 47%, and median NT‐proBNP 1971 pg/mL. HF therapies at baseline were among the most effectively employed in contemporary HF trials. GALACTIC‐HF randomized patients representative of recent HF registries and trials with substantial numbers of patients also having characteristics understudied in previous trials including more from North America (n = 1386), enrolled as inpatients (n = 2084), systolic blood pressure &lt; 100 mmHg (n = 1127), estimated glomerular filtration rate &lt; 30 mL/min/1.73 m2 (n = 528), and treated with sacubitril‐valsartan at baseline (n = 1594). Conclusions: GALACTIC‐HF enrolled a well‐treated, high‐risk population from both inpatient and outpatient settings, which will provide a definitive evaluation of the efficacy and safety of this novel therapy, as well as informing its potential future implementation

Genetically Encoded Red Photosensitizers with Enhanced Phototoxicity

Genetically encoded photosensitizers are increasingly used as optogenetic tools to control cell fate or trigger intracellular processes. A monomeric red fluorescent protein called SuperNova has been recently developed, however, it demonstrates suboptimal characteristics in most phototoxicity-based applications. Here, we applied directed evolution to this protein and identified SuperNova2, a protein with S10R substitution that results in enhanced brightness, chromophore maturation and phototoxicity in bacterial and mammalian cell cultures