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

Microaccelerometer on Surface Acoustic Waves with a Ring Resonator on Anisotropic Material

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

Diversity, Novelty, and Antimicrobial Activity of Endophytic Actinobacteria From Mangrove Plants in Beilun Estuary National Nature Reserve of Guangxi, China

Endophytic actinobacteria are one of the important pharmaceutical resources and well known for producing different types of bioactive substances. Nevertheless, detection of the novelty, diversity, and bioactivity on endophytic actinobacteria isolated from mangrove plants are scarce. In this study, five different mangrove plants, Avicennia marina, Aegiceras corniculatum, Kandelia obovota, Bruguiera gymnorrhiza, and Thespesia populnea, were collected from Beilun Estuary National Nature Reserve in Guangxi Zhuang Autonomous Region, China. A total of 101 endophytic actinobacteria strains were recovered by culture-based approaches. They distributed in 7 orders, 15 families, and 28 genera including Streptomyces, Curtobacterium, Mycobacterium, Micrococcus, Brevibacterium, Kocuria, Nocardioides, Kineococcus, Kytococcus, Marmoricola, Microbacterium, Micromonospora, Actinoplanes, Agrococcus, Amnibacterium, Brachybacterium, Citricoccus, Dermacoccus, Glutamicibacter, Gordonia, Isoptericola, Janibacter, Leucobacter, Nocardia, Nocardiopsis, Pseudokineococcus, Sanguibacter, and Verrucosispora. Among them, seven strains were potentially new species of genera Nocardioides, Streptomyces, Amnibacterium, Marmoricola, and Mycobacterium. Above all, strain 8BXZ-J1 has already been characterized as a new species of the genus Marmoricola. A total of 63 out of 101 strains were chosen to screen antibacterial activities by paper-disk diffusion method and inhibitors of ribosome and DNA biosynthesis by means of a double fluorescent protein reporter. A total of 31 strains exhibited positive results in at least one antibacterial assay. Notably, strain 8BXZ-J1 and three other potential novel species, 7BMP-1, 5BQP-J3, and 1BXZ-J1, all showed antibacterial bioactivity. In addition, 21 strains showed inhibitory activities against at least one “ESKAPE” resistant pathogens. We also found that Streptomyces strains 2BBP-J2 and 1BBP-1 produce bioactive compound with inhibitory activity on protein biosynthesis as result of translation stalling. Meanwhile, Streptomyces strain 3BQP-1 produces bioactive compound inducing SOS-response due to DNA damage. In conclusion, this study proved mangrove plants harbored a high diversity of cultivable endophytic actinobacteria, which can be a promising source for discovery of novel species and bioactive compounds