Neurotrophin receptor tyrosine kinases regulated with near-infrared light

Optical control over the activity of receptor tyrosine kinases (RTKs) provides an efficient way to reversibly and non-invasively map their functions. We combined catalytic domains of Trk (tropomyosin receptor kinase) family of RTKs, naturally activated by neurotrophins, with photosensory core module of DrBphP bacterial phytochrome to develop opto-kinases, termed Dr-TrkA and Dr-TrkB, reversibly switchable on and off with near-infrared and far-red light. We validated Dr-Trk ability to reversibly light-control several RTK pathways, calcium level, and demonstrated that their activation triggers canonical Trk signaling. Dr-TrkA induced apoptosis in neuroblastoma and glioblastoma, but not in other cell types. Absence of spectral crosstalk between Dr-Trks and blue-light-activatable LOV-domain-based translocation system enabled intracellular targeting of Dr-TrkA independently of its activation, additionally modulating Trk signaling. Dr-Trks have several superior characteristics that make them the opto-kinases of choice for regulation of RTK signaling: high activation range, fast and reversible photoswitching, and multiplexing with visible-light-controllable optogenetic tools.Peer reviewe

Smallest near-infrared fluorescent protein evolved from cyanobacteriochrome as versatile tag for spectral multiplexing

From a single domain of cyanobacteriochrome (CBCR) we developed a near-infrared (NIR) fluorescent protein (FP), termed miRFP670nano, with excitation at 645 nm and emission at 670 nm. This is the first CBCR-derived NIR FP evolved to efficiently bind endogenous biliverdin chromophore and brightly fluoresce in mammalian cells. miRFP670nano is a monomer with molecular weight of 17 kDa that is 2-fold smaller than bacterial phytochrome (BphP)-based NIR FPs and 1.6-fold smaller than GFP-like FPs. Crystal structure of the CBCR-based NIR FP with biliverdin reveals a molecular basis of its spectral and biochemical properties. Unlike BphP-derived NIR FPs, miRFP670nano is highly stable to denaturation and degradation and can be used as an internal protein tag. miRFP670nano is an effective FRET donor for red-shifted NIR FPs, enabling engineering NIR FRET biosensors spectrally compatible with GFP-like FPs and blue-green optogenetic tools. miRFP670nano unlocks a new source of diverse CBCR templates for NIR FPs.Peer reviewe

In vivo photoacoustic multi-contrast imaging and detection of protein interactions using a small near-infrared photochromic protein

Photoacoustic (PA) computed tomography (PACT) is a non-invasive imaging technique offering optical contrast, high resolution, and deep penetration in biological tissues. PACT, highly sensitive to optical absorption by molecules, is inherently suited for molecular imaging using optically absorbing probes. Genetically encoded probes with photochromic behavior dramatically increase detection sensitivity and specificity of PACT through photoswitching and differential imaging. Starting with a DrBphP bacterial phytochrome, we have engineered a near-infrared photochromic probe, DrBphP-PCM, which is superior to the full-length RpBphP1 phytochrome previously used in differential PACT. DrBphP-PCM has a smaller size, better folding, and higher photoswitching contrast. We have also developed an advanced PACT technique, which combines the reversibly-switchable photochromic probes with single-impulse panoramic PACT, termed RS-SIP-PACT. Using RS-SIP-PACT, we have characterized DrBphP-PCM both in vitro and in vivo as an advanced near-infrared photochromic probe for PACT. We introduce two phytochromes into the same mammalian cells, resulting in a distinctive decay characteristic in comparison with the cells expressing DrBphP-PCM only. By discriminating the different decay characteristics, we successfully separate multiple cell types in deep tissues. The simple structural organization of DrBphP-PCM allows engineering a bimolecular PA complementation reporter, a split version of DrBphP-PCM, termed DrSplit. DrSplit enables PA detection of protein-protein interactions in deepseated mouse tumors and livers, achieving 125-μm spatial resolution and 530-cell sensitivity in vivo. The combination of RS-SIP-PACT with DrBphP-PCM and DrSplit holds great potential for non-invasive multi-contrast deep-tissue functional imaging

Reflection of the philosophy of existentialism in literary work of Albert Camus

The aim of the study – to do identification of the ideas of the philosophy of existentialism in the artistic work of A. Camus, analysis of the possibility of considering his works without knowledge in the field of the writer's philosophy.Цель исследования - выявление идей философии экзистенциализма в художественном творчестве А. Камю, анализ возможности рассматривать его произведения без знаний в области философии писателя

A set of monomeric near-infrared fluorescent proteins for multicolor imaging across scales

Bright monomeric near-infrared (NIR) fluorescent proteins (FPs) are in high demand as protein tags for multicolor microscopy and in vivo imaging. Here we apply rational design to engineer a complete set of monomeric NIR FPs, which are the brightest genetically encoded NIR probes. We demonstrate that the enhanced miRFP series of NIR FPs, which combine high effective brightness in mammalian cells and monomeric state, perform well in both nanometer-scale imaging with diffraction unlimited stimulated emission depletion (STED) microscopy and centimeter-scale imaging in mice. In STED we achieve -40nm resolution in live cells. In living mice we detect -10(5) fluorescent cells in deep tissues. Using spectrally distinct monomeric NIR FP variants, we perform two-color live-cell STED microscopy and two-color imaging in vivo. Having emission peaks from 670nm to 720nm, the next generation of miRFPs should become versatile NIR probes for multiplexed imaging across spatial scales in different modalities.Peer reviewe

Actual philosophical problems

The article deals with main philosophical problems, usually named “immortal topics”, still actual for now.В статье рассмотрены некоторые основные проблемы философии, так называемые “вечные темы”, остающиеся актуальными и по сей ден

The psychedelic world of drugs and drug prevention in the literature of «cruel realism»

The aim of the study - to study the reasons for the emergence of the literary genre of "cruel realism" as a means of articulating existential attitudes and worldview principles.Цель исследования - изучение причины появления литературного жанра «жестокого реализма» как средства артикуляции экзистенциальных установок и мировоззренческих принципов

Figure of Borromean knot in Lacanian structural psychoanalysis

The article deals the interpretations of some Lacanian terms and the principles of structural psychoanalysis.В статье рассмотрены интерпретации некоторых лакановских терминов и принципы структурного психоанализа

Intuition in the medical profession

Each person understands the meaning of the term intuition in their own way. For some, intuition is something elusive, intangible, indefinable, but they answer without a doubt that they would like to develop it in themselves. For others, it is an inner voice. Many people define intuition as something supernatural, mystical, descended from above, from somewhere outside, as a kind of “sixth sense”. We can talk about intuition as an instant and immediate insight based on the already existing background knowledge. Among the various versions of the meaning of the word “intuition”, we can distinguish the reference one, given in the “Explanatory Dictionary of the Russian Language” by S. I. Ozhegov: INTUITION, 1. Intuition, subtle understanding, insight into the very essence of something. 2. Direct comprehension of the truth without justification by evidence.Каждый человек по-своему понимает значение термина интуиция. Для одних интуиция – это что-то неуловимое, неосязаемое, неопределяемое, но они без сомнений отвечают, что хотели бы в себе её развить. Для других — это внутренний голос. Многие определяет интуицию как нечто сверхъестественное, мистическое, снизошедшее сверху, откуда-то извне, как своеобразное “шестое чувство”. Можно говорить об интуиции, имея в виду инсайт, мгновенное и непосредственное озарение на основе уже имеющихся фоновых знаний. Среди разнообразных версий значения слова “интуиция” можно выделить эталонное, приведенное в “Толковом словаре русского языка” С.И. Ожегова: ИНТУИЦИЯ, 1. Чутьё, тонкое понимание, проникновение в самую суть чего-нибудь. 2. Непосредственное, без обоснования доказательствами постижение истины