PhOTO Zebrafish: A Transgenic Resource for In Vivo Lineage Tracing during Development and Regeneration

Background: Elucidating the complex cell dynamics (divisions, movement, morphological changes, etc.) underlying embryonic development and adult tissue regeneration requires an efficient means to track cells with high fidelity in space and time. To satisfy this criterion, we developed a transgenic zebrafish line, called PhOTO, that allows photoconvertible optical tracking of nuclear and membrane dynamics in vivo. Methodology: PhOTO zebrafish ubiquitously express targeted blue fluorescent protein (FP) Cerulean and photoconvertible FP Dendra2 fusions, allowing for instantaneous, precise targeting and tracking of any number of cells using Dendra2 photoconversion while simultaneously monitoring global cell behavior and morphology. Expression persists through adulthood, making the PhOTO zebrafish an excellent tool for studying tissue regeneration: after tail fin amputation and photoconversion of a ~100µm stripe along the cut area, marked differences seen in how cells contribute to the new tissue give detailed insight into the dynamic process of regeneration. Photoconverted cells that contributed to the regenerate were separated into three distinct populations corresponding to the extent of cell division 7 days after amputation, and a subset of cells that divided the least were organized into an evenly spaced, linear orientation along the length of the newly regenerating fin. Conclusions/Significance: PhOTO zebrafish have wide applicability for lineage tracing at the systems-level in the early embryo as well as in the adult, making them ideal candidate tools for future research in development, traumatic injury and regeneration, cancer progression, and stem cell behavior

Инфильтрация кожного лоскута местным анестетиком для послеоперационного обезболивания у детей с краниосиностозом после реконструктивных операций

The objective of the study is to evaluate the effectiveness of analgesia by infiltration of the skin flap with local anesthetic in children with craniosynostosis after reconstructive surgery.Materials and subjects. 50 children with craniosynostosis, who underwent reconstructive surgery on skull bones, were divided into two groups based on the method of postoperative anesthesia: in Group 1(experimental), the infiltration of the skin flap was used within multimodal anesthesia, while in Group 2, it was standard parenteral use of analgesic drugs. In the postoperative period, pain severity was assessed by FLACC scales, the amount of opioid and non-opioid analgesics consumed was assessed by the formalized Analgesiс Assessment Scale (FSA), and non-invasive hemodynamic monitoring (BP, HR) was performed.Results. The statistical analysis of the results revealed significant differences between groups in the assessment results of FSA and FLACC scales. In Group 1, the level of postoperative pain was significantly lower compared to Group 2. The amount of opioid and non-opioid analgesics consumed was also significantly lower in Group 1.Conclusion. The use of the infiltration of the skin flap as part of multimodal analgesia in children with craniosynostosis, after reconstructive surgery on skull bones significantly reduces the intensity of pain and the amount of opioid analgesics consumed in the postoperative period.Цель исследования: оценить эффективность обезболивания методом инфильтрации кожного лоскута местным анестетиком у детей с кра- ниосиностозом после реконструктивных операций.Материалы и методы. Проанализированы данные о последовательно оперированных 50 детях с диагнозом «краниосиностоз». Группа 1 (исследуемая) – в составе мультимодальной анальгезии использовали инфильтрацию кожного лоскута; группа 2 (контрольная) ‒ стан- дартное парентеральное применение анальгетических препаратов. В послеоперационном периоде оценивали интенсивность боли по шкале FLACC, количество потребленных наркотических и ненаркотических анальгетиков по формализированной шкале анальгезии (ФША), неинвазивный гемодинамический мониторинг.Результаты. Выявлены достоверные различия в группах по шкалам боли FLACC и ФША. В группе 1 уровень послеоперационной боли был значительно ниже, чем в группе 2. Количество потребленных наркотических и ненаркотических анальгетиков было также значительно ниже в группе 1.Вывод. Инфильтрация кожного лоскута местным анестетиком в составе мультимодальной анальгезии значительно снижает интенсивность боли у детей после реконструктивных операций по поводу краниосиностоза

ПАТОЛОГИЧЕСКИЕ МЕХАНИЗМЫ ВИЧ-АССОЦИИРОВАННЫХ НЕЙРОКОГНИТИВНЫХ РАССТРОЙСТВ

Significant communications of a condition of cognitive processes (memory, attention, thinking) and indicators of progressing of HIV infection are revealed (the HIV RNA level and CD4 lymphocytes in blood) that allows to assume existence of the direct mediated action of a virus on a functional condition of TsNS. Coherence of results of neuropsychological and neurovisualization techniques (EEG, MRT, PET) is shown. Involvement in pathological process at early stages of formation VICh-assotsiirovanykh of neurocognitive frustration of the following departments of a brain is revealed: forward zone crinkle, shell, mediobazavylny departments of a temporal share, premotorny departments, calloused body, retikulyarny formation.Выявлены значимые связи состояния когнитивных процессов (памяти, внимания, мышления) и показателей прогрессирования ВИЧ-инфекции (уровень РНК ВИЧ и CD4-лимфоцитов в крови), что позволяет предположить наличие прямого (или) опосредованного действия вируса на функциональное состояние ЦНС. Показана согласованность результатов нейропсихологических и нейровизуализационных методик (ЭЭГ, МРТ, ПЭТ). Выявлено вовлечение в патологический процесс на ранних этапах формирования ВИЧ-ассоциированых нейрокогнитивных расстройств следующих отделов головного мозга: передняя поясная извилина, скорлупа, медиобазальные отделы височной доли, премоторные отделы, мозолистое тело, ретикулярная формация

Single molecule tracking fluorescence microscopy in mitochondria reveals highly dynamic but confined movement of Tom40

Tom40 is an integral protein of the mitochondrial outer membrane, which as the central component of the Translocase of the Outer Membrane (TOM) complex forms a channel for protein import. We characterize the diffusion properties of individual Tom40 molecules fused to the photoconvertable fluorescent protein Dendra2 with millisecond temporal resolution. By imaging individual Tom40 molecules in intact isolated yeast mitochondria using photoactivated localization microscopy with sub-diffraction limited spatial precision, we demonstrate that Tom40 movement in the outer mitochondrial membrane is highly dynamic but confined in nature, suggesting anchoring of the TOM complex as a whole

A Comparison of Donor-Acceptor Pairs for Genetically Encoded FRET Sensors: Application to the Epac cAMP Sensor as an Example

We recently reported on CFP-Epac-YFP, an Epac-based single polypeptide FRET reporter to resolve cAMP levels in living cells. In this study, we compared and optimized the fluorescent protein donor/acceptor pairs for use in biosensors such as CFP-Epac-YFP. Our strategy was to prepare a wide range of constructs consisting of different donor and acceptor fluorescent proteins separated by a short linker. Constructs were expressed in HEK293 cells and tested for FRET and other relevant properties. The most promising pairs were subsequently used in an attempt to improve the FRET span of the Epac-based cAMP sensor. The results show significant albeit not perfect correlation between performance in the spacer construct and in the Epac sensor. Finally, this strategy enabled us to identify improved sensors both for detection by sensitized emission and by fluorescent lifetime imaging. The present overview should be helpful in guiding development of future FRET sensors

ЭЛЕКТРОФИЗИОЛОГИЧЕСКИЕ МЕТОДЫ В ДИАГНОСТИКЕ СУБКЛИНИЧЕСКИХ КОГНИТИВНЫХ НАРУШЕНИЙ У ВИЧ-ИНФИЦИРОВАННЫХ БОЛЬНЫХ

With a goal of the early detection of subclinical cognitive impairment and damage of the central nervous system in the early stages HIV infection the complex neurophysiological study (electroencephalogram, cognitive evoked potentials of auditory and visual modalities, complex sensory-motor reactions, visual evoked potentials) in 20 patients with HIV infection was performed. Electrophysiological signs of subclinical cognitive impairment and non-specific subclinical involvement of central nervous system were found. Application of the complex electrophysiological study by evaluating the function of widespread neuronal networks has allowed to increasing the sensitivity of early diagnosis of subclinical cognitive impairment in HIV-infected patients in 90% of cases.С целью раннего выявления субклинических когнитивных нарушений и поражения ЦНС у 20 пациентов с ВИЧ-инфекцией на ранних стадиях был проведен комплекс нейрофизиологических исследований: электроэнцефалограммы (ЭЭГ), когнитивных вызванных потенциалов мозга Р300 слуховой и зрительной модальностей, сложных сенсомоторных реакций, зрительных вызванных потенциалов и выявлены электрофизиологические признаки субклинических когнитивных нарушений и неспецифические электрофизиологические признаки субклинического поражения ЦНС. Применение расширенного комплекса электрофизиологических исследований с учетом специфики персистенции вируса в ЦНС позволило повысить чувствительность ранней диагностики субклинических когнитивных нарушений у ВИЧ-инфицированных больных до 90% случаев за счет оценки функции широко распространенных нейрональных сетей

Molecular Mechanism of a Green-Shifted, pH-Dependent Red Fluorescent Protein mKate Variant

Fluorescent proteins that can switch between distinct colors have contributed significantly to modern biomedical imaging technologies and molecular cell biology. Here we report the identification and biochemical analysis of a green-shifted red fluorescent protein variant GmKate, produced by the introduction of two mutations into mKate. Although the mutations decrease the overall brightness of the protein, GmKate is subject to pH-dependent, reversible green-to-red color conversion. At physiological pH, GmKate absorbs blue light (445 nm) and emits green fluorescence (525 nm). At pH above 9.0, GmKate absorbs 598 nm light and emits 646 nm, far-red fluorescence, similar to its sequence homolog mNeptune. Based on optical spectra and crystal structures of GmKate in its green and red states, the reversible color transition is attributed to the different protonation states of the cis-chromophore, an interpretation that was confirmed by quantum chemical calculations. Crystal structures reveal potential hydrogen bond networks around the chromophore that may facilitate the protonation switch, and indicate a molecular basis for the unusual bathochromic shift observed at high pH. This study provides mechanistic insights into the color tuning of mKate variants, which may aid the development of green-to-red color-convertible fluorescent sensors, and suggests GmKate as a prototype of genetically encoded pH sensors for biological studies

Single molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate

Photoswitchable fluorescent probes are central to localization-based super-resolution microscopy. Among these probes, fluorescent proteins are appealing because they are genetically encoded. Moreover, the ability to achieve a 1:1 labeling ratio between the fluorescent protein and the protein of interest makes these probes attractive for quantitative single-molecule counting. The percentage of fluorescent protein that is photoactivated into a fluorescently detectable form (i.e., the photoactivation efficiency) plays a crucial part in properly interpreting the quantitative information. It is important to characterize the photoactivation efficiency at the single-molecule level under the conditions used in super-resolution imaging. Here, we used the human glycine receptor expressed in Xenopus oocytes and stepwise photobleaching or single-molecule counting photoactivated localization microcopy (PALM) to determine the photoactivation efficiency of fluorescent proteins mEos2, mEos3.1, mEos3.2, Dendra2, mClavGR2, mMaple, PA-GFP and PA-mCherry. This analysis provides important information that must be considered when using these fluorescent proteins in quantitative super-resolution microscopy.Peer ReviewedPostprint (author's final draft