Red Form Research Articles

Combined Structural and Computational Study of the mRubyFT Fluorescent Timer Locked in Its Blue Form.

The mRubyFT is a monomeric genetically encoded fluorescent timer based on the mRuby2 fluorescent protein, which is characterized by the complete maturation of the blue form with the subsequent conversion to the red one. It has higher brightness in mammalian cells and higher photostability compared with other fluorescent timers. A high-resolution structure is a known characteristic of the mRubyFT with the red form chromophore, but structural details of its blue form remain obscure. In order to obtain insight into this, we obtained an S148I variant of the mRubyFT (mRubyFTS148I) with the blocked over time blue form of the chromophore. X-ray data at a 1.8 Å resolution allowed us to propose a chromophore conformation and its interactions with the neighboring residues. The imidazolidinone moiety of the chromophore is completely matured, being a conjugated π-system. The methine bridge is not oxidized in the blue form bringing flexibility to the phenolic moiety that manifests itself in poor electron density. Integration of these data with the results of molecular dynamic simulation disclosed that the OH group of the phenolic moiety forms a hydrogen bond with the side chain of the T163 residue. A detailed comparison of mRubyFTS148I with other available structures of the blue form of fluorescent proteins, Blue102 and mTagBFP, revealed a number of characteristic differences. Molecular dynamic simulations with the combined quantum mechanic/molecular mechanic potentials demonstrated that the blue form exists in two protonation states, anion and zwitterion, both sharing enolate tautomeric forms of the C=C-O- fragment. These two forms have similar excitation energies, as evaluated by calculations. Finally, excited state molecular dynamic simulations showed that excitation of the chromophore in both protonation states leads to the same anionic fluorescent state. The data obtained shed light on the structural features and spectral properties of the blue form of the mRubyFT timer.

Variability of morphological characteristics of red forms of amaranth with a high content of biologically active substances under conditions of the Moscow region

The study of variability of morphological features in red-colored varieties of amaranth and their correlations at different stages in the conditions of the Moscow region makes it possible to identify features that affect productivity, amount of amaranthine and other biologically active substances. After analyzing vegetative characteristics, the varietal features of amaranth plants were proved to have a smaller impact on root length, plant height and number of leaves characteristics than the cultivation conditions, especially at the initial stages (ISB (Influence share of the weather conditions) from 22 to 58 %). It follows from the phenotypic variability analysis that the genotypic component values varied significantly only at the last stages (Cvg 35 %). The maximum values of phenotypic variability were marked in all varieties at the stage of active growth considering the root length (Cve = 3247 %) and the number of leaves (Cve = 4885 %). The generative characteristics seemed to be significantly influenced by the varietal factor (ISA (Influence share of varietal characteristics) = 4088 %) starting from the third stage. The genotypic component of all varieties was high considering the inflorescence weight (Cvg = 7586 %). In signs of general productivity, it was studied that the leaves (93112 g/plant) contributed the most for amaranth forms, which mass in all varieties largely depended on weather conditions (ISB 55 %). The interrelation of the leaves productivity was noted high with all the vegetative characteristics on phase III-V (r = 0.710.92) and with inflorescence mass on phase V-VI.

Light-induced infrared difference spectroscopy on three different forms of orange carotenoid protein: focus on carotenoid vibrations.

Orange carotenoid protein (OCP) is a photoactive carotenoprotein involved in photoprotection of cyanobacteria, which uses a keto-catorenoid as a chromophore. When it absorbs blue-green light, it converts from an inactive OCPO orange form to an activated OCPR red form, the latter being able to bind the light-harvesting complexes facilitating thermal dissipation of the excess of absorbed light energy. Several research groups have focused their attention on the photoactivation mechanism, characterized by several steps, involving both carotenoid photophysics and protein conformational changes. Among the used techniques, time-resolved IR spectroscopy have the advantage of providing simultaneously information on both the chromophore and the protein, giving thereby the possibility to explore links between carotenoid dynamics and protein dynamics, leading to a better understanding of the mechanism. However, an appropriate interpretation of data requires previous assignment of marker IR bands, for both the carotenoid and the protein. To date, some assignments have concerned specific α-helices of the OCP backbone, but no specific marker band for the carotenoid was identified on solid ground. This paper provides evidence for the assignment of putative marker bands for three carotenoids bound in three different OCPs: 3'-hydroxyechineone (3'-hECN), echinenone (ECN), canthaxanthin (CAN). Light-induced FTIR difference spectra were recorded in H2O and D2O and compared with spectra of isolated carotenoids. The use of DFT calculations allowed to propose a description for the vibrations responsible of several IR bands. Interestingly, most bands are located at the same wavenumber for the three kinds of OCPs suggesting that the conformation of the three carotenoids is the same in the red and in the orange form. These results are discussed in the framework of recent time-resolved IR studies on OCP.

Blue-to-Red TagFT, mTagFT, mTsFT, and Green-to-FarRed mNeptusFT2 Proteins, Genetically Encoded True and Tandem Fluorescent Timers.

True genetically encoded monomeric fluorescent timers (tFTs) change their fluorescent color as a result of the complete transition of the blue form into the red form over time. Tandem FTs (tdFTs) change their color as a consequence of the fast and slow independent maturation of two forms with different colors. However, tFTs are limited to derivatives of the mCherry and mRuby red fluorescent proteins and have low brightness and photostability. The number of tdFTs is also limited, and there are no blue-to-red or green-to-far-red tdFTs. tFTs and tdFTs have not previously been directly compared. Here, we engineered novel blue-to-red tFTs, called TagFT and mTagFT, which were derived from the TagRFP protein. The main spectral and timing characteristics of the TagFT and mTagFT timers were determined in vitro. The brightnesses and photoconversions of the TagFT and mTagFT tFTs were characterized in live mammalian cells. The engineered split version of the TagFT timer matured in mammalian cells at 37 °C and allowed the detection of interactions between two proteins. The TagFT timer under the control of the minimal arc promoter, successfully visualized immediate-early gene induction in neuronal cultures. We also developed and optimized green-to-far-red and blue-to-red tdFTs, named mNeptusFT and mTsFT, which were based on mNeptune-sfGFP and mTagBFP2-mScarlet fusion proteins, respectively. We developed the FucciFT2 system based on the TagFT-hCdt1-100/mNeptusFT2-hGeminin combination, which could visualize the transitions between the G1 and S/G2/M phases of the cell cycle with better resolution than the conventional Fucci system because of the fluorescent color changes of the timers over time in different phases of the cell cycle. Finally, we determined the X-ray crystal structure of the mTagFT timer and analyzed it using directed mutagenesis.

Enhanced detection sensitivity of neuronal activity patterns using CaMPARI1 vs. CaMPARI2.

Calcium-modulated photoactivatable ratiometric integrator (CaMPARI) is a calcium ion (Ca2+)- and light-dependent genetically encoded fluorescent activity integrator that can capture snapshots of neuronal activity through an irreversible process known as photoconversion. This unique property was previously used to label neurons based upon their tuning properties in order to map synaptic connectivity and to record large-scale neuronal activity in freely moving mice without attaching any mechanical device to them. The latest version of CaMPARI (CaMPARI2) was engineered to enhance the contrast generated by photoconverting the green protein to the activity-dependent red form and to reduce the Ca2+-independent photoconversion rate compared to the first generation of CaMPARI (CaMPARI1). However, here we show that this optimization process also resulted in reduced photoconversion efficiency of active neurons in the mouse cortex and hippocampus. Through side-by-side comparison of the two CaMPARI sensors under several experimental conditions, we show that CaMPARI1 exhibits a substantially higher red-to-green ratio in active cells than CaMPARI2. In addition, we show that CaMPARI1 also functions as a more sensitive traditional Ca2+ sensor than CaMPARI2 by producing larger activity-driven dynamic fluorescence changes in the observed neurons. Therefore, we conclude that during the optimization process of CaMPARI2, some of the sensor's characteristics were not predicted properly by in vitro screening assays, and therefore in vivo screening and validation steps should be included in future optimization attempts to increase the predictability of screening pipelines.

Xanthoma in different species - an unusual tumor with many faces.

Xanthoma is an interesting, inflammatory, noncancerous lesion that is rarely diagnosed in companion animals, although it has been reported in a number of domestic and wild species. Macroscopically, it can range from a few millimeter plaques or nodules to even several-centimeter masses. It usually occurs within the skin, but has been diagnosed in almost all other internal systems and organs. As the name suggests, xanthoma most often is of yellow color, but whitish and red forms are also encountered. Although inconspicuous, the presence of xanthoma is often reflected in disorders of lipid and lipoprotein metabolism, which affects the entire body. Therefore, in patients with even a small xanthoma on the skin, coexisting metabolic and endocrine disorders should be excluded, such as: hyperlipidemia, hypercholesterolaemia, hypothyroidism, hyperadrenocorticism and diabetes.

Evaluation of safety and efficacy of fractional CO2 laser in treatment of post traumatic atrophic scars

Aim: This research evaluated the safety and efficacy of fractional CO2 LASER (FCOL) in treatment of post traumatic atrophic scarring. Research design: This was a prospective, randomized research.Place and duration of the research: Outpatient Dermatology and Venereology Clinic and Plastic Surgery Departments at Tanta University Hospitals, from December 2019 to June 2021.Methodology: This research was carried out on 20 participants with post traumatic atrophic scarring treated with FCOL. Follow up 3 months after treatment and evaluation of the improvement was done by Vancouver scar scale, 3 blinded dermatologists’ assessment, and patient satisfaction score. Results: There was improvement in all participants with variable degrees, there were no scarring showed excellent improvement, 4 scarring showed good improvement (20%), 10 scarring showed fair improvement (50%) and 6 scarring showed poor improvement (30%). Adverse reactions were in general mild and well tolerated, in the form of transient redness, mild hyperpigmentation, mild pain, that all resolved within few days. No relation between the degree of improvement and age of the participants, site of the scarring, scar duration, but in sex of the participants, it was more in males than in females. Conclusion: FCOL may be an effective approach for treating atrophic scarring, from both an aesthetic and a functional perspective.

An alternative key for identifying three prostrate species of the genus Euphorbia

Plants are a valuable source of different folk and modern medicines. Therefore, correctly identifying the plant species is a prerequisite for using them in the health care system. Euphorbia species are of great medicinal importance but are difficult to distinguish due to the occurrence of ecotypes and a high degree of polymorphism within the species. Therefore, the present study was planned to explore the extent of variations in morphological characters among three Euphorbia species, namely, Euphorbia prostrata Aiton, Euphorbia serpens Kunth and Euphorbia thymifolia L. The edaphic ecotypes red and green forms of Euphorbia prostrata growing separately or adjacent to each other were reported during field surveys. To ensure identification accuracy, illustrated online key was prepared as a supportive tool for the traditional key.

The Role of the 145 Residue in Photochemical Properties of the Biphotochromic Protein mSAASoti: Brightness versus Photoconversion.

Photoswitchable fluorescent proteins (FPs) have become indispensable tools for studying life sciences. mSAASoti FP, a biphotochromic FP, is an important representative of this protein family. We created a series of mSAASoti mutants in order to obtain fast photoswitchable variants with high brightness. K145P mSAASoti has the highest molar extinction coefficient of all SAASoti mutants studied; C21N/K145P/M163A switches to the dark state 36 times faster than mSAASoti, but it lost its ability to undergo green-to-red photoconversion. Finally, the C21N/K145P/F177S and C21N/K145P/M163A/F177S variants demonstrated a high photoswitching rate between both green and red forms.

Bridging the Gap - A Traffic Light System to Ease Communication Amongst Surgeons, Hand Therapists and Patients.

Communication in the healthcare setup is essential for patient safety and for seamless delivery of services to the patient. We have adopted a novel way of communication in the form of a colour-coded visual aid in the form of a traffic light system to guide the patients through the course of their treatment. This shows the treatment plan in the form of Red, Amber, Green and Blue. The Red (Stop) denotes complete immobilisation with Splints, Amber (Proceed with caution) denotes active movements only, Green (Go forward) denotes Passive and Active movements and Blue shows when the patient can undergo guided weight bearing and strengthening exercises without a splint. The implementation of this system has created a streamlining of our protocol and improvement in the quality of the care we deliver. Level of Evidence: Level V (Therapeutic).

Different properties of two types of red fluorescent proteins in octocoral, Scleronephthya spp. as Akane families.

We report the different properties of two types of red fluorescent proteins (RFP), undescribed species, extracted from two octocorals, Scleronephthya sp. 1 (S. sp. 1) and S. sp, 2 (Alcyonacea, Nephtheidae). S. sp. 1, named Alc-Orange, emits strong green emission at 492 nm and weak red emission at 590 and 630 nm when excited at 449 and 574 nm, respectively. S. sp. 2, LS-Red, emits strong deep red at 642 nm and weak green at 480 and 510 nm when excited at 574 nm and 434 nm, respectively. LS-Red has a very large Stokes shift of about 208 nm emitting at 642 nm when excited at 434 nm. Interestingly, LS-Red shows some emissions at 480 (blue emission), 514 (green emission), 563 (orange emission), and 642 nm (deep red emission) continuously at pH 7.5, which means multicolored fluorescence protein by one excitation at 434 nm. In pH dependence of fluorescence of Alc-Orange (pH 13 to 3.5), no relation between 'green and red FPs' was observed, whereas LS-Red showed the interconversion between 'green and red forms' depending on pH (11.5 to 4.5).

The location of the low-energy states in Lhca1 favors excitation energy transfer to the core in the plant PSI-LHCI supercomplex.

Lhca1 is one of the four pigment-protein complexes composing the outer antenna of plant Photosystem I-light-havesting I supercomplex (PSI-LHCI). It forms a functional dimer with Lhca4 but, differently from this complex, it does not contain 'red-forms,' i.e., pigments absorbing above 700nm. Interestingly, the recent PSI-LHCI structures suggest that Lhca1 is the main point of delivering the energy harvested by the antenna to the core. To identify the excitation energy pathways in Lhca1, we developed a structure-based exciton model based on the simultaneous fit of the low-temperature absorption, linear dichroism, and fluorescence spectra of wild-type Lhca1 and two mutants, lacking chlorophylls contributing to the long-wavelength region of the absorption. The model enables us to define the locations of the lowest energy pigments in Lhca1 and estimate pathways and timescales of energy transfer within the complex and to the PSI core. We found that Lhca1 has a particular energy landscape with an unusual (compared to Lhca4, LHCII, and CP29) configuration of the low-energy states. Remarkably, these states are located near the core, facilitating direct energy transfer to it. Moreover, the low-energy states of Lhca1 are also coupled to the red-most state (red forms) of the neighboring Lhca4 antenna, providing a pathway for effective excitation energy transfer from Lhca4 to the core.

Patient noncompliance as the cause of skin toxicity in capecitabine therapy

When using capecitabine, manifestations of toxicity in the form of palmar-plantar dysesthesia, redness, excessive sensitivity and peeling of the skin of the palms and feet are well known. However, in the scientific literature available to us, we did not find a single case of skin toxicity with capecitabine, in contrast to targeted therapy, for which skin manifestations are very characteristic. The dermal toxicity described in this paper developed as a result of patient noncompliance. These side effects reduced the adherence to therapy: the next courses of chemotherapy were postponed.

Acaricidal Activity and Field Efficacy Analysis of the Potential Biocontrol Agent Bacillus vallismortis NBIF-001 against Spider Mites.

In recent years, spider mites have caused considerable economic losses to global agriculture. However, currently available management strategies are limited because of the rapid development of resistance. In this study, Bacillus vallismortis NBIF-001 was isolated and evaluated for its acaricidal activity. NBIF-001 exhibited a significant lethal effect on spider mites within 48 h. The median lethal concentration (LC50) of the culture powders (3.2 × 1010 CFU/g) was 50.2 µg/mL for Tetranychus urticae (red form), 18.0 µg/mL for T. urticae (green form), and 15.7 µg/mL for Panonychus citri (McGregor). Cultivation optimisation experiments showed that when the number of spores increased, fermentation toxicity also increased. Moreover, field experiments demonstrated that NBIF-001 performed well in the biocontrol of P. citri, which showed a similar corrected field efficacy with the chemical control (67.1 ± 7.9% and 71.1 ± 6.4% after 14 days). Genomics analysis showed that NBIF-001 contains 231 factors and seven gene clusters of metabolites that may be involved in its acaricidal activity. Further bioassays of the fermentation supernatants showed that 50× dilution treatments killed 72.5 ± 5.4% of the mites in 48 h, which was similar with those of the broth. Bioassays of the supernatant proteins confirmed that various proteins exhibited acaricidal activity. Five candidate proteins were expressed and purified successfully. The bioassays showed that the small protein BVP8 exhibited significant acaricidal activity with an LC50 of 12.4 μg/mL (T. urticae). Overall, these findings suggest that B. vallismortis NBIF-001 is a potential biocontrol agent for spider mite management.

Is orange carotenoid protein photoactivation a single-photon process?

In all published photoactivation mechanisms of orange carotenoid protein (OCP), absorption of a single photon by the orange dark state starts a cascade of red-shifted OCP ground-state intermediates that subsequently decay within hundreds of milliseconds, resulting in the formation of the final red form OCPR, which is the biologically active form that plays a key role in cyanobacteria photoprotection. A major challenge in deducing the photoactivation mechanism is to create a uniform description explaining both single-pulse excitation experiments, involving single-photon absorption, and continuous light irradiation experiments, where the red-shifted OCP intermediate species may undergo re-excitation. We thus investigated photoactivation of Synechocystis OCP using stationary irradiation light with a biologically relevant photon flux density coupled with nanosecond laser pulse excitation. The kinetics of photoactivation upon continuous and nanosecond pulse irradiation light show that the OCPR formation quantum yield increases with photon flux density; thus, a simple single-photon model cannot describe the data recorded for OCP in vitro. The results strongly suggest a consecutive absorption of two photons involving a red intermediate with ≈100 millisecond lifetime. This intermediate is required in the photoactivation mechanism and formation of the red active form OCPR.

Substancje szkodliwe występujące w preparatach do koloryzacji [Noxious substances in hair dye cosmetics

Hair dyeing has been popular since ancient times, when pigments were mainly extracted from plants. With the development of technology and cultural trends, the search began not only for new pigments but also for new substances, which would make hair dyeing more durable and intense. The addition of synthetic compounds to the composition of hair coloring cosmetics, in addition to the indicated benefits also resulted in side effects. The most often listed substances with toxic properties are PPD (p-phenylenediamine), PTD (p-toluenediamine), persulfates (e.g. sodium persulfate), lavson, m-aminophenol or resorcinol. The adverse effects of these substances on the organism of the client or the hairdresser may manifest in the form of skin redness, swelling, purulent rash, urticaria or even chemical leucoderma, asthma or rhinitis.

Reliability of the Sexual Knowledge Picture Instrument: a potential diagnostic instrument for sexual abuse in young children

ObjectiveTo determine the intra-rater and inter-rater reliability of the Sexual Knowledge Picture Instrument (SKPI), a potential diagnostic instrument for young suspected victims of sexual abuse containing three scoring forms, that...

First biphotochromic fluorescent protein moxSAASoti stabilized for oxidizing environment

Biphotochromic proteins simultaneously possess reversible photoswitching (on-to-off) and irreversible photoconversion (green-to-red). High photochemical reactivity of cysteine residues is one of the reasons for the development of “mox”-monomeric and oxidation resistant proteins. Based on site-saturated simultaneous two-point C105 and C117 mutagenesis, we chose C21N/C71G/C105G/C117T/C175A as the moxSAASoti variant. Since its on-to-off photoswitching rate is higher, off-to-on recovery is more complete and photoconversion rates are higher than those of mSAASoti. We analyzed the conformational behavior of the F177 side chain by classical MD simulations. The conformational flexibility of the F177 side chain is mainly responsible for the off-to-on conversion rate changes and can be further utilized as a measure of the conversion rate. Point mutations in mSAASoti mainly affect the pKa values of the red form and off-to-on switching. We demonstrate that the microscopic measure of the observed pKa value is the C–O bond length in the phenyl fragment of the neutral chromophore. According to molecular dynamics simulations with QM/MM potentials, larger C–O bond lengths are found for proteins with larger pKa. This feature can be utilized for prediction of the pKa values of red fluorescent proteins.

Structure Insights Into Photosystem I Octamer From Cyanobacteria

The diversity of photosystem oligomers is essential to understanding how photosynthetic organisms adapt to light conditions. Due to its structural and physiological significance, the assembly of the PSI supercomplex has been of great interest recently in terms of both chloroplast and cyanobacteria. In this study, two novel photosystem I supercomplexes were isolated for the first time from the low light incubated culture of filamentous cyanobacterium Anabaena sp. PCC 7120. These complexes were defined as PSI hexamers and octamers through biochemical and biophysical characterization. Their 77K emission spectra indicated that the red forms of chlorophylls seemed not to be affected during oligomerization. By cryo-EM single-particle analysis, a near-atomic (7.0 Å) resolution structure of a PSI octamer was resolved, and the molecular assemblies of a stable PSI octamer were revealed.

Ultrafast excited state dynamics in the monomeric and trimeric photosystem I core complex of Spirulina platensis probed by two-dimensional electronic spectroscopy.

Photosystem I (PSI), a naturally occurring supercomplex composed of a core part and a light-harvesting antenna, plays an essential role in the photosynthetic electron transfer chain. Evolutionary adaptation dictates a large variability in the type, number, arrangement, and absorption of the Chlorophylls (Chls) responsible for the early steps of light-harvesting and charge separation. For example, the specific location of long-wavelength Chls (referred to as red forms) in the cyanobacterial core has been intensively investigated, but the assignment of the chromophores involved is still controversial. The most red-shifted Chl a form has been observed in the trimer of the PSI core of the cyanobacterium Spirulina platensis, with an absorption centered at ∼740nm. Here, we apply two-dimensional electronic spectroscopy to study photoexcitation dynamics in isolated trimers and monomers of the PSI core of S. platensis. By means of global analysis, we resolve and compare direct downhill and uphill excitation energy transfer (EET) processes between the bulk Chls and the red forms, observing significant differences between the monomer (lacking the most far red Chl form at 740nm) and the trimer, with the ultrafast EET component accelerated by five times, from 500 to 100fs, in the latter. Our findings highlight the complexity of EET dynamics occurring over a broad range of time constants and their sensitivity to energy distribution and arrangement of the cofactors involved. The comparison of monomeric and trimeric forms, differing both in the antenna dimension and in the extent of red forms, enables us to extract significant information regarding PSI functionality.