RESUMO
The spread of multidrug-resistant mycobacterium strains requires the development of new approaches to combat diseases caused by these pathogens. For that, photodynamic inactivation (PDI) is a promising approach. In this study, a tricarbocyanine (TCC) is used for the first time as a near-infrared (740 nm) activatable PDI photosensitizer to kill mycobacteria with deep light penetration. For better targeting, a novel tricarbocyanine dye functionalized with two trehalose units (TCC2Tre) is developed. The photodynamic effect of the conjugates against mycobacteria, including Mycobacterium tuberculosis, is evaluated. Under irradiation, TCC2Tre causes more effective killing of mycobacteria compared to the photosensitizer without trehalose conjugation, with 99.99% dead vegetative cells of M. tuberculosis and M. smegmatis. In addition, effective photoinactivation of dormant forms of M. smegmatis is observed after incubation with TCC2Tre. Mycobacteria treated with TCC2Tre are more sensitive to 740 nm light than the Gram-positive Micrococcus luteus and the Gram-negative Escherichia coli. For the first time, this study demonstrates the proof of principle of in vitro PDI of mycobacteria including the fast-growing M. smegmatis and the slow-growing M. tuberculosis using near-infrared activatable photosensitizers conjugated with trehalose. These findings are useful for the development of new efficient alternatives to antibiotic therapy.
Assuntos
Raios Infravermelhos , Mycobacterium smegmatis , Mycobacterium tuberculosis , Fármacos Fotossensibilizantes , Trealose , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/efeitos da radiação , Trealose/farmacologia , Trealose/química , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos da radiação , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/química , Carbocianinas/química , Carbocianinas/farmacologia , Fotoquimioterapia/métodosRESUMO
Mammalian spermatozoa are highly energized cells in which most of the proteins and activated signaling cascades are involved in the metabolic pathways. Flavin adenine dinucleotide (FAD) has one of the most important roles in the correct functional activity of spermatozoa since it acts as a cofactor for flavoenzymes, critical for proper metabolism and predominantly located in mitochondria. Non-invasive, vital and non-traumatic examination of sperm FAD level and microenvironment could be performed by fluorescence lifetime imaging microscopy (FLIM). In this study, we assessed the metabolic status of spermatozoa from healthy donors and found that FLIM could be used to segregate and separate the male germ cells according to the type of metabolic activity which corresponds with spermatozoa motility measured in standard spermogram tests.
Assuntos
Flavina-Adenina Dinucleotídeo , Sêmen , Espermatozoides , Humanos , Masculino , Flavina-Adenina Dinucleotídeo/metabolismo , Fluorescência , Microscopia de Fluorescência/métodos , Mitocôndrias/metabolismo , Sêmen/metabolismo , Espermatozoides/metabolismoRESUMO
During transition into a dormant state, Mycolicibacterium (Mycobacterium) smegmatis cells are able to accumulate free porphyrins that makes them sensitive to photodynamic inactivation (PDI). The formation of dormant cells in a liquid medium with an increased concentration of magnesium (up to 25 mM) and zinc (up to 62 µM) resulted in an increase in the total amount of endogenous porphyrins in dormant M. smegmatis cells and their photosensitivity, especially for bacteria phagocytosed by macrophages. To gain insight into possible targets for PDI in bacterial dormant mycobacterial cells, a proteomic profiling with SDS gel electrophoresis and mass spectrometry analysis were conducted. Illumination of dormant forms of M. smegmatis resulted in the disappearance of proteins in the separating SDS gel. Dormant cells obtained under an elevated concentration of metal ions were more sensitive to PDI. Differential analysis of proteins with their identification with MALDI-TOF revealed that 45.2% and 63.9% of individual proteins disappeared from the separating gel after illumination for 5 and 15 min, respectively. Light-sensitive proteins include enzymes belonging to the glycolytic pathway, TCA cycle, pentose phosphate pathway, oxidative phosphorylation and energy production. Several proteins involved in protecting against oxygen stress and protein aggregation were found to be sensitive to light. This makes dormant cells highly vulnerable to harmful factors during a long stay in a non-replicative state. PDI caused inhibition of the respiratory chain activity and destroyed enzymes involved in the synthesis of proteins and nucleic acids, the processes which are necessary for dormant cell reactivation and their transition to multiplying bacteria. Because of such multiple targeting, PDI action via endogenous porphyrins could be considered as an effective approach for killing dormant bacteria and a perspective to inactivate dormant mycobacteria and combat the latent form of mycobacteriosis, first of all, with surface localization.
Assuntos
Anti-Infecciosos , Proteômica , Mycobacterium smegmatis , Ciclo do Ácido Cítrico , Transporte de ElétronsRESUMO
Multimodality registration of optical and MR images in the same tissue volume in vivo may be enabled by MR contrast agents with an optical clearing (OC) effect. The goals of this study were to (a) investigate the effects of clinical MR contrast agent gadobutrol (GB) and its combinations as a potential OC agent assisting in fluorescence intensity (FI) imaging in vivo and (b) evaluate MRI as a tool for imaging of topical or systemic application of GB for the purpose of OC. Subcutaneous tumor xenografts expressing red fluorescent marker protein were used as disease models. MRI was performed at 1 T 1 H MRI using T1 -weighted 3D gradient-echo (T1w-3D GRE) sequences to measure time-dependent MR signal intensity changes by region of interest analysis after image segmentation. Topical application of 1.0 M or 0.7 M GB-containing OC mixture with water and dimethyl sulfoxide showed similar 30-40% increases of tumor FI during the initial 15 min. Afterwards, the OC effect of GB on FI and tumor/background FI ratio showed a decrease over time in the case of 1.0 M GB, unlike the 0.7 M GB mixture, which resulted in a steady increase of FI and tumor/background ratio for 15-60 min. The use of T1w-3D GRE MR pulse sequences showed that concentrated 1.0 M GB resulted in MR signal loss of the skin due to high magnetic susceptibility and that signal loss coincided with the OC effect. Intravenous injection of 0.3 mmol GB/kg resulted in a rapid but transient 40% increase of FI of the tumors. Overall, 1 T MRI enabled tracking of GB-containing OC compositions on the skin surface and tumor tissue, supporting the observation of a time-dependent FI increase in vivo.
Assuntos
Neoplasias , Compostos Organometálicos , Meios de Contraste , Humanos , Proteínas Luminescentes , Imageamento por Ressonância Magnética/métodos , Neoplasias/diagnóstico por imagem , Imagem Óptica , Proteína Vermelha FluorescenteRESUMO
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.
Assuntos
Corantes , Proteínas Luminescentes/metabolismo , Proteínas de Fluorescência Verde/químicaRESUMO
The spread of multi-drug-resistant bacterial strains causing serious infectious diseases dictates the development of new approaches to combat these diseases. In addition to drug resistance, the important causative agent of tuberculosis (Mycobacterium tuberculosis (Mtb)) is able to persist asymptomatically in individuals for many years, causing latent forms of tuberculosis. In such a dormant state, Mtb cells are also resistant to known antibiotics. In this regard, photodynamic inactivation (PDI) could be an effective alternative to antibiotics as its action is based on the generation of active forms of oxygen independently on the presence of specific antibiotic targets, thereby inactivating both drug-resistant and dormant bacteria. In this review, we summarise examples of the application of PDI for the elimination of representatives of the genus Mycobacteria, both in vitro and in vivo. According to published results, including photosensitisers in the PDI regime results in a significantly higher lethal effect. Such experiments were mainly performed using chemically synthesised photosensitisers, which need to be transported to the areas of bacterial infections, limiting PDI usage by surface (skin) diseases. In this regard, endogenous photosensitisers (mainly porphyrins) could be used to solve the problem of transportation. In vitro experiments demonstrate the effective application of PDI for mycobacteria, including Mtb, using endogenous porphyrins; the intracellular contents of these substances can be elevated by administration of 5-aminolevulenic acid, a precursor of porphyrin synthesis. Photodynamic inactivation can also be used for dormant mycobacteria, which are characterised by high levels of endogenous porphyrins. Thus, PDI can effectively eliminate drug-resistant mycobacteria. The exploitation of modern light-transmitting techniques opens new possibilities to use PDI in clinical settings. KEY POINTS: â¢The potential effects of photodynamic inactivation of mycobacteria are critically reviewed. â¢Approaches to photoinactivation of mycobacteria using exogenous and endogenous photosensitisers are described. â¢Prospects for the use of photodynamic inactivation in the treatment of tuberculosis are discussed.
Assuntos
Doenças Transmissíveis , Porfirinas , Antibacterianos , Humanos , Luz , Fármacos Fotossensibilizantes/farmacologia , Porfirinas/farmacologiaRESUMO
Mycobacterium tuberculosis is able to transition into a dormant state, causing a latent state of tuberculosis. Dormant mycobacteria acquire phenotypic resistance to all known antibacterial drugs; they are also able to maintain vitality in the host for decades and become active, causing the active form of the disease. In order to cure latent tuberculosis, new approaches should be developed. Earlier, we discovered accumulation in significant concentrations of porphyrins in dormant Mycobacterium smegmatis, which is a close, fast-growing relative of the causative agent of tuberculosis. In this study, we explore a new possibility to kill dormant mycobacteria by photodynamic inactivation (PDI) using accumulated porphyrins as endogenous photosensitisers. The dormant M. smegmatis were obtained under gradual acidification in Sauton's medium, for 14 days. Cells were exposed to light with different wavelengths emitted by three Spectra X light-emitting diodes (395/25, 470/24, 575/25 nm) and one separated 634-nm LED for 15 min. An increase in the concentration of coproporphyrin in M. smegmatis after 6 days of growth correlated with the beginning of a decrease in metabolic activity and formation of ovoid dormant forms. Dormant bacteria were sensitive to PDI and killed after 15-30 min of illumination, in contrast to active cells. The greatest inactivation of dormant mycobacteria occurred at 395 and 575 nm, which coincides with the main maximum of the absorption spectrum of extracted porphyrins. We, for the first time, demonstrate a successful application of PDI for inactivation of dormant mycobacteria, due to significant accumulation of endogenous photosensitisers-porphyrins.
Assuntos
Luz , Mycobacterium smegmatis/fisiologia , Mycobacterium smegmatis/efeitos da radiação , Fármacos Fotossensibilizantes/metabolismo , Porfirinas/metabolismo , Meios de Cultura/química , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos da radiação , Mycobacterium smegmatis/metabolismoRESUMO
SAASoti is a unique fluorescent protein (FP) that combines properties of green-to-red photoconversion and reversible photoswitching (in its green state), without any amino acid substitutions in the wild type gene. In the present work, we investigated its ability to photoswitch between fluorescent red ('on') and dark ('off') states. Surprisingly, generated by 400 nm exposure, the red form of SAASoti (R1) does not exhibit any reversible photoswitching behavior under 550 nm illumination, while a combination of prior 470 nm and subsequent 400 nm irradiation led to the appearance of another-R2-form that can be partially photoswitched (550 nm) to the dark state, with a very fast recovery time. The phenomenon might be explained by chemical modification in the chromophore microenvironment during prior 470 nm exposure, and the resulting R2 SAASoti differs chemically from the R1 form. The suggestion is supported by the mass spectrometry analysis of the tryptic peptides before and after 470 nm light exposure, that revealed Met164 oxidation, as proceeds in another dual phototransformable FP, IrisFP.
Assuntos
Corantes/química , Proteínas Luminescentes/química , Sequência de Aminoácidos , Animais , Antozoários/metabolismo , Cor , Espectrometria de Massas , OxirreduçãoRESUMO
Spectral diversity of fluorescent proteins, crucial for multiparameter imaging, is based mainly on chemical diversity of their chromophores. Recently we have reported, to our knowledge, a new green fluorescent protein WasCFP-the first fluorescent protein with a tryptophan-based chromophore in the anionic state. However, only a small portion of WasCFP molecules exists in the anionic state at physiological conditions. In this study we report on an improved variant of WasCFP, named NowGFP, with the anionic form dominating at 37°C and neutral pH. It is 30% brighter than enhanced green fluorescent protein (EGFP) and exhibits a fluorescence lifetime of 5.1 ns. We demonstrated that signals of NowGFP and EGFP can be clearly distinguished by fluorescence lifetime in various models, including mammalian cells, mouse tumor xenograft, and Drosophila larvae. NowGFP thus provides an additional channel for multiparameter fluorescence lifetime imaging microscopy of green fluorescent proteins.
Assuntos
Proteínas de Fluorescência Verde/química , Animais , Animais Geneticamente Modificados , Ânions/química , Drosophila , Escherichia coli , Fluorescência , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Microscopia Confocal , Mutação , Processos Fotoquímicos , Temperatura , Triptofano/química , Triptofano/metabolismoRESUMO
This article describes the genetically encoded caspase-3 FRET-sensor based on the terbium-binding peptide, cleavable linker with caspase-3 recognition site, and red fluorescent protein TagRFP. The engineered construction performs two induction-resonance energy transfer processes: from tryptophan of the terbium-binding peptide to Tb(3+) and from sensitized Tb(3+) to acceptor--the chromophore of TagRFP. Long-lived terbium-sensitized emission (microseconds), pulse excitation source, and time-resolved detection were utilized to eliminate directly excited TagRFP fluorescence and background cellular autofluorescence, which lasts a fraction of nanosecond, and thus to improve sensitivity of analyses. Furthermore the technique facilitates selective detection of fluorescence, induced by uncleaved acceptor emission. For the first time it was shown that fluorescence resonance energy transfer between sensitized terbium and TagRFP in the engineered construction can be studied via detection of microsecond TagRFP fluorescence intensities. The lifetime and distance distribution between donor and acceptor were calculated using molecular dynamics simulation. Using this data, quantum yield of terbium ions with binding peptide was estimated.
Assuntos
Técnicas Biossensoriais/métodos , Caspase 3/química , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas Luminescentes/química , Térbio/farmacologia , Sequência de Aminoácidos , Caspase 3/genética , Proteínas Luminescentes/genética , Metaloproteínas/química , Metaloproteínas/metabolismo , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Térbio/química , Proteína Vermelha FluorescenteRESUMO
Transition of Mycolicibacterium smegmatis (Msm) and Mycobacterium tuberculosis to dormancy in vitro is accompanied by an accumulation of free methylated forms of porphyrins (tetramethyl coproporphyrin - TMC) localized in the cell wall of dormant bacteria. A study of the fluorescence anisotropy of BODIPY based fluorescent probes on individual cell level using confocal microscope revealed significant changes in this parameter for BODIPY FL C16 from 0.05 to 0.22 for vegetative and dormant Msm cells correspondingly. Similarly, the increase of TMC concentration in vegetative Msm cells grown in the presence of 5-aminolevulinic acid (a known inducer of porphyrin synthesis) resulted in an increase of BODIPY FL C16 anisotropy. These changes in TMC concentration and membrane fluidity were accompanied by an inhibition of the activity of the respiratory chain measured by oxygen consumption and a reduction of the DCPIP redox acceptor. During the first 8 h of the reactivation of the dormant Msm cells, the porphyrin content and probe fluorescent anisotropy returned to the level for vegetative bacteria. We suggested that upon transition to dormancy, an accumulation of TMC in membranes leads to a decrease in membrane fluidity, resulting in an inhibition of the respiratory chain activity. However, direct interactions of TMC with membrane bound enzymes cannot also be excluded. This, in turn, may result in the down regulation of many metabolic energy-dependent reactions as a part of mechanisms accompanying the transition to a hypometabolic state of mycobacteria.
Assuntos
Compostos de Boro , Porfirinas , Transporte de Elétrons , Fluidez de Membrana , Ácidos Palmíticos/metabolismo , Mycobacterium smegmatis/metabolismoRESUMO
Wild-type SAASoti and its monomeric variant mSAASoti can undergo phototransformations, including reversible photoswitching of the green form to a nonfluorescent state and irreversible green-to-red photoconversion. In this study, we extend the photochemistry of mSAASoti variants to enable reversible photoswitching of the red form. This result is achieved by rational and site-saturated mutagenesis of the M163 and F177 residues. In the case of mSAASoti it is M163T substitution that leads to the fastest switching and the most photostable variant, and reversible photoswitching can be observed for both green and red forms when expressed in eukaryotic cells. We obtained a 13-fold increase in the switching efficiency with the maximum switching contrast of the green form and the appearance of comparable switching of the red form for the C21N/M163T mSAASoti variant. The crystal structure of the C21N mSAASoti in its green on-state was obtained for the first time at 3.0 Å resolution, and it is in good agreement with previously calculated 3D-model. Dynamic network analysis reveals that efficient photoswitching occurs if motions of the 66H residue and phenyl fragment of chromophore are correlated and these moieties belong to the same community.
Assuntos
Corantes , Proteínas Luminescentes/genética , Proteínas Luminescentes/química , Proteínas de Fluorescência Verde/genética , Mutagênese , FotoquímicaRESUMO
Mycobacterium tuberculosis (Mtb) is able to transition into a dormant state, causing the latent state of tuberculosis. Dormant mycobacteria acquire resistance to all known antibacterial drugs and can survive in the human body for decades before becoming active. In the dormant forms of M. tuberculosis, the synthesis of porphyrins and its Zn-complexes significantly increased when 5-aminolevulinic acid (ALA) was added to the growth medium. Transcriptome analysis revealed an activation of 8 genes involved in the metabolism of tetrapyrroles during the Mtb transition into a dormant state, which may lead to the observed accumulation of free porphyrins. Dormant Mtb viability was reduced by more than 99.99% under illumination for 30 min (300 J/cm2) with 565 nm light that correspond for Zn-porphyrin and coproporphyrin absorptions. We did not observe any PDI effect in vitro using active bacteria grown without ALA. However, after accumulation of active cells in lung macrophages and their persistence within macrophages for several days in the presence of ALA, a significant sensitivity of active Mtb cells (ca. 99.99%) to light exposure was developed. These findings create a perspective for the treatment of latent and multidrug-resistant tuberculosis by the eradication of the pathogen in order to prevent recurrence of this disease.
Assuntos
Mycobacterium tuberculosis , Porfirinas , Tuberculose , Humanos , Ácido Aminolevulínico/farmacologia , Macrófagos , ZincoRESUMO
In this paper, we propose a fluorescence-lifetime imaging microscopy (FLIM) multiplexing system based on the fluorogen-activating protein FAST. This genetically encoded fluorescent labeling platform employs FAST mutants that activate the same fluorogen but provide different fluorescence lifetimes for each specific protein-dye pair. All the proposed probes with varying lifetimes possess nearly identical and the smallest-in-class size, along with quite similar steady-state optical properties. In live mammalian cells, we target these chemogenetic tags to two intracellular structures simultaneously, where their fluorescence signals are clearly distinguished by FLIM. Due to the unique structure of certain fluorogens under study, their complexes with FAST mutants display a monophasic fluorescence decay, which may facilitate enhanced multiplexing efficiency by reducing signal cross-talks and providing optimal prerequisites for signal separation upon co-localized and/or spatially overlapped labeling.
Assuntos
Corantes Fluorescentes , Microscopia de Fluorescência , Microscopia de Fluorescência/métodos , Corantes Fluorescentes/química , Humanos , Animais , Fluorescência , MutaçãoRESUMO
Cell-to-cell transport of plant viruses through plasmodesmata (PD) requires viral movement proteins (MPs) often associated with cell membranes. The genome of the Hibiscus green spot virus encodes two MPs, BMB1 and BMB2, which enable virus cell-to-cell transport. BMB2 is known to localize to PD-associated membrane bodies (PAMBs), which are derived from the endoplasmic reticulum (ER) structures, and to direct BMB1 to PAMBs. This paper reports the fine structure of PAMBs. Immunogold labeling confirms the previously observed localization of BMB1 and BMB2 to PAMBs. EM tomography data show that the ER-derived structures in PAMBs are mostly cisterns interconnected by numerous intermembrane contacts that likely stabilize PAMBs. These contacts predominantly involve the rims of the cisterns rather than their flat surfaces. Using FRET-FLIM (Förster resonance energy transfer between fluorophores detected by fluorescence-lifetime imaging microscopy) and chemical cross-linking, BMB2 is shown to self-interact and form high-molecular-weight complexes. As BMB2 has been shown to have an affinity for highly curved membranes at cisternal rims, the interaction of BMB2 molecules located at rims of adjacent cisterns is suggested to be involved in the formation of intermembrane contacts in PAMBs.
RESUMO
Fluorescent Correlation Spectroscopy (FCS) allows us to determine interactions of labeled proteins or changes in the oligomeric state. The FCS method needs a low amount of fluorescent dye, near nanomolar concentrations. To control the amount of fluorescent dye, we used new photoconvertible FP SAASoti. This work is devoted to the proof of principle of using photoconvertible proteins to measure caspase enzymatic activity in a single live cell. The advantage of this approach is that partial photoconversion of the FP makes FCS measurements possible when studying enzymatic reactions. To investigate the process, in vivo we used HeLa cell line expressing the engineered FRET sensor, SAASoti-23-KFP. This FRET sensor has a cleavable (DEVD) sequence in the linker between two FPs for the detection of one of the key enzymes of apoptosis, caspase-3. Caspase-3 activity was detected by registering the increase in the fluorescent lifetimes of the sensor, whereas the diffusion coefficient of SAASoti decreased. This can be explained by an increase in the total cell viscosity during apoptosis. We can suppose that in the moment of detectible caspase-3 activity, cell structure already has crucial changes in viscosity.
RESUMO
Significance: Fluorescence molecular lifetime tomography (FMLT) plays an increasingly important role in experimental oncology. The article presents and experimentally verifies an original method of mesoscopic time domain FMLT, based on an asymptotic approximation to the fluorescence source function, which is valid for early arriving photons. Aim: The aim was to justify the efficiency of the method by experimental scanning and reconstruction of a phantom with a fluorophore. The experimental facility included the TCSPC system, the pulsed supercontinuum Fianium laser, and a three-channel fiber probe. Phantom scanning was done in mesoscopic regime for three-dimensional (3D) reflectance geometry. Approach: The sensitivity functions were simulated with a Monte Carlo method. A compressed-sensing-like reconstruction algorithm was used to solve the inverse problem for the fluorescence parameter distribution function, which included the fluorophore absorption coefficient and fluorescence lifetime distributions. The distributions were separated directly in the time domain with the QR-factorization least square method. Results: 3D tomograms of fluorescence parameters were obtained and analyzed using two strategies for the formation of measurement data arrays and sensitivity matrices. An algorithm is developed for the flexible choice of optimal strategy in view of attaining better reconstruction quality. Variants on how to improve the method are proposed, specifically, through stepped extraction and further use of a posteriori information about the object. Conclusions: Even if measurement data are limited, the proposed method is capable of giving adequate reconstructions but their quality depends on available a priori (or a posteriori) information. Further research aims to improve the method by implementing the variants proposed.
Assuntos
Fótons , Tomografia , Imagens de Fantasmas , Tomografia/métodos , Método de Monte Carlo , Algoritmos , Corantes FluorescentesRESUMO
Movement proteins (MPs) of plant viruses enable the translocation of viral genomes from infected to healthy cells through plasmodesmata (PD). The MPs functions involve the increase of the PD permeability and routing of viral genome both to the PD entrance and through the modified PD. Hibiscus green spot virus encodes two MPs, termed BMB1 and BMB2, which act in concert to accomplish virus cell-to-cell transport. BMB1, representing an NTPase/helicase domain-containing RNA-binding protein, localizes to the cytoplasm and the nucleoplasm. BMB2 is a small hydrophobic protein that interacts with the endoplasmic reticulum (ER) membranes and induces local constrictions of the ER tubules. In plant cells, BMB2 localizes to PD-associated membrane bodies (PAMBs) consisting of modified ER tubules and directs BMB1 to PAMBs. Here, we demonstrate that BMB1 and BMB2 interact in vitro and in vivo, and that their specific interaction is essential for BMB2-directed targeting of BMB1 to PAMBs. Using mutagenesis, we show that the interaction involves the C-terminal BMB1 region and the N-terminal region of BMB2.
Assuntos
Hibiscus , Vírus de Plantas , Vírus de RNA , Hibiscus/metabolismo , Vírus de Plantas/genética , Vírus de Plantas/metabolismo , Retículo Endoplasmático , Vírus de RNA/metabolismo , Proteínas do Movimento Viral em Plantas/genética , Proteínas do Movimento Viral em Plantas/metabolismo , Nicotiana , PlasmodesmosRESUMO
Pathogenic non-spore forming bacteria enter a dormant state under stressful conditions, which likely allows them to acquire resistance to various antibiotics. This work revealed the efficient formation of dormant "non-culturable" (NC) Corynebacterium jeikeium cells in stationary phase upon gradual acidification of the growth medium. Such cells were unable to form colonies and existed in a prolonged stationary phase. At an early stage of dormancy (approximately 14 days post-inoculation), dormant cells are able for resuscitation in liquid medium. However, those stored for long time in dormant state needed addition of supernatant taking from active C. jeikeium cultures for successful resuscitation. NC cells possessed low RNA synthesis and significant tolerance to antibiotics (rifampicin and vancomycin). They also accumulated free porphyrins, and 5-aminolevulinic acid addition enhanced free porphyrin accumulation which makes them potentially sensitive to photodynamic inactivation (PDI). PDI of dormant bacteria was accomplished by exposing cells to a 565 nm wavelength of light using a SOLIS-4C light-emitting diode for 60 min. This revealed that increased porphyrin concentrations were correlated with elevated PDI sensitivity. Results shown here demonstrate the potential utility of employing PDI to minimize levels of dormant, persistent corynebacteria and the C. jeikeium dormancy model developed here may be useful for finding new drugs and techniques for combatting persistent corynebacteria.
RESUMO
Skin optical clearing effect ex vivo and in vivo was achieved by topical application of low molecular weight paramagnetic magnetic resonance contrast agents. This novel feature has not been explored before. By using collimated transmittance the diffusion coefficients of three clinically used magnetic resonance contrast agents, that is Gadovist, Magnevist and Dotarem as well as X-ray contrast agent Visipaque in mouse skin were determined ex vivo as (4.29 ± 0.39) × 10-7 cm2 /s, (5.00 ± 0.72) × 10-7 cm2 /s, (3.72 ± 0.67) × 10-7 cm2 /s and (1.64 ± 0.18) × 10-7 cm2 /s, respectively. The application of gadobutrol (Gadovist) resulted in efficient optical clearing that in general, was superior to other contrast agents tested and allowed to achieve: (a) more than 12-fold increase of transmittance over 10 minutes after application ex vivo; (b) markedly improved images of skin architecture obtained with optical coherence tomography; (c) an increase of the fluorescence intensity/background ratio in TagRFP-red fluorescent marker protein expressing tumor by five times after 15 minutes application into the skin in vivo. The obtained results have immediate implications for multimodality imaging because many contrast agents are capable of simultaneously enhancing the contrast of multiple imaging modalities.