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Nowadays there is a growing interest worldwide in using bacteriophages for therapeutic purposes to combat antibiotic-resistant bacterial strains, driven by the increasing ineffectiveness of drugs against bacterial infections. Despite this fact, no novel commercially available therapeutic phage products have been developed in the last two decades, as it is extremely difficult to register them under the current legal regulations. This paper presents a description of the interaction between a bacteriophage manufacturer and a clinical institution, the specificity of which is the selection of bacteriophages not for an individual patient, but for the entire spectrum of bacteria circulating in the intensive care unit with continuous clinical and microbiological monitoring of efficacy. The study presents the description of three clinical cases of patients who received bacteriophage complex via inhalation for 28 days according to the protocol without antibiotic use throughout the period. No adverse effects were observed and the elimination of multidrug-resistant microorganisms from the bronchoalveolar lavage contents was detected in all patients. A decrease in such inflammatory markers as C-reactive protein (CRP) and procalcitonin was also noted. The obtained results demonstrate the potential of an adaptive phage therapy protocol in intensive care units for reducing the amount of antibiotics used and preserving their efficacy.
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BACKGROUND: Transcranial magnetic stimulation (TMS) allows non-invasive stimulation of the cortex. In multi-locus TMS (mTMS), the stimulating electric field (E-field) is controlled electronically without coil movement by adjusting currents in the coils of a transducer. OBJECTIVE: To develop an mTMS system that allows adjusting the location and orientation of the E-field maximum within a cortical region. METHODS: We designed and manufactured a planar 5-coil mTMS transducer to allow controlling the maximum of the induced E-field within a cortical region approximately 30 mm in diameter. We developed electronics with a design consisting of independently controlled H-bridge circuits to drive up to six TMS coils. To control the hardware, we programmed software that runs on a field-programmable gate array and a computer. To induce the desired E-field in the cortex, we developed an optimization method to calculate the currents needed in the coils. We characterized the mTMS system and conducted a proof-of-concept motor-mapping experiment on a healthy volunteer. In the motor mapping, we kept the transducer placement fixed while electronically shifting the E-field maximum on the precentral gyrus and measuring electromyography from the contralateral hand. RESULTS: The transducer consists of an oval coil, two figure-of-eight coils, and two four-leaf-clover coils stacked on top of each other. The technical characterization indicated that the mTMS system performs as designed. The measured motor evoked potential amplitudes varied consistently as a function of the location of the E-field maximum. CONCLUSION: The developed mTMS system enables electronically targeted brain stimulation within a cortical region.
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Corteza Motora , Estimulación Magnética Transcraneal , Electromiografía/métodos , Potenciales Evocados Motores , Humanos , Corteza Motora/fisiología , Técnicas Estereotáxicas , Estimulación Magnética Transcraneal/métodosRESUMEN
Safe application of nanoparticles in medicine requires full understanding of their pharmacokinetics including catabolism in the organism. However, information about nanoparticle degradation is still scanty due to difficulty of long-term measurements by invasive techniques. Here, we describe a magnetic spectral approach for in vivo monitoring of magnetic particle (MP) degradation. The method noninvasiveness has allowed performing of a broad comprehensive study of the 1-year fate of 17 types of iron oxide particles. We show a long-lasting influence of five parameters on the MP degradation half-life: dose, hydrodynamic size, ζ-potential, surface coating, and internal architecture. We observed a slowdown in MP biotransformation with an increase of the injected dose and faster degradation of the particles of a small hydrodynamic size. A comparison of six types of 100 nm particles coated by different hydrophilic polymer shells has shown that the slowest (t1/2 = 38 ± 6 days) and the fastest (t1/2 = 15 ± 4 days) degradations were achieved with a polyethylene glycol and polyglucuronic acid coatings, respectively. The most significant influence on the MP degradation was due to the internal architecture of the particles as the coverage of magnetic cores with a solid 39 nm polystyrene layer slowed down the half-life of the core-shell MPs from 48 days to more than 1 year. The revealed deeper insights into the particle degradation in vivo may facilitate rational design of nano- and microparticles with predictable long-term fate in vivo.
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Nanopartículas , Polietilenglicoles , Ratones , Animales , Polímeros , Fenómenos Físicos , Fenómenos Magnéticos , Tamaño de la PartículaRESUMEN
Blood circulation is the key parameter that determines the in vivo efficiency of nanoagents. Despite clinical success of the stealth liposomal agents with their inert and shielded surfaces, a great number of non-stealth nanomaterials is being developed due to their potential of enhanced functionality. By harnessing surface phenomena, such agents can offer advanced control over drug release through intricately designed nanopores, catalysis-propelled motion, computer-like analysis of several disease markers for precise target identification, etc. However, investigation of pharmacokinetic behavior of these agents becomes a great challenge due to ultra-short circulation (usually around several minutes) and impossibility to use the invasive blood-sampling techniques. Accordingly, the data on circulation of such agents has been scarce and irregular. Here, we demonstrate high-throughput capabilities of the developed magnetic particle quantification technique for nanoparticle circulation measurements and present a comprehensive investigation of factors that affect blood circulation of the non-stealth nanoparticles. Namely, we studied the following 9 factors: particle size, zeta-potential, coating, injection dose, repetitive administration, induction of anesthesia, mice strain, absence/presence of tumors, tumor size. Our fundamental findings demonstrate potential ways to extend the half-life of the agents in blood thereby giving them a better chance of achieving their goal in the organism. The study will be valuable for design of the next generation nanomaterials with advanced biomedical functionality.
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Nanopartículas , Animales , Cinética , Ratones , Tamaño de la PartículaRESUMEN
Prior to sensory experience spontaneous activity appears to play a fundamental role in the correct formation of prominent functional features of different cortical regions. The use of anaesthesia during pregnancy such as ketamine is largely considered to negatively affect neuronal development by interfering with synaptic transmission. Interestingly, the characteristics of spontaneous activity as well as the acute functional effects of maternal anaesthesia remain largely untested in the embryonic cortex in vivo. In the present work, we performed in vivo imaging of spontaneous calcium activity and cell motility in the marginal zone of the cortex of E14-15 embryos connected to the mother. We made use of a preparation where the blood circulation from the mother through the umbilical cord is preserved and fluctuations in intracellular calcium in the embryonic frontal cortex are acquired using two-photon imaging. We found that spontaneous transients were either sporadic or correlated in clusters of neuronal ensembles at this age. These events were not sensitive to maternal isoflurane anaesthesia but were strongly inhibited by acute in situ or maternal application of low concentration of the anaesthetic ketamine (a non-competitive antagonist of NMDA receptors). Moreover, simultaneous imaging of cell motility revealed a correlated strong sensitivity to ketamine. These results show that anaesthetic compounds can differ significantly in their impact on spontaneous early cortical activity as well as motility of cells in the marginal zone. The effects found in this study may be relevant in the etiology of heightened vulnerability to cerebral dysfunction associated with the use of ketamine during pregnancy.
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Anestesia/efectos adversos , Calcio/metabolismo , Corteza Cerebral/embriología , Receptores de N-Metil-D-Aspartato/genética , Anestésicos/efectos adversos , Animales , Movimiento Celular/efectos de los fármacos , Corteza Cerebral/efectos de los fármacos , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Humanos , Ketamina/administración & dosificación , Ketamina/efectos adversos , Neuronas/metabolismo , Neuronas/patología , Embarazo , Transmisión Sináptica/efectos de los fármacosRESUMEN
An interaction of recombinant hairpin-like cationic peptide EcAMP1 with conidia of plant pathogenic fungus Fusarium solani at the cellular level was studied by a combination of microscopic methods. EcAMP1 is from barnyard grass (Echinochloa crusgalli L.), and obtained by heterologous expression in Escherichia coli system. As a result, a direct relationship between hyphal growth inhibition and increasing active peptide concentration, time of incubation and fungal physiological condition has been determined. Dynamics of accumulation and redistribution of the peptide studied on fungal cellular cover and inside the conidia cells has been shown. The dynamics are dependent on time of coupling, as well as, a dissimilarity of EcAMP1 binding with cover of fungal conidia and its stepwise accumulation and diffuse localization in the cytoplasm. Correlation between structural disruption of fungal conidia and the presence of morphological changes has also been found. The correlation was found under the influence of peptide high concentrations at concentrations above 32 µM. The results indicate the presence of a binding of EcAMP1 with the surface of fungal conidia, thus, demonstrating a main specificity for its antifungal action at the cellular level. These results, however, cannot exclude the existence of attendant EcAMP1 action based on its intracellular localization on some specific targets. SCANNING 38:591-598, 2016. © 2016 Wiley Periodicals, Inc.
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Péptidos Catiónicos Antimicrobianos/farmacología , Echinochloa/química , Fusarium/efectos de los fármacos , Proteínas de Plantas/farmacología , Antifúngicos/farmacología , Péptidos Catiónicos Antimicrobianos/metabolismo , Microscopía , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/farmacología , Semillas/química , Esporas Fúngicas/efectos de los fármacosRESUMEN
AIM: To investigate porous silicon (PSi) nanoparticles (NPs) behavior in the embryonic brain. MATERIALS & METHODS: Fluorescently labeled PSi NPs were injected into the embryonic brains intraventricularly and to the mother intravenously (iv.). Brain histology from different time points up to 3 days was analyzed and live brains imaged with two-photon microscopy. RESULTS: PSi NPs were able to penetrate 80% of the embryonic cortical depth. Particle motility was confirmed in real-time in vivo. PSi NPs were able to penetrate the embryonic cortex after either iv. maternal or intraventricular injection. No developmental of macromorphological changes or increased cell apoptosis was observed. CONCLUSION: PSi NPs penetrate deep in the brain tissues of embryos after intraventricular injection and after iv. injection to the mother.
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Encéfalo/metabolismo , Intercambio Materno-Fetal , Nanopartículas/metabolismo , Silicio/metabolismo , Animales , Encéfalo/diagnóstico por imagen , Encéfalo/efectos de los fármacos , Difusión , Embrión de Mamíferos/metabolismo , Femenino , Humanos , Inyecciones Intravenosas , Inyecciones Intraventriculares , Ratones Endogámicos C57BL , Nanopartículas/química , Nanopartículas/toxicidad , Porosidad , Embarazo , Silicio/química , Propiedades de Superficie , Distribución TisularRESUMEN
The dynamics of intracellular calcium fluxes are instrumental in the proliferation, differentiation, and migration of neuronal cells. Knowledge thus far of the relationship between these calcium changes and physiological processes in the developing brain has derived principally from ex vivo and in vitro experiments. Here, we present a new method to image intracellular calcium flux in the cerebral cortex of live rodent embryos, whilst attached to the dam through the umbilical cord. Using this approach we demonstrate induction of calcium waves by laser stimulation. These waves are sensitive to ATP-receptor blockade and are significantly increased by pharmacological facilitation of intracellular-calcium release. This approach is the closest to physiological conditions yet achieved for imaging of calcium in the embryonic brain and as such opens new avenues for the study of prenatal brain development. Furthermore, the developed method could open the possibilities of preclinical translational studies in embryos particularly important for developmentally related diseases such as schizophrenia and autism.
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Nerve endings in skin are involved in physiological processes such as sensing(1) as well as in pathological processes such as neuropathic pain(2). Their close-to-surface positioning facilitates microscopic imaging of skin nerve endings in living intact animal. Using multiphoton microscopy, it is possible to obtain fine images overcoming the problem of strong light scattering of the skin tissue. Reporter transgenic mice that express EYFP under the control of Thy-1 promoter in neurons (including periphery sensory neurons) are well suited for the longitudinal studies of individual nerve endings over extended periods of time up to several months or even life-long. Furthermore, using the same femtosecond laser as for the imaging, it is possible to produce highly selective lesions of nerve fibers for the studies of the nerve fiber restructuring. Here, we present a simple and reliable protocol for longitudinal multiphoton in vivo imaging and laser-based microsurgery on mouse skin nerve endings.
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Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Terminaciones Nerviosas/ultraestructura , Piel/inervación , Animales , Proteínas Bacterianas/química , Terapia por Láser/métodos , Proteínas Luminiscentes/química , Ratones , Ratones Transgénicos , Microscopía de Fluorescencia por Excitación Multifotónica/instrumentaciónRESUMEN
Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.
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Lesiones Encefálicas/patología , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Animales , Modelos Animales de Enfermedad , Ratones , Ratones TransgénicosRESUMEN
It is widely acknowledged that the use of general anesthetics can undermine the relevance of electrophysiological or microscopical data obtained from a living animal's brain. Moreover, the lengthy recovery from anesthesia limits the frequency of repeated recording/imaging episodes in longitudinal studies. Hence, new methods that would allow stable recordings from non-anesthetized behaving mice are expected to advance the fields of cellular and cognitive neurosciences. Existing solutions range from mere physical restraint to more sophisticated approaches, such as linear and spherical treadmills used in combination with computer-generated virtual reality. Here, a novel method is described where a head-fixed mouse can move around an air-lifted mobile homecage and explore its environment under stress-free conditions. This method allows researchers to perform behavioral tests (e.g., learning, habituation or novel object recognition) simultaneously with two-photon microscopic imaging and/or patch-clamp recordings, all combined in a single experiment. This video-article describes the use of the awake animal head fixation device (mobile homecage), demonstrates the procedures of animal habituation, and exemplifies a number of possible applications of the method.
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Conducta Animal/fisiología , Electrofisiología/instrumentación , Microscopía/instrumentación , Análisis de la Célula Individual/instrumentación , Animales , Craneotomía/métodos , Electrofisiología/métodos , Femenino , Masculino , Ratones , Microscopía/métodos , Neuroimagen/métodos , Análisis de la Célula Individual/métodosRESUMEN
Visualization of individual cutaneous nerve endings has previously relied on laborious procedures of tissue excision, fixation, sectioning and staining for light or electron microscopy. We present a method for non-invasive, longitudinal two-photon microscopy of single nerve endings within the skin of anesthetized transgenic mice. Besides excellent signal-to-background ratio and nanometer-scale spatial resolution, this method offers time-lapse "movies" of pathophysiological changes in nerve fine structure over minutes, hours, days or weeks. Structure of keratinocytes and dermal matrix is visualized simultaneously with nerve endings, providing clear landmarks for longitudinal analysis. We further demonstrate feasibility of dissecting individual nerve fibers with infra-red laser and monitoring their degradation and regeneration. In summary, our excision-free optical biopsy technique is ideal for longitudinal microscopic analysis of animal skin and skin innervations in vivo and can be applied widely in preclinical models of chronic pain, allergies, skin cancers and a variety of dermatological disorders.