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The increase in bacterial resistance to antibiotics in recent years demands innovative strategies for the detection and combating of biofilms, which are notoriously resilient. Biofilms, particularly those on contact lenses, can lead to biofilm-related infections (e.g., conjunctivitis and keratitis), posing a significant risk to patients. Non-destructive and non-contact sensing techniques are essential in addressing this threat. Digital holographic tomography emerges as a promising solution. This allows for the 3D reconstruction of the refractive index distribution in biological samples, enabling label-free visualization and the quantitative analysis of biofilms. This tool provides insight into the dynamics of biofilm formation and maturation on the surface of transparent materials. Applying digital holographic tomography for biofilm examination has the potential to advance our ability to combat the antibiotic bacterial resistance crisis. A recent study focused on characterizing biofilm formation and maturation on six soft contact lens materials (three silicone hydrogels, three hydrogels), with a particular emphasis on Staphylococcus epidermis and Pseudomonas aeruginosa, both common culprits in ocular infections. The results revealed species- and time-dependent variations in the refractive indexes and volumes of biofilms, shedding light on cell dynamics, cell death, and contact lens material-related factors. The use of digital holographic tomography enables the quantitative analysis of biofilm dynamics, providing us with a better understanding and characterization of bacterial biofilms.
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Biofilmes , Lentes de Contato Hidrofílicas , Humanos , Bactérias , Antibacterianos , Hidrogéis , Lentes de Contato Hidrofílicas/microbiologia , Pseudomonas aeruginosa/fisiologiaRESUMO
Liposomal technologies are used in order to improve the effectiveness of current therapies or to reduce their negative side effects. However, the liposome-erythrocyte interaction during the intravenous administration of liposomal drug formulations may result in changes within the red blood cells (RBCs). In this study, it was shown that phosphatidylcholine-composed liposomal formulations of Photolon, used as a drug model, significantly influences the transmembrane potential, stiffness, as well as the shape of RBCs. These changes caused decreasing the number of stomatocytes and irregular shapes proportion within the cells exposed to liposomes. Thus, the reduction of anisocytosis was observed. Therefore, some nanodrugs in phosphatidylcholine liposomal formulation may have a beneficial effect on the survival time of erythrocytes.
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Composição de Medicamentos/métodos , Eritrócitos/citologia , Hemólise/efeitos dos fármacos , Lipossomos/química , Potenciais da Membrana , Porfirinas/farmacologia , Radiossensibilizantes/farmacologia , Animais , Forma Celular , Clorofilídeos , Eritrócitos/efeitos dos fármacos , Eritrócitos/fisiologia , Feminino , Fosfatidilcolinas/química , Porfirinas/química , Radiossensibilizantes/química , SuínosRESUMO
Quantifying changes in bacteria cells in the presence of antibacterial treatment is one of the main challenges facing contemporary medicine; it is a challenge that is relevant for tackling issues pertaining to bacterial biofilm formation that substantially decreases susceptibility to biocidal agents. Three-dimensional label-free imaging and quantitative analysis of bacteria-photosensitizer interactions, crucial for antimicrobial photodynamic therapy, is still limited due to the use of conventional imaging techniques. We present a new method for investigating the alterations in living cells and quantitatively analyzing the process of bacteria photodynamic inactivation. Digital holographic tomography (DHT) was used for in situ examination of the response of Escherichia coli and Staphylococcus aureus to the accumulation of the photosensitizers immobilized in the copolymer revealed by the changes in the 3D refractive index distributions of single cells. Obtained results were confirmed by confocal microscopy and statistical analysis. We demonstrated that DHT enables real-time characterization of the subcellular structures, the biophysical processes, and the induced local changes of the intracellular density in a label-free manner and at sub-micrometer spatial resolution.
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Escherichia coli/metabolismo , Holografia/métodos , Interpretação de Imagem Assistida por Computador/métodos , Fármacos Fotossensibilizantes/metabolismo , Staphylococcus aureus/metabolismo , Tomografia de Coerência Óptica/métodos , Escherichia coli/crescimento & desenvolvimento , Processamento de Sinais Assistido por Computador , Staphylococcus aureus/crescimento & desenvolvimentoRESUMO
Recently proposed methods of bacteria identification in optical biosensors based on the phenomenon of light diffraction on macro-colonies offer over 98% classification accuracy. However, such high accuracy relies on the comparable and repeatable spatial intensity distribution of diffraction patterns. Therefore, it is essential to eliminate all non-species/strain-dependent factors affecting the diffraction patterns. In this study, the impact of the bacterial colony and illuminating beam misalignment on the variation of classification features extracted from diffraction patterns was examined. It was demonstrated that misalignment introduced by the scanning module significantly affected diffraction patterns and extracted classification features used for bacteria identification. Therefore, it is a crucial system-dependent factor limiting the identification accuracy. The acceptable misalignment level, when the accuracy and quality of the classification features are not affected, was determined as no greater than 50 µm. Obtained results led to development of image-processing algorithms for determination of the direction of misalignment and concurrent alignment of the bacterial colonies' diffraction patterns. The proposed algorithms enable the rigorous monitoring and controlling of the measurement's conditions in order to preserve the high accuracy of bacteria identification.
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Algoritmos , Bactérias/classificação , Técnicas Biossensoriais , Bactérias/crescimento & desenvolvimento , Processamento de Imagem Assistida por ComputadorRESUMO
In our study, we describe the outcomes of the intercalation of different anthracycline antibiotics in double-stranded DNA at the nanoscale and single molecule level. Atomic force microscopy analysis revealed that intercalation results in significant elongation and thinning of dsDNA molecules. Additionally, using optical tweezers, we have shown that intercalation decreases the stiffness of DNA molecules, that results in greater susceptibility of dsDNA to break. Using DNA molecules with different GC/AT ratios, we checked whether anthracycline antibiotics show preference for GC-rich or AT-rich DNA fragments. We found that elongation, decrease in height and decrease in stiffness of dsDNA molecules was highest in GC-rich dsDNA, suggesting the preference of anthracycline antibiotics for GC pairs and GC-rich regions of DNA. This is important because such regions of genomes are enriched in DNA regulatory elements. By using three different anthracycline antibiotics, namely doxorubicin (DOX), epirubicin (EPI) and daunorubicin (DAU), we could compare their detrimental effects on DNA. Despite their analogical structure, anthracyclines differ in their effects on DNA molecules and GC-rich region preference. DOX had the strongest overall effect on the DNA topology, causing the largest elongation and decrease in height. On the other hand, EPI has the lowest preference for GC-rich dsDNA. Moreover, we demonstrated that the nanoscale perturbations in dsDNA topology are reflected by changes in the microscale properties of the cell, as even short exposition to doxorubicin resulted in an increase in nuclei stiffness, which can be due to aberration of the chromatin organization, upon intercalation of doxorubicin molecules.
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Antraciclinas/química , Antibióticos Antineoplásicos/química , DNA de Cadeia Simples/química , Núcleo Celular/genética , Simulação por Computador , Daunorrubicina/química , Doxorrubicina/química , Epirubicina/química , Humanos , Substâncias Intercalantes/química , Microscopia de Força Atômica , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Pinças ÓpticasRESUMO
BACKGROUND: Liposomes serve as delivery systems for biologically active compounds. Existing technologies inefficiently encapsulate large hydrophilic macromolecules, such as PVP-conjugated chlorin e6 (Photolon). This photoactive drug has been widely tested for therapeutic applications, including photodynamic reduction of atherosclerotic plaque. METHODS: A novel formulation of Photolon was produced using "gel hydration technology". Its pharmacokinetics was tested in Sus scrofa f. domestica. Its cellular uptake, cytotoxicity, and ability to induce a phototoxic reaction were demonstrated in J774A.1, RAW264.7 macrophages, and vascular smooth muscle (T/G HA-VSMC) as well as in vascular endothelial (HUVEC) cells. RESULTS: Developed liposomes had an average diameter of 124.7 ± 0.6 nm (polydispersity index (PDI) = 0.055) and contained >80% of Photolon). The half-life of formulation in S. scrofa was 20 min with area under the curve (AUC) equal to 14.7. The formulation was noncytotoxic in vitro and was rapidly (10 min) and efficiently accumulated by macrophages, but not T/G HA-VSMC or HUVEC. The accumulated quantity of photosensitizer was sufficient for induction of phototoxicity in J774A.1, but not in T/G HA-VSMC. CONCLUSIONS: Due to the excellent physical and pharmacokinetic properties and selectivity for macrophages, the novel liposomal formulation of Photolon is a promising therapeutic candidate for use in arteriosclerosis treatment when targeting macrophages but not accompanying vascular tissue is critical for effective and safe therapy.
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Lipossomos , Fotoquimioterapia , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Porfirinas/química , Porfirinas/farmacologia , Animais , Linhagem Celular , Clorofilídeos , Composição de Medicamentos , Humanos , Lipossomos/química , Lipossomos/ultraestrutura , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Fotoquimioterapia/métodos , Placa Aterosclerótica/etiologia , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologia , Placa Aterosclerótica/terapia , Espécies Reativas de OxigênioRESUMO
It is possible to identify bacteria species basing on their diffraction patterns followed by statistical analysis. The new approach exploits two steps: optimization of the recording conditions and introduction of new interpretable features for the identification. First, optimal diffraction registration plane, was determined. Next, results were verified by the analysis workflow based on ANOVA and Fisher divergence for feature selection, QDA and SVM models for classification and identification and CV with stratified sampling, sensitivity and specificity for performance assessment of the identification process. The proposed approach resulted in high sensitivity 0.9759 and specificity 0.9903 with very small identification error 1.34%.
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Algoritmos , Bactérias/química , Contaminação de Equipamentos/estatística & dados numéricos , Modelos Teóricos , Dispositivos Ópticos/microbiologiaRESUMO
The degeneration of Fraunhofer diffraction conditions in the optical system with converging spherical wave illumination for bacteria species identification based on diffraction patterns is analyzed by digital holographic methods. The obtained results have shown that the colonies of analyzed bacteria species act as biological lenses with the time-dependent light focusing properties, which are characterized and monitored by means of phase retrieval from sequentially captured digital holograms. This significantly affects the location of Fraunhofer patterns observation plane, which is continuously shifted across optical axis in time.
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Contagem de Colônia Microbiana/instrumentação , Escherichia coli/isolamento & purificação , Holografia/instrumentação , Consórcios Microbianos , Microscopia/instrumentação , Nefelometria e Turbidimetria/instrumentação , Processamento de Sinais Assistido por Computador/instrumentação , Algoritmos , Aumento da Imagem/instrumentação , Interferometria/instrumentaçãoRESUMO
It was demonstrated that statistical analysis of bacteria colonies Fresnel patterns recorded in the optical system with converging spherical wave illumination is suitable for highly effective bacteria species classification. The proposed method includes Fresnel patterns recording followed by image processing and the statistical analysis based on feature extraction, feature selection, classification and classification performance methods. Examination performed on various bacteria species (Salmonella enteritidis, Staphylococcus aureus, Staphylococcus intermedius, Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa and Citrobacter freundii) revealed that the proposed method achieved very high accuracy of over 98%.
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Algoritmos , Bactérias/isolamento & purificação , Carga Bacteriana/instrumentação , Carga Bacteriana/métodos , Interpretação Estatística de Dados , Refratometria/instrumentação , Desenho de Equipamento , Análise de Falha de EquipamentoRESUMO
Glioblastoma is one of the most aggressive tumours with a poor response to treatment and a poor prognosis for patients. One of the proteins expressed in glioblastoma tissue is CHI3L1 (YKL-40), which is upregulated and known for its angiogenesis-supporting and pro-tumour immunomodulatory effects in a variety of cancers. In this paper we present the anti-angiogenic, anti-migratory and immunomodulatory effects of the compound G721-0282, an inhibitor of CHI3L1. The inhibitor-induced changes were investigated using conventional techniques as well as the novel label-free digital holographic tomography (DHT), a quantitative phase imaging technique that allows the reconstruction of the refractive index (RI), which is used as an image contrast for 3D visualisation of living cells. DHT allowed digital staining of individual cells and intercellular structures based only on their specific RI. Quantitative spatially resolved analysis of the RI data shows that the concentration of G721-0282 leads to significant changes in the density of cells and their intracellular structures (in particular the cytoplasm and nucleus), in the volume of lipid droplets and in protein concentrations. Studies in the U-87 MG glioblastoma cell line, THP-1 monocytes differentiated into macrophages, human microvascular endothelial cells (HMEC-1) and in the spheroid model of glioblastoma composed of U-87 MG, HMEC-1 and macrophages suggest that inhibition of CHI3L1 may have potential in the antitumour treatment of glioblastoma. In this paper, we also propose a spheroid model for in vitro studies that mimics this type of tumour.
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Glioblastoma , Humanos , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Células Endoteliais/metabolismo , Refratometria , Diferenciação Celular , Imunidade , Linhagem Celular Tumoral , Proteína 1 Semelhante à Quitinase-3RESUMO
The novel optical system based on converging spherical wave illumination for analysis of bacteria colonies diffraction patterns, is proposed. The complex physical model of light transformation on bacteria colonies in this system, is presented. Fresnel diffraction patterns of bacteria colonies Escherichia coli, Salmonella enteritidis, Staphylococcus aureus grown in various conditions, were examined. It was demonstrated that the proposed system enables the characterization of morphological changes of colony structures basing on the changes of theirs Fresnel diffraction patterns.
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Bactérias/crescimento & desenvolvimento , Bactérias/efeitos da radiação , Luz , Dispositivos Ópticos , Absorção/efeitos dos fármacos , Absorção/efeitos da radiação , Bactérias/citologia , Bactérias/efeitos dos fármacos , Contagem de Colônia Microbiana , Meios de Cultura/farmacologia , Escherichia coli/citologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/efeitos da radiação , Salmonella/citologia , Salmonella/efeitos dos fármacos , Salmonella/crescimento & desenvolvimento , Salmonella/efeitos da radiação , Análise Espectral , Staphylococcus aureus/citologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/efeitos da radiação , Fatores de TempoRESUMO
The development of new diagnostics techniques and modalities is critical for early detection of microbial contamination. In this study, the novel integrated system for multi-parametric optical phenotyping and characterization of bacterial colonies, is presented. The system combines Mach-Zehnder interferometer with a spectral imaging system for capturing multispectral diffraction patterns and multispectral two-dimensional transmission maps of bacterial colonies, along with the simultaneous interferometric profilometry. The herein presented investigation was carried out on five representative bacteria species and nearly 3000 registered multispectral optical signatures. The interferograms were analyzed by four-step phase shift algorithm to reconstruct the colony profile to enable the obtaining of the comparable optical signatures. The dedicated image processing algorithms were used for extraction of quantitative features of these signatures. The random forest algorithm was applied for selection of the most predictive set of features, which were used in classification model based on Support-Vector Machine. Obtained results have shown that the use of multiple multispectral optical signatures provide a multi-parametric bacteria identification at an exceptionally high accuracy (99.4-100%), significantly better than in case of classification based on each of these signatures (multispectral diffraction patterns, two-dimensional transmission coefficient maps), separately. Obtained results revealed that analysis of multispectral signatures can also be applied for characterisation of physical, physicochemical and chemical properties of the bacterial colonies in the presence of the antimicrobial factors. Therefore, the proposed label-free, non-destructive optical technique has perspectives to be exploited in the multipurpose diagnostics and it can be used as a pre-screening tool in microbiological laboratories.
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Técnicas Biossensoriais , Algoritmos , Bactérias , Processamento de Imagem Assistida por ComputadorRESUMO
BACKGROUND: Thermoregulation is highly individual and predictive for potentially cascading pathologies. Altered and deficient thermoregulation is considered an important diagnostic indicator which can be of great clinical utility for specialized screening programs and individualized prediction and prevention of severe pathologies triggered early in life. WORKING HYPOTHESIS: Individual thermoregulation can be objectively assessed by thermovision camera before and after exercises in school children stratified by age and gender that may be of great clinical utility for personalized training early in life in the framework of 3P medicine. STUDY DESIGN: In this study, 60 female and male primary school children were exposed to physical exercises in the form of 45-min general fitness training. The subjects under examination were stratified by age: group 1 (7-year-olds), group 2 (9-year-olds), and group 3 (12-year-olds). Superficial body temperature patterns were measured by means of thermovision camera before and immediately after exercises, as well as after the 15-min recovery time. Temperature patterns were analyzed in 12 areas of the body front and back, covering trunk and upper and lower limbs. RESULTS: The obtained results revealed an individual and age-depended difference in response of the body to exercises. The first measurement prior to exercise (measurement 1) revealed no statistically significant differences in the mean surface temperature of all analyzed areas between 7- and 9-year-old children. Further, 7- and 9-year-old children did not differ significantly in the mean temperature recorded in the trunk compared to the 12-year-old children. However, in 12-year-old children, statistically significant higher values of the mean temperature of the upper and lower limbs, were observed compared to the group of 7-year-olds and significantly higher values of the mean temperature of the lower limbs compared to the group of 9-year-olds. Immediately after exercises (measurement 2), a statistically significant decrease in the temperature was noted in all groups and in all areas of the body. The greatest temperature change was observed in 12-year-olds, while the least one was measured in the youngest subjects. The statistically significant relation between the average trunk temperature of 7-year-old and 12-year-old children was observed: lower values of the mean temperature of the front and back of the trunk were noted in the group of 12-year-old children compared to the group of 7-year-olds. A significantly lower average temperature of the back of the trunk compared to the youngest group was also recorded in 9-year-old children. The study performed after the 15-min recovery time (measurement 3) showed an increase in the average temperature of all analyzed areas. In all subjects, the mean temperature recorded in measurement 3 did not differ significantly from the initial ones (measurement 1, prior to exercises). Only the mean temperature of the trunk back of 12-year-old children was significantly lower after the rest period compared to the initial examination. In all groups, the temperatures after exercises followed by a 15-min recovery returned to the initial ones, except of the trunk backs of 12-year-old children, where the temperature was lower than before exercises. CONCLUSIONS AND EXPERT RECOMMENDATIONS IN THE FRAMEWORK OF 3PM: Thermovision analysis is an effective tool to assess individual thermoregulation and to stratify school children for personalized exercise coaching. Body exercise-based disease prevention early in life is effective when tailored to the person: multi-parametric guidance for prescribing exercises individually is needed. Contextually, proposed individualized training approach should be adapted to the age-dependent particularities and individual thermoregulation.
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An increasing interest in a healthy lifestyle raises questions about optimal body weight. Evidently, it should be clearly discriminated between the standardised "normal" body weight and individually optimal weight. To this end, the basic principle of personalised medicine "one size does not fit all" has to be applied. Contextually, "normal" but e.g. borderline body mass index might be optimal for one person but apparently suboptimal for another one strongly depending on the individual genetic predisposition, geographic origin, cultural and nutritional habits and relevant lifestyle parameters-all included into comprehensive individual patient profile. Even if only slightly deviant, both overweight and underweight are acknowledged risk factors for a shifted metabolism which, if being not optimised, may strongly contribute to the development and progression of severe pathologies. Development of innovative screening programmes is essential to promote population health by application of health risks assessment, individualised patient profiling and multi-parametric analysis, further used for cost-effective targeted prevention and treatments tailored to the person. The following healthcare areas are considered to be potentially strongly benefiting from the above proposed measures: suboptimal health conditions, sports medicine, stress overload and associated complications, planned pregnancies, periodontal health and dentistry, sleep medicine, eye health and disorders, inflammatory disorders, healing and pain management, metabolic disorders, cardiovascular disease, cancers, psychiatric and neurologic disorders, stroke of known and unknown aetiology, improved individual and population outcomes under pandemic conditions such as COVID-19. In a long-term way, a significantly improved healthcare economy is one of benefits of the proposed paradigm shift from reactive to Predictive, Preventive and Personalised Medicine (PPPM/3PM). A tight collaboration between all stakeholders including scientific community, healthcare givers, patient organisations, policy-makers and educators is essential for the smooth implementation of 3PM concepts in daily practice.
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A novel method for evaluation of bacterial colonies number (Colony Forming Units--CFU), is described. Proposed algorithm, based on the Mellin transform, allows the CFU evaluation, invariant for the spatial orientation and scale changes. The proposed method involves image recording of bacteria grown in Petri dishes, calculation of the Fourier spectrum followed by coordinates transformation, and determination of the Mellin transform. It was proved that there is a high correlation between CFU and maxima of Mellin spectra. The method was practically implemented for evaluation of antibacterial activity of silver-based nanomaterials and the effect of an additional laser light irradiation.
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Contagem de Colônia Microbiana/métodos , Escherichia coli/isolamento & purificação , Análise de Fourier , Processamento de Imagem Assistida por Computador/métodos , Fenômenos Ópticos , Algoritmos , Antibacterianos/farmacologia , Simulação por Computador , Escherichia coli/citologia , Escherichia coli/efeitos dos fármacos , Nanopartículas Metálicas/química , Testes de Sensibilidade Microbiana , Prata/farmacologiaRESUMO
Current vascular stents, such as drug eluting stents (DES), have some serious drawbacks, like in stent restenosis and thrombosis. Therefore, other solutions are sought to overcome these post-implantations complications. These include the strategy of biofunctionalization of the stent surface with antibodies that facilitate adhesion of endothelial cells (ECs) or endothelial progenitor cells (EPCs). Rapid re-endothelialization of the surface minimizes the risk of possible complications. In this study, we proposed ammonium acryloyldimethyltaurate/vinylpyrrolidone co-polymer-based surface (AVC), which was mercaptosilanized in order to expose free thiol groups. The presence of free thiol groups allowed for the covalent attachment of CD133 antibodies by disulfide bridges formation between mercaptosilanized surface and cysteine of the protein molecule thiol groups. Various examinations were performed in order to validate the procedure, including attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) and Fourier transform Raman spectroscopy (FT-Raman), atomic force microscopy (AFM) and scanning electron microscopy (SEM). By means of ATR-FTIR spectroscopy presence of the CD133 antibody within coating was confirmed. In vitro studies proved good biocompatibility for blood cells without induction of hemolytic response. Thus, proposed biofunctionalized CD133 antibody AVC surface has shown sufficient stability for adapting as cardiovascular implant coating and biocompatibility. According to conducted in vitro studies, the modified surface can be further tested for applications in various biological systems.
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BACKGROUND: Cellulite is a common physiological condition of dermis, epidermis, and subcutaneous tissues experienced by 85 to 98% of the post-pubertal females in developed countries. Infrared (IR) thermography combined with artificial intelligence (AI)-based automated image processing can detect both early and advanced cellulite stages and open up the possibility of reliable diagnosis. Although the cellulite lesions may have various levels of severity, the quality of life of every woman, both in the physical and emotional sphere, is always an individual concern and therefore requires patient-oriented approach. OBJECTIVES: The purpose of this work was to elaborate an objective, fast, and cost-effective method for automatic identification of different stages of cellulite based on IR imaging that may be used for prescreening and personalization of the therapy. MATERIALS AND METHODS: In this study, we use custom-developed image preprocessing algorithms to automatically select cellulite regions and combine a total of 9 feature extraction methods with 9 different classification algorithms to determine the efficacy of cellulite stage recognition based on thermographic images taken from 212 female volunteers aged between 19 and 22. RESULTS: A combination of histogram of oriented gradients (HOG) and artificial neural network (ANN) enables determination of all stages of cellulite with an average accuracy higher than 80%. For primary stages of cellulite, the average accuracy achieved was more than 90%. CONCLUSIONS: The implementation of computer-aided, automatic identification of cellulite severity using infrared imaging is feasible for reliable diagnosis. Such a combination can be used for early diagnosis, as well as monitoring of cellulite progress or therapeutic outcomes in an objective way. IR thermography coupled to AI sets the vision towards their use as an effective tool for complex assessment of cellulite pathogenesis and stratification, which are critical in the implementation of IR thermographic imaging in predictive, preventive, and personalized medicine (PPPM).
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Rapid endothelialization of cardiovascular stents is critical to prevent major clinical complications such as restenosis. Reconstruction of the native endothelium on the stent surface can be achieved by the capture of endothelial progenitor cells (EPCs) or neighboring endothelial cells (ECs) in vivo. In this study, stainless steel cardiovascular stents were functionalized with recombinant scFv antibody fragments specific for vascular endothelial growth factor receptor-2 (VEGFR2) that is expressed on EPCs and ECs. Anti-VEGFR2 scFvs were expressed in glycosylated form in Escherichia coli and covalently attached to amine-functionalized, titania-coated steel disks and stents. ScFv-coated surfaces exhibited no detectable cytotoxicity to human ECs or erythrocytes in vitro and bound 15 times more VEGFR2-positive human umbilical vein ECs than controls after as little as 3 min. Porcine coronary arteries were successfully stented with scFv-coated stents with no adverse clinical events after 30 days. Endovascular imaging and histology revealed coverage of the anti-VEGFR2 scFv-coated stent with a cell layer after 5 days and the presence of a neointima layer with a minimum thickness of 80 µm after 30 days. Biofunctionalization of cardiovascular stents with endothelial cell-capturing antibody fragments in this manner offers promise in accelerating stent endothelialization in vivo. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:213-224, 2020.
Assuntos
Materiais Revestidos Biocompatíveis/farmacologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Anticorpos de Cadeia Única/farmacologia , Stents , Receptor 2 de Fatores de Crescimento do Endotélio Vascular , Animais , Linhagem Celular Transformada , Materiais Revestidos Biocompatíveis/química , Humanos , Anticorpos de Cadeia Única/química , Sus scrofaRESUMO
The potential use of a novel multichannel optical system towards fast and non-destructive bacteria identification and its application for environmental bacteria characterisation on the strain level is presented. It is the first attempt to use the proposed optical method to study various bacteria species (Gram-negative, Gram-positive) commonly present in the environment. The novel configuration of the optical system enables multichannel examination of bacterial colonies and provides additional functionality such as registration of two-dimensional (2D) distribution of monochromatic transmission coefficient of examined colonies, what can be used as a novel optical signature for bacteria characterization. Performed statistical analysis indicates that it is possible to identify representatives of environmental soil bacteria on the species level with the 98.51% accuracy and in case of two strains of Rahnella aquatilis bacteria on the strain level with the 98.8% accuracy. The proposed method is an alternative to the currently used preliminary bacteria examination in environment safety control with the advantage of being fast, reliable, non-destructive and requiring minimal sample preparation.
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We report a multistep strategy of biochemical surface modifications that resulted in the synthesis of new, effective and biocompatible intravascular implants coating with immobilized anti-CD133 antibodies, that proved to be the most effective in endothelial progenitor cells capture and reduced smooth muscle cells growth. Biomolecules were immobilized on differently functionalized surfaces. The distribution, nanostructural characteristics and intramolecular interactions of anti-CD133 molecules as well as their ability to bind EPCs was evaluated. We also tempted to build a molecular model of the CD133 protein to study antigen-antibody interactions. CD133 protein is expressed in endothelial progenitor cells (EPCs). Absence of preferential interaction site on CD133, but rather a presence of a small binding area, may be the specificity of reconnaissance sequence, thus importantly increasing the probability of CD133 protein binding. After all, regarding our molecular model, we are convinced that specific, and large enough interactions between anti-CD133 coating stent surface and CD133 present on EPCs will reduce risk of restenosis by favoring the endothelial growth. Additionally, the safety study of the vivo performance of modified titania based surface was performed using small animal models. No allergological or toxical local or systemic adverse effects of the developed coatings were noted.