RESUMO
Algae are key contributors to global carbon fixation and form the basis of many food webs. In nature, their growth is often supported or suppressed by microorganisms. The bacterium Pseudomonas protegens Pf-5 arrests the growth of the green unicellular alga Chlamydomonas reinhardtii, deflagellates the alga by the cyclic lipopeptide orfamide A, and alters its morphology [P. Aiyar et al., Nat. Commun. 8, 1756 (2017)]. Using a combination of Raman microspectroscopy, genome mining, and mutational analysis, we discovered a polyyne toxin, protegencin, which is secreted by P. protegens, penetrates the algal cells, and causes destruction of the carotenoids of their primitive visual system, the eyespot. Together with secreted orfamide A, protegencin thus prevents the phototactic behavior of C. reinhardtii A mutant of P. protegens deficient in protegencin production does not affect growth or eyespot carotenoids of C. reinhardtii Protegencin acts in a direct and destructive way by lysing and killing the algal cells. The toxic effect of protegencin is also observed in an eyeless mutant and with the colony-forming Chlorophyte alga Gonium pectorale These data reveal a two-pronged molecular strategy involving a cyclic lipopeptide and a conjugated tetrayne used by bacteria to attack select Chlamydomonad algae. In conjunction with the bloom-forming activity of several chlorophytes and the presence of the protegencin gene cluster in over 50 different Pseudomonas genomes [A. J. Mullins et al., bioRxiv [Preprint] (2021). https://www.biorxiv.org/content/10.1101/2021.03.05.433886v1 (Accessed 17 April 2021)], these data are highly relevant to ecological interactions between Chlorophyte algae and Pseudomonadales bacteria.
Assuntos
Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidade , Chlamydomonas reinhardtii/efeitos dos fármacos , Pseudomonas/metabolismo , Carotenoides , Técnicas de Cocultura , Genoma BacterianoRESUMO
Paclitaxel drug coated balloons (DCBs) should provide optimal drug transfer exclusively to the target tissue. The aim of this study was to evaluate the particle loss by handling during angioplasty. A robotic arm was developed for systematic and reproducible drug abrasion experiments. The contact force on eight different commercially available DCB types was gradually increased, and high-resolution microscopic images of the deflated and inflated balloons were recorded. Three types of DCBs were classified: no abrasion of the drug in both statuses (deflated and inflated), significant abrasion only in the inflated status, and significant abrasion in both statuses. Quantitative measurements via image processing confirmed the qualitative classification and showed changes of the drug area between 2.25 and 45.73% (13.28 ± 14.29%) in the deflated status, and between 1.66 and 40.41% (21.43 ± 16.48%) in the inflated status. The structures and compositions of the DCBs are different, some are significantly more susceptible to drug loss. Particle loss by handling during angioplasty leads to different paclitaxel doses in the target regions for same DCB types. Susceptibility to involuntary drug loss may cause side effects, such as varying effective paclitaxel doses, which may explain variations in studies regarding the therapeutic outcome.
Assuntos
Angioplastia , Materiais Revestidos Biocompatíveis , Sistemas de Liberação de Medicamentos/instrumentação , Paclitaxel/administração & dosagem , Angioplastia/instrumentação , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos/métodos , Paclitaxel/efeitos adversos , Resultado do TratamentoRESUMO
Microorganisms are constantly interacting in a given environment by a constant exchange of signaling molecules. In timber, wood-decay fungi will come into contact with other fungi and bacteria. In naturally bleached wood, dark, pigmented lines arising from confrontation of two fungi often hint at such interactions. The metabolites (and pigment) exchange was investigated using the lignicolous basidiomycete Schizophyllum commune, and co-occurring fungi and bacteria inoculated directly on sterilized wood, or on media. In interactions with competitive wood degrading fungi, yeasts or bacteria, different competition strategies and communication types were observed, and stress reactions, as well as competitor-induced enzymes or pigments were analyzed. Melanin, indole, flavonoids and carotenoids were shown to be induced in S. commune interactions. The induced genes included multi-copper oxidases lcc1, lcc2, mco1, mco2, mco3 and mco4, possibly involved in both pigment production and lignin degradation typical for wood bleaching by wood-decay fungi.
Assuntos
Schizophyllum/metabolismo , Bactérias/metabolismo , Pigmentos Biológicos/metabolismo , Metabolismo Secundário/fisiologia , Madeira/microbiologiaRESUMO
Dermal fibroblast cells can adopt different cell states such as proliferation, quiescence, apoptosis or senescence, in order to ensure tissue homeostasis. Proliferating (dividing) cells pass through the phases of the cell cycle, while quiescent and senescent cells exist in a non-proliferating cell cycle-arrested state. However, the reversible quiescence state is in contrast to the irreversible senescence state. Long-term quiescent cells transit into senescence indicating that cells age also when not passing through the cell cycle. Here, by label-free in vitro vibrational spectroscopy, we studied the biomolecular composition of quiescent dermal fibroblast cells and compared them with those of proliferating and senescent cells. Spectra were examined by multivariate statistical analysis using a PLS-LDA classification model, revealing differences in the biomolecular composition between the cell states mainly associated with protein alterations (variations in the side chain residues of amino acids and protein secondary structure), but also within nucleic acids and lipids. We observed spectral changes in quiescent compared to proliferating cells, which increased with quiescence cultivation time. Raman and infrared spectroscopy, which yield complementary biochemical information, clearly distinguished contact-inhibited from serum-starved quiescent cells. Furthermore, the spectra displayed spectral differences between quiescent cells and proliferating cells, which had recovered from quiescence. This became more distinct with increasing quiescence cultivation time. When comparing proliferating, (in particular long-term) quiescent and senescent cells, we found that Raman as well as infrared spectroscopy can separate these three cellular states from each other due to differences in their biomolecular composition. Our spectroscopic analysis shows that proliferating and quiescent fibroblast cells age by similar but biochemically not identical processes. Despite their aging induced changes, over long time periods quiescent cells can return into the cell cycle. Finally however, the cell cycle arrest becomes irreversible indicating senescence.
Assuntos
Senescência Celular , Fibroblastos/citologia , Apoptose , Proliferação de Células , Fibroblastos/metabolismo , Humanos , Espectrofotometria Infravermelho , Análise Espectral Raman , Fatores de TempoRESUMO
For more than two decades, Raman spectroscopy has found widespread use in biological and medical applications. The instrumentation and the statistical evaluation procedures have matured, enabling the lengthy transition from ex-vivo demonstration to in-vivo examinations. This transition goes hand-in-hand with many technological developments and tightly bound requirements for a successful implementation in a clinical environment, which are often difficult to assess for novice scientists in the field. This review outlines the required instrumentation and instrumentation parameters, designs, and developments of fiber optic probes for the in-vivo applications in a clinical setting. It aims at providing an overview of contemporary technology and clinical trials and attempts to identify future developments necessary to bring the emerging technology to the clinical end users. A comprehensive overview of in-vivo applications of fiber optic Raman probes to characterize different tissue and disease types is also given.
Assuntos
Diagnóstico por Imagem , Análise Espectral Raman , Algoritmos , Desenho de Equipamento , Humanos , Imagens de FantasmasRESUMO
Atherosclerosis is a process of thickening and stiffening of the arterial walls through the accumulation of lipids and fibrotic material, as a consequence of aging and unhealthy life style. However, not all arterial plaques lead to complications, which can lead to life-threatening events such as stroke and myocardial infarction. Diagnosis of the disease in early stages and identification of unstable atherosclerotic plaques are still challenging. It has been shown that the development of atherosclerotic plaques is an inflammatory process, where the accumulation of macrophages in the arterial walls is immanent in the early as well as late stages of the disease. We present a novel surface enhanced Raman spectroscopy (SERS)-based strategy for the detection of early stage atherosclerosis, based on the uptake of tagged gold nanoparticles by macrophages and subsequent detection by means of SERS. The results presented here provide a basis for future in vivo studies in animal models.The workflow of tracing the SERS-active nanoparticle uptake by macrophages employing confocal Raman imaging.
Assuntos
Macrófagos/metabolismo , Manose/química , Manose/metabolismo , Nanopartículas Metálicas/química , Placa Aterosclerótica/diagnóstico , Análise Espectral Raman , Transporte Biológico , Linhagem Celular , Diagnóstico Precoce , Ouro/química , Humanos , Placa Aterosclerótica/metabolismo , Dióxido de Silício/químicaRESUMO
Nowadays, cancer is one of the most dangerous and deadly disease all around the world. Cancer that is diagnosed at early stages is more likely to be treated successfully. Treatment of progressed cancer is very difficult, and generally surviving rates are much lower. Therefore, much research has been focused on developing non-invasive methods for detection of cancer and monitoring of its progress. Within this contribution, we present a novel strategy for selective isolation and detection of breast cancer cell lines (MCF-7 and BT-20) based on surface enhanced Raman scattering (SERS). A simplified protocol based on cell-aptamer interaction has been developed in which core-shell (Au@Fe3O4) nanoparticles (CSNs) were functionalized with a mucin 1 (MUC1) specific aptamer (Apt1) to capture cells through the interaction between Apt1 and overexpressed protein (MUC1) on the surface of the tumor cells. Meanwhile, a SERS nano-tag, synthesized by the conjugation of Apt1 to the surface of BSA coated and with 4-mercaptopyridine (4-Mpy) functionalized gold nanoparticles, was used to detect the isolated cells. As a conclusion, the proposed strategy can be extended to isolate and detect cells more precisely based on the detection of different kinds of biomarkers on the surface of cancer cells, simultaneously.
Assuntos
Neoplasias da Mama/patologia , Análise de Célula Única , Feminino , Humanos , Análise Espectral Raman , Propriedades de Superfície , Células Tumorais CultivadasRESUMO
New technologies to diagnose malaria at high sensitivity and specificity are urgently needed in the developing world where the disease continues to pose a huge burden on society. Infrared and Raman spectroscopy-based diagnostic methods have a number of advantages compared with other diagnostic tests currently on the market. These include high sensitivity and specificity for detecting low levels of parasitemia along with ease of use and portability. Here, we review the application of vibrational spectroscopic techniques for monitoring and detecting malaria infection. We discuss the role of vibrational (infrared and Raman) spectroscopy in understanding the processes of parasite biology and its application to the study of interactions with antimalarial drugs. The distinct molecular phenotype that characterizes malaria infection and the high sensitivity enabling detection of low parasite densities provides a genuine opportunity for vibrational spectroscopy to become a front-line tool in the elimination of this deadly disease and provide molecular insights into the chemistry of this unique organism.
Assuntos
Malária/diagnóstico , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Análise Espectral Raman/métodos , Animais , Eritrócitos/microbiologia , Eritrócitos/patologia , Heme/análise , Hemeproteínas/análise , Humanos , Plasmodium/crescimento & desenvolvimento , Espectroscopia de Infravermelho com Transformada de Fourier/instrumentação , Análise Espectral Raman/instrumentação , VibraçãoRESUMO
Basidiomycetes, that is, mushroom-type fungi, are known to produce pigments in response to environmental impacts. As antioxidants with a high level of unsaturation, these compounds can neutralize highly oxidative species. In the event of close contact with other microbes, the enzymatically controlled pigment production is triggered and pigment secretion is generated at the interaction zone. The identification and analysis of these pigments is important to understand the defense mechanism of fungi, which is essential to counteract an uncontrolled spread of harmful species. Usually, a detailed analysis of the pigments is time consuming as it depends on laborious sample preparation and isolation procedures. Furthermore, the applied protocols often influence the chemical integrity of the compound of interest. A possibility to noninvasively investigate the pigmentation is Raman microspectroscopy. The methodology has the potential to analyze the chemical composition of the sample spatially resolved at the interaction zone. After the acquisition of a representative spectroscopic library, the pigment production by basidiomycetes was monitored for during response to different fungi and bacteria. The presented results describe a very efficient noninvasive way of pigment analysis which can be applied with minimal sample preparation.
Assuntos
Basidiomycota/metabolismo , Pigmentos Biológicos/metabolismo , Análise Espectral Raman , Pigmentos Biológicos/químicaRESUMO
Intravascular imaging techniques provide detailed specification about plaque appearance and morphology, but cannot deliver information about the biochemical composition of atherosclerotic plaques. As the biochemical composition is related to the plaque type, important aspects such as the risk of a plaque rupture and treatment are still difficult to assess. Currently, various spectroscopic techniques are tested for potential applications for the chemical analysis of plaque depositions. Here, we employ Raman spectroscopy in combination with optical coherence tomography (OCT) for the characterization of plaques on rabbits in vivo. Experiments were carried out on New Zealand white rabbits treated with a fat- and cholesterol-enriched diet, using a Raman probe setup with a 785-nm multimode laser as an excitation source. Subsequently, OCT images were acquired with a swept source at 1305±55 nm at 22.6 mW. Raman spectra were recorded from normal regions and regions with early plaque formations. The probe positioning was monitored by x-ray angiography. The spectral information identified plaque depositions consisting of lipids, with triglycerides as the major component. Afterward, OCT images of the spectroscopically investigated areas were obtained. The spectral information correlates well with the observed intravascular morphology and is in good agreement with histology. Raman spectroscopy can provide detailed biochemical specification of atherosclerotic plaques.
Assuntos
Placa Aterosclerótica/diagnóstico por imagem , Análise Espectral Raman/métodos , Tomografia de Coerência Óptica/métodos , Animais , Artéria Carótida Primitiva/diagnóstico por imagem , Procedimentos Endovasculares , Desenho de Equipamento , Masculino , Coelhos , Análise Espectral Raman/instrumentaçãoRESUMO
Senescent cells contribute to tissue aging and dysfunction. Therefore, detecting senescent cells in skin is of interest for skin tumor diagnostics and therapy. Here, we studied the transition into senescence of human dermal fibroblasts (HDFs) in a three-dimensional (3D) human fibroblast-derived matrix (FDM). Senescent and proliferating cells were imaged by Raman spectroscopy (RS) and Fourier transform infrared (FTIR) spectroscopy. The obtained averaged spectra were analyzed using PLS-LDA. For these 3D cultured cells, RS and FTIR could clearly distinguish senescent from proliferating cells. For both techniques, we detected senescence-associated alterations in almost all cellular macromolecules. Furthermore, we identified different biochemical properties of 3D compared to two-dimensional (2D) cultured cells, indicating that cells in their natural, skin-like 3D environment act differently than in (2D) cell cultivations in vitro. Compared to 2D cultured cells, cells grown in 3D models displayed a sharper contrast between the proliferating and senescent state, also affecting the abundance of biomolecules including nucleic acids. The training accuracies of both vibrational spectroscopic techniques were >96%, demonstrating the suitability of these label-free measurements for detecting these cellular states in 3D skin models.
Assuntos
Proliferação de Células , Senescência Celular , Fibroblastos/citologia , Pele/citologia , Células Cultivadas , Humanos , Espectroscopia de Infravermelho com Transformada de Fourier , Análise Espectral RamanRESUMO
A primary amino-functionalized methyl methacrylate-based statistical copolymer is covalently coupled with retinoic acid (RA) and a fluorescent dye (DY590) in order to investigate the feasibility of the RA containing polymeric nanoparticles for Raman imaging studies and to study the possible selectivity of RA for hepatic stellate cells via intravital microscopy. Cationic nanoparticles are prepared by utilizing the nanoprecipitation method using modified polymers. Raman studies show that RA functional nanoparticles can be detectable in all tested cells without any need of additional label. Moreover, intravital microscopy indicates that DY590 is eliminated through the hepatobiliary route but not if used as covalently attached tracing molecule for nanoparticles. However, it is a suitable probe for sensitive detection of polymeric nanoparticles.
Assuntos
Canalículos Biliares/metabolismo , Células Estreladas do Fígado/metabolismo , Fígado/metabolismo , Nanopartículas/química , Polimetil Metacrilato/química , Tretinoína/química , Animais , Canalículos Biliares/ultraestrutura , Transporte Biológico , Portadores de Fármacos , Corantes Fluorescentes/química , Células Estreladas do Fígado/ultraestrutura , Humanos , Microscopia Intravital/métodos , Fígado/ultraestrutura , Camundongos , Nanopartículas/administração & dosagem , Microscopia Óptica não Linear/métodosRESUMO
Raman spectroscopic imaging was used to investigate the uptake of oxidized LDLs (oxLDLs) by human macrophages. To better understand the endocytic pathway and the intracellular fate of modified lipoproteins is of foremost interest with regard to the development of atherosclerotic plaques. To obtain information on the storage process of lipids caused by oxLDL uptake, Raman spectroscopic imaging was used because of its unique chemical specificity, especially for lipids. For the present study, a protocol was established to incorporate deuterated tripalmitate into oxLDL. Subsequently, human THP-1 macrophages were incubated for different time points and their chemical composition was analyzed using Raman spectroscopic imaging. ß-Carotene was found to be a reliable marker molecule for the uptake of lipoproteins into macrophages. In addition, lipoprotein administration led to small endocytic vesicles with different concentrations of deuterated lipids within the cells. For the first time, the translocation of deuterated lipids from endocytic vesicles into lipid droplets over time is reported in mature human THP-1 macrophages.
Assuntos
Gotículas Lipídicas/metabolismo , Lipoproteínas LDL/metabolismo , Macrófagos/citologia , Imagem Molecular , Análise Espectral Raman , Vesículas Transportadoras/metabolismo , Triglicerídeos/metabolismo , Transporte Biológico , Linhagem Celular , Humanos , Macrófagos/metabolismoRESUMO
Cellular senescence is a terminal cell cycle arrested state, assumed to be involved in tumor suppression. We studied four human fibroblast cell strains (BJ, MRC-5, IMR-90, and WI-38) from proliferation into senescence. Cells were investigated by label-free vibrational Raman and infrared spectroscopy, following their transition into replicative senescence. During the transition into senescence, we observed rather similar biomolecular abundances in all four cell strains and between proliferating and senescent cells; however, in the four aging cell strains, we found common molecular differences dominated by protein and lipid modifications. Hence, aging induces a change in the appearance of biomolecules (including degradation and storage of waste) rather than in their amount present in the cells. For all fibroblast strains combined, the partial least squares-linear discriminant analysis (PLS-LDA) model resulted in 75% and 81% accuracy for the Raman and infrared (IR) data, respectively. Within this validation, senescent cells were recognized with 93% sensitivity and 90% specificity for the Raman and 84% sensitivity and 97% specificity for the IR data. Thus, Raman and infrared spectroscopy can identify replicative senescence on the single cell level, suggesting that vibrational spectroscopy may be suitable for identifying and distinguishing different cellular states in vivo, e.g., in skin.
Assuntos
Proliferação de Células , Senescência Celular , Espectroscopia de Infravermelho com Transformada de Fourier , Análise Espectral Raman , Pontos de Checagem do Ciclo Celular , Células Cultivadas , Análise Discriminante , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Análise dos Mínimos QuadradosRESUMO
Monitoring living cells in real-time is important in order to unravel complex dynamic processes in life sciences. In particular the dynamics of initiation and progression of degenerative diseases is intensely studied. In atherosclerosis the thickening of arterial walls is related to high lipid levels in the blood stream, which trigger the lipid uptake and formation of droplets as neutral lipid reservoirs in macrophages in the arterial wall. Unregulated lipid uptake finally results in foam cell formation, which is a hallmark of atherosclerosis. In previous studies, the uptake and storage of different fatty acids was monitored by measuring fixed cells. Commonly employed fluorescence staining protocols are often error prone because of cytotoxicity and unspecific fluorescence backgrounds. By following living cells with Raman spectroscopic imaging, lipid uptake of macrophages was studied with real-time data acquisition. Isotopic labeling using deuterated palmitic acid has been combined with spontaneous and stimulated Raman imaging to investigate the dynamic process of fatty acid storage in human macrophages for incubation times from 45 min to 37 h. Striking heterogeneity in the uptake rate and the total concentration of deuterated palmitic acid covering two orders of magnitude is detected in single as well as ensembles of cultured human macrophages. SRS signal of deuterated palmitic acid measured at the CD vibration band after incorporation into living macrophages.
Assuntos
Ácidos Graxos/metabolismo , Metabolismo dos Lipídeos , Macrófagos/metabolismo , Análise Espectral Raman , Aterosclerose , HumanosRESUMO
Raman spectroscopy is an emerging technique in bioanalysis and imaging of biomaterials owing to its unique capability of generating spectroscopic fingerprints. Imaging cells and tissues by Raman microspectroscopy represents a nondestructive and label-free approach. All components of cells or tissues contribute to the Raman signals, giving rise to complex spectral signatures. Resonance Raman scattering and surface-enhanced Raman scattering can be used to enhance the signals and reduce the spectral complexity. Raman-active labels can be introduced to increase specificity and multimodality. In addition, nonlinear coherent Raman scattering methods offer higher sensitivities, which enable the rapid imaging of larger sampling areas. Finally, fiber-based imaging techniques pave the way towards inâ vivo applications of Raman spectroscopy. This Review summarizes the basic principles behind medical Raman imaging and its progress since 2012.
Assuntos
Materiais Biocompatíveis/química , Imagem Molecular , Análise Espectral Raman , Animais , HumanosRESUMO
In order to replace particularly biohazardous nematocides, there is a strong drive to finding natural product-based alternatives with the aim of containing nematode pests in agriculture. The metabolites produced by the fungal endophyte Fusarium oxysporum 162 when cultivated on rice media were isolated and their structures elucidated. Eleven compounds were obtained, of which six were isolated from a Fusarium spp. for the first time. The three most potent nematode-antagonistic compounds, 4-hydroxybenzoic acid, indole-3-acetic acid (IAA) and gibepyrone D had LC50 values of 104, 117 and 134 µg ml-1 , respectively, after 72 h. IAA is a well-known phytohormone that plays a role in triggering plant resistance, thus suggesting a dual activity, either directly, by killing or compromising nematodes, or indirectly, by inducing defence mechanisms against pathogens (nematodes) in plants. Such compounds may serve as important leads in the development of novel, environmental friendly, nematocides.
Assuntos
Antinematódeos/análise , Produtos Biológicos/análise , Endófitos/química , Fusarium/química , Tylenchoidea/efeitos dos fármacos , Animais , Antinematódeos/química , Produtos Biológicos/química , Meios de Cultura/química , Fusarium/crescimento & desenvolvimento , Fusarium/isolamento & purificação , Microscopia , Estrutura Molecular , Análise de Sobrevida , Tylenchoidea/anatomia & histologia , Tylenchoidea/fisiologiaRESUMO
In-vitro Raman micro-spectroscopy was used for diagnostics of the processes of uptake and biodegradation of porous silicon nanoparticles (SiNPs) in breast cancer cells (MCF-7 cell line). Two types of nanoparticles, with and without photoluminescence in the visible spectral range, were investigated. The spatial distribution of photoluminescent SiNPs within the cells obtained by Raman imaging was verified by high-resolution structured-illumination optical microscopy. Nearly complete biodegradation of SiNPs inside the living cells was observed after 13days of the incubation. The results reveal new prospects of multi-modal visualization of SiNPs inside cancer cells for theranostic applications.
Assuntos
Nanopartículas , Silício/farmacocinética , Humanos , Células MCF-7 , Imagem Óptica/métodos , Porosidade , Dióxido de Silício , Análise Espectral RamanRESUMO
Over the past years it had been demonstrated that multimodal imaging combining the nonlinear modalities coherent anti-Stokes Raman scattering (CARS), two-photon excited auto-fluorescence (TPEF) and second harmonic generation (SHG) show a great potential for tissue diagnosis and tumor identification. To extend the applicability of this multimodal imaging approach for in-vivo tissue screening of difficult to access body regions the development of suitable fiber optic probes is required. Here we report about a novel CARS imaging fiber probe consisting of 10,000 coherent light guiding elements preserving the spatial relationship between the entrance and the output of the fiber. Therefore the scanning procedure can be shifted from the distal to the proximal end of the fiber probe and no moving parts or driving current are required to realize in-vivo CARS endoscopy.