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26 pages, 17803 KiB

Open AccessArticle

A novel Microproteomic Approach Using Laser Capture Microdissection to Study Cellular Protrusions

by Karine Gousset, Ana Gordon, Shravan Kumar Kannan and Joey Tovar

Int. J. Mol. Sci. 2019, 20(5), 1172; https://doi.org/10.3390/ijms20051172 - 07 Mar 2019

Cited by 9 | Viewed by 4508

Cell–cell communication is vital to multicellular organisms, and distinct types of cellular protrusions play critical roles during development, cell signaling, and the spreading of pathogens and cancer. The differences in the structure and protein composition of these different types of protrusions and their [...] Read more.

Cell–cell communication is vital to multicellular organisms, and distinct types of cellular protrusions play critical roles during development, cell signaling, and the spreading of pathogens and cancer. The differences in the structure and protein composition of these different types of protrusions and their specific functions have not been elucidated due to the lack of a method for their specific isolation and analysis. In this paper, we described, for the first time, a method to specifically isolate distinct protrusion subtypes, based on their morphological structures or fluorescent markers, using laser capture microdissection (LCM). Combined with a unique fixation and protein extraction protocol, we pushed the limits of microproteomics and demonstrate that proteins from LCM-isolated protrusions can successfully and reproducibly be identified by mass spectrometry using ultra-high field Orbitrap technologies. Our method confirmed that different types of protrusions have distinct proteomes and it promises to advance the characterization and the understanding of these unique structures to shed light on their possible role in health and disease. Full article

(This article belongs to the Special Issue Laser Application in Life Sciences 2018)

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23 pages, 11939 KiB

Open AccessArticle

Red (635 nm), Near-Infrared (808 nm) and Violet-Blue (405 nm) Photobiomodulation Potentiality on Human Osteoblasts and Mesenchymal Stromal Cells: A Morphological and Molecular In Vitro Study

by Alessia Tani, Flaminia Chellini, Marco Giannelli, Daniele Nosi, Sandra Zecchi-Orlandini and Chiara Sassoli

Int. J. Mol. Sci. 2018, 19(7), 1946; https://doi.org/10.3390/ijms19071946 - 03 Jul 2018

Cited by 71 | Viewed by 6637

Abstract

Photobiomodulation (PBM) has been used for bone regenerative purposes in different fields of medicine and dentistry, but contradictory results demand a skeptical look for its potential benefits. This in vitro study compared PBM potentiality by red (635 ± 5 nm) or near-infrared (NIR, [...] Read more.

Photobiomodulation (PBM) has been used for bone regenerative purposes in different fields of medicine and dentistry, but contradictory results demand a skeptical look for its potential benefits. This in vitro study compared PBM potentiality by red (635 ± 5 nm) or near-infrared (NIR, 808 ± 10 nm) diode lasers and violet-blue (405 ± 5 nm) light-emitting diode operating in a continuous wave with a 0.4 J/cm² energy density, on human osteoblast and mesenchymal stromal cell (hMSC) viability, proliferation, adhesion and osteogenic differentiation. PBM treatments did not alter viability (PI/Syto16 and MTS assays). Confocal immunofluorescence and RT-PCR analyses indicated that red PBM (i) on both cell types increased vinculin-rich clusters, osteogenic markers expression (Runx-2, alkaline phosphatase, osteopontin) and mineralized bone-like nodule structure deposition and (ii) on hMSCs induced stress fiber formation and upregulated the expression of proliferation marker Ki67. Interestingly, osteoblast responses to red light were mediated by Akt signaling activation, which seems to positively modulate reactive oxygen species levels. Violet-blue light-irradiated cells behaved essentially as untreated ones and NIR irradiated ones displayed modifications of cytoskeleton assembly, Runx-2 expression and mineralization pattern. Although within the limitations of an in vitro experimentation, this study may suggest PBM with 635 nm laser as potential effective option for promoting/improving bone regeneration. Full article

(This article belongs to the Special Issue Laser Application in Life Sciences 2018)

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15 pages, 52582 KiB

Open AccessArticle

Phasor-Based Endogenous NAD(P)H Fluorescence Lifetime Imaging Unravels Specific Enzymatic Activity of Neutrophil Granulocytes Preceding NETosis

by Ruth Leben, Lennard Ostendorf, Sofie Van Koppen, Asylkhan Rakhymzhan, Anja E. Hauser, Helena Radbruch and Raluca A. Niesner

Int. J. Mol. Sci. 2018, 19(4), 1018; https://doi.org/10.3390/ijms19041018 - 29 Mar 2018

Cited by 25 | Viewed by 7017

Abstract

Time-correlated single-photon counting combined with multi-photon laser scanning microscopy has proven to be a versatile tool to perform fluorescence lifetime imaging in biological samples and, thus, shed light on cellular functions, both in vitro and in vivo. Here, by means of phasor-analyzed endogenous [...] Read more.

Time-correlated single-photon counting combined with multi-photon laser scanning microscopy has proven to be a versatile tool to perform fluorescence lifetime imaging in biological samples and, thus, shed light on cellular functions, both in vitro and in vivo. Here, by means of phasor-analyzed endogenous NAD(P)H (nicotinamide adenine dinucleotide (phosphate)) fluorescence lifetime imaging, we visualize the shift in the cellular metabolism of healthy human neutrophil granulocytes during phagocytosis of Staphylococcus aureus pHrodo™ beads. We correlate this with the process of NETosis, i.e., trapping of pathogens by DNA networks. Hence, we are able to directly show the dynamics of NADPH oxidase activation and its requirement in triggering NETosis in contrast to other pathways of cell death and to decipher the dedicated spatio-temporal sequence between NADPH oxidase activation, nuclear membrane disintegration and DNA network formation. The endogenous FLIM approach presented here uniquely meets the increasing need in the field of immunology to monitor cellular metabolism as a basic mechanism of cellular and tissue functions. Full article

(This article belongs to the Special Issue Laser Application in Life Sciences 2018)

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12 pages, 2296 KiB

Open AccessArticle

Photodynamic Inactivation Potentiates the Susceptibility of Antifungal Agents against the Planktonic and Biofilm Cells of Candida albicans

by Mu-Ching Huang, Mandy Shen, Yi-Jhen Huang, Hsiao-Chi Lin and Chin-Tin Chen

Int. J. Mol. Sci. 2018, 19(2), 434; https://doi.org/10.3390/ijms19020434 - 01 Feb 2018

Cited by 21 | Viewed by 3478

Abstract

Photodynamic inactivation (PDI) has been shown to be a potential treatment modality against Candida infection. However, limited light penetration might leave some cells alive and undergoing regrowth. In this study, we explored the possibility of combining PDI and antifungal agents to enhance the [...] Read more.

Photodynamic inactivation (PDI) has been shown to be a potential treatment modality against Candida infection. However, limited light penetration might leave some cells alive and undergoing regrowth. In this study, we explored the possibility of combining PDI and antifungal agents to enhance the therapeutic efficacy of Candida albicans and drug-resistant clinical isolates. We found that planktonic cells that had survived toluidine blue O (TBO)-mediated PDI were significantly susceptible to fluconazole within the first 2 h post PDI. Following PDI, the killing efficacy of antifungal agents relates to the PDI dose in wild-type and drug-resistant clinical isolates. However, only a 3-log reduction was found in the biofilm cells, suggesting limited therapeutic efficacy under the combined treatment of PDI and azole antifungal drugs. Using confocal microscopic analysis, we showed that TBO-mediated PDI could partially remove the extracellular polymeric substance (EPS) of biofilm. Finally, we showed that a combination of PDI with caspofungin could result in the complete killing of biofilms compared to those treated with caspofungin or PDI alone. These results clearly indicate that the combination of PDI and antifungal agents could be a promising treatment against C. albicans infections. Full article

(This article belongs to the Special Issue Laser Application in Life Sciences 2018)

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14 pages, 6359 KiB

Open AccessArticle

Variation on Molecular Structure, Crystallinity, and Optical Properties of Dentin Due to Nd:YAG Laser and Fluoride Aimed at Tooth Erosion Prevention

by Daísa L. Pereira, Anderson Z. Freitas, Luciano Bachmann, Carolina Benetti, Denise M. Zezell and Patricia A. Ana

Int. J. Mol. Sci. 2018, 19(2), 433; https://doi.org/10.3390/ijms19020433 - 01 Feb 2018

Cited by 23 | Viewed by 4653

Abstract

This in vitro study evaluated the compositional, crystalline, and morphological effects promoted by Nd:YAG laser on root dentin, and verified the effects of laser and topical acidulated phosphate fluoride application (APF-gel) on dentin erosion. 180 bovine dentin slabs were randomized into 4 groups [...] Read more.

This in vitro study evaluated the compositional, crystalline, and morphological effects promoted by Nd:YAG laser on root dentin, and verified the effects of laser and topical acidulated phosphate fluoride application (APF-gel) on dentin erosion. 180 bovine dentin slabs were randomized into 4 groups (n = 45): G1–untreated, G2–APF-gel (1.23% F⁻, 4 min), G3–Nd:YAG (1064 nm, 84.9 J/cm², 10 Hz), and G4–APF-gel application followed by Nd:YAG laser irradiation. The compositional, crystalline, and morphological effects promoted by treatments were investigated on five samples of each experimental group. The other samples were submitted to a 5-day, 10-day, or 15-day erosive and abrasive demineralization and remineralization cycling in order to create erosion lesions. The area and depth of lesions, as well as the optical attenuation coefficient, were assessed, and all data were statistically analysed (p < 0.05). Nd:YAG laser promoted the reduction of carbonate, the formation of tetracalcium phosphate, as well as the melting and recrystallization of the dentin surface. Laser significantly decreased the area and depth of erosion lesions and altered the optical attenuation coefficient when compared to untreated and APF-gel groups, but the association of APF-gel and laser did not promote an additional effect. Nd:YAG laser irradiation can be a promissory treatment to prevent dentin erosion and the abrasion process. Full article

(This article belongs to the Special Issue Laser Application in Life Sciences 2018)

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11 pages, 3101 KiB

Open AccessArticle

An in Vitro Study on the Effect of Combined Treatment with Photodynamic and Chemical Therapies on Candida albicans

by Yi-Hsuan Hsieh, Jun-Hui Zhang, Wen-Ching Chuang, Kun-Hua Yu, Xian-Bin Huang, Yao-Chang Lee and Cheng-I Lee

Int. J. Mol. Sci. 2018, 19(2), 337; https://doi.org/10.3390/ijms19020337 - 24 Jan 2018

Cited by 30 | Viewed by 5150

Abstract

Candida albicans is the most commonly encountered human fungal pathogen, and it is traditionally treated with antimicrobial chemical agents. The antimicrobial effect of these agents is largely weakened by drug resistance and biofilm-associated virulence. Enhancement of the antimicrobial activity of existing agents is [...] Read more.

Candida albicans is the most commonly encountered human fungal pathogen, and it is traditionally treated with antimicrobial chemical agents. The antimicrobial effect of these agents is largely weakened by drug resistance and biofilm-associated virulence. Enhancement of the antimicrobial activity of existing agents is needed for effective candidiasis treatment. Our aim was to develop a therapy that combined biofilm disruption with existing antimicrobial agents. Photodynamic therapy (PDT) utilizing curcumin and blue light was tested as an independent therapy and in combination with fluconazole treatment. Viability assays and morphology analysis were used to assess the effectiveness of C. albicans treatment. Results showed that fluconazole treatment decreased the viability of planktonic C. albicans, but the decrease was not as pronounced in adherent C. albicans because its biofilm form was markedly more resistant to the antimicrobiotic. PDT effectively eradicated C. albicans biofilms, and when combined with fluconazole, PDT significantly inhibited C. albicans to a greater extent. This study suggests that the addition of PDT to fluconazole to treat C. albicans infection enhances its effectiveness and can potentially be used clinically. Full article

(This article belongs to the Special Issue Laser Application in Life Sciences 2018)

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7500 KiB

Open AccessArticle

Challenges for Super-Resolution Localization Microscopy and Biomolecular Fluorescent Nano-Probing in Cancer Research

by Michael Hausmann, Nataša Ilić, Götz Pilarczyk, Jin-Ho Lee, Abiramy Logeswaran, Aurora Paola Borroni, Matthias Krufczik, Franziska Theda, Nadine Waltrich, Felix Bestvater, Georg Hildenbrand, Christoph Cremer and Michael Blank

Int. J. Mol. Sci. 2017, 18(10), 2066; https://doi.org/10.3390/ijms18102066 - 28 Sep 2017

Cited by 34 | Viewed by 9063

Abstract

Understanding molecular interactions and regulatory mechanisms in tumor initiation, progression, and treatment response are key requirements towards advanced cancer diagnosis and novel treatment procedures in personalized medicine. Beyond decoding the gene expression, malfunctioning and cancer-related epigenetic pathways, investigations of the spatial receptor arrangements [...] Read more.

Understanding molecular interactions and regulatory mechanisms in tumor initiation, progression, and treatment response are key requirements towards advanced cancer diagnosis and novel treatment procedures in personalized medicine. Beyond decoding the gene expression, malfunctioning and cancer-related epigenetic pathways, investigations of the spatial receptor arrangements in membranes and genome organization in cell nuclei, on the nano-scale, contribute to elucidating complex molecular mechanisms in cells and tissues. By these means, the correlation between cell function and spatial organization of molecules or molecular complexes can be studied, with respect to carcinogenesis, tumor sensitivity or tumor resistance to anticancer therapies, like radiation or antibody treatment. Here, we present several new applications for bio-molecular nano-probes and super-resolution, laser fluorescence localization microscopy and their potential in life sciences, especially in biomedical and cancer research. By means of a tool-box of fluorescent antibodies, green fluorescent protein (GFP) tagging, or specific oligonucleotides, we present tumor relevant re-arrangements of Erb-receptors in membranes, spatial organization of Smad specific ubiquitin protein ligase 2 (Smurf2) in the cytosol, tumor cell characteristic heterochromatin organization, and molecular re-arrangements induced by radiation or antibody treatment. The main purpose of this article is to demonstrate how nano-scaled distance measurements between bio-molecules, tagged by appropriate nano-probes, can be applied to elucidate structures and conformations of molecular complexes which are characteristic of tumorigenesis and treatment responses. These applications open new avenues towards a better interpretation of the spatial organization and treatment responses of functionally relevant molecules, at the single cell level, in normal and cancer cells, offering new potentials for individualized medicine. Full article

(This article belongs to the Special Issue Laser Application in Life Sciences 2018)

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Review

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18 pages, 10615 KiB

Open AccessReview

State-of-the-Art Fluorescence Fluctuation-Based Spectroscopic Techniques for the Study of Protein Aggregation

by Akira Kitamura and Masataka Kinjo

Int. J. Mol. Sci. 2018, 19(4), 964; https://doi.org/10.3390/ijms19040964 - 23 Mar 2018

Cited by 31 | Viewed by 6913

Abstract

Neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease, are devastating proteinopathies with misfolded protein aggregates accumulating in neuronal cells. Inclusion bodies of protein aggregates are frequently observed in the neuronal cells of patients. Investigation of the underlying [...] Read more.

Neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease, are devastating proteinopathies with misfolded protein aggregates accumulating in neuronal cells. Inclusion bodies of protein aggregates are frequently observed in the neuronal cells of patients. Investigation of the underlying causes of neurodegeneration requires the establishment and selection of appropriate methodologies for detailed investigation of the state and conformation of protein aggregates. In the current review, we present an overview of the principles and application of several methodologies used for the elucidation of protein aggregation, specifically ones based on determination of fluctuations of fluorescence. The discussed methods include fluorescence correlation spectroscopy (FCS), imaging FCS, image correlation spectroscopy (ICS), photobleaching ICS (pbICS), number and brightness (N&B) analysis, super-resolution optical fluctuation imaging (SOFI), and transient state (TRAST) monitoring spectroscopy. Some of these methodologies are classical protein aggregation analyses, while others are not yet widely used. Collectively, the methods presented here should help the future development of research not only into protein aggregation but also neurodegenerative diseases. Full article

(This article belongs to the Special Issue Laser Application in Life Sciences 2018)

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