Raman Spectroscopy Reveals Photobiomodulation-Induced α-Helix to ß-Sheet Transition in Tubulins: Potential Implications for Alzheimer's and Other Neurodegenerative Diseases.

In small clinical studies, the application of transcranial photobiomodulation (PBM), which typically delivers low-intensity near-infrared (NIR) to treat the brain, has led to some remarkable results in the treatment of dementia and several neurodegenerative diseases. However, despite the extensive literature detailing the mechanisms of action underlying PBM outcomes, the specific mechanisms affecting neurodegenerative diseases are not entirely clear. While large clinical trials are warranted to validate these findings, evidence of the mechanisms can explain and thus provide credible support for PBM as a potential treatment for these diseases. Tubulin and its polymerized state of microtubules have been known to play important roles in the pathology of Alzheimer's and other neurodegenerative diseases. Thus, we investigated the effects of PBM on these cellular structures in the quest for insights into the underlying therapeutic mechanisms. In this study, we employed a Raman spectroscopic analysis of the amide I band of polymerized samples of tubulin exposed to pulsed low-intensity NIR radiation (810 nm, 10 Hz, 22.5 J/cm2 dose). Peaks in the Raman fingerprint region (300-1900 cm-1)-in particular, in the amide I band (1600-1700 cm-1)-were used to quantify the percentage of protein secondary structures. Under this band, hidden signals of C=O stretching, belonging to different structures, are superimposed, producing a complex signal as a result. An accurate decomposition of the amide I band is therefore required for the reliable analysis of the conformation of proteins, which we achieved through a straightforward method employing a Voigt profile. This approach was validated through secondary structure analyses of unexposed control samples, for which comparisons with other values available in the literature could be conducted. Subsequently, using this validated method, we present novel findings of statistically significant alterations in the secondary structures of polymerized NIR-exposed tubulin, characterized by a notable decrease in α-helix content and a concurrent increase in ß-sheets compared to the control samples. This PBM-induced α-helix to ß-sheet transition connects to reduced microtubule stability and the introduction of dynamism to allow for the remodeling and, consequently, refreshing of microtubule structures. This newly discovered mechanism could have implications for reducing the risks associated with brain aging, including neurodegenerative diseases like Alzheimer's disease, through the introduction of an intervention following this transition.

The distinguishing electrical properties of cancer cells.

In recent decades, medical research has been primarily focused on the inherited aspect of cancers, despite the reality that only 5-10% of tumours discovered are derived from genetic causes. Cancer is a broad term, and therefore it is inaccurate to address it as a purely genetic disease. Understanding cancer cells' behaviour is the first step in countering them. Behind the scenes, there is a complicated network of environmental factors, DNA errors, metabolic shifts, and electrostatic alterations that build over time and lead to the illness's development. This latter aspect has been analyzed in previous studies, but how the different electrical changes integrate and affect each other is rarely examined. Every cell in the human body possesses electrical properties that are essential for proper behaviour both within and outside of the cell itself. It is not yet clear whether these changes correlate with cell mutation in cancer cells, or only with their subsequent development. Either way, these aspects merit further investigation, especially with regards to their causes and consequences. Trying to block changes at various levels of occurrence or assisting in their prevention could be the key to stopping cells from becoming cancerous. Therefore, a comprehensive understanding of the current knowledge regarding the electrical landscape of cells is much needed. We review four essential electrical characteristics of cells, providing a deep understanding of the electrostatic changes in cancer cells compared to their normal counterparts. In particular, we provide an overview of intracellular and extracellular pH modifications, differences in ionic concentrations in the cytoplasm, transmembrane potential variations, and changes within mitochondria. New therapies targeting or exploiting the electrical properties of cells are developed and tested every year, such as pH-dependent carriers and tumour-treating fields. A brief section regarding the state-of-the-art of these therapies can be found at the end of this review. Finally, we highlight how these alterations integrate and potentially yield indications of cells' malignancy or metastatic index.

Near-Infrared Photobiomodulation of Living Cells, Tubulin, and Microtubules In Vitro.

We report the results of experimental investigations involving photobiomodulation (PBM) of living cells, tubulin, and microtubules in buffer solutions exposed to near-infrared (NIR) light emitted from an 810 nm LED with a power density of 25 mW/cm2 pulsed at a frequency of 10 Hz. In the first group of experiments, we measured changes in the alternating current (AC) ionic conductivity in the 50-100 kHz range of HeLa and U251 cancer cell lines as living cells exposed to PBM for 60 min, and an increased resistance compared to the control cells was observed. In the second group of experiments, we investigated the stability and polymerization of microtubules under exposure to PBM. The protein buffer solution used was a mixture of Britton-Robinson buffer (BRB aka PEM) and microtubule cushion buffer. Exposure of Taxol-stabilized microtubules (~2 µM tubulin) to the LED for 120 min resulted in gradual disassembly of microtubules observed in fluorescence microscopy images. These results were compared to controls where microtubules remained stable. In the third group of experiments, we performed turbidity measurements throughout the tubulin polymerization process to quantify the rate and amount of polymerization for PBM-exposed tubulin vs. unexposed tubulin samples, using tubulin resuspended to final concentrations of ~ 22.7 µM and ~ 45.5 µM in the same buffer solution as before. Compared to the unexposed control samples, absorbance measurement results demonstrated a slower rate and reduced overall amount of polymerization in the less concentrated tubulin samples exposed to PBM for 30 min with the parameters mentioned above. Paradoxically, the opposite effect was observed in the 45.5 µM tubulin samples, demonstrating a remarkable increase in the polymerization rates and total polymer mass achieved after exposure to PBM. These results on the effects of PBM on living cells, tubulin, and microtubules are novel, further validating the modulating effects of PBM and contributing to designing more effective PBM parameters. Finally, potential consequences for the use of PBM in the context of neurodegenerative diseases are discussed.

Computational Study of Potential Galectin-3 Inhibitors in the Treatment of COVID-19.

Galectin-3 is a carbohydrate-binding protein and the most studied member of the galectin family. It regulates several functions throughout the body, among which are inflammation and post-injury remodelling. Recent studies have highlighted the similarity between Galectin-3's carbohydrate recognition domain and the so-called "galectin fold" present on the N-terminal domain of the S1 sub-unit of the SARS-CoV-2 spike protein. Sialic acids binding to the N-terminal domain of the Spike protein are known to be crucial for viral entry into humans, and the role of Galectin-3 as a mediator of lung fibrosis has long been the object of study since its levels have been found to be abnormally high in alveolar macrophages following lung injury. In this context, the discovery of a double inhibitor may both prevent viral entry and reduce post-infection pulmonary fibrosis. In this study, we use a database of 56 compounds, among which 37 have known experimental affinity with Galectin-3. We carry out virtual screening of this database with respect to Galectin-3 and Spike protein. Several ligands are found to exhibit promising binding affinity and interaction with the Spike protein's N-terminal domain as well as with Galectin-3. This finding strongly suggests that existing Galectin-3 inhibitors possess dual-binding capabilities to disrupt Spike-ACE2 interactions. Herein we identify the most promising inhibitors of Galectin-3 and Spike proteins, of which five emerge as potential dual effective inhibitors. Our preliminary results warrant further in vitro and in vivo testing of these putative inhibitors against SARS-CoV-2 with the hope of being able to halt the spread of the virus in the future.