Curcumin as a Perspective Protection for Retinal Pigment Epithelium during Autophagy Inhibition in the Course of Retinal Degeneration.

Defective autophagy in the retinal pigment epithelium (RPE) is involved in retinal degeneration, mostly in the course of age-related macular degeneration (AMD), which is an increasingly prevalent retinal disorder, eventually leading to blindness. However, most autophagy activators own serious adverse effects when administered systemically. Curcumin is a phytochemical, which induces autophagy with a wide dose-response curve, which brings minimal side effects. Recent studies indicating defective autophagy in AMD were analyzed. Accordingly, in this perspective, we discuss and provide some evidence about the protective effects of curcumin in preventing RPE cell damage induced by the autophagy inhibitor 3-methyladenine (3-MA). Cells from human RPE were administered the autophagy inhibitor 3-MA. The cell damage induced by 3-MA was assessed at light microscopy by hematoxylin & eosin, Fluoro Jade-B, and ZO1 immunohistochemistry along with electron microscopy. The autophagy inhibitor 3-MA produces cell loss and cell degeneration of RPE cells. These effects are counteracted dose-dependently by curcumin. In line with the hypothesis that the autophagy machinery is key in sustaining the integrity of the RPE, here we provide evidence that the powerful autophagy inhibitor 3-MA produces dose-dependently cell loss and cell degeneration in cultured RPE cells, while inhibiting autophagy as shown by LC3-II/LC3-I ratio and gold-standard assessment of autophagy through LC3-positive autophagy vacuoles. These effects are prevented dose-dependently by curcumin, which activates autophagy. These data shed the perspective of validating the role of phytochemicals as safe autophagy activators to treat AMD.

Autophagy Activation Promoted by Pulses of Light and Phytochemicals Counteracting Oxidative Stress during Age-Related Macular Degeneration.

The seminal role of autophagy during age-related macular degeneration (AMD) lies in the clearance of a number of reactive oxidative species that generate dysfunctional mitochondria. In fact, reactive oxygen species (ROS) in the retina generate misfolded proteins, alter lipids and sugars composition, disrupt DNA integrity, damage cell organelles and produce retinal inclusions while causing AMD. This explains why autophagy in the retinal pigment epithelium (RPE), mostly at the macular level, is essential in AMD and even in baseline conditions to provide a powerful and fast replacement of oxidized molecules and ROS-damaged mitochondria. When autophagy is impaired within RPE, the deleterious effects of ROS, which are produced in excess also during baseline conditions, are no longer counteracted, and retinal degeneration may occur. Within RPE, autophagy can be induced by various stimuli, such as light and naturally occurring phytochemicals. Light and phytochemicals, in turn, may synergize to enhance autophagy. This may explain the beneficial effects of light pulses combined with phytochemicals both in improving retinal structure and visual acuity. The ability of light to activate some phytochemicals may further extend such a synergism during retinal degeneration. In this way, photosensitive natural compounds may produce light-dependent beneficial antioxidant effects in AMD.

The Essential Role of Light-Induced Autophagy in the Inner Choroid/Outer Retinal Neurovascular Unit in Baseline Conditions and Degeneration.

The present article discusses the role of light in altering autophagy, both within the outer retina (retinal pigment epithelium, RPE, and the outer segment of photoreceptors) and the inner choroid (Bruch's membrane, BM, endothelial cells and the pericytes of choriocapillaris, CC). Here autophagy is needed to maintain the high metabolic requirements and to provide the specific physiological activity sub-serving the process of vision. Activation or inhibition of autophagy within RPE strongly depends on light exposure and it is concomitant with activation or inhibition of the outer segment of the photoreceptors. This also recruits CC, which provides blood flow and metabolic substrates. Thus, the inner choroid and outer retina are mutually dependent and their activity is orchestrated by light exposure in order to cope with metabolic demand. This is tuned by the autophagy status, which works as a sort of pivot in the cross-talk within the inner choroid/outer retina neurovascular unit. In degenerative conditions, and mostly during age-related macular degeneration (AMD), autophagy dysfunction occurs in this area to induce cell loss and extracellular aggregates. Therefore, a detailed analysis of the autophagy status encompassing CC, RPE and interposed BM is key to understanding the fine anatomy and altered biochemistry which underlie the onset and progression of AMD.

Pulsed blue light, saliva and curcumin significantly inactivate human coronavirus.

In a recent study, we showed that pulsed blue light (PBL) inactivates as much as 52.3% of human beta coronavirus HCoV-OC43, a surrogate of SARS-CoV-2, and one of the major strains of viruses responsible for the annual epidemic of the common cold. Since curcumin and saliva are similarly antiviral and curcumin acts as blue light photosensitizer, we used Qubit fluorometry and WarmStart RT-LAMP assays to study the effect of combining 405 nm, 410 nm, 425 nm or 450 nm wavelengths of PBL with curcumin, saliva or a combination of curcumin and saliva against human beta coronavirus HCoV-OC43. The results showed that PBL, curcumin and saliva independently and collectively inactivate HCoV-OC43. Without saliva or curcumin supplementation 21.6 J/cm2 PBL reduced HCoV-OC43 RNA concentration a maximum of 32.8% (log10 = 2.13). Saliva supplementation alone inactivated the virus, reducing its RNA concentration by 61% (log10 = 2.23); with irradiation the reduction was as much as 79.1%. Curcumin supplementation alone decreased viral RNA 71.1%, and a maximum of 87.8% with irradiation. The combination of saliva and curcumin reduced viral RNA to 83.1% and decreased the RNA up to 90.2% with irradiation. The reduced levels could not be detected with qPCR. These findings show that PBL in the range of 405 nm to 450 nm wavelength is antiviral against human coronavirus HCoV-OC43, a surrogate of the COVID-19 virus. Further, it shows that with curcumin as a photosensitizer, it is possible to photodynamically inactivate the virus beyond qPCR detectable level using PBL. Since HCoV-OC43 is of the same beta coronavirus family as SARS-CoV-2, has the same genomic size, and is often used as its surrogate, these findings heighten the prospect of similarly inactivating novel coronavirus SARS-CoV-2, the virus responsible for COVID-19 pandemic.

Pulsed blue light inactivates two strains of human coronavirus.

Emerging evidence suggests that blue light has the potential to inactivate viruses. Therefore, we investigated the effect of 405 nm, 410 nm, 425 nm and 450 nm pulsed blue light (PBL) on human alpha coronavirus HCoV-229 E and human beta coronavirus HCoV-OC43, using Qubit fluorometry and RT-LAMP to quantitate the amount of nucleic acid in irradiated and control samples. Like SARS-CoV-2, HCoV-229E and HCoV-OC43 are single stranded RNA viruses transmitted by air and direct contact; they have similar genomic sizes as SARS-CoV-2, and are used as surrogates for SARS-CoV-2. Irradiation was carried out either at 32.4 J cm-2 using 3 mW cm-2 irradiance or at 130 J cm-2 using 12 mW cm-2 irradiance. Results: (1) At each wavelength tested, PBL was antiviral against both coronaviruses. (2) 405 nm light gave the best result, yielding 52.3% (2.37 log10) inactivation against HCoV-OC43 (p < .0001), and a significant 1.46 log 10 (44%) inactivation of HCoV-229E (p < .01). HCoV-OC43, which like SARS-CoV-2 is a beta coronavirus, was more susceptible to PBL irradiation than alpha coronavirus HCoV-229E. The latter finding suggests that PBL is potentially antiviral against multiple coronavirus strains, and that, while its potency may vary from one virus to another, it seems more antiviral against beta coronaviruses, such as HCoV-OC43. (3) Further, the antiviral effect of PBL was better at a higher irradiance than a lower irradiance, and this indicates that with further refinement, a protocol capable of yielding 100% inactivation of viruses is attainable.

Structural membrane changes induced by pulsed blue light on methicillin-resistant Staphylococcus aureus (MRSA).

BACKGROUND: In a recent study we showed that blue light inactivates methicillin-resistant Staphylococcus aureus (MRSA) by perturbing, depolarizing, and disrupting its cell membrane. PURPOSE: The current study presents visual evidence that the observed biochemical changes also result in cell metabolic changes and structural alteration of the cell membrane. METHODS: Cultures of MRSA were treated with 450 nm pulsed blue light (PBL) at 3 mW/cm2 irradiance, using a sub lethal dose of 2.7 J/cm2 radiant exposure three times at 30-min intervals. Following 24 h incubation at 37 °C, irradiated colonies and control non-irradiated colonies were processed for light and transmission electron microscopy. RESULTS: The images obtained revealed three major effects of PBL; (1) disruption of MRSA cell membrane, (2) alteration of membrane structure, and (3) disruption of cell replication. CONCLUSION: These signs of bacterial inactivation at a dose deliberately selected to be sub-lethal supports our previous finding that rapid depolarization of bacterial cell membrane and disruption of cellular function comprise another mechanism underlying photo-inactivation of bacteria. Further, it affirms the potency of PBL.

Blue light absorbing pigment in Streptococcus agalactiae does not potentiate the antimicrobial effect of pulsed 450 nm light.

INTRODUCTION: Recently, it was shown that Group B Streptococcus (GBS) COH1 strain, which has granadaene-an endogenous chromophore known to absorb blue light-is not susceptible to 450 nm pulsed blue light (PBL) inactivation unless the bacterium is co-cultured with exogenous porphyrin. PURPOSE: To confirm or refute the finding, we studied the effect of blue light on NCTC, another strain of GBS with more granadaene than COH1, to determine if the abundance of granadaene-and by implication more absorption of blue light-fosters GBS susceptibility to PBL. METHODS: We irradiated cultures of the bacterium with or without protoporphyrin, coproporphyrin, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD) or NADH. After 24-h incubation, bacterial colonies were enumerated, log10 CFU/mL computed, and descriptive and inferential data analyzed and compared. RESULTS: (1) The rich amount of granadaene in NCTC did not enhance its susceptibility to antimicrobial pulsed blue light (PBL). (2) Adding exogenous porphyrin fostered NCTC susceptibility to irradiation, resulting in 100% bacterial suppression. (3) Exogenous FMN or FAD, which strongly absorb 450 nm light, did not promote the antimicrobial effect of PBL, neither did exogenous NAD or NADH, two weak blue light-absorbing photosensitizers. CONCLUSION: These results strengthen our previous assertion that an endogenous chromophore with the capacity to absorb and transform light energy into a biochemical process that engenders bacterial cell death, is essential for 450 nm PBL to suppress GBS.

The viability of human cells irradiated with 470-nm light at various radiant energies in vitro.

Blue light is known to be antimicrobial, but its effect on normal cutaneous and subcutaneous cells remains unclear. Therefore, we studied the effect of 470-nm light on the viability of adult and neonatal human dermal fibroblasts, Jurkat T-cells, and THP-1 monocytes in vitro. Each culture was irradiated with 0, 3, 55, or 110 J/cm2 of 470-nm light and subjected to trypan blue assay to ascertain viability. Further, MTT, neutral red, and fluorescence assays of fibroblasts were performed, and cell morphology visualized using bright field and fluorescence microscopy. At each dose and in each of the four cell lines, there was no significant difference in cell concentration between irradiated and non-irradiated cultures, even though irradiation with 55 J/cm2 or 110 J/cm2 slightly decreased cell count. Light microscopy showed progressive morphological changes in the fibroblasts as energy fluence increased from 55 to 110 J/cm2. Irradiation at 3 J/cm2 produced a slight but non-significant increase in the viability of Jurkat T-cells and THP-1 monocytes. In contrast, at 110 J/cm2 radiant exposure, irradiation slightly decreased the viability of all four cells. While 3 J/cm2 appears stimulatory, our finding that 110 J/cm2 produces a slight decrease in viability and engenders morphological changes in fibroblasts, suggesting that such high doses should be avoided in blue light treatments.

The importance of porphyrins in blue light suppression of Streptococcus agalactiae.

It is well documented that blue light absorption by bacterial chromophores triggers downstream production of reactive oxygen species (ROS), which in turn results in bacterial cell death. To elucidate the importance of chromophores in the bactericidal effect of blue light, and to determine whether blue light absorption per se or the presence of porphyrins known to engender ROS is crucial in blue light treatment, we studied the effect of 450 nm pulsed light on Streptococcus agalactiae, also known as Group B Streptococcus (GBS) strain COH1. GBS does not synthesize porphyrins but has a blue light-absorbing chromophore, granadaene. We irradiated planktonic cultures of GBS with or without exogenous chromophore supplementation using either protoporphyrin IX (PPIX), coproporphyrin III (CPIII), Nicotinamide adenine dinucleotide (NAD), reduced nicotinamide adenine dinucleotide (NADH), Flavin adenine dinucleotide (FAD), or Flavin mononucleotide (FMN). Quantification of surviving bacterial colonies, presented as percent survival and CFU/mL (log10), showed that (1) 450 nm blue light does not suppress the growth of GBS, even though its endogenous chromophore, granadaene, absorbs light in the 450 nm spectrum. (2) The addition of either of the two exogenous porphyrins, PPIX or CPIII, significantly suppressed GBS, indicating the importance of porphyrins in the antimicrobial action of blue light. (3) Adding exogenous FMN or FAD, two known absorbers of 450 nm light, minimally potentiated the bactericidal effect of blue light, again confirming that mere absorption of blue light by chromophores does not necessarily result in bacterial suppression. (4) Irradiation of GBS with or without NAD+ or NADH supplementation-two weak absorbers of 450 nm light-minimally suppressed GBS, indicating that a blue light-absorbing chromophore is essential for the bactericidal action of blue light. (5) Collectively, these findings show that in addition to the presence of a blue light-absorbing chromophore in bacteria, a chromophore with the right metabolic machinery and biochemical structure, capable of producing ROS, is necessary for 450 nm blue light to suppress GBS.

Light as a potential treatment for pandemic coronavirus infections: A perspective.

The recent outbreak of COVID-19, which continues to ravage communities with high death tolls and untold psychosocial and catastrophic economic consequences, is a vivid reminder of nature's capacity to defy contemporary healthcare. The pandemic calls for rapid mobilization of every potential clinical tool, including phototherapy-one of the most effective treatments used to reduce the impact of the 1918 "Spanish influenza" pandemic. This paper cites several studies showing that phototherapy has immense potential to reduce the impact of coronavirus diseases, and offers suggested ways that the healthcare industry can integrate modern light technologies in the fight against COVID-19 and other infections. The evidence shows that violet/blue (400-470 nm) light is antimicrobial against numerous bacteria, and that it accounts for Niels Ryberg Finsen's Nobel-winning treatment of tuberculosis. Further evidence shows that blue light inactivates several viruses, including the common flu coronavirus, and that in experimental animals, red and near infrared light reduce respiratory disorders, similar to those complications associated with coronavirus infection. Moreover, in patients, red light has been shown to alleviate chronic obstructive lung disease and bronchial asthma. These findings call for urgent efforts to further explore the clinical value of light, and not wait for another pandemic to serve as a reminder. The ubiquity of inexpensive light emitting lasers and light emitting diodes (LEDs), makes it relatively easy to develop safe low-cost light-based devices with the potential to reduce infections, sanitize equipment, hospital facilities, emergency care vehicles, homes, and the general environment as pilot studies have shown.

Pulsed 450 nm blue light suppresses MRSA and Propionibacterium acnes in planktonic cultures and bacterial biofilms.

In our recent study, we showed that pulsed blue light (PBL) suppresses the growth of Propionibacterium acnes more than continuous wave (CW) blue light in vitro, but it is not known that other bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), respond similarly to PBL. The high potency of PBL relative to CW blue light makes it a suitable antimicrobial for suppressing bacterial growth in biofilms as well. Therefore, we determined if MRSA-a deadly bacterium of global concern-is susceptible to 450 nm PBL irradiation in vitro, and ascertained whether the bactericidal effect of PBL on planktonic P. acnes culture can be replicated in biofilms of P. acnes and MRSA. In three series of experiments, we irradiated P. acnes and MRSA respectively, either in planktonic cultures, forming biofilms or formed biofilms. Compared to controls, the results showed 100% bacterial suppression in planktonic cultures of MRSA irradiated with 3 mW/cm2 irradiance and 7.6 J/cm2 radiant exposure three times at 30-minute intervals, and also in P. acnes cultures irradiated with 2 mW/cm2 irradiance 5 J/cm2 radiant exposure thrice daily during each of 3 days. Irradiation of biofilms with the same irradiances and radiant exposures that gave 100% bacterial suppression in planktonic cultures resulted in disruption and disassembly of the architecture of MRSA and P. acnes biofilms, more so in forming biofilms than formed biofilms. The antimicrobial effect on each bacterium was minimal in forming biofilms, and even less in formed biofilms. Increasing radiant exposure slightly from 7.6 J/cm2 to 10.8 J/cm2 without changing any other parameter, yielded more disruption of the biofilm and fewer live MRSA and P. acnes, suggesting that 100% bacterial suppression is possible with further refinement of the protocol. In both planktonic cultures and biofilms, PBL suppressed MRSA more than P. acnes.

Pulsed 450 nm blue light significantly inactivates Propionibacterium acnes more than continuous wave blue light.

Infection with Propionibacterium acnes is ubiquitous, and drug resistant strains have been on the rise as the use of pharmaceutical antimicrobials continues to engender the emergence of further resistant strains. In previous studies, we showed that treatment with blue light serves as an alternative to pharmaceutical intervention. As a part of our ongoing effort to improve the antimicrobial efficacy of blue light, we studied the effect of pulsed 450 nm light on P. acnes in vitro and compared two pulsed rates with continuous wave irradiation. We irradiated cultures of P. acnes at various irradiances and radiant energies either singly or repeatedly at various time intervals, using printed micro-LEDs, with the goal of finding the lowest combination of irradiance and radiant energy that would yield 100% bacterial suppression. Our results show that treatment with 33% pulsed light gave the best result compared to 20% pulsed wave or continuous wave. Timing irradiation to coincide with the replication cycle of P. acnes produced a significantly better antimicrobial effect. Furthermore, repeated irradiation at 3-h or 4-h interval enabled significant bacterial suppression even at lower irradiances; thus, making single irradiation at high irradiances unnecessary. Moreover, combining repeated irradiation with appropriate duration of treatment and 33% irradiation pulse rate gave optimal 100% [7 log10] bacterial suppression.

Optimizing the bactericidal effect of pulsed blue light on Propionibacterium acnes - A correlative fluorescence spectroscopy study.

Propionibacterium acnes infection is the eighth most prevalent disease, affecting 80% of people worldwide. Resistance to antibiotics has been on the rise; over 40% of acne infections now resist commonly used topical and oral anti-acnes antibiotics, making treatment difficult. In our effort to refine blue light as an alternative safe clinically effective treatment, we determined if 100% bacterial suppression is attainable at ultralow irradiances and radiant energies, and explored the relationship between bacterial suppression and fluorescence during treatment. P. acnes were irradiated in vitro repeatedly three times per day at 3- or 4-hour intervals over three or more days, using 3 or 5 J/cm2 radiant energy of 450 nm pulsed blue light (PBL) at irradiances as low as 2 mW/cm2. In another series of experiments, we measured changes in P. acnes fluorescence as bacteria were repeatedly irradiated at various radiant exposures over three to four days. Our results showed that (1) 33% PBL, applied three times per day at 3-hour intervals each day over a three-day period at 2 mW/cm2 irradiance and 5 J/cm2 radiant exposure, resulted in100% bacterial suppression (7 log10 reduction), (2) the absorbed 450 nm light caused P. acnes to fluoresce predominantly in the red spectrum, with the fluorescence diminishing correlatively as treatment was repeated at 3-hour intervals and rising significantly during long periods of no treatment, and (3) treatment at 3-hour intervals gave better results than treatment at 4-hour intervals.

Rare mutations in Pfmdr1 gene of Plasmodium falciparum detected in clinical isolates from patients treated with anti-malarial drug in Nigeria.

BACKGROUND: Plasmodium falciparum, the deadliest causative agent of malaria, has high prevalence in Nigeria. Drug resistance causing failure of previously effective drugs has compromised anti-malarial treatment. On this basis, there is need for a proactive surveillance for resistance markers to the currently recommended artemisinin-based combination therapy (ACT), for early detection of resistance before it become widespread. METHODS: This study assessed anti-malarial resistance genes polymorphism in patients with uncomplicated P. falciparum malaria in Lagos, Nigeria. Sanger and Next Generation Sequencing (NGS) methods were used to screen for mutations in thirty-seven malaria positive blood samples targeting the P. falciparum chloroquine-resistance transporter (Pfcrt), P. falciparum multidrug-resistance 1 (Pfmdr1), and P. falciparum kelch 13 (Pfk13) genes, which have been previously associated with anti-malarial resistance. RESULTS: Expectedly, the NGS method was more proficient, detecting six Pfmdr1, seven Pfcrt and three Pfk13 mutations in the studied clinical isolates from Nigeria, a malaria endemic area. These mutations included rare Pfmdr1 mutations, N504K, N649D, F938Y and S967N, which were previously unreported. In addition, there was moderate prevalence of the K76T mutation (34.6%) associated with chloroquine and amodiaquine resistance, and high prevalence of the N86 wild type allele (92.3%) associated with lumefantrine resistance. CONCLUSION: Widespread circulation of mutations associated with resistance to current anti-malarial drugs could potentially limit effective malaria therapy in endemic populations.

Understanding the antimicrobial activity of selected disinfectants against methicillin-resistant Staphylococcus aureus (MRSA).

Disinfectants and biocidal products have been widely used to combat Methicillin-resistant Staphylococcus aureus (MRSA) infections in homes and healthcare environments. Although disruption of cytoplasmic membrane integrity has been documented as the main bactericidal effect of biocides, little is known about the biochemical alterations induced by these chemical agents. In this study, we used Fourier transform infrared (FT-IR) spectroscopy and chemometric tools as an alternative non-destructive technique to determine the bactericidal effects of commonly used disinfectants against MRSA USA-300. FTIR spectroscopy permits a detailed characterization of bacterial reactivity, allowing an understanding of the fundamental mechanism of action involved in the interaction between bacteria and disinfectants. The disinfectants studied were ethanol 70% (N = 5), isopropanol (N = 5), sodium hypochlorite (N = 5), triclosan (N = 5) and triclocarban (N = 5). Results showed less than 5% colony forming units growth of MRSA treated with triclocarban and no growth in the other groups. Nearly 70,000 mid-infrared spectra from the five treatments and the two control (untreated; N = 4) groups of MRSA (bacteria grown in TSB and incubated at 37°C (Control I) / at ambient temperature (Control II), for 24h) were pre-processed and analyzed using principal component analysis followed by linear discriminant analysis (PCA-LDA). Clustering of strains of MRSA belonging to five treatments and the discrimination between each treatment and two control groups in MRSA (untreated) were investigated. PCA-LDA discriminatory frequencies suggested that ethanol-treated spectra are the most similar to isopropanol-treated spectra biochemically. Also reported here are the biochemical alterations in the structure of proteins, lipid membranes, and phosphate groups of MRSA produced by sodium hypochlorite, triclosan, and triclocarban treatments. These findings provide mechanistic information involved in the interaction between MRSA strains and hygiene products; thereby demonstrating the potential of spectroscopic analysis as an objective, robust, and label-free tool for evaluating the macromolecular changes involved in disinfectant-treated MRSA.

Blue/violet laser inactivates methicillin-resistant Staphylococcus aureus by altering its transmembrane potential.

The resistance of methicillin-resistant Staphylococcus aureus to antibiotics presents serious clinical problems that prompted the need for finding alternative or combination therapies. One such therapy is irradiation with blue light. To determine the alterations in metabolic processes implicated in the observed antimicrobial effects of blue light, we investigated the changes in membrane potential and the presence of free-radical-producing photo-acceptor molecules. Bacterial cultures irradiated with one or two doses of 405nm laser light (each consisting of 121J/cm2) were imaged with spectrally resolved laser-scanning microscopes to detect endogenous fluorescent species as well as the voltage sensitive dye 3,3'-Diethyloxacarbocyanine iodide. The endogenous fluorescence indicated the presence of photosensitizers (i.e., porphyrins, NADH, FAD) in the cells, while the exogenous signal allowed us to monitor rapid changes in transmembrane potential following treatment with light. The changes were drastic within the first 5min after irradiation with the first dose and continued slowly after the second irradiation. These results suggest that the early antimicrobial activity of blue light results from alteration of membrane integrity with a consequent decrease in membrane polarization and rapid alteration of vital cellular functions. The observation of an early antimicrobial activity of light is very encouraging, as it suggests that treatment does not necessarily have to be administered over a long period of time.

Spectrally resolved infrared microscopy and chemometric tools to reveal the interaction between blue light (470nm) and methicillin-resistant Staphylococcus aureus.

Blue light inactivates methicillin-resistant Staphylococcus aureus (MRSA), a Gram-positive antibiotic resistant bacterium that leads to fatal infections; however, the mechanism of bacterial death remains unclear. In this paper, to uncover the mechanism underlying the bactericidal effect of blue light, a combination of Fourier transform infrared (FTIR) spectroscopy and chemometric tools is employed to detect the photoreactivity of MRSA and its distinctive pathway toward apoptosis after treatment. The mechanism of action of UV light and vancomycin against MRSA is also investigated to support the findings. Principal component analysis followed by linear discriminant analysis (PCA- LDA) is employed to reveal clustering of five groups of MRSA samples, namely untreated (control I), untreated and incubated at ambient air (control II), irradiated with 470nm blue light, irradiated with 253.5 UV light, and vancomycin-treated MRSA. Loadings plot from PCA-LDA analysis reveals important functional groups in proteins (1683, 1656, 1596, 1542cm-1), lipids (1743, 1409cm-1), and nucleic acids region of the spectrum (1060, 1087cm-1) that are responsible for the classification of blue light irradiated spectra and control spectra. Cluster vector plots and scores plot reveals that UV light-irradiated spectra are the most biochemically similar to blue light- irradiated spectra; however, some wavenumbers experience a shift. The shifts between blue light and UV light irradiated loadings plot at νasym PO2- band (from 1228 to 1238cm-1), DNA backbone (from 970 to 966cm-1) and base pairing vibration of DNA (from 1717 to 1712cm-1) suggest distinctive changes in DNA conformation in response to irradiation. Our findings indicate that irradiation of MRSA with 470nm light induces A-DNA cleavage and that B-DNA is more resistant to damage by blue light. Blue light and UV light treatment of MRSA are complementary and distinct from the known antimicrobial effect of vancomycin. Moreover, it is known that UV-induced cleavage of DNA predominantly targets B-DNA, which is in agreement with the FTIR findings. Overall the results suggest that the combination of light and vancomycin could be a more robust approach in treating MRSA infections.

Blue light does not impair wound healing in vitro.

Irradiation with red or near infrared light promotes tissue repair, while treatment with blue light is known to be antimicrobial. Consequently, it is thought that infected wounds could benefit more from combined blue and red/infrared light therapy; but there is a concern that blue light may slow healing. We investigated the effect of blue 470nm light on wound healing, in terms of wound closure, total protein and collagen synthesis, growth factor and cytokines expression, in an in vitro scratch wound model. Human dermal fibroblasts were cultured for 48h until confluent. Then a linear scratch wound was created and irradiated with 3, 5, 10 or 55J/cm(2). Control plates were not irradiated. Following 24h of incubation, cells were fixed and stained for migration and fluorescence analyses and the supernatant collected for quantification of total protein, hydroxyproline, bFGF, IL-6 and IL-10. The results showed that wound closure was similar for groups treated with 3, 5 and 10J/cm(2), with a slight improvement with the 5J/cm(2) dose, and slower closure with 55J/cm(2) p<0.001). Total protein concentration increased after irradiation with 3, 5 and 10J/cm(2), reaching statistical significance at 5J/cm(2) compared to control (p<0.0001). However, hydroxyproline levels did not differ between groups. Similarly, bFGF and IL-10 concentrations did not differ between groups, but IL-6 concentration decreased progressively as fluence increased (p<0.0001). Fluorescence analysis showed viable cells regardless of irradiation fluence. We conclude that irradiation with blue light at low fluence does not impair in vitro wound healing. The significant decrease in IL-6 suggests that 470nm light is anti-inflammatory.

A comparison of four methods for determining viability in human dermal fibroblasts irradiated with blue light.

INTRODUCTION: Several tests are available for assessing the viability of cells; however, there is a dearth of studies comparing the results obtained with each test. We compared the capability of four viability assays (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), neutral red, trypan blue and live/dead fluorescence), to detect potential toxicity in fibroblasts irradiated with 470nm blue light. METHODS: Cells were irradiated at 3, 55, 110 and 220J/cm(2), incubated for 24h and viability assessed using each test. RESULTS: MTT assay showed significant decreases in viability when cells were irradiated with 110 and 220J/cm(2) energy fluence (dose) (89% and 57% viable cells, respectively; p<0.0001, compared to control); likewise the trypan blue assay showed 42% and 46% viable cells (p<0.0001). Neutral red assay revealed significant decrease in viability when cells were irradiated with 220J/cm(2) (84% viable cells; p=0.0008, compared to control). The live/dead fluorescence assay was less sensitive, evincing 91% and 95% viable cells after irradiation with 110 and 220J/cm(2) respectively. DISCUSSION: (1) The four assays differed in their levels of sensitivity to cell viability. (2) The adverse effect of increasing doses seems to manifest as alteration of mitochondrial metabolism, followed by lysosomal dysfunction, membrane disruption and finally loss of cell membrane integrity. (3) Overall, irradiation with 3J/cm(2) or 55J/cm(2) did not adversely affect cell viability. Thus, doses below 110J/cm(2) appear safe.