Low-dose photodynamic therapy effect on closure of scratch wounds of normal and diabetic fibroblast cells: An in vitro study

Chronic wounds such as diabetic ulcers are a serious public health problem. Extensive research is needed to find new alternatives for wound treatment. Photodynamic therapy (PDT) is a non-invasive method, which has been studied for several decades to treat cancer, infections, and other diseases. PDT involves the administration of a photosensitizer compound followed by irradiation with using light at specific wavelength to produce reactive oxygen species (ROS) using molecular oxygen. It is possible that low dose photodynamic therapy (LDPDT) could improve wound healing and stimulates the cell repair process. This study we explored the effect of LDPDT on wound healing in vitro using normal and diabetic cellular wound models. The effects of different concentrations of 5-ALA and different energy densities (dark or light) on the cell viability of human fibroblast cells were studied using the MTT assay. After ascertaining the optimum parameters, a scratch wound assay was performed on both normal and diabetic cells and then cells treated with 1 and 5 μg/mL of 5-ALA at 1 J/cm² energy density. ROS production and morphological alteration of the cells were studied. The mortality of normal fibroblast cells increased with increasing 5-ALA concentration and also increasing energy density (up to 3 J/cm²). However, in diabetic cells, the mortality rate did not decrease. Diabetic cells showed increased migration and closure of the scratch compared to normal cells under similar conditions. A low concentration of 5-ALA (5 μg/mL) and low energy density of 1 J/cm² in both normal and diabetic cells gave a small increase in ROS levels compared to controls. This may explain the positive effects of LDPDT on wound healing. The findings of this study suggest that LDPDT may have a potential effect on the wound healing of diabetic wounds.     

Safety of light emitting diode‐red light on human skin: Two randomized controlled trials

Therapeutic applications of light emitting diode‐red light (LED‐RL) are expanding, yet data on its clinical effects are lacking. Our goal was to evaluate the safety of high fluence LED‐RL (≥160 J/cm²). In two phase I, single‐blind, dose escalation, randomized controlled trials, healthy subjects received LED‐RL or mock irradiation to the forearm thrice weekly for 3 weeks at fluences of 160‐640 J/cm² for all skin types (STARS 1, n = 60) and at 480‐640 J/cm² for non‐Hispanic Caucasians (STARS 2, n = 55). The primary outcome was the incidence of adverse events (AEs). The maximum tolerated dose was the highest fluence that did not elicit predefined AEs. Dose‐limiting AEs, including blistering and prolonged erythema, occurred at 480 J/cm² in STARS 1 (n = 1) and 640 J/cm² in STARS 2 (n = 2). AEs of transient erythema and hyperpigmentation were mild. No serious AEs occurred. We determined that LED‐RL is safe up to 320 J/cm² for skin of color and 480 J/cm² for non‐Hispanic Caucasian individuals. LED‐RL may exert differential cutaneous effects depending on race and ethnicity, with darker skin being more photosensitive. These findings may guide future studies to evaluate the efficacy of LED‐RL for the treatment of various diseases.     

The Use of New Methylene Blue in Pseudomonas aeruginosa Biofilm Destruction

A Pseudomonas aeruginosa biofilm was produced in a model system using the bacterial strain NCIMB 8295, grown on silicone tubing (bore size 0.75 cm). Destruction of the biofilm was attempted using either ampicillin or a combination of white light (light dose=7.2 J cm^{m 2}) and the phenothiazinium photosensitiser new methylene blue, and damage, both to extra-cellular polymeric substance (EPS) and to the organism, was monitored. It was found that although little damage to the EPS occurred with ampicillin, NMB caused both cell death and breakdown of the EPS, suggesting the use of photodynamic antimicrobial chemotherapy (PACT) in the disinfection of pathogenic biofilms, e.g. at external catheter surfaces.     

Antimicrobial activity of amphiphilic nanomicelles loaded with curcumin against Pseudomonas aeruginosa alone and activated by blue laser light

The aim of this work was to assess the antimicrobial efficacy on Pseudomonas aeruginosa of nanomicelles loaded with curcumin (CUR) alone and activated by blue laser light in an antimicrobial photodynamic therapy (APDT) approach. First, free CUR in liquid suspension and loaded in three amphiphilic nanomicelles (CUR-DAPMA, CUR-SPD and CUR-SPM) were tested both on bacteria and keratinocytes. While free CUR exerted limited efficacy showing moderate cytotoxicity, a strong inhibition of bacterial growth was obtained using all three nanosystems without toxicity on eukaryotic cells. CUR-SPM emerged as the most effective, and was therefore employed in APDT experiments. Among the three sublethal blue laser (λ 445 nm) protocols tested, the ones characterized by a fluence of 18 and 30 J/cm² further decreased the antimicrobial concentration to 50 nM. The combination of blue laser APDT with CUR-SPM nanomicelles results in an effective synergistic activity that represents a promising novel therapeutic approach on resistant species.     

Antimicrobial blue light for decontamination of platelets during storage

Platelet (PLT) storage is currently limited to 5 days in clinics in the United States, in part, due to an increasing risk for microbial contamination over time. In light of well‐documented antimicrobial activity of blue light (405‐470 nm), we investigated potentials to decontaminate microbes during PLT storage by antimicrobial blue light (aBL). We found that PLTs produced no detectable levels of porphyrins or their derivatives, the chromophores that specifically absorb blue light, in marked contrast to microbes that generated porphyrins abundantly. The difference formed a basis with which aBL selectively inactivated contaminated microbes prior to and during the storage, without incurring any harm to PLTs. In accordance with this, when contamination with representative microbes was simulated in PLT concentrates supplemented with 65% of PLT additive solution in a standard storage bag, all “contaminated” microbes tested were completely inactivated after exposure of the bag to 405 nm aBL at 75 J/cm² only once. While killing microbes efficiently, this dose of aBL irradiation exerted no adverse effects on the viability, activation or aggregation of PLTs ex vivo and could be used repeatedly during PLT storage. PLT survival in vivo was also unaltered by aBL irradiation after infusion of aBL‐irradiated mouse PLTs into mice. The study provides proof‐of‐concept evidence for a potential of aBL to decontaminate PLTs during storage.      

Blue LED light exposure develops intracellular reactive oxygen species,lipid peroxidation,and subsequent cellular injuries in cultured bovine retinal pigment epithelial cells

Abstract

The effects of blue light emitter diode (LED) light exposure on retinal pigment epithelial cells (RPE cells) were examined to detect cellular damage or change and to clarify its mechanisms. The RPE cells were cultured and exposed by blue (470 nm) LED at 4.8 mW/cm². The cellular viability was determined by XTT assay and cellular injury was determined by the lactate dehydrogenase activity in medium. Intracellular reactive oxygen species (ROS) generation was determined by confocal laser microscope image analysis using dihydrorhodamine 123 and lipid peroxidation was determined by 4-hydroxy-2-nonenal protein-adducts immunofluorescent staining (HNE). At 24 h after 50 J/cm² exposures, cellular viability was significantly decreased to 74% and cellular injury was significantly increased to 365% of control. Immediately after the light exposure, ROS generation was significantly increased to 154%, 177%, and 395% of control and HNE intensity was increased to 211%, 359%, and 746% of control by 1, 10, and 50 J/cm², respectively. These results suggest, at least in part, that oxidative stress is an early step leading to cellular damage by blue LED exposure and cellular oxidative damage would be caused by the blue light exposure at even lower dose (1, 10 J/cm²).     

Sialolithiasis: Application parameters for an optimal laser therapy

In‐vitro experimental parametric studies of laser ablation using natural sialoliths and artificial stones have been performed toward an efficient laser treatment of sialolithiasis. Surface microstructure and water adsorption become critical for coupling high power pulsed Ho:YAG laser radiation (λ = 2080 nm, τ ～250 μsec), inducing ablative interactions and stone fragmentation. Results reveal a generic trend, with single pulse laser energy density threshold for sialolith ablative erosion at ～200 J cm^?2 (corresponding to intensity ～800 kW cm^?2) and fragmentation rates reaching ～1 mm/pulse at ～2400 J cm^?2. This process shows no saturation, suggesting that very high energy density irradiation at low pulse repetition rate is an efficient approach. Such operation facilitates rapid cooling and minimal thermal loading of the oral and maxillofacial area, thus causing negligible adverse effects. The method is expected to contribute to the establishment of an easy and optimal therapeutic protocol for sialolithiasis pathology.     

Blue light (470 nm) effectively inhibits bacterial and fungal growth

Blue light (470 nm) LED antimicrobial properties were studied alone against bacteria and with or without the food grade photosensitizer, erythrosine (ERY) against filamentous fungi. Leuconostoc mesenteroides (LM), Bacillus atrophaeus (BA) or Pseudomonas aeruginosa (PA) aliquots were exposed on nutrient agar plates to Array 1 (AR1, 0·2 mW cm^?2) or Array 2 (AR2, 80 mW cm^?2), which emitted impure or pure blue light (0–300 J cm^?2), respectively. Inoculated control (room light only) plates were incubated (48 h) and colonies enumerated. The antifungal properties of blue light combined with ERY (11·4 and 22·8 μmol l^?1) on Penicillium digitatum (PD) and Fusarium graminearum (FG) conidia were determined. Conidial controls consisted of: no light, room light‐treated conidia and ERY plus room light. Light‐treated (ERY + blue light) conidial samples were exposed only to AR2 (0–100 J cm^?2), aliquots spread on potato dextrose agar plates, incubated (48 h, 30°C) and colonies counted. Blue light alone significantly reduced bacterial and FG viability. Combined with ERY, it significantly reduced PD viability. Blue light is lethal to bacteria and filamentous fungi although effectiveness is dependent on light purity, energy levels and microbial genus.

Significance and Impact of the Study

Light from two arrays of different blue LEDs significantly reduced bacterial (Leuconostoc mesenteroides, Bacillus atrophaeus and Pseudomonas aeruginosa) viabilities. Significant in vitro viability loss was observed for the filamentous fungi, Penicillium digitatum and Fusarium graminearum when exposed to pure blue light only plus a photosensitizer. F. graminearum viability was significantly reduced by blue light alone. Results suggest that (i) the amount of significant loss in bacterial viability observed for blue light that is pure or with traces of other wavelengths is genus dependent and (ii) depending on fungal genera, pure blue light is fungicidal with or without a photosensitizer.     

Synergic Effect of Photodynamic Therapy with Methylene Blue and Surfactants in the Inhibition of Candida albicans

Photodynamic therapy (PDT) has been originally developed for the treatment of cancer, but it has been successfully employed in the treatment of infectious diseases, including fungal infections. Surfactants are amphiphilic compounds that also have antifungal properties. The present work demonstrates the synergic effect of PDT with methylene blue (MB) and LED combined with four different surfactants in the killing of Candida albicans. Subinhibitory concentrations of CTAC, HPS, SDS and Triton X-100 were tested with MB PDT. The combined therapies proved to be more efficient than PDT or surfactants separately. The best results were obtained with CTAC and HPS and PDT with MB at the concentration of 32 μg/mL. In conclusion, the combination of surfactants and PDT is an alternative antifungal treatment that can achieve more effective performance with minimal discomfort to the patient.     

Effect of light quality on Bacillus amyloliquefaciens JBC36 and its biocontrol efficacy

Light is one of the most important environmental signals regulating physiological processes of many microorganisms. However, very few studies have been reported on the qualitative or quantitative effects of light on control of postharvest spoilage using antagonistic bacteria. In this study, we investigated the effects of white, red, green, and blue light at photon flux densities of 40, 240, and 360 μmol m^?2 s^?1 on Bacillus amyloliquefaciens JBC36 (JBC36), which has been reported as a promising candidate for biocontrol of green and blue mold on mandarin fruit. With the exception of blue light at 240 and 360 μmol m^?2 s^?1, light generally stimulated growth of JBC36 compared to the controls grown in the dark. Red light increased swarming motility irrespective of intensity and significantly enhanced biofilm formation at 240 μmol m^?2 s^?1. Production of antifungal metabolites and antifungal activity on Penicillium digitatum was also affected by light quality. Interestingly, antifungal activity was significantly increased when JBC36 and P. digitatum was co-incubated under red and green light at an intensity of 240 μmol m^?2 s^?1. We also demonstrated that the quality of light resulted in changes in colonization of JBC36 on mandarin fruit and control of green mold. In particular, red light increased the population level on mandarin fruit and biocontrol efficacy against green mold. These results represent the first report on the effect of light quality on an antagonistic bacterium for the control of postharvest spoilage. We believe that an improved understanding of the JBC36 response to light quality may help in the development of strategies to increase biocontrol efficacy of postharvest spoilage.     

The effect of radiation with polychromatic visible and infrared light on the tumorigenicity of murine hepatoma 22A cells and their sensitivity to lysis by natural killers

The tumorigenicity of murine hepatoma cells (MH-22a) and their sensitivity to lysis by natural killers (NKs) have been studied after exposure to polychromatic visible and infrared light (VIS-IR, 480–3400 nm, 40 mW/cm²), similar to the terrestrial solar spectrum without its minor UV component, with the aim of clarifying the participation of this important environmental and physiotherapeutic factor in regulation of antitumor protective system. MH-22 cells were exposed in vitro to VIS-IR light and their sensitivity to lytic activity of NKs was evaluated. It was found that, after exposure to VIS-IR light at a dose of 4.8 J/cm², the sensitivity of MH-22a cells to lysis by NKs increased by 1.5–2 times, while after exposure at a dose of 9.6 J/cm² it did not change at all the ratios of the NKs-number (effectors) to that of hepatoma cells — targets (1 : 5–1 : 50). An increase of the hepatoma cell sensitivity to NKs was accompanied by structural changes of cell surface: the capability of supramembranous glycoproteins (glycocalyx) to sorb the vital dye alcian blue (AB) was significantly lower than in the case of unexposed cells of the control group. However, no changes in AB sorption was revealed in hepatoma cells exposed to light at a dose of 9.6 J/cm². The tumorigenicity of photoirradiated MH-22a cells has been studied in the experiments in vitro. For 25 days after transplantation of light-exposed hepatoma cells to C3HA syngene mice, the tumor volume proved to be smaller after exposure to light at both doses of 4.8 and 9.6 J/cm² than in the control group (by 4–4.5 times and 2.5–4 times, respectively), which correlated with an increase of sensitivity to lysis by NKs and with a decrease of AB sorption after light exposure only at a dose of 4.8 J/cm². Using the flow-cytometry method, we could show that VIS-IR light at the doses used did not interfere with the distribution of hepatoma cells over the cell-cycle phases and, thus, deceleration of the tumor growth was not associated with a cytostatic effect of the VIS-IR light. To evaluate the effect of polychromatic light on growth of the preformed tumors, a 5-day course of daily light exposure of C3HA tumor-bearing mice was performed on the 10th day after subcutaneous transplantation of 2 × 10⁵ cells of syngene hepatoma, when tumors developed in all (100%) animals. As in the case of transplantation of light-exposed cells, irradiation of tumor-bearing mice at doses 4.8–9.6 J/cm² resulted in a deceleration of tumor growth (by 2.1–2.9 and 2,2 times, respectively) for 4 weeks compared with unirradiated mice.     

In Vitro Photodynamic Inactivation of Plant-Pathogenic Fungi Colletotrichum acutatum and Colletotrichum gloeosporioides with Novel Phenothiazinium Photosensitizers

The increasing tolerance to currently used fungicides in both clinical and agricultural areas is of great concern. The nonconventional light-based approach of antimicrobial photodynamic treatment (APDT) is a promising alternative to conventional fungicides. We evaluated the effects of APDT with four phenothiazinium derivatives (methylene blue [MB], new methylene blue N [NMBN], toluidine blue O [TBO], and the novel pentacyclic phenothiazinium photosensitizer [PS] S137) on conidia of three fungal species (Colletotrichum acutatum, Colletotrichum gloeosporioides, and Aspergillus nidulans). The efficacy of APDT with each PS was determined, initially, based on photosensitizer MICs. Additionally, the effects of APDT with two selected PSs (NMBN and S137) on survival of conidia were evaluated. The subcellular localization of the PS in C. acutatum conidia was determined. The effects of photodynamic treatments on leaves of the plant host Citrus sinensis were also investigated. APDT with S137 showed the lowest MIC. MICs for S137 were 5 μM for the three fungal species when a fluence of 25 J cm⁻² was used. APDT with NMBN (50 μM) and S137 (10 μM) resulted in a reduction in the survival of the conidia of all species of approximately 5 logs with fluences of ≥15 J cm⁻². Washing of the conidia before light exposure did not prevent photodynamic inactivation. Both NMBN and S137 accumulated in cytoplasmic structures, such as lipid bodies, of C. acutatum conidia. No damage to orange tree leaves was observed after APDT.     

The effect of low-intensity laser irradiation in the blue, green, and red spectral ranges on healing of experimental skin wounds in rats

The effects of low-intensity laser irradiation in the red (632.8 nm), green (532 nm), and blue (441.2 nm) spectral ranges on wound healing has been studied in rats. The effect of the traditionally used red laser irradiation has been compared with the effect caused by laser irradiation in other spectral ranges, aiming to support the provisional hypothesis that a similar healing effect could be achieved at lower doses of wound irradiation by lasers emitting in the blue and green spectral ranges. The following parameters have been used to characterize healing of the experimental wounds: the functional activity of phagocytes in the wound exudate, which was determined from luminol-dependent chemiluminescence, the phagocyte number; the wound exudates’ antioxidant activity; and the rate of healing, which was determined as the change of the wound surface area. It was found that in all cases the laser irradiation accelerated the healing of wounds. Exposure to red laser irradiation at the dose of 1.5 J/cm²), and to blue or green laser irradiation at a dose of 0.75 J/cm² shortened the time of the wound healing from 22 to 17 and 19 days, respectively. The functional activity of leukocytes in irradiated groups increased by day 5 after surgery, whereas in the control group it decreased. The superoxide dismutase activity increased in all experimental groups by day 5 after surgery. Laser irradiation in the red spectral range at a dose of 1.5 J/cm² resulted in a larger increase in superoxide dismutase activity, as compared to that found after exposure to laser irradiation in the blue and green spectral ranges at a dose of 0.75 J/cm².     

Antifungal activity of magnoflorine against <Emphasis Type="Italic">Candida</Emphasis> strains

Candida albicans is a major invasive pathogen, and the development of strains resistant to conventional antifungal agents has been reported in recent years. We evaluated the antifungal activity of 44 compounds against Candida strains. Magnoflorine showed the highest growth inhibitory activity of the tested Candida strains, with a minimum inhibitory concentration (MIC) of 50 μg/mL based on microdilution antifungal susceptibility testing. Disk diffusion assay confirmed the antifungal activity of magnoflorine and revealed that this activity was stable over 3 days compared to those of berberine and cinnamaldehyde. Cytotoxicity testing showed that magnoflorine could potentially be used in a clinical setting because it didn’t have any toxicity to HaCaT cells even in 200 μg/mL of treatment. Magnoflorine at 50 μg/mL inhibited 55.91?±?7.17% of alpha-glucosidase activity which is required for normal cell wall composition and virulence of Candida albicans. Magnoflorine also reduced the formation of C. albicans’ biofilm. Combined treatment with magnoflorine and miconazole decreased the amount of miconazole required to kill various Candida albicans. Therefore, magnoflorine is a good candidate lead compound for novel antifungal agents.     

Effective photodynamic treatment of Trichophyton species with Rose Bengal

Photodynamic therapy (PDT) with Rose Bengal has previously achieved eradication of Trichophyton rubrum infections causing toenail onychomycosis; however, its antifungal activity against other clinically relevant dermatophytes has yet to be studied. Here, we test the efficacy of PDT using Rose Bengal (140 μM) and 532 nm irradiation (101 J/cm²) against Trichophyton mentagrophytes and Trichophyton interdigitale spores, in comparison to T. rubrum. A significant reduction (>99%) of T. mentagrophytes and T. interdigitale was observed, while actual eradication of viable T. rubrum was achieved (99.99%). Laser irradiation alone inhibited growth of T. rubrum (55.2%) and T. mentagrophytes (45.2%) significantly more than T. interdigitale (25.5%) (P = .0086), which may indicate an increased presence of fungal pigments, xanthomegnin and melanin. The findings suggest that Rose Bengal-PDT can act against a broader spectrum of fungal pathogens, and with continued development may be employed in a wider range of clinical antifungal applications.     

Proliferation and tumorigenity of murine hepatoma cells irradiated with polychromatic visible and infrared light

In experiments in vitro, the effects of polychromatic visible (VIS) light combined with polychromatic infrared light (VIS-IR, 480–3400 nm) and the effects of the entire spectrum of VIS radiation (385–750 nm) on viability and proliferative activity of the murine hepatoma cells MH22a were studied. In experiments in vivo, changes in the tumorigenic properties of cells MH22a were studied after the same kinds of light exposure. It was shown that irradiation of hepatoma cells with two kinds of polychromatic light at a wide range of doses (4.8–38.4 J/cm²) did not lead to an increase in the number of dead cells for 24–72 h of cultivation and did not cause deceleration of the hepatoma cell proliferation; moreover, the VIS-IR light at a dose of 4.8 J/cm² and the VIS light at a dose 38.4 J/cm² even promoted more intense cell proliferation after 24 h. In cells irradiated with VIS-IR and VIS light, the proliferation index rose by 1.6 and 1.4 times, respectively, and the time of the cells’ number doubling decreased as compared with control. Studying the tumorigenic properties of irradiated tumor cells has shown that, for 30 days after transplantation to syngenic mice C3HA of hepatoma cells 24 h after their irradiation with VIS-IR light at a dose of 4.8 J/cm², the tumor volume decreased significantly (2.6–4.1 times) at all periods of observation, while the incidence of tumor formation decreased, whereas the survival of the tumor-bearing mice did not change. Transplantation of cells irradiated with the same light at a dose of 9.6 J/cm² did not lead to significant changes in the tumor volume, the tumor formation incidence, and animal survival. The main contribution to the antitumor effect of VIS-IR light seems to be made by the VIS component, as transplantation into mice of cells irradiated with VIS light alone at a dose of 38.4 J/cm² also stimulating proliferation of hepatoma cells in vitro resulted in a decrease of their tumorigenic properties. However, the IR component in the combined VIS-IR radiation enhanced the antitumor effect of the VIS light; as a result, it was manifested after use of doses eight times lower (4.8 J/cm²) than in the case of VIS light alone (38.4 J/cm²). Mechanisms of the decrease of tumorigenic properties of hepatoma cells after irradiation with polychromatic light at doses stimulating their proliferation in vitro are studied.     

Effect of intermittency factor on singlet oxygen and PGE2 formation in azulene-mediated photodynamic therapy: A preliminary study

In photodynamic therapy, intermittent irradiation modes that incorporate an interval between pulses are believed to decrease the effect of hypoxia by permitting an interval of re-oxygenation. The effect of the irradiation intermittency factor (the ratio of the irradiation pulse time to the total irradiation time) on singlet oxygen formation and inflammatory cytokine production was examined using azulene as a photosensitizer. Effects of difference intermittency factor on singlet oxygen formation and inflammatory cytokine were examined. Azulene solutions (1/10 μM) were irradiated with a 638-nm 500 mW diode laser in fractionation (intermittency factor of 5 or 9) or continuous mode using 50 mW/cm² at 4 or 8 J/cm². Singlet oxygen measurement was performed using a dimethyl anthracene probe. Peripheral blood mononuclear cells (PBMC) were stimulated by 10 ng/ml rhTNF-α for 6 h, before addition of 1 and 10 μM azulene solutions and irradiation. PGE₂ measurement was undertaken using a human PGE₂ ELISA kit. Kruskal-Wallis with Dunn Bonferroni test was used for statistical analyses at p < 0.05.Irradiation of 1 μM azulene+4 J/cm²+intermittency factor of 9 increased singlet oxygen 3-fold (p < 0.0001). Irradiation of 10 μM azulene at either 4 J/cm²+intermittency of 9 or 8 J/cm²+intermittency factor of 5 reduced PGE₂ expression in PBMCs to non-inflamed levels. Thus, at 50 mW/cm², 10 μM azulene-mediated photodynamic therapy with a high intermittency factor and a low energy density generated sufficient singlet oxygen to suppress PGE₂ in Inflamed PBMCs.     

High-fluence light emitting diode-red light inhibits cell cycle progression in human dermal fibroblasts

Skin fibrosis is a debilitating feature of several systemic and dermatologic diseases. While current treatment options carry significant risk of side effects and recurrence, high-fluence light emitting diode-generated red light (LED-RL) is an alternative therapeutic that is safe, non-invasive, and accessible. We previously demonstrated LED-RL decreases fibroblast proliferation, a key pathogenic component of fibrosis. However, the cellular mechanism by which high fluence LED-RL modulates fibroblast proliferation is unclear. Herein, we explored the effects of high fluence LED-RL on human dermal fibroblast cell cycle progression. We demonstrate that LED-RL at 640 J/cm² induced significant arrest of cells in G₀/G₁ compared to temperature-matched control. This was accompanied by a corresponding increase in expression of checkpoint regulator p53 in irradiated cells. These data demonstrate high fluence LED-RL may exert its anti-proliferative effect on fibroblasts by inducing G₀/G₁ arrest. Further, this study provides insight into the molecular mechanism underlying LED-RL as an anti-fibrotic therapeutic.     

Studies on the relationship between pulsed UV light irradiation and the simultaneous occurrence of molecular and cellular damage in clinically-relevant Candida albicans

This constitutes the first study to report on the relationship between pulsed UV light (PL) irradiation and the simultaneous occurrence of molecular and cellular damage in clinical strains of Candida albicans. Microbial protein leakage and propidium iodide (PI) uptake assays demonstrated significant increases in cell membrane permeability in PL-treated yeast that depended on the amount of UV pulses applied. This finding correlated well with the measurement of increased levels of lipid hydroperoxidation in the cell membrane of PL-treated yeast. PL-treated yeast cells also displayed a specific pattern of intracellular reactive oxygen species (ROS) generation, where ROS were initially localised in the mitochondria after low levels of pulsing (UV dose 0.82 μJ/cm²) before more wide-spread cytosolic ROS production occurred with enhanced pulsing. Intracellular ROS levels were measured using the specific mitochondrial peroxide stain dihydrorhodamine 123 and the cytosolic oxidation stain dichloroflurescin diacetate. Use of the dihydroethidium stain also revealed increased levels of intracellular superoxide as a consequence of augmented pulsing. The ROS bursts observed during the initial phases of PL treatment was consistent with the occurrence of apoptotic cells as confirmed by detection of specific apoptotic markers, abnormal chromatin condensation and externalisation of cell membrane lipid phosphatidylserine. Increased amount of PL-irradiation (ca. UV does 1.24-1.65 μJ/cm²) also resulted in the occurrence of late apoptotic and necrotic yeast phenotypes, which coincided with the transition from mitochondrial to cytosolic localisation of ROS and with irreversible cell membrane leakage. Use of the comet assay also revealed significant nuclear damage in similarly treated PL samples. Although some level of cellular repair was observed in all test strains during sub-lethal exposure to PL-treatments (≤ 20 pulses or UV dose 0.55 μJ/cm²), this was absent in similar samples exposed to increased amounts of pulsing. This study showed that PL-irradiation inactivates C. albicans test strains through a multi-targeted process with no evidence of microbial ability to support cell growth after ≤ 20 pulses. Implications of our findings in terms of application of PL for contact-surface disinfection are discussed.