A Cell-Permeable Photosensitizer for Selective Proximity Labeling and Crosslinking of Aggregated Proteome.

Intracellular proteome aggregation is a ubiquitous disease hallmark with its composition associated with pathogenicity. Herein, this work reports on a cell-permeable photosensitizer (P8, Rose Bengal derivative) for selective photo induced proximity labeling and crosslinking of cellular aggregated proteome. Rose Bengal is identified out of common photosensitizer scaffolds for its unique intrinsic binding affinity to various protein aggregates driven by the hydrophobic effect. Further acetylation permeabilizes Rose Bengal to selectively image, label, and crosslink aggregated proteome in live stressed cells. A combination of photo-chemical, tandem mass spectrometry, and protein biochemistry characterizations reveals the complexity in photosensitizing pathways (both Type I & II), modification sites and labeling mechanisms. The diverse labeling sites and reaction types result in highly effective enrichment and identification of aggregated proteome. Finally, aggregated proteomics and interaction analyses thereby reveal extensive entangling of proteostasis network components mediated by HSP70 chaperone (HSPA1B) and active participation of autophagy pathway in combating proteasome inhibition. Overall, this work exemplifies the first photo induced proximity labeling and crosslinking method (namely AggID) to profile intracellular aggregated proteome and analyze its interactions.

Heterogeneous photocatalytic degradation of antiviral drug didanosine mediated by rose bengal and TiO2 nanoparticles.

There is a great concern among the researcher to remove the problem of the persistent organic pollutants in wastewater. Pharmaceutical agrochemical and personal care products are generally considered Persistent organic pollutants. Therefore, it is a matter of concern to develop new techniques how to remove these pollutants safely at low cost. This study mainly focuses on the commonly used antiviral drug didanosine and one most commonly used dye rose bengal. In this study, an organic dye rose bengal and TiO2 nanoparticles have been used in combination with UV light to achieve the photodegradation of selected pharmaceutical products and the dye was also degraded by using TiO2 Nanoparticles. The formation of three oxidation products was detected by using a very popular separation technique thin layer and column chromatography. The isolated photoproduct was characterized by using advanced characterization techniques like FTIR (Fourier transform infrared spectroscopy), UV Spectroscopy, and Proton and 13C NMR (Nuclear Magnetic Resonance spectroscopy). The role of singlet oxygen as an active species in this reaction was confirmed by using D2O as a reaction medium. The role of singlet oxygen in this photochemical reaction was also established by the addition of sodium azide. The TiO2 nanophotocatalyst efficiently degrade the didanosine and rose bengal in the presence of the UV light. In the TiO2-induced photocatalytic degradation of didanosine and dyes, the hydroxyl and superoxide radical anion play a prominent role. The finding of this manuscript is very useful to develop an efficient low-cost method for the treatment of wastewater contaminated by antiviral drugs, similar pharmaceutical products and dyes. This study was also very helpful to establish a plausible mechanism behind the phototoxicity of the didanosine.

Cellular uptake of rose bengal is mediated by OATP1B1/1B3 transporters.

Due to its fluorescent properties and high yield of singlet oxygen, rose bengal (RB) is one of the most promising photosensitizers for cancer treatment. However, the negative charge of RB molecule may significantly hamper its intracellular delivery by passive diffusion through the cell membrane. Thus, specific membrane protein transporters may be needed. The organic anion transporting polypeptides (OATPs) are a well-characterized group of membrane protein transporters, responsible for cellular uptake of a number of drugs. To our knowledge, this is the first study that evaluates cellular transport of RB mediated by the OATP transporter family. First, electrified liquid-liquid interface, together with biophysical analysis and molecular dynamics simulations were used to characterize the interaction of RB with several models of a cellular membranes. These experiments proved that RB interacts only with the membrane's surface, without spontaneously crossing the lipid bilayer. Evaluation of intracellular uptake of RB by flow cytometry and confocal microscopy showed significant differences in uptake between liver and intestinal cell line models differing in expression of OATP transporters. The use of specific pharmacological inhibitors of OATPs, together with Western blotting and in silico analysis, indicated that OATPs are crucial for cellular uptake of RB.

Astrocyte Progenitors Derived From Patients With Alzheimer Disease Do Not Impair Stroke Recovery in Mice.

BACKGROUND: Species-specific differences in astrocytes and their Alzheimer disease-associated pathology may influence cellular responses to other insults. Herein, human glial chimeric mice were generated to evaluate how Alzheimer disease predisposing genetic background in human astrocytes contributes to behavioral outcome and brain pathology after cortical photothrombotic ischemia. METHODS: Neonatal (P0) immunodeficient mice of both sexes were transplanted with induced pluripotent stem cell-derived astrocyte progenitors from Alzheimer disease patients carrying PSEN1 exon 9 deletion (PSEN1 ΔE9), with isogenic controls, with cells from a healthy donor, or with mouse astrocytes or vehicle. After 14 months, a photothrombotic lesion was produced with Rose Bengal in the motor cortex. Behavior was assessed before ischemia and 1 and 4 weeks after the induction of stroke, followed by tissue perfusion for histology. RESULTS: Open field, cylinder, and grid-walking tests showed a persistent locomotor and sensorimotor impairment after ischemia and female mice had larger infarct sizes; yet, these were not affected by astrocytes with PSEN1 ΔE9 background. Staining for human nuclear antigen confirmed that human cells successfully engrafted throughout the mouse brain. However, only a small number of human cells were positive for astrocytic marker GFAP (glial fibrillary acidic protein), mostly located in the corpus callosum and retaining complex human-specific morphology with longer processes compared with host counterparts. While host astrocytes formed the glial scar, human astrocytes were scattered in small numbers close to the lesion boundary. Aß (beta-amyloid) deposits were not present in PSEN1 ΔE9 astrocyte-transplanted mice. CONCLUSIONS: Transplanted human cells survived and distributed widely in the host brain but had no impact on severity of ischemic damage after cortical photothrombosis in chimeric mice. Only a small number of transplanted human astrocytes acquired GFAP-positive glial phenotype or migrated toward the ischemic lesion forming glial scar. PSEN1 ΔE9 astrocytes did not impair behavioral recovery after experimental stroke.

The Impact of Different Rose Bengal Formulations on Corneal Thickness and the Efficacy of Rose Bengal/Green Light Corneal Cross-linking in the Rabbit Eye.

PURPOSE: To examine central corneal thickness (CCT) changes during in vivo rose bengal-green light corneal cross-linking (RG-CXL) and compare the CXL efficacy of different rose bengal formulations. METHODS: After epithelium removal, the right eyes of rabbits were immersed in rose bengal solution prepared by different solvents (water, phosphate buffered saline, dextran, and hydroxypropyl methylcellulos [HPMC]) for 2 or 20 minutes, then the rose bengal distribution in the corneal stroma was analyzed by confocal fluorescence detection. During the RG-CXL process, the CCT was measured at seven time points. The left eyes served as the untreated control group. Corneal enzymatic resistance and corneal biomechanics were tested to compare the RG-CXL efficacy. RESULTS: The rose bengal infiltration depths were 120 and 200 µm for the 2- and 20-minute groups, respectively. CCT increased significantly after infiltration, then decreased significantly in the first 200 seconds of irradiation and decreased slowly for the next 400 seconds. The CCT of the 20-minute groups was significantly thicker than that of the 2-minute groups (P < .0001). All RG-CXL treatments improved the corneal enzymatic resistance and corneal biomechanics, with the effects being greater in the 20-minute groups. The inclusion of 1.1% HPMC in the rose bengal formulation helped to maintain CCT during irradiation while not affecting either the infiltration of rose bengal or the efficacy of RG-CXL. CONCLUSIONS: Within the range studied, RG-CXL efficacy increased with infiltration time. The incorporation of a 20-minute infiltration of 0.1% rose bengal-1.1% HPMC into the RG-CXL procedure may further improve the safety of the treatment and its prospects for clinical use. [J Refract Surg. 2022;38(7):450-458.].

Ultrasound-dependent RNAi using TatU1A-rose bengal conjugate.

Tat-U1A-rose bengal conjugate (TatU1A-RB) was prepared as an ultrasound-sensitive RNA carrier molecule. This molecule consists of Tat cell-penetrating peptide, U1A RNA-binding protein, and rose bengal as a sonosensitizer. We demonstrated that TatU1A-RB delivered RNA via the endocytosis pathway, which was followed by ultrasound-dependent endosomal escape and cytosolic dispersion of the RNA. A short hairpin RNA (shRNA) delivered by TatU1A-RB mediated RNA interference (RNAi) ultrasound-dependently. Even by ultrasound irradiation through blood cells, RNAi could be induced with TatU1A-RB and the shRNA. This ultrasound-dependent cytosolic RNA delivery method will serve as the basis for a new approach to nucleic acid therapeutics.

A Novel In Situ Dendritic Cell Vaccine Triggered by Rose Bengal Enhances Adaptive Antitumour Immunity.

Dendritic cell- (DC-) based vaccination has emerged as a promising antitumour immunotherapy. However, overcoming immune tolerance and immunosuppression in the tumour microenvironment (TME) is still a great challenge. Recent studies have shown that Rose Bengal (RB) can effectively induce immunogenic cell death (ICD) in cancer cells, presenting whole tumour antigens for DC processing and presentation. However, the synergistic antitumour effect of combining intralesional RB with immature DCs (RB-iDCs) remains unclear. In the present study, we investigated whether RB-iDCs have superior antitumour effects compared with either single agent and evaluated the immunological mechanism of RB-iDCs in a murine lung cancer model. The results showed that intralesional RB-iDCs suppressed subcutaneous tumour growth and lung metastasis, which resulted in 100% mouse survival and significantly increased TNF-α production by CD8+ T cells. These effects were closely related to the induction of the expression of distinct ICD hallmarks by RB in both bulk cancer cells and cancer stem cells (CSCs), especially calreticulin (CRT), thus enhancing immune effector cell (i.e., CD4+, CD8+, and memory T cells) infiltration and attenuating the accumulation of immunosuppressive cells (i.e., Tregs, macrophages, and myeloid-derived suppressor cells (MDSCs)) in the TME. This study reveals that the RB-iDC vaccine can synergistically destroy the primary tumour, inhibit distant metastasis, and prevent tumour relapse in a lung cancer mouse model, which provides important preclinical data for the development of a novel combinatorial immunotherapy.

Optimization of the decolorization conditions of Rose Bengal by using Aspergillus niger TF05 and a decolorization mechanism.

Aspergillus niger TF05 was applied to decolorize Rose Bengal dye. The effects of carbon source, nitrogen source, metal ion and spore concentration on Rose Bengal treatment with A. niger TF05 were studied. A Plackett-Burman design (PBD) and a uniform design (UD) were used to optimize the decolorization conditions of A. niger TF05 and enhance its decolorization effect. The mechanism of Rose Bengal decolorization by A. niger TF05 was examined by analysing degradation products via UV-visible light spectroscopy, IR spectroscopy and GC-MS. The best decolorization effect was achieved in the single factor test with glucose and ammonium chloride as carbon and nitrogen sources, respectively. Mg2+ was an essential ion that could improve the mould ball state and adsorption efficiency if the spore concentration was maintained at 106 spores ml-1. The optimal decolorization conditions obtained using the PBD and UD methods were 11.5 g l-1 glucose, 6.5 g l-1 ammonium chloride, 0.4 g l-1 magnesium sulphate, pH 5.8, 28 °C, 140 r.p.m. rotational speed, 0.18 g l-1 dye concentration, 0.5 ml of inocula and 120 h decolorization time. Under these conditions, the maximum decolorization rate was 106%. Spectral analysis suggested that the absorption peak of the product changed clearly after decolorization; GC-MS analysis revealed that the intermediate product tetrachlorophthalic anhydride formed after decolorization. The combined use of the PBD and UD methods can optimize multi-factor experiments. A. niger TF05 decolorized Rose Bengal during intracellular enzymatic degradation after adsorption.

Noncovalent Interactions with PAMAM and PPI Dendrimers Promote the Cellular Uptake and Photodynamic Activity of Rose Bengal: The Role of the Dendrimer Structure.

Rose bengal is an anionic dye considered as a potential photosensitizer for anticancer photodynamic therapy. The clinical utility of rose bengal is hampered by its short half-life, limited transmembrane transport, aggregation, and self-quenching; consequently, efficient drug carriers that overcome these obstacles are urgently required. In this study, we performed multilevel in vitro and in silico characterization of interactions between rose bengal and cationic poly(amidoamine) (PAMAM) and poly(propyleneimine) (PPI) dendrimers of the third and fourth generation and assessed the ability of the resultant complexes to modulate the photosensitizing properties of the drug. We focused on explaining the molecular basis of this phenomenon and proved that the generation- and structure-dependent binding of the dye by the dendrimers increases the cellular uptake and production of singlet oxygen and intracellular reactive oxygen species, leading to an increase in phototoxicity. We conclude that the application of dendrimer carriers could enable the design of efficient photodynamic therapies based on rose bengal.

Designer exosomes for targeted and efficient ferroptosis induction in cancer via chemo-photodynamic therapy.

Background: Efficient and specific induction of cell death in liver cancer is urgently needed. In this study, we aimed to design an exosome-based platform to deliver ferroptosis inducer (Erastin, Er) and photosensitizer (Rose Bengal, RB) into tumor tissues with high specificity. Methods: Exosome donor cells (HEK293T) were transfected with control or CD47-overexpressing plasmid. Exosomes were isolated and loaded with Er and RB via sonication method. Hepa1-6 cell xenograft C57BL/6 model was injected with control and engineered exosomes via tail vein. In vivo distribution of the injected exosomes was analyzed via tracking the fluorescence labeled exosomes. Photodynamic therapy was conducted by 532 nm laser irradiation. The therapeutic effects on hepatocellular carcinoma and toxic side-effects were systemically analyzed. Results: CD47 was efficiently loaded on the exosomes from the donor cells when CD47 was forced expressed by transfection. CD47 surface functionalization (ExosCD47) made the exosomes effectively escape the phagocytosis of mononuclear phagocyte system (MPS), and thus increased the distribution in tumor tissues. Erastin and RB could be effectively encapsulated into exosomes after sonication, and the drug-loaded exosomes (Er/RB@ExosCD47) strongly induced ferroptosis both in vitro and in vivo in tumor cells after irradiation of 532 nm laser. Moreover, compared with the control exosomes (Er/RB@ExosCtrl), Er/RB@ExosCD47 displayed much lower toxicity in liver. Conclusion: The engineered exosomes composed of CD47, Erastin, and Rose Bengal, induce obvious ferroptosis in hepatocellular carcinoma (HCC) with minimized toxicity in liver and kidney. The proposed exosomes would provide a promising strategy to treat types of malignant tumors.

Characterization of Nannochloropsis oceanica Rose Bengal Mutants Sheds Light on Acclimation Mechanisms to High Light When Grown in Low Temperature.

A barrier to realizing Nannochloropsis oceanica's potential for omega-3 eicosapentaenoic acid (EPA) production is the disparity between conditions that are optimal for growth and those that are optimal for EPA biomass content. A case in point is temperature: higher content of polyunsaturated fatty acid, and especially EPA, is observed in low-temperature (LT) environments, where growth rates are often inhibited. We hypothesized that mutant strains of N. oceanica resistant to the singlet-oxygen photosensitizer Rose Bengal (RB) would withstand the oxidative stress conditions that prevail in the combined stressful environment of high light (HL; 250 µmol photons m-2 s-1) and LT (18°C). This growth environment caused the wild-type (WT) strain to experience a spike in lipid peroxidation and an inability to proliferate, whereas growth and homeostatic reactive oxygen species levels were observed in the mutant strains. We suggest that the mutant strains' success in this environment can be attributed to their truncated photosystem II antennas and their increased ability to diffuse energy in those antennas as heat (non-photosynthetic quenching). As a result, the mutant strains produced upward of four times more EPA than the WT strain in this HL-LT environment. The major plastidial lipid monogalactosyldiacylglycerol was a likely target for oxidative damage, contributing to the photosynthetic inhibition of the WT strain. A mutation in the NO10G01010.1 gene, causing a subunit of the 2-oxoisovalerate dehydrogenase E1 protein to become non-functional, was determined to be the likely source of tolerance in the RB113 mutant strain.

Non-Polymeric Nanogels as Versatile Nanocarriers: Intracellular Transport of the Photosensitizers Rose Bengal and Hypericin for Photodynamic Therapy.

The use of nanocarriers for intracellular transport of actives has been extensively studied in recent years and represents a central area of nanomedicine. The main novelty of this paper lies on the use of nanogels formed by a low-molecular-weight gelator (1). Here, non-polymeric, molecular nanogels are successfully used for intracellular transport of two photodynamic therapy (PDT) agents, Rose Bengal (RB) and hypericin (HYP). The two photosensitizers (PSs) exhibit different drawbacks for their use in clinical applications. HYP is poorly water-soluble, while the cellular uptake of RB is hindered due to its dianionic character at physiological pH values. Additionally, both PSs tend to aggregate precluding an effective PDT. Despite the different nature of these PSs, nanogels from gelator 1 provide, in both cases, an efficient intracellular transport into human colon adenocarcinoma cells (HT-29) and a notably improved PDT efficiency, as assessed by confocal laser scanning microscopy and flow cytometry. Furthermore, no significant dark toxicity of the nanogels is observed, supporting the biocompatibility of the delivery system. The developed nanogels are highly reproducible due to their non-polymeric nature, and their synthesis is easily scaled up. The results presented here thus confirm the potential of molecular nanogels as valuable nanocarriers, capable of entrapping both hydrophobic and hydrophilic actives, for PDT of cancer.

Binding of rose bengal to lysozyme modulates photooxidation and cross-linking reactions involving tyrosine and tryptophan.

This work examined the hypothesis that interactions of Rose Bengal (RB2-) with lysozyme (Lyso) might mediate type 1 photoreactions resulting in protein cross-linking even under conditions favoring 1O2 formation. UV-visible spectrophotometry, isothermal titration calorimetry (ITC), and docking analysis were employed to characterize RB2--Lyso interactions, while oxidation of Lyso was studied by SDS-PAGE gels, extent of amino acid consumption, and liquid chromatography (LC) with mass detection (employing tryptic peptides digested in H218O and H2O). Docking studies showed five interaction sites including the active site. Hydrophobic interactions induced a red shift of the visible spectrum of RB2- giving a Kd of 4.8 µM, while data from ITC studies, yielded a Kd of 0.68 µM as an average of the interactions with stoichiometry of 3.3 RB2- per Lyso. LC analysis showed a high consumption of readily-oxidized amino acids (His, Trp, Met and Tyr) located at different and diverse locations within the protein. This appears to reflect extensive damage on the protein probably mediated by a type 2 (1O2) mechanism. In contrast, docking and mass spectrometry analysis provided evidence for the generation of specific intra- (Tyr23-Tyr20) and inter-molecular (Tyr23-Trp62) Lyso cross-links, and Lyso dimer formation via radical-radical, type 1 mechanisms.

Comparative performance of lateral flow immunochromatography, iELISA and Rose Bengal tests for the diagnosis of cattle, sheep, goat and swine brucellosis.

BACKGROUND: Brucellosis is a world-wide extended zoonosis that causes a grave problem in developing economies. Animal vaccination and diagnosis are essential to control brucellosis, and the need for accurate but also simple and low-cost tests that can be implemented in low-infrastructure laboratories has been emphasized. METHODOLOGY: We evaluated bovine, sheep, goat and swine lateral flow immunochromatography assay kits (LFA), the Rose Bengal test (RBT) and a well-validated protein G indirect ELISA (iELISA) using sera of Brucella culture-positive and unvaccinated brucellosis free livestock. Sera from cattle vaccinated with S19 and RB51 brucellosis vaccines were also tested. Finally, we compared RBT and LFA using sera of white Fulani cattle of unknown bacteriological status from a brucellosis endemic area of Nigeria. RESULTS AND CONCLUSIONS: Although differences were not statistically significant, RBT showed the highest values for diagnostic sensitivity/specificity in cattle (LFA, 96.6/98.8; RBT, 98.9/100; and iELISA, 96.6/100) and the iELISA yielded highest values in sheep (LFA, 94.0/100; RBT, 92.0/100; iELISA, 100/100), goats (LFA, 95.7/96.2; RBT, 97.8/100; iELISA, 100/100) and pigs (LFA, 92.3/100; RBT, 92.3/100; iELISA, 100/100). Vaccine S19 administered subcutaneously interfered in all tests but conjunctival application minimized the problem. Although designed not to interfere in serodiagnosis, vaccine RB51 interfered in LFA and iELISA but not in the RBT. We found closely similar apparent prevalence results when testing the Nigerian Fulani cattle by RBT and LFA. Although both RBT and LFA (showing similar diagnostic performance) are suitable for small laboratories in resource-limited areas, RBT has the advantage that a single reagent is useful in all animal species. Considering these advantages, its low cost and that it is also useful for human brucellosis diagnosis, RBT might be a good choice for resource-limited laboratories.

Rose Bengal attached and dextran coated gadolinium oxide nanoparticles for potential diagnostic imaging applications.

We report here, reverse micelle mediated synthesis of multifunctional dextran (dex) coated Gd2O3 nanoparticles (NPs) carrying rose bengal (RB) dye for magnetic resonance and optical imaging. The diameter of these RB attached dex coated Gd2O3 NPs (Gd-dex-RB NPs) was found to be ~17 nm as measured by TEM. NMR line broadening effect on the surrounding water protons affirmed the paramagnetic nature of these NPs. Optical properties of Gd-dex-RB NPs were validated by UV-Vis and fluorescence spectroscopy. Time dependent release profile of RB from NPs at two different pH of 7.4 and 5.0 revealed that these NPs behave as slow releasing system. In-vitro study revealed that NPs are efficiently taken up by cells and show optical activity in cellular environment. In vitro cell viability (SRB) assay was performed on cancerous (A-549, U-87) and normal (HEK-293) cell lines, showed the absence of cytotoxic effect of Gd-dex-RB NPs. Therefore, such multifunctional NPs can be efficiently used for bio-imaging and optical tracking.

Deamidation of pseudopeptidic molecular hydrogelators and its application to controlled release.

HYPOTHESIS: The incorporation of a succinic acid-derived moiety in amino acid derivatives would favor an intramolecular catalysis of a deamidation reaction. Such reaction would permit controlled disassembly of molecular hydrogelators and the use of the hydrogels for controlled release of actives. EXPERIMENTAL: Low molecular weight hydrogelators containing a succinic acid-derived moiety were prepared by conventional organic synthesis procedures. Hydrogels were examined by electron microscopy and 1HNMR studies were carried out to evaluate the solubility in water of the hydrogelators and the deamidation reaction. Liberation of Rose Bengal entrapped in the hydrogels was monitored by UV-Vis spectroscopy. FINDINGS: Molecular hydrogels formed by pseudopeptidic derivatives of l-valine suffer a thermal deamidation reaction, leading to partial disassembly. The succinic acid-derived moiety present in the gelators is responsible of intramolecular catalysis of a deamidation reaction. Such neighboring group effect is reminiscent of biochemical processes such as protein deamidation and self-excision of inteins. It has been found that the thermodynamic equilibrium of the deamidation reaction is regulated by the efficiency of hydrogelation. As a proof of concept, the thermally promoted deamidation is applied to controlled release of Rose Bengal.

Serological survey for Brucella antibodies in donkeys of north-eastern Nigeria.

A cross-sectional epidemiological study was conducted to determine seroprevalence and risk factors influencing the presence of Brucella antibodies in donkeys of Borno State, north-eastern Nigeria. The study aimed at providing baseline information that may be used in planning a control policy against equine brucellosis. Blood samples were collected from 601 donkeys, comprised of 374 males and 227 females from the six agricultural zones of the state between March 2013 and September 2014. The sera obtained were tested for Brucella antibodies using Rose Bengal plate test (RBPT) and competitive enzyme-linked immunosorbent assay (cELISA). Of the 601 donkeys tested, 43 (7.2%) and 40 (6.7%) were seropositive by RBPT and cELISA, respectively. A seroprevalence of 8.6% was obtained in male and 3.5% in female donkeys. According to age, the highest seroprevalence of 9.6% was obtained from donkeys of age group 4-6 years. With respect to pregnancy status, a higher seroprevalence (6.8%) was obtained from pregnant donkeys compared to 3.8% obtained from the non-pregnant ones. There were statistically significant associations between the presence of antibodies and sex (p < 0.05) and the presence of antibodies and age (p < 0.05) of the studied donkeys. However, no statistically significant association (p > 0.05) was observed between the pregnancy status and presence of antibodies. The study concludes that Brucella infection is present in donkeys in all the agricultural zones of the state. The relatively high seroprevalence (7.2%) obtained is of public health concern because of the close interaction between donkeys, ruminants, and humans in the study area.

Luciferase-Rose Bengal conjugates for singlet oxygen generation by bioluminescence resonance energy transfer.

Conjugates of Rose Bengal and Renilla luciferase generated singlet oxygen upon binding with coelenterazine via bioluminescence resonance energy transfer (BRET). Since the applications of conventional PDT have been limited to superficial lesions due to the limited light penetration in tissue, BRET activated PDT which does not require external light illumination may overcome the limitations of conventional PDT.

Rose bengal uptake by E. faecalis and F. nucleatum and light-mediated antibacterial activity measured by flow cytometry.

Antibacterial photodynamic therapy (aPDT) using rose bengal (RB) and blue-light kills bacteria through the production of reactive oxygen derivates. However, the interaction mechanism of RB with bacterial cells remains unclear. This study investigated the uptake efficiency and the antibacterial activity of blue light-activated RB against Enterococcus faecalis and Fusobacterium nucleatum. Spectrophotometry and epifluorescence microscopy were used to evaluate binding of RB to bacteria. The antibacterial activity of RB after various irradiation times was assessed by flow cytometry in combination with cell sorting. Uptake of RB increased in a concentration dependent manner in both strains although E. faecalis displayed higher uptake values. RB appeared to bind specific sites located at the cellular poles of E. faecalis and at regular intervals along F. nucleatum. Blue-light irradiation of samples incubated with RB significantly reduced bacterial viability. After incubation with 10µM RB and 240s irradiation, only 0.01% (±0.01%) of E. faecalis cells and 0.03% (±0.03%) of F. nucleatum survived after treatment. This study indicated that RB can bind to E. faecalis and F. nucleatum in a sufficient amount to elicit effective aPDT. Epifluorescence microscopy showed a yet-unreported property of RB binding to bacterial membranes. Flow cytometry allowed the detection of bacteria with damaged membranes that were unable to form colonies on agars after cell sorting.

Randomized, Multicenter, Double-Blind Study of the Safety and Efficacy of 1%D-3-Hydroxybutyrate eye drops for Dry Eye Disease.

In a previous study, we demonstrated that topical D-beta-hydroxybutyrate ameliorates corneal epithelial erosion and superficial punctate keratopathy in a rat model of dry eye disease. In the current investigation, we performed a prospective, randomized, multicentre, double-blind, placebo-controlled study to assess the safety and efficacy of 1% D-3-hydroxybutyrate eye drops in patients with dry eye disease. A total of 65 patients were randomly assigned to either the placebo group or the 1% D-3-hydroxybutyrate group, and the treatments were administered 6 times a day for 4 weeks. We then evaluated corneal fluorescein staining, corneal and conjunctival rose Bengal staining, tear film break-up time (BUT), Schirmer score, and subjective symptoms. At both 2 and 4 weeks, the corneal rose Bengal score was significantly better in the 1% D-3-hydroxybutyrate group than in the placebo group. Among patients with an initial Schirmer score of ≤5 mm, the corneal fluorescein staining score was significantly better in the 1% D-3-hydroxybutyrate group than in the placebo group at two weeks. Mild ocular symptoms occurred in both groups, and these spontaneously resolved. The present study suggested that 1% D-3-hydroxybutyrate eye drops are safe and effective in treating ocular surface disorders in patients with tear-deficient dry eye disease.