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Angiosperms germinated in the dark develop etioplasts, the chloroplast precursors, in cotyledon cells. Etioplasts contain lattice membrane structures called prolamellar bodies (PLBs) and lamellar prothylakoids as internal membrane systems. PLBs accumulate the chlorophyll intermediate protochlorophyllide (Pchlide) in a complex with NADPH and light-dependent NADPH:Pchlide oxidoreductase (LPOR). Two galactolipids, monogalactosyldiacylglycerol and digalactosyldiacylglycerol (DGDG), are major constituents of etioplast membranes. We previously reported that monogalactosyldiacylglycerol facilitates the synthesis of Pchlide and the formation of the Pchlide-LPOR-NADPH complex in etioplasts, but the importance of DGDG in etioplasts is still unknown. To determine the role of DGDG in etioplast development and functions, we characterized a knockout mutant (dgd1) of Arabidopsis (Arabidopsis thaliana) DGD1, which encodes the major isoform of DGDG synthase, in the etioplast development stage. In etiolated dgd1 seedlings, DGDG content decreased to 20% of the wild-type level, the lattice structure of PLBs was disordered, and the development of prothylakoids was impaired. In addition, membrane-associated processes of Pchlide biosynthesis, formation of the Pchlide-LPOR-NADPH complex, and dissociation of the complex after the photoconversion of Pchlide to chlorophyllide were impaired in dgd1, although the photoconversion reaction by LPOR was not affected by the DGDG deficiency. Total carotenoid content also decreased in etiolated dgd1 seedlings, but the carotenoid composition was unchanged. Our data demonstrate a deep involvement of DGDG in the formation of the internal membrane structures in etioplasts as well as in membrane-associated processes of pigment biosynthesis and pigment-protein complex organization.

Antimicrobial resistance in Cutibacterium acnes is a growing concern, limiting treatment options for acne vulgaris (AV) and increasing the risk of opportunistic infections. Photodynamic therapy (PDT), using an effective photosensitizer (PS) and optimized light parameters, is a potent broad-spectrum antimicrobial strategy with minimal risk of resistance development. This study aimed to assess the efficacy of PDT using sodium iron chlorophyllin (CHL-Fe) as an in-house photosensitizer (CH-PDT) against 10 clinically isolated, drug-resistant C. acnes strains. The isolates, obtained from patients with mild to severe AV in a dermatology referral center, were resistant to one or more commonly prescribed antibiotics. CH-PDT was conducted using varying concentrations of CHL-Fe and red light (RL) fluences in laboratory conditions. A consistent bactericidal activity (> 3 log CFU/mL reduction) was achieved across all resistance profiles with a fixed dose of CHL-Fe at 7.5 mg/dL combined with RL at 3.5 J/cm². Neither CHL-Fe nor RL alone, even at higher doses, produced similar results. The study included pan-, extensively, multi-, and single-drug-resistant strains, making the findings broadly applicable to clinical practice. These findings underscore CH-PDT as a promising nonantibiotic therapy for drug-resistant AV, with potential implications for eradicating C. acnes colonization and preventing infections in surgical and implant-related settings.

3,8-Divinyl (proto)chlorophyll(ide) a 8-vinyl reductase (DVR) catalyzes the reduction of 8-vinyl group on the tetrapyrrole to an ethyl group, which is indispensable for monovinyl chlorophyll (Chl) synthesis. So far, three 8-vinyl reductase genes (DVR, bciA, and slr1923) have been characterized from Arabidopsis (Arabidopsis thaliana), Chlorobium tepidum, and Synechocystis sp. PCC6803. However, no 8-vinyl reductase gene has yet been identified in monocotyledonous plants. In this study, we isolated a spontaneous mutant, 824ys, in rice (Oryza sativa). The mutant exhibited a yellow-green leaf phenotype, reduced Chl level, arrested chloroplast development, and retarded growth rate. The phenotype of the 824ys mutant was caused by a recessive mutation in a nuclear gene on the short arm of rice chromosome 3. Map-based cloning of this mutant resulted in the identification of a gene (Os03g22780) showing sequence similarity with the Arabidopsis DVR gene (AT5G18660). In the 824ys mutant, nine nucleotides were deleted at residues 952 to 960 in the open reading frame, resulting in a deletion of three amino acid residues in the encoded product. High-performance liquid chromatography analysis of Chls indicated the mutant accumulates only divinyl Chl a and b. A recombinant protein encoded by Os03g22780 was expressed in Escherichia coli and found to catalyze the conversion of divinyl chlorophyll(ide) a to monovinyl chlorophyll(ide) a. Therefore, it has been confirmed that Os03g22780, renamed as OsDVR, encodes a functional DVR in rice. Based upon these results, we succeeded to identify an 8-vinyl reductase gene in monocotyledonous plants and, more importantly, confirmed the DVR activity to convert divinyl Chl a to monovinyl Chl a.

To improve the tumor-targeting efficacy of photodynamic therapy, biotin was conjugated with chlorin e6 to develop a new tumor-targeting photosensitizer, Ce6-biotin. The Ce6-biotin had good water solubility and low aggregation. The singlet-oxygen generation rate of Ce6-biotin was slightly increased compared to Ce6. Flow cytometry and confocal laser scanning microscopy results confirmed Ce6-biotin had higher binding affinity toward biotin-receptor-positive HeLa human cervical carcinoma cells than its precursor, Ce6. Due to the BR-targeting ability of Ce6-biotin, it exhibited stronger cytotoxicity to HeLa cells upon laser irradiation. The IC50 against HeLa cells of Ce6-biotin and Ce6 were 1.28 µM and 2.31 µM, respectively. Furthermore, both Ce6-biotin and Ce6 showed minimal dark toxicity. The selectively enhanced therapeutic efficacy and low dark toxicity suggest that Ce6-biotin is a promising PS for BR-positive-tumor-targeting photodynamic therapy.

Pancreatic cancer (PC) has high lethality due to multiple reasons, and its limited response to conventional chemotherapy like gemcitabine (GEM) is a non-negligible one. Therefore, our study introduces Chlorophyllin (CHL) as an effective therapeutic candidate to enhance the therapeutic efficacy of GEM. Our results demonstrate that the combination of CHL and GEM exhibits a significant synergistic anti-tumor effect by targeting multiple oncogenic processes in PC, including inhibiting cell proliferation, invasion, and migration, as well as inducing cell apoptosis. Further investigations of mechanism have revealed that CHL induces cuproptosis in PC cells through a multifaceted process, involving depleting cellular intracellular glutathione (GSH), increasing reactive oxygen species (ROS) levels, and subsequently upregulating the HSP70 protein in response to heightened oxidative stress. Additionally, CHL releases free Cu 2+ , binds to the Ferredoxin 1 (FDX1) protein, and ultimately leads to the oligomerization of Dihydrolipoamide S-Acetyltransferase (DLAT) proteins to amplify the copper toxicity within PC cells. Moreover, in vivo experiments have demonstrated that the combination of CHL and GEM effectively inhibits the growth of subcutaneously transplanted tumors while maintaining a favorable biosafety profile. In conclusion, our study identifies CHL as a potent enhancer of GEM’s anti-tumor effects in PC through the induction of cuproptosis, thus providing a novel therapeutic avenue for patients with PC.

Photodynamic therapy (PDT) can eradicate not only cancer cells but also stimulate an antitumor immune response. Herein, we describe two efficient synthetic methodologies for the preparation of Chlorin e6 (Ce6) from Spirulina platensis and address the phototoxic effect of Ce6 in vitro along with antitumor activity in vivo. Melanoma B16F10 cells were seeded and phototoxicity was monitored by the MTT assay. The C57BL/6 mice were subcutaneously inoculated on the left and right flank with B16F10 cells. The mice were intravenously injected with Ce6 of 2.5 mg/kg and then exposed to red light (660 nm) on the left flank tumors 3 h after the injection. The immune response was studied by analyzing Interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and Interleukin-2 (IL-2) of the right flank tumors through qPCR. Our results revealed that the tumor was suppressed not only in the left flank but also in the right flank, where no PDT was given. The upregulated gene and protein expression of IFN-γ, TNF-α, and IL-2 revealed antitumor immunity due to Ce6-PDT. The findings of this study suggest an efficient methodology of Ce6 preparation and the efficacy of Ce6-PDT as a promising antitumor immune response.

Generally, bacteriochlorophyllides were responsible for the photosynthesis in bacteria. Seven types of bacteriochlorophyllides have been disclosed. Bacteriochlorophyllides a/b/g could be synthesized from divinyl chlorophyllide a. The other bacteriochlorophyllides c/d/e/f could be synthesized from chlorophyllide a. The chemical structure and synthetic route of bacteriochlorophyllides were summarized in this review. Furthermore, the potential applications of bacteriochlorophyllides in photosensitizers, immunosensors, influence on bacteriochlorophyll aggregation, dye-sensitized solar cell, heme synthesis and for light energy harvesting simulation were discussed.

Chlorophyllase (CLH) is a common plant enzyme that catalyzes the hydrolysis of chlorophyll to form chlorophyllide, a more hydrophilic derivative. For more than a century, the biological role of CLH has been controversial, although this enzyme has been often considered to catalyze chlorophyll catabolism during stress-induced chlorophyll breakdown. In this study, we found that the absence of CLH does not affect chlorophyll breakdown in intact leaf tissue in the absence or the presence of methyl-jasmonate, which is known to enhance stress-induced chlorophyll breakdown. Fractionation of cellular membranes shows that Arabidopsis (Arabidopsis thaliana) CLH is located in the endoplasmic reticulum and the tonoplast of intact plant cells. These results indicate that CLH is not involved in endogenous chlorophyll catabolism. Instead, we found that CLH promotes chlorophyllide formation upon disruption of leaf cells, or when it is artificially mistargeted to the chloroplast. These results indicate that CLH is responsible for chlorophyllide formation after the collapse of cells, which led us to hypothesize that chlorophyllide formation might be a process of defense against chewing herbivores. We found that Arabidopsis leaves with genetically enhanced CLH activity exhibit toxicity when fed toSpodoptera lituralarvae, an insect herbivore. In addition, purified chlorophyllide partially suppresses the growth of the larvae. Taken together, these results support the presence of a unique binary defense system against insect herbivores involving chlorophyll and CLH. Potential mechanisms of chlorophyllide action for defense are discussed.

Chlorophyllin (CHL) effectively decreases the side effects of radiotherapy (RT) by scavenging radiation-induced free radicals and reactive oxygen species in preclinical trials. This study aims to assess the efficacy of oral CHL for the treatment of brain radionecrosis in patients with diffuse glioma. This is a phase 2 trial prospective, interventional study. Adults (> 18 years) with a histological diagnosis of diffuse glioma developing radionecrosis will be eligible for the study. Radionecrosis will be identified using standard imaging protocols with magnetic resonance imaging (MRI) with or without positron emission tomography (PET). Patients will be accrued in two strata: symptomatic (stratum A) and asymptomatic (stratum B). Chlorophyllin will be prescribed to all patients using a morning oral dose of 750 mg before breakfast for 3 months. In addition, participants in stratum A will be given a tapering dose of dexamethasone for 1 month, while stratum B will not be receiving any steroids. Imaging with an MRI brain protocol and PET scan will be planned at 1 month and MRI at 3 months after starting CHL. The primary endpoint is the clinical-radiological response at 1 month. Secondary endpoints include response at 3 months, biological responses, survival analysis, and quality-of-life scores. The total sample size is 118 (60 and 58 in stratum A and B, respectively), with one interim analysis planned. Radionecrosis leads to significant morbidity and is usually treated with corticosteroids, which can lead to several side effects from both acute and long-term use. Refractory radionecrosis requires treatment with bevacizumab or surgical resection. Chlorophyllin is a cheap, safe, and readily available phytopharmaceutical drug, which is being investigated in the phase 2 study and, if proven effective, can be considered an alternative for treating radionecrosis. Clinical Trial Registry India (CTRI): CTRI/2023/08/056166; ClinicalTrials.gov: NCT06016452.

Photodynamic therapy (PDT) damages cancer cells via photosensitization using harmless laser irradiation. We synthesized a new photosensitizer, mannose-conjugated-chlorin e6 (M-chlorin e6), which targets mannose receptors that are highly expressed on M2-like tumor-associated macrophages (M2-TAMs) and cancer cells. In our previous study, we demonstrated that M-chlorin e6 PDT reduces tumor volume and decreases the proportion of M2-TAMs. Whether M-chlorin e6 PDT-treated cancer cells activate tumor immunity remains unclear, although the decrease in M2-TAMs is thought to be a direct injurious effect of M-chlorin e6 PDT. Calreticulin (CRT) is exposed at the surface of the membrane of cancer cells in response to treatment with chemotherapeutic agents such as anthracycline and oxaliplatin. Surface-exposed CRT induces phagocytosis of CRT receptor-positive cells, including macrophages, inducing anticancer immune responses. In the present study, we found that M-chlorin e6 PDT increases CRT on the surface of cancer cells, leading to macrophage phagocytosis of cancer cells. Furthermore, M-chlorin e6 PDT increases CD80 CD86 macrophages. These results suggest that M-chlorin e6 PDT exerts anti-tumor effects by both enhancing the phagocytosis of cancer cells and strengthening the anti-tumor phenotype of macrophages.