Pressure-enhanced sensing of tissue oxygenation via endogenous porphyrin: Implications for dynamic visualization of cancer in surgery.

Fluorescence guidance is routinely used in surgery to enhance perfusion contrast in multiple types of diseases. Pressure-enhanced sensing of tissue oxygenation (PRESTO) via fluorescence is a technique extensively analyzed here, that uses an FDA-approved human precursor molecule, 5-aminolevulinic acid (ALA), to stimulate a unique delayed fluorescence signal that is representative of tissue hypoxia. The ALA precontrast agent is metabolized in most tissues into a red fluorescent molecule, protoporphyrin IX (PpIX), which has both prompt fluorescence, indicative of the concentration, and a delayed fluorescence, that is amplified in low tissue oxygen situations. Applied pressure from palpation induces transient capillary stasis and a resulting transient PRESTO contrast, dominant when there is near hypoxia. This study examined the kinetics and behavior of this effect in both normal and tumor tissues, with a prolonged high PRESTO contrast (contrast to background of 7.3) across 5 tumor models, due to sluggish capillaries and inhibited vasodynamics. This tissue function imaging approach is a fundamentally unique tool for real-time palpation-induced tissue response in vivo, relevant for chronic hypoxia, such as vascular diseases or oncologic surgery.

Non-native Pathway Engineering with CRISPRi for Carbon Dioxide Assimilation and Valued 5-Aminolevulinic Acid Synthesis in Escherichia coli Nissle.

Carbon dioxide emission and acidification during chemical biosynthesis are critical challenges toward microbial cell factories' sustainability and efficiency. Due to its acidophilic traits among workhorse lineages, the probiotic Escherichia coli Nissle (EcN) has emerged as a promising chemical bioproducer. However, EcN lacks a CO2-fixing system. Herein, EcN was equipped with a simultaneous CO2 fixation system and subsequently utilized to produce low-emission 5-aminolevulinic acid (5-ALA). Two different artificial CO2-assimilating pathways were reconstructed: the novel ribose-1,5-bisphosphate (R15P) route and the conventional ribulose-5-phosphate (Ru5P) route. CRISPRi was employed to target the pfkAB and zwf genes in order to redirect the carbon flux. As expected, the CRISPRi design successfully strengthened the CO2 fixation. The CO2-fixing route via R15P resulted in high biomass, while the engineered Ru5P route acquired the highest 5-ALA and suppressed the CO2 release by 77%. CO2 fixation during 5-ALA production in EcN was successfully synchronized through fine-tuning the non-native pathways with CRISPRi.

Radiolabeling Heme and Porphyrin with 14C-Glycine or 14C δ-Aminolevulinic Acid.

Radiolabeling enables the quantitation of newly synthesized heme and porphyrin, allowing us to distinguish heme synthesis rates from total cellular heme. Here, we describe a protocol for labeling heme with 14C-glycine or ALA and the sequential extraction of heme and porphyrin from the same samples for quantitation by liquid scintillation.

Localization of protoporphyrin IX during glioma-resection surgery via paired stimulated Raman histology and fluorescence microscopy.

The most widely used fluorophore in glioma-resection surgery, 5-aminolevulinic acid (5-ALA), is thought to cause the selective accumulation of fluorescent protoporphyrin IX (PpIX) in tumour cells. Here we show that the clinical detection of PpIX can be improved via a microscope that performs paired stimulated Raman histology and two-photon excitation fluorescence microscopy (TPEF). We validated the technique in fresh tumour specimens from 115 patients with high-grade gliomas across four medical institutions. We found a weak negative correlation between tissue cellularity and the fluorescence intensity of PpIX across all imaged specimens. Semi-supervised clustering of the TPEF images revealed five distinct patterns of PpIX fluorescence, and spatial transcriptomic analyses of the imaged tissue showed that myeloid cells predominate in areas where PpIX accumulates in the intracellular space. Further analysis of external spatially resolved metabolomics, transcriptomics and RNA-sequencing datasets from glioblastoma specimens confirmed that myeloid cells preferentially accumulate and metabolize PpIX. Our findings question 5-ALA-induced fluorescence in glioma cells and show how 5-ALA and TPEF imaging can provide a window into the immune microenvironment of gliomas.

Ultra-Performance Liquid Chromatography (UPLC) Analysis of Heme Biosynthesis Intermediates.

The utilization of ultra-performance liquid chromatography (UPLC) to analyze the various intermediates in the heme biosynthetic pathway is presented. The first product, ALA, was derivatized to a highly fluorescent pyrrolizine; PBG, the second intermediate, was enzymatically converted to uroporphyrinogen, and all the porphyrinogen intermediates were oxidized in acid to form fluorescent porphyrins. Heme was measured as hemin. The stable porphyrin forms of the intermediates, are then resolved and quantified by UPLC. Further details about the various methods are discussed to promote successful UPLC analyses. Method variations that may be preferable in certain situations are also presented.

Production of 17α-hydroxyprogesterone using an engineered biocatalyst with efficient electron transfer and improved 5-aminolevulinic acid synthesis coupled with a P450 hydroxylase.

17α-Hydroxyprogesterone (17α-OH-PROG) is an important intermediate with a wide range of applications in the pharmaceutical industry. Strategies based on efficient electron transfer and cofactor regeneration were used for the production of 17α-OH-PROG. Here, CYP260A1, Fpr and Adx were expressed using a double plasmid system, resulting in higher biotransformation efficiency. Further optimization of reaction conditions and addition of polymyxin B increased the production of 17α-OH-PROG from 12.52 mg/L to 102.37 mg/L after 12 h of biotransformation. To avoid the addition of external 5-aminolevulinic acid (ALA) as a heme precursor for the P450 enzyme, a modified C5 pathway was introduced into the engineered strain, further reducing the overall process cost. The resulting whole-cell biocatalyst achieved the highest biotransformation yield of 17α-OH-PROG reported to date, offering a promising strategy for commercial application of P450 enzymes in industrial production of hydroxylated intermediates.

Modularized Engineering of Shewanella oneidensis MR-1 for Efficient and Directional Synthesis of 5-Aminolevulinic Acid.

Shewanella oneidensis MR-1 has found widespread applications in pollutant transformation and bioenergy production, closely tied to its outstanding heme synthesis capabilities. However, this significant biosynthetic potential is still unexploited so far. Here, we turned this bacterium into a highly-efficient bio-factory for green synthesis of 5-Aminolevulinic Acid (5-ALA), an important chemical for broad applications in agriculture, medicine, and the food industries. The native C5 pathway genes of S. oneidensis was employed, together with the introduction of foreign anti-oxidation module, to establish the 5-ALA production module, resulting 87-fold higher 5-ALA yield and drastically enhanced tolerance than the wild type. Furthermore, the metabolic flux was regulated by using CRISPR interference and base editing techniques to suppress the competitive pathways to further improve the 5-ALA titer. The engineered strain exhibited 123-fold higher 5-ALA production capability than the wild type. This study not only provides an appealing new route for 5-ALA biosynthesis, but also presents a multi-dimensional modularized engineering strategy to broaden the application scope of S. oneidensis.

Gene expression regulation by modulating Hfq expression in coordination with tailor-made sRNA-based knockdown in Escherichia coli.

The tailor-made synthetic sRNA-based gene expression knockdown system has demonstrated its efficacy in achieving pathway balancing in microbes, facilitating precise target gene repression and fine-tuned control of gene expression. This system operates under a competitive mode of gene regulation, wherein the tailor-made synthetic sRNA shares the intrinsic intracellular Hfq protein with other RNAs. The limited intracellular Hfq amount has the potential to become a constraining factor in the post-transcription regulation of sRNAs. To enhance the efficiency of the tailor-made sRNA gene expression regulation platform, we introduced an Hfq expression level modulation-coordinated sRNA-based gene knockdown system. This system comprises tailor-made sRNA expression cassettes that produce varying Hfq expression levels using different strength promoters. Modulating the expression levels of Hfq significantly improved the repressing capacity of sRNA, as evidenced by evaluations with four fluorescence proteins. In order to validate the practical application of this system, we applied the Hfq-modulated sRNA-based gene knockdown cassette to Escherichia coli strains producing 5-aminolevulinic acid and L-tyrosine. Diversifying the expression levels of metabolic enzymes through this cassette resulted in substantial increases of 74.6% in 5-aminolevulinic acid and 144% in L-tyrosine production. Tailor-made synthetic sRNA-based gene expression knockdown system, coupled with Hfq copy modulation, exhibits potential for optimizing metabolic fluxes through biosynthetic pathways, thereby enhancing the production yields of bioproducts.

Exogenous 5-Aminolevulinic acid improved low-temperature tolerance tomato seedling by regulating starch content and phenylalanine metabolism.

Tomato is an important horticultural cash crop, and low-temperature stress has seriously affected the yield and quality of tomato. 5-Aminolevulinic acid (ALA) is widely used in agriculture as an efficient and harmless growth regulator. It is currently unclear whether exogenous ALA can cope with low-temperature stress by regulating tomato starch content and phenylalanine metabolism. In this study, exogenous ALA remarkably improved the low-temperature tolerance of tomato seedlings. RNA-sequencing results showed that exogenous ALA affected starch metabolism and phenylalanine metabolism in tomato seedling leaves under low-temperature stress. Subsequently, we used histochemical staining, observation of chloroplast microstructure, substance content determination, and qRT-PCR analysis to demonstrate that exogenous ALA could improve the low-temperature tolerance of tomato seedlings by regulating starch content and phenylalanine metabolism (SlPAL, SlPOD1, and SlPOD2). Simultaneously, we found that exogenous ALA induced the expression of SlMYBs and SlWRKYs under low-temperature stress. In addition, dual luciferase, yeast one hybrid, and electrophoretic mobility shift assays indicate that SlMYB4 and SlMYB88 could regulate the expression of SlPOD2 in phenylalanine metabolism. We demonstrated that exogenous ALA could improve the low-temperature tolerance of tomato seedlings by regulating starch content and phenylalanine metabolism.

Construction of 5-Aminolevulinic Acid Microbial Cell Factories through Identification of Novel Synthases and Metabolic Pathway Screens and Transporters.

5-Aminolevulinic acid (5-ALA) plays a pivotal role in the biosynthesis of heme and chlorophyll and has garnered great attention for its agricultural applications. This study explores the multifaceted construction of 5-ALA microbial cell factories. Evolutionary analysis-guided screening identified a novel 5-ALA synthase from Sphingobium amiense as the best synthase. An sRNA library facilitated global gene screening that demonstrated that trpC and ilvA repression enhanced 5-ALA production by 74.3% and 102%, respectively. Subsequently, efflux of 5-ALA by the transporter Gdx increased 5-ALA biosynthesis by 25.7%. To mitigate oxidative toxicity, DNA-binding proteins from starved cells were employed, enhancing cell density and 5-ALA titer by 21.1 and 4.1%, respectively. Combining these strategies resulted in an Escherichia coli strain that produced 5-ALA to 1.51 g·L-1 in shake flask experiments and 6.19 g·L-1 through fed-batch fermentation. This study broadens the repertoire of available 5-ALA synthases and transporters and provides a new platform for optimizing 5-ALA bioproduction.

5-Aminolevulinic acid treatment mitigates pesticide stress in bean seedlings by regulating stress-related gene expression and retrotransposon movements.

Overdoses of pesticides lead to a decrease in the yield and quality of plants, such as beans. The unconscious use of deltamethrin, one of the synthetic insecticides, increases the amount of reactive oxygen species (ROS) by causing oxidative stress in plants. In this case, plants tolerate stress by activating the antioxidant defense mechanism and many genes. 5-Aminolevulinic acid (ALA) improves tolerance to stress by acting exogenously in low doses. There are many gene families that are effective in the regulation of this mechanism. In addition, one of the response mechanisms at the molecular level against environmental stressors in plants is retrotransposon movement. In this study, the expression levels of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), and stress-associated protein (SAP) genes were determined by Q-PCR in deltamethrin (0.5 ppm) and various doses (20, 40, and 80 mg/l) of ALA-treated bean seedlings. In addition, one of the response mechanisms at the molecular level against environmental stressors in plants is retrotransposon movement. It was determined that deltamethrin increased the expression of SOD (1.8-fold), GPX (1.4-fold), CAT (2.7-fold), and SAP (2.5-fold) genes, while 20 and 40 mg/l ALA gradually increased the expression of these genes at levels close to control, but 80 mg/l ALA increased the expression of these genes almost to the same level as deltamethrin (2.1-fold, 1.4-fold, 2.6-fold, and 2.6-fold in SOD, GPX, CAT, and SAP genes, respectively). In addition, retrotransposon-microsatellite amplified polymorphism (REMAP) was performed to determine the polymorphism caused by retrotransposon movements. While deltamethrin treatment has caused a decrease in genomic template stability (GTS) (27%), ALA treatments have prevented this decline. At doses of 20, 40, and 80 mg/L of ALA treatments, the GTS ratios were determined to be 96.8%, 74.6%, and 58.7%, respectively. Collectively, these findings demonstrated that ALA has the utility of alleviating pesticide stress effects on beans.

Single-cell analysis of 5-aminolevulinic acid intraoperative labeling specificity for glioblastoma.

OBJECTIVE: Glioblastoma (GBM) is the most common and aggressive malignant primary brain tumor, and resection is a key part of the standard of care. In fluorescence-guided surgery (FGS), fluorophores differentiate tumor tissue from surrounding normal brain. The heme synthesis pathway converts 5-aminolevulinic acid (5-ALA), a fluorogenic substrate used for FGS, to fluorescent protoporphyrin IX (PpIX). The resulting fluorescence is believed to be specific to neoplastic glioma cells, but this specificity has not been examined at a single-cell level. The objective of this study was to determine the specificity with which 5-ALA labels the diversity of cell types in GBM. METHODS: The authors performed single-cell optical phenotyping and expression sequencing-version 2 (SCOPE-seq2), a paired single-cell imaging and RNA sequencing method, of individual cells on human GBM surgical specimens with macroscopically visible PpIX fluorescence from patients who received 5-ALA prior to surgery. SCOPE-seq2 allowed the authors to simultaneously image PpIX fluorescence and unambiguously identify neoplastic cells from single-cell RNA sequencing. Experiments were also conducted in cell culture and co-culture models of glioma and in acute slice cultures from a mouse glioma model to investigate cell- and tissue-specific uptake and secretion of 5-ALA and PpIX. RESULTS: SCOPE-seq2 analysis of human GBM surgical specimens revealed that 5-ALA treatment resulted in labeling that was not specific to neoplastic glioma cells. The cell culture further demonstrated that nonneoplastic cells could be labeled by 5-ALA directly or by PpIX secreted from surrounding neoplastic cells. Acute slice cultures from mouse glioma models showed that 5-ALA preferentially labeled GBM tumor tissue over nonneoplastic brain tissue with significant labeling in the tumor margins, and that this contrast was not due to blood-brain barrier disruption. CONCLUSIONS: Together, these findings support the use of 5-ALA as an indicator of GBM tissue but question the main advantage of 5-ALA for specific intracellular labeling of neoplastic glioma cells in FGS. Further studies are needed to systematically compare the performance of 5-ALA to that of potential alternatives for FGS.

Construction of the Rhodobacter sphaeroides strain overproducing 5-aminolevulinic acid by insertion of endogenous promoter.

5-Aminolevulinic acid (ALA) is a precursor of heme and a natural amino acid synthesized in the cells of most living organisms. Currently, ALA is used as an ingredient in pharmaceuticals, supplements, cosmetics, feed, fertilizers, and other products. ALA is mainly produced by industrial fermentation by the photosynthetic bacterium Rhodobacter sphaeroides. In this study, we tried to improve the ALA productivity by R. sphaeroides using a genetic strategy to highly express ALA synthase (ALAS) genes. We inserted a constitutive promoter (PrrnB or Prsp_7571) upstream of genes encoding ALAS (hemA and/or hemT) to construct strains that constitutively express ALAS. The highest transcript levels of hemA were observed in the strain where PrrnB was inserted into the hemA promoter region and were 3.5-fold higher than those in the wild-type. The highest transcript levels of hemT were observed in the strain where PrrnB was inserted into the hemT promoter region and were 46-fold higher than those in the wild-type. The maximum ALAS activity was observed in crude cell extracts of the strain where PrrnB was inserted into the hemT promoter region under optimized growth conditions that was 2.7-fold higher than that in the wild type. This strain showed 12-fold accumulation of ALA compared to the wild-type. Thus, we improved ALA productivity without using exogenous DNA sequences. In the future, further improvement in ALA productivity may be expected by applying this approach to current industrial ALA-producing bacteria.

A novel tetratricopeptide-repeat protein, TTP1, forms complexes with glutamyl-tRNA reductase and protochlorophyllide oxidoreductase during tetrapyrrole biosynthesis.

The biosynthesis of the tetrapyrrole end-products chlorophyll and heme depends on a multifaceted control mechanism that acts primarily at the post-translational level upon the rate-limiting step of 5-aminolevulinic acid synthesis and upon light-dependent protochlorophyllide oxidoreductase (POR). These regulatory processes require auxiliary factors that modulate the activity, stability, complex formation, and subplastidal localization of the relevant proteins. Together, they ensure optimal metabolic flow during the day and at night. As an Arabidopsis homolog of the POR-interacting tetratricopeptide-repeat protein (Pitt) first reported in Synechocystis, we characterize tetrapyrrole biosynthesis-regulating tetratricopeptide-repeat protein1 (TTP1). TTP1 is a plastid-localized, membrane-bound factor that interacts with POR, the Mg protoporphyrin monomethylester cyclase CHL27, glutamyl-tRNA reductase (GluTR), GluTR-binding protein, and FLUORESCENCE IN BLUE LIGHT. Lack of TTP1 leads to accumulation of GluTR, enhanced 5-aminolevulinic acid synthesis and lower levels of POR. Knockout mutants show enhanced sensitivity to reactive oxygen species and a slower greening of etiolated seedlings. Based on our studies, the interaction of TTP1 with GluTR and POR does not directly inhibit their enzymatic activity and contribute to the control of 5-aminolevulinic acid synthesis. Instead, we propose that TTP1 sequesters a fraction of these proteins on the thylakoid membrane, and contributes to their stability.

Transcription factor MdNAC33 is involved in ALA-induced anthocyanin accumulation in apples.

5-Aminolevulinic acid (ALA), as a new natural plant growth regulator, has a significant function in promoting anthocyanin accumulation in many species of fruits. However, the mechanisms underlying remain obscure. In a transcriptome study of our group, it was found that many transcription factors (TFs) including NACs responsive to ALA treatment during anthocyanin accumulation. In the present study, we found a NAC of apple, MdNAC33 was coordinatively expressed with anthocyanin accumulation after ALA treatment in the apple fruits and leaves, suggesting that this TF may be involved in anthocyanin accumulation induced by ALA. We found that the MdNAC33 protein was localized in the nucleus and exhibited strong transcriptional activity in both yeast cells and plants, where its C-terminal contributed to the transcriptional activity. Functional analysis showed that overexpression of MdNAC33 promoted the accumulation of anthocyanin, while the silencing vector of MdNAC33 (RNAi) significantly impaired the anthocyanin accumulation induced by ALA. Yeast one-hybrid (Y1H), luciferase assay and electrophoretic mobility shift assay (EMSA) indicated that MdNAC33 could bind to promoters of MdbHLH3, MdDFR and MdANS to activate the gene expressions. In addition, MdNAC33 specifically interacts with MdMYB1, a positive regulator of anthocyanin biosynthesis, which was then in turn binding to its target genes MdUFGT and MdGSTF12, to promote anthocyanin accumulation in apples. Taken together, our data indicate that MdNAC33 plays multiple roles in ALA-induced anthocyanin biosynthesis. It provides new insights into the mechanisms of anthocyanin accumulation induced by ALA.

ALA-PDT promotes the death and contractile capacity of hypertrophic scar fibroblasts through inhibiting the TGF-ß1/Smad2/3/4 signaling pathway.

BACKGROUND: Hypertrophic scars, an abnormal wound-healing response to burn injuries, are characterized by massive fibroblast proliferation and excessive deposition of extracellular matrix and collagen. 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT) is a promising therapy for hypertrophic scar, details of the mechanisms remain to be elucidated. In this study, we aimed to investigate the molecular mechanisms involved in ALA-PDT against hypertrophic scar fibroblasts. METHODS: The morphologies of hypertrophic scar fibroblasts (HSFs) treated with ALA-PDT were observed under a light microscopy. The viability of HSFs was detected using the CCK-8 assay. HSFs-populated collagen gel contraction assays were conducted to examine the fibroblast contractility and the cytotoxicity of HSFs in 3D collagen tissues were observed using confocal microscopy. The effect of ALA-PDT on TGF-ß1/Smad2/3/4 signaling pathway activation and effector gene expression were verified by immunoprecipitation, western blot and real-time quantitative PCR analysis. RESULTS: We observed significant changes in cell morphology after ALA-PDT treatment of HSFs. As ALA concentration and light dose increased, the viability of HSFs significantly decreased. ALA-PDT can significantly alleviate the contractile capacity and promote the death of HSFs induced by TGF-ß1 treatment in a three-dimensional collagen culture model. TGF-ß1 treatment of HSFs can significantly induce phosphorylation of Smad2/3 (p-Smad2/3) in whole cells, as well as p-Smad2/3 and Smad4 proteins into the nucleus and increase the mRNA levels of collagen 1/3 and α-SMA. ALA-PDT hampers the TGF-ß1-Smad2/3/4 signaling pathway activation by inducing K48-linked ubiquitination and degradation of Smad4. CONCLUSIONS: Our results provide evidence that ALA-PDT can inhibit fibroblast contraction and promote cell death by inhibiting the activation of the TGF-ß1 signaling pathway that mediates hypertrophic scar formation, which may be the basis for the efficacy of ALA-PDT in the treatment of hypertrophic scars.

Modified 5-aminolevulinic acid photodynamic therapy induces cutaneous squamous cell carcinoma cell pyroptosis via the JNK signaling pathway.

Modified 5-aminolevulinic acid photodynamic therapy (M-PDT) is a novel therapeutic modality for cutaneous squamous cell carcinoma (cSCC) that is reported to be effective and well tolerated. However, the mechanisms underlying its antitumor effects are not fully understood. In this research, we investigated the effects of M-PDT on pyroptosis, a form of programmed cell death characterized by cell swelling, ruptures of cell membrane, and inflammatory cytokine release, in two human cSCC cell lines, SCL-1 and HSC-5. We found that M-PDT triggered pyroptosis in a dose-dependent manner, as evidenced by increased lactate dehydrogenase release, propidium iodide staining, and expression of pyroptosis-related proteins, such as NLR family pyrin domain containing 3 (NLRP3), N-terminal of gasdermin D (N-GSDMD), cleaved caspase-1, and mature interleukin 1 beta (IL-1B) in both cell lines. This process was inhibited by treatment with MCC950, an NLRP3-specific inhibitor, suggesting the involvement of the NLRP3 inflammasome in M-PDT-induced pyroptosis. We also demonstrated that M-PDT activated c-Jun N-terminal kinase (JNK) signaling, which is required for pyroptosis induction, as treatment with SP600125, a JNK inhibitor, suppressed the expression of pyroptosis-related proteins after M-PDT. JNK activation enhanced M-PDT-induced pyroptosis, highlighting the significance of the JNK pathway in M-PDT. Moreover, M-PDT increased intracellular reactive oxygen species (ROS) levels, which are responsible for JNK activation and pyroptosis induction. In summary, our results revealed that M-PDT triggers pyroptosis through ROS-mediated JNK activation and subsequent NLRP3 inflammasome activation in cSCC cells, providing a better understanding of the molecular mechanism of M-PDT and promoting its clinical application.

Exogenously applied 5-aminolevulinic acid modulates growth, yield, and physiological parameters in lentil (Lens culinaris Medik.) under rain-fed and supplemental irrigation conditions.

Lentils are a significant source of plant protein and are cultivated across Asia, Europe, and North Africa. Plants are subjected to various environmental stresses, which can hinder growth, yield, and productivity. 5-aminolevulinic acid (ALA) is a compound that acts as a precursor in the biosynthesis of tetrapyrroles and can increase plant tolerance to different abiotic stressors. However, the effects of exogenously applied ALA on lentil growth, yield, and physiological parameters under rain-fed and supplemental irrigation conditions are not well-known. In this study, a split plot experiment was conducted to investigate the impact of ALA foliar application and supplemental irrigation on lentil (Lens culinaris Medik.). The experiment was designed based on a randomized complete block with three replications. The main plot included four levels of supplemental irrigation [(supplementary irrigation in the flowering and early seed-filling stages, supplementary irrigation in the flowering stage, supplementary irrigation in the early seed-filling along with rain-fed conditions (no irrigation)]. The subplot considered foliar application of ALA at varying levels [(0 (control), 50 and 100 ppm)]. The results showed that water regimes and foliar spray with ALA significantly (P Ë‚ 0.01) affected plant height, number of pods per plant, pod weight, number of seeds per pod and weight of 1000 seeds, biological yield, seed yield, and harvest index. The highest total chlorophyll content was observed in plants that were subjected to supplementary irrigation in flowering and early seed filling stages and foliar sprayed with 100 ppm ALA. The study also found that exogenous ALA improved drought tolerance in lentil plants under rain-fed conditions mainly by regulating antioxidant enzymes, which ultimately protected the cellular membranes against overproduction of H2O2. Furthermore, ALA application increased total carbohydrate contents at all supplemental irrigation levels, but the rate was higher in complementary irrigation conditions during flowering and early seed-filling stages. Malondialdehyde (MDA), H2O2, and proline contents were increased in field-grown plants under rain-fed conditions without exogenous ALA application. In conclusion, this study sheds light on the effects of ALA foliar spray and supplemental irrigation on lentil growth, yield, and physiological parameters. The findings suggest that exogenous ALA can improve plant tolerance to various abiotic stressors and enhance plant growth, yield, and physiological parameters.

Oral 5-aminolevulinic acid combined with sodium ferrous citrate prevents blood-aqueous barrier breakdown after anterior chamber paracentesis in healthy beagle dogs.

This study investigated the preventive effect of 5-aminolevulinic acid combined with sodium ferrous citrate (5-ALA/SFC) on blood-aqueous barrier (BAB) breakdown induced after anterior chamber paracentesis (ACP) in beagles. 5-ALA/SFC (1/0.64 mg/kg or 3/1.92 mg/kg) or carprofen (4.0 mg/kg) was orally administered daily for 7 days prior to ACP. Then, a sample of the aqueous humor (AH) was collected from one eye via ACP (first sample) and again 60 min later (second sample). The protein and prostaglandin E2 (PGE2) concentrations in both samples were measured. Compared with the control group, high-dose 5-ALA/SFC and carprofen significantly reduced the AH protein and PGE2 concentrations in the second sample. Our findings suggest that 5-ALA/SFC suppresses BAB breakdown in dogs.

Regulating Photosensitizer Metabolism with DNAzyme-Loaded Nanoparticles for Amplified Mitochondria-Targeting Photodynamic Immunotherapy.

Mitochondria-specific photosensitizer accumulation is highly recommended for photodynamic therapy and mitochondrial DNA (mtDNA) oxidative damage-based innate immunotherapy but remains challenging. 5-Aminolevulinic acid (ALA), precursor of photosensitizer protoporphyrin IX (PpIX), can induce the exclusive biosynthesis of PpIX in mitochondria. Nevertheless, its photodynamic effect is limited by the intracellular biotransformation of ALA in tumors. Here, we report a photosensitizer metabolism-regulating strategy using ALA/DNAzyme-co-loaded nanoparticles (ALA&Dz@ZIF-PEG) for mitochondria-targeting photodynamic immunotherapy. The zeolitic imidazolate framework (ZIF-8) nanoparticles can be disassembled and release large amounts of zinc ions (Zn2+) within tumor cells. Notably, Zn2+ can relieve tumor hypoxia for promoting the conversion of ALA to PpIX. Moreover, Zn2+ acts as a cofactor of rationally designed DNAzyme for silencing excessive ferrochelatase (FECH; which catalyzes PpIX into photoinactive Heme), cooperatively promoting the exclusive accumulation of PpIX in mitochondria via the "open source and reduced expenditure" manner. Subsequently, the photodynamic effects derived from PpIX lead to the damage and release of mtDNA and activate the innate immune response. In addition, the released Zn2+ further enhances the mtDNA/cGAS-STING pathway mediated innate immunity. The ALA&Dz@ZIF-PEG system induced 3 times more PpIX accumulation than ALA-loaded liposome, significantly enhancing tumor regression in xenograft tumor models.