Streptomyces sp. strain MBCN152-1, isolated from cabbage, has potential as a biocontrol agent for Alternaria brassicicola on cabbage seedlings. The present study examined its mode of action. Light microscopy showed that appressorium formation by A. brassicicola was significantly suppressed on cabbage seedlings bacterized with MBCN152-1. Furthermore, scanning electron microscopy revealed that the mycelia of MBCN152-1, which were epiphytically growing on the cotyledon leaves of cabbage seedlings, intensively coiled around the germinating conidia of A. brassicicola. In vitro co-culture experiments demonstrated that MBCN152-1 is an aggressive mycoparasite of A. brassicicola, but not of A. brassicae or Colletotrichum higginsianum. Biocontrol experiments indicated that MBCN152-1 did not control diseases caused by A. brassicae or C. higginsianum. These results suggest that mycoparasitism is the primary mode of action for MBCN152-1. This is the first study to clearly demonstrate the significance of mycoparasitism in the biocontrol efficacy of endophytic Streptomyces. Alternaria species infect a variety of plants, including vegetables, cereals, and fruit trees, and affect plant productivity, resulting in reduced quality and yields  , and Colletotrichum higginsianum MAFF305635 were used as the pathogens. A. brassicicola and A. brassicae were cultured at 25°C for 1 week on potato dextrose agar and half-strength V8 juice agar containing 0.004% rose bengal , respectively. C. higginsianum was cultured on potato sucrose agar at 25°C for 1 week. The conidia of each pathogen were harvested in sterile distilled water (SDW) and filtered through two layers of sterile Kimwipes to remove mycelia. The conidial suspension of each pathogen was adjusted to the required concentrations using a hemocytometer and was then used for inoculation.In the present study, A. brassicicola conidial suspension (1×105 conidia mL–1) was spotted on the surface of each cotyledon leaf and was then incubated in the same environmental control chamber. As a control, non-bacterized seedlings were similarly inoculated with A. brassicicola. Conidial germination (%), germ tube length (μm), and appressorium formation (%) by the pathogen were assessed up to 48 h post-inoculation (hpi) by microscopic observations using aniline blue staining according to previously described procedures  glutaraldehyde (buffered to pH 7.0 with 0.1 M phosphate buffer) for 3 h. After being washed with 0.1 M phosphate buffer (pH 7.0) for 1 h, samples were fixed in 5% (v/v) osmium tetroxide (buffered to pH 7.3 with 0.1 M phosphate buffer) for 3 h, washed with 0.1 M phosphate buffer (pH 7.3) for 1 h, and dehydrated in an ethanol series . Dehydrated samples were immersed in various ratios of t-butyl alcohol to ethanol  for 30 min each and then lyophilized using a t-butyl alcohol freeze-drying device . These specimens were attached to specimen stubs and observed with SEM  after gold coating.Cabbage seeds (cv. Matsunami) were bacterized with MBCN152-1 and then inoculated with in vitro interactions between MBCN152-1 and three fungal Brassica pathogens, namely, A. brassicicola, A. brassicae, and C. higginsianum, equal volumes of an MBCN152-1 spore suspension (5×104 spores mL–1) and a conidial suspension (5×103 conidia mL–1) of A. brassicicola, A. brassicae, or C. higginsianum were thoroughly mixed. A 50-μL aliquot of each mixed suspension was dropped onto the surface of a 1% (w/v) water agar plate and cultured in the dark at 27°C. Fungal pathogens cultured alone served as controls. At 3 and 8 days post-inoculation (dpi), agar blocks were excised from the inoculated points and mounted on glass slides. Fungal hyphae on the agar blocks were stained with calcofluor white MR2  according to the manufacturer’s instructions and then observed under a fluorescence microscope.To investigate A. brassicicola cells, fungal germ tubes and hyphae on agar blocks excised from the agar plates at 3 dpi were stained with 0.05% Evans blue by placing 100 μL of the solution on agar blocks, incubating for 5 min, and then gently washing the blocks with SDW. Stained fungi on the agar blocks were observed under a light microscope.To examine the viability of 5 spores mL–1) and the conidial suspension (5×104 conidia mL–1) of either A. brassicicola, A. brassicae, or C. higginsianum were dropped onto the surface of Pridham and Gottlieb’s basal mineral salts agar medium  Congo red solution for 15 min to assess chitinase activity. After the Congo red solution was discarded, 1 M NaCl solution was added to the plates and discarded 15 min later. Chitinase activity was indicated by a clear zone (Fifty microliters of a mixture of the MBCN 152-1 spore suspension (5×10r medium  suppleme7 spores (g soil mix)–1. Surface-sterilized seeds of cv. Matsunami were sown into 128-cell plug trays filled with a MBCN152-1–blended soil mix and grown in a glasshouse (23–29°C). Seven days after sowing, cabbage seedlings were spray-inoculated with a conidial suspension of A. brassicae (5×104 conidia mL–1) or C. higginsianum (5×105 conidia mL–1) until run-off, and were then incubated for 24 h in a humid chamber  maintained at >90% relative humidity and 27°C for 24 h and then for 7 days in the aforementioned glasshouse. Seedlings grown in the untreated soil mix were also inoculated with the pathogens as controls. At the end of the experiment, the disease severity of the cabbage seedlings was evaluated according to the following scale: 0=no symptoms, 1=slightly infected , 2=severely infected (large necrotic lesions formed on cotyledon leaves), and 3=dead seedlings. Each treatment comprised two replicates and each replicate used 128 plug seedlings, thereby making 256 seedlings per treatment, and the experiment was repeated three times.A spore suspension of MBCN152-1 was blended thoroughly with an autoclaved commercial soil mix  at a concentration of approximately 1×10A. brassicicola conidia on seedlings bacterized with MBCN152-1 was similar to that on control seedlings , A. brassicicola germinated and expanded thick hyphae at 3 dpi , its production of chitinase did not increase. These results suggest that MBCN152-1 recognizes A. brassicicola as its prey, actively secretes chitinase to attack and lyse the fungal cell wall, and feeds on dead cell contents. In Trichoderma species, the production of cell wall-degrading enzymes was induced prior to and during contact with the host fungus during mycoparasitism  The Significance of Mycoparasitism by https://doi.org/10.1264/jsme2.ME22048Supplementary Material


Puberty is associated with transient insulin resistance that normally recedes at the end of puberty; however, in overweight children, insulin resistance persists, leading to an increased risk of type 2 diabetes. The mechanisms whereby pancreatic β cells adapt to pubertal insulin resistance, and how they are affected by the metabolic status, have not been investigated. Here, we show that puberty is associated with a transient increase in β cell proliferation in rats and humans of both sexes. In rats, β cell proliferation correlated with a rise in growth hormone (GH) levels. Serum from pubertal rats and humans promoted β cell proliferation, suggesting the implication of a circulating factor. In pubertal rat islets, expression of genes of the GH/serotonin (5-hydroxytryptamine [5-HT]) pathway underwent changes consistent with a proliferative effect. Inhibition of the pro-proliferative 5-HT receptor isoform HTR2B blocked the increase in β cell proliferation in pubertal islets ex vivo and in vivo. Peripubertal metabolic stress blunted β cell proliferation during puberty and led to altered glucose homeostasis later in life. This study identifies a role of GH/GH receptor/5-HT/HTR2B signaling in the control of β cell mass expansion during puberty and identifies a mechanistic link between pubertal obesity and the risk of developing type 2 diabetes. Puberty is a period of considerable dynamic hormonal changes characterized by activation of the hypothalamic/pituitary/gonadal (HPG) axis with subsequent secretion of sex steroids and an increase in growth hormone (GH) release at its highest rate in life. Linked to the action of these hormones, puberty is marked by an accumulation of fat mass and a decrease in insulin sensitivity associated with hyperinsulinemia –5. WhileExpansion of β cell mass occurs under physiological  or pathological  conditions of insulin resistance, and these demand a commensurate rise in insulin output from the pancreatic β cells to maintain glucose homeostasis , 17. In At the onset of puberty, gonadotropin-releasing hormone 1–expressing (GNRH1-expressing) neurons in the hypothalamus secrete GNRH1, which activates receptors in the anterior pituitary to produce luteinizing hormone (LH) and follicle-stimulating hormone (FSH). LH and FSH then stimulate gonadal steroid secretion of estrogens from the ovaries and testosterone from the testes. In addition to their direct endocrine actions on target tissues, sex steroids stimulate GH production from the pituitary to activate the GH/insulin-like growth factor 1 (IGF1) axis , 22. By The present study was designed to accomplish the following: (a) determine whether β cells undergo a wave of replication during puberty in rodents and humans, (b) investigate the role of the GH/GHR/5-HT/HTR2B signaling pathway in controlling β cell proliferation during puberty, and (c) assess whether high-fat feeding during puberty in rats compromises the β cell adaptive response and leads to abnormal glucose homeostasis in adulthood.https://doi.org/10.1172/jci.insight.160854DS1). Based on the clinical evaluation of the secondary sex characteristics  and changes in body weight, we defined the peripubertal period in males between 4.5 and 7.5 weeks (wk) of age and puberty onset in females beginning at 4 wk of age  we detected several MKI67To investigate whether β cell proliferation at puberty onset is mediated by a circulating factor, we exposed rat islets to decomplemented weaning, pubertal, or adult rat serum for 72 hours and measured islet cell proliferation by flow cytometry. Pubertal serum significantly increased α and β cell proliferation in islets isolated at puberty compared with serum collected at weaning . In isleThese data suggest that β cell proliferation during puberty in rodents and humans is triggered by a circulating factor.Ghr, Tph1, and the stimulatory 5-HT receptor isoform Htr2b was increased in islets at the onset of puberty, whereas that of the inhibitory 5-HT receptor isoform Htr1d was diminished  from 4 to 8 wk of age followed by normal chow diet until 12 wk of age (HFD-CHOW), or they were exposed to chow diet throughout (CHOW-CHOW) . Body weTo assess whether HFD during puberty impacts β cell mass expansion, we measured β cell proliferation and mass in HFD-CHOW and CHOW-CHOW groups . β Cell Overall, these data indicate that pubertal HFD blunts the normal β cell proliferative response and leads to glucose intolerance and lower β cell mass in adulthood.Ghr and Htr2b mRNA expression was decreased compared with controls, while Tph1 and Htr1d were similar between groups , the selective HTR2B receptor antagonist SB204741 and the estrogen receptor antagonist ICI 182,780 were from Tocris . Recombinant rat GH protein (rGH) was from Abcam . BrdU, the androgen receptor antagonist flutamide, and the DYRK1A inhibitor harmine were from Sigma-Aldrich . The insulin receptor antagonist S961 was from Phoenix Pharmaceuticals . The Amicon Ultra-0.5 Centrifugal Filter Units were from MilliporeSigma . Fatty acid–free BSA was obtained from Equitech-Bio.A Wistar rat (Charles River Laboratories) colony was maintained in-house. Animals were housed under controlled temperature on a 12-hour light-dark cycle with free access to water and fed ad libitum with normal chow diet  or HFD . The animals received HFD or CHOW diet from 4 to 8 wk of age, and they then received CHOW diet until 12 wk of age. Body weight, food intake, and fed blood glucose were monitored weekly during the HFD study. Clinical parameters , metabolic tests, β cell proliferation, and mass were assessed at different time points from weaning to adulthood. Plasma insulin, C-peptide, testosterone, GH, and IGF1 were measured by ELISA .Male rats were injected i.p. from D25 ± 1 day to D37 ± 1 day with Cetrorelix (100 μg/day in 0.4 mL of 0.9% of NaCl), SB204741 , or vehicle. At the end of the treatment, body and testicular weight, plasma testosterone, and IGF1 levels and β cell proliferation in pancreatic sections were measured. BrdU (50 mg/kg/day) was injected i.p. during the last 7 days of treatment. IPGTT and β cell proliferation were assessed at D38 ± 1 day.IPGTT were performed on 4- to 5-hour–fasted rats by measuring tail blood glucose  at t = 0, 15, 30, 45, and 60 minutes and measuring plasma insulin levels at t = 0, 15, and 30 min after i.p. dextrose administration (1 g/kg).IPITT were performed on 4- to 5-hour–fasted rats by measuring tail blood glucose at t = 0, 15, 30, 45, 60, 90, and 120 minutes after i.p. insulin administration .One-step HGC were performed in conscious, ad libitum–fed animals as described . Briefly+ and INS+ or MKI67+ and NKX6-1+ cells over the total INS+ or NKX6-1+ cells, respectively, and double-positive BrdU+ and INS+ over the total INS+ cells. At least 1,500 β cells were manually counted per condition in 1–2 sections. The experimenter was blind to group assignments. β Cell mass and islet size were measured on paraffin sections  prepared as described  and cryoprotected overnight in 30% sucrose. Tissue was then embedded in OCT compound, frozen, sectioned at 8 μm, and mounted on Superfrost Plus slides (Invitrogen). Antigen retrieval was performed using sodium citrate buffer (pH 6). β Cell proliferation was measured by immunofluorescent staining for MKI67 and insulin (INS) or NKX6-1, or BrdU and INS, as described . Primaryescribed  using anescribed .+ and CPEP+ cells over the total CPEP+ cells as described above for rats. Primary antibodies and dilutions are listed in Formalin fixed, paraffin embedded sections were deparaffinized and rehydrated. Antigen retrieval was performed using sodium citrate buffer (pH 6). β Cell proliferation was measured by immunofluorescent staining for MKI67 and C-peptide and expressed as a percentage of double-positive MKI67g for 10 minutes at 4°C in BD Vacutainer SST tubes . Prepubertal (4- to 8- year-old) or pubertal (11- to 14-year-old) sera from boys and girls were obtained from Innovative Research Inc. All sera were decomplemented by heating at 56°C ± 2°C during 30 minutes prior to use.Blood was collected from male rats following abdominal aorta puncture at weaning (D21) or during puberty (D37–D39) and centrifuged at 1,100Peripubertal (D37 ± 1 day) rat islets were isolated from male rats by collagenase digestion and dextran density gradient centrifugation as described previously  and alloUpon reception, human islets were maintained in cGMP Prodo Islet Media  with 5% (v/v) human AB serum and 1% (v/v) glutamine/glutathione mixture prior to use.Batches of 200 peripubertal male rat islets (D37 ± 1 day) were cultured in RPMI-1640 in the presence of 7.5 mM glucose for 4 hours prior to the addition of rat serum (10% v/v) for an additional 72 hours. Adult human islets were cultured in Prodo media (5.8 mM glucose) with decomplemented sex-matched human serum (10% v/v) or with human AB serum (5% v/v) for 72 hours. Harmine (10 μM); SB204741 (35 μM); rGH (1 μg/mL); S961 (100 nM); ICI 182,780 (10 nM); and flutamide (10 nM) were used and EdU (10 μM) was added throughout. Media were replaced daily.https://www.flowjo.com/solutions/flowjo). Dead cell stain–, EdU-, INS-, and glucagon-labeled (GCG-labeled) cells were detected using the 405, 488, 640, and 561 nm lasers coupled with 525/50, 530/30, 670/14, and 586/15 nm BP filters, respectively. Proliferation was calculated as the fold-change of the percentage of double-positive EdU+ and INS+ cells over the total INS+ cell population, over the control condition. At least 2,300 INS+ cells were counted in each sample.Following treatment, islets were dissociated into single cells with accutase  for 10 minutes at 37°C, and β cell proliferation was assessed by flow cytometry as described previously . Dead ceFollowing islet isolation from rats at prepuberty (D23–D24), puberty (D37–D38) or young adulthood (D64–D66), islets were incubated in Krebs-Ringer Bicarbonate Buffer (KRBH) (pH 7.4) with 0.1% (w/v) fatty acid-free BSA and 1.0 mM of glucose for 20 minutes. Triplicate batches of 10 islets each were then incubated for an additional 20 minutes in KRBH, 0.1% fatty acid–free BSA, and 1.0 mM glucose, followed by a 1-hour static incubation in KRBH in the presence of 1.0, 8.3, or 16.7 mM glucose. Secreted insulin was measured in the supernatant by RIA. Intracellular insulin content was measured after acid-alcohol extraction.g for 5 min at 4°C. 5-HT was measured in supernatants using an ELISA kit (LDN). Total protein content was measured using the BCA assay (Thermo Fisher Scientific). 5-HT content was normalized to total protein.Following isolation, rat islets were allowed to recover in RPMI-1640 supplemented with 10% (v/v) FBS at 11.1 mM glucose overnight, after which islets were lysed with radioimmunoprecipitation assay (RIPA) buffer (0.5 μL/islet), sonicated, and centrifuged at 12,000Ppia) RNA levels and normalized to control islets. Primer sequences are listed in Total RNA was extracted from 150 to 200 islets after an overnight recovery in RPMI-1640 supplemented with 10% (v/v) FBS at 11.1 mM glucose using the RNeasy Micro kit (Qiagen). RNA was quantified by spectrophotometry using a NanoDrop 2000 (Invitrogen) and reverse transcribed (1 g). Real-time PCR was performed using the Rotor-Gene SYBR Green PCR kit (Qiagen). Results are expressed as the ratio of target RNA to cyclophilin A  were provided by the Alberta Diabetes Institute Islet Core and the Pathology Department of Ste-Justine Hospital  . Islets ALC conceived and designed the study, performed experiments, analyzed data, provided intellectual input, and wrote the manuscript. CG performed experiments and analyzed data. GF performed experiments and analyzed data. ME performed experiments and analyzed data. CT performed experiments and analyzed data. MB performed experiments and analyzed data. DDS provided samples and analyzed data. JG designed the study, provided intellectual input, wrote the manuscript, and supervised the project. VP conceived and designed the study, provided intellectual input, wrote the manuscript, acquired funding, and supervised and administered the project. VP is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis


Official Method of AnalysisSM2020.07.A simple, accurate, and reliable method to measure available carbohydrate components of food products, including cereal and dairy products, fruits, vegetables, processed food, food ingredients, and animal foods, was developed by Megazyme . A single-laboratory validation of the enzymatic method resulted in First Action status as Official Method 2020.07 for the measurement of available carbohydrates, including digestible starch, lactose, sucrose, isomaltose, maltose, glucose, fructose, and galactose in a broad range of food and feed products.A collaborative study was conducted to evaluate the repeatability and reproducibility of Samples are defatted if containing >10% fat content, and incubated with pancreatic α-amylase and amyloglucosidase under conditions that simulate those in the small intestine . The reaction solution is clarified and diluted, and an aliquot is incubated with sucrase, maltase, oligo-1,6-α-glucosidase, and β-galactosidase to hydrolyze sucrose, maltose, isomaltose, and lactose to glucose, fructose, and galactose, which are then measured enzymatically. The multi-laboratory validation (MLV) matrixes included cereal, animal feeds, fruit, vegetables, infant formula, powdered milk drink, a dessert product, and mushrooms. Additional materials were analyzed by collaborators as “practice samples.”r) <3.91% and reproducibility relative standard deviations (RSDR) ranging from 3.51 to 11.58% with 9 of the 10 matrixes having RSDR of <6.19%. For the practice samples, the RSDR ranged from 2.7 to 11.4% with 7 of the 8 samples having RSDR of <4.4%.All MLV matrixes resulted in repeatability relative standard deviations (RSDOfficial Method 2020.07 meets the AOAC requirements for repeatability and reproducibility, and the data support Final Action status.Official Method 2020.07 is a robust, simple to use, and reproducible method for the analysis of available carbohydrates in a wide range of matrixes. Available carbohydrates are carbohydrates that are digested in the human small intestine. A method for direct analysis of glucose, fructose, sucrose, lactose, maltose, and starch in foods was introduced by McCance and Lawrence in 1929 . The McCOfficial MethodSM2020.07), and a multi-laboratory collaborative study involving 17 laboratories and 10 matrixes was conducted and is discussed herein.A single-laboratory validation was previously reported  with anaOfficial Method 2020.07 required enzymes, a control sample, data reporting sheets, and an Excel calculator. Each laboratory was asked to perform a single analysis of each sample, to ask questions regarding the method, and to provide feedback to the Method Author. The samples included quick oats, infant formula powder , red-skinned, yellow-fleshed potatoes, powdered cappuccino drink mix, granular RS4 resistant wheat starch, IMO mixture from non-genetically modified corn starch, and rye-based crispbread crackers.Prior to the collaborative study, all participating laboratories were provided practice samples to familiarize themselves with the method and to ensure adequate method performance. Laboratories were shipped seven matrix samples along with Quick oats and rye crackers.—Products were purchased from a local supermarket. Approximately 300 g of material was ground in a grinding mill until 100% passed through a 0.5 mm sieve. The ground material was collected in a plastic bag, mixed thoroughly by inversion, and then transferred to 1 L Duran bottles, well-sealed and stored at room temperature away from direct sunlight.Potatoes.—Potatoes were purchased from a local supermarket. They were steamed until tender, drained, and cooled to room temperature, chopped finely, weighed, and lyophilized, with wet and dry weights recorded. The recovered dry weight of potatoes was 18.1%.Infant formula and cappuccino drink mix.—Products were purchased from a local supermarket and transferred to well-sealed Duran glass bottles and stored at room temperature away from direct sunlight. These samples were analyzed as purchased.Modified wheat starch and IMO.—Modified wheat starch  was purchased from MGP Ingredients , and IMO mixture was purchased from Top Health Ingredients . These samples were analyzed as purchased.Test portions of ∼5 g of each sample type were transferred to prelabeled glass vials, which were sealed with rubber grommets and screw caps. Upon receipt, the collaborators were instructed to store all test portions at room temperature away from direct sunlight until use.Moisture content of practice samples.—Moisture contents were determined using an Ohaus MB45 Moisture analyzer. Values obtained were: quick oats 9.4%, infant formula 4.4%, dried potato 4.2%, cappuccino drink mix 2.3%, rye crackers 5.6%, phosphate cross-linked wheat starch 11.6%, and IMO 7.1%.Ten pre-prepared foods were selected for the collaborative study to cover a broad range of food categories. These included bran cereal, chocolate digestive biscuits, small-dog adult kibble, chicken feed, ripe banana, cauliflower, mushrooms (closed cap), infant formula powder, milk-based hot chocolate drink mix, and instant pudding mix.Test portions of ∼5 g of each sample type were transferred to prelabeled glass vials, which were sealed with rubber grommets and screw caps. Two randomly selected test portion vials from each matrix preparation were packaged for shipment. Samples, a control bran cereal sample, copy of the method, a link to a video of the method, Excel-based data report forms, Mega-Calc™ Data Calculator v.12/20 , sample storage instructions, and an adequate supply of enzymes in the K-AVCHO test kit as well as details on how to prepare and store them were distributed to collaborating laboratories by express overnight shipment. Upon receipt, the collaborators were instructed to store all test portions at room temperature away from direct sunlight until the start of the study and to store kit components as described on the individual bottle labels.Bran cereal.—The product was purchased from a local supermarket. Approximately 300 g of material was ground in a grinding mill until 100% passed through a 0.5 mm sieve. The ground material was collected in a plastic bag, mixed thoroughly by inversion, and then transferred to 1 L Duran bottles, well-sealed and stored at room temperature away from direct sunlight.Chocolate digestive biscuits, small-dog adult kibble, and chicken feed.—Products were purchased from a local supermarket and homogenized with a high-speed blender . Portions of approximately 100 g were transferred to 2 L beakers, and approximately 800 mL of petroleum ether (or hexane) was added to each and stirred intermittently with a spatula in a well-ventilated fume cupboard over 15 min. The solids were allowed to settle over ∼4 h, and the supernatant solution was carefully decanted and discarded. This process was repeated a further two times. The solids were transferred to a flat polypropylene tray and allowed to dry in a well-ventilated fume hood over ∼3 h and then weighed. The content of fat remaining in the samples was determined using the ANKOM XT15 extractor. The dry material was ground until 100% passed a 0.5 mm sieve and then thoroughly mixed in a plastic bag and transferred and stored in well-sealed Duran glass bottles at room temperature away from direct sunlight. Chocolate biscuits, which had a fat content of 26.7%, were defatted as described above. Subsequent analysis of the dried product using the ANKOM XT15 extractor gave a residual fat content of 0.85%. Small-dog adult kibble feed, with a fat content of 8.0%, was extracted as described above, and subsequent analysis of the dried product using the ANKOM defatting equipment gave a residual fat content of 2.6%. Chicken feed, with a fat content of 10%, was extracted as described above. Subsequent analysis of the dried product using the ANKOM defatting equipment gave a residual fat content of 3.9%. These partially defatted samples were directly analyzed in the available carbohydrates assay procedure.Banana, cauliflower, and mushrooms.—Fresh produce was procured from a local supermarket. Cauliflower was steamed until tender, drained, and cooled to room temperature. Banana, steamed cauliflower, and mushrooms were chopped finely, weighed, and lyophilized, with wet and dry weights recorded. The recovered dry weight of mushrooms was 8.0%, steamed cauliflower was 11.1%, and ripe banana was 25.7%. Samples (∼300 g) were ground in a Nutri-Bullet homogenizer followed by further grinding in a mill until 100% passed through a 0.5 mm sieve. The ground material was thoroughly mixed in a plastic bag and transferred and stored in well-sealed Duran glass bottles at room temperature away from direct sunlight.Infant formula powder, chocolate drink powder, and instant pudding powder.—Samples were purchased from a local supermarket and transferred to well-sealed Duran glass bottles and stored at room temperature away from direct sunlight. These samples were analyzed as purchased.Moisture content of collaborative study samples.—Moisture contents were determined using an Ohaus MB45 Moisture analyzer. Values obtained were: bran cereal 4.3%, dried mushrooms 5.7%, defatted chocolate digestive biscuits 5.0%, defatted small-dog adult kibble 7.3%, infant formula 3.0%, dried cauliflower 5.4%, hot chocolate drink powder 3.0%, instant pudding mix 3.9%, dried ripe bananas 3.0%, and defatted chicken pellet feed 8.8%.r) and reproducibility (sR) standard deviations, relative standard deviations of repeatability (RSDr) and reproducibility (RSDR), and measurement uncertainty (U) values.Results were submitted by collaborators using supplied Excel-based spreadsheets and evaluated according to AOAC guidelines  using anOfficial MethodSM 2020. 07AOAC Determination of Available Carbohydrates in Cereal and Cereal Products, Dairy Products, Vegetables, Fruit, and Food Productsand Animal FeedsFirst Action 2020Final Action 2022.SeeTest samples to be analyzed are suspended in maleate buffer (pH 6.0) containing PAA and AMG and continually mixed or stirred at 37°C for 4 h. During this time, digestible starch is hydrolyzed to glucose plus traces of remaining maltose as shown in Samples of the solutions are then removed, diluted, and centrifuged. Aliquots of this solution are incubated with a mixture of sucrase, maltase, β-galactosidase, and oligo-1,6-α-glucosidase at pH 6.5 and 30°C for 30 min, during which time sucrose is specifically hydrolyzed to glucose and fructose by sucrase enzyme, maltose and isomaltose are hydrolyzed to glucose, and lactose is hydrolyzed to galactose and glucose by β-galactosidase , and 5.+) to D-galactonate with the formation of reduced nicotinamide-adenine dinucleotide phosphate (NADPH)  and galactose mutarotase , β-D-galactose is oxidized by nicotinamide-adenine dinucleotide phosphate (NADP (NADPH) .The amount of NADPH formed in this reaction is stoichiometric with the amount of D-galactose. It is the NADPH that is measured by the increase in absorbance at 340 nm.Glucose and fructose are phosphorylated by the enzyme hexokinase (HK) and adenosine-5′-triphosphate (ATP) to glucose-6-phosphate (G-6-P) and fructose-6-phosphate (F-6-P) with the simultaneous formation of adenosine-5′-diphosphate (ADP)  and 8.+) to gluconate-6-phosphate with the formation of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) (In the presence of the enzyme glucose-6-phosphate dehydrogenase (G6P-DH), G-6-P is oxidized by nicotinamide-adenine dinucleotide phosphate (NADP (NADPH) .The amount of NADPH formed in this reaction is stoichiometric with the amount of glucose. It is the NADPH that is measured by the increase in absorbance at 340 nm.On completion of +, forming gluconate-6-phosphate and NADPH, leading to a further rise in absorbance that is stoichiometric with the amount of fructose.The G-6-P formed reacts in turn with NADPThe method is simple to use, and the absorbance response for galactose, glucose, and fructose is the same 2020.07A.EtOH.—95% v/v.Imidazole buffer  containing magnesium chloride (100 mM) and sodium azide (0.02% w/v) as a preservative.—Stable for >2 years at 4°C. Use as supplied.+ (0.84 mM) plus ATP (2.5 mM).—NADPStable for >5 years stored dry below −10°C. Dissolve the contents of the vial in 12 mL of distilled water and store in ∼4 mL aliquots at <−10°C between use.Stock PAA plus AMG powder.—PAA (40 KU/g) plus AMG (17 KU/g) as a freeze-dried powder mixture. Note: One unit of AMG activity is the amount of enzyme required to release one µmole of d-glucose from soluble starch per minute at 40°C and pH 4.5; one unit of PAA activity is the amount of enzyme required to release one µmole of p-nitrophenyl from Ceralpha reagent per minute at 40°C and pH 6.9 . PAA/AMG preparations should be essentially devoid of β-glucanase, β-xylanase, and detectable levels of free d-glucose. Stable for >4 years at −20°C.PAA/AMG solution.—(1) Immediately before use, dissolve 0.5 g of PAA/AMG powder [B(d)] in 25 mL of sodium maleate buffer  [B(l)] and stir for approximately 5 min. This preparation [B(e)(1)] contains PAA at 0.8 KU/mL and AMG at 0.34 KU/mL. Store on ice during use. Use on the day of preparation. (2) Alternatively: Some individuals are allergic to powdered PAA and/or AMG. In this instance, engage an analyst who is not allergic to prepare the powdered enzymes as an ammonium sulfate suspension as follows: gradually add 2.5 g of PAA/AMG powder mix (PAA 40 KU/g plus AMG 17 KU/g) to 35 mL sodium maleate buffer  [C(l)] in a 100 mL beaker on a magnetic stirrer in a laboratory hood and stir until the enzymes are completely dissolved (∼5 min). Add 17 g of granular ammonium sulfate and dissolve by stirring. Adjust the volume to 50 mL with ammonium sulfate solution [B(n)] and store at 4°C. This preparation [B(e)(2)] contains PAA at 2 KU/mL and AMG at 0.85 KU/mL. Stable at 4°C for 3 months.Sucrase (85 U/mL on sucrose), maltase (500 U/mL on p-nitrophenyl α-glucoside), β-galactosidase  plus oligo-a-1,6-glucosidase (500 U/mL on on p-nitrophenyl α-glucoside).—Suspension in 3.2 M ammonium sulfate plus sodium azide . Stable for >2 years at 4°C. Use as supplied.Galactose dehydrogenase (200 U/mL) plus galactose mutarotase suspension (4.1 mg/mL).—Suspension in 3.2 M ammonium sulfate plus sodium azide . Stable for >2 years at 4°C. Use as supplied.Hexokinase (425 U/mL) plus glucose-6-phosphate dehydrogenase (110 U/mL) .—Suspension in 3.2 M ammonium sulfate plus sodium azide . Stable for >2 years at 4°C. Use as supplied.Phosphoglucose isomerase suspension (1000 U/mL).—Suspension in 3.2 M ammonium sulfate plus sodium azide . Stable for >2 years at 4°C. Use as supplied.D-Glucose, D-fructose plus D-galactose standard solution .—Stable for >2 years at 4°C. Use as supplied.Available carbohydrates control (∼10 g).—Available carbohydrates value shown on the label. Stable for >2 years at 4°C.2 (2 mM).Sodium maleate buffer  plus CaCl—Dissolve 11.6 g of maleic acid in 1600 mL of deionized water and adjust the pH to 6.0 with 4 M (160 g/L) NaOH solution. Add 0.6 g of calcium chloride dihydrate (CaCl2·2H2O), dissolve, and adjust the volume to 2 L. Store in a well-sealed bottle , and add two drops of toluene to prevent microbial infection. Stable for ∼1 year at 4°C.Sodium maleate buffer  containing BSA (0.5 mg/mL).—Dissolve 5.8 g of maleic acid in 800 mL of deionized water and adjust the pH to 6.5 with 4 M (160 g/L) NaOH solution. Add 0.5 g of BSA  and 0.2 g of sodium azide and dissolve by stirring. Adjust the volume to 1 L. Store in a well-sealed bottle . Stable for ∼1 year at 4°C.Ammonium sulfate solution, 50% w/v.—Add 50 g of ammonium sulfate to 80 mL of distilled water and dissolve by stirring. Adjust volume to 100 mL with distilled water. Store in a well-sealed bottle . Stable for >2 years at room temperature.B(b) to B(k) are supplied in the Available Carbohydrates Assay Kit (K-AVCHO) available from Megazyme, Bray Business Park, Southern Cross Road, Bray, County Wicklow, Ireland.Items Grinding mill.—Centrifugal, with 12-tooth rotor and 0.5 mm sieve. Alternatively, a cyclone mill can be used for small test laboratory samples provided they have sufficient air flow or other cooling to avoid overheating samples.Meat mincer.—Hand-operated or electric, fitted with a 4.5 mm screen.Water bath.—Linear motion, shake speed 200 strokes/min, 35 mm stroke length, capable of maintaining temperature of 37 ± 1°C. With clips or springs to allow attachment of tubes. http://www.keison.co.uk/grantinstruments_ols200.shtml  (Germany) 2020.07C.Submersible magnetic stirrer.—2mag Mixdrive 15® submersible magnetic stirrer  or equivalent set at 170 rpm.Spectrophotometer.—Capable of operating at 340 nm, 10 mm path length, and maintaining 30 ± 1°C; e.g., MegaQuantTM Wave Spectrophotometer (Megazyme cat. no. D-MQWAVE).Analytical balance.—0.1 mg readability, accuracy, and precision.Freeze-drier.—Virtis Genesis® 25XL Biopharma Process Systems, Biopharma House, Winchester, UK, or similar.Microfuge centrifuge.—Capable of 13 000 rpm (∼15 500 × g).Disposable microfuge tubes.—2.0 mL polypropylene with attached snap cap.pH meter.Vortex mixer.Moisture analyzer.—OHAUS MB45, or equivalent.Fat extraction system.—ANKOM XT15 Extractor, or equivalent.Magnetic stirrer.Magnetic stirring bars.—Polytetrafluoroethylene, 20 × 6 mm, ridged.Digestion bottles.—250 mL soda-lime glass, wide-mouth bottles with polyvinyl lined cap .Laboratory timer.Micro-pipettors.—Capable of dispensing 1.00 mL, 100 µL, and 20 µL for dispensing 1.0 mL and 100 µL of sample solutions and 20 µL of enzyme preparations.Positive displacement pipettors.—With a 5 mL tip to dispense 0.5 mL of 95% v/v and to dispense 0.1 mL sucrase/β-galactosidase, 0.1 mL imidazole buffer, and 0.1 mL NADP+ solution; with a 25 mL tip to dispense 2.5 mL aliquots of PAA/AMG; and with a 12.5 mL tip to remove 1.0 mL aliquots from incubation solutions.Dispensers.—Brand HandyStep dispensette® S Digital 2.5–25 mL Cat. No. 4600351, or equivalent, to dispense 17.5 mL of sodium maleate buffer [B(l)] and 25 mL of distilled water.Polypropylene tubes.—30 mL, 30 × 84 mm, flat-bottom centrifuge tubes with screw cap.Polypropylene sheet with precision cut holes.—To hold and align 30 mL polypropylene tubes on the stirrer plate of the 2mag Mixdrive 15 submersible magnetic stirrer ( stirrer 2020.07C.https://www.megazyme.com/available-carbohydrates-assay-kit?c=110). Some individuals are allergic to powdered pancreatic α-amylase and/or amyloglucosidase. This enzyme preparation should be weighed and dissolved in a well-ventilated fume hood. The preparation can be stabilized with ammonium sulfate to reduce handling of the powder product.The general safety measures that apply to all chemical substances should be adhered to. For more information regarding the safe use and handling of the Available Carbohydrates Assay Kit reagents (K-AVCHO), refer to the K-AVCHO SDS document that is downloadable from the Megazyme website  materials. Pour canned vegetables onto a strainer and wash with demineralized water, freeze-dry, and mill to pass a 0.5 mm screen. Homogenize dried fruit materials in a high-speed blender  and then dry the paste in a forced-air oven at 40°C overnight. Further grind samples to be analyzed using a mortar and pestle. Homogenize high-fat samples (>5% fat content) such as malted-milk biscuits, chocolate digestive biscuits, jam and cream biscuits, shortbread finger biscuits, dairy milk chocolate, and pet foods using a high-speed blender . In a well-ventilated fume hood, transfer a sample of the homogenized material  to a preweighed 200 mL beaker and add 50 mL of petroleum ether. Stir the mixture with a spatula for 20 s, and allow the solids to settle. Carefully decant the supernatant solution and then repeat this process a further two times. Allow the solids in the beaker to dry in a well-ventilated fume hood and weigh. Calculate fat content. Store materials in the presence of a desiccant.® assay procedure employing nonreducing end-blocked p-nitrophenyl maltohepatoside in the presence of excess levels of thermostable α-glucosidase. Perform incubations in sodium maleate buffer at pH 6.9 and 40°C as described in the α-Amylase Assay Kit  booklet . One unit of enzyme activity is defined as the amount of enzyme that releases one µmole of p-nitrophenol per minute under the defined assay procedure. Report the α-amylase activity at the optimal pH of 6.9. Incubations for the measurement of digestible starch, resistant starch, and available carbohydrates are performed at pH 6.0. α-Amylase activity at pH 6.0 is ∼ 77% of that at pH 6.9. Assay AMG by incubating 0.2 mL of suitably diluted enzyme in 100 mM sodium acetate buffer (pH 4.5) with 0.5 mL of soluble starch (10 mg/mL) in 100 mM sodium acetate buffer (pH 4.5) at 40°C. At various time intervals, heat reaction tubes to ∼100°C in a boiling water bath to terminate the reaction, and measure released glucose using GOPOD reagent . One unit of AMG is defined as the amount of enzyme required to release one µmole of D-glucose per minute at pH 4.5 and 40°C. When in admixture with PAA, assay AMG using AMG Assay Reagent (Megazyme Cat. No. R-AMGR3) and calculate units of activity on starch using a conversion factor. Record the AMG activity at the optimal pH of 4.5. Incubations for the measurement of digestible starch, resistant starch, and available carbohydrates are performed at pH 6.0. The AMG activity at pH 6.0 is ∼60% of that at pH 4.5.Measure the activity of α-amylase in PAA using the CeralphaHydrolysis of digestible starch.—(1) Weigh a 0.500 ± 0.005 g test portion accurately into a 30 mL tube [C(v)]. (2) Wet the test portion with 0.5 mL of ethanol  [C(a)], add 17.5 mL of 50 mM sodium maleate buffer, pH 6.0 [B(l)], using a positive displacement dispenser [C(u)], and vortex mix the tube for a few minutes to ensure all sample is suspended, and incubate the tubes at 37°C for 5 min. (3) If incubations are to be performed in a shaking water bath, add 2.5 mL of PAA/AMG solution [C(e)(1)] to the tube, cap the tube, and place the tubes horizontally in the line of direction of shaking [C(c)] ((4) If incubations are to be performed by stirring of the tube contents, add a magnetic stir bar [C(p)] and 2.5 mL of PAA/AMG solution [B(e)(1)] to the tube, cap the tube, and incubate the reaction solution with stirring at 170 rpm on a submersible magnetic stirrer [C(e)] at 37°C for 4 h ([C(w)] to hold the tubes in place.g [C(c)] 2020.07B, for 4 h 2020.07C Note: If using an (NH4)2SO4 suspension of this enzyme preparation [B(e)(2)], add 19 mL of 50 mM sodium maleate buffer [B(l)] to the test portion, mix the contents on a vortex mixer, and incubate the tubes at 37°C for 5 min. Then add 1 mL of enzyme suspension. (5) After 4 h, remove the tubes from the shaking or stirring water bath and filter the reaction solution through glass fiber filter paper. Alternatively, store the reaction solutions at room temperature for 5 min to allow solids to partially settle and then transfer 2 mL of the supernatant solution [G(a)(5)] to a 2.0 mL polypropylene microfuge tube [C(j)] and centrifuge at 13 000 rpm (15 500 × g) for 5 min. Accurately transfer 1.0 mL of the filtered solution or supernatant on centrifugation to 25 mL of distilled water in a 30 mL polypropylene tube [C(v)]. Cap the tube, mix the contents thoroughly, and store at 4°C awaiting analysis. For materials containing <10% available carbohydrate content, carefully transfer 1.0 mL of the reaction solution to 5 mL of distilled water and mix well.Measurement of available carbohydrates.—Analyze a 0.1 mL aliquot of the supernatant solution as described in 2-A1) for both blank and sample. Subtract the absorbance difference of the blank from the absorbance difference of the sample, thereby obtaining ΔAgalactose.Determine the absorbance difference (A3-A2) for both blank and sample. Subtract the absorbance difference of the blank from the absorbance difference of the sample, thereby obtaining ΔAglucose.Determine the absorbance difference (A4-A3) for both blank and sample. Subtract the absorbance difference of the blank from the absorbance difference of sample, thereby obtaining ΔAfructose.Determine the absorbance difference  (or 6-fold for samples with <10% available carbohydrates)where: V = final volume [mL]; MW = molecular weight of D-galactose, D-glucose, or D-fructose [g/mol]; ε = extinction coefficient of NADPH at 340 nm = 6300 [l × molIt follows for galactose:for glucose:for fructose:Calculate the content (g/100 g) in solid and semi-solid materials as follows:Content of galactose:Content of glucose:Content of fructose:galactose [g/L] = concentration of galactose per L of undiluted extraction solution; cglucose [g/L] = concentration of glucose per L of undiluted extraction solution; cfructose [g/L] = concentration of fructose per L of undiluted extraction solution; EV = volume of solution used in the initial extraction ; EV/1000 = adjustment from g/L of undiluted extraction solution to g/volume of extraction solution actually used; W = weight of test portion analyzed in g.where: cAvailable carbohydrates (g/100 g)galactose (g/100 g) + glucose (g/100 g) + fructose (g/100 g)= Note: These calculations can be simplified by using the Megazyme Mega-CalcTM downloadable workbook from the Megazyme website (https://www.megazyme.com/available-carbohydrates-assay-kit?c=110).Indicative controls are used as a check on assay conditions. Available carbohydrates, galactose, glucose, and fructose values for the control sample included in the Available Carbohydrates Assay Kit should be within 3% of that stated on the label. Inulin, levan, and galactosyl-sucrose oligosaccharides should not be hydrolyzed and thus should contribute nothing to the available carbohydrates value. Sucrose in FOS will be hydrolyzed by the sucrase and, thus, correctly measured as available carbohydrates.Samples were selected to cover a wide range of foods, feeds, and ingredients for which available carbohydrate values would be useful. The samples chosen contained various levels of lactose, sucrose, starch, and nonresistant starch to ensure complete and reproducible hydrolysis of these components to the monosaccharides, galactose, glucose, and fructose and to demonstrate quantitative measurement of the three monosaccharides. All materials were prepared, dried, dispensed into sealed tubes, and blind-coded before dispatch to avoid possible deterioration during shipping.R) ranged from 1.37 to 3.19 g/100 g, and the reproducibility relative standard deviation (RSDR) ranged from 2.7 to 11.4%, with 7 of the 8 samples having an RSDR of <4.4%. The higher reproducibility standard deviation for RS4 (phosphate crosslinked wheat starch) most likely reflects the different incubation/suspension conditions of the samples with pancreatic α-amylase plus amyloglucosidase employed by some of the collaborators. The reproducibility values are consistent with those reported for analyses of similar samples with dietary fiber methods.The results of the analyses of the practice samples for total available carbohydrates are shown in seeAll 17 laboratories that analyzed practice samples completed the study and reported a full set of results. Collaborating laboratory data were evaluated statistically according to AOAC International Guidelines  using anr for total available carbohydrates ranged from 0.08 to 2.23 g/100 g, and sR ranged from 0.25 to 4.24 g/100 g. RSDR ranged from 3.47 to 11.58%, with 9 of the 10 samples having RSDR of <6.19%. Measurement uncertainty (U) ranged from 0.50 to 8.48%  derived from a collaborative study as a measure of the combined standard uncertainty of the method is appropriate on the basis that: this is an empirical method whereby the measurement is dependent on the method used, there is no available certified reference material (CRM), the method has been subject to a collaborative study whereby laboratory collaborators performed all stages of the method.Measurement uncertainty of the Available Carbohydrates (AVCHO) procedure was assessed and calculated in accordance with the guidelines specified by Eurachem CITAC Guide CG4 (QUAM : 2012.P1). The use of the reproducibility standard deviation (sThe method was applied to collaborative study using 10 samples matrixes (20 homogeneous test samples as 10 blind duplicates) analyzed by 17 collaborators.U) is calculated using a coverage factor (k) of 2 on the basis that all mean results have a degrees of freedom greater than 25. The coverage factor (k) of 2 provides a level of confidence of approximately 95%.Expanded uncertainty  can be explained either by the fact that the measurement values were close to zero, and below the limit of detection for the given assay, or by an obvious single outlier. Outside of this reasoning the RSDR for any of the monosaccharides across the samples ranged from 3.4 to 14.7%.The total available carbohydrates value of a given sample is the sum total of the individual monosaccharides fructose, glucose, and galactose from each of the carbohydrates analyzed. Raw data for each of the individual monosaccharides  reported by the collaborating laboratories are shown in Some of the values reported for fructose and galactose were negative in value but extremely close to zero. The negative values are a result of a very slightly negative absorbance difference between two absorbance readings of the same reaction tube at different stages and is an artifact of the assay. Since it is not physically possible for a sample to have a negative content of any of the monosaccharides these negative values should theoretically be taken as being zero. The contribution by any of the negative values to the total carbohydrates measurement was negligible, versus using a corrected value of zero; therefore, none of the values were corrected, and raw data were used for the calculation of total available carbohydrates. Correcting negative values to zero would also complicate the results analysis process.In addition, any variability associated with the individual monosaccharide measurements contributes to repeatability, reproducibility, and measurement uncertainty for the whole of the total available carbohydrates method. As previously mentioned, each of these statistical parameters for the whole total available carbohydrates method was well within the range of performance typically expected for similar methods or analytical formats.4 wheat starch and cooked potato. In these analyses, no specific problems were identified by the collaborators; however, several suggestions were received that helped improve the clarity of the method. Collaborators reported the procedures they used in the initial incubation step with PAA/AMG. These included the recommended procedures of mixing or stirring in a temperature-controlled water bath, intermittent regular mixing followed by incubation in a water bath at 37°C, or mixing and incubation in a temperature-controlled oven. Each of the methods performed adequately, but intermittent, manual mixing is very labor-intensive and not recommended. Various questions concerning the exact conditions required and flexibility in performing the cuvette incubations were asked, including temperature control and the method for mixing the cuvette contents. Collaborators were advised that the various reactions would be completed if the cuvettes were incubated for the stated time within the temperature range of 20–30°C. The preferred method for mixing cuvette contents is by covering the cuvette with Parafilm® and inverting the cuvette a few times.The pretrial practice sample analyses identified sections of the method description that needed improvement and provided insights into the analysis of samples containing resistant starch, including RSB(e)(1) and (2) (preparation of PAA/AMG for use in incubations) and section G(a)  were adopted, and the procedures were clarified as a result. In tabulating the results from the MLV, data for some samples were clearly inconsistent with that from other laboratories, so to identify the reasons for this inconsistency, the collaborators were contacted. The data submitted by Laboratory 17 were, in general, consistent with that obtained from other laboratories except for data for samples D and N. The available carbohydrates values reported were 7.7% for sample D and 7.4% for sample N. The collaborator was contacted and said, “Finally we found that the lab technician had a mistake on pipetting among samples.” Consequently, we accepted a second set of data for these samples (seeWhen analyzing the practice samples, helpful comments were made by several collaborators, and these suggestions were incorporated into the method text before the full MLV was performed. Comments from collaborators, particularly those related to section sections mples see. Laborat2020.07 for the measurement of available carbohydrates be adopted as Final Action Official method.Based on the results obtained from this study, it is recommended that the AVCHO method


ABCA4 gene, many of which affect ABCA4 splicing. In this study, nine antisense oligonucleotides (AONs) were designed to correct pseudoexon (PE) inclusion caused by a recurrent deep-intronic variant in ABCA4 (c.769-784C>T). First, the ability of AONs to skip the PE from the final ABCA4 mRNA transcript was assessed in two cellular models carrying the c.769-784C>T variant: a midigene assay using HEK293T cells and patient-derived fibroblasts. Based on the splicing-correcting ability of each individual AON, the three most efficacious AONs targeting independent regions of the PE were selected for a final assessment in photoreceptor precursor cells (PPCs). The final analysis in the PPC model confirmed high efficacy of AON2, -5, and -7 in promoting PE exclusion. Among the three AONs, AON2 is chosen as the lead candidate for further optimization, hereby showcasing the high potential of AONs to correct aberrant splicing events driven by deep-intronic variants.Stargardt disease is an inherited retinal disease caused by biallelic mutations in the  ABCA4, MIM 601691) gene give rise to ABCA4-associated retinopathies of which Stargardt disease  is the most common, with an estimated incidence between 1 in 8,000 and 1 in 10,000   on allele 1 and c.1822T>A (p.(Phe608Ile)) on allele 2, whereas the healthy control carried an ABCA4 WT gene. To assess the efficacy of the AONs, fibroblast cells were seeded in six-well plates at 0.35 × 106 cells/well. After overnight incubation, the cells were transfected with 0.5 µM of the corresponding AONs or SON. Five to six hours after AON transfection an additional 1 mL of the medium was added to all wells. Fibroblasts were incubated for 24 h. After 24 h, cycloheximide (CHX)   was used to suppress the nonsense-mediated decay (NMD) pathway and to visualize the transcript containing the PE. The transfection protocol was followed, as described elsewhere [Fibroblast cell lines derived from individuals with 6 cells were seeded per well on a 12-well plate coated with Matrigel . Seeded iPSCs were cultured with Essential-Flex E8 medium until 100% confluency was reached. At this point the Essential-Flex E8 medium was changed for differentiation medium (Cl). The CI medium consisted of DMEM/F12 supplemented with nonessential amino acids , B27 supplements , N2 supplements , 100 ng/µL of insulin growth factor-1 , 10 ng/µL of recombinant fibroblast growth factor basic , 10 µg/µL of heparin , 200 µg/mL of recombinant human COCO protein , and 100 µg/mL of primocin . In each well, half of the medium was replaced daily. AONs were delivered on day 28 of differentiation, CHX was added to the medium on day 29, and PPCs were harvested on day 30.Control (iPS15-00001) and patient (iPS15-00021) iPSCs were reprogrammed and characterized by the Stem Cell Technology Center , as previously described . iPSCs wOn day 28 of differentiation, PPCs were treated with AONs (0.5 µM and 1 µM) by mixing the AONs directly with the differentiation medium. After 24 h of incubation CHX was added at a final concentration of 0.1 mg/µL and incubated overnight. Forty-eight hours after AON delivery, the cells were ready for collection. Medium was collected from all wells and centrifuged, and the cell pellet was used for RNA isolation. RNA extracted from HEK293T, fibroblasts, and PPC was isolated using the Nucleospin RNA kit  following the manufacturer’s instructions. For HEK293T cells, 1 µg of total RNA was used for cDNA synthesis using the iScript cDNA Synthesis kit  according to the provider’s protocol. For fibroblasts and PPCs, 1 µg of total RNA was used for all cDNA synthesis reactions using the SuperScript VILO Master Mix  and following the manufacturer’s instructions.−(ΔΔCt) method [RNA isolation and cDNA synthesis were performed on undifferentiated iPSC and differentiated PPCs, as described above. Primers for the quantitative PCR analysis are listed in ) method .RHO exon 3 (forward) and ABCA4 exon 7 (reverse). Exon 5 of RHO was used to assess the transfection efficiency of the midigene constructs. For the RT-PCR analysis of splicing in patient-derived fibroblasts and patient-derived PPC models, primers were located in exon 5 (forward) and exon 7 (reverse) of ABCA4. Actin (ACTB) primers were used as a control. Primer sequences are listed in RT-PCR was used to assess the splicing correction in all three models. For the HEK293T-midigene model, primers were located in ABCA4 reactions and for the RHO reaction. All reaction mixtures (25 μL) contained 10 μM of each primer pair, Taq DNA Polymerase 1 U/μL , 1× PCR buffer with MgCl2, 1.5 mM MgCl2, 2 mM dNTPs, and 40 ng cDNA. PCR conditions for the RHO-ABCA4 midigene fragment from RHO exon 3 to ABCA4 exon 7 were as follows: 94 °C for 3 min, 35 cycles of 30 s at 94 °C, 30 s at 58 °C, and 90 s at 72 °C, followed by a final step of 2 min at 72 °C. RHO PCR on exon 5 was performed under the same conditions except for an elongation time of 30 s. Ten µL of the RHO-ABCA4 PCR product and 10 μL of the RHO amplicon were resolved on a 2% (w/v) agarose gel.For the HEK293T-midigene model, 40 ng of cDNA was used for all the ABCA4 reactions and 40 ng for the ACTB analysis. All reaction mixtures (25 μL) contained 10 μM of each primer pair, Taq DNA Polymerase 1 U/μL , 1× PCR buffer with MgCl2, 1.5 mM MgCl2, 2 μM dNTPs, and 80 or 40 ng cDNA. PCR conditions for ABCA4 fragments from exons 5 to 7 were as follows: 94 °C for 3 min, 35 cycles of 30 s at 94 °C, 30 s at 58 °C, and 90 s at 72 °C, followed by a final step of 2 min at 72 °C. PCR was performed in the same conditions, and 25 μL of the ABCA4 PCR product and 10 μL of the actin amplicon were resolved on a 2% (w/v) agarose gel.For patient-derived fibroblasts and patient-derived PPCs, 80 ng of cDNA was used for all the Semi-quantitative analysis of the bands was performed by using Fiji software   Table S. Percentp-values smaller than 0.05 were considered statistically significant.Data were represented as means ± SD and analyzed with GraphPad Prism 9 software . To study the differences between treated and untreated conditions, we implemented the one-way ANOVA test. The c.769-784C>T variant strengthens a pre-existing splice acceptor site in intron 6 that leads to the insertion of the PE, as depicted in p = 0.70, p = 0.29, and p = 0.30, respectively, when compared to AON-untreated, patient-derived cycloheximide control). Additionally, an AON-artifact was observed with AON1, which has been already described previously [p < 0.05). AON7 did not reach statistically significant splicing correction (p = 0.0596); however, it was the only AON targeting a strong exonic splicing enhancer (ESE) motif located in the PE, as shown in To study the potential of these AONs in patient-derived cells, fibroblasts were used. The same nine AONs were transfected into patient-derived fibroblasts that were then treated with CHX to inhibit the NMD pathway. This allowed for visualization of all the transcripts and accurate assessment of the ratio between correct transcript and PE transcript. In control fibroblasts, the PE represents ~16% of the total transcript. In fibroblasts carrying the c.769-784C>T variant, the PE transcript represented ~69% of the total transcript. After the AONs were transfected, we observed a various effectiveness on splicing correction, as captured by gel image and semi-quantification in eviously . MoreoveABCA4 wild-type sequence . PE insertion in the CHX-treated mutant line represented 34% of the total transcript. Statistically significant PE exclusion was achieved with all AONs at both concentrations (p < 0.05). AON2 delivered at 0.5 µM and AON7 delivered at 0.5 µM and 1 µM were the most potent, whereas AON5 delivered at 0.5 µM was the least efficacious  and the extremely high allelic heterogeneity  have maIn this study, we used 2′-MOE and the PS backbone modification. Historically, the combination of 2′-OMe and PS modifications yielded optimistic results in splicing correction in IRDs contributing genes by facilitating resistance to nucleases and increased target binding ,34,35,36ABCA4 gene against which the AONs were tested. The midigene used here contained parts of intron 6 and exon 7 only, as depicted in ABCA4 fragment present in the midigene drastically simplifies the complexity of the 3D folding, therefore making it easier for the AON to exert its effect. Despite these limitations, the HEK293T-midigene system is still considered useful because it allows for performing splice assays on hypothetical variants in genes with restricted expression for which a patients’ biopsy is not available, and as depicted here and in other studies, it allows for mass screening of AONs [ABCA4, which resolves the problem of simplified RNA folding. The result of added genetic complexity revealed AON1, -4, and -6 failed to reach statistically significant splicing correction and therefore, were excluded from further studies. Another important feature observed in the fibroblasts model used was the exaggerated effect of the variant on splicing. Looking at the rate of PE containing transcript to WT transcript in fibroblasts, it is clear over 50% of transcript is faulty ), especially in Dutch cohorts, or c.5882G>A (p.(Gly1961Glu)). In both cases, those complex alleles have been associated, in general, with late-onset STGD1 ,48. HoweABCA4 c.769-784C>T variant was corrected with high efficacy at the pre-mRNA level using AONs. We recommend a detailed protein and toxicity workup to provide evidence of AON-mediated rescue of ABCA4 activity and safety. Among the AONs developed for severe ABCA4 variants, our AON is the first to address complex heterozygous alleles of which only one variant is suitable for correction.Taken together, in this study, we showed that PE insertion due to


Halorubrum tebenquichense loaded with curcumin , which mediated photodynamic therapy (PDT) on A549 cells and on THP-1-macrophages, two cell types found in airway cancers. The effect of nATC- PDT on vessels modeled with a chicken embryo chorioallantoic membrane (CAM), after dropping the formulations on its surface covered with mucins, was also determined. nATCs are known to efficiently trap CUR for at least six months, constituting easy-to-prepare, stable formulations suitable for nebulization. CUR instead, is easily released from carriers such as liposomes made of ordinary phospholipids and cholesterol after a few weeks. Irradiated at 9 J/cm2, nATC  was phototoxic (3.7 ± 0.5 μM IC50), on A549 cells after 24 h. The irradiation reduced mitochondrial membrane potential (ΔΨm), ATP levels and lysosomal functionalism, and caused early apoptotic death and late necrosis of A549 cells upon 24 h. nATC induced higher extra and intracellular reactive oxygen species (ROS) than free CUR. nATC-PDT impaired the migration of A549 cells in a wound healing assay, reduced the expression of CD204 in THP-1 macrophages, and induced the highest levels of IL-6 and IL-8, suggesting a switch of macrophage phenotype from pro-tumoral M2 to antitumoral M1. Moreover, nATC reduced the matrix metalloproteinases (MMP), −2 and −9 secretion, by A549 cells with independence of irradiation. Finally, remarkably, upon irradiation at 9 J/cm2 on the superficial vasculature of a CAM covered with mucins, nATC caused the vessels to collapse after 8 h, with no harm on non-irradiated zones. Overall, these results suggest that nebulized nATC blue light-mediated PDT may be selectively deleterious on superficial tumors submerged under a thick mucin layer.This study aimed to determine the damage mechanisms caused by naturally targeted nanoarchaeosomes made of diether lipids from  The loading of considerable amounts of CUR (150 μg CUR/15 mg lipids/mL) required the addition of Tween 80 to the lipid bilayers. By SAXS and Raman, it was determined that CUR, surrounded by Tween 80 molecules, experienced a tight immobilization interacting with the terminal side of diether archaeolipid chains of high microviscosity and low lateral mobility. Such interaction was absent in liposomes, which slowly released CUR along storage. nATC resulted not only in being structurally stable in front of nebulization stress, but importantly, also against dilution and storage for months. Nebulized at 5 μg CUR/mL on an inflamed air–liquid interface made of epithelial A549 cells  model), nATC increased TEER, normalized the permeation of Lucifer Yellow, and decreased the release of IL-6, TNF-α, and IL-8 [Our group has previously reported the performance of nATC, nebulized nanovesicles solubilizing ~600 µM CUR, prepared with polar lipids from the archaea and IL-8 . In otheCUR, cholesterol (chol), 3--2,5-diphenyl tetrazolium bromide (MTT), phorbol 12-13-acetate (PMA), 2-mercaptoethanol, trizma base, Tween 80, Tween 20, Triton X-100, sodium dodecyl sulfate (SDS), and ammonium persulfate were from Sigma-Aldrich . 1,2-hydrogenated-L-α-phosphatidylcholine (HSPC) was from Northern Lipids Inc. . Roswell Park Memorial Institute medium (RPMI), penicillin–streptomycin sulphate, glutamine, sodium pyruvate, and trypsin/ethylenediamine tetraacetic acid were from Thermo Fisher Scientific . Acrylamide, bis-acrylamide, and TEMED were obtained from Bio-Rad Laboratories . Fetal bovine serum (FBS) was from Internegocios . Yeast extract, Griess Reagent Solution “A” and Griess Reagent Solution “B” were from Laboratorios Britania S.A. . The other reagents were of analytic grade from Anedra, Research AG .H. tebenquichense archaea, isolated from soil samples of Salina Chica, Peninsula de Valdés, Chubut, Argentina were grown in basal medium supplemented with yeast extract and glucose [ glucose  in a 25- glucose . Around  glucose  and elec glucose .w:w, nLT), nLT loaded with CUR , TPA-nanovesicles containing Tween 80  and nAT loaded with CUR  were prepared by the film hydration method, and were homogenized by sonication and extrusion as described before [Conventional nanovesicles containing Tween 80 (HSPC: chol: Tween 80 1:0.33:0.53 d before . The resPhospholipids were quantified by a colorimetric phosphate microassay . CUR was2 (A homemade LED irradiation device with a wavelength of 420 nm was used at a power of 30 mW/cm2 . The cel® CCL-185™) was maintained in RPMI supplemented with 10 % FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamine. The human monocyte cell line THP-1 (ATCC TIB-202™) was maintained in RPMI supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, 2 mM L-glutamine, 0.05 mM 2-mercaptoethanol and 1 mM sodium pyruvate. THP-1 monocytes were differentiated into macrophages by treatment with 100 ng/mL PMA for 24 h. All cell lines were grown in a humidified atmosphere of 5% CO2 at 37 °C.Human epithelial lung cell line A549 (ATCC2) were obtained with Cytation™ 5 .Fluorescence emission measurements λex = 425 nm and absorption measurements of free CUR and CUR nanovesicles at 40 μM in RPMI supplemented with 5% SFB before and after samples irradiation ,  was deacetylated with 0.01 M NaOH at 37 °C for 30 min protected from light . Free CU4 cells per well and grown for 24 h. Then, cells were incubated with nATC, nLTC or free CUR at concentrations of 20 and 40 μM in RPMI supplemented with 5% FBS. Cells were incubated at 37 °C to allow nanovesicles uptake, then plates were irradiated with a fluence of 9 J/cm2 or were incubated in the dark (non-irradiated). After 24 h incubation at 37 °C in a 5% CO2 atmosphere, supernatants were collected for RNS quantification and cells were washed with PBS. ROS was quantified in culture cells by adding 4.4 μg/mL CM-H2DCFDA dye in PBS. After 30 min incubation at 37 °C in a 5% CO2 atmosphere, cells were washed. Fluorescence intensity at λex = 492 nm and λem = 517 nm was determined in a Cytation™ 5. A positive control of ROS production was carried out by incubating cells with 400 μM of hydrogen peroxide. RNS was quantified in culture supernatants after precipitating cells and debris, by adding 1 volume of Griess Reactive Solution “A” and 1 volume of Griess Reactive Solution “B”. After 15 min incubation protected from light, the absorbance was measured at 525 nm in a Cytation™ 5 instrument. RNS concentrations were calculated by comparison with absorbance at 525 nm of standard calibration solutions made with sodium nitrite from 2.5 to 30 μM.ROS or RNS generation by A549 cells after PDT was measured using CM-H2DCFDA dye and Griess method, respectively. Briefly, A549 cells were seeded in 48-well plates with a density of 2 × 104 cells per well and grown for 24 h. Then, cells were incubated with CUR nanovesicles or free CUR, ), at concentrations of 0.625, 1.25, 2.5, 5, 10, 20, and 40 μM of CUR in RPMI supplemented with 5% FBS. DMSO at 0.3% v/v and empty nanovesicles at total lipid concentrations of 0.25, 0.5, 1, and 3 mg/mL were also tested. Cells were incubated at 37 °C for 1 h to allow nanovesicle cellular uptake and then plates were irradiated for 1, 5, or 10 min, corresponding to a fluence of 1.8, 9, and 18 J/cm2, respectively. For the non-irradiated control, plates were incubated without irradiation. For washing the control, after 1 h incubation with samples, cell supernatants were removed, washed twice with PBS and plates were irradiated with a fluence of 9 J/cm2. After irradiation, cells were incubated for 24 h at 37 °C in a 5% CO2 atmosphere, then the medium was removed, cells were washed with PBS and 100 µL of 5 mg/mL MTT solution were added to each well. After 2 h incubation at 37 °C, MTT solution was removed, the insoluble formazan crystals were dissolved in DMSO, and absorbance was measured at 570 nm using Cytation™ 5. The cell viability was expressed as a percentage of cells untreated and IC50 values were determined for each condition applying a non-linear regression model of dose-response curve, (Inhibitor) vs. normalized response with GraphPad Prism 8.0.1 .Cell viability upon 24 h of incubation with nanovesicles was measured by the MTT assay. Briefly, A549 cells were seeded in 96-well plates with a density of 1 × 104 cells per well and grown for 24 h. Then, cells were incubated with nATC, nLTC or free CUR at concentrations of 5, 10, 20, and 40 μM in RPMI supplemented with 5% FBS. Cells were incubated at 37 °C to allow nanovesicles uptake, then plates were irradiated with a fluence of 9 J/cm2 or incubated in the dark (non-irradiated). After 24 h incubation, supernatants were discarded, cells were washed with PBS and incubated with 2.5 μg/mL JC-1 staining mixture for 15 min at 37 °C. Upon supernatant removal and PBS washing, the fluorescence intensity of each well was measured in Cytation™ 5. The fluorescence of JC-1 monomers was determined at λex = 490 nm and λem = 530 nm and JC-1 aggregates at λex = 525 nm and λem = 590 nm. The positive control was carried out by incubating cells with a solution of 100 ng/mL of valinomycin.The effect of CUR nanovesicles on ΔΨm of A549 was determined using JC-1 dye mitochondria staining kit  according to the manufacturer guidelines. Briefly, A549 cells were seeded in 48-well plates with a density of 2 × 105 cells per well in 12-well plates and grown for 24 h. Then, cells were incubated with CUR nanovesicles or free CUR at concentrations of 20 and 40 μM in RPMI supplemented with 5% FBS for 1 h at 37 °C and cells were irradiated with a fluence of 9 J/cm2 and incubated for 1, 6 or 24 h. For Annexin V/PI staining, after 1 and 6 h incubation, cells were trypsinized, washed with PBS, and resuspended in 20 μL of binding buffer. Then, cells were incubated with fluorescein-conjugated Annexin V reagent for 15 min at room temperature in the dark. Thereafter, cells were washed and resuspended on binding buffer with PI for 15 min at 4 °C. A total of 1 × 104 cells were analyzed using a FACSCalibur flow cytometer  and data analysis was performed with the FlowJo 10.8.0 software . Annexin V-positive cells were considered apoptotic, whereas Annexin V and PI-positive cells were considered necrotic. For YO-PRO™-1 and PI stanning, after 24 h incubation, cell supernatants were discarded, washed, and incubated at 0.5 μM YO-PRO™-1 and incubated for 15 min at room temperature. After that, PI was added to each well and incubated for 15 min at 4 °C and washed with PBS. Fluorescence microscopies were made with Cytation™ 5. YO-PRO™-1 positive cells were considered apoptotic, whereas YO-PRO™-1 and PI-positive cells were considered necrotic.Induction of apoptosis/necrosis on A549 cells by PDT was determined by staining with Annexin V, propidium iodide (PI) or YO-PRO™-1 dyes . A549 cells were seeded at a density of 3.2 × 10® Luminescent Cell Viability Assay  according to manufacturer guidelines. Briefly, A549 cells were seeded and incubated with nATC, nLTC, or free CUR as stated in 2 atmosphere, supernatants were discarded, and cells were washed with PBS and incubated with fresh medium for 30 min at room temperature. Then, one volume of CellTiter-Glo® reactive was added to cell media in each well, stirred for 2 min in an orbital shaker, and incubated for 10 min at room temperature until signal stabilization. The luminescence of each well was measured in a Cytation™ 5.The intracellular ATP content of A549 cells after PDT was determined with CellTiter-Glo® Non-Radioactive Cytotoxicity Assay , according to manufacturer guidelines. Briefly, A549 cells were seeded and incubated with nATC, nLTC, or free CUR as stated in 2 or incubated in the dark (non-irradiated). After 24 h incubation at 37 °C in a 5% CO2 atmosphere an aliquot of 35 μL supernatants was transferred to fresh 96-well plates, 35 μL of the CytoTox 96® Reagent were added to each well and incubated for 30 min at room temperature. The reaction was terminated by adding 35 μL of Stop solution. The absorbance was measured at λ 490 nm in Cytation™ 5.The LDH release on A549 cells supernatant after PDT was determined with CytoTox 965 cells per well in 12-well plates with rounded cover slips on the bottom and allowed to attach overnight. Then, cells were incubated with CUR nanovesicles or free CUR at concentrations of 40 μM in RPMI supplemented with 5% FBS for 1 h or 6 h at 37 °C. For LR assays samples were incubated for 1 h, irradiated with a fluence of 9 J/cm2, and incubated for an additional 5 h. Then supernatants were discarded, cells were washed with PBS, and incubated with 3.8 µM LR for 30 min at 37 °C for endosomal–lysosomal staining. Afterward cells were washed and fixed for 5 min with formaldehyde 3.75% (w/v) in PBS. For cellular uptake determinations, samples were incubated for 1 h at 37 °C, washed with PBS and cells were fixed. Finally, preparations were washed three times in PBS and mounted in a 90% (w/v) glycerol solution. Fluorescence microscopies were made with a Leica laser-scanning spectral confocal microscope TCS SP8; . Pearson’s correlation coefficient (Rr), which describes the correlation of intensity distribution between channels, was calculated using Fiji Software [CUR cellular uptake was measured by fluorescence confocal microscopy. LysoTracker™ Red DND-99 (LR), , a fluorescent probe that accumulates in acidic compartments such as lysosomes, was used as an indicator of lysosomal health, as well as a marker to study CUR colocalization in lysosomes. A549 cells were seeded at a density of 1.2 × 10Software .4 cells per well in RPMI supplemented with 10% FBS, pyruvate sodium 1% (w/v), 100 ng/mL of PMA, and incubated for 24 h to allow macrophage transformation. A549 cells were seeded at a density of 2 × 105 cells per well in 48-well plates and grown for 24 h. Then, A549 cells were incubated with CUR nanovesicles or free CUR at concentrations of 5 μM and 40 μM for 1 h at 37 °C and were irradiated with a fluence of 9 J/cm2 or left in the dark (non-irradiated), and incubated for additional 24 h. Then 250 μL of A549 supernatants were transferred to each THP-1 well and 50 μL of fresh RPMI medium was added. After 24 h, the supernatants were stored at −20 °C for cytokine determination. THP-1 cells were washed with PBS, trypsinized, washed again, and labeled with the CD204-APC antibody  for 30 min, except for the control cells. Cells were then washed with PBS and fixed with formaldehyde 3.75% (w/v) in PBS for 10 min at 4 °C. A total of 2 × 104 cells were analyzed using a FACSCalibur flow cytometer , and data analysis was performed with FlowJo software. Human IL-8 and IL-6 concentrations were measured by enzyme-linked immunosorbent assay BD OptEIA™  following the manufacturer’s instructions. Absorbance measurements were carried out at 450 nm on a microplate reader in a microplate reader.TAM-like macrophages were generated by cultivating THP-1 macrophages with A549 cell culture supernatant . THP-1 c5 cells per well in 12-well plates and grown for 48 h to complete confluence in RPMI supplemented with 10% FBS. Scratch wounds were created in confluent monolayers using a sterile p200 pipette tip. Following washing, cells were cultured in RPMI without SFB and incubated with CUR nanovesicles or free CUR at concentrations of 5 μM. After that, cells were irradiated with a fluence of 9 J/cm2 and incubated at 37 °C. After 24 h incubation, cells were washed with PBS and incubated with RPMI without SFB for an additional 72 h. Repopulation of the wounded areas was observed under phase-contrast microscopy with Cytation™ 5. The size of the scratch wound area was determined at 0, 24, and 96 h using Fiji Software.A549 cells were seeded at a density of 3.2 × 10The effect of nanovesicles on MMP activity was determined using the MMP-substrate fluorogenic peptide FS-6 (Mca-Lys-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2)  according to a method previously described . Brieflyw/v) polyacrylamide containing 0.1% (m/v) SDS and 0.2 mg/mL gelatin from porcine skin type A . On each electrophoretic lane, the same amount of total protein supernatants (1000 μg each) was electrophoresed. Electrophoresis was performed using running buffer (0.025 M Tris/0.192 glycine pH 8.3 containing 0.1% w/v SDS) at a constant voltage of 120 V. The gels were washed for 1 h in washing buffer  to remove SDS. Then, gels were incubated for 48 h at 37 °C in incubation buffer . After that, gels were stained for 48 h with 0.5% (w/v) Coomassie Brilliant Blue R250 to visualize bands of proteolytic activity. Gels were washed out in 5% acetic acid and 10% methanol. Sites of gelatinolytic activity were detectable as clear bands against a background of uniform staining. The intensity of the gelatinolytic bands was measured with Fiji Software. The bands of pro- and active MMP-2 were detected at 72 kDa and 62 kDa and those of pro and active MMP-9 at 92 kDa and 82 kDa, respectively.The gelatinolytic activity of MMP-2 and MMP-9 was analyzed by gelatin zymography . BrieflyThe chicken CAM was used as a model to determine vascular damage produced by photodynamic therapy. Fertilized, specific pathogen-free White Leghorn eggs were supplied by Instituto Rosenbusch S.A.  on incubation day 7. Undamaged eggs were incubated at 38.0 ± 0.1 °C in a relative humidity of 65 ± 2% and under automatic rotation in an egg incubator . Between three and six eggs weighing 45–75 g were used to study each condition.w/v) NaCl, and removed. A picture of the CAM (time 0) was obtained with a Leica S8APO stereoscopic magnifying glass and recorded with a Leica DMC2900 camera . Then, 30 μL of 2 mg/mL solution of mucins from porcine stomach type II  was applied on the CAM, immediately after 300 μL of nATC, nLTC, nAT, or nLT at CUR 40 μM and 1 mg/mL of total lipids or PBS were applied over the mucin layer and irradiated with a fluence of 9 J/cm2 or kept in the dark (non-irradiated). Control eggs were incubated with PBS and left in the dark or irradiated with a fluence of 9 J/cm2 for an irradiation control.On days 8–9, the air chamber of the eggs was marked and a perforation of 15 mm was made on the shell, then, the internal white membrane was exposed, moistened with 0.9% . Statistical analyses for vascular damage assays were performed by the Kruskal–Wallis non-parametric test followed by Dunn’s multiple comparisons. Differences were considered statistically significant at a p-value of < 0.05.Statistical analyses were performed by one-way analysis of variance followed by Dunnet’s test using Prisma 8.0.1 Software. The 1O2 [2, the absorbance and fluorescence of CUR formulations significantly decreased as follows: free CUR ~60%, nATC ~50%, and nLTC ~20%  . It was 1O2   by all formulations (50 ~3.6 μM CUR (50 15.3 μM post washing vs. 3.7 μM without washing); the cytotoxicity of nLTC and of free CUR followed a less pronounced similar trend. This suggests that extracellular, non-internalized CUR nanovesicles exerted considerable phototoxicity  used as the control a. In theThe decreased production of ATP is directly related to mitochondrial dysfunction and is indicative of cell death. After internalization by A549 cells, irradiated CUR nanovesicles strongly decreased the ATP levels, whereas free CUR induced a less pronounced decrease. In non-irradiated conditions, no significant changes in ATP levels were registered b.The dyes Annexin V and PI were employed to determine early A549 cell death mechanisms. Once internalized, no signs of cell death were observed 1 h post-irradiation; after 6 h, 40, and 20 μM CUR nATC induced higher levels of early apoptosis (13%) whereas 40 μM free CUR induced higher levels of secondary necrosis (11%) a. At 24 The cell uptake of CUR formulations, and further lysosomal location after 1 h incubation, were quantified by fluorescence microscopy by CUR and LR dye fluorescence, respectively. By this means, the lysosomal integrity was also assessed since the LR dye accumulates in healthy lysosomes only. Surprisingly, the amount of intracellular CUR delivered by nATC and free CUR were comparable, and two-fold higher than that delivered by nLTC a,b. ThusTHP-1 macrophages were cultivated in a conditioned medium from A549 cells previously incubated with nATC and nLTC at 5–40 μM CUR and irradiated. The conditioned media was found to decrease the expression of the CD204 cell membrane receptor on THP-1 macrophages a. RemarkThe MMP are molecules needed for tissue invasion as well as a marker of metastasis. The extracellular release of MMP by A549 cells was determined by enzymatic kinetics employing the FS-6 peptide as a fluorogenic substrate. nATC only decreased the MMP release, independently from irradiation, achieving a complete inhibition of MMP at 40 μM CUR. Surprisingly, free CUR stimulated the release of MMP, after cell irradiation, whereas nLTC had no inhibitory activity a. In addThe oxygen reactive species generated by PDT induce irreversible damage in endothelial cells and vascular cell membranes; such damage may collapse the tumoral microvasculature, leading to hemorrhages and tumor destruction by lack of oxygen and nutrients . In thisCUR is a poorly hydrosoluble and poorly bioavailable drug, and its particulate formulations are mostly designed to increase its oral bioavailability, or, less frequently, to act as a controlled release depot after parenteral administration ,47. The 2, the order of damage was modified and the IC50 values were substantially reduced by 30-fold for nATC and nLTC, resulting in the nanovesicles being more cytotoxic than free CUR. This signified that nanovesicles, independently of their chemical nature, would favor the exertion of intracellular phototoxicity. This may be related to the higher ability of CUR nanovesicles to produce ROS after irradiation and RNS in the dark. The only effect that could be ascribed solely to the archaeolipids matrix was the fact that non-irradiated, 40 μM CUR nATC impaired the production of MMP-2 and MMP-9 by A549 cells. This chemical effect is in line with the previously reported ability of nATC in repairing epithelia [Our results must be analyzed in terms of feasible in vivo future uses, this is the nebulization suitability of each formulation, and the ability to retain CUR over time. Since free CUR cannot be nebulized, and nLTC releases CUR crystals during storage, the present comparison between nATC and nLTC and free CUR was performed only to assess the role of the archaeolipid matrix and to compare the effects of nanovesicles vs. those of free CUR after cell uptake. In such context, it was found that the archaeolipid matrix of nATC did not exert any additional cyto or phototoxicity compared to the liposomal matrix; after 24 h of irradiated, free CUR, nLTC, and nATC caused the same lysosomal damage, and a decrease in ΔΨm, induced the same extent of ATP consumption, whereas the extra and intracellular generation of ROS induced by 40 or 20 μM of CUR nanovesicles, was practically the same. The cytotoxicity of non-irradiated formulations followed the trend: CUR> nATC> nLTC; upon irradiation at 9 J/cmApart from A549 phototoxicity, two remarkable effects of nATC were caused. One was that upon internalization of 5 uM nATC and irradiation, their migration was impaired. The other was that conditioned media from A549 cells treated with 5 μM nATC and irradiated reduced the expression of the CD204 marker on macrophages derived from THP-1 monocytes. Used as models for TAM, the reduced expression of CD204 suggests a reduction of their pro-tumoral M2 phenotype. This is important since the tumoral microenvironment of epithelial tumors such as NSCLC predominantly contains M2 phenotypic TAM , which i2), which first induced shrinkage and ended in a complete collapse of small vessels after 30 min [CAM has been previously used to model vessels submitted to CUR-PDT. The CAM surface received a high CUR concentration  and was irradiated at high fluence  . After 8Sulfolobus acidocaldarius) [S. acidocaldarius is a thermoacidophile microorganism thriving in extreme conditions of temperature (80 °C) and acidity (pH < 2); its lipids are highly thermostable caldarchaeols containing pentacycles, that form monolayers [In addition, recently, the group of Bakowski explored the performance of nebulized CUR nanovesicles to perform antitumoral PDT, consisting of bilayers lacking Tween 80 and containing 90% weight DPPC (synthetic hydrogenated ordinary phospholipid) and 10% TEL (tetraether from the archaea ldarius) . S. acidnolayers . In therBecause of their simpler growth conditions and lower costs, halophilic archaea are of the outmost biotechnological potential as sources of polar and non-polar archaeolipids . Importa