BRAF mutations are associated with high mortality and are a substantial factor in therapeutic decisions. Therapies targeting BRAF-mutated tumors, such as vemurafenib (PLX), have significantly improved the overall survival of melanoma patients. However, patient relapse and low response rates remain challenging, even with contemporary therapeutic alternatives. Highly proliferative tumors often rely on glycolysis to sustain their aggressive phenotype. 3-bromopyruvate (3BP) is a promising glycolysis inhibitor reported to mitigate resistance in tumors. This study aimed to evaluate the potential of 3BP as an antineoplastic agent for PLX-resistant melanoma treatment. (2) The effect of 3BP alone or in combination with PLX on viability, proliferation, colony formation, cell death, migration, invasion, epithelial-mesenchymal marker and metabolic protein expression, extracellular glucose and lactate, and reactive species were evaluated in two PLX-resistant melanoma cell lines. (3) 3BP treatment, which was more effective as monotherapy than combined with PLX, disturbed the metabolic and epithelial-mesenchymal profile of PLX-resistant cells, impairing their proliferation, migration, and invasion and triggering cell death. (4) 3BP monotherapy is a potent metabolic-disrupting agent against PLX-resistant melanomas, supporting the suppression of the malignant phenotype in this type of neoplasia.(1)  BRAF gene is dominant among melanoma patients, occurring in approximately 50% of cases [Melanoma is the deadliest form of skin cancer . The incof cases . Drugs tof cases . Currentof cases ,9,10 andof cases ,12,13, wof cases  and neoaof cases  contextsof cases , low resof cases ,12,13 reBRAF-mutated melanomas often undergo metabolic changes toward aerobic glycolysis to supply their demands for growth and division [Similar to other types of cancer , metaboldivision . Since tdivision , resistadivision , increasdivision ,22, as wdivision ,24. Altodivision . Interesdivision —a metabodivision . The ovedivision  and withdivision ,29,30. Adivision . MCT1 hadivision , a halogdivision . Howeverdivision , its abidivision , its potdivision ,36, and division ,38. Howedivision , as welldivision ,40, no cTherefore, to explore possible therapeutic alternatives for melanoma treatment, this study aimed to evaluate the effect of 3BP as an antineoplastic agent in PLX-resistant melanomas.To investigate possible differences in the metabolic profiles of melanoma cell lines, Western blot analysis of proteins related to glycolysis and glutaminolysis pathways was performed a,b. Both50 values (60 µM for A375R and 80 µM for SKMEL28R in 3BP monotherapy and 45 µM for A375R and 90 µM for SKMEL28R in 3BP/PLX combined therapy) and IC25 values (45 µM for A375R and 65 µM for SKMEL28R in 3BP monotherapy and 35 µM for A375R and 80 µM for SKMEL28R in 3BP/PLX combined therapy) obtained after 24 h of treatment were used for the functional assays. A PLX viability curve comparing resistant and naïve cells confirmed the resistant phenotype of the studied melanoma cells  markers. As depicted in Further investigations of the effect of 3BP were performed concerning the migration and invasion of PLX-resistant melanoma cells . The resTumors present high bioenergetic and biosynthetic demands . For thi50 values among those cell lines were 1.37-fold for 3BP monotherapy and 1.85-fold for 3BP/PLX combined therapy. Importantly, although A375R cells exhibited a lower IC50 than SKMEL28R cells, both cell lines were responsive to 3BP treatment in the functional assays. This response corroborates the protein expression characterization data, which showed a high expression of MCT1 in those cell lines. This transporter and its chaperone CD147 are dominant 3BP uptake mechanisms that can modulate cell sensitivity to this drug [Melanoma is one of the most heterogeneous types of cancer , presenthis drug . High MChis drug  and therInterestingly, 3BP decreased proliferation and colony formation, in addition to triggering cell death regardless of PLX presence, indicating that 3BP presents cytostatic and cytotoxic effects on melanoma cells. These results agree with the literature, which shows that 3BP can both arrest the cell cycle ,51,52 anMetabolic reprogramming controls EMT , an even25 treatment (indicating low interference of cell death in this process). Since the same was not encountered in SKMEL28R, it is hypothesized that complex metabolic/proliferative interactions should be occurring.Studies have indicated that prolonged exposure to BRAF/MEK inhibitors renders melanomas less dependent on glycolysis but addicted to mitochondrial metabolism and anaplerotic pathways, such as glutaminolysis ,22. The Remarkably, 4 h of treatment with 3BP increased glucose uptake in A375R cells. Although the same outcome was not found in SKMEL28R cells, 24 h of exposure to 3BP decreased extracellular glucose in both cell lines. These responses contrast with the inhibitory activity already described for this drug on HK2 , an enzyAs a limitation in this study, it is of paramount importance to emphasize that neither studied cell line was obtained from PLX-resistant melanoma samples. Since PLX resistance was achieved by continuous exposure to PLX from established cell lines, this inhibitor was maintained in the culture medium throughout the entire cell culture period to avoid acquired resistance issues, such as resistance loss at the time of the experiments. That continuous exposure seems to have contributed to a more aggressive phenotype in the control group  after 24 h of PLX absence indicates that those cells did not become addicted to this drug. This effect, observed in the increase in viability in the absence of PLX (likely due to intensified proliferation), contrasts with the literature that claims that drug holidays in melanoma tumor cells with acquired resistance to PLX cause a growth disadvantage upon drug withdrawal, leading to tumor regression . These r2. Their PLX-resistant counterparts, A375R and SKMEL28R, were established as previously described [4 cells and treated with 0.5–6 µM PLX every 3 days for 4–6 weeks. Clonal colonies were then isolated, and PLX (4.5 µM for A375R and 6.0 µM for SKMEL28R) was replenished every 2–3 days to sustain their resistance phenotype. Short tandem repeat (STR) analysis was performed to ensure cell authenticity according to international standards [A375 melanoma naïve cells were kindly donated by Keiran Smalley , and SKMEL28 melanoma naïve cells were donated by Dr. Marisol Soengas . Melanoma cells were maintained in Dulbecco’s modified Eagle’s medium  with 4500 mg/L glucose and 4 mM L-glutamine supplemented with 10% fetal bovine serum  and 1% penicillin-streptomycin solution  at 37 °C and 5% COescribed ,81,82. Itandards . Biweekl3-bromopyruvate (Sigma Aldrich) was dissolved in DPBS  at room temperature immediately before use. Vemurafenib  was dissolved in dimethyl sulfoxide  to prepare stock solutions of 10 µM, which were stored at 4 °C until use.3 cells/well. Then, the cells were exposed for 24 h or 48 h to increasing concentrations of PLX  or 3BP  in the presence or absence of PLX. Subsequently, cell viability was measured by sulforhodamine B  assay, according to the manufacturer’s instructions. In short, cells were fixed with 50% trichloroacetic acid (Sigma Aldrich) for 1 h at 4 °C. Then, the cells were stained with 0.4% SRB for 30 min and washed with 1% acetic acid . Finally, the incorporated stain was diluted in 10 mM Tris (Sigma Aldrich) and read by a spectrophotometer  at 565 nm (λref = 690 nm). For IC50 and IC25 determination, the results from the cell viability assay were normalized by the viability percentage of the drug vehicle. GraphPad Prism software, version 8.0.1 , was used for nonlinear regression analysis and the dose–response sigmoidal equation (variable slope). The obtained IC50 and IC25 values were used in the functional assays.Cells were plated onto 96-well plates at a density of 5 × 10The combined effect of PLX and 3BP was evaluated by CalcuSyn software, version 2.0  based on the Chou–Talalay method. The effect of each drug on cell viability was calculated by SRB assay as follows: 3BP alone (range between 5 and 150 µM), PLX alone (range between 2 and 14 µM), 3BP + PLX combination for A375 and A375R (fixed 4.5 µM concentration for PLX and range between 5 and 150 µM for 3BP), and 3BP + PLX combination for SKMEL28 and SKMEL28R (fixed 6.0 µM concentration for PLX and range between 5 and 150 µM for 3BP). The resulting combination index (CI), calculated by the software, was used to determine drug interactions, namely additive effect (CI = 1), synergism (CI < 1), and antagonism (CI > 1) .3 cells/well and allowed to grow until 80% confluence. Then, the cells were maintained under treatment conditions with 10% BrdU for 24 h. Subsequently, the cells were incubated in a DNA denaturant for 30 min, and the peroxidase-conjugated anti-BrdU antibody was then added and incubated for 90 min. Next, the cells were held in substrate solution for 30 min, and the final solution was analyzed by a spectrophotometer  at 370 nm (λref = 492 nm). The relative proliferation of the cells was determined as a percentage of the proliferation of the DPBS group.Cell proliferation was determined by 5-bromo-2-deoxyuridine  incorporation according to the manufacturer’s instructions. In brief, the cells were plated onto a 96-well plate at a density of 5 × 103 cells/well and exposed to different treatment conditions in DMEM supplemented with 10% FBS for 20 days. The colonies formed were stained with crystal violet in 5% paraformaldehyde and photographed . Colony formation capacity was evaluated by solubilizing stained colonies in acetic acid and analyzing the resulting solution in a spectrophotometer  at 570 nm. The relative colony formation capacity was determined as a percentage of the absorbance of the DPBS group.Cells were plated onto 6-well plates at a density of 5 × 105 cells/well, allowed to grow until 80% confluence, and exposed to treatment conditions for 24 h. Subsequently, both the supernatant and adherent cells were collected, washed in DPBS and incubated with annexin V and propidium iodide for 15 min at room temperature in the dark. The percentage of early or late apoptotic cells was determined by flow cytometry , collecting 50,000 events for each condition.Cell death was evaluated by annexin V and propidium iodide assays (Roche) according to the manufacturer’s instructions. In short, cells were plated onto 6-well plates at a density of 3 × 105 cells/well, allowed to grow until 80% confluence, and then exposed to different treatment conditions for 24 h. Then, both adherent and supernatant cells were collected and treated with 5 µM dihydroethidium  for 1 h and 30 min at room temperature while protected from light. Concomitantly, the negative control group was treated with 20 nm N-acetylcysteine . One hour after exposure to DHE, the positive control group was treated with antimycin A . Subsequently, the cells were centrifuged and homogenized in DBPS. The percentage of positive DHE cells was evaluated by flow cytometry , collecting 50,000 events for each condition.Cells were plated onto 6-well plates at a density of 6 × 104 cells were plated onto 24-well 0.4-µm Cell Culture Inserts . For the invasion assay, 5 × 105 cells were plated onto previously rehydrated 24-well 0.8-µm Biocoat Matrigel Invasion Chambers . The cells were maintained under treatment conditions on the upper chamber in culture medium without FBS and allowed to migrate/invade for 24 h to the lower portion of the insert, which contained culture medium with 10% FBS. Migrating/invading cells were then fixed in methanol and stained with hematoxylin/eosin. Membranes were photographed , and migratory/invasive cells were counted by OpenCFU software, version 3.8 . Relative cell migration/invasion was determined as a percentage of the number of migratory cells in the DPBS group.For the migration assay, 5 × 105 cells/well onto 6-well plates, allowed to grow until 80% confluence, and then exposed to different treatment conditions for 24 h. Then, both adherent and supernatant cells were homogenized in cold lysis buffer  for 15 min and centrifuged at 13,000 rpm for 15 min at 4 °C. The supernatant was collected, and the soluble protein concentration was quantified by Bradford . Aliquots containing 20 µg of total protein were separated on polyacrylamide gels by SDS–PAGE and transferred onto nitrocellulose membranes  in 25 mM Tris-base/glycine buffer using the mini TransBlot Turbo Transfer System (BioRad). The membranes were blocked with 5% milk powder in TBS/0.1% Tween  for 1 h at room temperature and then incubated in primary antibody overnight at 4 °C. The antibodies used were GLS  from Atlas Antibodies ; CA9 , GLUT1 , HK2  and LDH5  from Abcam ; ASMA , ECAD , GLUD1/2 , NCAD , SLUG , SNAIL , TGFB , and VIM  from Cell Signaling Technology ; ASCT2  from Merck Millipore ; and CD147 , MCT1 , MCT4  and PDK  from Santa Cruz Biotechnology . Subsequently, the membranes were washed in TBS-T and incubated in either anti-mouse  or anti-rabbit  antibody diluted 1:5000 in 5% milk powder in TBST. The blots were detected using chemiluminescence with either SignalFire ECL Reagent  or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific). The chemiluminescent signal was detected using ImageQuant LAS 4000 mini , and densitometry analysis was performed using ImageJ software, version 1.4, . Β-actin  from Cell Signaling Technology was used as the loading control.Cells were plated at a density of 6 × 103 cells/well and allowed to grow until 80% confluence. Then, cell media before (T0) and after 24 h (T24) of exposure to different treatment conditions were collected and stored at –20 °C. Glucose Colorimetric Assay and Lactate Colorimetric Assay  were used to quantify glucose and lactate, respectively, according to the manufacturer’s instructions. The formula T0-T24 was used to calculate the extracellular glucose variation, while the extracellular lactate variation was obtained from the formula T24-T0. The obtained values were normalized to the total biomass, which was determined by a cell viability assay. Data are expressed as total µmol of metabolite/total biomass.Cells were plated onto 96-well plates at a density of 5 × 105 cells/well and allowed to grow until 80% confluence. Then, the cells were exposed to 10 µM 2-amino)-2-deoxyglucose  in DMEM without glucose under treatment conditions for 4 h. Cells were treated with 5 µM apigenin as a positive control. After treatment, both adherent and suspended cells were collected, washed in DPBS, and exposed to 5 µM 7-aminoactinomycin D  for 15 min at room temperature protected from light. The determination of viable and positive 2NBDG cell percentages was evaluated by flow cytometry (Accuri C6 Plus Flow Cytometer—BD Biosciences), collecting 50,000 events for each condition.Cells were plated onto 6-well plates at a density of 6 × 10t-test was used to evaluate significant differences between DPBS and 3BP and between the DPBS + PLX and 3BP + PLX groups, as well as significant differences between cell lines, considering p-values ≤ 0.05 significant.GraphPad Prism software, version 8.0.1, was utilized for statistical analysis. Student’s The present study provides insights into the possible mechanisms of action by which an antimetabolic agent acts on PLX-resistant melanoma cell lines. It was found that 3BP can decrease proliferation, induce necrosis, generate ROS, and dysregulate EMT proteins in PLX-resistant melanomas. However, a metabolic characterization was shown to be a primordial step in screening patients who might benefit from 3BP therapy. Factors such as MCT1 expression and glycolytic activity seem to contribute to 3BP sensitivity, but evidence indicates that glutaminolytic activity and antioxidant capacity might be disadvantageous for 3BP activity. According to the obtained results, 3BP and PLX present a competitive interaction. Nevertheless, PLX-resistant melanomas were shown to be sensitive to 3BP monotherapy, posing it as a promising alternative for this type of cancer.


P68 + DQA nanoformulations enhanced the mean passage across the BBB model of curcumin, NAC and DFO by 49%, 28% and 49%, respectively . Live cell mitochondrial staining analysis showed consistent co-location of the nanocarriers within the mitochondria. P68 + DQA nanocarriers also increased the ability of curcumin and NAC, alone or combined with DFO, to protect against rotenone induced cytotoxicity and oxidative stress by up to 19% and 14% , as measured by the MTT and mitochondrial hydroxyl radical assays respectively. These results indicate that the P68 + DQA nanocarriers were successful at enhancing the protective effects of curcumin, NAC and/or DFO by increasing the brain penetrance and targeted delivery of the associated bioactives to the mitochondria in this model. This study thus emphasises the potential effectiveness of this nanocarrier strategy in fully utilising the therapeutic benefit of these antioxidants and lays the foundation for further studies in more advanced models of PD.As the blood-brain barrier (BBB) prevents most compounds from entering the brain, nanocarrier delivery systems are frequently being explored to potentially enhance the passage of drugs due to their nanometer sizes and functional characteristics. This study aims to investigate whether Pluronic Parkinson’s disease (PD) is one of the most common progressive neurodegenerative diseases ,2,3,4. Iin vitro and in vivo studies have indicated neuroprotective effects of DFO, curcumin and NAC individually [Compounds such as curcumin and n-acetylcysteine (NAC) have generated interest as potential disease-modifying treatments for PD due to their antioxidant capabilities ,21,22,23vidually ,25,26, avidually . Howevervidually ,28,29,30® F68 (P68) combined with the mitochondria targeting agent, dequalinium (DQA), have been successfully used to develop micellar nanocarriers of curcumin and NAC, alone or in combination with DFO [Nanocarriers have demonstrated potential as targeted delivery systems to counter issues of bioavailability and to enhance delivery across biological membranes, including the blood brain barrier (BBB) ,32. Polywith DFO ,39. Prevwith DFO ,39. Nanowith DFO ,41. The with DFO ,14,15,42in vivo, where the ability of drugs to treat neurological conditions are limited due to the primary challenge of permeating across the BBB. The BBB is known to prevent the passage and activity of 98% of potential neuropharmaceuticals [However, as SH-SY5Y cells were directly exposed to the treatments in these studies, it is not established whether the effects would be replicated euticals  and alth® hCMEC/D3 BBB and SH-SY5Y cellular model. It will also evaluate whether the protective effects of the free and formulated drugs against rotenone induced cytotoxicity and oxidative stress are retained following passage across the BBB model. A significant advantage of using such a co-culture in-vitro model is that it allows quantitative estimation of the relative bioaccesibility of the nanoformulations and qualitative real time evaluation of their mitochondrial targeting ability thus laying the foundation for more specific future in vivo studies utilising the most optimum nanoformulated preparation(s).Therefore, this study aims to investigate whether P68 + DQA nanoformulations improve the ability of curcumin and NAC, alone and in combination with DFO, to access the brain and target mitochondria in a biomimetic manner by evaluating the effects of each drug in a combined Transwell®, Minimum Essential Media (MEM), 100× antibiotic-antimycotic and poloxomer 68 (Pluronic® F68), as well as the MitoTracker Red CMXRos kit and NucBlue™ Live ReadyProbes™ reagent. Dimethyl sulfoxide (DMSO), Thiazolyl Blue Tetrazolium Blue (MTT), Dequalinium chloride hydrate (95%), Rotenone (≥95%), Curcumin , Deferoxamine mesylate salt (92.5%) and N-Acetyl-L-cysteine (99%) were obtained from Sigma-Aldrich . Abcam  was the supplier of the mitochondrial hydroxyl radical detection assay kit (cat no. ab219931). Millipore  provided the Millex-MP sterile filters and flasks were from Nunc . Corning  supplied the plasticware such as culture plates, pipettes, eppendorf tubes and stripettes. Purified water with a resistivity of 18.2 MΩ (Milli-Q water) was used to prepare experimental reagents.Unless otherwise stated, chemicals were either analytical grade or cell culture grade. SH-SY5Y cells were from the American Type Culture Collection  and hCMEC/D3 cells were kindly gifted from Dr Simon McArthur . The hCMEC/D3 cells originated from VHBio Ltd.  . EGM-2 MV Microvascular Endothelial Cell Growth Medium-2 BulletKitTM, Hank’s Buffered Saline Solution (HBSS), HEPES Buffer (1 M), Dulbecco’s phosphate buffered saline (DPBS), fibronectin (bovine plasma) and collagen from calf skin, were supplied by Lonza . Fisher Scientific  supplied methanol (HPLC grade), L-glutamine, foetal bovine serum (FBS), Dulbecco’s Modified Eagle Medium (DMEM) GlutamaxNanoformulations were prepared using a modified thin-film hydration method ,40. DiffAve hydrodynamic diameter and polydispersity index. Surface charge was measured as zeta potential. These characteristics were assessed using the Zetasizer Nano ZS .Nanoformulation particle size distribution was measured as ZNanoformulation drug loading and association efficiency were studied using UV-Visible (UV-Vis) spectroscopy and calculated from the calibration curves of the free drugs, as previously described ,39,44. Iin vitro model of the BBB as previously described [w/v ascorbic acid, 5% v/v foetal bovine serum (FBS), 0.1% v/v gentamicin sulfate-amphotericin, 0.4% v/v human basic fibroblast growth factor, 0.1% v/v human recombinant insulin-like growth factor, 0.04% v/v hydrocortisone and 0.1% v/v vascular endothelial growth factor, in 5% CO2 and 37 °C environment until ~70% confluent. Following trypsinisation, the cells were seeded at 300,000 cells/cm2 into polycarbonate membrane inserts (3.0 μm pore size) of 6 and 96-well Costar Transwell® plates according to the experiment being conducted. Inserts were precoated with 1:20 type 1 collagen from calf skin: DPBS for 1 h followed by 1:100 fibronectin from bovine plasma: DPBS for a further 1 h. 72 h before testing, hCMEC/D3 cells were incubated in VEGF-free media to aid promote tight junction formation [The hCMEC/D3 cerebral microvascular endothelial cell line was used to create an escribed . Brieflyormation ,46,47,482) and therefore expressed as Ω.cm2. High TEER values are desired as the presence of tight junctions increases the resistance [2 [® plates, TEER values were therefore measured each day until a resistance in the region of 300 Ω.cm2 was reached before carrying out any of the BBB model passage experiments. Trans-endothelial electrical resistance (TEER) was measured to assess the resistance of the BBB model as previously described ,49. An esistance . In the tance [2 ,48,51,522 was reached, hCMEC/D3 cells were incubated at 37 °C for 1 h with 1.5 mL 0.1 mg/mL Lucifer yellow in HBSS + 10 mM HEPES in the apical upper compartment and 2.5 mL of HBBS in the lower basolateral chamber. Following this, the medium in the basolateral compartment and samples from the treatments added to the apical compartment were aliquoted in duplicate into a black 96 well plate in order to carry out fluorometric analysis using the Fluostar Optima Fluorescence Plate Reader  at excitation 485 nm, emission at 535 nm. The results were expressed in terms of permeability in 10–3 cm/min with the aim of achieving a permeability between 1.2 and 0.6 × 10–3 cm/min as previously reported [2), and T is the time of transport (min) [The integrity of the BBB model was also assessed using the Lucifer yellow permeability assay . reported ,56,57, art (min) ,53.A transport assay was carried out to assess the flux of nanoformulations across the model BBB as previously described ,58,59,60® plates precoated with 1:20 type 1 collagen from calf skin: DPBS (1 h) and 1:100 fibronectin from bovine plasma: DPBS (1 h), at a seeding density of 300,000 cells/cm2. SH-SY5Y cells were grown in parallel. Once 70% confluent, adherent SH-SY5Y cells were detached from the surface of the T75 flasks via trypsinisation and seeded at 1,000,000 cells/cm2 into 6-well or 96-well plates. The Transwell® inserts containing hCMEC/D3 cells at a membrane potential of 300 Ω.cm2 were placed into the relevant culture plates once the SH-SY5Y cells were confluent. The treatments and assays outlined below were then immediately carried out using this co-culture system.As described above, the hCMEC/D3 cells were grown in T75 flasks until sufficiently confluent (~70%). The adherent cells were then seeded into the 3.0 μm pore polycarbonate membrane inserts of 6-well or 96-well Costar Transwell2. The basolateral inserts were then removed and the SH-SY5Y cells were incubated for a further 2 h at 37 °C to allow the treatments to be internalised. Following the incubation, the treatment media was aspirated, and cells were washed with DPBS and incubated for 30 min with 100 nM solution of red fluorescent mitochondrial dye Invitrogen™ MitoTracker™ Red CMXRos in MEM. The cells were then washed with DPBS and cell nuclei were stained with NucBlue™ Live ReadyProbes™ reagent diluted in MEM. The samples were examined under a fluorescent microscope  to assess mitochondrial targeting properties of curcumin-loaded nanocarriers. These experiments were carried out using curcumin and curcumin-loaded nanocarriers only due to the ability of curcumin to auto-fluoresce and be identified using a fluorescent microscope. The nuclei, mitochondria and curcumin were visualised using the DAPI , CY5  and GFP  objectives respectively. The ability of the curcumin nanoformulations to target mitochondria was assessed as previously described by Zupančič et al. . The basThe basolateral chamber containing confluent SH-SY5Y cells was treated with MEM (200 μL). 150 μL of nanoformulated or corresponding free curcumin, NAC and/or DFO treatments (in HBSS) was added to the apical chamber containing hCMEC/D3 cells at a range of concentrations and incubated for 1 h at 37 °C. Following removal of the inserts, SH-SY5Y cells were incubated at 37 °C for a further 2 h. The cells then underwent a 24 h 100 μM rotenone treatment. To assess the protective effects of the treatments against rotenone induced cytotoxicity and oxidative stress following passage across the BBB model, the MTT and mitochondrial hydroxyl radical assays were carried out, as previously described . RotenonThe protective properties of drug-loaded nanocarriers against rotenone-induced reduction in cell viability was assessed using the MTT Assay. 20 μL of 5 mg/mL solution of MTT DPBS solution was added to the cells at 37 °C for 4 h. The resulting formazan crystals were dissolved in 100 μL of DMSO, following aspiration. The MaxQ 4000 benchtop orbital shaker  was used at 75 rpm for 15 min to ensure the DMSO was mixed well. A spectrophotometer  was then used to read the absorbance at 570 nm. The mitochondrial hydroxyl radical detection assay was carried out in the co-culture model according to manufacturer’s protocol . After the nanoformulation treatments and once the hCMEC/D3 inserts had been removed (described above), DPBS was used to wash the SH-SY5Y cells, and they were then treated at 37 °C for 1 h with 100 µL of the hydroxyl radical specific OH580 probe. The cells were then incubated with 100 µM rotenone for 24 h at 37 °C. Once washed, the cells were read on the Fluostar Optima Fluorescence Plate Reader  at 540/590 nm excitation/emission.The data is provided as mean ± standard deviation (S.D.), for all experiments the mean of six replicates was calculated for each treatment. Statistical analysis of the TEER and BBB model passage data was carried out using two-way analysis of variance (ANOVA) with the Šidák multiple comparisons post hoc test. One-way ANOVA with the Dunnett’s T3 post hoc test was used for the results of each MTT and mitochondrial hydroxyl radical assay .p < 0.0001) (All drug loaded nanoformulations exhibited high association efficiency (82–98%) with NAC formulations exhibiting 6% and 16% higher mean association efficiencies than curcumin formulations in the antioxidant only and combined antioxidant and DFO formulations (respectively), however there was no significant difference . All dru 0.0001) . The add 0.0001) . The mea 0.0001) . The add 0.0001) . All nan 0.0001) . The mea 0.0001) .® inserts was shown to peak at 320 Ω.cm2 on day five post seeding, falling to 304 Ω.cm2 by day seven (−3 cm/min (0.91 ± 0.13 × 10−3 cm/min).The mean TEER of hCMEC/D3 cell monolayers grown on Transwellay seven . On day p = 0.0108, A significant reduction in TEER was observed following treatment with 100 µM free DFO (p < 0.0001) , with the formulations resulting in between 19.8–48.8% more curcumin compared to the free drug treat-ments  = 235.4,  0.0001) A. In theat-ments A. The laat-ments A. Howeveat-ments A.p < 0.0001) were observed following passage across the hCMEC/D3 monolayer   = 44.73, onolayer B. The P6 0.0079) B. The hi 0.0079) B. p < 0.0001) , with the highest increase of DFO observed when using the P68 + DQA 100 µM DFO treatment  = 222.0,  0.0001) . In all reatment .Due to the ability of curcumin to autofluoresce, free and P68 + DQA nanoformulated curcumin was used to assess the mitochondrial targeting properties of the P68 + DQA nanocarriers. The fluorescent microscopy imaging presented in p < 0.0001) . Pre-treatment with P68 + DQA preparations of 10 µM curcumin + 100 µM DFO resulted significantly higher cell viability  compared to the free drug preparation  = 95.47,  0.0001) A. 3 h prparation A. The P6paration A. p < 0.0001) . However, P68 + DQA 1000 µM NAC and 1000 µM NAC + 100 µM DFO conditions resulted in significantly higher cell viabilities compared to the corresponding free drug conditions , in both cases maintaining cell viability at more than 90% of control  = 44.8,  0.0001) B. All fr control B.® model to evaluate the ability of the free and nanoformulated treatments to protect against rotenone induced oxidative stress. Significant differences were observed between the different free and P68 + DQA curcumin and curcumin + DFO treatments  = 71.23, p < 0.0001)   A. All cul cases) A. p = 0.004), 10 µM curcumin (p = 0.0091), 5 µM curcumin + 50 µM DFO (p = 0.0169) and 10 µM curcumin + 100 µM DFO (p = 0.0003) compared to the corresponding free drug conditions  A, with Pnditions A. p < 0.0001) to protect against rotenone induced oxidative stress in the Transwell® model , where there was a difference of 7.5%, with the percentage of hydroxyl radical following the P68 + DQA treatment reaching −3.1% relative to control levels  = 83.49, l® model B. All frl levels B.in vitro model of the BBB [in vivo effects [® co-culture system to evaluate the passage of P68 + DQA nanoformulated curcumin, NAC, and/or DFO treatments across the model BBB as well as the effectiveness of these treatments at protecting against PD-related neurotoxicity and oxidative stress following permeation of the membrane. For a preclinical screening study of this nature, the model has the advantage of allowing quantitative permeability assessments in combination with real-time live cell determinations while limiting use of animals. Although not a replacement for animal models, this approach allows selection and refinements of the most optimal delivery systems which can then be fully validated in in vivo studies.The human derived brain endothelial cell line, hCMEC/D3, cultured on permeable cell culture plate inserts is a well-established  the BBB ,51,61,62 effects . This st2. This, along with the mean permeability of 0.91 ± 0.13 × 10−3 cm/min, was consistent with previous studies which have reported acceptable permeability as less than 1.2 × 10−3 cm/min [2 when cultured in the presence of hydrocorti-sone to modulate the expression of tight junctional proteins and prevent endothelial barrier breakdown [The TEER of the hCMEC/D3 monolayers peaked at 320 Ω.cm3 cm/min ,56,57 anreakdown ,51,52,56in vivo and to evaluate whether the protective effects of these treatments against rotenone induced oxidative stress  were® model by up to 49% for curcumin (with 10 µM curcumin) and 28% for NAC (with 1000 µM NAC and 1000 µM NAC + 100 µM DFO)  . This tr µM DFO) . ® model. Although it was previously reported that DQA is a mitochondrial targeting agent ,40,41; tin vitro models of PD [® compartment and where for the most part the formulations were only at least as good as the free drug conditions at protecting against rotenone, when using the Transwell® model the P68 + DQA nanoformulated treatments were found to be generally more protective than the free drugs. In line with the live cell mitochondrial staining analysis, this suggests that the P68 + DQA formulations were able to mostly stay intact until reaching the mitochondria within the SH-SY5Y cells where they could then exert their effects, highlighting the value of the targeted delivery approach. Overall, the cell viability and mitochondrial hydroxyl radical assay results were consistent with previous studies which have shown protective effects of curcumin, NAC and DFO against rotenone in ls of PD ,21,38,39There was, however, little change compared to previous reports using the SH-SY5Y model alone in terms of which concentrations of these treatments were the most successful at protecting against rotenone ,39. The ® system. This is supported by the live cell mitochondrial staining analysis results which demonstrated for the first time mitochondrial-specific accumulation of curcumin when using the P68 + DQA nanocarriers in a Transwell® hCMEC/D3—SH-SY5Y co-culture system. The improved biological effects of protecting against rotenone observed with the P68 + DQA nanoformulations compared to the free drug treatments in this system is supportive of these nanocarriers increasing passage across the model BBB. This nanocarrier strategy may therefore be an effective approach to fully utilise the therapeutic benefit of these antioxidants. The use of lipid nanoparticles to deliver the Pfizer-BioNTech [In summary, taken together, these results indicate that the P68 + DQA nanoformulations were successful at enhancing the protective effects of curcumin and NAC, alone and in combination with DFO, by increasing the BBB permeability and targeted delivery of the associated drugs within the cellular TranswellBioNTech  and the BioNTech  COVID-19


Lonicera japonica Thunb.) is a new departure for environmental remediation. The effects of four electric field conditions on Cd accumulation, growth, and photosynthesis of L. japonica under four Cd treatments were investigated. Under 25 and 50 mg L−1 Cd treatments, Cd accumulation in L. japonica was enhanced significantly compared to the control and reached 1110.79 mg kg−1 in root and 428.67 mg kg−1 in shoots influenced by the electric field, especially at 2 V cm−1, and with higher bioaccumulation coefficient (BC), translocation factor (TF), removal efficiency (RE), and the maximum Cd uptake, indicating that 2 V cm−1 voltage may be the most suitable electric field for consolidating Cd-hyperaccumulator ability. It is accompanied by increased root and shoots biomass and photosynthetic parameters through the electric field effect. These results show that a suitable electric field may improve the growth, hyperaccumulation, and photosynthetic ability of L.japonica. Meanwhile, low Cd supply (5 mg L−1) and medium voltage (2 V cm−1) improved plant growth and photosynthetic capacity, conducive to the practical application to a plant facing low concentration Cd contamination in the real environment.The multi-system of electro-phytotechnology using a woody ornamental cadmium (Cd) hyperaccumulator ( With the rapid development of industrialization and urbanization in the past few decades, a growing number of heavy metals are deposited in soil, mainly derived from mining activities, vehicle emissions, and industrial dust, have caused severe harm to human health and the environment ,2,3,4,5.−1) [Phytoremediation—hyperaccumulator or accumulator absorption of toxic heavy metals from soils to plant organs—has become a promising technique that is inexpensive, easily applied, and eco-friendly ,20,21. T−1) ,23,24. H−1) ,26. Rece−1) ,30,31,32−1) ,33,34,35−1) ,38,39,40−1) ,46,47,48−1) ,54,55,56Lonicera japonica Thunb.—a popular woody ornamental plant—has become established in temperate and tropical regions worldwide in the past 150 years [L. japonica was chosen in the study based on our previous findings, which showed that it is a new-found woody Cd-hyperaccumulator [L. japonica as a model plant to show the effect of different electric fields on Cd accumulation and transport, and investigate the responses of plant growth and photosynthesis under different Cd concentrations. The specific objectives are to confirm the phytoremediation potential of a woody ornamental Cd-hyperaccumulator assisted by electric fields and develop a practical multi-system of electro-phytotechnology used to prevent contaminated soils.Nowadays, more and more ornamental plants are widely used for gardening and greening across the human living environment ,58,59. O50 years . The pla50 years . L. japoumulator ,64,65. TL. japonica were collected from the non-contaminated experimental field of Shenyang Agricultural University and propagated in sterilized sand with a nutrient medium. The nutrient medium was Hoagland solution modified by the following composition (mmol L−1): Ca(NO3)2 × 4 H2O 5.00, MgSO4 × 7 H2O 2.00, KNO3 5.00, KH2PO4 1.00, H3BO3 0.05, ZnSO4 × 7 H2O 0.80 × 10−3, MnCl2 × 4 H2O 9.00 × 10−3, CuSO4 × 5 H2O 0.30 × 10−3, (NH4)6Mo7O24 × 4 H2O 0.02 × 10−3, Fe-EDTA 0.10 [−2 s−1 PPFD, 16/8 h light/dark, 70–80% relative humidity).Seedlings of DTA 0.10 ,66. The L. japonica were transferred into adumbral containers (45 × 22 × 15 cm3) with 6 L Hoagland nutrient medium, 4 plants for each. The nutrient medium was renewed once every 3 days. Subsequently, Cd2+  was added into the nutrient medium to get: 0, 5, 25 and 50 (mg L−1), respectively. Additionally, a homogeneous electric field with a pair of graphite electrodes  connected to a DC power supply  was applied for 6 h per day, and the electrical setting of the experiment is shown in −1) are shown in After 8 weeks of cultivation, −2 s−1), stomatal conductance , transpiration rate , and intercellular CO2 concentration . Light level, CO2 concentration, and leaf temperature inside the leaf chamber were kept constant at 1000 μmol m−2 s−1 PPFD, 25 ± 0.3 °C, and 380 ± 5 μmol CO2 mol−1, respectively. Eight leaves per treatment were used for the determination.Photosynthetic parameters were measured in fully expanded leaves under the electric field using a portable photosynthesis system . The photosynthetic parameters contained net photosynthetic rate . The Cd concentration in plant tissues was determined using an atomic absorption spectrophotometer .Dried plant materials were ground to a fine powder. The powders were digested with a concentrated acid mixture of HNOThe bioaccumulation coefficient (BC) indicated the ability of plants to accumulate cadmium in the medium. It was shown as:The translocation factor (TF) reflected the different abilities of plants to translocate cadmium from a different portion of plants. It was described as:shoot is the contents (mg kg−1) of cadmium in the harvested shoots of plants; Metalmedium is the initial cadmium content (mg kg−1) of the medium; Massshoot and Massmedium are the masses (g) of the shoots and medium of the harvested plants, respectively.To assess the effects of cadmium on phytoextraction by plants, the removal efficiency (RE) was described as ,67.(3)R1 and M2 are the cadmium concentrations in the plant tissue, and W1 and W2 are the plant biomass at time T1  and T2 .The cadmium uptake was measured as .(4)Uptap < 0.05. Multiple comparison was also determined using the least significant difference (LSD) test.All experimental measurements were set for three replicates. Average values and standard deviations (SD) were calculated by Microsoft Office Excel 2016 for all the data in the present study. The experimental data were presented as the means ± SD. The statistical analysis of variance was carried out with the SPSS 22.0 software tool. The significant difference was performed between treatments at L.japonica was shown in 1–T4 treatments (under Cd stress without electric field), Cd concentration in roots had a slightly increasing trend which ranged from 103.72 to 657.58 mg kg−1. Under T5–T16 treatments, the electric field significantly enhanced the concentrations of Cd in roots compared with the control, especially exposed to high concentrations (25 and 50 mg L−1) Cd. The Cd concentrations in roots were enhanced significantly by 2 and 3 V cm−1 voltages, which reached 1110.79 and 1608.24 mg kg−1 (T11 and T12), 1291.95 and 1692.37 mg kg−1 (T15 and T16), respectively. Despite that, Cd concentrations in shoots were enhanced significantly in the electric field. The different voltages promoted Cd concentrations in shoots above 180 mg kg−1 under 25 and 50 mg L−1 Cd stress. The maximum Cd concentrations in shoots increased by 2 V cm−1 voltage (exposed to 50 mg L−1 Cd) were added to 428.67 mg kg−1, 2.44 times of T4 treatment (V0-Cd50). Other studies also reported that heavy metal  concentrations in plants increased with the application of an electric field [2+ into cytosol and enzyme cascades.Under different treatments, Cd accumulation in roots and shoots of ic field . The benic field . Our preic field ,46, whicL. japonica. The electric fields enhanced root BC significantly and shoots BC of L. japonica exposed to different concentrations of Cd compared with T1–T4 treatments (under Cd stress without electric fields). Under different concentrations of Cd stress, root BC and shoots BC were promoted significantly by 2 V cm−1 voltage and 3 V cm−1 voltage, which reached above 32.16 (T12) and 8.11 (T16). When exposed to higher concentrations of Cd, the TF of the plants was enhanced significantly by 1 V cm−1 voltage and 2 V cm−1 voltage. Exposed to 5 mg L−1 Cd stress, the RE of the plants was increased from 14.02 under no voltage (T2) treatment to 36.78 under 3 V cm−1 voltage treatment (T14). With the increase of Cd concentration in the medium, the maximum RE in the plants was 4.00 and 2.61 times of T3 (V0-Cd25) and T4 treatment (V0-Cd50). Compared with the treatments (under Cd stress without electric field), different voltages enhanced the uptake of Cd in the plants exposed to different concentrations of Cd. The maximum Cd uptake of the plants reached 53.02 μg plant−1 day−1 promoted by 2 V cm−1 voltage (T11).The measured results of the bioaccumulation coefficient (BC), translocation factor (TF), removal efficiency (RE), and heavy metal uptake are shown in L.japonica in terms of root and shoots biomass dry weight under different treatments are shown in 1–T4 treatments (under Cd stress without electric field), the dry weight of root biomass increased exposed to 5 and 25 mg L−1 Cd, and decreased slightly exposed to 50 mg L−1 Cd, indicating that the plants had a good tolerance to Cd stress. Under T5, T9, and T13 treatments (under electric field without Cd stress), the dry weight of root biomass significantly increased, especially under 2 V cm−1 voltage (T9), 22.02% higher than the control. Under T6–8, T10–12, and T14–16 treatments (under electric field and Cd stress), different voltages enhanced the dry weight of root biomass exposed to different concentrations of Cd compared to the control. Furthermore, under 2 V cm−1 voltage, the dry weight of root biomass exposed to 5 (T10) and 25 mg L−1 Cd (T11) were enhanced significantly by 52.38% and 37.50% higher than the control. A similar phenomenon [−1 Cd stress, different voltages promoted the dry weight of root biomass compared with T4 treatment (V0-Cd50).The root and shoots biomass of the plant are considered highly sensitive indicators in their response to heavy metal and other environmental stress ,73,74,75enomenon  also sho1–T4 treatments (under Cd stress without electric field), the dry weight of shoots biomass increased when exposed to different concentrations of Cd. The earlier study [−1 voltage (T9), 13.21% higher than the control. It agrees with the study [Brassica rapa L. showed fast growth and biomass production with a low or moderate voltage gradient. Under T6–8, T10–12, and T14–16 treatments (under electric field and Cd stress), there is a similarly increased trend regarding the dry weight of root biomass. Furthermore, under 2 V cm−1 voltage, the dry weight of shoots biomass exposed to 5 mg L−1 Cd (T10) was enhanced significantly by 25.91% higher than the control. When the plants were exposed to 50 mg L−1 Cd stress, the dry weight of shoots biomass was enhanced by different voltages compared with T4 treatment (V0-Cd50), which showed the electric field could promote the tolerance responses of the plants to high concentrations of Cd.By comparison, under Ter study  showed the study , which rL.japonica. In the present study, under 2 V cm−1 voltage, the dry weight of root and shoots biomass was significantly enhanced to 5 mg L−1 Cd compared with the control. Our present results agree with previous reports proving that the electric field stimulated plant biomass production and growth by regulating the transport and distribution of plant growth hormones [Above all, applying a medium-strength electric field enhanced the root and shoots growth of hormones ,33,42,462 concentration (Ci) in the leaves of L.japonica under different treatments were evaluated. The significant stimulating effect of Pn exposed to low concentrations of Cd resulted in the improvement of gas exchange and transpiration in terms of the increase in Gs, Tr, and Ci, indicating the positive effect of low Cd concentrations on the contents of Rubisco [−2 s−1, 1.19 mol m−2 s−1, and 3.33 mmol m−2 s−1, and all were enhanced significantly by 2 V cm−1 voltage . It is coincident with root and shoots biomass dry weight, indicating the combination of the low Cd supply (5 mg L−1) and medium voltage (2 V cm−1) was beneficial to elevate the photosynthetic capacity and growth of the plants. A similar phenomenon is also described as the hormesis effect [−1), different voltages significantly enhanced the Pn, Gs, Tr, and Ci of the plants. However, with the increase of Cd concentration in the medium, the effects were not obvious compared with T3 treatment . Some studies reported that Cd negatively affected photosynthesis [L.japonica did not suffer metal toxicity and produced metabolites for absorption, defense, growth, and development, which may be related to good tolerance and hyperaccumulation characteristics of the plant to stress [13-T16 treatments, the increased Ci with the increase of Cd concentration in the medium may result from inhibiting chloroplast metabolism via hampering light and dark reactions of photosynthesis or Calvin cycle enzymes and the photosynthetic electron transport chain [Photosynthesis, one of the most important biological processes for plant growth and food production, is especially sensitive to Cd stress ,77,78,79 Rubisco . A simil Rubisco . The Pn,s effect . When thynthesis ,81,82, wynthesis ,83,84. Ho stress . Under Trt chain .−1) Cd. The Cd concentrations in roots were enhanced significantly by 2 and 3 V cm−1 voltages, which reached above 1110.79 mg kg−1 (T11 and T12) and nearly 1700.00 mg kg−1 (T15 and T16), respectively. By comparison, the different voltages promoted Cd concentrations in shoots above 180 mg kg−1 under 25 and 50 mg L−1 Cd stress. The maximum Cd concentrations in shoots increased by 2 V cm−1 voltage (exposed to 50 mg L−1 Cd) were added to 428.67 mg kg−1, which was 2.44 times higher than T4 treatment (V0-Cd50). Root BC, shoots BC, TF, and the maximum Cd uptake of the plant were also promoted significantly by 2 V cm−1 voltage. The above results showed that 2 V cm−1 voltage might be the most suitable electric field for improving the hyperaccumulation ability of the plant.In the present study, the electric field significantly enhanced the concentrations of Cd in roots and shoots compared with the control, especially exposed to high concentrations , which indicated that the synergistic benefits of low Cd supply (5 mg L−1) and medium voltage (2 V cm−1) contributed to the elevated plant growth and photosynthetic capacity. This characteristic is conducive to the practical application of the plant facing low concentration Cd-contamination in the real environment. Under T1-T4 treatments (under Cd stress without electric field), the dry weight of root biomass decreased slightly exposed to 50 mg L−1 Cd, indicating the good tolerance of the plant to Cd. However, under T6–8, T10–12, and T14–16 treatments (under electric field and Cd stress), different voltages promoted the dry weight of root biomass compared with T4 treatment (V0-Cd50), indicating the electric field could promote the tolerant ability of the plant to higher concentration Cd.At the same time, under 2 V cm−1 Cd. With the increase of Cd concentration in the medium, the effects were not obvious compared with T3 treatment (V0-Cd25). Increased or unimpacted photosynthesis showed that L. japonica did not suffer metal toxicity and produced metabolites for absorption, defense, growth, and development, along with the improved growth tolerance and hyperaccumulation ability of the plant in the electric field under external Cd stress.Moreover, different voltages significantly enhanced the Pn, Gs, Tr, and Ci of the plants exposed to 25 mg LL. japonica) to remediate Cd contamination. On the other hand, L. japonica, as a popular ornamental, had dual merits of phytoremediation and decoration, which will bring social and environmental benefits. The present study would provide a reference for promoting large-scale soil remediation by electric field-assisted phytoremediation, and future researchers need to consider the economic feasibility.According to the results above, on the one hand, it is feasible to develop a multi-system of electro-phytotechnology using a woody Cd-hyperaccumulator (


Candida albicans Rap1 has been previously shown to be involved in telomeric regulation, but its other functions are still mostly unknown. In this study, we found that the deletion of the RAP1 gene altered cell wall properties, composition, and gene expression. In addition, deletion of RAP1 affected C. albicans biofilm formation and modulated phagocytosis and cytokine release by host immune cells. Finally, the RAP1 gene deletion mutant showed attenuation of C. albicans virulence in a Galleria mellonella infection model. Therefore, these findings provide new insights into Rap1 functions that are particularly relevant to pathogenesis and virulence of C. albicans.Rap1 (repressor activator protein 1) is a multifunctional protein, playing important roles in telomeric and nontelomeric functions in many eukaryotes. IMPORTANCEC. albicans is an important fungal pathogen of humans. The cell wall is the outermost layer of C. albicans and is important for commensalism and infection by this pathogen. Moreover, the cell wall is also an important target for antifungals. Studies of how C. albicans maintains its cell wall integrity are critical for a better understanding of fungal pathogenesis and virulence. This work focuses on exploring unknown functions of C. albicans Rap1 and reveals its contribution to cell wall integrity, biofilm formation, and virulence. Notably, these findings will also improve our general understanding of complex machinery to control pathogenesis and virulence of fungal pathogens. Candida albicans is a commensal yeast inhabiting multiple body sites of healthy individuals, normally being innocuous  plays an important role in telomere regulation by directly binding to double-stranded telomeric DNA. The molecular events regulated by ScRap1 are exemplified by subtelomeric gene silencing  is a conserved DNA-binding protein identified in yeasts, protozoa, and mammalian cells . In the ilencing , telomerilencing , and telilencing –25. Nevelencing –, 27. In tructure –30. HoweC. albicans Rap1, particularly those related to C. albicans pathogenesis. We demonstrated that deletion of C. albicansRAP1 alters cell wall properties, composition, and expression of genes related to cell wall biosynthesis and remodeling. Moreover, the deletion of RAP1 promoted activation of Mkc1 and Cek1 kinases and impacted C. albicans biofilm formation. Finally, the RAP1 gene deletion (rap1Δ/Δ) mutant also affected C. albicans-macrophage interaction and exhibited attenuated virulence in a Galleria mellonella infection model.In this study, we have begun to reveal other functions of C. albicans Rap1, we generated the rap1Δ/Δ mutant and RAP1-reintegrated strains. The successful strain construction was verified by PCR analysis of genomic DNA . Moreover, the rap1Δ/Δ mutant was viable, indicating C. albicansRAP1 is not essential, which is consistent with previous findings  contributes to C. albicans aggregation and adhesion  analysis. The results show that contents of all three polysaccharides were increased in the rap1Δ/Δ mutant  real-time quantitative PCR (qPCR) analysis was performed to measure expression of genes encoding enzymes for cell wall biosynthesis and remodeling , modulation of many other genes was detected in the rap1Δ/Δ mutant compared to the wild-type and RAP1-reintegrated strains , which encode exo-1,3-β-glucanase, glycosidase, and 1,3-β-glucosyltransferase, respectively . Together, these results and the findings that RAP1 deletion affects susceptibility to cell wall-perturbing drugs, cell wall properties and composition, and cell wall-related gene expression -2H-tetrazolium-5-carboxanilide] reduction method was used. Indeed, the rap1Δ/Δ mutant formed a robust biofilm compared to those of the wild-type and RAP1-reintegrated strains . When macrophages were infected with the rap1Δ/Δ mutant, the levels of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) secretion were lower than those in macrophages infected with the wild-type and RAP1-reintegrated strains  accumulation and dispersal . Therefore, to examine whether deletion of RAP1 has an impact on cell adhesion, ECM synthesis, and biofilm dispersal is of interest for future research.Cell wall components, e.g., Als1, and CSH have been linked to ormation , 69. Accispersal , 71. Hypormation  to C, inC. albicans cell wall represent the major pathogen-associated molecular patterns (PAMPs) that can be recognized by diverse pattern recognition receptors (PRRs) to trigger host immune responses  and transcription factors  can also regulate CWI and biofilm formation in C. albicans . Cells were routinely maintained at −80°C and plated on YPD agar  before each experiment. A single colony was inoculated into YPD broth and grown at 30°C overnight (~16 h) with shaking (180 rpm). The overnight culture was subcultured in synthetic complete (SC) medium  and grown at 30°C with shaking to the exponential phase. For the induction of the MAL2 promoter, YPM  was used  were used for a second round of deletion cassette integration and excision to knock out the second allele of RAP1.The r method . The priS2AdRAP1 . The DNAn by PCR . Then, tRAP1-reintegrated strains, the DNA fragment comprising the RAP1 promoter along with the full-length RAP1 coding sequence was amplified from the SC5314 genome using the primer pair Rap1-UR-F-ApaI and Rap1-DR-R-SacI. This fragment was cloned into pSFS2AdRAP1 upstream of the SAT1-flipper cassette to replace the original ApaI-SacI fragment, generating pRAP1R. The DNA fragment carrying the full-length RAP1 gene, the SAT1-flipper cassette, and the 5′ and 3′ flanking regions of RAP1 was excised from pRAP1R, purified, and transformed into the homozygous RAP1 deletion mutant (rap1Δ/Δ) strains. Nourseothricin selection and pop-out of the SAT1-flipper cassette were performed as described previously . Ten microliters of 10-fold serial dilutions was spotted onto SC agar plates with or without calcofluor white (180 μg/mL), Congo red (20 μg/mL), or tunicamycin . Cell viability was recorded after incubation at 30°C for 3 days.600) was measured (A0). Then, 200 μL of xylene was mixed with the cell suspension and held at 30°C for 30 min to allow phase separation. The aqueous layer was transferred to a polystyrene tube, and its OD600 was measured (A1). The percentage of CSH was calculated as [(A0 − A1)/A0] × 100.The CSH assay was performed as previously described  with somC. albicans cell wall, a phenol-sulfuric acid method was used as previously described 2 2 . The samACT1 transcripts were used as the internal control (TC) values were determined. Finally, the relative fold change in gene expression was calculated using the 2−ΔΔCT method . Cells were disrupted by vortexing with acid-washed glass beads, and total proteins were extracted as previously described -polyacrylamide gel electrophoresis (PAGE) as previously described . For Wes600 of ~0.01 with 250 μL of SC broth in each well of a 24-well flat-bottom polystyrene microplate. For the assay of biofilm formation, cells were incubated at 37°C with 5% CO2 for 48 h  that was kept in each well of a 24-well microplate containing 1 mL of SC medium. After incubation at 37°C with 5% CO2 for 48 h, biofilm formed on the coverslip was washed, fixed, dehydrated, and dried as described before . Additionally, C. albicans cells were stained overnight at 4°C with 50 μg/mL fluorescein isothiocyanate . For the phagocytosis assay, 2 × 105 J774A.1 cells were seeded on each well of a 6-well plate  containing a sterile coverslip with 2 mL of DMEM. The FITC-stained C. albicans cells were then added into each well containing J774A.1 cells at a multiplicity of infection (MOI) of 3 and coincubated at 37°C with 5% CO2 for 30 min, followed by washing and formaldehyde fixation. Moreover, nonphagocytosed cells were stained with 5 μg/mL calcofluor white at room temperature for 10 min. The percentage of C. albicans cells engulfed was assessed by analyzing at least 100 macrophages per well, using an Axio Imager A1 fluorescence microscope .The murine macrophage cell line J774A.1 was grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with Gibco GlutaMAX, 10% Gibco heat-inactivated fetal bovine serum (FBS), and antibiotics-antimycotic  at 37°C with 5% COC. albicans cells were collected, washed three times with PBS, and resuspended in DMEM without FBS. Additionally, 3 × 106 J774A.1 cells were seeded in each 60-mm culture dish  containing 3 mL of DMEM. C. albicans cells were then added to each dish of J774A.1 cells (MOI of 3) and coincubated at 37°C with 5% CO2 for 24 h. The supernatant was collected and used to detect and quantify different secreted cytokines with cytokine ELISA kits . For the detection of IL-6, priming of macrophages with 0.5 ng/mL LPS  for 24 h was performed before C. albicans infection  in the final instar stage were used , incubated at 37°C, and monitored and recorded daily. Survival curves were plotted using the Kaplan-Meier method.To study the role of Rap1 in ere used , 89. Lar5 cells of C. albicans (suspended in PBS) were injected into G. mellonella larvae, incubated at 37°C for 24 h. Twenty microliters of hemolymph was collected from individual larva of each infection group (containing 10 randomly chosen larvae), mixed with 20 mL of double-distilled water (ddH2O) to lyse the hemocyte, and plated onto YPD agar. The plates were incubated at 37°C for 3 to 4 days, and numbers of CFU were counted.To assess fungal load, 6 × 10t test was used to determine significant differences between samples. Statistical significance was indicated with a P value of <0.05.The two-tailed Student


Origanum dictamnus L. is a medicinal local endemic to the Island of Crete, Greece. Its propagation through biotechnological tissue culture techniques is essential due to its augmented multi-industrial sector demand. For direct organogenesis, among different culture media variants , and cytokinins , the MS + added with BA (2.2 μM) was the most effective treatment for shoots and roots formation. For indirect organogenesis, all explant types  showed a 100% callusing rate after 2 months in all media variants tested; ODK1: 20 μM thidiazuron (TDZ) + 5 μM indole-3-butyric acid (IBA) or ODK2: 0.5 μM kinetin + 5 μM 2,4-dichlorophenoxy acetic acid . The leaves and petiole explants assured a low rate of shoot regeneration (20%) in ODK1. Afterwards, leaf-, petiole-and root-callus derived from both media were transferred to four new media plant growth regulators—free or with BA + IBA + gibberellic acid (GA3). After 10 months from callus transferring, the petiole callus gave rise to roots (20–75%) while the leaf callus exhibited 10–30% shoot or 30% root regeneration. In this study, indirect organogenesis of O. dictamnus was carried out for the first time, thus various organs can be used for plant regeneration, and the developed protocol may be applicable in the horticulture industry. Origanum dictamnus L. (Lamiaceae) or Cretan dittany is a native and range-restricted Greek chamaephyte endemic to the island of Crete  that grows in habitats such as cliffs, rocks, walls, ravines, and boulders [O. dictamnus represents the most promising case of an endemic neglected and underutilized plant due to the very high potential (94.4%) in the medicinal–cosmetic sector coupled with very high feasibility for sustainable exploitation (91.67%), outlining the extant value chain and the sustainable commercial exploitation associated with this taxon, as already achieved mostly in Crete but also abroad [boulders . The conboulders . O. dicto abroad . Finalizo abroad . Dittanytential 9.4% in thOriganum growing as wild populations in the Mediterranean basin have frequently become the object of predatory exploitation because of overharvest practices seriously threatening the sustainability of these resources, therefore cultivation of these species is highly recommended to lessen their overexploitation [Origanum species, allowing their cultivation and further sustainable use of biological diversity [Many species of the genus oitation . This suiversity . Plant tiversity  and the iversity . Howeveriversity . Organogiversity .O. dictamnus L. In this context, the main aim of this research was to study the direct and indirect organogenesis potential of the Cretan dittany. In terms of direct organogenesis, the specific objectives were (1) to identify an improved medium, thus two basal culture media were included: Murashige and Skoog (MS) [Based on the literature, there is no report on the application of tissue cultures for indirect organogenesis of oog (MS) , and Gamoog (MS)  and (2) oog (MS) , herein Among the 14 treatments tested, the MS medium containing 2.2 μM BA exhibited the highest proliferation rate (3.15) and root number (16.5 roots/rooted explant), while the B5 medium enriched with 2.2 μM BA gave the highest percentage in the formation of new multiple shoots (90%); as reported in the previous work . The MS Taking simultaneously into consideration all proliferation and rooting parameters, the MS medium was the ideal one, BA the preferred cytokinin type, and 2.2 μM its optimum concentration, promoting better direct organogenesis of explants in a single 30-day culture stage  and ODR0 media, accordingly. Shoot regeneration from leaf callus was evident at a rate of 10% and 30% when leaf callus transferred from ODK1 (20 μM TDZ + 5 μM IBA) to ODR0 and ODR2 (1.1 μM BA + 0.25 μM IBA + 0.3 μM GA3) medium, respectively. Root regeneration from leaf callus (30%) was recorded when explants derived from ODK2 and cultured afterwards in ODR0. Petiole callus originated from ODK2 were afterwards differentiated and gave regeneration to roots at a 20–75% rate in ODR0 (75%), ODR1: 0.25 μM IBA + 0.3 μM GA3 (20%), ODR2 (20%) and ODR3 (70%) media. Among the three callus explant types, petiole callus had the best root regeneration performance followed by leaf callus. Root callus did not have any shoot and/or root organogenetic response, while leaf callus led to shoot or root regeneration when derived from ODK1 or ODK2, respectively  to ODR0 (PGR’s-free) medium while petiole callus exhibited 60% and 75% new callus formation rates when transferred from ODK2 to ODR3  in MS, and 25.74 mM in B5 (24.73 mM KNO3 + 1.01 mM NH42SO4) [Origanum species as well including O. onites [O. syriacum L. [O. majorana L. [Origanum vulgare x applii [O. vulgare [Shoot proliferation and rooting in vitro is highly contingent on the composition of the basal medium in salts including type, concentration and macronutrients/micronutrients ratio, source of iron, vitamins and surrounding conditions such as photoperiod and temperature , therefoNH42SO4) . The MS . onites , O. syriiacum L. , O. majox applii , and O. O. dictamnus microshoots under study was better enhanced by 2.2 μM BA followed by 2.32 μM kinetin and afterwards by 2.46 μM 2-iP. The height of the in vitro grown shoots, the increase in the length of the internodes, the rate of the proliferated microshoots, and above all, the genotype are parameters which are highly affected by the individual and combined application of the type and concentration of the added cytokinin to the culture medium [Origanum species, including O. majorana L. [O. sipyleum [O. syriacum and O. ehrenbergii [O. vulgare [O. minutiflorum [O. vulgare x applii [O. vulgare [Except the basal culture medium composition, the setting up of a competent in vitro direct regeneration protocol is also strongly dependent on the quest of the best cytokinin type and its best concentration as a crucial success coefficient whichever the plant species is . Shoot pe medium . Differee medium . The supe medium . Secondle medium . Thirdlye medium . Finallye medium . Numerouorana L. , O. sipysipyleum , O. syri vulgare , O. minutiflorum , O. vulgx applii  and O. v vulgare .O. dictamnus microshoots under study was better stimulated by the cytokinin BA in the MS medium followed by kinetin and subsequently by 2-iP, while in B5 medium kinetin was the most preferred cytokinin, BA the least preferred and 2-iP of moderate ability, each cytokinin applied with 0.25 μM IBA + 0.3 μM GA3. Different responses have been outlined in other Origanum species where rooting of microshoots was better promoted either in a full-strength MS medium PGRs-free such as in the case of O. syriacum and O. ehrenbergii [O. sipyleum L. [O. acutidens (Hand.-Mazz.) Ietswaart [O. syriacum [O. heracleoticum L. [O. vulgare [v/v) substrate mixture after 2 months in the greenhouse mist gave a mean 80.4% ex vitro survival rate, regardless of previous in vitro conditions. A wide range of survival rates (48–98%) of rooted in vitro plantlets to ex vitro conditions during acclimatization and gradual hardening process is reported for other Origanum species including O. sipyleum L. [O. majorana L. [O. syriacum L. [v/v) [v/v) [v/v) [v/v) [3 promoting the best proliferation and rooting in vitro of shoot-node explants of O. dictamnus in a single 30-day culture stage ensuring 100% ex vitro survival success. The high ex vitro acclimatization and hardening survival rate of in vitro plantlets and their following vegetative and root system growth is of paramount importance in every plant tissue culture system since it can contribute to cost-savings, marketing trade cycle abridge, speeding up the installation of propagation material, and the further introduction of the plants into cultivation in field conditions [Rooting performance of enbergii  or in diyleum L. ,30 and Oetswaart , O. syrisyriacum ,31, O. hticum L.  and O. v vulgare . The per vulgare . Rooted orana L. , and O. iacum L.  as well L. [v/v) ,30,31,32v) [v/v) ; peat mov) [v/v) ; and peanditions .3, the MS medium + 2.22 μM BA found to be superior for shoot proliferation and rooting in vitro as well as for ex vitro survival success of the rooted microshoots. The further advancements of this study were the effect of two other cytokinins types except of BA, that of kinetin and 2-iP and the fact that all these three cytokinins were supplemented in MS and B5 media at two different and marginal concentrations, the lower of 0 μM, (cytokinin-free) and the higher of 4.4 μM, which had not been tested in the previous work of Sarropoulou et al. [3 combination, as well as in the ex vitro survival rate, can be the result of an optimal division and elongation reinforcement due to the role of BA as a cytokinin mainly in cell division/proliferation and less to cell expansion, while IBA as an auxin in both cell division and cell enlargement [Origanum species, in O. acutidens (Hand.-Mazz.) Ietswaart, each increase in BA concentration (0.6–2.4 mg/L) with 0.2 mg/L NAA led consistently to increased root numbers, shoot and root lengths up to 1.8 mg/L BA, however a sharp decline in all the above-mentioned parameters was observed when higher BA concentration (2.4 mg/L) was used [In a previous study on the same plant species  as preseu et al. . The finargement , comparewas used  and thiswas used . It becowas used , thus bewas used . As concwas used  since rowas used . The augwas used .Origanum spp.  [O. majorana L.) [The process of plant organs regeneration is controlled by the appropriate concentration ratio between auxins and cytokinins, the two main PGRs groups that are essential to drive specific organogenesis responses from different tissue parts , thus the interplay actions between cytokinins and auxins are complex and roots, shoots and callus formation are highly affected by different combinations and concentrations of PGRs , explantyriacum) , the MS yriacum) ,46 or frrana L.) .O. dictamnus, a simultaneous occurrence of callus formation (100%) and shoot regeneration (20%) was obtained by leaf and petiole explants in ODK1 (20 μM TDZ + 5 μM IBA) medium. TDZ is a phenylurea that impels a high morphogenesis activity in plant tissues especially in leaves [The medium composition and explant source as a combined factor exerted a significant effect on callus initiation and plant regeneration . In the n leaves  and actin leaves , displayn leaves . In tissn leaves . TDZ hasn leaves  and thisn leaves , ensurinn leaves .O. dictamnus, after 10 months of culture, there was new secondary callus formation over the initial primary ones only by root callus when transferred from ODK2  to ODR0 medium (PGR’s-free), and by petiole callus from ODK2 to ODR3 (2.2 μM BA + 0.25 μM IBA + 0.3 μM GA3) or ODR0.The regeneration ability of callus is dependent on plant species, explant type, medium components and endogenous concentrations of PGRs as an outcome of their uptake from extracellular sources, and their metabolism and endogenous interaction . Even th3) medium. The low shoot regeneration rate from leaf callus (10–30%) might be due to the long-term maintenance period (10 months) of the explants in culture [O. dictamnus as presented herein and the reasons for this response might be (1) the higher juvenility and the lower number of vascular tissue in leaf-callus explants [O. dictamnus leaf-callus derived explants was observed in ODK2-ODR0 (PGRs-free) transition combination media whereas petiole-callus explants differentiated into roots in all tested regeneration media, both in the absence of PGR’s and enriched with BA + IBA + GA3. The in vitro shoot regeneration of O. dictamnus through leaf-derived callus and petiole-derived callus and not through callus of root origin is a very important step because the vegetative parts of a plant are desirable explants for tissue culture systems due to their ability to preserve the genetic homozygosity of the parent genotype [Previous studies have demonstrated that the cytokinin BA at a certain concentration can boost callus subculture, differentiation, and regeneration in a large number of plant species . In this culture . In vitr culture . Ehsanda culture  pointed explants , (2) youexplants  and (3) explants . Root regenotype .O. dictamnus, root callus explants in the ODR0 (PGRs-free) medium after successive subcultures became dark-brown, then black, and finally collapsed. In consistency with our findings, hypocotyl callus of marjoram (O. majorana L.) retained high proliferation rate for two subcultures but afterwards the callus grew slower, turned brown, and did not survive in subsequent subcultures [O. dictamnus desiccated shoots regenerated from leaf-callus in ODK1 and then transferred to ODR0 or ODR2 media.In this study with cultures . Such recultures  that inhcultures . This exO. dictamnus plants conserved ex situ in the premises of the Balkan Botanic Garden of Kroussia, Institute of Plant Breeding and Genetic Resources, Hellenic Agricultural Organization—DIMITRA, seed collection took place from mature inflorescences and these seeds were given thereafter the unique IPEN  accession number GR-1-BBGK-03,2108. All information regarding the disinfection protocol  followed, the initial establishment conditions of 7-year-old seeds after long-term storage  and 37.93% maximum germination rate obtained under 16 h photoperiod and 21–23 °C temperature after 12 days of in vitro culture in MS medium enriched with 20 g L−1 sucrose and 6 g L−1 Plant Agar are elaborately described in a previous study [TM B-caps were used for the placement of the nutrient medium and the culture of the explants. Each Magenta vessel was filled with 25 mL of medium. A growth chamber under controlled laboratory conditions of temperature 22 ± 2 °C, photoperiod 16 h light/8 h dark, illumination type of cool white fluorescent lamps (WFLs)  and 40 μmol m−2 s−1 light quantity was used for the incubation of the in vitro plant tissue cultures.After taxonomic identification of us study . The sub3) for shoot elongation, and 20 g L−1 sucrose  as the main energy carbon source and afterwards were gelled with Plant Agar  at 6 g L−1. The basic environmental requirements for incubation of the in vitro plant cultures inside the controlled laboratory growth chamber were set as follows: 22 ± 2 °C temperature, 16 h photoperiod, WFLs of 40 μmol m−2 s−1 light irradiance. The experiment was terminated after 30 days, wherein eight different macroscopic attributes for shoot proliferation and rooting potential were evaluated.For direct organogenesis, internodal shoot segments 1–1.5 cm long that bore two buds were the experimental explants stem from already established in vitro cultures. The concurrent application of two basal nutrient media  with three cytokinin types, i.e., BA, Kin and 2-iP was evaluated. Each cytokinin was incorporated into the basal culture medium, either MS or B5 at three concentrations, i.e., BA: 0, 2.2 and 4.4 μM, Kin: 0, 2.32 and 4.65 μM, and 2-iP: 0, 2.46 and 4.92 μΜ. All media were filled with 0.25 μM indole-3-butyric acid (IBA) as an auxin type boosting root formation, 0.3 μM gibberellic acid  inside the greenhouse mist system were 18 °C base T for plantlets root system, 15 °C air T for the vegetative part of the plants and RH ranging between 80% and 100% under conditions of thermal curtains. After 60 days period time in the mist for gradual acclimatization, on early March, the survival rate of the successfully undergo hardening plants was recorded. The following step included the transplantation of the plants into bigger pots of 0.33 L volume capacity (dimensions: 8 cm length × 8 cm width × 7 cm height) supplemented with peat moss  and perlite mixture substrate at 3:1 v/v, respectively. The newly transplanted plants were then transferred outside the indoor mist system in the bench of the same greenhouse without heating where the prevailing environmental conditions were scheduled to be 17–24 °C temperature and 55–70% RH range providing a progressively decrement of RH about 5% per day and a gradual increment of light intensity for a period of 3 months for further growth. In the subsequent stage during the onset of summer, particularly in early June due to increased temperatures inside the greenhouse, the vegetative and root system developed plants were transplanted into 2.5 L higher volume pots filled with peat (TS2), perlite and soil (2:1:½v/v) and placed in the natural environment, in the outdoor nursery under 50% direct sun exposure provided by a shading net for ex situ conservation purposes and future sustainable exploitation.In early January, microshoots with well-developed root systems after washing with tap water to remove any adhering solidifying agent were then transferred in the internal heated mist of the greenhouse within multiple propagation trays each of 100 mL volume composed of peat  and perlite (Geoflor) substrate mixture at a 1:1 −1 sucrose and 6 g L−1 Plant Agar. After two months, callus, shoot and root regeneration rates (%) were recorded.Three different explant types were used: (1) leaves, (2) petioles, and (3) roots, cultured in MS medium with two different PGRs type and concentration combinations; ODK1: 5 μM IBA + 20 μM thidiazuron (TDZ), and ODK2: 5 μM 2,4-dichlorophenoxy acetic acid  + 0.5 μM Kin. These two media (ODK1 and ODK2) were also enriched with 30 g L−1 sucrose and 6 g L−1 Plant Agar. The composition of the four new regeneration media was: ODR0 (PGR’s-free), ODR1: 0.25 μM IBA + 0.3 μM GA3, ODR2: 1.1 μM BA + 0.25 μM IBA + 0.3 μM GA3, and ODR3: 2.2 μM BA + 0.25 μM IBA + 0.3 μM GA3. Aggregates of brittle and very soft texture sponge-like callus were used as experimental explants, cultured under 24 h darkness at 21–23 oC. After 10 months, the following data were recorded: new callus formation percentage (%): the number of initial callus explants with formation of new callus over initial ones/the total number of initial callus explants × 100%, shoot regeneration percentage (%): the number of callus explants with multiple shoot induction/the total number of callus explants × 100%, and root regeneration percentage (%): the number of callus explants with root formation/the total number of callus explants × 100%. Detailed chemical compositions of the different culture media tested for indirect in vitro organogenesis subsequent stages are presented in In the following stage, the callus derived from the three explant types  in ODK1 and ODK2 media were then transferred in four new PGRs-supplemented MS media for further regeneration , all enriched with 20 g Lp ≤ 0.05 (p ≤ 0.05) and error bars in each bar per graph are standard errors. For each figure graph, a horizontal scale bar on the X-axis is provided in its respective caption in parenthesis . In Tables and figure graphs, the analysis of variance (one-way ANOVA) and the Duncan multiple-range test at a 5% level were deployed for mean values ± standard errors (S.E.) variability and comparison among treatments for each parameter per experiment conducted. In tables, means ± S.E. with different letters within a column are statistically significant different from each other according to the Duncan’s multiple range test at p ≤ 0.05 . In figuenthesis c. Furtheenthesis a–g.The micropropagation experiment included 14 treatments with 12 repetitions/treatment (four explants/vessel × three vessels/treatment), therefore it was a 2 × 3 × 3 factorial one with two culture media , three cytokinin types  and three cytokinin concentrations.The first stage of the indirect organogenesis experiment (six treatments) was a 3 × 2 factorial one including three explant types  and two culture media  with 25 explants per treatment and per explant type .The second stage of indirect organogenesis experiment was a 3 × 4 × 2 factorial one consisting of 24 treatments corresponding to three callus explant types , four new tested culture media , and two media initially used  prior to callus formation, i.e., 3 groups (vessels) × 10 calluses per vessel, thus 30 leaf calluses, 30 petiole calluses and 30 root calluses per treatment.O. dictamnus are deployed and underlined. In particular, from the results, it was observed that MS medium provides a better response for micropropagation and ex vitro survival of O. dictamnus when compared with Gamborg B5. It is clearly illustrated that between different cytokinin types and concentrations tested, there is a strong inter-correlation and interaction. Among the 14 combination treatments tested for direct organogenesis, the 2.2 μM BA + 0.25 μM IBA + 0.3 μM GA3 was the most cost- and time-effective one within a single-one stage (30 days). Among the explant types and the culture media tested, shoot regeneration occurred only by leaf- and petiole explants cultured in medium containing 20 μM TDZ + 5 μM IBA. Petiole callus had the best root regeneration performance followed by leaf callus, and leaf callus resulted in shoot or root regeneration. O. dictamnus callus can be utilized for obtaining active constituents, nevertheless, significant scope exists in establishing cell cultures for enhanced production of the desired metabolite in a controlled environment. Further studies could be carried out on the analysis of secondary metabolites produced in vitro. Selected clones with higher bioactive compounds content could satisfy pharmaceutical industry needs, reducing overexploitation of this range-restricted/threatened Greek endemic from nature for plant material sourcing.An efficient in vitro indirect regeneration protocol of the Cretan dittany was carried out and is reported for the first time in the present study. The unravelling and accomplishments of direct and indirect regeneration potential of various plant tissues and organs of


DL) applied to female pelvic MRI, using standard T2-weighted TSE (T2S) as reference.To investigate the feasibility of a deep learning-accelerated T2-weighted turbo spin echo (TSE) sequence  and contrast-to-noise ratio (CNR). Image geometric distortion was evaluated by measuring the diameters in all three directions of the uterus and lesions. Qualitative image evaluation including overall image quality, artifacts, boundary sharpness of the uterine zonal layers, and lesion conspicuity were assessed by three radiologists using a 5-point Likert scale, with 5 indicating the best quality. Comparative analyses were conducted for the two sequences.In total, 24 volunteers and 48 consecutive patients with benign uterine diseases were enrolled. Patients in the menstrual phase were excluded. T2DL resulted in a 62.7% timing reduction . Compared to T2S, T2DL had significantly higher SNR (p ≤ 0.001) and CNR (p ≤ 0.007), and without geometric distortion (p = 0.925–0.981). Inter-observer agreement regarding qualitative evaluation was excellent . T2DL provided superior image quality , boundary sharpness of the uterine zonal layers , lesion conspicuity , and fewer artifacts  in sagittal, axial, and coronal imaging.T2Compared with standard TSE, deep learning-accelerated T2-weighted TSE is feasible to reduce acquisition time of female pelvic MRI with significant improvement of image quality. DL resulted in a 62.7% reduction in acquisition time compared to T2ST2DL can provide higher SNR and CNR, and without geometric distortionT2DL is feasible for reducing the motion-induced blurring and improving image qualityT2 Due to its excellent soft-tissue contrast, MRI is widely used to evaluate malignant and benign diseases of the female pelvis –3. T2-weMultiple strategies have been employed to optimize acquisition of images in a limited timeframe while maintaining sufficient diagnostic value. Efforts to reduce motion artifacts include patient preparation  and scanning techniques . However, these methods do not completely eradicate artifacts and may increase the cost and duration of MR examinations , 10, 11.DL) with that of a standard TSE sequence (T2S) for gynecological T2WI by evaluating examination time, image quality, and lesion conspicuity of benign uterine diseases.With recent advances in artificial intelligence, a deep learning (DL) reconstruction method involving an unrolled variational network allows for a reduction in acquisition time while preserving image quality and realistic textures . The parDL) and standard T2-weighted TSE sequence (T2S). Sample size estimation was conducted using G*Power 3 (version 3.1.9.7), and the statistical test was set as “matched pairs—different between two dependent means/Wilcoxon signed-rank test”. The error was set at 0.05, and the power level was set to 80–95%. Therefore, a total sample size of 22–38 was estimated when considering the difference in quantitative parameter means between T2DL and T2S, while a sample size of 23–39 was obtained when considering the difference in qualitative evaluation score between T2DL and T2S. The latter was selected as the target sample size for the study because it was larger. Then, participants in the study were enrolled in two stages: volunteers enrolled for feasibility evaluation and patients enrolled for clinical application  or qualitative evaluation scores (using Wilcoxon signed-rank test) between the DL-accelerated turbo spin echo (TSE) sequence (T2S) in axial, sagittal, and coronal planes; T2WI acquired with a prototypic DL-accelerated TSE sequence (T2DL)  in axial, sagittal, and coronal planes; axial T1-weighted imaging (T1WI); and DWI with two b values in the axial plane (50 and 800 s/mm2). T2DL was acquired after T2s to ensure the standard-of-care practice for clinical MRI. The detailed parameters for both T2W protocols are listed in Table Participants received a 10-mL glycerin enema in the rectum 30 min before MRI to reduce air in the rectum and sigmoid. A moderately full bladder was required . MRI stuS and T2DL) were randomized, anonymized, and independently evaluated using image-viewing software (RadiAnt DICOM Viewer 2020.1). Quantitative image evaluation was performed by a radiologist with 3 years of experience interpreting pelvic MR images. Whenever possible, operator-defined ROIs were placed on sagittal T2w images in the myometrium, junctional zone, gluteal muscle, and leiomyoma. The ROIs were as large as possible and were devoid of severe artifacts. The ROIs in the gluteal muscle were relatively small because care was taken to avoid areas of macroscopic fat.All axial, sagittal, and coronal T2W images  of images was measured as the noise value. The SNR for the myometrium, junctional zone, and leiomyoma is calculated according to Eq. entclass1pt{minimaDL and T2S images. For the 24 healthy volunteers, the long , short , and transverse  diameters of the uterus were measured on both T2DL and T2S slices in which the uterus appeared largest. Given the relative ease and accuracy of uterine leiomyoma as compared to adenomyosis and scarred uterus measurements, for each affected patient (n = 27), T2DL and T2S slices with the maximum lesion area were examined for the largest lesion, followed by measurements of the anteroposterior, vertical, and transverse diameters of this lesion.Geometric image distortion was evaluated by measuring the diameters in all three directions of the uterus and lesions depicted on both T2All anonymized image datasets were independently evaluated in a random order by three radiologists with 3, 5, and 11 years of experience in interpreting pelvic MR images. The readers were blinded to the sequence types, as image information was concealed in the viewing software. Overall image quality, artifacts, boundary sharpness of the uterine zonal layers, and lesion conspicuity on axial, coronal, and sagittal images were rated by the radiologists using a 5-point Likert scale, with 1 and 5 representing the poorest and best performance, respectively (Table https://www.ibm.com). After testing for normality using the Kolmogorov–Smirnov test, continuous variables conforming to a normal distribution are presented as means ± standard deviations, otherwise as median (interquartile range [IQR]). Ordinal scaled variables are presented as median (IQR). The results of quantitative analysis were compared using a paired t-test for normally distributed variables, or Wilcoxon signed-rank test for nonnormally distributed variables. The distribution and concordance of uterine and lesion diameters in all three directions were obtained on both T2DL and T2S images and were presented in Bland–Altman plots. Qualitative scores were compared using the Wilcoxon signed-rank test. Interobserver agreement was evaluated using the Kendall W test . Two-tailed p values < 0.05 were considered statistically significant.Statistical analysis was performed using SPSS version 21.0.0.0 . Among the 48 patients with benign pelvic diseases, 27 had uterine leiomyoma, 9 had adenomyosis, and 12 had a scarred uterus.In total, 72 participants  were included in this study. The body mass index of participants was 21.58 (19.73–23.81) kg/cmDL and T2S, respectively. The total acquisition time, including all T2-weighted sequences, was 5:42 min and 15:18 min for T2DL and T2S, respectively. Overall, the acquisition time of T2DL images was reduced by 62.7% compared to that of T2S images. A total of 432 image datasets were independently evaluated. The median (IQR) or mean ± SD areas of ROIs in the myometrium, junctional zone, gluteal muscle, leiomyoma, and image background were 69.10 (52.83–76.93) mm2, 11.30 (8.10–17.65) mm2, 25.51 ± 10.69 mm2, 579.75 (153.70–1286.73) mm2, and 1052.03 ± 586.69 mm2, respectively. The mean SNRs of the myometrium, junctional zone, and leiomyoma were significantly higher on T2DL images than on T2S images  . The mean ± SD anteroposterior, vertical, and transverse diameters of the leiomyoma lesions were 44.22 ± 23.87 mm, 43.37 ± 26.65 mm, and 44.28 ± 23.51 mm, respectively, for T2DL and 44.41 ± 24.17 mm, 44.04 ± 27.17 mm, and 44.12 ± 23.63 mm, respectively, for T2S . Bland–Altman plots revealed minimal geometric distortion in the T2DL images. The mean difference in percentage and limits of agreement between T2DL and T2S was − 0.67% to 0.15% . Images from 72 participants were used to evaluate the overall image quality, artifacts, and boundary sharpness of the uterine zonal layers and images from 37 patients with visible lesions were used to evaluate lesion conspicuity. The overall quality of sagittal, axial, and coronal images was rated higher for T2DL (median [IQR] of 5 (4–5), 5 (4–5), and 5 (4–5), respectively) than for T2S (median [IQR] of 4 (4–5), 4 (4–4), and 4 (4–4); all p < 0.001).Interobserver agreement on qualitative image evaluation among the three readers was excellent   scores for T2DL and T2S were 5 (5–5) and 4 (4–5), 5 (4–5) and 4 (4–4), and 5 (4–5) and 4 (4–4), respectively in sagittal, axial, and coronal images . Furthermore, lesion conspicuity was rated superior for T2DL compared with T2S, with a median (IQR) of 5 (4–5) versus 4 (4–4), respectively, for both sagittal and axial images and a median (IQR) of 5 (4–5) versus 4 (4–5) for coronal images . The results of quantitative image evaluation of T2DL and T2S images by the three readers are presented in Table Ghost artifacts were detected in T2were 4 4– and 5 [41) Figs. . In addins Figs. . Scores DL with that of a T2S for gynecological MRI in healthy volunteers and patients with benign uterine disease. Compared to T2S, T2DL produced images with markedly improved SNR and CNR without geometric distortion. Further, T2DL reduced the acquisition time by 62.7%. In addition, T2DL images displayed superior image quality, boundary sharpness of the uterine zonal layers, and lesion conspicuity of benign uterine disease, as well as fewer artifacts in the three planes.This prospective study first compared the feasibility of a T2DL and T2S. Some previously published studies revealed that such measurement method may degrade the SNR performance in parallel imaging, as noise is not evenly distributed in accelerated images reconstructed with parallel imaging [DL vs. T2S) both with parallel imaging reconstruction, and the acquisition parameters, including repetition time, echo time, echo train length, and parallel acceleration factor used in T2DL are identical to those used in T2S. The ROIs on the image background were placed as large as possible (mean area: 1052.03 mm2) and were devoid of artifacts to reduce the impact of noise heterogeneity on the quantitative analysis. We found that although the mean SIs within the ROIs of the two sequences were similar in this study, T2DL images demonstrated considerably lower background noise intensity compared to T2S images. Therefore, the TSE sequence with DL reconstruction resulted in higher SNR and CNR values relative to the conventional approach. In addition, a higher CNR between the myometrium and junctional zone may have enhanced the boundary sharpness of the uterine zonal layers, as T2DL images were rated superior to T2S images on boundary sharpness evaluation by all three readers.In previous studies of the DL reconstruction technique for accelerated abdominal and prostate MR acquisitions, noise was subjectively rated as superior to that of standard sequence images , 24. In  imaging , 26. HowDL images. Consequently, the effect of motion artifacts was smaller for T2DL than for T2S, resulting in more clearly delineated lesion edges and internal structures. Typical findings of adenomyosis, including poorly demarcated low-SI area, intramyometrial cysts, and small high-SI areas on T2W images, were more sharply depicted on T2DL than on T2S, as shown in Fig. DL images were evaluated as superior to T2S images suggests that these accelerated images may be clinically acceptable.Motion artifacts pose a major challenge in pelvic MRI. Periodic motion from the lower anterior abdominal wall and pulsatile vessels, as well as the random motion of the peristaltic viscera are key sources of artifact generation , 28. TheGynecological MRI has been recognized as the optimal imaging modality for the assessment of pelvic diseases in women. Accordingly, the DL reconstruction technique, which accelerates gynecological imaging, offers relevant clinical benefits , 29, 31.DL images. Second, since varying noise distributions resulting from the deployed neural networks may exist, the validity of the SNR and CNR measurement in the present study is not clear. Measurement methods applicable for MR images with the DL reconstruction should be proposed for quantitative evaluation in future studies. Third, patients we included had different benign lesion types, which could potentially affect subjective and objective analyses. Future studies involving patients with the same lesion type are recommended. Finally, the conclusions may have limited generalizability, because only healthy volunteers and patients with benign uterine diseases were enrolled. The image quality and diagnostic performance of T2DL images in women with other pelvic diseases, especially gynecologic malignancies, remain to be elucidated. In this regard, the clinical value of this DL reconstruction technique should be further explored for other gynecological diseases.This study has several limitations. First, as the study involved preliminary experiments using a DL reconstruction technique for gynecological MRI, the study population was small and was derived from a single center. Future multicenter studies involving larger cohorts are needed to evaluate the robustness and diagnostic ability of T2In conclusion, the DL-accelerated T2w TSE sequence is an effective and promising approach that reduces the acquisition time of female pelvic MRI compared to the standard TSE sequence, with the added benefits of significantly improved image quality, boundary sharpness, and lesion conspicuity in patients with benign uterine disease.