G. lucidum, G. tropicum, and C. indica grown in Bangladesh for phenolic and flavonoid content and their antioxidant properties. Moreover, the phenolic composition of the extracts was analyzed by using the HPLC-DAD system. G. lucidum extract exhibited the highest antioxidant potential as evidenced by its lowest IC50 value in all the tested assay models  along with the highest phenolic content (81.34 ± 0.68 GAE g−1 extract). G. tropicum and C. indica extracts also showed significant antioxidant properties and a good amount of phenolic content, 52.16 ± 0.25 GAE g−1 extract, and 47.1 ± 0.26 GAE g−1 extract, respectively. The scavenging activity increased with the increasing concentration of extracts in all cases. The total phenolic content of the ethanolic extracts of mushroom species was highly correlated with antioxidant effects with Pearson's correlation coefficient (r) values ranging from 0.8883–0.9851. The α-amylase inhibitory and antibacterial activity of G. lucidum was evaluated by using 3,5-dinitrosalicylic acid and disc diffusion method, respectively. The maximum inhibitory activity recorded against α-amylase was 70.98 ± 0.042% at a concentration of 500 μg/mL. G. lucidum extract exhibited the highest antibacterial activity against Pseudomonas aeruginosa with 23.00 ± 1.00 mm clear zone of inhibition and an MIC value of 3.5 mg/mL. The results indicate that the mushroom species tested in this study could serve as a potential source of natural antioxidants in the development of nutraceuticals and herbal drugs for the management of oxidative stress-associated diseases as well as infectious diseases.Functional foods such as mushrooms are rich in polyphenolic compounds and secondary metabolites with health-promoting properties such as antioxidant, antimicrobial, antidiabetic and immunostimulatory effects. The present study is aimed to investigate the ethanolic extracts of three varieties of mushrooms, namely,  Reactive oxygen species (ROS) are crucial for cellular activities at physiological concentration. However, excess production of reactive oxygen species and other free radicals may lead to oxidative stress—the imbalance between the generation and accumulation of reactive oxygen species. In addition, human beings are continuously exposed to free radicals produced from chemicals, radiation, and other environmental toxins . OxidatiThe consumption of exogenous antioxidants is pivotal to support the antioxidant defense system by maintaining a sufficient level of antioxidants. Synthetic antioxidants such as butylated hydroxy anisole (BHA), butylated hydroxytoluene (BHT), and propyl gallate (PG) have been used extensively. However, over time, safety concerns have been raised due to their associated adverse effects such as skin allergies, GI issues, and cancer . This haIn recent times, diverse antioxidant compounds have been detected in the Fungi kingdom , 11. MusC. indica, commonly known as the milky mushroom, might be therapeutically effective against oxidative stress and immunodeficiency disorders [G. lucidum or Reishi mushroom is a popular medicinal mushroom due to its health-promoting effects such as antitumor, antimicrobial, anti-inflammatory, antidiabetic, and antioxidant activities [G. tropicum is one of the major wild Ganoderma species recorded in Chinese Pharmacopeia to treat cardiovascular diseases [Calocybe indica, Ganoderma lucidum, and Ganoderma tropicum. The antioxidant potential has been evaluated using multiple in vitro models combined with HPLC analysis. In addition, two in vitro assays, the α-amylase inhibitory activity and the antibacterial activity, were performed to assess the biological activity of G. lucidum extract. Results obtained from the free radical scavenging activities have been examined for their correlation with phenolic contents towards the exploration of possible uses of this plant in the development of herbal drugs and nutraceuticals.Mushrooms are cultivated and consumed worldwide due to their high nutritional content and therapeutic benefits . Reportsisorders . G. lucitivities , 18. G. diseases . It is kdiseases , 21 and diseases . As commdiseases . AgainstC. indica, G. tropicum, and G. lucidum are ARMT 08, ARMT 04, and ARMT 018, respectively.Three species of mushrooms used in this study were collected from the National Mushroom Development & Extension Center, Sobhanbag, Dhaka-Aricha Highway, Savar, Bangladesh. The samples were identified and authenticated by the experts in the National Mushroom Development & Extension center. The Accession no. of the three species, The fruiting bodies of mushrooms were cleaned and washed with distilled water to remove any residual compost. These were then air-dried to constant weight and ground into a coarse powder using a laboratory scale mill and blender. Dried and powdered samples (50.0 g) were extracted with 250 mL ethanol. The extraction was carried out for 7 days accompanied by occasional shaking and stirring. The extracts were then filtered first through a piece of clean, cotton material and finally through Whatman No. 1 filter paper. The residues were extracted again with 100 mL of ethanol. The combined extracts were evaporated to dryness under reduced pressure by a rotary vacuum evaporator. The dried extracts were stored at 4°C in a refrigerator until used for analysis.C. indica, G. tropicum, and G. lucidum were screened for the presence of phytoconstituents such as phenolic compounds, flavonoids, tannins, saponins, and alkaloids according to standard phytochemical screening methods [3, respectively [The ethanolic extracts of  methods . Wagner'ectively . Salkowsectively . Unless ectively .μg/mL) of gallic acid as standard. The results were expressed as mg of gallic acid equivalents (GAE) per gram extract.Total phenolic content in the extracts was determined by the Folin-Ciocalteu assay . Briefly3 colorimetric method was used to estimate the total flavonoid content of the extracts [The AlClextracts . BrieflyThe scavenging activity of extracts was determined using DPPH free radical-scavenging assay . BrieflyA0 is the absorbance of the control and A1 is the absorbance in the presence of the sample.2O2 scavenging activity of the extracts was evaluated as described by Reddy et al. [2O2 (40 mM) was prepared in 0.1 M phosphate buffer (pH 7.4), and 3.4 mL of each extract at varying concentrations was mixed with 0.6 mL H2O2 solution. The mixtures were incubated at room temperature and the absorbance of these mixtures was recorded at 230 nm. Ascorbic acid was used as the positive control. The H2O2 scavenging activity of the extracts and standard compounds was calculated as follows:The Hy et al. . In brieA0 was the absorbance of the control, and A1 was the absorbance in the presence of the sample of extract/standard.Nitric oxide scavenging activity of the extracts was estimated based on the Griess Illosvoy reaction . BrieflyA0 was the absorbance of the control, and A1 was the absorbance in the presence of the sample of extract/standard.C. indica, G. lucidum, and G. tropicum extracts as described previously [μm) Dionex column (4.6 x 250 mm) at 30°C with a flow rate of 1 mL/min and an injection volume of 20 μl. The mobile phase consisted of acetonitrile (solvent A), acetic acid solution pH 3.0 (solvent B), and methanol (solvent C) with the gradient elution program of 5%A/95%B (0-5 min), 10%A/90%B (6-9 min), 15%A/75%B/10%C (11-15 min), 20%A/65%B/15%C (16-19 min), 30%A/50%B/20%C (20-29 min), 40%A/30%B/30%C (30-35 min), and 100%A (36-40 min). The UV detector was set to 280 nm for 22.0 min, changed to 320 nm for 28.0 min, again changed to 280 nm for 35 min, and finally to 380 nm for 36 min and held for the rest of the analysis period while the diode array detector was set at an acquisition range from 200 nm to 700 nm. For the preparation of calibration curve, a standard stock solution was prepared in methanol containing arbutin (AR), (-)-epicatechin (ECA) (5 μg/mL each), gallic acid (GA), hydroquinone (HQ), vanillic acid (VA), rosmarinic acid (RA), myricetin (MC) (4 μg/mL each), caffeic acid (CA), Syringic acid (SA), vanillin (VL), trans-ferulic acid (FA) (3 μg/mL each), p-coumaric acid (PCA), quercetin (QU), kaempferol (KF) (2 μg/mL each), (+)-catechin hydrate (CH), ellagic acid (EA) (10 μg/mL each), trans-cinnamic acid (TCA) (1 μg/mL), rutin hydrate (RH) (6 μg/mL), and benzoic acid (BA) (8 μg/mL). A solution of the extract (10 mg/mL) was prepared in ethanol prior to HPLC analysis and all the solutions  were filtered through 0.20 μm syringe filter  and then degassed in an ultrasonic bath  for 15 min. Data acquisition, peak integration, and calibrations were calculated with Dionex Chromeleon software (Version 6.80 RS 10).HPLC-DAD analysis method was utilized to determine the phenolic composition of eviously . It was G. lucidum extract was determined by a modified Bernfeld's method [μL of the test extract at concentrations ranging from 31.25-500 μg/mL was mixed with 200 μL of α-amylase enzyme solution and 200 μL of 2 mM of phosphate buffer (pH −6.9). After 20 minutes of incubation at room temperature, 200 μL of 1% starch solution was added to the reaction mixture and incubated further for 10 minutes at room temperature. To terminate the reaction, 200 μL of 3,5-dinitrosalicylic acid (DNSA) reagent was added and the tubes are incubated for 5 minutes in a boiling water bath. The mixture was allowed to cool at room temperature and diluted with 5 mL of distilled water. The absorbance was measured by a UV-spectrophotometer at 540 nm and the α-amylase inhibitory activity was calculated as percent inhibition using the following formula:The in vitro alpha amylase inhibitory activity of s method . BrieflyA0 is the absorbance of the control, and A1 is the absorbance of the sample of extract/standard. Commercially available acarbose (Sugatrol 50 mg) was used as a standard.G lucidum extract was determined by the disc diffusion method against five bacterial species. The strains were maintained on agar slants at 4°C and activated by subculturing at 37°C for 24 h before screening [6CFU/mL following a 0.5 McFarland turbidity standard. Using a cotton swab, a suspension of the test microorganisms was swabbed on the Muller Hinton Agar (MHA) medium and the plates were allowed to dry for 5 min. For screening, sterile 6 mm diameter filter paper discs were impregnated with filter sterilized test extracts of G. lucidum (2 mg/disc) and placed on the surface of the seeded agar plates followed by incubation at 37°C for 24 h. Commercially available kanamycin discs (30 μg/disc) were used as reference standard. Discs prepared using the solvent (DMSO) served as the negative control. Antibacterial activity was evaluated by measuring the diameters of zones of inhibition.Antibacterial activity of creening . The oveG. lucidum extract was determined by the broth dilution method in the Mueller Hinton broth supplemented with 10% glucose and 0.5% phenol red [6CFU/mL. The culture tubes were incubated aerobically at 37°C for 24 h. The MIC values were taken as the lowest concentration of the extract that inhibited bacterial growth or change in color from red to yellow, resulting from the formation of acidic metabolites that corresponds to microbial growth [The MIC of enol red , 31. Thel growth , 31. Cull growth .p < 0.05. For evaluation of the possible correlation between phenolic content and the free radical activities of the extracts, Pearson's correlation analysis was carried out. The IC50 values were determined by linear regression analysis.All analyses were carried out in triplicates and the data are expressed as mean ± standard deviation (SD). GraphPad Prism software and Microsoft Excel 2007 were used for statistical analyses. One-way ANOVA and appropriate post hoc tests were performed and differences were considered significant when Qualitative phytochemical screening of the mushroom extracts showed the presence of various secondary metabolites in ethanolic extracts .G. lucidum extract (81.34 ± 0.68 mg GAE/g) was significantly higher than that in C. indica (52.16 ± 0.03 mg GAE/g) and G. tropicum (47.13 ± 0.23 mg GAE/g). TPC found in the ethanolic extract of locally grown G. lucidum was higher than that reported by Sheikh et al. (71.43 ± 0.94 mg GAE/g) [G. lucidum could be used as a potential source of natural antioxidants. Total flavonoid content in the ethanolic extract of G. tropicum (29.31 ± 0.38 mg QE/g) was slightly higher than that in C. indica and G. lucidum  content was highest in G. lucidum extract (11.93 mg/100 g extract), followed by C. indica (8.85 mg/100 g extract) and G. tropicum (8.09 mg/100 g extract) extracts. TFA is known to exhibit anti-inflammatory, cytoprotective, and antioxidant effects and also prevents lipid peroxidation in biological systems [G. tropicum extract (10.53 mg/100 g extract) whereas epicatechin was highest in G. lucidum extract (8.67 mg/100 g extract). Due to their potent antioxidant properties, catechins are found to be beneficial in preventing and protecting from pathologies caused by oxidative stress [Total phenolic and flavonoid contents in the mushroom extracts, expressed as mg GAEs/g of extract and mg QEs/g of extract, respectively, are shown in g GAE/g)  and by Cg GAE/g) . This su lucidum . Polyphe. indica . High amiposomes . Trans-f systems . Catechie stress . These r2O2 scavenging assay, and nitric oxide scavenging assay, have been utilized to assess the antioxidant capacity of ethanolic extracts of C. indica, G. lucidum, and G. tropicum. The scavenging potential of the extracts was expressed in terms of IC50 values . The scavenging activity of the extracts followed the sequence G. lucidum > G. tropicum > C. indica. At a concentration of 500 μg/mL, the scavenging ability of the G. lucidum extract is 93.78 ± 0.42%, whereas the value was 81.43 ± 0.67% for C. indica and 87.01 ± 0.30% for G. tropicum. The lowest activity was observed in G. tropicum having 30.76 ± 0.24% scavenging at the concentration of 31.25 μg/mL. G. lucidum extract also showed lowest IC50 value (40.44 ± 2.1 μg/mL) among the mushroom extracts tested . Varied scavenging effectiveness of G. lucidum extracts has been reported previously. Sheikh et al. from India reported DPPH scavenging efficiency for ethanolic extract of G. lucidum with IC50 of 13.615 μg/mL [G. lucidum [μg/mL. Celik et al. [50 value of 7.03 ± 0.07 μg/mL with ethanolic extract of G. lucidum. Such variations in scavenging activity could be attributed to geographical origin, environmental growth conditions and the type of solvents used for extraction.DPPH scavenging assay is based on the ability of DPPH to react with proton donors. DPPH, the stable free radical, possesses a characteristic absorption at 517 nm which decreases when it reacts with the antioxidants. This decrease in absorption is determined as a measure of the extent of free radical scavenging capability. The use of DPPH radical is advantageous as this free radical is unaffected by side reactions including enzyme inhibition .  Figures tested , indicat15 μg/mL  while thk et al.  from TurR2 = 0.7953 − 0.8975). As shown in G. lucidum showed the highest free radical inhibition at all the tested concentrations whereas C. indica extract exhibited the lowest efficiency. Hydrogen peroxide results in tissue damage in living systems [2O2 indicates their cytoprotective activity, which could be of potential use in the development of herbal medicines. IC50 value of G. lucidum extract . The highest NO scavenging activity was exhibited by G. lucidum extract  [G. lucidum. The results demonstrate that G. lucidum possesses strong NO scavenging property. In this study, G. lucidum extract scavenged 52% nitric oxide radical at 100 μg/mL, which is lower than that reported by Sheikh et al. (63%) [Nitric oxide, an unstable free radical, is known to induce various inflammatory responses leading to tissue toxicity, carcinoma, and pathological conditions such as multiple sclerosis, arthritis, and ulcerative colitis. Nitric oxide reacts with oxygen and generates stable nitrate and nitrites in cells. The free radical scavengers compete with nitric oxide for oxygen and inhibit the overproduction of nitrite ions which mitigates the propagation of inflammation . All the extract  with IC585 μg/mL , which i (μg/mL)  for NO sl. (63%)  from Indr) and coefficient of determination (R2) were determined for each assay model. As shown in 50 values of extracts in antioxidant assays with the Pearson value (r) ranging from 0.8883 to 0.9851. Strong positive correlation was found between TPC and DPPH scavenging  and H2O2 scavenging  activity. Relatively lower correlation was observed with NO scavenging  activity.Total phenols are considered as the major natural antioxidant components and contributors to the biological activities of medicinal plants including mushrooms , 32, 41.G. lucidum extract demonstrated the highest scavenging activity compared to other extracts in all assay models used in this study, which could be attributed to its high total phenolic content. In order to further explore the medicinal effects of G. lucidum extract, two in vitro biological assays were performed, namely, the α-amylase inhibitory activity and antibacterial activity. Use of antioxidants has been found effective in reducing the severity of complications linked with oxidative stress-associated diseases such as diabetes [G. lucidum supplementation has exhibited reduced fasting blood sugar, glycosylated hemoglobin, and improved insulin resistance in animal models [α-glucosidase, and α-amylase inhibitory activities of G. lucidum [α-amylase enzyme is normally found in the pancreatic juice and saliva which helps to break down starch into absorbable molecules. Inhibitors of this enzyme delay the breakdown of carbohydrates in the small intestine and help to control the postprandial blood glucose elevation [G. lucidum showed 70.98 ± 0.042% of α-amylase inhibition (μg/mL). The extract exhibited a dose-dependent increase in the percentage of enzyme inhibition. The standard, acarbose, showed an IC50 value of 88.05 ± 2.96 μg/mL, whereas the G. lucidum extract inhibited the enzyme with an IC50 value of 124.41 ± 3.40 μg/mL. This value found for G. lucidum is lower than that reported by Uddin et al. [μg/mL) indicating stronger α-amylase inhibitory activity of the ethanolic extract. The results obtained from α-amylase inhibition assay suggest that G. lucidum can be used as a functional food for diabetics and also serve as a natural source of antidiabetic drugs.The results obtained in this study are in line with published studies that report correlation between the antioxidant activities and total phenolics, thereby suggesting phenolics as the major contributor towards the antioxidant activity , 43, 44 diabetes . G. lucil models . Previou lucidum , 46–48. levation , hence ahibition  at the hG. lucidum extract was evaluated against five bacterial strains  and Shigella flexneri (16.33 ± 1.15 mm), and the results were comparable with kanamycin  cultivated in Bangladesh reported MIC values ranging from 1 mg/mL to 9 mg/mL against test organisms [G. lucidum.The antibacterial activity of  strains  by disc ruginosa , a gram-anamycin . However studies , 52 that et. al,  which ineviously  against rganisms . VariatiCalocybe indica, Ganoderma lucidum, and Ganoderma tropicum possess significant antioxidant and free radical scavenging activities which could be correlated to their phenolic content. Among the three local varieties of mushroom tested, G. lucidum exhibited the highest antioxidant potential and also demonstrated significant α-amylase inhibitory and antibacterial activities. This suggests that locally grown varieties of mushrooms especially, G. lucidum could serve as a potential source of natural antioxidants and antibacterial agents. Inclusion of these mushrooms in diet and as a functional food could help in alleviating pathologies linked with oxidative stress-associated diseases such as diabetes, degenerative disorders, and aberrant immune response. The significant antibacterial activity demonstrated by G. lucidum extracts against gram-negative bacteria used in this study indicates that it can be exploited as a natural drug in the treatment of several infectious diseases. However, isolation and identification of the specific compounds responsible for the biological activities and in vivo studies are needed for better clinical applications.The present study revealed that


To compare the efficacy of open surgery (OS), endovascular interventions (EIs), and ultrasound‐guided thrombin injection (UGTI) for the treatment of peripheral arterial pseudoaneurysms (PAs).From January 1, 2001, to February 10, 2021, 38 patients diagnosed with traumatic and iatrogenic PAs treated with OS, EI, and UGTI were retrospectively analyzed. There were 18 females and 20 males, with an age of 56.47 ± 14.08 years . Anesthesia modality, operation duration, blood transfusion, duration of hospital stay, primary and secondary success rates, and complication rate were used to evaluate the surgical outcomes.χ2 = 39.80, p < 0.05). The blood tranfusion amount (units) were 3.6 ± 6.0, 0.8 ± 2.5, 0.0 ± 0.0 for OS, EI, and UGTI groups, respectively, with significant difference between OS and UGTI groups . The operation duration (minutes) of OS, EI, and UGTI groups were 80.0 ± 41.9, 56.0 ± 8.4, and 22.7 ± 5.3, respectively, with significant difference between any two groups . The duration of hospital stay (days) were 47.7 ± 39.0, 31.5 ± 17.6, and 16.3 ± 9.5, repectively, with significant difference between any two groups . The primary clinical success rates were 80% (12/15), 90% (9/10), and 92.3% (12/13) in OS,EI, and UGTI groups, respectively, with no significant difference between any two groups . The secondary clinical success rates were 100% for all three groups. The overall complication rates of OS, EI, and UGTI groups were 20% (3/15), 10% (1/10), and 7.7% (1/13), respectively, with no significant difference between any two groups . The infection rates were 13.3% (2/15), 10% (1/10), and 0% (0/13) in OS, EI, and UGTI groups respectively, with no significant difference between any two groups . The reintervention rates were 6.7% (1/15), 0% (0/10), 7.7% (1/13) in OS, EI, and UGTI groups, respectively, with no significant difference between two groups . Neuralgia was relieved in all patients.There were 11 cases under regional anesthesia and 4 under general anesthesia in OS group, 9 under regional anesthesia and 1 under general anesthesia in EI group, and no regional or general anesthesia was required in UGTI group. There was no significant differences between any two groups (OS, EI, and UGTI are efficacious and safe options for the treatment of appropriate patients with traumatic and iatrogenic PAs. UGTI would be considered as a first‐line therapy for this condotion. The open surgery, endovascular intervention, and UTGI are efficacious and safe for treatment of appropriate patients with peripheral artery PA. The patients should be treated individually. Peripheral arterial pseudoaneurysm (PA) is particularly rare, with a reported incidence of 0.06% to 7.7%Treatment options vary from conservative treatment to open surgery (OS), and in recent years, to minimally invasive interventions such as endovascular intervention (EI) and ultrasound‐guided thrombin injection (UGTI)et al. reported on 14 PA cases treated with OS following orthopedic trauma and recognized this treatment option as the standard therapy for PAOS is an important treatment option for PAs with the advantage of a high rate of long‐term vascular patency and the disadvantages of major blood loss and infectionAlthough OS is considered the standard of care, treatment for PA has evolved in recent years from OS to minimally invasive interventions, such as UGTI and EI. Minimally invasive interventions have the advantages of decreased morbidity and mortality rates. UGTI has a high success rate of more than 90% and a low complication rateet al. reported that iatrogenic crural PAs treated with intraluminal stent grafting had poor long‐term patencyet al. reported all covered stents remained patent at 2–19 months of follow‐up in five PA patients treated with endovascular repairEI is an option for treating PAs with varying success rates. Gratl To date, there is still controversy over the optimal treatment for PATaking this into consideration, we retrospectively analyzed the data of OS, EI, and UGTI for PA treatment in our hospital. This study aims to: (i) compare the efficacy and safety of the three aforementioned methods for treating traumatic and iatrogenic PA; (ii) discuss the choice of operation; and (iii) attempt to propose a pragmatic treatment strategy.The study was approved by the Clinical Research Ethical Committee of Tianjin Hospital, with an approval number of 2022‐076. From January 1, 2001, to February 10, 2021, 49 patients diagnosed with traumatic and iatrogenic PA undergoing OS, EI, and UGTI were identified. A total of 38 patients were enrolled in the study, according to the inclusion and exclusion criteria. The patients were divided into three groups based on the treatment modality: OS, EI, and UGTI.The inclusion criteria are as follows: (i) age > 15 years; (ii) PA caused by trauma or iatrogenic injury; (iii) diameter of sac >2 cm; and (iv) treated with UGTI, EI, or OS.The exclusion criteria are as follows: (i) asymptomatic patients; (ii) PA sites at the wrist, hand, foot, or ankle: (iii) infected PA: (iv) ruptured PA; and (v) accompanying arteriovenous fistula.Duplex ultrasonography was used as the first‐line imaging modality for screening and surveillance. This technique can delineate the size of PA, the thrombus within the sac, and shows the patency of the donor arteries. On B‐mode imaging, PA shows an echolucent sac adjacent to the artery. The color Doppler may demonstrate a characteristic “yin‐yang sign” of a whirling flow pattern. The diagnosis relies on the image of sac's neck and “to‐and‐for” waveform between the donating artery and the sac, indicating flow in and out of the pseudoaneurysm sac.Each PA was assumed to be an ellipsoid and the volume was estimated by the formula:3 .The mean size of the PAs was 160 cmOur workflow of treating PA patients is shown in Fig. All of the surgeries were performed by senior doctors: J.L. Zhang, M. Huang, and J. Li independently.OS was performed under regional or general anesthesia. The surgical option was decided individually. Excision with arterial ligation was indicated for expendable minor arteries with good collateral circulation and no distal ischemia. Excision with end‐to‐end anastomosis was performed for vital arteries with a defect size ≦2 cm, while excision with lateral repair was performed for minor injuries of local artery walls. Excision with autogenous saphenous vein graft interposition was preferred for vital arteries with a defect size >2 cm . Results are presented as mean ± standard deviation or proportions. ANOVA was used for continuous variables, such as age, diagnostic period, sac size, volume of blood transfusion, operation duration, and duration of hospital stay. Categorical variables included sex, affected side, location, donor artery, anesthesia modality, success and complication rates of the treatment options. These variables were calculated and compared with Fisher's exact test. A The patients were followed up with a duration of 14 months . The patients underwent UGTI were followed up with Duplex ultrasound the next day and 1 month after operation. The patients treated with EI and OS were examined with color Doppler ultrasound at 1 day, 1 month, 3 months, and 1 year after operation. All patients were followed by telephone thereafter and consulted with a doctor if necessary.There were 18 females and 20 males, with an average age of 56.47 ± 14.08 years . There were 15, 10, and 13 patients in the OS, EI, and UGTI groups, respectively. Among them, 10 PAs resulted from trauma and 28 were iatrogenic. Thirty PAs were located in the lower extremities and eight in the upper limbs, 19 cases on left side and 19 on another side. The patients were diagnosed with an average time of 45 days  delayed after initial injury. Patients typically presented with a progressively enlarging and pulsatile mass, accompanied with pain in most cases. There were 25 patients with comorbidities. Demographic characteristics are shown in Table χ2 = 39.80, p < 0.05).The comparison of results of the different procedures are shown in Table χ2 = 39.80, p < 0.05).The patients in the OS and EI groups required blood transfusion during operation, while those in the UGTI group did not need any transfusion. The volumes of blood transfusion (Units) were 3.6 ± 6.00, 8.0 ± 2.5, and 0.0 ± 0.0 respectively. There was significant difference only between the OS and UGTI groups . The mean duration of OS was the longest, followed by EI, and then UGTI.The operation durations (minutes) were 80.0 ± 41.9, 56.0 ± 8.4, and 22.7 ± 5.3 in the OS, EI, and UGTI groups, respectively, with significant differences between any two groups .The hospital stay (days) was 47.7 ± 39.0, 31.5 ± 17.6, and 16.3 ± 9.5 in the OS, EI, and UGTI groups, respectively. The patients undergoing OS had to stay in the hospital the longest, while those treated with UGTI stayed the shortest. There were significant differences between any two groups . Secondary clinical success rates were 100% for all the groups. There was no PA recurrence in any groups. Neuralgia was relieved in all patients. All of the repaired arteries, covered stents, vascular anastomoses, and vein graft were patent throughout the follow‐up period.Primary clinical success was observed in 12, 9, and 11 patients in the OS, EI, and UGTI groups. The primary clinical success rates were 80%, 90%, and 92.3% in the OS, EI, and UGTI groups, respectively, with no significant difference between any two groups . Wound infection was documented in three patients postoperatively: two in the OS group and one in the EI group. The infection rates were 13.3% (2/15), 10% (1/10), and 0% (0/13) in the OS, EI, and UGTI groups, respectively, with no significant difference between any two groups . Among the three patients, one patient underwent further surgery, while the other two were treated with wound dressing changes and medication. Reinterventions were performed for two patients, one with autogenous vein graft who developed anastomotic breakdown and one due to UGTI failure. The reintervention rates of the OS, EI, and UGTI groups were 6.7% (1/15), 0% (0/10), and 7.7% (1/13), respectively, with no significant difference between any two groups . One patient treated with EI had a mild stenosis 1 year after operation. However, the patient had no signs of distal ischemia and did not require reintervention. No patient presented with ischemia of the affected extremity. There was no procedure‐related mortality in any groups.A total of five patients (13.1%) had complications—three in the OS group, one in the EI group, and one in the UGTI group—with complication rates of 20%, 10%, and 7.7%, respectively. There was no significant difference between any two groups , have yielded promising results and become preferred treatments for PA in recent yearsThe most common complications of PAs are neurologic dysfunction, distal embolization, and ruptureet al. reported their treatment protocol for femoral artery PAs. They presented their experience with patients who had iatrogenic PAs treated with UGTI and OS. They recommended UGTI as the first‐line option for PAset al.et al.Although the traumatic and iatrogenic PAs can be treated with all the three aforementioned methods, there remains no consensus on the practice guidelines for the management due to the high variability of presentation and the limited availability of relevant high‐quality literature. To date, most opinions arise from case series in literature. The treatment strategy depends on several factors, such as anatomic location, neck width, sac size, features of the donor artery, rupture risk, patient comorbidity, and physician's decision. Because of their unpredictable nature, surgical treatment is warranted for asymptomatic PAs with or without spontaneous thrombosisThis study has several limitations. First, this is a retrospective analysis and the treatment options are not randomized. Second, the follow‐up is relatively short, and long‐term outcomes should be evaluated in future studies. Finally, the size of the study cohort is relatively small.Traumatic and iatrogenic PAs remain a challenging clinical problem. OS, EI, and UGTI are efficacious and safe options for PA management. The correct treatment should be based on a thorough understanding of PA pattern and its characteristics. UGTI is simpler, faster, and more effective than the other two methods. EI and OS may be reserved for those with failed UGTI. OS can be utilized as a final solution. Satisfactory clinical outcomes can be achieved when PAs are correctly approached. We recommend a considerably pragmatic workflow for treatment.The authors have no conflicts of interest to declare.


Vitamin D [25(OH)D] is essential for normal bone development and maintenance. Furthermore, its deficiency has been associated with obesity, cardiovascular diseases, insulin resistance, autoimmune diseases, and certain cancers.To determine the incidence of serum 25(OH)D deficiency (<20 ng/ml) among apparently healthy Chilean children (4–14 years old) from three Chilean geographic areas during May–September 2018.Serum 25(OH)D levels were measured by a competitive protein-binding ELISA assay in 1134 children, and correlations between serum 25(OH)D levels, BMI, and geographic area were calculated. Individuals were grouped according to their serum 25-hydroxyvitamin D levels (ng/ml): severe deficiency: <5; moderate deficiency: 5–10.9; mild deficiency: 11–20.9; insufficiency: 21–29.9 and sufficiency: 30–100.p = 0.038). In Antofagasta, there were more overweight children and also a higher percentage of children with VitD deficiency (<30 ng/ml) compared to Santiago or Concepción.We found 80.4% of children had serum 25(OH)D deficiency, with 1.7% severe, 24.6% moderate, and 54.1% mild. In the three cities, the percentage of serum 25(OH)D deficit was increased when comparing overweight or obesity with a healthy weight. Additionally, an interaction effect was observed between geographic area, nutritional status, and serum 25(OH)D levels using the factorial ANOVA test D deficiency in children between 4 and 14 years old in Chile (80.4%) during May–September 2018. Obese and overweight children had the highest prevalence of serum 25(OH)D deficiency. Vitamin D (VitD) is essential for developing and maintaining a healthy skeleton, and it has been linked to reduced risk for acute and chronic illnesses . VitD deIn Chile, a limited number of studies conducted on healthy children have shown evidence of the impact of nutritional status and sunlight exposure on VitD levels .Childhood obesity is a public health problem in Chile. The latest data published by the OECD show that 74% of the adult Chilean population are overweight or obese. Furthermore, the FAO report indicates that Chile has one of the highest rates of childhood overweight (9.3%) in Latin America and the Caribbean .The objective of this study was to analyze serum 25(OH)D levels in a representative sample of children between 4 and 14 years of age located in the Santiago, Antofagasta, and Concepción.The study was conducted from May to September 2018 with 1134 children aged 4–14 years from different latitudes and UV index (UVI): Antofagasta , Santiago , and Concepción . Study participants were recruited independently of nutritional status according to gender, age group, and socioeconomic status. Children were grouped according to their serum 25(OH)D levels [Anthropometric measurements were made with children in light clothing, weight, and height were measured with a portable stadiometer (SECA 813/213). The nutritional status classification was carried out according to the WHO standards for the age and gender of the children. Children with BMI/Age < +1 DS were classified as healthy weight, those with BMI/Age between +1 SD and +2 SD were classified as overweight, and those with BMI/Age ≥ +2 SD as obese. Children with chronic diseases, such as diabetes, cancer, or chronic kidney disease, were excluded. Informed consent was obtained from all subjects. The study protocol was reviewed and approved by the Ethics Committee of the University of Chile.Serum 25(OH)D was measured in triplicate using an Enzyme-Linked Immunosorbent Assay kit . Sensitivity of 2 ng/ml with an intra-assay variation coefficient of 5.1% and an inter-assay variation coefficient of 5.9%.p < 0.05 was considered significant.Results are presented as percentages (%) and means ± SD. All calculations were performed using the software package SPSS version 15.0.1 . Shapiro–Wilk normality test was performed to determine the distribution of the variables. Pearson correlation test, factorial ANOVA, one-way ANOVA, and Tukey posthoc analysis were used. In all cases, a value of p = 0.041). In the three cities, the serum 25(OH)D deficit was increased when comparing overweight or obesity with a healthy weight. An interaction effect was observed between geographic area, nutritional status, and VitD levels using the factorial ANOVA test (p = 0.038). In Antofagasta, there were more overweight children and also a higher percentage of children with VitD deficiency (<30 ng/ml) compared to Santiago or Concepción.Antofagasta’s children presented both the highest weight, height, waist circumference, BMI, and height/age ratio relative to the other cities evaluated in this study. We showed a high level of serum 25(OH)D deficiency in the three cities. The serum 25(OH)D levels were significantly higher in Antofagasta compared to Concepción Table . In Antop < 0.0001).Only 22.4% in Antofagasta, 20.9% in Santiago, and 15.5% in Concepción reached levels of serum 25(OH)D > 20 ng/ml , Antofagasta 73–84% (13–24 °C), and Concepción 40–60% (6–16 °C). Despite this variation, we believe that the high prevalence of overweight/obesity in these regions is the primary condition for VitD sequestration or dilution. In the southern areas of Chile (Punta Arenas), a high prevalence of obesity has been described in school children groups, accompanied by severe VitD deficiencies. Obesity and lack of exposure to sunlight are likely to cause these severe deficiencies . While o


ALP, AST, ALT, MDA, NO, IL-6, TNF-α, and caspase-3 increased significantly in the RIR group compared to the control group (p < 0.05). GSH, GPX, and CAT decreased significantly in the RIR group compared to the control group (p < 0.05). TE caused a decrease in ALP, AST, ALT, MDA, NO, IL-6, and TNF-α compared to the RIR group and caused an increase in the amount of GSH, GPX, and CAT in the RIR group (p < 0.05). This study showed that TE has antioxidant and anti-inflammatory properties that reduce liver damage induced by RIR.Liver damage occurs following renal ischemia-reperfusion (RIR) that can cause inflammation and inflammatory cytokines activated after kidney injury. In this study, thyme essential oil (TE) with antioxidant and anti-inflammatory properties was used to reduce liver damage induced by renal IR. 32 male rats were randomly divided into 4 equal groups: (1) control, (2) RIR, (3) RIR+TE, and (4) TE. Rats received TE as a pretreatment at a dose of 0.5 ml/kg for one week. Then, under anesthesia for 45 minutes for ischemia, the kidneys of the animals were closed with clamps, and reperfusion was performed for 24 hours. Animal serum was isolated to evaluate alkaline phosphatase (ALP), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) parameters. The liver of rats was examined for the measurement of malondialdehyde (MDA), nitric oxide (NO), glutathione (GSH), glutathione peroxidase (GPX), catalase (CAT), and expression of genes such as interleukin-6 (IL-6), tumor necrosis factor- The mortality rate is 11%, and if it is associated with dysfunction of other organs, it increases to 45-60% . RIR may (TNF-α) . Also, s (TNF-α) . Due to Thymus vulgaris Leaf is an aromatic plant that grows mainly in the Mediterranean region. Thyme essential oil (TE) consists of thymol, carvacrol, eugenol, saponins, steroids, alkaloids, flavonoids, polyunsaturated fatty acids, cinnamon, paracetamol, and vitamins A and C –10. The α, and IL-6 were investigated.The aim of our study was to investigate the effect of thyme oil on oxidative stress, apoptosis, and inflammatory of the liver following RIR. In this study, parameters such as AST, alanine aminotransferase (ALT), ALP, oxidative stress tests, liver antioxidant enzymes, expression of caspase-3, TNF-Zataria multiflora Boiss (Shirazi thyme leaves) was purchased from Shiraz markets and powdered by grinding machine after drying. 100 gr of thyme powder along with 500 ml distilled water was poured into an Erlenmeyer flask and heated at 100°C for 2 hours. After that, its essential oil was extracted through steam distillation by a clevenger device (brand: BORO G). This device has a section known as condenser which converts steam and essential molecules into liquid while passing through it. This liquid, essential oil, is then stored in the containers away from light at 4°C in the refrigerator until injection.In this study, 32 male Wistar rats weighing approximately 200-220 gr were kept in the animal house of Dezful University of Medical Sciences in the same conditions, with a temperature of 22-24°C and a humidity of 45-55% with 12 hours of darkness and light. Throughout the treatment protocol, water and food were freely available to the animals. This research was performed with the approval and based on the guidelines of Animal Care and Ethics Committee of Dezful University of Medical Sciences, Dezful, Iran .Thirty-two male Wistar rats were randomly divided into four groups of eight: group 1—control, under anesthesia, laparotomy without renal ischemia-reperfusion surgery; group 2—ischemia reperfusion (RIR), 45 minutes of ischemia and then 24 hours of perfusion; group 3—RIR + TE, RIR group with injection of 0.5 ml/kg of TE intraperitoneally , 12 for Animals were not allowed to eat something by mouth, and they were kept NPO for 8 hours before surgery. Rats were anesthetized with 75 mg/kg ketamine and 8 mg/kg xylazine. A midline incision was made in the abdomen, the kidney pedicle was closed for 45 minutes, and then reperfusion was performed for 24 hours. Blood was taken from the hearts of animals under anesthesia, and their livers were isolated for biochemical analysis and gene expression.2 (cm2) [The body weight in rats was measured at the beginning of treatment and before death. Body mass index (BMI) was calculated through this formula: BMI = body weight (grams)/length2 (cm2) . Also, tThe daily water and food consumption of each cage was measured for one week, and the average water and food intake was calculated .The blood sample was centrifuged to separate serum at 3000 rpm for 10 minutes. ALP, AST, and ALT in the serum were measured with Pars Azmon commercial kits by autoanalyzer tool.μl of sample and 500 μl of 2 M AcONa buffer (pH = 3.5) with 0.2% TBA were mixed and incubated for 1 h at 95°C. Then, 500 μl of 50 mM KH2PO4 buffer (pH = 6.8) was added to each sample and centrifuged for 5 minutes at 4°C; and finally, absorption of the supernatant was read by spectrophotometry at 532 μm.Liver MDA was performed by thiobarbituric acid method . Brieflyμl of 1% sulfonamide (in 5% H3PO4) and 50 μl of a solution of 0.1% naphthylethylenediamine and 100 μl of samples was prepared; then, its absorption at 560 nm was read by the ELISA reader instrument [Liver nitric oxide was measured by Griess method. In summary, a mixture of 50 strument .2O2 (75 mM) was added to 1.5 ml of 0.1 M phosphate buffer (pH = 7) and 50 μl of sample in the reaction mixture. Its absorption reduction was read at 240 nm for 1 minute with a spectrophotometer instrument.Liver catalase was performed by the Aebi method . Brieflyμl of a homogenized liver sample with 1.25 ml of reagent , 100 μl cumene hydroperoxide (0.18 mmol) was prepared, and the reduction of NADPH adsorption at 470 nm was read by ELISA reader instrument.Liver GPX was performed by a fielding method . In summLiver GSH was performed by Zhang and Kirkham method . Brieflyα, interleukin-6, and caspase-3 in the liver was detected using RT-qPCR. Each 20 μl reaction consisted of 1 μl of cDNA, 10 μl SYBR Green qPCR Mix (1 X), 1 μl of primer, and 8 μl of DEPC water. The reactions were subjected to an initial denaturation of 95°C for 10 minutes before thawing, then 40 cycles at 95°C for 10 seconds, and annealing for 30 seconds at 60°C, and then final extension step at 72°C for 10 seconds. Calculation of gene-specific efficiencies and normalization to the mean expression of β-actin were calculated [Using Trizol reagent, RNA of all liver samples was extracted according to the manufacturer's instructions (AnCell Iran). The concentration and purity of RNA were determined by Thermo NanoDrop. Using the CDNA synthesis kit , the complementary DNA of the first strand was synthesized according to the manufacturer's instructions. Expression of TNF-lculated . Used prp value < 0.05. Value was expressed as mean ± standard error of the mean (SEM). The diagrams were drawn with GraphPad Prism software.Data were analyzed by SPSS 23 software and one-way analysis of variance (ANOVA). The significance level of the data was considered F  = 0.46, p = 0.71). One-way ANOVA showed no significant difference between the groups in final body weight  = 0.58, p = 0.63)  .F  = 4.78, p = 0.008). Liver weight in the RIR group showed a significant increase compared to the control group (p < 0.05). Liver weight in the RIR+TE and TE groups showed a significant decrease compared to the RIR group (p < 0.05). No significant difference was found between sham and TE groups in liver weight (p > 0.05)  .F  = 0.26, p = 0.85). No significant difference in food intake was observed between different groups  = 0.33, p = 0.8). No significant statistical difference was observed between groups in water intake  = 0.15, p = 0.91) (There was no significant difference in BMI between different groups ( = 0.91) .F  = 274.97, p < 0.0001). Serum ALP in the RIR group showed a significant increase compared to the control group (p < 0.0001). Serum ALP in the RIR+TE and TE groups showed a significant decrease compared to the RIR group (p < 0.0001). In this study, no significant difference was found between sham and TE groups in serum ALP (p > 0.05) (A significant difference in serum ALP level was observed between different groups ( > 0.05) .F  = 69.84, p < 0.0001). Serum AST in the RIR group showed a significant increase compared to the control group (p < 0.0001). Serum AST in the RIR+TE and TE groups showed a significant decrease compared to the RIR group (p < 0.0001). In this study, no significant difference was found between the control and TE groups in serum AST (p > 0.05)  .F  = 5.5, p = 0.004). Serum ALT in the RIR group showed a significant increase compared to the control group (p < 0.05). Serum ALT in the RIR+TE and TE groups showed a significant decrease compared to the RIR group (p < 0.05). In this study, no significant difference was found between sham and TE groups in serum ALT (p > 0.05)  .F  = 184.22, p < 0.0001). MDA levels in the RIR group showed a significant increase compared to the control group (p < 0.0001). MDA levels in the RIR+TE and TE groups showed a significant decrease compared to the RIR group (p < 0.0001). In this study, MDA level in TE group showed a significant increase compared to control group (p < 0.0001)  .F  = 116.72, p < 0.0001). NO levels in the RIR group showed a significant increase compared to the control group (p < 0.0001). NO levels in the RIR+TE and TE groups showed a significant decrease compared to the RIR group (p < 0.0001). NO level in the TE group showed a significant increase compared to control group (p < 0.0001)  .F  = 431.36, p < 0.0001). CAT activity in the RIR group showed a significant decrease compared to the control group (p < 0.0001). CAT activity in the RIR+TE group showed a significant increase compared to the RIR group (p < 0.001). CAT activity in the TE group showed a significant decrease compared to that in the control group (p < 0.0001)  .F  = 183.95, p < 0.0001). Activity of GPX in the RIR group showed a significant decrease compared to the control group (p < 0.0001). Activity of GPX in the RIR+TE and TE groups showed a significant decrease compared to the RIR group (p < 0.0001). GPX activity in the TE group showed a significant decrease compared to the control group (p < 0.0001)  .F  = 84.68, p < 0.0001). GSH levels in the RIR group showed a significant decrease compared to the control group (p < 0.0001). GSH levels in the RIR+TE group showed a significant increase compared to the RIR group (p < 0.0001) and GSH levels in the TE group showed a significant decrease compared to the RIR group (p < 0.05). There was a significant decrease in GSH level in the TE group compared to control group (p < 0.0001)  .F  = 20.58, p < 0.0001). The mRNA expression of IL-6 in the RIR group compared to the control group showed a significant increase (p < 0.0001). The mRNA expression of IL-6 in the RIR+TE and TE groups compared to that in the RIR group showed a significant decrease (p < 0.0001)  = 23.17, p < 0.0001). The mRNA expression of TNF-α in the RIR group compared to the control group showed a significant increase (p < 0.0001). The mRNA expression of TNF-α in the RIR+TE and TE groups compared to the RIR group showed a significant decrease (p < 0.05)  . A signi < 0.05) .F  = 65.38, p < 0.0001). The mRNA expression of caspase-3 in the RIR group compared to the control group showed a significant increase (p < 0.0001). The mRNA expression of caspase-3 in the RIR+TE and TE did not show significant differences with the RIR group (p > 0.05). The mRNA expression of caspase-3 in the TE group showed a significant increase compared to that in the control group (p < 0.0001) (There was a significant difference between different groups regarding mRNA expression of caspase-3 in liver ( 0.0001) .α, IL-1β, and IL-6 [α in the liver tissue. With reducing oxidative stress and inflammation and increasing antioxidant activity [Liver injury induced by RIR can cause damage to other organs and their death . Currentand IL-6 . In the activity , 33, in activity . TE, witactivity . Accordiactivity . Therefoactivity . Crocin activity . Followiactivity . In anotactivity . Carvacractivity . Carvacractivity . 200 mg/activity . Thymol The results of our study showed that pretreatment with thyme essential oil for one week can protect the liver from damage caused by renal ischemia-reperfusion due to its antioxidant and anti-inflammatory effects. In this study, thyme oil showed apoptotic activity in liver tissue, in which it is suggested to investigate different doses of thyme oil in future studies.


Klebsiella pneumoniae is a major cause of nosocomial infection and is considered a clinically important bacterium with antibiotic-resistant strains. There are few reports of K. pneumoniae infections in cultured aquatic animals, and no natural infection has been reported in amphibians. From September to October 2021, a high-mortality disease outbreak occurred in a pond-raised American bullfrog farm in Guangzhou, China. The infected bullfrogs were characterized by multiple organ congestive enlargement and inflammation. A pathogenic bacterium was isolated from the viscera of infected bullfrogs and confirmed to be K. pneumoniae by morphological, biochemical, and phylogenetic analyses. Infection experiments confirmed the virulence of the pathogenic strain against bullfrogs and tadpoles. A histopathological examination showed that the strain was harmful to multiple organs. Antibiotic resistance experiments indicated the isolate was a carbapenemase-producing multidrug-resistant K. pneumoniae (MDR-KP) strain. This study is the first report of K. pneumoniae infected American bullfrogs (Rana catesbeiana) and amphibians. These results will shed light on the pathogenicity of K. pneumoniae and help prevent and control K. pneumoniae infections in bullfrogs.IMPORTANCEKlebsiella pneumoniae is recognized as the most common multidrug-resistant bacterial pathogen in humans, and little is known about its pathogenicity in aquatic animals. Recently, K. pneumoniae was found to cause substantial mortality and morbidity in American farm frogs. This was the first report of K. pneumoniae infecting amphibians. In this study, we analyzed the biochemical, growth, and phylogenetic characteristics of the K. pneumoniae strain and described the symptoms and pathological features of infected bullfrogs and tadpoles; this will provide useful data for the prevention and control of infectious diseases, which has been suggested to decrease economic losses in bullfrog farming and reduce the potential threat to public health posed by K. pneumoniae. Klebsiella pneumoniae is a Gram-negative bacterium within the Enterobacteriaceae family, first described in 1882  is a semiaquatic frog first described in 1802 .Fifteen purified isolates were obtained from diseased bullfrogs. The 16S rRNA gene sequences of all isolates were amplified and sequenced. Sequencing analyses showed that the 16S rRNA genes of forty-five isolates were 99.9% to 100% homologous. One bacterial isolate was selected and named NW202109 for further identification and detailed investigation. The isolate NW202109 produced large, dome-shaped, sticky colonies with diameters of 0.8 to 2.2 mm on 5% sheep blood medium . TransmirpoB, and gyrB partial gene sequences of NW202109 were determined and submitted to the GenBank database . The sequences of 16S rRNA, rpoB, and gyrB were most similar to those of K. pneumoniae, with 97.8% (CP052449.1), 99.04% (CP054303.1), and 99.87% (CP072422.1) identity, respectively. Therefore, the phylogenetic trees were constructed based on the same gene sequences and a homologous series of NW202109, as shown in Klebsiella. In the phylogenetic trees derived from the rpoB and gyrB gene sequences, NW202109 clustered with K. pneumoniae.The 16S rRNA, NW 202109 survived at a pH of 3 to 11 and grew best at a pH of 6 to 9, with an optical density (OD) of 1.7 to 1.75 . Limited6 CFU/g) showed abdominal distention, slow movement, and one acute death 2 days after artificial infection. High mortality occurred between 10 and 12 days after infection, and the cumulative mortality rate reached 80%. When the bacterial challenge dose dropped to 4 × 105 CFU/g, the bullfrogs showed similar signs, one acute death occurred, and the final cumulative mortality decreased to 30% (4 CFU/g) died on the fourth day, and no significant signs were observed in the remaining infected bullfrogs. No deaths or visible changes were observed in the control groups. Artificially infected bullfrogs were dissected and showed congestive enlargement of multiple organs, similar to that of the naturally infected bullfrogs. K. pneumoniae was isolated and identified from all dying infected bullfrogs.Bullfrogs with the highest bacterial challenge dose (4 × 10d to 30% . In cont7 CFU/mL) began to bloat and swim in rolls, and one died on the first day after the immersion experiment ; intermediately susceptible to aztreonam and doxycycline; and resistant to penicillin, ampicillin, imipenem, neomycin, erythrocin, medemycin, and vancomycin. These results were the same at both 35°C and 28°C. In addition, NW202109 was sensitive to kanamycin at 35°C and intermediate at 28°C, and sensitive to chloramphenicol at 35°C and resistant at 28°C .K. pneumoniae (NW202109) was isolated from diseased American bullfrogs. Infection experiments showed that the strain was highly virulent to bullfrogs, and the clinical signs in infected bullfrogs were similar to those observed in naturally diseased bullfrogs. The same pathogenic bacteria were then isolated from the tissues of experimentally infected bullfrogs, fulfilling Koch’s postulates  for bacterial identification. Sequence analysis based on the 16S rRNA gene is a standard method of bacterial identification and was once considered the “gold standard” for bacterial identification .Fifteen moribund bullfrogs from affected farms were dissected using aseptic techniques. Tissue samples were taken from the liver, kidney, and spleen. The tissues were used to inoculate a 5% sheep blood medium  to culture the bacterium for 18 h at 28°C for isolation and purification. The resulting single colonies were subcultured on the same medium to check the purity of the isolated strain.Before the experiment, the isolates were subcultured overnight in brain-heart infusion (BHI) broth  at 28°C. The biochemical characteristics, hydrolytic activity, and carbon source utilization of isolated bacteria were evaluated using the BD Phoenix Automated Microbiology System . Morphology was characterized using a HT7800 transmission electron microscope .7 CFU/mL) and incubated at 28°C. The salinity of sterile BHI broth was adjusted to 0% to 6% over 13 gradients. For salinity, 5 mL sterile BHI broth was inoculated with 50 μL of bacterial suspension (2 × 107 CFU/mL) and incubated at 28°C for each gradient. All samples were cultured at 200 rpm/min for 18 h, and the concentration of the culture was detected by measuring the absorbance of the culture at 600 nm (A600).In this study, we verified the effects of pH and salinity on the survival and growth of the bacterium. The sterile BHI culturing broths varied from pH 3 to 11. At each pH gradient, 5 mL of sterile BHI broth was inoculated with 50 μL of bacterial suspension , 21.5 μL sterile double-distilled water, 1 μL DNA template (100 ng), and 1.25 μL of each specific primer (10 mm).Thermal cycler conditions were set as follows: 94°C for 5 min; 32 cycles of 95°C for 25 s, 56°C for 25 s, 72°C for 30 s, and 72°C for 5 min; and storage at 4°C. The PCR products were sequenced directly by Tianyi Huiyuan Gene Technology Co., Ltd., with sample preparation following the manufacturer’s instructions. Sequence analysis was performed using the BLAST database network service and MEGA v7.0.20. The adjacency neighbor-joining tree algorithm, Kimura 2-parameters, and 1,000 bootstrap copies were used to infer the unrooted evolutionary tree.DNA from the isolated bacterium was extracted with a genomic DNA extraction kit  following the manufacturer's guidelines. The DNA was stored at −80°C until required for genetic analysis. PCR amplification primers for the 16S rRNA, u et al. . The PCRTo determine the pathogenicity of the isolated bacterium, challenge experiments were conducted using healthy bullfrogs and tadpoles. Both bullfrogs and tadpoles were split into four groups: three groups were challenged, and one served as a negative control.9, 1 × 108, and 1 × 107 CFU/mL, respectively; the negative-control group received a 0.65% NS injection. For the tadpole groups, 10 tadpoles from each group were immersed in 2 L bacterial suspension for 20 min at concentrations of 1 × 107, 1 × 106, and 1 × 105 CFU/mL, respectively. Tadpoles in the control group were immersed in 0.65% NS water. After the immersion challenge, all the tadpoles were maintained in aerated freshwater. Clinical signs and mortality were observed daily for 14 days, and bacteria were isolated and identified from infected bullfrogs and tadpoles.Ten bullfrogs were each challenged with 200 μL of the bacterium suspended in 0.65% sterile normal saline (NS) through intramuscular injection. For each bullfrog group, the concentration of the bacterial solution was adjusted to 1 × 10The brain, heart, liver, spleen, kidney, intestine, and lung of the infected bullfrogs with obvious signs were fixed with 4% (wt/vol) paraformaldehyde for 96 h, dehydrated with 80%, 90%, 95%, and 100% graded concentrated ethanol, made transparent with xylene, and embedded in paraffin, and sections were made from 3 to 5 μm. Sections were stained by Mayer’s Hematoxylin and Eosin and observed by light microscopy.9 CFU/mL was tiled onto Mueller-Hinton agar plates prepared with drug-sensitive tablets . The inoculated plates were cultured at 35°C and 28°C for 24 h to determine the inhibition zone diameter. According to the M100 Performance Standards for Antimicrobial Susceptibility Testing, 31st edition (The isolated strain was screened for sensitivity to 18 antibiotics using the Kirby Bauer disk-diffusion method . For the edition , the res


Pseudomonas resinovorans. The oil–extracted SCGs (OESCGs) were hydrolyzed and used for biohydrogen production through Clostridium butyricum DSM10702. The oil extraction yield through n–hexane was 14.4%, which accounted for 97% of the oil present in the SCGs. OESCG hydrolysate (OESCGH) had a sugar concentration of 32.26 g/L, which was 15.4% higher than that of the SCG hydrolysate (SCGH) (27.96 g/L). Hydrogen production using these substrates was 181.19 mL and 136.58 mL in OESCGH and SCGH media, respectively. The consumed sugar concentration was 6.77 g/L in OESCGH and 5.09 g/L in SCGH media. VFA production with OESCGH (3.58 g/L) increased by 40.9% compared with SCGH (2.54 g/L). In addition, in a fed–batch culture using the extracted oil, cell dry weight was 5.4 g/L, PHA was 1.6 g/L, and PHA contents were 29.5% at 24 h.Coffee waste is an abundant biomass that can be converted into high value chemical products, and is used in various renewable biological processes. In this study, oil was extracted from spent coffee grounds (SCGs) and used for polyhydroxyalkanoate (PHA) production through  Coffee is the world’s most-consumed drink, and about 167.17 million metric tons of coffee were produced in 2021 . Based oOrganic waste and biomass resources  usually have a large amount of carbohydrates that can be used by microorganisms, so they are used in the form of monosaccharides or oligomers through hydrolysis ,6,7. ThiRecently, interest in hydrogen energy and bioplastics is increasing because of global issues such as environmental pollution and fossil fuel depletion ,13. HydrPseudomonas sp. is a representative strain known to produce mcl–PHA [Pseudomonas sp. is able to use fatty acids such as olive oil and waste cooking oil, thereby enhancing the value of various organic waste resources [Bioplastics are eco–friendly plastics that can be produced or decomposed by microorganisms . Similar mcl–PHA . Severalesources .Therefore, this study aims to (i) investigate SCG oil extraction and yield, (ii) hydrolyze oil–extracted SCGs and use them as hydrogen production substrates, (iii) investigate PHA production possibilities using extracted SCG oil, and (iv) investigate the possibility of mass culture using a 10 L fermenter.2SO4, n-hexane, HCl, NH4Cl, NaOH, methanol, NaCl, sodium acetate, sodium bicarbonate, palmitic acid, oleic acid, stearic acid, linoleic acid, l–cysteine hydrochloride and antifoam were purchased from Daejung Chemical & Metals . LB media, yeast extract, peptone, beef extract, soybean extract and urea were purchased from BD-DIFCO .All reagents and chemicals were procured at the highest purity. M9 minimal salts (5×), 3-Hydroxyoctanoic acid, 3-Hydroxydecanoic acid, and 3-Hydroxydodecanoic acid were purchased from Sigma Aldrich . Chloroform, Hn–hexane as a solvent, and 25 g of SCGs was put into a weighed cellulose filter for oil extraction for 1.5 h. The heating mantle was kept at 70 °C. After oil extraction was completed, the filter containing SCGs was dried in a fume hood for 24 h to evaporate the n–hexane and then weighed. The oil mixed with the n–hexane was purified by evaporation at 50 °C and 50 rpm for 30 min using a rotary evaporator. The concentrated SCGs oil was weighed to confirm the amount of the n–hexane residue and compared with the SCGs weight change. Mass extraction was carried out for future experiments with the products sterilized at 121 °C for 15 min in an autoclave and then refrigerated.SCGs were obtained from a local cafe in Cheonan-si, South Korea. To dry the SCGs, they were spread widely and sun–dried for 8 h and then dried in a drying oven at 105 °C for 24 h. Oil was extracted from the dried SCGs using a Soxhlet extractor with 200 mL of Gas chromatography–mass spectrometry  analysis was performed to analyze the main components of the extracted SCGs oil, and a gas chromatograph  was used for quantitative analysis.n–hexane. Then, hydrolysis was performed with a 10% solid/liquid (S/L) ratio and 1% H2SO4 at 130 °C for 1 h. OESCG hydrolysate (OESCGH) was centrifuged at 3500 rpm for 30 min, and the supernatant was recovered. The composition of the OESCGH was analyzed through high–performance liquid chromatography . The OESCGH was mass–produced and then sterilized in an autoclave for 15 min at 121 °C before being refrigerated for use in subsequent experiments.The oil–extracted SCGs (OESCGs) were used as a substrate for hydrogen production. OESCGs were dried in the fume hood for 12 h to remove residual Clostridium butyricum DSM10702 was used for hydrogen production and was inoculated in the anaerobic chamber under 99.9% of N2. The culture conditions were pH 5.5 (adjusted by HCl), 37 °C, and 150 rpm. Gas production, hydrogen production, and metabolite change were measured for 32 h. Defined RCM was composed as follows: 3 g/L yeast extract, 10 g/L peptone, 10 g/L beef extract, 0.5 g/L L–cysteine hydrochloride, 3 g/L sodium acetate, 5 g/L sodium chloride, 5 g/L sodium bicarbonate, and 100 μL/L antifoam.An experiment was conducted to compare the hydrogen productivity of general SCG hydrolysate (SCGH) and OESCGH. The culture medium was defined–reinforced clostridium medium (RCM). Here, SCGH or OESCGH was added and diluted to a total sugar concentration of 10 g/L. Pseudomonas resinovorans, which is known to produce mcl–PHA using oil as a substrate, was used as the inoculum. A stock stored in a deep freezer was pre–cultured in 5 mL of lysogeny broth (LB) media. Pre–cultured bacteria were inoculated into the main culture medium after 24 h. The medium was M9 salt medium, and 2% oil was added as a substrate. After that, for C/N ratio optimization, various N sources were added at 0.1% and 0.5%, and productivity was confirmed. A fed–batch culture was performed using ammonium chloride as an N source and SCGs oil as a substrate. The reactor was a 10 L fermenter , with a working volume of 3 L, in duplicate, and inoculation volume of 10%. The medium was M9 salt medium, initial SCGs oil concentration was 2%, and SCGs oil was fed at 5% at 4 h and at 18 h. An amount of 1 M HCl and 3 M NaOH were used to adjust the pH to 6.8 ± 0.2, and aeration was maintained at 10 L/3 L/1 min to adjust to 30% dissolved oxygen (DO). The reactor was operated at 600 rpm and 30 °C for 96 h, and samples were taken to analyze cell dry weight (CDW) and PHA contents. The culture medium was centrifuged at 4 °C and 3500 rpm, for 1 h. The supernatant was discarded, and the cell pellets were freeze–dried after washing. Acetone was added to the freeze–dried cells to dissolve PHA, and methanol was treated at 1:10 to recover PHA precipitated at the bottom.A PHA production test was conducted to confirm the suitability of the SCGs oil. http://www.palisade.com, accessed on 12 December 2022) and NIST .GC/MS was used for qualitative analysis using the method described in previous studies . After oFor quantitative analysis of the SCGs oil, fatty acid (for standard curve), and PHA, each sample was analyzed using the GC. Samples of 10 mg of SCGs oil or fatty acid were contained in a 15 mL glass round bottom tube for pretreatment for FAME analysis.For quantitative analysis of PHA, 1 mL of culture medium was centrifuged at 13,000 rpm for 5 min. Afterwards, the cell pellet was washed twice with deionized water (DW), and the resuspended cells were placed in a 15 mL glass round bottom tube that was sealed with teflon, frozen in a freezer, and freeze–dried. Afterwards, the CDW was measured, and the dried cells were treated for FAME analysis. For FAME, 1 mL of chloroform and 1 mL of sulfuric acid–methanol solution (15:85) were added and the sample was reacted at 100 °C for 2 h on a heating block. The sample was then cooled at room temperature for 10 min and then chilled on ice for 10 min. After adding 1 mL of iced cold water, the sample was shaken and the organic phase below was analyzed. Of the prepared sample, 1 µL was injected through an auto–sampler and analyzed with a flame ionization detector (FID).The analysis conditions were as follows: a 30 m × 0.25 mm DB–FFAP capillary column, hydrogen 40 mL/min as the carrier gas, air zero flow 450 mL/min and high purity nitrogen gas 45 mL/min as the makeup flow, inlet temperature 250 °C, detector temperature 250 °C, and the oven held at 90 °C for 5 min, heated to 220 °C with a heating rate of 20 °C/min, and then held at 220 °C for 7 min.2 gas (99.999%) was used as the carrier gas, the inlet temperature was 100 °C, the detector temperature was 250 °C, and the oven was held at 80 °C.For analysis of bio-hydrogen productivity, the gas produced in the serum bottle was collected through a 1 mL syringe  and injected into the GC. Hydrogen measurement was performed through a thermal conductivity detector (TCD) with a 10 m × 0.3 mm CP–Molsieve 5A capillary column. The analysis conditions were as follows: high purity N2SO4 as a mobile phase, 60 °C for column temperature, 300 mm × 7.8 mm Aminex HPX–87H ion excursion column, 55 °C for detector temperature, refractive index detector, 25 μL of injection volume, 0.6 mL/min of flow rate, and 55 min of running time.HPLC was used to measure metabolic product changes in the bio-hydrogen production. A 1 mL sample was centrifuged for 5 min at 4 °C and 13,000 rpm. Then, the supernatant was filtered through a PTFE membrane filter with 0.45 μm pore size. The HPLC analysis conditions were as follows: 5 mM HThe measured hydrogen and biogas were converted to values at standard pressure and temperature before being applied to the modified Gompertz model below :(4)H=P×eP is the hydrogen production potential (mL), Rm is the maximum hydrogen production rate (mL/h), λ is the lag phase (h), t is the time (h), and e is 2.7182 [H is the estimated hydrogen production (mL), s 2.7182 .n–hexane, ethanol, methanol, pentane, acetone, and isopropanol [n–hexane was used [n–hexane and extraction time used were 25 g, 200 mL, and 1.5 h, respectively [In general, oil extraction from oilseed includes press extraction and organic solvent extraction. The organic solvent extraction method was selected because SCGs are not suitable for the press extraction method as they are finely ground. Solvents used for oil extraction include propanol ,27. Accowas used ,27,28. Aectively .n–hexane and SCGs oil was fractionally distilled, and the concentrated SCGs oil was 4.03 g. There was a difference of 0.41 g between the concentrated SCGs oil and the SCGs weight variation. It was found that about 10.4% of n–hexane in SCGs oil remained even after fractional distillation (data not shown). Therefore, it is important to select strains that can operate efficiently considering that n–hexane is known to be toxic to a relatively large number of bacterial species [The total weight of the cellulose filter and SCGs was 30.05 g . The totThe SCGs oil extraction yield was 14.4%, and the fat content in the SCGs was 14.9% based on the dry weight . Based ow/w), H2SO4 1.0% (w/w), 130 °C, and 1 h). The hydrolysis results of general SCGs and OESCGs are shown in For hydrogen production experiments, hydrolysis was performed using OESCGs. The OESCGs were hydrolyzed under conditions derived from previous experiments (S/L ratio 10% (w/w) and H2SO4 1.0% (w/w) (35.93 g/L), which is thought to be because of an increase in carbohydrate content after oil is extracted. The carbohydrate content of dried SCGs was 69.2%  was 40.4%, and that of the OESCGs (80.8%) was 39.9%. It was also found that the ratio (3.1:6.9) of galactose and mannose, which are the main sugars of the SCGH, remained the same as that of the general SCGs hydrolysis (3.1:6.9). These results show that hydrolysates of the same composition with higher sugar concentrations can be obtained through oil extraction.The concentration of short chain fatty acids (SCFAs) tended to be increased, similar to the sugar concentration, with the SCFA concentration of the OESCGH at 2.61 g/L, higher than that of the SCGH (1.86 g/L), whereas the concentrations of the furan derivatives were 0.27 g/L and 0.28 g/L, respectively, with no increasing tendency B,C. ThisIt is known that the concentration of furan derivatives that inhibits the growth of microorganisms is low, and it has been shown that bacterial growth is inhibited above 0.5 g/L 5–hydroxyl methyl furfural or above 2.0 g/L furfural ,34. In tHydrogen production experiments were conducted using the SCGs hydrolysates prepared above, and hydrogen productivity determined for the SCGs hydrolysates from general SCGs and OESCGs . When usThere was a large difference in VFA production and sugar consumption between SCGH and OESCGH D. In OES2SO4. One of these factors is believed to have caused the difference in productivity. When SCGH and OESCGH were put in the RCM media in the same volume, there was no significant difference in hydrogen production, sugar consumption, or lag time  was used to investigate the thermal properties of mcl-PHA, including the glass transition temperature (Tg), melting temperature (Tm), crystallization temperature (Tc), and melting enthalpy (ΔHm). The results obtained for the scl-co-mcl-PHA were Tg = −59.79 °C and Tc = 86.8 °C with ΔHm = 38.6 J/g, and Tm = 11.239 °C with ΔHm = 41.2 J/g .Pseudomonas sp. PAMC28620, composed of poly(25.5% 3HO-co-52.1% 3HD-co-5.7% 3HdD-co-16.7% 3HtD) with Tm = 172.8 °C, Tg = 3.99 °C, and Tc = 54.61 °C [Pseudomonas sp. MPC6. Composed of poly(89.5% 3HB-co-1.8% 3HHx-co-3.3% 3HO-co-4.4% 3HD-co-1.1% 3HdD) with Tm = 163.5 °C, Tg = 2.3 °C, and Tc = 46.0 °C [The thermal characteristics of our mcl-PHA differed both in Tm and Tg from those of the previously-reported mcl-PHA from 54.61 °C . The the 46.0 °C .P. resinovorans may have potential in applications that require a sticky and relatively low-temperature modeling polymer, as well as one that is biodegradable.As highly crystalline polymers have limited application in industrial and medical fields , the mclP. resinovorans differed from PHAs produced by other species [The molecular weight of the obtained PHA was characterized by Gel Permeation chromatography (GPC), which demonstrated a retention time peak start at 14.07 min, peak maximum at 15.86 min, and peak end at 18.93 min . The mcl species . Therefo4/g COD using SCG hydrolysates reported in another anaerobic fermentation study [The mass balance was calculated on a carbohydrate basis to confirm the chemical oxygen demand (COD) reduction of SCGs . The 738on study . In addiIt is believed that this amount could be increased later through optimization of the culture process and production process. One of the important constituents in coffee is caffeine. Gokulakrishnan et al. reported that caffeine concentrations above 2.5 mg/mL can affect microbial growth . AccordiHowever, after dark fermentation for hydrogen production, caffeine in effluent was 0.15 mg/g, which is about 6.5 times higher than the caffeine concentration of 23 mg/L at a sewage treatment plant in Seoul . CaffeinP. resinovorans, which can use n–hexane remaining in the oil, produced 5.6 g/L of CDW and 29.7% of PHA content at 24 h in a fed–batch culture. In summary, it was shown that waste disposal costs can be reduced by performing both processes to increase the utilization of SCGs that were previously only hydrolyzed or had oil extracted. In addition, hydrogen and PHA were produced as useful resources. These results show that fossil fuel–energy and petroleum–based plastics can be replaced and enable the proposal of an environmental resource circulation model.This study produced eco–friendly energy and biodegradable plastics to induce the valorization of SCGs that are produced in large quantities every year. For bioplastic production, 97% oil was extracted from total oil in the SCGs. OESCGs were hydrolyzed and showed a sugar concentration of 32.26 g/L. OESCGH exhibited a low inhibitor concentration, and the post–treatment process could be omitted. Hydrogen production was 15% higher than that of SCGH. This shows that the process of extracting oil is more advantageous in the next utilization step. The extracted oil was used as a substrate for PHA production.


Aspergillus fumigatus can cocolonize CF patients with Pseudomonas aeruginosa, which has been associated with lung function decline. P. aeruginosa produces several compounds with inhibitory and antibiofilm effects against A. fumigatusin vitro; however, little is known about the fungal compounds produced in counterattack. Here, we annotated fungal and bacterial secondary metabolites (SM) produced in mixed biofilms under normoxia and hypoxia conditions. We detected nine SM produced by P. aeruginosa. Phenazines and different analogs of pyoverdin were the main compounds produced by P. aeruginosa, and their secretion levels were increased by the fungal presence. The roles of the two operons responsible for phenazine production (phzA1 and phzA2) were also investigated, and mutants lacking one of those operons were able to produce partial sets of phenazines. We detected a total of 20 SM secreted by A. fumigatus either in monoculture or in coculture with P. aeruginosa. All these compounds were secreted during biofilm formation in either normoxia or hypoxia. However, only eight compounds  were detected during biofilm formation by the coculture of A. fumigatus and P. aeruginosa under normoxia and hypoxia conditions. Overall, we showed how diverse SM secretion is during A. fumigatus and P. aeruginosa mixed culture and how this can affect biofilm formation in normoxia and hypoxia.In cystic fibrosis (CF), mucus plaques are formed in the patient’s lungs, creating a hypoxic condition and a propitious environment for colonization and persistence of many microorganisms. There is clinical evidence showing that  Pseudomonas aeruginosa is a Gram-negative bacterium that grows aerobically and under anaerobic conditions in certain specific circumstances. The species is ubiquitous in nature and has been found inhabiting soil and water and also colonizing humans, where it sometimes acts as an opportunistic pathogen .pathogen . In immufections , 2. P. aP. aeruginosa  disorders to invasive and life-threatening diseases , due to bacteria-produced compounds  located in two nearly identical redundant operons (phz1 and phz2) that have different promoters and flanking regions  produced by bacteria with broad physiological functions, including acting as antibiotics  and anti22–ruginosa , such as regions . Another operons .P. aeruginosa compounds with inhibitory and antibiofilm effects against A. fumigatus have been revealed, little is known about the compounds produced by the fungus during the interaction. Furthermore, most of the studies about P. aeruginosa SM during interactions were done by using mutants lacking important genes for biosynthesis of such compounds and/or by measuring the effect of adding purified compounds or bacteria-filtered supernatant to the coculture or a monoculture. There is a lack of studies identifying fungal and bacterial SM produced throughout coculturing and mixed biofilms and their roles in the fungus-bacterium interaction, especially regarding fungal metabolites.Although P. aeruginosa and A. fumigatus in single or mixed biofilms during normoxia and hypoxia conditions. We detected 10 SMs produced by P. aeruginosa. Phenazines and different analogs of pyoverdin were the main compounds produced by P. aeruginosa, and their secretion levels were increased by the fungal presence. The contributions of the two operons that regulate phenazine production (phzA1 and phzA2) are still controversial and were also investigated. The results showed that ΔphzA1 and ΔphzA2 mutants can produce a subset of phenazines when in hypoxia and in the presence of the fungus. In contrast, we were able to detect 20 SM produced by A. fumigatus, but only 8 of them  were produced in the presence of P. aeruginosa.Here, we show the SM produced by P. aeruginosa and A. fumigatus biofilm formation under normoxia and hypoxia by using 105 CFU/mL from exponential-phase P. aeruginosa cultures and 106A. fumigatus conidia/mL (P. aeruginosa wild type and ΔphzA1 (A1) and ΔphzA2 (A2) mutant strains were comparable in biofilm formation. Mixed biofilms of A. fumigatus-P. aeruginosa, A. fumigatus-P. aeruginosa ΔphzA1 (AfA1), and AfA2 had more biomass than bacteria-only biofilms, in either normoxia or hypoxia  using P. aeruginosaecfX  and gyrB (encoding a DNA gyrase) and A. fumigatus 18S DNA  to identify SM in the A. fumigatus and P. aeruginosa biofilm supernatants. We were able to annotate a total of 29 SM, 9 from P. aeruginosa and 20 from A. fumigatus, in the supernatants produced under either condition (https://doi.org/10.6084/m9.figshare.19620702). PhZA1-G1 and PhZA2-G2 pathways use chorismic acid as the precursor for transformation into PCA (phzA1 mutant (A1 biofilm) under normoxia conditions –high-resolution tandem mass spectrometry  in both normoxia and hypoxia, except for pyoverdine E under normoxia .These results indicated that interaction with A. fumigatus (http://vm-trypanocyc.toulouse.inra.fr/META/NEW-IMAGE?type=PATHWAY&object=PWY-7525&orgids=LEISH) , demethoxyfumitremorgin C (in normoxia and hypoxia), and fumitremorgin C (in normoxia and hypoxia); there were no differences between A. fumigatus-P. aeruginosa wild type, AfA1, and Af2  . Severals=LEISH)  to G. AlA. fumigatus biofilm formation under normoxia and hypoxia conditions (A. fumigatus in biofilms only under normoxia (A. fumigatus-only biofilms (P. aeruginosa wild type and the mutant strains (A1 and A2) behaved in similar patterns regarding the production of those compounds, with a few exceptions: AfA1 biofilms did not produce ferrichrome in hypoxia or triacetylfusigen in normoxia.We annotated several metabolites relevant for iron assimilation, such as ferrichrome, ferricrocin, and triacetylfusigen, produced during nditions  to C. Menormoxia , while cnormoxia  and F. Fbiofilms , and C. A. fumigatus can still produce several metabolites important for iron chelation during biofilm formation in both normoxia and hypoxia conditions, notably, ferrichrome, ferricrocin, and triacetylfusigen. Nevertheless, the results also showed that P. aeruginosa strongly affects the overall production of all iron chelators detected in this approach, suggesting that competition for this micronutrient is a key point in the A. fumigatus-P. aeruginosa interaction in biofilms.Taken together, these results strongly indicate that A. fumigatus biofilm formation under normoxia and hypoxia conditions  and GT-modified forms, such as bis(methylthio) GT, GT E, and GT G, are produced during nditions  to D. GTnditions . Curiousmpletely . Pyripyrbiofilms , indicat hypoxia .A. fumigatus-P. aeruginosa biofilm formation and suggest that A. fumigatus uses GT as a defense against P. aeruginosa.Taken together, these results emphasize the importance of GT, pyripyropene A, and fumiquinazoline F and G during A. fumigatus SM are important for the establishment of A. fumigatus-P. aeruginosa biofilm formation under normoxia and hypoxia conditions, we tested several mutants impaired in the production of (i) gliotoxin , (ii) pseurotin , (iii) fumiquinazoline , and (iv) fumagillin and pseurotin   . All thenditions .A. fumigatus ΔpsoF and ΔfmqA had increased biofilm formation during normoxia in the absence and presence of P. aeruginosa 2; 0.42 mM], which could support P. aeruginosa growth in hypoxia. P. aeruginosa can also survive in oxygen-limited conditions using pyocyanin as an electron acceptor to regenerate NAD+   , 22. Allal death . 1-HP isproperty . P. aerupression . Two redoduction –49. Earloduction . Recent P. aeruginosa-produced compound that is induced upon iron starvation is pyoverdine  produced during biofilm formation by the coculture of A. fumigatus and P. aeruginosa upon either normoxia or hypoxia conditions. Interestingly, brevianamide F and fumiquinazoline F and G were produced only upon hypoxia conditions, while methyl ferrichrome was produced only upon normoxia conditions. These results indicate that these SM are important for the interaction between A. fumigatus and P. aeruginosa, and the production of some of them is regulated by the oxygen condition. Of note, gliotoxin was the only SM produced in higher levels in mixed biofilms compared to A. fumigatus-only biofilms, suggesting that the fungus specifically overproduces this compound in response to the bacterial antagonist. Gliotoxin has been the most well-studied and characterized SM from A. fumigatus, and it is also important for the interaction with P. aeruginosa and for biofilm formation  involved in iron starvation adaptation in response to P. aeruginosa 1-HP has been demonstrated , and A. fumigatus CEA17, ΔgliT, ΔpsoF, ΔfmqA, and ΔfapR. P. aeruginosa was grown from frozen stocks ([LB] medium plus 20% glycerol) in solid LB for 24h at 37°C. A single colony was transferred to 30 mL of LB and cultured overnight at 37°C, 200 rpm. The culture was centrifuged at 4,000 × g, for 5 min, and the pellet was washed with 10 mL of phosphate-buffered saline (PBS). After centrifugation, the pellet was resuspended in LB and the inoculum was adjusted, using a spectrophotometer, to an optical density at 600 nm (OD600) of 0.07 to 0.075. This inoculum was grown in 30 mL of LB at 37°C, 200 rpm, for 5 h, and the centrifugation and PBS washing processes were repeated. The final pellet was resuspended in RPMI-HEPES, and the inoculum was adjusted to an OD of 0.07 to 0.075 (approximately 5 × 108 to 8 × 108 CFU/mL).The following species and strains were used in this work: ild type ), ΔphzA1zA1::Mr7 ), and ΔpA. fumigatus strains were grown from frozen stocks, and conidia suspensions were obtained by harvesting grown mycelia on minimal medium plates as described by Ries and colleagues  under hypoxic  or normoxic (approximately 20% O2 and 0.04% CO2) conditions, at 37°C. After 5 days, the supernatant was collected, the plate was washed with 10 mL of ultrapure water to collect the cells, and both were transferred to a 50-mL tube. This mixture was centrifuged at 4,000 × g for 15 min at 4°C to obtain the pellet, which was used for qPCR assays, and the supernatant (20 mL), which was filtered through a 0.22-μm filter, was frozen and lyophilized for SM extraction.To measure the interaction between On the bottom of the small Petri dishes used in this experiment, biofilm production was measured by the crystal violet (CV) method. The biofilm was dried at 37°C for 30 min and then stained with 5 mL of 0.05% (wt/vol) CV for 10 min. The plates were washed with 50 mL of PBS, and the CV was solubilized with 3 mL of 95% ethanol. Samples of 100 μL were transferred to 96-well plates, and the absorbance at 595 nm was determined, as a measure for biofilm formation.P. aeruginosa-A. fumigatus interaction were frozen and lyophilized before being triturated by adding 2-mm glass beads and 0.1-mm zirconia-silica beads and vortexing for 5 min. To the resulting powder, 1 mL of extraction buffer was added, and the tubes were vortexed for 5 min. Tubes were incubated in a water bath at 70°C for 45 min; every 10 to 15 min, the tubes were removed from the water bath and vortexed for 5 min. One milliliter of phenol-chloroform (1:1) was added to the mixture, and tubes were vortexed for 5 min. The content was transferred to 2-mL tubes and centrifuged at 14,000 × g for 15 min at room temperature. The supernatants were collected and transferred to 1.5-mL tubes, and 600 μL of isopropanol (Merck) was added. Samples were incubated at 4°C for 1 h before being centrifuged at 14,000 × g for 15 min at 4°C. The supernatant was discarded, the pellet was washed with 200 μL of 70% ethanol, and air dried for 15 min at room temperature, after which the pellet was resuspended in deionized water and treated with RNase (Promega).For DNA extraction, pellets obtained from cultures for evaluating the P. aeruginosa DNA was specifically quantified by qPCR with primers for ecfX  and gyrB  . The 10-μL qPCR mixture was composed of 5 μL SYBR green PCR master mix , 2.5 pmol of each primer, and 100 ng DNA. Cycling was performed on the ABI 7500 fast real-time PCR system with an initial hold at 95°C for 15 min, followed by 45 cycles at 95°C for 15 s and 60°C for 1 min, with a cycle threshold of 35. Negative controls without DNA were included in each qPCR run.CGCC-3′) , 30. A. SM were extracted from 50 mg freeze-dried sample of the entire supernatant of each sample by resuspension in 1 mL of HPLC-grade methanol (MeOH), followed by 1 h of sonication in an ultrasonic bath. For sample preparation, 500 μL of each obtained extract was filtered (0.22-μm filter), transferred to vials, and diluted with HPLC-grade MeOH to a total volume of 1 mL.2 positive-mode analysis was performed in a Thermo Scientific QExactive hybrid Quadrupole-Orbitrap mass spectrometer coupled to a Dionex UltiMate 3000 RSLCnano UHPLC system. For the stationary phase, a Thermo Scientific column, Accucore C18 2.6 μm (2.1 mm by 100 mm) was used. The mobile phase was 0.1% formic acid (A) and acetonitrile plus 0.1% formic acid (B). Eluent profiles (A/B percentages) were 95/5 up to 2/98 within 10 min, maintaining 2/98 for 5 min, down to 95/5 within 1.2 min, and maintaining for 8.8 min. Total run time was 20 min for each run, and flow rate was 0.3 mL · min−1. The injection volume was 5 μL. MS spectra were acquired with m/z ranges from 100 to 1,500, with 70,000 for mass resolution. Ionization parameters were a sheath gas flow rate of 45 L.h−1, auxiliary gas flow rate of 10 L.h−1, sweep gas flow rate of 2 L.h−1, spray voltage of 3.5 kV, capillary temperature of 250°C, S-lens RF level of 50, and auxiliary gas heater temperature of 400°C. MS2 spectra were acquired in data-dependent acquisition mode. Normalized collision energy was applied stepwise  V, and the 5 most intense precursors per cycle were measured with 17,500 resolution.UHPLC-HRMS2 data were converted into mzXML format files using MSConvert   workflow  on GNPS csd.edu) . The datibraries , 76. Theibraries . The molibraries . Resulti2 database via the FBMN and Dereplicator Plus workflows available on the GNPS platform. Other metabolites were manually searched against natural products databases such as the Dictionary of Natural Products, and the acquired MS2 spectra were compared to spectra deposited either on the GNPS database or previously published in the literature. The compounds gliotoxin, gliotoxin E, pseurotin E, and spirotryprostatin A were annotated based on their exact masses.For SM dereplication, metabolites were annotated based on the GNPS MShttps://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=0b027bfab9064518945341d3c24ba2a9, and the Dereplicator Plus job can be accessed at https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=415264520a41492a9b43b47e623028ed for hypoxia conditions and at https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=08a9ebb5609745d5a6f805896e527b67 and https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=e59e11f719cf4ae5beb406090a987f01 for normoxia.The molecular networking job can be publicly accessed at