A novel encochleated amphotericin B deoxycholate (cAMB) formulation has oral bioavailability, efficacy in an animal model, and minimal toxicity due to targeted drug delivery into macrophages, where intracellular fungi reside. We conducted a phase I, ascending-dose trial of cAMB administered at 1.0 g, 1.5 g, or 2.0 g per day in 4 to 6 divided doses among HIV-positive survivors of cryptococcosis ( n = 9 per cohort). We assessed the tolerability of cAMB and the adverse events (AEs) associated with cAMB treatment over 3 days. A second trial (n = 9) assessed the tolerability of 1.5 g/day given for 7 days. In the single-ascending-dose study, all subjects received their full daily dose without vomiting (100% tolerability). The cohort receiving 1.0 g had 4 transient clinical AEs in 2 subjects within 48 h and 8 laboratory AEs . The cohort receiving 1.5 g had 7 clinical AEs in 1 subject attributed to acute gastroenteritis (n = 4 grade 2) and 5 laboratory AEs (n = 1 grade 2). The cohort receiving 2.0 g had 20 clinical AEs among 5 subjects within 48 h (n = 3 grade 2) and 11 laboratory AEs . From a qualitative survey, 26 of 27 subjects (96%) preferred their experience with oral cAMB over their prior experience with intravenous (i.v.) AMB. The second, multiple-dose cohort received 1.5 g/day for 1 week, with 98.4% (248/252) of the doses being taken. Overall, 5 clinical AEs (n = 5 grade 1) and 6 laboratory AEs (n = 6 grade 1) occurred without kidney toxicity. Oral cAMB was well tolerated when given in 4 to 6 divided daily doses without the toxicities commonly seen with i.v. AMB. Amphotericin B deoxycholate (AMB) has substantial toxicities. A novel encochleated amphotericin B deoxycholate (cAMB) formulation has oral bioavailability, efficacy in an animal model, and minimal toxicity due to targeted drug delivery into macrophages, where intracellular fungi reside. We conducted a phase I, ascending-dose trial of cAMB administered at 1.0 g, 1.5 g, or 2.0 g per day in 4 to 6 divided doses among HIV-positive survivors of cryptococcosis ( Intravenously (i.v.) administered amphotericin B deoxycholate (AMB) is traditionally considered the vanguard antifungal therapy for serious fungal infections due to its broad activity and low resistance rates. However, i.v. AMB has numerous significant toxicities, including anemia, nephrotoxicity, and infusion-related reactions. Further, i.v. AMB has historically been available only as an intravenous medication, as oral absorption in humans is limited and causes toxicity . EncochlThe cAMB cochleates are comprised of a solid lipid bilayer sheet rolled into a spiral in which amphotericin molecules are trapped, along with calcium . The druin vitro activity against Cryptococcus neoformans, Candida spp., and Aspergillus fumigatus (Matinas investigator brochure). In a cryptococcosis mouse model experiment by Williamson and colleagues, mice given cAMB at 25 mg/kg of body weight plus flucytosine (5FC) had rates of survival equivalent to those of mice injected with AMB plus 5FC  recorded in different dosing cohorts. Based on our phase I data, we plan to move cAMB to a phase II efficacy trial for HIV-associated cryptococcal meningitis.In the initial single-ascending-dose trial, a total of 35 HIV-positive survivors of cryptococcal meningitis were screened. We enrolled 9 participants into each of the three sequential dosing cohorts: 1.0 g, 1.5 g, and 2.0 g per day. All participants were survivors of prior cryptococcal meningitis  and had previously received i.v. AMB for ∼14 days. The median age was 36 years , with 41% (11/27) of the participants being women. All participants were currently receiving antiretroviral therapy (ART), with 15 (42%) taking atazanavir and 37% (10/27) actively taking fluconazole at 200 mg as prophylaxis. The baseline characteristics are summarized in To meet the tolerability criteria, the participants were required to take the full daily dose without vomiting within 30 min after taking any dose, to report AEs of grade 2 or less, and to not discontinue the cAMB due to an AE or participant choice. For the cohorts receiving 1.0 g and 1.5 g, all 18 participants met the full tolerability criteria. In the cohort receiving 2.0 g, all 9 participants completed the dosing, but 1 participant experienced a grade 3 laboratory AE of transient thrombocytopenia at 48 h of unclear etiology. Therefore, only 89% (8/9) of the participants met the full tolerability criteria with dosing at 2.0 g.n = 5), nausea (n = 5), and dizziness (n = 4). Of the same 27 participants, 15 (56%) experienced at least one laboratory AE. A total of 24 laboratory AEs were reported, with 14 (58%) being grade 1 and 9 (38%) being grade 2. There was one grade 3 adverse event, in which a participant in the cohort receiving 2.0 g had platelet counts of 163,000 and 160,000 cells/μl at baseline and 24 h, respectively, which then dropped to 31,000 cells/μl at 48 h. No platelet clumping was reported. The platelet count was repeated several times to ensure AE resolution; the count rebounded to 172,000 cells/μl by 2 weeks. The participant had no clinical deterioration or evidence of bleeding at the time of the documented thrombocytopenia. No grade 4 or serious laboratory AEs occurred. The most common laboratory AEs were an elevation in the total bilirubin level (n = 6), a mild isolated elevation in the aspartate transaminase (AST) level (n = 5), and mild sodium abnormalities  which met the National Institute of Allergy and Infectious Diseases (NIAID) Division of AIDS (DAIDS) criteria for a grade 1 AE (146 to 149 meq/liter) but which was within the normal range of the local College of American Pathologists (CAP)-certified laboratory for the population (138 to 150 meq/liter). Of the subjects with elevations in AST levels, only one was accompanied by an elevation in the alanine transaminase level, which occurred in a subject with chronic hepatitis B. The AE prevalence is summarized in Of the 27 participants in the phase IA study, 8 (30%) experienced at least one clinical AE. A total of 31 unique clinical AEs were reported, with 24 (77%) being grade 1 and 7 (23%) being grade 2. Increasing numbers of AEs correlated with increasing cAMB dose, with 65% (20/31) being recorded in the cohort receiving 2.0 g. No grade 3, grade 4, or serious AEs occurred. The most common clinical AEs were abdominal pain  did not significantly differ across the three dosing cohorts, with the overall median being 2,200 ng · h/ml (P = 0.58) (Cmax) averaged across the three cohorts was 59.2 ng/ml . In pharmacokinetic analyses of the phase IB cohort, the plasma concentration at 24 h (74.8 ng/ml) was 77% of the day 7 concentration of 97.7 ng/ml. At 72 h, the plasma concentration of 91.1 ng/ml was 93% of the day 7 concentration.The plasma concentrations of cAMB were measured at 0, 6, 12, 24, and 48 h from the time of cAMB initiation, and the area under the curve (AUC) was calculated. Interestingly, the plasma AUC from 0 to 48 h (AUC = 0.58) . Based oUsing a qualitative scale, we assessed the impressions of the participants in the phase IA cohort of their experience with cAMB compared with their prior experience with i.v. AMB. In the preparticipation survey, 40% (10/25) indicated that they would be willing to receive i.v. AMB again for their cryptococcal meningitis versus an alternative therapy. After completing the study follow-up, 96% (26/27) of the participants indicated the they would prefer cAMB to i.v. AMB for cryptococcal meningitis treatment. Overall, 93% (25/27) thought that the multiple doses of cAMB were very convenient for therapy, whereas 65% (15/23) of the respondents thought that i.v. AMB was very convenient for therapy. One participant could not recall the initial i.v. AMB therapy. Nausea severity scoring was rated as “none” by 8 of 9 participants in the 1-g and 1.5-g cohorts and 7 of 9 participants in the 2.0-g cohort. In comparison, 25% (6/24) recalled severe nausea with i.v. AMB.We found that cAMB administered in 4 to 6 divided daily doses was generally safe and tolerated in healthy HIV-positive Ugandans at doses of up to 2.0 g/day. While mild AEs were more frequently encountered as the dose was increased, 95% (34/36) of the participants completed all prescribed doses without a grade 3 or worse AE. It is worth noting that we found that 2017 DAIDS laboratory AE criteria occasionally graded a laboratory value  as a grade 1 event when the number fell within the reference range of the Ugandan population studied at our CAP-certified laboratory. Therefore, some of our grade 1 events may have been overreported. Based on our data, 1.5 g daily may represent the sweet spot of optimal tolerability versus dosing escalation.cAMB appears to have a favorable toxicity profile compared to that of i.v. AMB. Data taken from the 177-participant COAT  trial  recordedWe found that smaller, frequent dosing helped mitigate the gastrointestinal side effects seen in early studies that utilized larger, one-time dosing. For example, a prior phase I study (CAM-102) of healthy volunteers given cAMB at 800 mg daily in a single dose reported that 58% (7/12) of participants developed a gastrointestinal AE (Matinas investigator brochure), whereas only 11% (2/18) of our participants developed any gastrointestinal AEs, despite receiving 1,000 and 1,500 mg daily with dosing divided 4 and 5 times daily, respectively. Further, our participants overwhelmingly declared that their subjective experience with cAMB was better than that with i.v. AMB.0–24) was 636 ng · h/ml and the mean Cmax was 40.8 ng/ml (an ∼7% increase compared to the Cmax of 38.1 ng/ml achieved after a single 400-mg dose) (Matinas investigator brochure). In contrast, giving 1,000 mg in 4 to 6 divided doses increased the AUC0–24 to 990 ng · h/ml and the Cmax to 64.9 ng/ml (∼60% more than that after a single 800-mg dose). Interestingly, we saw negligible differences in Cmax or AUC between our cohorts receiving divided doses of 1.0 g, 1.5 g, and 2.0 g   review, we are moving forward with a phase II randomized trial evaluating the efficacy of cAMB in HIV-positive Ugandans with cryptococcal meningitis using early fungicidal activity as the primary endpoint. Persons will be given stage-based incremental decreases in the i.v. AMB duration while concurrently increasing the cAMB duration in order to determine cAMB efficacy while prioritizing participant safety. Based on our phase I data, we plan to use cAMB at 2.0 g daily with flucytosine for cryptococcal meningitis induction therapy, followed by cAMB at 1.5 g with fluconazole for 4 weeks of consolidation therapy. Persons with acute cryptococcal meningitis and elevated intracranial pressure commonly have significant nausea and vomiting, and therefore, the tolerability will be an ongoing area of monitoring. However, 2-week cryptococcal meningitis survivors discharged home with improving symptoms may find 1.5 g to be more tolerable for the additional 4 weeks of therapy. We began the phase 2 EnACT  trial enrollment in July 2020. cAMB remains a promising novel antifungal in the fight against serious fungal infections, including cryptococcosis.We conducted a phase I safety and tolerability trial for cAMB in HIV-positive Ugandans from October 2019 to January 2020 in Kampala, Uganda . The trial was designed to occur in two sequential phases, with 27 participants enrolled in phase IA and 9 participants enrolled in phase IB. The participants in phase IA received a single ascending dose of cAMB sequentially administered at 1.0 g, 1.5 g, and 2.0 g per day in 4 to 6 divided doses, with 9 participants being used in each dose group. At least 6 of 9 participants had to tolerate the dose before proceeding to the next dosing cohort. Dose-limiting intolerability was defined as experiencing a grade 3 or higher AE, vomiting within 30 min after taking a dose, or discontinuing the medication due to an AE or toxicity. The participants in phase IB received the cAMB dose that was 100% tolerated in phase IA for 7 days to verify safety and tolerability with continuous dosing. Participants in phases IA and IB had the same inclusion and exclusion criteria.2 (measured within 3 months). Participants were excluded if they exhibited a current illness, had a known untreated significant health problem, or had received amphotericin B therapy in the past 90 days. Women who were pregnant or breast-feeding were excluded.Participants were recruited from a prior cryptococcal meningitis trial cohort  . All recTrial endpoints included (i) the proportion of the daily dose received and tolerated without vomiting within 30 min; (ii) the cumulative score of nausea, vomiting, and diarrhea by Common Terminology Criteria AE (CTCAE)-graded criteria ; (iii) tWe collected plasma for pharmacokinetic analyses at 0, 6, 12, 24, and 48 h in phase IA. We monitored complete blood count, chemistries, and kidney and liver function tests at 0, 24, and 48 h, with follow-up occurring thereafter for any abnormal results. For phase IB, the same laboratory monitoring occurred at +1 day, +3 days, and +7 days for safety assessment and pharmacokinetic analyses. All laboratory measurements were performed at the IDI Core Laboratory, which is a College of American Pathologists (CAP)-certified laboratory and which participates in external quality assurance for all laboratory assays. Plasma drug quantification was conducted at Medpace Bioanalytical Laboratories  using an internally validated assay (MBL 17301). Briefly, 100 μl of plasma was placed in a 2-ml tube and fortified with internal standard working solution. The analyte and internal standard were extracted via protein precipitation using acetonitrile. The supernatant was then dried under a stream of nitrogen, and the resulting residue was reconstituted in 150 μl of 0.1% formic acid in methanol-water (1:1) before being submitted to liquid chromatography-tandem mass spectrometry analysis on a Sciex Qtrap or Triple Quad mass spectrometer.Statistical analyses were primarily descriptive. The median and interquartile ranges or the number and proportions are reported, where appropriate. Area-under-the-curve (AUC) and half-life calculations were made with Phoenix WinNonlin (version 8.2) software , using a linear-up/logarithm-down method, with comparisons for determination of statistical significance being performed by the Kruskal-Wallis analysis of variance (ANOVA) in SAS (version 9.4) software . A visual analog scale was used to assess the participants’ subjective experience with various aspects of the trial. The numbers of clinical and laboratory adverse events (AE) were summarized by the Division of AIDS (DAIDS) toxicity table, version 2017.The Mulago Hospital Research and Ethics Committee, the Ugandan National Council of Science and Technology, the Uganda National Drug Authority, and the University of Minnesota Institutional Review Board approved the study. The study was performed under U.S. Food and Drug Administration (FDA) Investigational New Drug (IND) application number 072807. All participants provided informed consent in their preferred language. An internal review was performed after the study with each dosing cohort to ensure tolerability. A data safety monitoring board (DSMB) reviewed all safety data after completion of phase I data collection.


Passiflora edulis) production worldwide, including China. In this study, the complete genome sequence (excluding the poly (A) tail) of two TeMV isolates, Fuzhou and Wuyishan, were determined to be 10,050 and 10,057 nucleotides, respectively. Sequence analysis indicated that Fuzhou and Wuyishan isolates share 78–98% nucleotide and 83–99% amino acid sequence identities with two TeMV isolates of Hanoi and GX, and a proposed new potyvirus, tentatively named PasFru. Phylogenetic analysis indicated that these TeMV isolates and PasFru were clustered into a monophyletic clade with high confidences. This indicated that PasFru and the four TeMV isolates should be considered as one potyvirus species. Two recombination breakpoints were identified within the CI and NIb genes of the Fuzhou isolate, and also within the P1 gene of the Wuyishan isolate. To the best of our knowledge, this is the first report of TeMV recombinants worldwide.Telosma mosaic virus (TeMV) is an important plant virus causing considerable economic losses to passion fruit ( Passiflora edulis), originating in South America, is an important fruit crop that comprises a variety of cultivars and it is consumed globally  in Vietnam (Pogostemon cablin) in Indonesia , collected from Haikou city in Hannan Province, China, was identified and proposed to be a new member of the Potyvirus genus based on analyses of the complete genome sequence (NC_009742), although TeMV has been identified in many countries. One nearly complete genome of TeMV isolate from Guangxi Province in China  is also available in GenBank. To date, no recombinant TeMV isolate has been reported worldwide.During a survey of passion orchards in 2017 in Fujian Province, China, plants exhibiting virus-like mosaic and crinkle symptoms were prevalent. The disease causes a serious reduction in production and decreases the quality of passion fruit . In 2018sequence . In addiThe objectives of this study were (i) to identify two new TeMV isolates from passion fruit in China using transmission electron microscopy, indirect ELISA and RT-PCR; (ii) to obtain their complete genome sequences and characterize their genomic structure; and (iii) to clarify the current confusion surrounding the taxonomic status of some of these TeMV isolates, particularly the proposed new potyvirus PasFru.Two passion fruit samples showing mosaic and crinkle symptoms on the leaves  were colTotal RNA was extracted from the leaf tissue which was positive with virus infection by ELISA using an RNA extraction kit . The quantity and quality of the extracted RNA were determined by measuring absorptions at 260–280 nm with a NanoDrop 2000c . The first-strand cDNA was synthesized using Moloney murine leukemia virus (M-MLV) reverse transcriptase  following the manufacturer’s protocol. For the reaction, in total of 11 μl mixture containing three μl RNA (~1 μg), one μl random primer (100 μm) and seven μl DEPC-treated water were incubated at 70 °C for 10 min. Then, the mixture was transferred immediately to an ice bath for 5 min. Finally, five μl 5× buffer , two μl dNTP mix , one μl RNAsin Plus RNase inhibitor  and one μl M-MLV reverse transcriptase  were added to the mixture of primer and RNA. The RT reaction was carried out at 42 °C for 60 min followed by at 70 °C for 10 min. The cDNA was chilled on ice and stored at −70 °C.Potyvirus, a set of universal potyvirus primer LegPotyF 5′-GCWKCHATGATYGARGCHTGGG-3′ and LegPotyR 5′-AYYTGYTYMTCHCCATCCATC-3′ , one µl of each primer (10 µM), two µl of cDNA and 8.5 µl DEPC-treated water. PCR conditions were as follows: initial denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 45 °C for 45 s and an extension step at 72 °C for 1 min. The amplification program was followed by a final extension step at 72 °C for 10 min.For the detection of the genus CCATC-3′  was used2 (25 mM), one µl of dNTPs (2.5 mM), one µl of each primer (10 µM), 0.2 µl of Taq DNA polymerase (5 U/μl), three µl of cDNA and 13.8 µl DEPC-treated water. PCR conditions were as follows: initial denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 1 min and an extension step at 72 °C for 1 min. A final 10 min elongation step at 72 °C was performed at the end of the 35 cycles.Telosma mosaic virus was detected by using specific primers TeMV-CPf 5′-TCAAGTAAGGTGGATGATGTT-3′ and TeMV-CPr 5′-CTGCACAGAGCCAACCCCAA-3′ as previously described . The priEscherichia coli DH5α competent cell. The positive clones containing the insert fragment were identified by PCR. Three of the positive clones were sequenced by Shanghai Sangon Biological Engineering Technology and Service Co., Ltd.Polymerase chain reaction products were analyzed by 1.5% agarose gel electrophoresis, stained with GelRed and photographed under UV-light. The target fragments of PCR products were purified by using Agarose Gel DNA Purification Kit . The purified products were ligated to pMD18-T vector  and then transformed into the MK340754) and Wuyishan isolate (accession number: MK340755) were deposited in GenBank.To amplify and clone the full-length genome sequences, five overlapping fragments covering the coding regions of the TeMV genome were amplified using RT-PCR with 5 pairs of specific primers designed from the highly conservative region of the TeMV genome . RACE PChttp://www.dpvweb.net/potycleavage/index.html).The complete genome was assembled from overlapping RT-PCR clones after removal of the vector and primer sequence. To identify the closest relatives of the Fuzhou and Wuyishan isolates, we performed a BLASTn search against the nt/nr databases and a sequence identity matrix using BioEdit. The putative cleavage site patterns in the polyprotein were identified using online website  by codon and removed poorly aligned regions using TranslatorX . Maximump-value cutoff of 0.01 was calculated. To reduce the presence of false positives, recombination events supported by at least four methods with an associated p-value of < 10−6 were considered to be significant.Recombination analysis were conducted using seven different methods  implemented in the RDP4 package . For eacThe results of electron microscopy showed the presence of potyvirus-like flexuous rod particles of about 750–770 nm in length . PresencKJ789129). These results suggested that TeMV was present in passion fruit plants showing mosaic and crinkle leaves from Fuzhou and Wuyishan.A BLASTn search against GenBank indicated that the RT-PCR sequences obtained here share more than 98% nucleotide identities with TeMV  tail) of Fuzhou is 10,050 nucleotides (nts) in length and of Wuyishan is 10,057 nts in length. Their 5′-untranslated regions (5′-UTRs) are 169 and 177 nts, respectively, while the 3′-UTRs were both 251 nts . Both coThe polyproteins of Fuzhou and Wuyishan were predicted to be proteolytically processed into ten mature peptides. Their cleavage sites are in consensus to those of other TeMV isolates, whose dipeptides are Y/S, G/G, Q/G, Q/S, Q/S, Q/G, E/S, Q/S and Q/S . DetailsPotyvirus, Fuzhou, Wuyishan, Hanoi, GX and PasFru should be considered as one species of Potyvirus. Interestingly, the N-terminal region of P1 sequences of Fuzhou and Wuyishan are divergent to both length and amino acid sequences, especially with Hanoi , together with GX, Hanoi and PasFru , suggest species . Hanoi, p ≤ 7.70E × 10−134, Bootscan, p ≤ 1.34 × 10−138; Maxchi, p ≤ 2.19 × 10−45; Chimaera, p ≤ 5.65 × 10−47; Siscan, p ≤ 7.25 × 10−54 and 3Seq, p ≤ 4.44 × 10−15) (p ≤ 5.53 × 10−6), Bootscan (p ≤ 5.21 × 10−23), Maxchi (p ≤ 4.12 × 10−18), Chimaera (p ≤ 2.13 × 10−10), Siscan (p ≤ 5.92 × 10−9) and 3Seq (p ≤ 1.11 × 10−14)  . A short× 10−15)  and PasF× 10−14) .Recombination is an important evolutionary factor that generated genetic variation in viral populations. The phenomenon is prevalent in potyviruses, for example, in turnip mosaic virus , potato Potyviridae, the species demarcation criteria for the complete ORF are less than 76% nucleotide and 82% amino acid identity. The thresholds for species demarcation are <58% nucleotide identity for the P1 coding region, and <74–78% nucleotide identity for other coding regions. However, for the CP, the optimal species demarcation criteria are <76–77% nucleotide and <80% amino acid identity, respectively. According to this criterion, Fuzhou, Wuyishan, GX, PasFru and Hanoi should be considered as isolates of TeMV. In Potyvirus, P1 is the most divergent protein varying in length and its amino acid sequences (In the family equences , which iequences . Yang etThe results of this study identified PasFru as the common progenitor of Fuzhou and Wuyishan. This suggested that PasFru possibly emerged more ancient than other TeMV isolates in China. However, the temporal dynamics of TeMV could not be estimated since the TeMV isolates in our study are relatively limited, particularly the earlier isolates were not sampled. In addition, TeMV tended to cluster according to their geographical or host species origin; this could be explained, in part, as geography-specific or host-driven adaptation . HoweverIn summary, this study represents one of serval attempts to reveal the taxonomic status of TeMV isolates. Our sequence analyses suggest that PasFru and other TeMV isolates should be considered as one potyvirus species. In addition, we found Fuzhou and Wuyishan were TeMV recombinants. To the best of our knowledge, this is the first report of TeMV recombinants worldwide.10.7717/peerj.8576/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.8576/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.8576/supp-3Supplemental Information 3Click here for additional data file.


However, in the primary and secondary roots, CAT activity was increased but APX decreased. Similarly, peroxidase (POD) and superoxide dismutase (SOD) activities were enhanced in the median leaves but a declining trend was observed in the terminal leaves, primary roots and secondary roots. Glutathione reductase (GR) activity, ascorbic acid (AsA) contents and GSH concentrations were increased in all parts except AsA in the median leaves. Transmission electron micrographs of Pb-treated plants exhibited deformed cell wall and cell membrane, disfigured chloroplasts and irregularly shaped mitochondria in the terminal and median leaves. Further, cell membrane, mitochondria, nucleus and other cell organelles in root cells were severely affected by the Pb. Thus their identification was little bit difficult through ultramicroscopy. External GSH stabilized leaf and root ultramorphology by stabilizing cell membranes, stimulating formation of multivesicular body vesicles, and by maintaining structural integrity of other organelles. Evidently, GSH played major alleviatory role against Pb toxicity in upland cotton.Heavy metals-based changes in the plants and their alleviation through eco-friendly agents including reduced glutathione (GSH) have been widely studied. In the present experiment, we tested the alleviatory role of reduced glutathione (GSH) in seedlings of upland cotton cultivar, TM-1 under lead (Pb) toxicity. Plants were grown in the Hoagland solution containing Pb (0 μM), Pb (500 μM), GSH (50 μM), and GSH + Pb (50 μM + 500 μM). Lead exposure exacerbated hydrogen peroxide (H Its uptolecules . Plants olecules .•O–, produced at the sites of electron transport chain, to form less toxic H2O2 and O2. Both, CAT and APX neutralize H2O2, however; former is more effective scavenger due its higher affinity for H2O2 (2O2 as a substrate to produce H2O and O2. Glutathione reductase reduces oxidized glutathione (GSSG) back into GSH to maintain balanced levels of the later in cells  control (0 μM Pb), (2) 500 μM Pb as Pb (NO3)2, (3) 50 μM GSH, and (4) 50 μM GSH + 500 μM Pb. These treatment levels were selected based on earlier findings from screening tests done in our lab and cotton’s seedling threshold level. After 10 days of stress period, harvested plants were separated into terminal leaves , median leaves (excluding cotyledonary leaves), primary roots and secondary roots. Seedling stage is the best time to study biochemical and morphological changes in leaves and roots of plants. Plant samples were either freshly used for biochemical assays or stored at −80°C till further analyses.Upland cotton variety TM-1 was selected for this study which is a standard genetic line and comparatively a heavy metal tolerant variety. Its seeds were obtained from Cotton Germplasm Lab, Zhejiang University, Hangzhou, China, and grown hydroponically according to 2HPO4.12H2O L–1 and 0.663 NaH2PO4.2H2O L–1), adjusted to pH 6.5, was added to 0.5 g plant sample in a pre-cooled mortar and pestle. Thoroughly homogenized samples were centrifuged at 12000 × g for 20 min at 4°C and supernatants were collected in 10 mL tubes, and kept at −30°C till further use.Enzyme extracts (EE) were obtained from plant samples to determine ROS, antioxidant enzymes and AsA content. Briefly, samples were treated with 10 M Na-EDTA to remove unbound Pb, and thoroughly washed with distilled water. Then 8 mL of pre-cooled phosphate buffer solution  and 1 mL EE were added and absorbance was recorded at 390 nm. Molar extinction coefficient (ε) of 0.028 μM cm–1 was used to calculate H2O2 contents and expressed as μM g–1 fresh weight. For MDA, 1.5 mL EE and 2.5 mL mixture of 5% trichloroacetic acid (TCA) + 5% thiobarbituric acid (TBA) was incubated at 95°C for 15 min and then immediately cooled on ice. After short centrifugation at 4800 × g, the absorbance of MDA-TBA adduct was measured at 532 nm and 600 nm. The MDA content was determined using ε = 155 mM cm–1 and expressed as μM g–1 fresh weight. For OH• determination, the reaction mixture comprised of 0.7 mL EE, 3 mL 0.5% TBA and 1 mL glacial acetic acid. After incubation at 100°C for 30 min, the reaction mixture was immediately cooled at 4°C and absorbance was measured at 532 nm. The OH• concentration in the sample was calculated using extinction coefficient, ε = 155 mM cm–1 and values were expressed as μM g–1 fresh weight.Reactive oxygen species such as H2O2, 0.1 mL EE and 2.8 mL PBS. The reaction mixture was shaken gently before taking reading at 240 nm. One unit of CAT activity was considered equal to the amount of enzyme required to catalyze one micro mole of H2O2 per min. For APX, the reaction mixture contained 0.1 mL H2O2 (300 mM), 0.1 mL EE, 0.1 mL AsA (7.5 mM) and 2.7 mL PBS. The absorbance was measured at 290 nm. One unit of APX activity was defined as the amount of enzyme needed to oxidize one micro mole of ascorbate per minute. The POD and SOD activities were determined according to 2O2 (300 mM), and 2.7 mL PBS. The absorbance was measured at 470 nm. For SOD activity, a reaction solution of 75 μM nitroblue tetrazolium (NBT), 20 μM riboflavin, 100 μM Na-EDTA and 130 mM methionine was prepared. Then, 2.725 mL reaction solution, 0.25 mL ddH2O and 0.025 mL EE were mixed and kept under light (4000 Lux) for 20 min, followed by recording absorbance at 560 nm. One unit of SOD activity was equal to the amount of enzyme required to inhibit 50% of NBT reduction. The GR activity was determined as decline in NADPH levels at 390 nm absorbance using extinction coefficient of 6.2 mM cm–1  were grinded with 4 mL trichloroacetic acid (5% v/v) and the homogenate was centrifuged at 4000 × g for 10 min at 4°C to get supernatant. To 2.6 mL NaH2PO4 , 0.25 mL supernatant was added and shaked. Then, 0.18 mL DNTP, dissolved in phosphate buffer, was added and mixture was kept at 300°C for 5 min. Absorbance was measured at 412 nm and GSH was quantified against standard curve.Lead contents in leaves and roots of upland cotton were determined according to + root scanner (Regent Instruments) using WinRhizo software for image acquisition. Transmission electron microscopy of terminal and median leaves and primary roots and secondary roots was done according to 4 for 1.5 h. Then graded series of ethanol i.e., 50–100% and absolute acetone were used to dehydrate samples. Samples were then put in 1:1 and 3:1 mixture of absolute acetone and final spur resin for 1 and 3 h, respectively. After, keeping the samples overnight in final spur resin, 100 nm thin samples, cut on a ultramicrotome , were stained with 6% aqueous uranyl acetate and Renold’s lead citrate, and mounted on copper grids. Micrographs were taken on transmission electron microscope model-7650, Hitachi Japan operating at 80 kv.Cotton roots were scanned by MIN MAC, STD 1600Data obtained from three replications were subjected to ANOVA, using statistical software package, SPSS 16.0 . Significant differences among various treatment means were determined at 5% probability by Duncan’s multiple range test. The data were presented as mean ± SD (standard deviation) of three independent biological replicates.2O2 than roots. Pb caused a significant percent increase of 52, 46, 41 and 53 in H2O2 in terminal leaves, median leaves, primary roots and secondary roots respectively as compared with control. GSH alone treatment increased H2O2 levels in all studied plant parts except secondary roots. GSH + Pb treatment maximally reduced H2O2 in primary roots than terminal and median leaves, while less reduction was found in secondary roots. Similarly, highest increase (vs. respective controls) in the OH• due to Pb was found in primary roots (98%) and secondary roots (80%) as compared to terminal (67%) and median leaves (60%). The highest decline in OH• was caused by GSH in terminal leaves in the presence of Pb. Although, GSH reduced OH• levels in the terminal and median leaves, its levels were still higher than primary and secondary roots. Notably, after Pb exposure, primary roots faced more lipid peroxidation (MDA) than terminal and median leaves, and secondary roots. GSH effectively controlled lipid peroxidation in all plant sections except median leaves.Data revealed that ROS levels in leaves and roots of upland cotton were elevated upon Pb exposure, followed by reduction through GSH application . In contResults demonstrated changes in the activities of antioxidant enzymes upon Pb, and Pb + GSH exposures . The CATResults revealed that maximum Pb uptake was observed in the primary roots followed by secondary roots . FurtherRepresentative photographs clearly showed the adverse effects of Pb and the alleviating impact of GSH on the Pb-treated cotton seedlings . As a whRoot scanning revealed that Pb stress reduced density of cotton roots; however, GSH increased root density in primary and secondary roots . Like lePrevious research has shown that cotton can accumulate significant amounts of heavy metals including Pb in seeds, leaves and roots, thereby bringing physiological, metabolic and ultrastructural changes . However2O2, OH• and MDA are stress indicators (2O2 generation and lipid peroxidation (MDA) was observed in the cotton’s terminal and median leaves as compared with primary and secondary roots , for example cadmium (Cd)-chl complex (Chl complex but by generation of O∙− also, which is triggered by this complex. Further, Pb2+ being analogous to Mg2+ may replace the later in chlorophylls, causing further deterioration in the chloroplast ultrastructure. A recent study, conducted on Lemna trisulca L. has reported rearrangement of chloroplast as an avoidance strategy to Pb stress (2O2 as stimulant in chloroplasts (Changes in cell wall ultrastructure of terminal and median leaves , 5 and pelements . Moreoveelements . In earlelements . Conformelements . In addielements . These r complex . Chloropb stress  which isroplasts , thus pl∙− and other ROSs (∙− in mitochondria, that’s why SOD activity was increased in GSH + Pb group. Moreover, O∙− may cause H2O2 production, which can be neutralized by CAT, but the later is present in peroxisomes only, providing strong evidence for H2O2 neutralization through GSH redox system (2O2 is reduced to H2O by GPX mainly using GSH as a substrate (In this study, mitochondria were seen deformed both in terminal and median leaves, and primary and secondary roots of upland cotton under Pb exposure. These changes may be attributed to excess ROS generation, as mitochondria release free electrons in electron transport chain to form Oher ROSs . As a fix system , and allubstrate . FurtherAllium cepa L. root tips cells upon exposure to Pb (Pb also altered cell nuclear matrix in the terminal and median leaves, and primary and secondary roots of upland cotton in current study. Previous research has shown some release of nuclear proteins such as nucleophosmin, nucleolin and fibrillarin from nucleolus to the cytoplasm in re to Pb . These nre to Pb . We obsere to Pb . Even unre to Pb . Thus, iLead is an unavoidable toxic heavy metal in the agro-ecosystems. In this multi-organ study, Pb increased ROS generation and decreased the activities of CAT, POD, SOD, APX, and SOD but increased GR activity in the terminal and median leaves, and primary and secondary roots of upland cotton. External GSH neutralized excess ROS levels, optimized activities of antioxidant enzymes and reduced Pb uptake in leaves and roots. However, maximum decline in the Pb uptake and peak GSH contents were observed in the terminal leaves. Moreover, Pb deteriorated ultrastructural configuration in both type of leaves and root system by affecting cell wall, plasma membrane, chloroplasts, mitochondria, and nucleus. GSH application maintained better cell morphology in the terminal leaves and primary roots as compared to other parts, mainly by regulating antioxidant machinery and restricted uptake of Pb. However, how DNA is regulated under external GSH supply in cotton leaves and roots in the presence of Pb needs further investigation.The datasets generated for this study are available on request to the corresponding author.MK, SS, and YZ equally contributed to the lab work. ZM and MDK prepared figures, tables and conducted transmission electron microscopy. SZ acquired funding, supervised this research work and critically revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer MF declared a shared affiliation, with no collaboration, with several of the authors ZM, SS, YZ, and SZ to the handling Editor at the time of review.


In both situations, chronic intermittent hypoxia (CIH) exposure produces carotid body (CB) hyperactivation generating an increased input to the brainstem which originates sympathetic hyperactivity, followed by hypertension that is abolished by CB denervation. CB has dopamine (DA) receptors in chemoreceptor cells acting as DA-2 autoreceptors. The aim was to check if blocking DA-2 receptors could decrease the CB hypersensitivity produced by CIH, minimizing CIH-related effects. Domperidone (DOM), a selective peripheral DA-2 receptor antagonist that does not cross the blood-brain barrier, was used to examine its effect on CIH (30 days) exposed rats. Arterial pressure, CB secretory activity and whole-body plethysmography were measured. DOM, acute or chronically administered during the last 15 days of CIH, reversed the hypertension produced by CIH, an analogous effect to that obtained with CB denervation. DOM marginally decreased blood pressure in control animals and did not affect hypoxic ventilatory response in control or CIH animals. No adverse effects were observed. DOM, used as gastrokinetic and antiemetic drug, could be a therapeutic opportunity for hypertension in SAHS patients’ resistant to standard treatments.The sleep apnea-hypopnea syndrome (SAHS) involves periods of intermittent hypoxia, experimentally reproduced by exposing animal models to oscillatory PO Current epidemiologic studies show that patients with SAHS have increased risk of hypertension, but the mechanisms are not well defined . Experimerations ,4 like terations . It is currently accepted that chronic intermittent hypoxia (CIH) produces carotid body (CB) sensitization generating an increased input to the brain stem which originates sympathetic hyperactivity, followed by hypertension. The CB is involved in the origin of hypertension and the functional elimination of CB after denervation or ablation prevents the development of hypertension ,7,8,9,10The study was designed to test the hypothesis that blocking D2R on peripheral chemoreceptors could modify the CB hypersensitivity produced by chronic exposure to intermittent hypoxia and consequently, to minimize CIH related effects. Domperidone (DOM) is a selective peripheral dopaminergic D2R antagonist that only marginally crosses the blood brain barrier and therefore lacks central activity . It has −1 DOM intraperitoneal (ip) was assessed in both groups of animals. p < 0.001 vs. CIH) after ip administration of DOM in CIH rats. In part C of the figure the effect of the drug on systolic and diastolic pressure values from CIH animals is represented, showing the decrease of both: systolic pressure from 158 ± 15 to 106 ± 26 mm Hg (*** p < 0.001) and diastolic pressure from 132 ± 15 to 75 ± 28 mm Hg (*** p < 0.001) from 9 animals. No significant changes were observed in control animals. p < 0.001 CIH vs. C) and with DOM treatment (* p < 0.05 CIHD vs. CD). −1, also without statistical differences between C and CIH or after the injection of DOM in each condition.Mean arterial blood pressure (MAP) is the most common and easy cardiocirculatory parameter to establish CIH effects . The acup < 0.01 vs. C) and the treatment with both doses of DOM during the last 2 weeks induced a MAP decreased to values of 95 ± 28 (* p < 0.05) and 82 ± 25 mg Hg (** p < 0.01) vs CIH respectively, reducing MAP to control values in CIHD group in a dose dependent manner.The effect of DOM chronic treatment is shown in 2/N2; 3 min), and recovery, breathing air again. No significant differences were observed in response to the hypoxic test among groups, using DOM (0.75 mg/day) treatment. A similar drop in MAP was observed in all animal groups breathing the hypoxic mixture, recovering previous values promptly after returning to air breathing. The percentage of hypoxia-induced MAP decrease in the four group of animals was between 52.6% ± 13.5 and 56.3% ± 7.2. Similarly, there were no statistical differences in Fulton index . C rats treated with both doses of DOM had a DA content 245 ± 28 and 206 ± 33 pmol/mg, respectively. However, the higher dose of DOM decreased DA content in CIHD group  to a lower value than that of C animals. ++p < 0.01 vs. CIH).In an attempt to define the participation of CB in the observed effect of DOM on arterial pressure we measured the influence of blocking D2R in CB endogenous CA content. In 2) on in vitro CB from C and CIH animals. DOM did not modify 3H-CA secretion induced by acute hypoxic stimulus in C rats but significantly decreased it in CIH animals, as represented in parts E and F in which the accumulative 3H-CA hypoxia-evoked release from C and CD and from CIH and CIHD, respectively, are shown.It is known that hypoxia and other natural and pharmacological stimuli activate CB chemoreceptors inducing the release of CA ,24. Figu2; 10 min) and hypercapnia  tests were assessed by continuous recording of respiratory frequency , tidal volume , and minute volume  in the four groups of animals. DOM  did not significantly modify any of the parameters in C or CIH animals. 2) and the different atmospheres. Respiratory response to acute hypoxia . MV values from CD were not significantly different compared to C animals at any hypoxia or hypercapnia stimuli tested. In every hypoxic test, CD animals shown a slight increase in MV and the opposite in the case of the hypercapnia stimulus (1151 ± 136 vs. 1041 ± 200 ml/min−1/kg−1). CIH animals showed a no significantly ventilatory response decrease at any atmosphere tested when compared to C rats. CIH reduced both, breathing frequency and/or tidal volume, producing approximately a 5–10% decrease in MV, as previously published [−1/kg−1, also reaching the maximum values when animals breathed 7% O2 (886 ± 126 vs. 953 ± 182 ml/min−1/kg−1). Values of MV in the hypercapnia test were 969 ± 97 vs. 984 ± 152 ml/min−1/kg−1 in CIH and CIHD. The decrease of MV observed in CIH was maintained in CIHD animals at every intensity of hypoxia test. Ventilatory responses in normoxia, acute hypoxia, and hypercapnia tests revealed that DOM treatment does not affect hypoxic respiratory response in C or CIH animals.In room air breathing rats  MV was 532 ± 288 in C and 564 ± 178 ml/minublished ,25,26. C3H-DA synthesis rate (1.24 ± 0.36 and 1.65 ± 0.43 pmol/mg RA/h) in C and CD respectively. In animals exposed to CIH, 3H-DA synthesis rate was 1.16 ± 0.29 pmol/mg RA/h and the treatment with DOM gave values of 1.33 ± 0.28 pmol/mg RA/h. Synthesis of 3H-NE is represented in part D with values of 0.65 ± 0.23 and 0.68 ± 0.23 pmol/mg/h in C and CD animals. In CIH animals 3H-NE synthesis rate was 0.80 ± 0.33 pmol/mg/h and DOM treatment marginally reduced it (0.64 ± 0.19 pmol/mg/h) although no statistical differences in CA synthesis rate were observed.To evaluate sympathetic activity, CA content and CA rate of synthesis in RA sympathetic nerve endings were measured. In control conditions A content−1 DOM on the hypertension induced by CIH showed a decrease of MAP of approximately 30% and no significant variation of blood pressure was observed in control animals with the same treatment. The fast effect of DOM in lowering blood pressure when administered ip could suggest a direct effect on D2R in blood vessels, which are not well described but have been found in the rat artery walls [2 consumption, CO2 production, or respiratory exchange ratio, highlighting the importance of endogenous DA in the normal cardiopulmonary response to exercise. DOM, a drug with therapeutic safety that has been clinically used to control gastro-esophageal motility and emesis, could be a therapeutic option for hypertension in SAHS patient’s resistant to other treatments. Animal models have demonstrated that CIH produces CB hyperactivation followed by secretion of DA and other neurotransmitters . Pre andry walls . Like otry walls  that thery walls , not obs2S) synthetized and released by chemoreceptor cells [As previously reported ,17, CB cor cells ,34,35 woor cells  that couor cells  studyingor cells  that DOMor cells  did not or cells ,41. It hor cells  that acu2+ and K+ channels, and consequently produce vasoconstriction. Additionally, DA is produced and released by the vascular endothelium and hypoxia stimulates CA release in those cells [It is currently accepted that the CB is the origin of the sympathetic hyperactivity observed in chronic exposure to intermittent hypoxia in animal models ,43,44,45se cells ,54. Renase cells . Resultsse cells  the lackse cells  and it hse cells ,57. The In summary, this study demonstrated that DOM can reverse the hypertension induced by chronic exposure to intermittent hypoxia in rats, and the CB could be involved in this modulation through the blocking of D2R. The underlying mechanisms implicated in the effect of DOM on lowering blood pressure in the CIH rat model need further investigation.Experiments were carried out in compliance with the applicable international laws and policies , and were reviewed and approved by the University of Valladolid Institutional Committee for Animal Care and Use .−1 body weight; i.p.) or ketamine plus diazepam . At the end of experiments, animals were euthanized by the administration of a lethal dose of sodium pentobarbital. Young adult male Wistar rats were used, with free access to standard food and water and maintained under controlled conditions of temperature, humidity and a stationary light–dark cycle. Rats were randomly distributed in four different groups: Control (C); control with DOM administered in the drinking water at doses of 1.5 or 0.75 mg/day during the last 15 days before the experiments (CD); chronic exposure to intermittent hypoxia  during 30 days as described previously  (CIH); a−1; i.p. anesthetized animals, tracheostomized and ventilated with room air  (60 cycles. min-1 and a positive end-expiratory pressure of 2 cm H2O) or with the hypoxia gas mixture . Arterial blood pressure was continuously monitored by a catheter located in the right common carotid artery and connected to a pressure transducer . Signals were stored  for later analysis. After arterial blood pressure measurements were completed, a 0.2 ml arterial blood sample was collected for hematocrit determination. After rats were euthanized, heart dissection was performed and right (RV) and left ventricle plus septum (LV+S) were weighed to calculate the Fulton Index (RV/LV+S). Arterial blood pressure and heart rate were recorded from ketamine 100 mg/Kg-1 and diazepam 2 mg/Kg2, reminder nitrogen) and hypercapnic gas mixture (5% CO2 in air) were used as previously described [Respiratory parameters as tidal volume , respiratory frequency , and minute ventilation  were obtained by whole-body, unrestrained plethysmography. Methacrylate-walled chambers  continuously fluxed (1.5 L/min) with air, hypoxic gas mixtures  content, in CB and renal artery (RA), were analyzed after removing the tissues from anesthetized animals, glass to glass homogenized (0.1N perchloric acid (PCA) and 0.1 mM EDTA), centrifuged and processed for HPLC-ED analysis as described before .2, 1.1 MgCl2, 10 HEPES, 5 glucose, 24 NaCO3H) equilibrated with gas mixtures containing 21 or 7% O2 and 5% CO2 (pH 7.40). Incubating solutions were changed every 10 min to measure 3H-CA content by scintillation counter as previously described [Isolated CB were incubated 2 hours with 3,5-3H-tyrosine (30 µM) of high specific activity  and cofactors for tyrosine hydroxylase and dopamine beta hydroxylase, 100 μM 6-methyl-tetrahydropterine and 1 mM ascorbic acid, respectively, to evaluate stimulus-evoked secretory response. Later, CB were moved to vials with Tyrode-bicarbonate solution . Mean value comparisons were made using unpaired Student’s t-test, One-Way Analysis of Variance (ANOVA) or Two-way ANOVA with Tukey’s or Sidak’s multi-comparison test, according to the structure of data, considering a p-value < 0.05 as statistically significant.


Aspergillus flavus is a saprophytic cosmopolitan fungus, capable of infecting crops both pre- and post-harvest and exploiting different secondary metabolites, including aflatoxins. Aflatoxins are known carcinogens to animals and humans, but display no clear effect in host plants such as maize. In a previous study, we mined the genome of A. flavus to identify secondary metabolite clusters putatively involving the pathogenesis process in maize. We now focus on cluster 32, encoding for fungal effectors such as salicylate hydroxylase , and necrosis- and ethylene-inducing proteins (npp1 domain protein) whose expression is triggered upon kernel contact. In order to understand the role of this genetic cluster in maize kernel infection, mutants of A. flavus, impaired or enhanced in specific functions , were studied for their ability to cause disease. Within this frame, we conducted histological and histochemical experiments to verify the expression of specific genes within the cluster , the production of salicylate, and the presence of its dehydroxylated form. Results suggest that the initial phase of fungal infection (2 days) of the living tissues of maize kernels  coincides with a significant increase of fungal effectors such as SalOH and Npp1 that appear to be instrumental in eluding host defences and colonising the starch-enriched tissues, and therefore suggest a role of cluster 32 to the onset of infection. Aspergillus is one of the best-described fungal genera, mainly because of its negative impact on economy and agriculture through production of highly toxic secondary metabolites +. Chromatographic separation was performed with a Zorbax Eclipse XDB-C18, 50 × 4.6 mm inner diameter, 1.8 µm particle size,  at 25 °C and the injection volume was 5 µL. The mobile phases consisted of A: methanol/water/acetic acid 10:89:1 (v/v/v) and B: methanol/water/acetic acid 97:2:1 (v/v/v) while both contained 5 mM ammonium acetate. The total runtime was 20 min, with a flow rate of 0.4 mL/min. The gradient elution was as follows: 0–2 min 1% B, 3–14 min 99% B, 15–18 min 99% B, 19–20 min 1% B. The gradient was followed by 4 min for re-equilibration. The acquisition was in MRM negative ion mode [M − H]+. The elution gradient was carried out with binary solvent system consisting of 5 mM NH4AcO in H2O (solvent A) and 5 mM NH4AcO in MeOH (solvent B) at a constant flow-rate of 600 mL·min−1. The elution gradient was as follows: 0–2 min 0% B, 0–14 min 100% B, 14–18 min 100% B, with 2 min for re-equilibration. The MRM fragmentation patterns for each compound are listed in For rutin and quercetin, the acquisition was in MRM positive ion mode [M-H]® 7100- Haraeus-Kulzer, Wehrheim, Germany)/absolute ethanol (1/3), two hours in resin/absolute ethanol (1/1), and two hours in resin/ absolute ethanol (3/1). Overnight impregnation  was performed at 4 °C and then replaced with resin plus catalyst 2 (20:1) at room temperature for resin inclusion. The included samples were then cut with an automatic microtome HM 350  to 8 mm as thickness and stained with toluidine blue for 2 min.Maize kernels (infected and non-infected) were immersed in 70% ethanol for two hours, then 90% ethanol for two hours and next 100% ethanol overnight. Two hours in resin (TechnovitA. flavus growth was not detectable. The microscopic observation was carried out with objective 40×.Maize kernels (infected and non-infected) were transferred in cooled (−20 °C) 80% acetone (dilution with distilled water) and chilled for 20 min at −20 °C. Acetone was removed and samples were washed three times with distilled water before the addition of GUS buffer to the samples. An infiltration of 15 min under vacuum was performed, followed by incubation at 37 °C in darkness for at least 2 h. To fix the reaction, the GUS buffer was removed, then replaced with 70% ethanol and preserved at 4 °C. The time points of withdrawals were 4 h, 8 h, 16 h, 24 h, 48 h, 72 h, 96 h and 7 d. No histochemical data is available for the 4 and 8 h time points because Programmed cell death was evaluated using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labelling (TUNEL assay) using a TACS XL™ detection kit . Procedures were performed according to the manufacturer’s instructions on kernel sections (6 µm thick) immediately observed under a light microscope (Leica DMRB), and photographs were captured using a by DC500 . Both positive and negative controls were performed according to the manufacturer. The microscopic observation was carried out with objective 100×.A. flavus [2Cys6Zn∆ strain  , the necrosis and ethylene inducing protein (npp1), and quercetin dehydrogenase (AFLA_096260). The elected genes are shown to enable infection by preventing effective plant defence  . During  evident . In our  strains : data sh defence . Zn2Cys6SalOH  was validated also by HPLC/MS-MS. We observed an increased level of cathecol in comparison to the starting level of salicylic acid in all A. flavus strains (A. flavus is able to bypass the plant defences, namely those regulated by SA, by taking advantage of the activity of genes specifically encoded by cluster 32. NLPs are non-specific toxins capable of inducing host tissue necrosis and defence responses [npp1 is involved in A. flavus virulence on maize kernels. Experiments on two strains, nepA-OE-GUS B5-12 and nepA∆-GUS B9-5, allowed us to show induced necrotrophy through histochemical assays (nepA∆-GUS B9-5 is similar to colonisation by nepA-OE-GUS B5-12, which supports the idea that reduced pathogenicity of the nepA∆-GUS B9-5 strain is not the direct consequence of a decreased growth rate, and suggest a central role of npp1 in the inter process that sees the saprophytic lifestyle of A. flavus.The activity of  strains . This evesponses . We provl assays . The infA. flavus-maize competition, cluster 32 appears to represent the “winning hand” of the pathogen over the host and, by extension, the genetic element that enables the lifestyle switch from biotrophic to necrotrophic. We can thus argue that cluster 32 has acquired a centrality in A. flavus ecology, which makes it an interesting topic in future research efforts, especially those aimed at providing the host plant with genetic elements to confer innate resistance.In


Paper-based microfluidic analysis devices (μPADs) have attracted attention as a cost-effective platform for point-of-care testing (POCT), food safety, and environmental monitoring. Recently, three-dimensional (3D)-μPADs have been developed to improve the performance of μPADs. For accurate diagnosis of diseases, however, 3D-μPADs need to be developed to simultaneously detect multiple biomarkers. Here, we report a 3D-μPADs platform for the detection of multiple biomarkers that can be analyzed and diagnosed with a smartphone. The 3D-μPADs were fabricated using a 3D digital light processing printer and consisted of a sample reservoir (300 µL) connected to 24 detection zones (of 4 mm in diameter) through eight microchannels (of 2 mm in width). With the smartphone application, eight different biomarkers related to various diseases were detectable in concentrations ranging from normal to abnormal conditions: glucose (0–20 mmol/L), cholesterol (0–10 mmol/L), albumin (0–7 g/dL), alkaline phosphatase (0–800 U/L), creatinine (0–500 µmol/L), aspartate aminotransferase (0–800 U/L), alanine aminotransferase (0–1000 U/L), and urea nitrogen (0–7.2 mmol/L). These results suggest that 3D-µPADs can be used as a POCT platform for simultaneous detection of multiple biomarkers. Paper-based microfluidic analysis devices (μPADs) have attracted attention as a cost-effective tool for point-of-care testing (POCT), food safety, and environmental monitoring ,2,3,4. TMost recently, we have reported on a method to fabricate 3D-µPADs for the colorimetric detection of biomarkers using 3D digital light processing (DLP) printing . By expoSmartphones can provide easy detection and quick analysis results for POCT kits ,17,18. FIn this study, we report on 3D-µPADs for smartphone-based detection of multiple biomarkers. The 3D-µPADs were fabricated using a 3D DLP printer with ultraviolet (UV) light. They consisted of a single reservoir connected with 24 detection zones through eight microchannels, allowing eight different biomarkers to be simultaneously and repeatedly detected in a sample. Colored signals from the 3D-µPADs were imaged and analyzed using a smartphone.D-(+)-Glucose, glucose oxidase (GOx) from Aspergillus niger, 4-aminoantipyrine (4-AAP), albumin from human serum, tetrabromophenol blue (TBPB), citric acid, cholesterol (Chol.) ALP, alkaline phosphatase yellow (p-nitrophenyl phosphate), an AST activity assay kit, an alanine aminotransferase (ALT) activity assay kit, a creatinine assay kit, and ethyl alcohol were purchased from Sigma-Aldrich Korea . A urea nitrogen (UN) colorimetric detection kit was purchased from Thermo Fisher Scientific . Horseradish peroxidase (HRP), cholesterol oxidase (CO), and cholesterol esterase (CE) were obtained from Toyobo Co. . N-Ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline sodium salt monohydrate (MAOS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline, sodium salt, and dihydrate (ADOS) were purchased from Dojindo . ® Professional  and saved as different STL files. The files were then sent to the DLP printer  using a universal serial bus (USB) drive.The top and bottom structures of the 3D-µPADs were separately designed using the Student Edition of InventorBefore printing, a layer of filter paper  from Whatman International Ltd.  was soaked with UV-curable resin  in a petri dish for 10 s. The resin-soaked paper was moved to a tray filled with resin in the printer. Then, its bottom side was exposed for 2 s to the UV patterns for the bottom structures projected through the tray b. After The reagents for the detection of glucose were prepared by dissolving MAOS (1 mM), 4-AAP 10 mM), HRP (1 mg/mL), and GOx (10 mg/mL) in phosphate-buffered saline (PBS: pH 7.4) ,25. The  mM, HRP To test whether the 3D-µPADs were able to simultaneously detect multiple biomarkers, eight biomarkers for metabolic diseases such as diabetes and cardiovascular, liver, and renal diseases were selected. Glucose is a biomarker for diabetes , cholestTo obtain a dose-response curve for biomarkers, standard solutions of the biomarkers were prepared by mixing the stock solution of each biomarker with different volumes of PBS (pH 7.4). The stock solution was prepared by dissolving a certain concentration of a biomarker in PBS. The concentrations of the standard solutions for the biomarkers varied, depending on the optimal detection range of each biomarker: 0–20 mmol/L for glucose, 0–10 mmol/L for Chol., 0–7 g/dL for albumin, 0–800 U/L for ALP, 0–500 µmol/L for creatinine, 0–800 U/L for AST, 0–1000 U/L for ALT, and 0–7.2 mmol/L for UN. To generate the standard curve of a biomarker, 10 µL of the standard solution of the biomarker was dropped on the detection zone. At 1 min after the dropping, the gray intensity in the detection zone was measured using ImageJ . To simultaneously detect the biomarkers in PBS, a mixture of the biomarkers at different concentrations was prepared by mixing their stock solutions with PBS at various ratios. Furthermore, 300 µL of the mixture was dropped onto the sample reservoir of the 3D-µPADs. At 3 min after the dropping, the gray intensity of each detection zone was measured using ImageJ.Once the names of the biomarkers were marked on the start screen, the linearization equation calculated from the dose-response curve was specified with its normal range. Images of the control and test samples were imported into the application. The detection zones in the images were designated as circles. The gray intensity in each circle was measured. Then, the gray intensity value for each biomarker was obtained by subtracting the gray intensity of the sample from the intensity of the control. Eventually, the concentration and level (low to high) of the eight biomarkers appeared.To reduce the effect of reflecting light on the measurement of the color intensity of the detection zones , a switcFor fast printing, UV light instead of visible light  was usedThe 3D-µPADs consist of a sample reservoir  in the center connected with 24 detection zones (diameter: 4 mm) through eight channels  c. At lean = 48), indicating that all channels of the 3D-µPADs were uniformly fabricated.The flow rate of a sample solution affects signal generation in 3D-µPADs . TherefoTo test whether solutions in the reservoir reached the detection zones at a similar flow rate, the flow speed of ink in each channel was measured after dropping 300 µL of red ink into the reservoir over the channels in the 3D-µPADs at different times (0–150 s) e. It too2) values were all >0.96. According to the dose-response curves, the limits of detection were 0.3 mmol/L for glucose, 0.3 mmol/L for cholesterol, 0.1 g/dL for albumin, 10 U/L for ALP, 40 μmol/L for creatinine, 10 U/L for AST, 50 U/L for ALT, and 0.04 mmol/L for UN. The linear range of each biomarker was obtained from the dose–response curve, including its respective normal range  CoefficThe 3D-µPADs have 24 detection zones in total, with three detection zones in a pair allocated to each biomarker, allowing the biomarker to be measured three times a. When aFor fast and convenient detection of multiple biomarkers on the 3D-µPADs, a smartphone application was developed and tested with 300 µL of PBS containing the eight biomarkers . Once the colors were generated from the detection zones on the 3D-µPADs, which took ~3 min a,b, the In this study, 3D-µPADs were fabricated using a 3D DLP printer for multiple detections. The reservoir on 3D-µPADs can store enough sample for multiple detections. Sequentially, the concentration can be obtained three times per biomarker in one experiment to reduce errors. Eight biomarkers  in PBS and serum could be detected simultaneously on the 3D-μPADs through a smartphone. In this experiment, the size of the detection zone was set to 4 mm to easily analyze the selected area using a smartphone camera. However, using a high-quality smartphone, we could minimize the detection zone. This also reduces the amount of the sample, which leads to the detection of more biomarkers. In previous research, we succeeded in integrating plasma separation membrane with 3D-µPADs . This su


One of the challenges in the emerging field of synthetic biology is engineering organelle genomes. Creating synthetic organelle genomes can open the door to a wide range of applications, such as improving crop yields, treating mitochondrial diseases, or manufacturing high-value chemicals in an environmentally sustainable way. Organelles are tiny biological machines that work inside of living cells. Mitochondria, for example, are responsible for harvesting sugar to create energy for the cell. In previous work, we demonstrated a method to make copies of an alga mitochondrial genome using yeast and bacteria. Algae are of industrial interest for their potential to produce and store large quantities of biofuels and nutritional ingredients. Here, we applied the same approach to copy the mitochondrial genome of a related alga. Although the cloning of this mitochondrial genome in yeast using the previously developed method was possible, the properties of this genome may make it more susceptible to mutations during propagation in bacteria. This work expands our understanding of potential hurdles that can be encountered when cloning and propagating synthetic organelle genomes in host organisms.Phaeodactylum tricornutum can be cloned and engineered in Saccharomyces cerevisiae and Escherichia coli. Here, we show that the same approach can be used to clone mitochondrial genomes of another microalga, Thalassiosira pseudonana. We have demonstrated that these genomes can be cloned in S. cerevisiae as easily as those of P. tricornutum, but they are less stable when propagated in E. coli. Specifically, after approximately 60 generations of propagation in E. coli, 17% of cloned T. pseudonana mitochondrial genomes contained deletions compared to 0% of previously cloned P. tricornutum mitochondrial genomes. This genome instability is potentially due to the lower G+C DNA content of T. pseudonana (30%) compared to P. tricornutum (35%). Consequently, the previously established method can be applied to clone T. pseudonana’s mitochondrial genome, however, more frequent analyses of genome integrity will be required following propagation in E. coli prior to use in downstream applications.Algae are attractive organisms for biotechnology applications such as the production of biofuels, medicines, and other high-value compounds due to their genetic diversity, varied physical characteristics, and metabolic processes. As new species are being domesticated, rapid nuclear and organelle genome engineering methods need to be developed or optimized. To that end, we have previously demonstrated that the mitochondrial genome of microalgae  Thalassiosira pseudonana. This model centric diatom is naturally found in oceanic water and plays a significant role in global carbon cycling and combatting climate change [T. pseudonana, its nuclear, mitochondrial, and plastid genomes were sequenced [T. pseudonana include selectable markers [T. pseudonana’s chloroplast and mitochondria have been developed and proteomic data made available [T. pseudonana nuclear genome; however, engineering of its organelle genomes is still challenging. There are several advantages to engineering organelle genomes, including polycistronic gene organization, the lack of transgene silencing, reduced positional gene expression effects, and the compartmentalization of biosynthetic pathways, each of which simplifies engineering [Recent advancements in DNA sequencing and synthesis resulted in the development of a powerful set of biotechnology tools that can help to address global challenges in food and water sustainability, medicine production, and eco-friendly energies. Many potential organisms are under investigation for desirable properties useful for biotechnology applications. One attractive candidate is e change ,2. In ade change ,4. Due tequenced ,6,7, enaequenced ,9. Addit markers , promote markers ,12 and c markers ,13,14. Fvailable . Most ofineering . In prepineering ,21,22,23Phaeodactylum tricornutum, a model diatom algae species, in baker’s yeast Saccharomyces cerevisiae and Escherichia coli [. S. cerevisiae has proven to be an excellent host for cloning large DNA fragments or whole genomes [E. coli [T. pseudonana because of the unique characteristics of its mitochondrial genome. First, the T. pseudonana mitochondrial genome is compact (~44 kbp), harboring a relatively small repeat region (~5 kbp) compared to the repeat region of P. tricornutum (~35 kbp). Second, T. pseudonana has a lower G+C content mitochondrial genome (30%) than P. tricornutum (35%). Third, T. pseudonana’s mitochondrial genome uses an alternative genetic code, which substitutes a typical stop codon (UGA) for a tryptophan residue [We recently demonstrated the cloning of the mitochondrial genome of hia coli . S. cere genomes ,25,26,27[E. coli . To test residue .T. pseudonana‘s mitochondrial genome in yeast and demonstrate that it can also be propagated in E. coli. In the first iteration (Design 1), the mitochondrial genome was cloned in its entirety ; in the second iteration (Design 2), ~3.8 kbp of the ~5 kbp repetitive region was excluded . Growth experiments performed on yeast in liquid media revealed that yeast strains carrying plasmids with cloned mitochondrial genomes had a slightly increased growth rate; however, after 24 hours, the yeast strains grew to the same (Design 1) or slightly lower (Design 2) end-point densities as control strains. When these genomes were propagated in E. coli on a low copy number plasmid, they had the same growth rate and end-point densities compared to the control strains. However, when grown with arabinose to increase the copy number of these genomes, all samples grew to significantly lower end-point densities after 11.5 h. Also, analysis of plasmids containing mitochondrial genomes following propagation in E. coli over 60 generations showed that about 17% of T. pseudonana mitochondrial genomes were mutated compared to 0% identified for equivalent experiments conducted using the P. tricornutum mitochondrial genome. Finally, RNA sequencing performed on E. coli harboring either alga’s mitochondrial genome found that expression can be detected for T. pseudonana and P. tricornutum mitochondrial genes.Here, we report the successful cloning of Thalassiosira pseudonana (Culture Collection of Algae and Protozoa CCAP 1085/12) was grown in synthetic seawater (L1 medium) supplemented with 200 μM of sodium silicate (Na2SiO3-9H20)  at 18 °C under cool white fluorescent lights (75 μE m−2 s−1) and a photoperiod of 16 h light: 8 h dark. L1 media was made as previously described [0) was grown at 30 °C in rich yeast medium , 40 g L−1 peptone , 40 g L−1 glucose , and 200 mg L−1 adenine hemisulfate ), or synthetic complete medium lacking either histidine or both uracil and histidine . After yeast spheroplast transformation, all complete minimal media used contained 1 M sorbitol  [−1) . Canada) . EscheriE. coli, yeast, and algae was isolated as previously described [DNA from escribed .T. pseudonana genomic DNA. The mitochondrial genome was amplified as eight overlapping fragments , and the respective forward and reverse primers each at a final concentration of 0.2 μM. The thermocycler  conditions for fragments 2, 4–9, and 11–12 were as follows: 25 cycles of 98 °C for 10 s, 60 °C for 15 s, and 68 °C for 600 s, and one cycle of 68 °C for 600 s, finishing with an infinite hold at 12 °C. The thermocycler for fragment 1 was programmed as follows: 5 cycles of 98 °C for 10 s, 50 °C for 15 s, and 68 °C for 420 s, followed by 20 cycles of 98 °C for 10 s, 60 °C for 15 s, and 68 °C for 420 s, and one cycle of 68 °C for 600 s, finishing with an infinite hold at 12 °C. The thermocycler for fragment 3 was programmed as follows: 25 cycles of 98 °C for 10 s, 50 °C for 15 s, and 68 °C for 660 s, and one cycle of 68 °C for 600 s, finishing with an infinite hold at 12 °C. The thermocycler for fragment 10 was programmed as follows: 25 cycles of 98 °C for 10 s, 55 °C for 15 s, and 68 °C for 60 s, and one cycle of 68 °C for 600 s, finishing with an infinite hold at 12 °C. PCR product amplification was confirmed by performing agarose gel electrophoresis with 2 μL of PCR product on a 1.4% agarose (w/v) gel. To eliminate plasmid template DNA, PCR products were treated with DpnI restriction enzyme as described in Reference [Each fragment was individually amplified in a 50 μL PCR reaction using 1 μL of PrimeSTAR GXL polymerase , 1 μL of template DNA , and the respective forward and reverse primers each at a final concentration of 0.2 μM. The thermocycler for fragments 1, 3–5, and 7–9 was programmed as follows: 25 cycles of 98 °C for 10 s, 60 °C for 15 s, and 68 °C for 600 s, and one cycle of 68 °C for 600 s, finishing with an infinite hold at 12 °C. The thermocycler for fragments 2 and 6 was programmed as follows: 30 cycles of 98 °C for 10 s, 50 °C for 15 s, and 68 °C for 660 s, followed by one cycle of 68 °C for 600 s, finishing with an infinite hold at 12 °C. PCR product amplification was confirmed by performing agarose gel electrophoresis with 2 μL of PCR product on a 1.4% agarose (w/v) gel. To eliminate plasmid template DNA, PCR products were treated with DpnI restriction enzyme as described in Reference [Each fragment was individually amplified in a 50 μL PCR reaction using 1 μL of PrimeSTAR GXL, 1 μL of template DNA , 2 μL of NEBuffer™ 3.1 restriction buffer , 0.4 μL PvuI , and 10.6 μL of water. For pTP-PCR C3.1/4.1, digestion reactions were generated using 7 μL of DNA (~5000 ng μL−1), 2 μL of CutSmart® Buffer , 0.4 μL PmeI , 0.4 μL BamHI-HF , and 10.2 μL of water. Reaction mixtures were transferred to a Bio-Rad thermocycler and incubated either at 37 °C for 90 min, or 37 °C for 90 min followed by 65 °C for 20 min for pTP-PCR C1.1/2.1 and C3.1/4.1, respectively. After confirmation by multiplex PCR and diagnostic restriction enzyme digestion, the four plasmids underwent next-generation whole plasmid sequencing at CCIB DNA Core .To identify positive clones, individual eference  with theS. cerevisiae strains harboring pTP-PCR C1, C2, C3, and C4 plasmids, or pPtGE31 and pAGE3.0 control plasmids , as described in Reference [600) measurements taken every 15 min for 24 h. This experiment was performed with three biological replicates, each with four technical replicates, therefore, 12 readings were obtained and averaged for each strain, the standard error of the mean was calculated, and the curves were plotted. End-point densities at 1440 min were averaged for each strain and the standard error of the mean was calculated. The doubling time (td) of each replicate was determined using the R package Growthcurver  [Growth rates were evaluated for eference , with abr, 2016) . The td E. coli strains harboring pTP-PCR C1.1, C2.1, C3.1, and C4.1 plasmids or pPtGE31 and pAGE3.0 control plasmids , as described in Reference [600 measurements were taken every 15 min for 11.5 h. This experiment was performed with three biological replicates, each with four technical replicates, therefore, 12 readings were obtained and averaged for each strain, the standard error of the mean was calculated, and the curves were plotted. End-point densities at 705 min were averaged for each strain and the standard error of the mean was calculated. The doubling time (td) of each replicate was determined using the R package Growthcurver  [Growth rates were evaluated for eference , except r, 2016) . The td E. coli harboring the pTP-PCR C2.1, pPT-PCR C2.1 [−1) from frozen strain stocks. In the morning, 1 mL of cells was diluted into 25 mL of LB media supplemented with chloramphenicol (15 μg mL−1), and grown for 120 min at 37 °C until optical density (OD600) reached 1. Subsequently, the RNA stabilization of the culture was performed using RNAprotect Bacteria Reagent . Briefly, 400 μL (2 × 108 cells) of culture was transferred to a 15 mL Falcon tube containing 800 μL of RNAprotect Bacteria Reagent, and the suspension was vortexed for 5 s and incubated at room temperature (RT) for 5 min. Total RNA was isolated using the RNeasy Mini Kit  according to the manufacturer’s instructions. Following treatment with DNase using RNase-free DNase Set , the RNA concentration was determined using DeNovix  and the integrity was verified by running 400 ng of RNA on a 1% agarose (w/v) gel. RNA samples were stored at −80 °C until use.PCR C2.1 , or pPtGThe quality of isolated RNA Methods  was valiE. coli harboring either pTP-PCR C2.1 or pPT-TAR C1 [E. coli, isolating single colonies on LB plates, and then grown overnight in liquid before freezing) overnight at 37 °C (225 rpm) in 5 mL of LB media supplemented with chloramphenicol (15 μg mL−1). The saturated overnight culture was adjusted to an OD600 of 0.1 and frozen strain stocks were generated (we defined this as G0). Next, 1 μL of the adjusted culture was added to 50 mL of LB media supplemented with chloramphenicol (15 μg mL−1). Cultures were grown at 37 °C (225 rpm) to approximately an OD600 of 3. Four repetitions (~60 generations) of diluting grown cultures to an OD600 of 0.1 and passaging 1 μL of adjusted culture into 50 mL of LB media supplemented with chloramphenicol (15 μg mL−1) were performed. After four serial passages, frozen strain stocks of each bacterial strain adjusted to an OD600 of 0.1 were generated for analysis of plasmid stability (G60).T-TAR C1  plasmids−1) and grown at 37 °C for 24 h to obtain single colonies. Thirty single colonies of each construct were struck onto selective LB agar plates supplemented with chloramphenicol (15 μg mL−1) and grown for 12 h at 37 °C. Next, each streak was resuspended in a PCR tube containing 100 μL of TE buffer. The cell mixture was transferred to a Bio-Rad thermocycler and incubated at 95 °C for 15 min to lyse the cells. Using a microfuge, the cellular debris was pelleted to the bottom of the PCR tube and 1 μL of supernatant was used as a template for diagnostic multiplex PCR. Two additional diagnostic primer sets . To test plasmid stability, primer sets P 19-24F/R were used to generate 140, 229, 300, 440, 540, and 606 bp amplicons, respectively. Then, 2 μL of the PCR products were loaded onto a 2% agarose (w/v) gel for electrophoresis and analyzed.Strain stocks of pTP-PCR C2.1 or pPT-TAR C1 from the start (G0) and end (G60) of propagation were thawed on ice for 20 min and then diluted 1:5000 with LB media in 1.5 mL microcentrifuge tubes. Next, 100 μL of each diluted culture was plated separately onto selective LB agar plates supplemented with chloramphenicol  as well as a reduced version lacking 3.8 kbp of the ~5.0 kbp repetitive sequence   and the pPtGE31 plasmid [E. coli. In total, 12 DNA fragments were amplified  . For Des plasmid  backbonemplified C and asscolonies . For Desmplified D and assmplified . For eace digest E, F. ForT. pseudonana mitochondrial genomes, or variations between our strain and the sequenced genome. If desired, individual fragments could be cloned and confirmed by sequencing before use in yeast assembly.Sequences obtained for the pTP-PCR plasmids were aligned to reference sequences . Upon anS. cerevisiae and E. coli were used as host organisms to clone and store the T. pseudonana mitochondrial genome. We measured the growth of E. coli and yeast strains in liquid media using a 96-well plate reader. Growth experiments performed for yeast revealed that strains carrying plasmids with cloned mitochondrial genomes had a slightly increased growth rate; however, after 24 h, the yeast strains grew to the same (Design 1) or slightly lower (Design 2) end-point densities as compared to control strains  copy number. When these genomes were propagated in E. coli without induction of plasmid copy number, there was no significant difference in the growth rate as compared to the control strain , we observed that a small fraction of genomes were being mutated, as was evident by an absent PCR amplicon when clones were evaluated with multiplex PCR (data not shown). To further investigate this, we evaluated one cloned mitochondrial genome (pTP-PCR C2.1) directly after transferring from yeast to E. coli (“G0”) or after approximately 60 generations (“G60”). Since we did not observe similar mutations in our previous work cloning the P. tricornutum mitochondrial genome, we used our cloned P. tricornutum mitochondrial genome (pPT-TAR C1) as a control [T. pseudonana (from clone pTP-PCR C2.1) and P. tricornutum (from clone pPT-TAR C1) were evaluated at G0 and G60 using a higher resolution multiplex PCR screen with six amplicons.Additionally, when propagated in  control . In totaE. coli clones harboring either plasmid showed successful amplification of all six amplicons. At G60, all 30 E. coli clones harboring pPT-TAR C1 had a complete genome as analyzed by multiplex PCR, suggesting that, over 60 generations, this plasmid is stably maintained. However, only 25 of 30 E. coli clones containing pTP-PCR C2.1 had complete genomes at G60, as analyzed by multiplex PCR . As expected, no reads from pPtGE31 mapped against the algal mitochondrial genomes, while genes from the pTP-PCR C2.1 and pPT-PCR C2.1 samples showed low read coverage. Low coverage of the mitochondrial genomes was likely obtained because the rRNA depletion kit selected failed to deplete the E. coli host’s rRNA. The bacterial selection marker CAT (providing resistance to chloramphenicol) was detected in all samples as expected; however, low read coverage was obtained for many of the genes that are on the pTP-PCR C2.1 and pPT-PCR C2.1 mitochondrial genomes  could be cloned [It has been observed previously that cloning low G+C-content DNA into bacteria can be problematic. As the G+C-content decreases, the probability of any sequence producing a spontaneous promoter or origin of replication becomes more likely, which can result in plasmid toxicity and instability . The adde cloned . Future E. coli hosts, the low read coverage obtained prevented us from performing a reliable differential expression analysis. However, the data does demonstrate that genes of the algal mitochondrial genomes are expressed in the E.coli hosts. To determine what genes of the mitochondrial genomes are more strongly expressed in the E. coli hosts, we will need to obtain higher coverage of the mitochondrial genome by successfully depleting the host’s rRNA in a future experiment.Although we can confirm that most of the mitochondrial genes on the pTP-PCR C2.1 and pPT-PCR C2.1 plasmids are expressed in Chlamydomonas reinhardtii [P. tricornutum [T. pseudonana’s mitochondria will be designed using alternative genetic code (UGA will be used for a tryptophan instead of a stop codon), allowing antibiotic resistance proteins to be expressed only in the mitochondrial compartment. These two design features will generate a powerful system for developing the genetic tools required for mitochondrial DNA delivery in T. pseudonana.Now that two algal mitochondrial genomes have been cloned in host strains, we have a better understanding of potential hurdles that can be encountered when applying this method to other species. As the next step, a robust method for delivery of these genomes to mitochondria will need to be developed. First, the mitochondrial genomes will need to be engineered with mitochondria-specific selectable markers. Using antibiotics that target organelle-specific processes, previous studies have demonstrated an increased efficacy of antibiotic resistance proteins when they are localized to the organelle compartment ,39. Furtnhardtii  and the cornutum , respectT. pseudonana in its entirety  or lacking a repetitive region . The cloned genomes imposed no substantial growth burden on S. cerevisiae and E. coli when these host organisms were used to propagate the plasmids. In E. coli, some plasmid instability was observed after 60 generations, likely attributable to the low G+C-content of the mitochondrial genome. RNA sequencing was performed, and it was found that mitochondrial genes were being expressed from the plasmids harbored in E. coli. In this study, we replicated the previous methods for cloning and manipulating algal mitochondrial genomes using T. pseudonana. Subsequent work will focus on developing the technologies required for efficient mitochondrial DNA delivery.We have demonstrated that a previously developed method for cloning and manipulating mitochondrial genomes can be applied to additional microalga. With a PCR-based approach, we cloned the mitochondrial genome of


Red fruits and their juices are rich sources of polyphenols, especially anthocyanins. Some studies have shown that such polyphenols can inhibit enzymes of the carbohydrate metabolism, such as α-amylase and α-glucosidase, that indirectly regulate blood sugar levels. The presented study examined the in vitro inhibitory activity against α-amylase and α-glucosidase of various phenolic extracts prepared from direct juices, concentrates, and purees of nine different berries which differ in their anthocyanin and copigment profile. Generally, the extracts with the highest phenolic content—aronia , pomegranate , and red grape —showed also one of the highest inhibitory activities against α-amylase  and α-glucosidase  and, partially, were even more potent inhibitors than acarbose . Additionally, the investigation of single anthocyanins and glycosylated flavonoids demonstrated a structure- and size-dependent inhibitory activity. In the future in vivo studies are envisaged. Small fruits and their juices are rich sources of polyphenols, especially anthocyanins. The anthocyanins are a sub-group of flavonoids that primarily occur in violet, blue, and red fruits. They are water-soluble plant pigments and serve as an attractant, as well as a protector against oxidative stress and excessive light. Their hue is determined by the pH and the presence of copigments . CopigmeDiabetes mellitus is a major global health problem: in 2019 463 million people suffered from diabetes worldwide, and its global prevalence is increasing. It is commonly caused by impaired insulin secretion, with or without insulin resistance, and is characterized by hyperglycemia . There aThe inhibition of carbohydrate digestive enzymes such as α-amylase and α-glucosidase is one of the therapeutic strategies to control postprandial hyperglycemia and also the basis of the antidiabetic effects of some plant foods . The digSome inhibitors of these digestive enzymes have been approved for use as medicines to treat type I and II diabetes, namely acarbose, miglitol, and voglibose . In addiThis study focused on extracts prepared from red fruit juices. The extracts were tested in an enzyme inhibition assay to determine their inhibitory activity against α-amylase and α-glucosidase. Additionally, isolated anthocyanins and copigments were investigated to create a link between the molecular structure and the inhibitory potential of individual phenolic compounds and also to examine possible synergistic and antagonistic mechanism.Thirty-eight fruit products derived from aronia, bilberry, blackcurrant, cranberry, elderberry, lingonberry, pomegranate, red grape, and sour cherry were extracted and investigated with regard to their inhibition of α-glucosidase and α-amylase activity, including direct juices , concentrates , and purees.In a recently published article, we presented the phenolic composition of different extracts in detail . Here, wThe total phenolic contents (TPC) of the extracts are presented in The extracts with the highest total phenolic contents determined by the Folin-Ciocalteu method were those derived from pomegranate, aronia, and red grape. In these three extracts, the composition of polyphenols determined by HPLC-MS analysis was quite different. Aronia juice extracts contained mostly anthocyanins and procyanidins, as reported previously . Pomegra®, was used as the positive control (PC); its IC50 value  was determined to be 441 ± 30 μg/mL. 50 values for the fruit product extracts (amounts of samples: 1–4). All of the aronia products  had lower IC50 values than the positive control, as had the extracts prepared from lingonberry concentrate (IC50 = 361 μg/mL) and cranberry puree (IC50 = 424 μg/mL). These products were the strongest inhibitors among the studied red fruit extracts. In general, the extracts of the juice concentrates exhibited stronger inhibitory activity than those of the direct juices. However, extracts of aronia direct juice (273 ± 57 μg/mL) were marginal more potent inhibitors than the juice concentrate extracts (381 ± 57 μg/mL). In the α-amylase inhibition screening, all of the puree extracts were more active than the corresponding direct juice extracts, although only purees of cranberry, blackcurrant, and bilberry were investigated . The direct juice extracts of sour cherry (IC50 = 1943 ± 615 μg/mL), elderberry (IC50 = 1373 ± 320 μg/mL), and bilberry (IC50 = 1088 ± 192 μg/mL) were the weakest α-amylase inhibitors among the studied products. In this assay there were no significant differences observed between the investigated red grapes varieties, except for one sample juice concentrate from Italian grapes which demonstrated half the inhibitory activity than the others.The pseudotetrasaccharide acarbose, which is a well-known α-amylase inhibitor marketed under the brand name Glucobay50 values of the red juice extracts used in the α-glucosidase assay are shown in 50 = 1439 ± 85 μg/mL) was used as the positive control (PC). All the studied extracts other than those derived from elderberries  had lower IC50 values than the positive control and thus had higher inhibitory activities. The puree extracts  had very similar inhibition potentials to the corresponding direct juice extracts, whose IC50 values were 208 ± 44 μg/mL, 230 ± 53 μg/mL and 512 ± 337 μg/mL, respectively. Interestingly, although the juice concentrate extracts were generally stronger inhibitors of α-amylase than the corresponding direct juices, the reverse was true for α-glucosidase inhibition. The only exceptions were lingonberry and red grape, whose concentrates had mean IC50 values of 118 μg/mL and 112 μg/mL, respectively, whereas the direct juices had IC50 values of 205 ± 59 μg/mL and 209 ± 43 μg/mL, respectively. Aside from these two juice concentrate extracts, the NFC extracts from aronia and pomegranate, which had IC50 values of 93 ± 31 μg/mL and 113 ± 32 μg/mL, were the most active amongst the studied fruit products. The examined red grape varieties showed no difference in their inhibitory potential.The ICBoth, the α-amylase and α-glucosidase inhibition assay showed that the extracts made from purees were the most potent inhibitors for each fruit type. The increased inhibitory activity compared to those of direct juice extracts was reflected in higher phenolic contents. In the α-amylase inhibition assay, the extracts from concentrates were generally more potent than the corresponding direct (NFC) juices; except for aronia. Controversially, in the α-glucosidase inhibition assay, extracts from concentrates were generally less active than those from direct juices; the only fruits for which this trend did not hold were lingonberry and bilberry.50 values has already been described. It varied from 50 μg/mL to 10 mg/mL for α-amylase [Extracts of sour cherry, elderberry, and bilberry exhibited comparatively weak α-amylase inhibition, whereas those of aronia, lingonberry, cranberry, and red grape were more potent. The strongest α-glucosidase inhibitors were extracts from aronia, pomegranate, lingonberry, and red grape, whereas extracts of bilberry, sour cherry, and elderberry were less active. This is consistent with literature reports indicating that aronia extracts showed higher α-glucosidase and α-amylase inhibitory activity than extracts of other red fruits such as sour cherry . In both-amylase . The hig-amylase .50 value was higher than 2 mM. Therefore, the % inhibition of 2 mM of each single substance was compared as shown in To identify the active ingredients in the extracts as well as to investigate possible synergistic effects, different commercially available standards from anthocyanins and copigments were examined. For all tested samples the ICThe results of the commercially available copigment standards tested in both assays are shown in Generally, very low inhibition (%) could be determined for all investigated anthocyanins and copigments for both enzymes. For α-amylase there was even no inhibitory activity measurable for some anthocyanins. The investigation of the anthocyanins showed a certain structure dependency of the inhibition potential. With more hydroxyl groups being present in a molecule, higher inhibitory activity was observed. The same tendency was found for the copigments. This is because the hydroxyl groups are able to strongly bind to the catalytic center and following from this inhibit this center ,23,24. Bd-maltotrioside (CNPG3), 4-nitrophenyl-α-d-glucopyranoside (pNGP), α-amylase from porcine pancreas, α-glucosidase from Saccharomyces cerevisiae, ethanol (≥ 99.8%) and Amberlite® XAD-7 were purchased from Sigma-Aldrich . Acarbose was obtained as Glucobay® (50 mg acarbose/tablet) from Bayer Pharmaceuticals . Gallic acid monohydrate and sodium carbonate were purchased from Fluka . The Folin-Ciocalteu reagent was obtained from Merck . Anthocyanin and copigment standards were obtained from Phytolab  and Extrasynthèse .2-Chloro-4-nitrophenyl-α-Products derived from juices and juice products from nine different red fruits were screened, including aronia (Aronia melanocarpa), bilberry (Vaccinium myrtillus L.), blackcurrant (Ribes nigrum L.), cranberry (Vaccinium macrocarpon), elderberry (Sambucus nigra L.), lingonberry (Vaccinium vitis-idaea L.), pomegranate (Punica granatum L.), red grape (Vitis vinifera L.), and sour cherry (Prunus cerasus L.). As different varieties of red grapes are marketed, we investigated the inhibitory potential of varieties such as Vitis labrusca “Concord” and Vitis vinifera “Lambrusco” as well as samples and extracts originating from French, Spanish, and Italian grapes, respectively. In total, the study investigated twenty-six samples of direct juices, nine from concentrates, and three from purees.v/v (stirring overnight at room temperature). After filtration, solvent was evaporated under reduced pressure at 40 °C and the samples were lyophilized.The puree samples were subjected to batch extraction with ethanol/water 19:1 ® XAD-7 column. The column was washed with water in order to eliminate carbohydrates, proteins and minerals. The phenolic compounds were then eluted with a mixture of ethanol/water 19:1 v/v. Solvents were evaporated under reduced pressure at 40 °C and the XAD-7 extracts were freeze-dried.Each of the puree samples, direct juices and juice concentrates were applied onto an Amberlitev/v/v (eluent A) and water/acetonitrile/formic acid 48:50:2 v/v/v (eluent B) at a flow rate of 200 μL/min. Gradient elution was performed, starting with 6% eluent B and rising to 35% over 30 min. The level of eluent B was then set to 40% until minute 35 and then 90% until 45 min. This level was maintained for 5 min before being reduced to 30% until 55 min. Finally, the initial conditions (6% eluent B) were restored until 70 min. The ESI source was operated in positive mode (anthocyanins), negative mode (copigments) and alternating mode (+/− 3000 V), using nitrogen as the nebulizer gas (60 psi) and the drying gas . The sample extracts were dissolved in 2 mL eluent A. An aliquot of 5 μL was analyzed by the HPLC/DAD/ESI-MSn method described above using the Bruker Hystar V.3.2, Bruker ESI-Compass 1.3 for HCT/Esquire, and Data Analysis Version 3.0 software packages .The HPLC system  consisted of a binary pump (G1312A), an autosampler (G1329B), and a DAD-detector (G1316A). It was coupled to a HCT Ultra Ion Trap mass spectrometer  with an electrospray ionization source (ESI). The anthocyanins and copigments were separated on a Luna C18(2) 3 μm column  using water/acetonitrile/formic acid 95:3:2 2CO3 solution was added at the same time to each mixture and incubated for 2 h at room temperature. The extinction was then measured at λ 760 nm using a spectrophotometer . The TPC was calculated with a calibration curve constructed using gallic acid solutions. Each sample was measured in triplicates, and the results were expressed as grams of gallic acid equivalents per 100 g extract (g GAE/100 g).The total phenolic content (TPC) of the samples was determined using the Folin-Ciocalteu method as described previously with slight modifications . Brieflyd-maltotrioside) was added, and the resulting mixtures were incubated at 37 °C for 8 min. Their absorbance was then measured at λ 405 nm using a microplate reader . Each sample was measured in triplicate.The α-amylase inhibition assay was based on a previously described spectrophotometric method . Acarbosd-glucopyranoside as substrate. 20 μL aliquots of the sample (in five different concentrations) or negative and positive control solutions were each mixed with 70 μL enzyme solution (1 U/mL) in 0.1 M PBS (pH 6.8). After incubation at 25 °C for 10 min, 100 μL of 4 mM pNPG solution in PBS was added and incubated in a 96-well microplate for 5 min at 25 °C. The absorbance was measured at λ 405 nm using a microplate reader . Each sample was measured in triplicate.The α-glucosidase inhibition assay used 4-nitrophenyl-α-It has been reported that the inhibition of α-glucosidase activity by fruit products is mainly due to anthocyanins . Our res