Moreover, in Var/H92 under water stress, both higher proline and ascorbate concentration were observed. Consequently, less membrane lipid peroxidation was quantified in Var/H92.Currently, limited water supply is a major problem in many parts of the world. Grafting peppers onto adequate rootstocks is a sustainable technique used to cope with water scarcity in plants. For 1 month, this work compared grafted peppers by employing two rootstocks (H92 and H90), with different sensitivities to water stress, and ungrafted plants in biomass, photosynthesis, and antioxidant response terms to identify physiological–antioxidant pathways of water stress tolerance. Water stress significantly stunted growth in all the plant types, although tolerant grafted plants  had higher leaf area and fresh weight values. Var/H92 showed photosynthesis and stomata conductance maintenance, compared to sensitive grafted plants (Var/H90) and ungrafted plants under water stress, linked with greater instantaneous water use efficiency. The antioxidant system was effective in removing reactive oxygen species (ROS) that could damage photosynthesis; a significant positive and negative linear correlation was observed between the rate of CO Abiotic stresses pose a huge threat for crops by limiting their growth and development, which eventually leads to poor productivity and low yields ,2,3. OneMany kinds of approaches have been tested to deal with this threat, including agricultural practices focused on irrigation management, but also the introgression of drought resistance traits by molecular breeding. On the one hand, agricultural practices are not always suitable, given the peculiarities of each growing area and the unpredictability of drought periods. On the other hand, molecular and conventional breeding strategies have faced many difficulties when assessing drought tolerance because it is achieved by combining quantitative characters. These, in turn, are controlled by many minor genes with additive effects ,4,5,6,9.Given all these different approaches, vegetable grafting emerges as a sustainable effective technique, particularly in Solanaceae and Cucurbitaceae species, to overcome water deficit and other abiotic stresses by preventing harmful impacts on shoots ,12,13,142 assimilation and intrinsic water use efficiency  [2 assimilation rate and less transpiration, and is joined with stomata regulation [N can be less severe than the drop in gs, which causes WUE to increase. These results imply that stomatal limitations are responsible for reducing AN under drought stress, which is associated with a down-regulated photosynthetic metabolism [N values drop more than gs values and, consequently, WUE usually lowers. This situation implies the existence of stomatal, but mainly non-stomatal limitations (inhibition of metabolic processes) connected with photosynthesis damage, which thus affects physiological adaptation to water stress [Several studies have confirmed positive physiological traits when employing tolerant rootstocks under water starvation conditions compared to ungrafted, self-grafted or plants grafted on susceptible rootstocks ,18,19,20/E, WUE) ,13 when gulation ,24,25. Hgulation ,27. Thesgulation ,29. Genetabolism ,25,30. Nr stress .2; which would result in photo-oxidative stress by reactive oxygen species (ROS) overproduction [Maintaining photosynthesis under water limitation levels is essential because its suppression could increase excitation energy excess and electron flux to Ooduction ,32. ROS oduction . ROS detoduction ), but aloduction ).Increasing antioxidant system activities is necessary to enhance plant protection and for reaching tolerance below non optimal water levels . In recePepper is one of the most important crops grown in the Mediterranean climate, where water scarcity is a relevant constraint . NeverthC. annuum × C. annuum) that resulted in hybrids with more uniform germination, vigor, and growth. These hybrids were tested and screened under water stress conditions based on photosynthetic parameters, leading to identify both tolerant and sensitive hybrids under water stress (unpublished data). Of these, one tolerant rootstock (H92) with higher CO2 assimilation and one sensitive rootstock (H90) were chosen to test the hypothesis herein posed.Our previous studies performed classical breeding assays with pepper accessions (The aim of the present study is to determine whether the maintenance of photosynthesis observed in the tolerant hybrid rootstock (H92) under water stress is associated with the protection of the photosynthetic apparatus, mediated by the activation of the antioxidant system components. For this purpose, several parameters, such as biomass, photosynthetic parameters, ascorbate, proline, catalase activity, and lipid peroxidation were determined in grafted plants using tolerant and sensitive rootstocks and ungrafted plants, grown both in water stress and control conditions.Capsicum annuum hybrids , obtained through traditional breeding by crossing pepper accessions in previous studies [Two  studies ,40,41, w3−, 1.0 H2PO4−, 2.0 SO42−, 1.0 NH4+, 6.0 K+, 4.0 Ca2+, and 2.0 Mg2+. Micronutrients were also provided : 15 Fe2+, 10 Mn2+, 5 Zn2+, 30 B3+, 0.75 Cu2+, and 0.6 Mo6+) [−1 and pH was 6.7. To apply the same fertilizer doses in the control and water stress treatments, nutrient solution was applied to both treatments when irrigation to stressed plants was administered. To meet irrigation water requirements, only water was applied to the other irrigation events in the non-stressed treatment.After allowing pepper plants to adapt to the greenhouse for 1 week, they were distributed for two treatments (six plants per treatment). Under the water stress conditions, water irrigation was lowered to 55% of total irrigation under the control conditions, which was calculated based on the weekly crop evapotranspiration (ETc). The number of irrigations was scheduled to maintain drainage between 20% and 30%. Plants were irrigated with Hoagland’s no. 2 nutrient solution containing : 14 NOAll of the measurements were taken 10 days after treatment (10 DAT) started, 20 DAT, and 30 DAT. During each measurement event, four measurements (one plant/replication) per plant type and treatment combination were taken. Photosynthetic parameters and physiological analyses were measured on the third or fourth leaves from the apex. For the physiological analysis, leaves were previously frozen with liquid nitrogen and stored at −80 °C. Samples were ground by a mixer mill  with liquid nitrogen to prevent melting.Osmotic potential of leaf sap  and relative water content (RWC) were measured at 30 DAT in leaves. RWC was determined by weighing leaves before and after a 24 h rehydration process performed with distilled water, obtaining the fresh weigh (FW) and turgid weight (TW), respectively. To obtain the dry weight (DW), leaves were dried at 65 °C for 72 h and then weighed. RWC was determined as: RWC (%) = (FW − DW)/(TW − DW) × 100.−1) was converted into MPa by the Van’t Hoff equation [The Ψs was measured by an osmometer . Leaves were detached, placed inside 1 mL tubes, and quickly frozen at −20 °C. After melting, sap was collected by centrifugation for 1 min at 9.000 rpm in 1.5 mL tubes. Osmolyte content of leaf sap , stomatal conductance to water vapor , substomatal CO2 concentration  and transpiration rate  were measured by a portable LI-COR 6400 infrared gas analyzer . The AN/E parameter was calculated as instantaneous WUE. Measurements were taken under saturating light conditions (1000 µmol quanta m−2 s−1), 400 µmol CO2 mol−1 of reference CO2, at 27 °C (27 ± 2 °C) and 75% RH. For each measurement time, data were collected on two consecutive days from 09:00 h to 11:00 h (UT + 01:00 h).The COt = AsA + DHA) were determined in parallel according to [w/v) trichloroacetic acid (TCA) and centrifuged for 5 min at 15,000× g and 4 °C. Then 50 µL of the supernatant were mixed with 150 µL of 0.2 M phosphate buffer (pH 7.4) and 50 µL of distilled H2O2 for the AsA determination. For AsAt, 50 µL of the supernatant were mixed with 50 µL of 10 mM dithiothreitol (DTT) and 100 µL of 0.2 M phosphate buffer (pH 7.4), and incubated in a water bath at 42 °C for 15 min. Then 50 µL of 0.5% (w/v) N-ethylmaleimide (NEM) were added and samples were incubated for 1 min at room temperature. Both AsA and AsAt tubes were mixed with 250 µL of 10% (w/v) TCA, 200 µL of 42% (v/v) H3PO4, 200 µL of 4% (w/v) 2,2′-dipyridyl and 100 µL of 3% (w/v) FeCl3, and incubated in a water bath for 40 min at 42 °C. Absorbance was recorded at 525 nm in both cases. The DHA concentration was determined as: AsAt − AsA.Ascorbic acid (AsA), dehydroascorbate (DHA) and total AsA (AsArding to  with somw/v) polyvinylpolypyrrolidone (PVPP), and was centrifuged for 30 min at 10,000× g and 4 °C. The supernatant was conserved under ice conditions until measurements were taken. To start the reaction, 1960 µL of 50 mM potassium phosphate (pH 7) were mixed with 20 µL of sample and 20 µL of H2O2. Reduction of H2O2 by catalase activity was monitored spectrophotometrically at 240 nm for 4 min at room temperature. U (µmol/min) g−1 FW was calculated using the Lambert–Beer equation with the H2O2 extinction coefficient (ε = 39.4 mM−1 cm−1).Catalase enzyme activity (EC 1.11.1.6) was measured as in , but witw/v) TCA and centrifuged for 5 min at 10,000× g and 4 °C. Later, 1 mL of supernatant was mixed with 2 mL of reaction buffer (20% TCA + 0.5% TBA) and samples were incubated for 30 min at 95 °C in a water bath. The non-specific background absorbance reading at 600 nm was subtracted from the specific absorbance reading at 532 nm.Lipid peroxidation was determined by the malondialdehyde (MDA) procedure using the thiobarbituric acid (TBA) reaction according to , and modProline quantification was performed as reported in  with sliThe biomass parameters were measured at the end of the experiment (30 DAT). Leaves and stems were weighed and the number of leaves per plant was recorded. The total leaf area was measured with a LI-3100 Area Meter . Later leaves and stems were exposed to dry heat (for 72 h at 70 °C) in a laboratory oven and dry weight (DW) was recorded.p < 0.05. No significant differences were found in the replicates for each measured parameter. The correlation analyses between AN and AsA/AsAt and AN and proline were performed with the above-mentioned Statgraphics software and the correlation coefficient (r) was obtained.The results of all the parameters were subjected to a two-way analysis of variance (ANOVA) with Statgraphics Centurion 18  after including two factors, namely plant type (PT) and treatment (T), and by considering their interaction. The physiological parameters were analyzed separately for each measurement time . The means of all the parameters were compared by Fisher’s least significance difference (LSD test) at p < 0.01). Plants under control conditions maintained RWC between 90 and 100% at the end of the experiment without significant differences among them. Water stress reduced significantly the RWC in all PT. Var plants were the most sensitive (77% RWC) followed by Var/H90 (83%) and Var/H92 (86%).Leaf RWC values A showed p < 0.01. Under WS, all PT showed significant lower values compared to control conditions; Var/H90 displayed the largest decrease and Var/H92 the lowest drop in Ψs with significant differences among them.Leaf osmotic potential (Ψs) values at 30 DAT are showed in s at 30 DAT and AN/E at 20 DAT) exhibited a statistically significant interaction between treatment (T) and plant type (PT). For this interaction, AN at 30 DAT, and gs at 10 DAT presented p < 0.05, with p < 0.01 for the remaining photosynthetic parameters and DAT.All the measured photosynthetic parameters  did not reveal any significant differences at 20 DAT when the control and WS plants were compared for all the plant types .Instantaneous WUE  were observed throughout the experiment. AsA presented p < 0.01, while AsA/AsAt showed p < 0.05 at 10 DAT and p ≤ 0.01 at 20 DAT and 30 DAT.For both the evaluated parameters, AsA (reduced ascorbate) and the AsA/AsAt only differed statistically in Var, with a lower ratio under the WS conditions . All plant types showed significant differences between treatments while testing  were found during the experiment  were found between T and PT throughout the experiment. Var/H92 appeared to be undisturbed when subjected to WS all along the experiment. At 10 DAT, only Var showed significant differences and the lowest MDA content for the stress conditions (Interactions for MDA content (nditions A. At 30 nditions B.p < 0.01) between PT and T while testing. The whole set of plants had a significantly higher proline content when water stress was applied throughout the experiment  . p < 0.01 and p < 0.05, respectively . The plants under water stress had a smaller number of leaves than the plants under the control conditions  following Var/H90 (16%) and Var/H92 (12%), which showed the better RWC conservation. According to Hsiao , the decN decreased in Var/H90 and Var, but not in Var/H92, which showed no significant differences with control plants. At the end of the experiment, AN significantly decreased in all PT respect to their control, despite Var/H92 got the highest values under WS. These results agree with the ones obtained in grafted plants using tolerant rootstocks, which improved photosynthesis under WS (see reviews in [N showed linear correlation (p < 0.0001) with gs in all PT at 10, 20 and 30 DAT , which could mean that AN reduction was mainly due to stomata limitations [Alterations in water relations under WS affected photosynthesis process in all PT. However, differential behavior was observed among them. After 10 DAT, Aviews in ,12). In itations ,30.2 assimilation rate and the amelioration of stomata performance. In Var and Var/H90 in response to water stress, higher RWC reduction and Ψs can be related with stomatal closure and lower WUE, according to [Cucurbita maxima × C. moschata [Grafting has been identified as an effective tool for increasing WUE in water stress situations ,27. A sirding to . This rerding to  in pepperding to  or in mimoschata . It is nN and proline concentration (p < 0.01) was measured at 10 DAT, 20 DAT, and 30 DAT . This implies that while AN decreased, proline increased with the same trend. This relation constituted a coupled response to water stress by minimizing its impact on plant performance, and it has been observed in pepper-grafted plants under salt stress [p < 0.01) was found at 10 and 30 DAT between AN and AsA/AsAt (r = 0.84 for 10 DAT and r = 0.55 for 30 DAT). Indeed, a marked significant increase in AsA and the AsA/AsAt ratio was measured in Var/H92 at the end of experiment, when WS pressure on plants was higher. Higher AsA levels have been observed in tolerant tomato- and pepper-grafted plants under both salinity and water stress [Despite the benefits of vegetable grafting on photosynthesis maintenance, and the antioxidant system role under water stress are both widely described in the scientific bibliography ,12,53, ft stress  and sunft stress . The higr stress ,61. Withr stress ,63. No A2O2, which is detoxified by catalase enzyme [2O2 in Var/H92. Plants under water stress overproduce ROS, one of them is He enzyme . In our Water-limiting conditions result in impaired growth, and reduce both the number of leaves and individual leaf size . Accordi2 fixation could limit this process in Var/H90 and Var, and to a lesser extent in Var/H92 [2 by single-leaf measurements. Nonetheless, FW considering PT factor was higher in the Var/H92 plants, associated with higher water conservation or acquisition capacity. Leaf expansion normally depends on turgor pressure and assimilates supply. Thus, slow CO Var/H92 . Neverth2 uptake observed using the tolerant rootstock H92 seems to involve minor oxidative stress, as observed by lesser membrane lipid peroxidation related to higher proline content and AsA concentration. Nonetheless, to validate these results, rootstock H92 should be tested under long-term experiments to evaluate its effects in terms of productivity and biomass under water stress.In conclusion, the higher maintained rate of CO


GcgR antagonist restored neutrophil migration, reduced CFU numbers in the peritoneal cavity and improved survival rate of diabetic mice after CLP procedure, however, the treatment did no alter hyperglycemia, CXCL1/KC plasma levels and blood neutrophilia. In addition, glucagon inhibited CXCL1/KC-induced neutrophil migration to the peritoneal cavity of non-diabetic mice. Glucagon also decreased the chemotaxis of neutrophils triggered by CXCL1/KC, PAF, or fMLP in vitro. The inhibitory action of glucagon occurred in parallel with the reduction of CXCL1/KC-induced actin polymerization in neutrophils in vitro, but not CD11a and CD11b translocation to cell surface. The suppressor effect of glucagon on CXCL1/KC-induced neutrophil chemotaxis in vitro was reversed by pre-treatment with GcgR antagonist and adenylyl cyclase or PKA inhibitors. Glucagon also increased PKA phosphorylation directly in neutrophils in vitro. Furthermore, glucagon impaired zymosan-A-induced ROS production by neutrophils in vitro. Human neutrophil chemotaxis and adherence to endothelial cells in vitro were inhibited by glucagon treatment. According to our results, this inhibition was independent of CD11a and CD11b translocation to neutrophil surface or neutrophil release of CXCL8/IL-8. Altogether, our results suggest that glucagon may be involved in the reduction of neutrophil migration and increased susceptibility to sepsis in diabetic mice. This work collaborates with better understanding of the increased susceptibility and worsening of sepsis in diabetics, which can contribute to the development of new effective therapeutic strategies for diabetic septic patients.Sepsis is one of the most common comorbidities observed in diabetic patients, associated with a deficient innate immune response. Recently, we have shown that glucagon possesses anti-inflammatory properties. In this study, we investigated if hyperglucagonemia triggered by diabetes might reduce the migration of neutrophils, increasing sepsis susceptibility. 21 days after diabetes induction by intravenous injection of alloxan, we induced moderate sepsis in Swiss-Webster mice through cecum ligation and puncture (CLP). The glucagon receptor (GcgR) antagonist des-his1-[Glu9]-glucagon amide was injected intraperitoneally 24h and 1h before CLP. We also tested the effect of glucagon on CXCL1/KC-induced neutrophil migration to the peritoneal cavity in mice. Neutrophil chemotaxis  Diabetes is frequently associated with the development and worsening of sepsis, with about 22% of septic patients presenting a medical history of diabetes , 2. AlthType 1 diabetes is associated with increased circulating levels of glucagon . Furtherin vivo and chemotaxis in vitro -4 inhibitors are associated with reduced recruitment in vitro . Inhibitin vitro . Since gin vitro , this wo+2 or Mg+2 (HBSS), mouse CXCL1/KC, human CXCL8/IL-8, N-formyl methionyl-leucyl-phenylalanine (fMLP), H-89, SQ 22536, DMSO, LPS, Penicillin, Zymosan A and Streptomycin were purchased from Sigma Chemical Co . Ficoll-Paque PLUS (density 1.077 g/mL) was purchased from GE Healthcare Bio-Sciences , platelet-activating factor (PAF) was purchased from Calbiochem , and fetal bovine serum (FBS) was purchased from Gibco . Meropenem was purchased from ABL – Antibióticos do Brasil  and Luminol was donated by Prof. Margareth Queiroz from Oswaldo Cruz Institute/Fiocruz . All the solutions were freshly prepared immediately before use.Alloxan, Glucagon, Des-his1-[Glu9]-glucagon amide, Rolipram, Percoll, RPMI medium, Hank’s balanced salt solution without CaPeripheral blood samples were obtained from healthy volunteers of both sexes  and aged 25 to 30 years. Donors who reported any kind of infection, inflammation, and/or use of anti-inflammatory or antibiotic medication were excluded from the work.ad libitum access to food and water. All animal procedures were performed in the laboratory beginning between 8:00 and 9:00 a.m. Animals euthanasia was performed with ketamine   and xylazine   as previously reported  were obtained from the Oswaldo Cruz Foundation breeding colony. Mice were housed in groups of five in a temperature- humidity- and light-controlled (12h light: 12h darkness cycle) colony room. Mice were given reported . Detailsad libitum. Mice injected with vehicle and submitted to similar experimental conditions were used as negative controls. Blood glycemia was determined using a glucose monitor  in samples obtained from the tail vein. After three days of alloxan injection, mice with blood glucose levels below 11 mmol/l were excluded from further experiments.Diabetes was induced by a single intravenous injection of alloxan monohydrate (65 mg/kg) , dilutedSixteen mice were randomly assigned into 2 experimental groups: non-diabetic mice (n = 8) and Diabetic mice (n = 8). Twenty-one days after diabetes induction, animals were killed, and blood was immediately collected from the abdominal aorta in vials containing heparin (40 U/mL) for glucagon and insulin quantification. Plasma was obtained after sample centrifugation for 10 min at 1000 ×g. Glucagon and insulin levels were quantified by radioimmunoassay following manufacturer’s guidelines , using a gamma counter .Twenty-one days after alloxan administration, sepsis was induced by CLP as previously described . BrieflyFourteen healthy mice were randomly assigned in 3 experimental groups: Control group mice (n = 4); CXCL1/KC-stimulated mice (n = 5); Glucagon treated and CXCL1/KC-stimulated mice (n = 5). Animals were pretreated with glucagon   or its vCells were recovered from the peritoneal cavity through lavage with 3 mL of sterile saline in the laminar flow cabinet. Total leukocyte counts were performed in Neubauer chambers using optical microscopy , after diluting the samples (40X) in Turk solution (2% acetic acid). Differential cell counts were carried out on panoptic-stained  cytospin preparations under the oil immersion objective to determine the percentage of mononuclear cells and neutrophils .The number of CFU in the peritoneal lavage was determined by diluting the samples 10 or 100 times, seeding on culture plates, and incubating under aerobic and sterile conditions on Difco tryptic soy agar  for 24h at 37°C. The number of bacteria colonies were counted and expressed as CFU/cavity .The blood was obtained from the tail vein of mice. Neutrophil count was performed in blood smears stained with May-Grunwald–Giemsa dye using optical microscopy  under the oil immersion objective .g, 10 min, 4°C). Blood was collected from abdominal aorta in tubes containing 100 µL of 3.2% sodium citrate, and plasma was obtained after centrifugation . Then, IL-6, CXCL1/KC, and CXCL2/MIP-2 were quantified from the supernatant using a commercial ELISA kit according to the manufacturer’s instructions .The peritoneal lavage was recovered and centrifuged (1500 x Bone marrow (BM)-neutrophils were obtained after flushing the femur and tibia of both posterior paws of mice, as previously described . Then, n5 cells/well) were incubated in RPMI medium without FBS and treated with PDE-4 inhibitor rolipram (5 μM), glucagon (0.3-3 μM), or vehicle  for 3h in vitro at 37°C in a 5% CO2 atmosphere. For the evaluation of neutrophils viability, aliquots of cell suspensions were mixed with trypan blue solution (0.2%) and were counted using a light microscope . Neutrophils that presented blue color in their interior were considered non-viable. The cells presented at least 85% or 95% of viability in all groups studied for neutrophils obtained from mice and humans, respectively.Neutrophils obtained from humans’ blood or mice BM  was performed in a 96 well Neuroprobe ChemoTx® 101-5 chemotaxis plate , using chemotactic stimuli CXCL1/KC (25 nM), fMLP (1 μM), PAF (20 μM) or medium for 40 min in vitro. After that period, the lower compartment of the chemotaxis plate was read in a plate reader  at an excitation wavelength of 485nm and emission wavelength of 530nm  or medium for 2h in vitro at 37°C in a 5% CO2 atmosphere  for 30 min. After washing with HBSS, neutrophils were pretreated with glucagon (0.03-3 μM), rolipram (5 μM), or vehicle  for 30 min or 1h for murine and human cells, respectively, of 530nm . The nummosphere . The res+ cells. For evaluation of GcgR expression in human neutrophils, first these cells were isolated from human blood using Ficollpaque and then labeled with polyclonal antibody anti-GcgR (PE 1:20)  were treated with glucagon (3 μM) or rolipram (5 μM) for 30 or 40 min for BM-murine and human cells, respectively, in vitro. Next, murine BM-neutrophils were activated with CXCL1/KC (10 nM) for 40 min, while human neutrophils were stimulated with CXCL8/IL-8 (12 nM) for 1h in vitro. Then, the cells were labeled using monoclonal antibodies anti-Ly6G (clone 1A8) (FITC 1:400), anti-CD11a (PE 1:100), and anti-CD11b (PECY7 1:200) (Thermo Fisher Scientific) for 30 min. The results of CD11a and CD11b translocation to neutrophils surface were represented as a percentage of median fluorescence intensity (MFI) values compared to a 100% baseline MFI values attributed to the group of cells pretreated with vehicle and stimulated with CXCL1/KC, for murine, or CXCL8/IL-8, for human cells, in vitro. In all experiments, before incubation with the respective antibodies, all the cells were exposed to sheep serum for 15 min at 4°C to block nonspecific binding, and then washed with PBS 1X. In addition, as control of nonspecific binding, we used isotype-matched antibodies, or the primary polyclonal rabbit antibody anti-GcgR (Santa Cruz Biotechnology) was omitted. All the results were acquired by flow cytometry  and analyzed using FlowJo software . Gating strategies for the identification of neutrophils in these experiments are shown in Blood neutrophils from non-diabetic or diabetic mice were stained with monoclonal antibody anti-Ly6G (clone 1A8) (FITC 1:400) , a specific marker for neutrophils , and pri5 cells/well) were pretreated with glucagon (3 μM), rolipram (5 μM) or vehicle  for 30 min and, then, stimulated with CXCL1/KC (10 nM) or medium for 40 min in vitro at 37°C in a 5% CO2 atmosphere. Neutrophil slides were prepared by cytospin centrifuge, F-actin was stained with TRITC-labeled phalloidin (Sigma), and actin polymerization was evaluated by fluorescence microscopy, as previously described  were treated with glucagon (3 μM) or vehicle (medium) at several time intervals  in vitro at 37°C in a 5% CO2 atmosphere. The cells were homogenized in cold RIPA buffer containing a cocktail for protease and phosphatase inhibitors . Cell supernatant was collected, denatured and the total proteins were quantified by using the BCA method (Sigma). For western blot analysis, equal amounts of proteins were resolved on SDS–polyacrylamide gel electrophoresis and subsequently transferred onto nitrocellulose membranes, as previously described  were pretreated with glucagon (0.3-3 μM), rolipram (5 μM) or vehicle  for 30 min in vitro at 37°C in a 5% CO2 atmosphere. Then, the cells were stimulated with zymosan A (0.1 mg/mL) opsonized with mouse serum, in the presence of luminol (1 mM). The chemiluminescence reaction was monitored by a plate reader  for 1h at 37°C. ROS production was represented as the area under the curve (AUC) of the time-course.ROS was measured through the kinetics of the chemiluminescence of luminol  after st6 U/mL, 0.2 g/mL streptomycin, and 10% FBS at 37°C in a 5% CO2 atmosphere. Cells were grown to 90% confluence in 96-wells plates and were stimulated with LPS  for 4 h prior to the adhesion assay in vitro. After stimulation, the cells were washed with PBS 1X. In parallel, neutrophils were isolated from human blood (5x105 cells/well) and labeled with calcein-AM (5 μM) for 30 min. After washing with HBSS, these cells were pretreated with glucagon (0.3-3 μM) or vehicle (medium) for 40 min in vitro at 37°C in a 5% CO2 atmosphere. Next, neutrophils were stimulated with CXCL8/IL-8 (12 nM) or vehicle (medium) for 1h in vitro, and then the wells were washed with HBSS.Human umbilical vein endothelial cells (HUVECs) were cultured in RPMI medium supplemented with penicillin 1 × 105 cells/well) to wells containing LPS-provoked-HUVECs for 1h at 37°C in a 5% CO2 atmosphere. Non-adherent cells were then removed by gentle aspiration and washing with PBS 1X, and the number of adherent neutrophils was determined with a fluorescence plate reader  using an excitation wavelength of 485 nm and an emission wavelength of 538 nm  were treated with glucagon (0.3-3 μM) for 1h and, then, stimulated with LPS (10 ng/mL)  according to the manufacturer’s instructions. The results are expressed as the percent of IL-8 release compared to a 100% baseline for cells pre-treated with vehicle and stimulated with CXCL8/IL-8 in vitro.Neutrophils isolated from human blood (5x100 ng/mL)  for 2h ipost hoc test. In the case of the survival rate, the χ2-test (Fisher’s exact test) was used. All statistical analysis was performed with GraphPad Prism 8 software . Probability values (P) of 0.05 or less were considered significant.The data are reported as the mean ± standard error of the mean (SEM). All data were evaluated to ensure normal distribution and statistically analyzed by one-way ANOVA, followed by a Student-Newman–Keuls P < 0.05). The values of circulating glucose of untreated non-diabetic mice were 8 ± 0.3 mmol/L .Twenty-one days after alloxan-induced diabetes, mice presented increased blood glucose levels with a simultaneous reduction of body weight and plasma insulin levels Table 1,Diabetic mice subjected to CLP showed decreased accumulation of total leukocytes, mainly neutrophils, into the peritoneal cavity compared to non-diabetic mice subjected to CLP  treatment, which was used as a positive control of migration inhibition, effectively inhibited the chemotactic response of neutrophils towards CXCL1/KC, PAF, and fMLP  and rolipram (5 µM) inhibited ROS production induced by zymosan A by mice BM-neutrophils in vitro, however, rolipram significantly reduced the presence of both integrins in cell surface  in peritoneal cavity and circulation, or pro-inflammatory cytokine, IL-6, in peritoneum. Therefore, the restoration of neutrophil migration to the peritoneal cavity induced by GcgR antagonist treatment appears to be independent on the production of molecules that stimulate neutrophil activation. Together, our data suggest that glucagon reduces neutrophil migration to the injured tissue of diabetic mice by a direct effect on neutrophil migration machinery and not by the inhibition of neutrophil specific chemoattractants production. In addition, the GcgR antagonist improved diabetic mice survival rate without altering glycemia nor systemic inflammation induced by sepsis. These results strongly suggest that GcgR pathway might be over-activated, and thus, contributing to sepsis pathophysiology in diabetic mice.in vivo and in vitro. In this work, we showed that glucagon inhibited CXCL1/KC-induced neutrophil migration to the peritoneal cavity of mice. In addition, we also noted that glucagon decreased the chemotaxis of murine neutrophils induced by CXCL1/KC, PAF, or fMLP in vitro as well as CXCL8/IL-8-induced chemotaxis of human circulating neutrophils in vitro. Since glucagon inhibited neutrophil chemotaxis toward several chemoattractant in vitro, it probably inhibited neutrophil migration through some molecular mechanisms independent of chemokine receptor down-regulation, as it has been previously reported for neutrophils obtained from diabetic mice , protocol number 1.057.012. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Committee on Use of Laboratory Animals of the Oswaldo Cruz Institute .DI and MF contributed to conception and design of the study, performed experiments, analyzed the data, and wrote the manuscript. CG-d-A contributed to design of the study, performed experiments, analyzed the data, and contributed to preparation of the manuscript. AC and AYOS performed experiments and analyzed the data. HC-F-N contributed to the conception and design of the study, with essential reagents or tools, analyzed the data, and contributed to preparation of the manuscript. RS contributed to design of the study, performed experiments, and analyzed the data. TF analyzed the data and contributed with essential reagents or tools. PRS and MM analyzed the data, contributed with essential reagents or tools and to preparation of the manuscript. ARS contributed to conception and design of the study, performed experiments, analyzed the data, contributed with essential reagents or tools and to preparation of the manuscript. VC contributed to conception and design of the study, with essential reagents or tools, analyzed the data, and wrote the manuscript. All authors contributed to the article and approved the submitted version.This study was financial supported by Fundação Carlos Chagas de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Programa de Auxílio à Pesquisa (PAPESVI/FIOCRUZ), Programa INOVA FIOCRUZ, and Ministério da Saúde, Brazil and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES).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.


TgLOX4 and TgLOX5, showed significant changes in expression following tulip bulb growth. Ectopic overexpression of TgLOX4 and TgLOX5 in Arabidopsis enhanced endogenous JA content, improved plant growth, and increased lateral root numbers. Silencing of these two genes in tulip repressed the growth of daughter bulbs. Furthermore, exogenous JA treatment promoted tulip bulb growth, whereas the JA biosynthesis inhibitor sodium diethyldithiocarbamate (DIECA) inhibited this process. This study offers supporting evidence for the involvement of tulip TgLOX4 and TgLOX5 in the regulation of daughter bulb growth and development.Tulip bulbs are modified underground stems that originate from axillary meristems of mother bulb scales. Hormones, including jasmonic acids (JAs), play key roles in the regulation of tulip bulb development. Here, we compared variations in daughter bulb development through transcriptomic profiling analysis and characterized the functions of JA biosynthesis-related genes during daughter bulb enlargement. The results showed that tulip cultivars exhibited contrasting bulb size variations. Transcriptomic analyses revealed that genes involved in plant hormones and development, including the two lipoxygenase genes  Tulipa gesneriana L.) is an ornamental bulbous plant that is widely used for landscaping and cut flowers [Tulipa genus is distributed in the Mediterranean, Central Asia, Europe and Northern Africa [Tulip  by a lipoxygenase (LOX), an allene oxide synthase (AOS), and an allene oxide cyclase (AOC) [Plant hormones are key regulators of plant growth and development , 13. Jas flowers . The sigse (AOC) , 17.Arabidopsis 13-LOXs, especially LOX2, LOX3, LOX4, and LOX6, can produce JA precursors in leaves [LOX gene family members were identified and exhibited differential associations with growth, development, and fruit ripening [LOX1 gene is highly expressed in newly formed tubers. Suppression of LOX1 class activity resulted in reduced tuber yields and disruption of normal tuber morphology [LOX mRNAs and proteins were detected in nodules, mainly in the developing stage, but their expression and activity levels decreased in nodules of complete size [LOX genes and changes in JA content contribute to the promotion of plant growth and development.LOXs (EC 1.13.1.13) are non-heme, nonsulfur oxidoreductases that are widely present in living organisms, including mammals, plants, fishes, mosses, bacteria, yeast, fungi, corals, algae, and mushrooms . Plant Ln leaves , 21. In ripening . In potaripening , 24. Therphology . Legume ete size . All theLOX genes in the JA pathway were also dissected through ectopic expression in Arabidopsis plants and repressed expression in tulip by virus-induced gene silencing (VIGS). The results provide new clues for understanding the mechanisms of tulip bulb development.In tulip, MeJA treatment induced fatty acid and sterol concentrations in stems . ExogenoIn this study, natural variations in tulip bulb size were first investigated. The daughter bulb perimeters of 66 tulip cultivars varied from 6.7 cm to 13.4 cm . The perDaughter bulbs of the two tulip cultivars at four developmental stages were collected for RNA sequencing analysis. In cultivar “Ad Rem”, a total of 5845, 7728, and 11 105 unigenes showed significant expression changes in S2_vs_S1, S3_vs_S1, and S4_vs_S1, respectively. In cultivar “Red Power”, the numbers of changed unigenes were 8815, 8957, and 16 786 for S2_vs_S1, S3_vs_S1, and S4_vs_S1, respectively . OverlapGO term enrichment analysis indicated that GO terms including regulation of cell proliferation, meiotic cell cycle, cell wall, macromolecule, and metabolic process were over-represented only in “Ad Rem” . Other GPathway enrichment analysis was performed using MapMAN software. The results indicated that twelve pathways were overrepresented in S2 to S4 relative to S1 in the two tulip cultivars: fermentation, major CHO metabolism, biodegradation of xenobiotics, amino acid metabolism, minor CHO metabolism, TCA/org transformation, cell wall, transport, secondary metabolism, lipid metabolism, cell, and hormone metabolism . AnotherTgLOX4 (F01.PB33674) and TgLOX5 (F01.PB63464) had higher FPKM values in “Ad Rem” than in “Red Power”. We then verified the expression level changes in TgLOX4 and TgLOX5 by real-time qRT-PCR. The results showed that both genes had significantly higher expression in “Ad Rem” than in “Red Power” at four developmental stages, except for TgLOX4 at the S4 stage  (TgLOX4 or TgLOX5 transgenic lines (TgLOX4 and TgLOX5 transgenic plants. The results showed that both TgLOX4 and TgLOX5 transgenic lines had significantly higher JA content than the WT (CORONATINE INSENSITIVE1 (AtCOI1) increased 2.6–42-fold in transgenic Arabidopsis compared with the WT  DOMAIN-CONTAINING PROTEIN genes (LBDs), including AtLBD13, AtLBD14, AtLBD16, AtLBD18 and AtLBD29, was upregulated in TgLOX4 and TgLOX5 transgenic Arabidopsis  . Howeveric lines . We thenn the WT . Expressh the WT . The baspression . Moreovebidopsis ; Fig. S935::TgLOX4 and WT plants, but 35::TgLOX5 transgenic plants showed significantly larger rosette diameters than the WT  inhibited tulip plant growth, as evidenced by reduced plant height  LOX3 transgenic Arabidopsis exhibited faster root growth under osmotic stress conditions compared with the WT [lox3 reduced root length and plant height [Arabidopsis TgLOX4 and TgLOX5 overexpression lines exhibited more lateral roots and branches and greater plant height compared with the WT, indicating that TgLOX genes are involved in stem and root development, at least in Arabidopsis. In addition, VIGS data showed that silencing of TgLOX4 and TgLOX5 repressed tulip bulb growth. Therefore, data from Arabidopsis and tulip were consistent, partially illuminating the roles played by TgLOX4 and TgLOX5 in tulip bulb growth. However, we cannot rule out the possibility that several other LOXs function as JA biosynthetic enzymes. The functions of TgLOX4 and TgLOX5 in the JA biosynthesis pathway are worthy of further discussion.Plant LOXs are involved in diverse functions, including growth and development, stress response, senescence, seed germination, fruit ripening, and synthesis of JA and ABA . LOX genand MeJA , 40. Durand MeJA . Therefoand MeJA . LOX actand MeJA , 43. Thrultivars . In tomaripening . In tulid leaves , 5. Silend bulbs , indicatormation . Persimmh the WT , whereast height . In thisTgLOX4 and TgLOX5 in Arabidopsis increased endogenous JA content and improved plant and root growth, whereas silencing of these genes inhibited tulip bulb development. We propose that TgLOX4 and TgLOX5 enhance JA biosynthesis, activate JA signaling pathways, and possibly promote tulip bulb growth and development by upregulating the expression of LBD and JA-responsive genes  and stored at 5°C for 12 weeks. The tulip bulbs were planted in a glasshouse at the Ornamental Plants Research Farm of Huazhong Agricultural University . The conditions in the greenhouse were maintained at 20°C day/15°C night, with a relative humidity of 60–70% and a 16-h light/8-h dark photoperiod.Moreno-Pachon et al. classified the developmental stages of tulip plants and bulblets from the storage period (October to December) through the growing season (February to July) under field conditions in the Netherlands . In thisArabidopsis thaliana Columbia-0 (Col-0) ecotype was used in this study to generate transgenic plants. Arabidopsis seeds were sterilized for 4 min using 2% (v/v) sodium hypochlorite (NaClO) containing 0.1% (v/v) Triton X-100. The seeds were then washed five times with sterile water. Seeds were stored at 4°C for 5 days under dark conditions for vernalization. After planting on MS plates, all seeds were cultured in a growth chamber maintained at 22 ± 1°C with 60% relative humidity and a 16-h light/8-h dark photoperiod. The chamber was supplemented with 100 μmol photons m−2 s−1 light intensity.The 2O: concentrated HCl = 2:1:0.002, v/v/v). Then 100 μL of the 2H-JA working solution was added as an internal standard. The samples were mixed well and centrifuged at 4°C and 13 000 g for 5 min. After centrifugation, 900 μL of the solvent from the lower phase was transferred and concentrated using a nitrogen evaporator with nitrogen flow. The samples were re-dissolved in 0.1 mL methanol, and 50 μL of sample solution was injected into a reverse-phase C18 Gemini HPLC column for HPLC-ESI-MS/MS analysis.Plant tissues were frozen with liquid nitrogen and ground into fine powder in a mortar. Approximately 100-mg samples were transferred to 2-mL tubes containing 1 mL extraction solvent . A NanoDrop 2000 spectrophotometer  and a Bioanalyzer 2100 system  were used to assess RNA purity and integrity, respectively. A total amount of 1 μg RNA per sample was used for cDNA library construction with the NEBNext Ultra RNA Library Prep Kit for Illumina  following the manufacturer’s recommendations. The libraries were sequenced on an Illumina HiSeq platform, and 150-bp paired-end reads were generated. Clean data (clean reads) were obtained by removing low-quality reads and reads that contained adapters and poly-N from the raw data. Trinity was used for transcriptome assembly based on the left.fq and right.fq files . Gene fuhttp://bonsai.hgc.jp/~mdehoon/software/cluster/) with an uncentered matrix and complete linkage methods [http://jtreeview.sourceforge.net/) as described previously [Hierarchical clustering analysis was performed using the CLUSTER 3.0 program ( methods . The reseviously .Arabidopsis homologs. Corresponding Arabidopsis Genome Initiative (AGI) locus codes for differentially expressed unigenes were used as input to the Classification SuperViewer Tool (http://bar.utoronto.ca/ntools/cgi-bin/ntools_classification_superviewer.cgi) for pathway enrichment analyses [http://mapman.gabipd.org/home) was selected as a classification source. The normalized frequency (NF) was calculated as described previously [Differentially expressed tulip unigenes were annotated based on their analyses . MapMan eviously : NF = saTgLOX4) and F01.PB63464 (TgLOX5) were identified. The coding regions of TgLOX4 and TgLOX5 were amplified using the specific primers listed in TgLOX4 and TgLOX5 were cloned into the pCAMBIA1300 vector using the XbaI and KpnI restriction sites, and the resulting plasmids were introduced into Agrobacterium tumefaciens strain GV3101. Transgenic Arabidopsis lines were generated by the floral-dip method [TgLOX4 and TgLOX5 transgenic plants were screened and verified by qPCR analysis with the primers listed in Based on tulip RNA sequencing data (GEO database accession number GSE167530), the full-length sequences of F01.PB33674 . Multiple protein sequence alignments and phylogenetic tree construction for TgLOX4 and TgLOX5 were performed using MEGA 7 with the maximum likelihood method.Amino acids sequence alignment of Arabidopsis were planted directly in soil for leaf and stem measurements. Each pot contained only one seedling at 1 week after planting. Leaf length, leaf width and seedling diameter were measured at 21 days after planting, and branch number, plant height and silique length were measured at 42 days after planting.Transgenic (T2 generation) and wild-type Arabidopsis seeds were sown on MS plates. One-week-old seedlings of identical size were transferred to fresh MS plates. Primary root length was measured and lateral root numbers were counted after 7 d of growth. For each genotype, three replicates of at least 30 seedlings each were measured, and the whole experiment was repeated three times.To investigate root length and lateral root number, wild-type and transgenic TgLOX4 and TgLOX5 genes was silenced through virus-induced gene silencing (VIGS) as described by Zhong et al. [TgLOX4 and a 394-bp fragment of TgLOX5 were amplified by PCR and inserted into the pTRV2 vector to generate the pTRV2-TgLOX4 and pTRV2-TgLOX5 constructs, respectively. A. tumefaciens strain GV3101 was used for construct transformation. Tulip bulbs were immersed in infiltration buffer containing Agrobacterium cells transformed with equal amounts of pTRV1 and pTRV2 or pTRV2-target genes. Tulip bulbs submerged in the bacterial suspension were infiltrated under a vacuum at 0.8 MPa for 30 min to promote infection efficiency. After infiltration, the bulbs were kept in the dark at 22°C for 48 h, then planted in a greenhouse at 22°C with a relative humidity of 60–70% and a 16-h light/8-h dark cycle.The expression of g et al.  and Wangg et al. . A 394-bTgLOX4, TgLOX5, and WT Arabidopsis were collected for qPCR analysis. Total RNA was extracted from tulip bulbs and Arabidopsis using the EASYspin Plus Complex Plant RNA Kit . Equal amounts (1 μg) of total RNA were used for reverse transcription with the HiScript II 1st Strand cDNA Synthesis Kit  following the manufacturer’s instructions. AtACT2 (AT3G18780) was used as the reference gene for Arabidopsis, and tulip TgACTIN (unigene ID PB13161) was used as the reference gene for tulip. The web tool GenScript (https://www.genscript.com/ssl-bin/app/primer) was used to design real-time qRT-PCR primers. The relative transcription levels were calculated using the 2−ΔΔCt method [Tulip bulbs at four developmental stages and whole seedlings of 2-week-old transgenic t method . Primer −1 sucrose, 1.0 g l−1 casein hydrolysate, 1.0 mg l−1 thiamine and 200 mg l−1 L-Gln. Different concentrations of JA and the JA biosynthesis inhibitor DIECA were added to the MS medium. After incubation at 22°C with a 16-h light/8-h dark cycle for three months, fresh weights and perimeters of daughter bulbs were measured.“Ad Rem” daughter bulbs (axillary buds) were separated from mother bulbs after three months of storage at 5°C. The bulblets were sterilized in 70% ethanol for 1 min, then soaked in 20% (v/v) sodium hypochlorite (NaClO) for 20 min and finally washed with sterilized water 5 times. The sterile buds were cultured on solid MS medium containing 60 g l−4 M, 10−5 M, and 10−7 M) of JA solution were used based on preliminary results. Both the water control and the JA solutions were applied as foliar spray at the S2 and S3 stages once every two days, three times in total at each stage. For each replicate (30 plants), 2 L of solution containing the indicated concentration of JA or water was used.For exogenous JA treatment, JAs at the indicated concentrations and water (control) were sprayed on both sides of the plant leaves. Three concentrations  of each replicate. At least 50 bulbs or plants were used for each treatment. Duncan’s multiple range test (DMRT) was used to assess differences between the means. Different letters above the columns in each figure indicate significant differences at *This research was supported by the National Key Research and Development Program (2020YFD1000402) and the Huazhong Agricultural University Scientific & Technological Self-innovation Foundation to Zhulong Chan (2662016QD026 and 2662016RC010) and Yanping Wang (2662020YLPY010).Q.S. and Z.C. conceived the experiments. Q.S. and B.Z. conducted the experiments. C.Y. and W.W. helped with data collection. Q.S. and Z.C. wrote the manuscript. Y.W. and L.X. guided the research and revised the manuscript. All authors read and approved the manuscript.TgLOX4 and TgLOX5 from two tulip cultivars have been deposited at NCBI GenBank under accession numbers MW582299, MW582300, MW582301 and MW582302.The raw RNA sequencing data have been deposited at the NCBI Gene Expression Omnibus (GEO) under accession number GSE167530. The sequences of The authors declare no competing interests.Horticulture Research online.Web_Material_uhac006Click here for additional data file.


Histoplasma capsulatum lipid metabolic pathway by incorporating proteomic and lipidomic analyses. We performed genetic complementation and overexpression of H. capsulatum genes in Saccharomyces cerevisiae to validate reactions identified in the map and to determine enzymes responsible for catalyzing orphan reactions. The map led to the identification of both the fatty acid desaturation and the sphingolipid biosynthesis pathways as targets for drug development. We found that the sphingolipid biosynthesis inhibitor myriocin, the fatty acid desaturase inhibitor thiocarlide, and the fatty acid analog 10-thiastearic acid inhibit H. capsulatum growth in nanomolar to low-micromolar concentrations. These compounds also reduced the intracellular infection in an alveolar macrophage cell line. Overall, this lipid metabolic map revealed pathways that can be targeted for drug development.Lipids play a fundamental role in fungal cell biology, being essential cell membrane components and major targets of antifungal drugs. A deeper knowledge of lipid metabolism is key for developing new drugs and a better understanding of fungal pathogenesis. Here, we built a comprehensive map of the  Histoplasma capsulatum, the causative agent of histoplasmosis, is an important pathogen in this context, being associated with HIV infections and causing morbidity and mortality worldwide    . The most diverse subclasses of lipids in terms of the number of identified species were triacylglycerols (TG), phosphatidylcholines (PC), and phosphatidylethanolamine (PE), with 130, 57, and 51 individual species, respectively (https://osf.io/ku8ta/). To provide a measurement of the protein abundances in the cells, we calculated the relative copy number of each protein and scaled them into high, moderate, low, and very low abundance  that were subdivided into 19 subclasses  , along with previous literature on H. capsulatum. We used the H. capsulatum genomic, proteomic, and lipidomic information to further restrict or add reactions that are present in this organism. The H. capsulatum map was incorporated with the relative abundance of the lipid species within the same subclass and the protein abundances .To provide a global view of ic model  structurundances . In termundances . Similarundances . In the undances . Out of undances . The numundances . In termundances , low 3)H. capsulundances . Like thundances . Consist in each . To incrlication , which sH. capsulatum acyltransferase Ale1 , because its homolog in S. cerevisiae has been shown to modify different classes of lipids , which would allow curation of a large portion of the metabolic map. A lipidomic analysis was performed in the Lpt1 knockout strain of S. cerevisiae complemented or not with the Hc-Lpt1 homolog  , using a plasmid with a galactose-inducible promoter. This allows specific expression of Hc-Lpt1 in the presence of galactose but not glucose. Both gene knockout and recombinant expression were validated by proteomic analysis . The Hc-LPT1 complementation also significantly reduced the levels of 77 lipids, including 5 LPC, 3 lysophosphatidylethanolamines (LPE), 2 sphinganines, and 3 DGs (see Table S12 at https://osf.io/ku8ta/). On the other hand, disruption of the Sc-LPT1 gene reduced the levels of 30 potential products, including PC, PE, PI, PS, and TG. Notably, the proportion of acylated products between LPT1s from the two species was very different, with PC, PE, PI, and PS being more acylated by the S. cerevisiae homolog and PA, DG, TG, Cer, and MIPC by the H. capsulatum counterpart (https://osf.io/ku8ta/). Sc-LPT1 showed a preference for production of lipid species containing short fatty acyl chains, with all the identified products having chains with ≤14 carbons  and longer (≥C18) fatty acyl chains , PC, PE, phosphatidylinositol (PI), PS, DG, TG, Cer, and mannosyl-inositolphosphoceramide (MIPC)  to D inositolphosphoceramides and humans produce complex glycosphingolipids, whereas H. capsulatum produces both (glyco)inositolphosphoceramides and simpler glycosphingolipids  compared to humans to allow specificity and reduce the chances of side effects. Therefore, we searched the lipid metabolic map for lipid pathways with enzymes that were present in tabolism  and B. Sgolipids .S. cerevisiae has only one fatty acid desaturase gene  in its genome (H. capsulatum has a gene annotated as delta-12 desaturase (FAD2) and two uncharacterized desaturases (HCBG_08514 and HCBG_05092), which collectively might be responsible for the production of fatty acids with 2 and 3 double bonds (H. capsulatum and S. cerevisiae lipids. The results showed that both fungi incorporate fatty acyl groups with different chain lengths into their lipids. S. cerevisiae incorporates significantly more fatty acyl chains with fewer than 16 carbons (by Fisher’s exact test), whereas H. capsulatum has significantly more lipids with fatty acids longer than 17 carbons , whereas H. capsulatum Ole1 increased the ratios of C14:1/C14:0, C16:1/C16:0, and C18:1/C18:0 by 11%, 24%, and 81%, respectively . The expression of HCBG_08514 and HCBG_05092 had almost no impact on the saturation levels of the fatty acids , which could be due to the lack of the precursors in S. cerevisiae. C16:2 , C18:2 , and C18:3  were only detected when H. capsulatum FAD2 was expressed . A composition analysis of H. capsulatum fatty acids revealed two low-abundance peaks of C18:3, one being α-linolenic acid and a second peak not matching any available standard (https://osf.io/ku8ta/). These results showed that H. capsulatum FAD2 is indeed a bifunctional delta-12/delta-15 desaturase, which helped to further curate the lipid metabolic map , the C18:1/C18:0 ratio from 4.5 to 3.7 (18% reduction), and the C20:1/C20:0 ratio from 1.3 to 0.8 (39% reduction) (16:1/C16:0 ratio had a slight increase from 0.036 to 0.044 (22% increase) , 10-thiastearic acid, and thiocarlide (two fatty acid desaturase inhibitors) on ectively . To confectively , suggestduction) . Conversncrease) . We alsoncrease) . As a concrease) .H. capsulatum drug targets, we tested the ability of these inhibitors to reduce intracellular infection. We chose alveolar macrophages, as they are the primary cells targeted by H. capsulatum in infection  and long-chain (≥C18) fatty acids. Our experiments with LPT1 also helped to curate the lipid map, since the S. cerevisiae homolog has a different specificity for substrates, having a major impact on producing phospholipids with short fatty acyl chains. In S. cerevisiae, the substrates of LPT1 have been shown in vitro using radioactive precursors to acylated LPC, LPG, LPA, LPE, LPI, and LPS  is a potent inhibitor (nanomolar range) of Mycobacterium tuberculosis delta-9 stearoyl desaturase and was used as a second line of antituberculosis drugs in the 1960s (34in vitro (Trypanosoma cruzi (T. cruzi growth on a low micromolar range and reduced the levels of unsaturated fatty acids by approximately 30% (at 10 μM concentration)  . These npsulatum  and B. Tar fungi . Such dear fungi , 42.Our data also showed that 10-thiastearic acid might target a pathway other than fatty acid desaturation. This is expected to some extent, since 10-thiastearic acid is a fatty acid analog; therefore, it is more likely to inhibit other processes of fatty acid metabolism. However, this type of molecule should still be considered a drug candidate due to its antifungal activity and potential low toxicity to humans. Our data showed no toxicity in AMJ2 alveolar macrophages with up to a 64-fold MIC of 10-thiastearic acid. In addition, the 10-thiastearic acid analog tetradecylthioacetic acid has a low toxicity in humans up to 1 g/day . EngineeCandida albicans, C. auris, and Aspergillus fumigatus in nanomolar to micromolar concentrations  and excellent antifungal drug candidates . Strain-dependent variations regarding lipid and protein profiles are expected among different fungal strains of the same species, which needs to be further studied in future work.One limitation of this study is that profiles . TherefoHistoplasma capsulatum G217B strain and murine alveolar macrophage AMJ2 were purchased from the American Type and Culture Collection . S. cerevisiae deficient in the LPT1 gene  and wild-type strains  were purchased from Dharmacon .H. capsulatum acyltransferase LPT1 (UniProt ID C0NZS2) or desaturases Ole1 (UniProt ID C0NLE5), FAD2 (UniProt ID C0NKL1), HCBG_08514 (UniProt ID C0NZD4), and HCBG_05092 (UniProt ID C0NPL2) genes and S. cerevisiae Ole1 (UniProt ID P21147) that were codon optimized, synthesized, and cloned into the Gal-inducible plasmid pESC-URA by GenScript . Briefly, 500 μl of cells grown in YPD for 16 h was spun down and the supernatant removed. Added to the BY4742 and 12431 cells were 5 μg of herring sperm DNA (5 μl of 10 mg/ml stock), 400 ng of GenScript plasmid DNA, 500 μl of PLATE buffer , and 57 μl dimethyl sulfoxide. Cells were incubated at room temperature for 15 min, followed by 15 min at 42°C. Cells were collected by brief centrifugation of 5 s at 8,000 × g, and supernatant was removed, resuspended in phosphate-buffered saline (PBS), and plated on YNB-2% glucose-LLH 2% agar plates without uracil. Resulting colonies were screened for plasmid presence by PCR of a portion of the pESC-URA-lpt1 plasmid  and preserved as glycerol stocks.Strains were maintained on YPD  or YNB with amino acid supplements . The strains were transformed by a lithium-acetate heat shock method  with a pg for 2 min with a swinging-bucket rotor fitting 50-ml conical tubes. Pellets were collected into preweighted tubes and washed 2× with TBS before flash freezing in liquid nitrogen. All samples were made in 4 replicates as input for downstream omics analysis.For the Hc_LPT1 and desaturase expression assays, cells were precultured overnight in YNB 2% glucose LLH with or without uracil to accommodate the nonplasmid strain auxotrophy as a negative control. For biomass collection during induction, which often slows or prevents cell division, cells were inoculated to an optical density of 0.5 into YNB-2% galactose LLH with or without uracil and collected after induction of plasmid expression for 17 h. LPT1 cultures were also analyzed after 17 h of growth in glucose LLH with or without uracil (uninduced). Cultures were collected by spinning 25 ml of culture in a tabletop centrifuge (VWR symphony 4417 R) at 2,000 × H. capsulatum G217B were grown in Ham’s F12 medium at 37°C under constant shaking and used 3 days after its inoculation, as previously described  as previously described  and S. cerevisiae S288c (downloaded 11 January 2018) sequences from UniProt Knowlegdgebase, considering protein N-terminal acetylation and oxidation of methionine as variable modifications and cysteine carbamidomethylation as a fixed modification. The remaining parameters were set as the software default. Protein abundances were estimated by the intensity-based absolute quantification (iBAQ) method  method . The int) method  to the r) method .P ≤ 0.05 by t test considering equal variance and unpaired samples. The distribution of fatty acyl groups was done by counting individual fatty acyl groups and was considered significantly different with a P value of ≤0.05 by Fisher’s exact test.Total lipid extracts and phospholipid fraction from the Silica 60 column were resuspended in methanol and subjected to LC-MS/MS analysis as previously described . To asseFatty acids were methylated with anhydrous methanolic HCl (1.2 N) for 1 h at 100°C and extracted by adding equal volumes of water and hexane. Sterols were treated with 30 mg/ml methoxyamine in pyridine for 90 min at 37°C with shaking and derivatized with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) (Sigma-Aldrich) with 1% trimethylchlorosilane (TMCS) (Sigma-Aldrich) at 37°C with shaking for 30 min . DerivatH. capsulatum yeasts were washed with PBS, suspended in Ham’s F12, and loaded into plates with serially diluted compounds at a final cellular density of 2.5 × 104 cells/ml in 4 replicates. Cells were grown at 37°C under constant shaking for 7 days, and minimal drug concentration with no visual growth was considered the MIC. To evaluate the effect of lipid synthesis inhibition of H. capsulatum, triplicates of 106 cells/ml were incubated in Ham’s F12 at 37°C with shaking at 250 rpm for 48 h with half the MIC of myriocin, 10-thiasteric acid, and thiocarlide. Cells were washed with PBS and pellets were frozen for further lipid extraction and subsequent fatty acid and lipidomics analyses. To assess the ability of the compounds to kill intracellular fungi, murine alveolar macrophage cell line AMJ2 was plated onto 96-well plates and incubated overnight at 37°C with 5% CO2. The macrophages were challenged with H. capsulatum (ratio of 2 yeasts per macrophage cell) in the presence or absence of myriocin, 10-thiasteric acid, and thiocarlide for 48 h (see the figure legends for concentrations). Macrophages were lysed with distilled water and the yeast suspensions were diluted and plated onto brain heart infusion agar supplemented with 5% sheep’s blood. Plates were incubated at 37°C and CFU were counted after growth. To address cytotoxicity of the inhibitors on alveolar macrophages, AMJ2 cells were plated (105 cells/well) in 96-well plates and incubated overnight at 37°C and 5% CO2. Cells were washed with PBS and incubated with serially diluted inhibitors. Solvent controls were made with the same solvent amount present in the highest concentration for each inhibitor. Plates were incubated for 24 h at 37°C and 5% CO2. MTT  was added to each well (50 μg/well) and incubated for 4 h at 37°C and 5% CO2. After supernatant removal, the formazan crystals were solubilized with isopropanol and read with a plate reader at 570 nm. All values were normalized using the negative control (absence of drug or solvent).S. cerevisiae model  under accession number PXD017734. The code for the lipidomics visualization tool is available under the BSD 2-Clause License at GitHub at https://github.com/wichne/LipidomicsMapTools.Proteomics data were deposited into the Pride repository (


With the standard of HBsAg ≤ 4.699 log10 IU/mL, the sensitivity and specificity of HBsAg for predicting the three levels of significant hepatitis activity were successively 75.81% and 81.82%, 79.23% and 78.57% and 80.82% and 67.44%. (4) Conclusion: Quantitative HBsAg instead of HBV DNA is valuable in predicting significant hepatitis activity of HBeAg-positive chronic HBV infection.(1) Background: As specialparameters in predicting significant hepatitis activity of hepatitis B e antigen (HBeAg)-positive chronic hepatitis B virus (HBV) infection, the quantitative standard of HBV DNA has not been agreed and that of hepatitis B surface antigen(HBsAg) has not been formed. Our objective is to evaluate the validity of HBsAg and HBV DNA in predicting the significant hepatitis activity of HBeAg-positive patients. (2) Methods: A population of 516 patients with HBeAg-positive chronic HBV infection was enrolled. Serum ALT was measured using an Abbott Architect c16000 autoanalyzer; diagnoses of liver pathological grade and stage referred to the Scheuer standard. Three levels of significant hepatitis activity were preset, which were successively “ALT ≥ 20 IU/L or Grade > G1 or Stage > S1”, “ALT ≥ 30 IU/L or Grade > G1 or Stage > S1” and “ALT ≥ 40 IU/L or Grade > G1 or Stage > S1”. (3) Results: A subpopulation of 288 patients with possible high HBV replication was selected based on locally weighted scatterplot smoothing regression curves between ALT and HBsAg, HBeAg and HBV DNA. In the subpopulation with possible high HBV replication, areas under receiver operating characteristic curves of HBsAg for predicting the three levels of significant hepatitis activity were successively 0.868, 0.839 and 0.789, which were all significantly greater than those of HBV DNA, as those were successively 0.553, 0.550 and 0.574 ( Chronic hepatitis B virus (HBV) infection remains a major public health issue affecting human health . Liver cWith reference to serum hepatitis B e antigen (HBeAg) status and alanine transferase (ALT) and HBV DNA levels, the natural history of chronic HBV infection is generally divided into four consecutive but possibly reciprocating phases See : (1) HBeHowever, the upper limit of normal (ULN) for ALT is still controversial ,3,4,5, aThe established quantitative serum markers of HBV include hepatitis B surface antigen (HBsAg), HBeAg, hepatitis B core-related antigen (HBcrAg), antibodies against hepatitis B core antigen (anti-HBc), HBV DNA and HBV RNA ,12. AmonThe quantitative standard for HBsAg in predicting the significant hepatitis activity phase of HBeAg-positive chronic HBV infection has not been formed ,3,4. HBsThe purpose of this study was to delimit the subpopulation with possible high and possible low HBV replication in a population with HBeAg-positive chronic HBV infection, and on this basis, to evaluate the performance of HBsAg, HBeAg and HBV DNA for predicting significant HBeAg-positive hepatitis activity.HBeAg-positive patients with chronic HBV infection who were hospitalized and underwent liver biopsy in Shanghai Public Health Clinical Center of Fudan University from January 2017 to September 2020 were screened. A total of 516 patients with complete hospitalization records, detailed descriptions of liver pathology and matching laboratory data were enrolled for this study. Among them, 313 were males and 203 were females, and age ranged from 10 to 68 years with a median (interquartile range) of 34 (29–39) years; the proportions of patients with age <25 years and <30 years in males were 9.3% (29/313) and 30.0% (94/313), and in females were 6.4% (13/203) and 27.1% (55/203). Patients with the following conditions were excluded: coinfection with other hepatotropic viruses , Epstein–Barr virus, cytomegalovirus; Schistosomiasis japonica liver disease, nonalcoholic/metabolic fatty liver disease (steatosis > 5%), drug-induced liver injury, excessive drinking , autoimmune diseases, endocrine and metabolic diseases, gallstones and bile duct stones, liver tumors and decompensated liver disease; antiviral therapy with interferon-α/peg-interferon-α or nucleosides/nucleotides, hepato-protective therapy with glycyrrhizates or oxymatrine/matrine or bicyclol/bifendate or traditional Chinese medicine prescriptions (including Chinese patent medicines) within 6 months before liver biopsy; length of biopsy specimen less than 1.0 cm or number of portal areas less than 6.2 to 2 × 109 IU/mL. ALT was measured using an Abbott Architect c16000 automatic biochemical analysis system  and auxiliary reagents. Based on related literature [Fasting venous blood was collected and serum was separated on the morning of liver biopsy. HBsAg and HBeAg were measured by microparticle enzyme immunoassay using an Abbott Architect i2000 automatic immunoassay system  and auxiliary reagents. The detection range of HBsAg is 0.05 to 250 IU/mL, and a sample was diluted by 500 times and re-measured if HBsAg exceeded the upper limit of detection; the lower limit of detection of HBeAg is 1.0 SCO. HBV DNA was measured by PCR probe assay using a Roche LightCycler 480 qPCR system , the reagents were purchased from Sansure Biotech Inc.  and the detection range of HBV DNA is 1 × 10terature ,4,5, thrPercutaneous suction liver biopsies assisted by ultrasonography were performed using a 16G biopsy needle after written informed consent forms were signed. The biopsy specimens were immediately transferred into plastic tubes, quickly frozen and processed within 36 h. The quality assessments and pathological diagnoses of biopsy specimens were performed independently by one experienced pathologist, who was ignorant of the laboratory information. The pathological diagnoses of liver specimens referred to the Scheuer scoring system , in whicU non-parametric test was used to compare the differences in ALT, HBsAg, HBeAg and HBV DNA levels between subpopulations with possible high and with possible low HBV replication. Spearman rank correlation analysis was used to analyze the correlations between ALT, liver pathological grade and stage and HBsAg, HBeAg and HBV DNA. Fisher Z non-parametric test was used to compare the differences in Spearman correlation coefficients between ALT, liver pathological grade and stage and HBsAg, HBeAg and HBV DNA between each other. Receiver operating characteristic (ROC) curve was used to evaluate the performance of HBsAg, HBeAg and HBV DNA for predicting “biochemical or pathological” significant hepatitis activity in the subpopulation with possible high HBV replication and for predicting biochemically significant hepatitis activity in the subpopulation with possible low HBV replication. Dependent sample Hanley and McNeil Z nonparametric test was used to compare the differences in areas under ROC curves (AUCs) between HBsAg, HBeAg and HBV DNA for predicting significant hepatitis activity. p < 0.05 was defined as statistically significant.MedCalc version 15.8  was used for statistical analyses and graph productions. Locally weighted scatterplot smoothing (LOESS) regression analysis was used to explore the evolving trend between ALT and HBsAg, HBeAg and HBV DNA, and to delimit the subpopulations with possible high and possible low HBV replication. Mann–WhitneyHBsAg, HBeAg and HBV DNA were used as independent variables, ALT was used as a dependent variable and LOESS regression analyses were performed with a span of 60%. The LOESS regression curves between ALT and HBsAg, HBeAg and HBV DNA are illustrated in 10 IU/mL, HBeAg > 2.875 and ≤2.875 log10 SCO and HBV DNA > 7.500 and ≤7.500 log10 IU/mL. The proportions of ALT ≥ 20 IU/L, ≥ 30 IU/L and ≥ 40 IU/L in patients with high levels of HBsAg were 92.0% (161/175), 79.4% (139/175) and 70.9% (124/175), with high levels of HBeAg were 93.6% (221/236), 84.7% (200/236) and 77.1% (182/236), and with high levels of HBV DNA were 94.4% (184/195), 85.1% (166/195) and 77.4% (151/195).According to the LOESS regression curves, HBsAg, HBeAg and HBV DNA levels could be divided into two strata of high and medium–low levels: HBsAg > 4.250 and ≤4.250 log10 IU/mL or HBeAg > 2.875 log10 SCO or HBV DNA > 7.500 log10 IU/mL” and “HBsAg ≤ 4.250 log10 IU/mL and HBeAg ≤ 2.875 log10 SCO and HBV DNA ≤ 7.500 log10 IU/mL”. In the subpopulation with possible high HBV replication, the proportions of patients with high levels of HBsAg, HBeAg and HBV DNA were 60.8% (175/288), 81.9% (236/288) and 67.7% (195/288).With the standard of the quantitative stratification of HBsAg, HBeAg and HBV DNA, the subpopulations with possible high HBV replication and possible low HBV replication were defined as “HBsAg > 4.250 logThe demographic, laboratory and pathological characteristics of the overall population and of the subpopulations with possible high and possible low HBV replication are summarized in In the subpopulation with possible high HBV replication, the proportions of patients with age < 25 years and < 30 years in males were 8.4% (15/179) and 33.0% (59/179), and in females were 6.4% (7/109) and 24.8% (27/109). In the subpopulation with possible low HBV replication, the proportions of patients with age < 25 years and < 30 years in males were 10.4% (14/134) and 26.1% (35/134), and in females were 6.4% (6/94) and 29.8% (28/94).The Spearman correlation coefficients between HBsAg, HBeAg and HBV DNA and ALT, pathological grade and stage in the overall population and in the subpopulations with possible high and possible low HBV replication are summarized in Z = 2.143, p = 0.0321), the AUC of HBsAg and HBeAgfor predicting “ALT ≥ 30 IU/L or Grade > G1 or Stage > S1” was significantly greater than that of HBV DNA , and the AUC of HBsAg for predicting “ALT ≥ 40 IU/L or Grade > G1 or Stage > S1” was significantly greater than that of HBeAg and HBV DNA .In the overall population, the AUCs of HBsAg, HBeAg and HBV DNA for predicting “ALT ≥ 20 IU/L or Grade > G1 or Stage > S1” were 0.660, 0.573 and 0.538, for predicting “ALT ≥ 30 IU/L or Grade > G1 or Stage > S1” were 0.682, 0.600 and 0.504, and for predicting “ALT ≥ 40 IU/L or Grade > G1 or Stage > S1” were 0.654, 0.580 and 0.522; among them, the AUC of HBsAg for predicting “ALT ≥ 20 IU/L or Grade > G1 or Stage > S1” was significantly greater than that of HBV DNA , the AUC of HBsAg and HBeAg for predicting “ALT ≥ 30 IU/L” was significantly less than that of HBV DNA , and the AUC of HBsAg for predicting “ALT ≥ 40 IU/L” was significantly less than that of HBeAg and HBV DNA .In the overall population, the AUCs of HBsAg, HBeAg and HBV DNA for predicting “ALT ≥ 20 IU/L” were 0.551, 0.569 and 0.661, for predicting “ALT ≥ 30 IU/L” were 0.533, 0.575 and 0.647, respectively, and for predicting “ALT ≥ 40 IU/L” were 0.507, 0.592 and 0.635; among them, the AUC of HBsAg and HBeAg for predicting “ALT ≥ 20 IU/L” was significantly less than that of HBV DNA (In the subpopulations with possible high and possible low HBV replication, the ROC curves and AUCs of HBsAg, HBeAg and HBV DNA for predicting “ALT ≥ 20 IU/L or Grade > G1 or Stage > S1” and “ALT ≥ 20 IU/L”, “ALT ≥ 30 IU/L or Grade > G1 or Stage > S1” and “ALT ≥ 30 IU/L”, and “ALT ≥ 40 IU/L or Grade > G1 or Stage > S1” and “ALT ≥ 40 IU/L” are illustrated in Based on the Youden index, an optimal cutoff was determined. With reference to the minimum difference between the specificity of HBsAg and HBeAg for predicting “ALT ≥ 20 IU/L or Grade > G1 or Stage > S1” and the sensitivity of HBsAg and HBeAg for predicting “ALT ≥ 40 IU/L or Grade > G1 or Stage > S1” in the subpopulation with possible high HBV replication, and between the specificity of HBV DNA and HBeAg for predicting “ALT ≥ 20 IU/L” and the sensitivity of HBV DNA and HBeAg for predicting “ALT ≥ 40 IU/L” in the subpopulation with possible low HBV replication, a practical cutoff that was easy to remember was chosen. The optimal cutoffs and practical cutoffs with corresponding diagnostic parameters are summarized in 10 IU/mL or HBeAg > 3.154 log10 SCO or HBV DNA ≤ 4.362 log10 IU/mL or HBeAg ≤ 0.861 log10 SCO alone and in combination with ALT < 20 IU/L, < 30 IU/L or < 40 IU/L are summarized in The proportions of liver pathological grades and stages in patients with HBsAg > 4.699 logIn this study, based on a subpopulation with possible high and possible low HBV replication, we explored the role of HBsAg, HBeAg and HBV DNA in the persistence and onset of HBeAg-positive chronic HBV infection, and with reference to the criteria of “the preset ULNs for ALT” and “Grade > G1 or Stage > S1”, we evaluated the performance of HBsAg, HBeAg and HBV DNA in predicting significant hepatitis activity of HBeAg-positive chronic HBV infection. We also chose the practical cutoffs of HBsAg and HBeAg in predicting significant hepatitis activity in a subpopulation with possible high HBV replication and of HBV DNA and HBeAg in predicting significant hepatitis activity in a subpopulation with possible low HBV replication, and evaluated the reliability of HBsAg, HBeAg or HBV DNA alone and in combination with ALT in predicting liver pathological grades and stages.So far, the nomenclature of four natural history phases of chronic HBV infection has not been fully unified ,3,4. ConThe virological mechanisms of the persistence and onset of chronic HBV infection have not been thoroughly elucidated ,30,31. TThe results of this study also support the inferences on the virological pathogenesis of chronic HBV infection based on experimental studies: high and medium–low levels of serum HBsAg, by inducing immune exhaustion and immune ignorance ,33,34, mTo date, there is no consensus on ULNs for ALT, which can be used to predict significant hepatitis activity in chronic HBV infection ,3,4. TheIn patients with HBeAg-positive chronic HBV infection, non-significant hepatitis activity is not only related to high levels but also low levels of HBV replication. Among them, non-significant hepatitis activity related to high levels of HBV replication belongs to “persistent inactivity” of the“immune tolerance” phase, which is manifested as non-significant serum biochemical abnormalities and non-significant liver histological changes, while non-significant hepatitis activity related to low levels of HBV replication belongs to “transient inactivity” of the “immune activation” phase, which is manifested as non-significant serum biochemical abnormalities and unconfined liver histological changes See . Therefo10 IU/mL, the proportions of liver pathological stage > S1 and > S2 were 31.2% and 1.3%, and in combination with ALT ≥ 40 IU/L, the proportions of liver pathological stage > S1 and > S2 were 17.1% and 0.0%. However, in patients with HBV DNA ≤ 4.362 log10 IU/mL, the proportions of liver pathological stage > S1 and > S2 were 65.8% and 38.4%, and in combination with ALT ≥ 20 IU/L, the proportions of liver pathological stage > S1 and > S2 were 68.8% and 25.0%. These suggested that the ability of ALT based on the three preset ULNs and of HBV DNA with reference to the practical cutoff alone and in combination with ALT for excluding extensive liver fibrosis and cirrhosis is all very limited; however, the performances of HBsAg with the standard of the practical cutoff alone and in combination with ALT for excluding extensive liver fibrosis and cirrhosis are both almost excellent.In this study, we chose the practical cutoffs of HBsAg and HBeAg and of HBV DNA and HBeAg, which were convenient for clinical application, for predicting significant hepatitis activity in subpopulations with possible high and low HBV replication. Based on information of this study population, in patients with ALT ≥ 20 IU/L, ≥ 30 IU/L and ≥ 40 IU/L, the proportions of liver pathological stage > S1 and > S2 were 44.4% and 11.2%, 47.3% and 17.6% and 47.1% and 21.0%. In patients with HBsAg > 4.699 logThis study had some limitations. First, the study population was all patients undergoing liver biopsy, and most of the patients were adults, so the selection bias of patients cannot be completely excluded. Second, the sample size was relatively small, and the gender differences in the normal references of ALT were not considered, so the findings of this study may need to be further confirmed in different genders by expanding the sample size. Third, this study was a retrospective cross-sectional study, and its findings may also need to be further verified by retrospective or prospective follow-up studies.10 IU/mL and of ALT < 40 IU/L, HBsAg alone and in combination with ALT had excellent performance in excluding HBeAg-positive extensive liver fibrosis and cirrhosis of HBeAg-positive chronic HBV infection.In the subpopulation with possible high HBV replication, HBsAg and HBeAg instead of HBV DNA were significantly negatively correlated with ALT, and had very good and good performance in predicting “biochemical or pathological” significant hepatitis activity; in the subpopulation with possible low HBV replication, HBV DNA and HBeAg instead of HBsAg were significantly positively correlated with ALT, and had very good and good performance in predicting biochemically significant hepatitis activity. With the standard of HBsAg > 4.699 log


Lemna minor (duckweed). The effectiveness of this biogenic synthesis was evidenced for comparison with non-biogenic ZnO-NPs (obtained without using the plant extract), which have been synthesized in this research. XRD (X-ray diffraction), FE-SEM (field emission gun electron scanning microscopy), EDX (energy dispersive x-ray spectroscopy), TEM (transmission electron microscope) and UV-vis (ultraviolet-visible spectrophotometry) showed the biogenic approach effectiveness. The duckweed extract was subjected to UHPLC-ESI/QTOF-MS (ultra high-pressure liquid chromatography quadrupole time of flight mass spectrometry) phenolic profiling. This untargeted characterization highlighted a high and chemically diverse content in the duckweed extract of compounds potentially implicated in nanoparticulation. From an application standpoint, the effect of biogenic nanoparticles was investigated on some traits of maize subjected to seed priming with a wide range of biogenic ZnO-NPs concentrations. Inductive effects on the shoot and root biomass development were ascertained concerning the applied dosage. Furthermore, the biogenic ZnO-NPs stimulated the content of chlorophylls, carotenoids, and anthocyanin. Finally, the study of malondialdehyde content (MDA) as a marker of the oxidative status further highlighted the beneficial and positive action of the biogenic ZnO-NPs on maize.The need to increase crop productivity and resistance directs interest in nanotechnology. Indeed, biogenic metal oxide nanoparticles can promote beneficial effects in plants, while their synthesis avoids the environmental impacts of conventional synthetic procedures. In this context, this research aimed to synthesize biogenic zinc oxide nanoparticles (ZnO-NPs) using, for the first time, an extract of a wild and spontaneous aquatic species,  Nanotechnology is attracting increasing interest due to the many possible applications in various scientific areas. Several nanomaterials of different origin can be used for a variety of technological applications . DiverseNPs synthesis can be carried out using some plant extracts as reducing, ligation and capping agents, thanks to the considerable content of these biological materials of certain classes of compounds ,9. Indee2+, thus stabilizing the NPs and preventing excessive growth [NPs have also been used in agriculture as, in small concentrations, they can improve physiological, morphological and biochemical traits in the treated species ,5,14. See growth . TherefoUlva Lactuca, an alga, has been used to synthesize ZnO-NPs with a size of 18–27 nm and spherical shape [Carissa edulis, for the biogenic synthesis of zinc oxide NPs with an average size of 50–55 nm and flower-shaped morphology. Soto-Robles et al. [Justicia spicigera, an evergreen shrub. Álvarez-Chimal et al. [Aloe barbadensis Mill ZnO-NPs of 35 nm size in the form of spherical aggregates. All these studies indicated the plants used, and their metabolites substantially impact particles size and shape. However, the research for the biogenic synthesis of metal oxide NPs has generally focused on terrestrial plants or algal extracts. To date, no studies have been done on biomasses from freshwater aquatic plants. Several freshwater plants may be of interest as they are incredibly productive, fast-growing, and adapted to very different and even hostile conditions, indicating high amounts of antioxidants and protective compounds [Several published studies have demonstrated the effectiveness of extracts obtained from some plants for ZnO-NPs synthesis. To mention a few examples, recently, al shape . Fowsiyaal shape  used a ms et al.  obtainedl et al.  used a pl et al.  synthesiompounds .Lemna minor (duckweed), a small floating species used in ecological and phytoremediation studies, is of particular interest [−2 day−1 dry weight [In this respect, interest ,25,26. Tinterest . Indeed,y weight . Therefoy weight . Besidesy weight , thanks y weight ,25.Based on these premises, a study was carried out to obtain biogenic ZnO-NPs from a duckweed extract and test them on plant growth promotion potential on maize. Considering that duckweed is particularly rich in phenolic compounds , the stu3, 99%) calcium nitrate tetrahydrate (Ca(NO3)2 × 4H2O, 98%), magnesium sulphate heptahydrate , potassium sulphate , potassium chloride , potassium phosphate monobasic , di-potassium hydrogen phosphate , boric acid , manganese sulphate monohydrate , copper sulphate , zinc sulphate heptahydrate , ammonium molybdate tetrahydrate ((NH4)6Mo7O24 × 4H2O, 99%), ethylenediaminetetraacetic acid ferric sodium salt (Fe-EDTA), trichloroacetic acid (99%) and thiobarbituric acid (>98%) were provided by Sigma-Aldrich . Solvents for UHPLC-ESI/QTOF-MS were LCMS grade from Merck-VWR . Sodium hydroxide, acetone and ethanol were provided by Carlo Erba . All of the chemicals were used as received without additional purification.Sodium hypochlorite , potassium nitrate (KNOw/v) for 2 min, then rinsed with copious amounts of distilled water, and transferred into polyethylene trays (35 × 28 × 14 cm) containing a nutrient solution composed of 3.46 mmol L−1 KNO3, 1.25 mmol L−1 Ca(NO3)2 × 4H2O, 0.66 mmol L−1 KH2PO4, 0.071 mmol L−1 K2HPO4, 0.41 mmol L−1 MgSO4 × 7H2O, 0.28 mmol L−1 K2SO4, 1.94 μmol L−1 H3BO3, 0.63 μmol L−1 ZnSO4 × 7H2O, 0.18 μmol L−1 (NH4)6Mo7O24 × 4H2O × 2H2O, 1 μmol L−1 MnSO4 × H2O, 21.80 μmol L−1 FeEDTA, 1 μmol L−1 CuSO4. These trays were transferred into a growth chamber and submitted to 120 μmol m−2 s−1 of light intensity (photoperiod: 12/12 h—light/dark) at 24 ± 2 °C. The growth media was renewed every two weeks.Duckweed was collected from a small freshwater lake located in Perugia . The harvested plants were disinfected with an aqueous sodium hypochlorite solution (0.5%—v/v) aqueous solution. The suspension was stirred at 80 °C for 15 min, then centrifuged for 20 min at 8000 rpm and filtered through a 0.22 μm regenerated cellulose syringe membrane into amber glass vials.Twenty grams of duckweed were washed repeatedly with distilled water and dried in an oven at 50 °C until a constant weight was reached. One gram of dried duckweed was reduced to powder, extracted in 40 mL of a 25% ethanol  as previously reported . Brieflyhttp://phenol-explorer.eu/, accessed on 24 February 2021) was used as the reference in the annotation. Features were annotated only when present within 100% of replications. After that, phenolics were grouped in sub-classes (according to Phenol-Explorer 3.6 annotations), and each cumulative abundance quantified against calibration curves  prepared from pure standard solutions , as reported in previous work [The annotation of phenolics from raw mass features was done in Profinder B.07  by combining monoisotopic accurate mass (mass accuracy < 5 ppm) with isotopic spacing and isotopic ratio, following mass and retention time alignment ,33. The ous work .4 × 7H2O, 30 mL of the plant extract, obtained as described previously, was added drop by drop. The resulting suspension was kept at 80 °C for 2 h. The solid phase was then recovered by centrifugation, repeatedly washed and centrifuged, and left for 16 h in an oven at 80 °C. After that, the solid was powdered and calcined in a muffle at 400 °C for 4 h. The ZnO-NPs obtained by applying this procedure, based on the use of the duckweed extract, are referred to throughout the text as biogenic ZnO-NPs. The efficiency of the biogenic synthesis was estimated according to Król et al. [To 50 mL of a solution containing 1.40 M ZnSOl et al. .4 × 7H2O. After the reaction, the suspension was washed and centrifuged repeatedly. The white solid obtained was left for 16 h at 80 °C, then reduced to a fine powder and calcinated in a muffle for 4 h at 400 °C. The ZnO-NPs obtained by applying this procedure, thus without using the duckweed extract, are referred to throughout the text as non-biogenic ZnO-NPs.The same procedure was used to synthesize zinc oxide NPs without the plant extract, using different volumes. 100 mL of 2 M NaOH were added drop by drop to 50 mL of the solution 1.4 M ZnSOPowder X-Ray Diffraction (PXRD) patterns were collected in reflection geometry in the 10–80° 2θ range, with a 40 s step-1 counting time and with a step size of 0.016° on a PANalytical X’PERT PRO diffractometer , PW3050 goniometer , equipped with an X’Celerator detector  by using the Cu Kα radiation. The long fine focus (LFF) ceramic tube operated at 40 kV and 40 mA. The UV-vis spectra of samples were measured in the range 250–700 nm.The morphology and composition were examined by field emission gun electron scanning microscopy (FE-SEM) LEO 1525 ZEISS . Elemental composition and chemical mapping were determined using a Bruker Quantax EDS . The samples were deposited on conductive carbon adhesive tape and metalized with chromium (8 nm).TEM images were obtained using a Philips 208 transmission electron microscope . The samples were prepared by putting one drop of an ethanol dispersion of the sample powder on a copper grid pre-coated with a Formvar film and dried in air.v/v). Subsequently, the seeds were rinsed with distilled water. For seed-priming with the biogenic NPs, suspensions containing biogenic ZnO-NPs were prepared at the concentrations of 25 mg L−1 (T25), 50 mg L−1 (T50), 100 mg L−1 (T100) and 200 mg L−1 (T200) and sonicated for 5 min. Seeds were immersed in 10 mL of the above solutions for 8 h under slow agitation. Then, the seeds were placed in Petri dishes on paper (10 seeds/plate), added with 10 mL of distilled water, covered and put into a growth chamber in the dark (22 ± 2 °C). Control seeds were obtained for priming with distilled water.Maize (cv Belgrano) seeds were surface-sterilized for three minutes with NaClO 2 × 4H2O, 0. 5 mmol L−1 MgSO4 × 7H2O, 0.7 mmol L−1 K2SO4, 0.1 mmol L−1 KCl, 0.1 mmol L−1 KH2PO4, 1 μmol L−1 H3BO3, 0.5 μmol L−1 MnSO4 × H2O, 0.5 μmol L−1 CuSO4, 0.5 μmol L−1 ZnSO4 × 7H2O, 0.01 μmol L−1 (NH4)6Mo7O24 × 4H2O, and 100 μmol L−1 Fe-EDTA. Fourteen days after sowing (14 DAS—second leaf stage), plants were harvested and subjected to the following determinations.For days after priming and sowing, seedlings were transferred to hydroponic solutions containing a nutrient solution 2 mmol Lv/v). The suspensions were filtered, and the absorbance determined at 452.5, 644, and 663 nm [At 14 DAS (days after sowing), the shoot and root lengths and fresh weight were recorded for samples of maize subjected to the different treatments. Further, the contents of chlorophyll a, chlorophyll b and carotenoids were assessed. In detail, 1.0 g of plant sample was extracted with 85% acetone in water (d 663 nm .Maize shoots were harvested at 14 DAS (1 g) and extracted with ethanol (95%) using pestle and mortar to determine the anthocyanin content. The resulting suspension was filtered and centrifuged, and the anthocyanin content was determined spectrophotometrically at 535 and 650 nm .w/v) trichloroacetic acid and 0.25% (w/v) thiobarbituric acid. The suspensions were then centrifuged at 10,000× g for 15 min. The supernatants were heated in a water bath (95 °C). After quick cooling, the MDA content was quantified spectrophotometrically [The lipid peroxidation was determined in maize seedlings treated with T25, T50, T100 and T200, collected at 14 DAS (days after sowing). Seedlings (0.5 g) were homogenized in a solution containing 10% . The most represented compounds in this class were tyrosol and p-HPEA-AC, 4-hydroxycoumarin, the phenolic terpene carnosic acid and the alkylphenol 5-nonadecylresorcinol. Interestingly, the extract was also rich in flavonoids, with anthocyanins, flavonols, flavones, and flavonols being 24.5, 9.6, 31.2, and 110.1 mg 100 g−1 equivalents, respectively. In particular, the most abundant flavonoids were several glycosylated forms of kaempferol and quercetin, followed by peonidin and delphinidin derivatives, among others. Regarding phenolic acid contents (181.9 mg 100 g−1), the hydroxycinnamic, ferulic, caffeic, and coumaric acids were the most abundant. The whole list of phenolic compounds annotated in duckweed extracts is provided as According to an untargeted metabolomics approach, phenolic compounds were profiled in duckweed extracts. As provided in The concentration of the biogenic ZnO-NPs differently influenced the shoot and root length of maize seedlings at 14 DAS . Shoot lAs for carotenoids, T25, T50 and T100 determined substantial increases in their content. Moreover, T200 did not affect carotenoids b. ConcerThe malondialdehyde content was determined in maize seedlings submitted to the different treatments with the biogenic ZnO-NPs . The higNanotechnology can play a role in enhancing crop performance. Some research demonstrated that NPs could lead to highly positive and beneficial effects on crops, even if subjected to abiotic stress ,22,39,40In this context, a duckweed extract was used to carry out the biogenic synthesis of zinc oxide NPs. The NPs obtained applying the biogenic approach were then compared to those synthesized without the plant extract. In general, the XRD patterns showed by the ZnO-NPs indicated as both the biogenic and non-biogenic syntheses allowed obtaining nanometric structures . The bioThe UV-vis spectra showed the typical absorption peak of ZnO-NPs in the range 370–380 nm . The maxCarissa edulis, Justicia spicigera and Medicago sativa [FE-SEM and TEM analyses indicated results in line with the XRD patterns . The moro sativa ,21,34. Io sativa ,7,34.As for the results of the untargeted metabolomic profile of the duckweed extract, the high number of phenolic compounds might have played a significant role in controlling the size and shape of the biogenic NPs . Indeed,No detailed mechanisms explain the effect of the phenolic moiety in NPs formation, but its contribution to reducing capacity and stabilization has been postulated . The proInterestingly, it was reported that the specific plant source  could influence the characteristics of the NPs ,47,48. T2+ released to roots from the NPs dissolution [Successively, the effect of the biogenic ZnO-NPs on maize was tested by direct application on seeds. In general, ZnO-NPs application to crops can prompt some beneficial effects for their ability to enhance defensive mechanisms and protect cell membrane structures, even though the mode of action of ZnO-NPs remains still unknown . Seed-prsolution . Such ansolution . Figure solution .−1 up to several thousand [In contrast, at the root level, the only effective treatment was T25, whereas T200 exerted an inhibiting effect. The T200 result can be explained based on the ZnO-NPs toxicity on crops resulting from excessive concentrations. Some studies documented the phytotoxic effects of NPs if applied in the range of 200 µg Lthousand ,52.As for the content of chlorophyll a and b found in maize samples subjected to the various treatments, the intermediate doses induced significant increases in chlorophyll content compared to the control samples a. In conIn contrast, T200, by providing excessive zinc to the crop, inhibited chlorophyll formation a. Such aConcerning carotenoids, ZnO-NPs showed some interesting effects b. The imThe biological role of anthocyanin is of pivotal importance as they can act against ROS in the vacuole, being considered antioxidants and inhibitors of lipid peroxidation ,61. In gFinally, the MDA content displayed by maize plants treated with the biogenic ZnO-NPs was ascertained . In gene−1 [−1 [On the contrary, this conclusion cannot be extended to T200. In this case, the reduction of MDA compared to control samples  may resu−1  and soyb−1 [−1 . LikewisTherefore, when appropriate dosage and target uses have been defined, duckweed-based NPs synthesis can represent a promising approach for producing nanosized materials with potential biostimulant activity in crops . This abThis study has proven for the first time that by using an extract from duckweed, it is possible to obtain zinc oxide NPs. Then, we intend to highlight how unexplored natural resources available in nature can be exploited to synthesise valuable materials. Even in the context of green chemistry, the exploitation of such natural resources is a sustainable way forward as it has no environmental impact and is in line with the paradigm of the transition to a circular economy. Therefore, our research candidates the world of aquatic plants as an attractive environment to be better explored for finding biological entities that allow practical and sustainable synthesis of nanomaterials.In detail, this research showed how duckweed could be successfully used to control ZnO-NPs, and this effect is evident when comparing the biogenic NPs with those synthesized without the plant extract. The biogenic ZnO-NPs were characterized by spherical shape and dimension of 11.7 nm (referred to as XRD determinations). Further, the plant-mediated synthesis showed satisfying efficiency in the ZnO-NPs synthesis of about 83.8%. The distinctive features of the duckweed were related to its high content of phenolic metabolites, capable of promoting efficient biogenic synthesis. Moreover, the untargeted metabolomics approach applied in this study, which is rarely used to screen the suitability of metabolites present in plant material for biogenic synthesis, has proved to be an advantageous technique for testing the adequacy of the biological entity for biogenic synthesis. Finally, it should also be emphasized as the approach developed in this work is noteworthy for being non-pathogenic and easily scalable, representing a valuable option when NPs are intended to be used for their biological activity.Subsequently, maize seed priming revealed, at particular dosages, beneficial effects on development, pigments and antioxidants, which explain the low lipid peroxidation index. In light of the above, this research evidenced the possibility of using the obtained NPs to improve maize traits through seed nano-priming. This aspect may become strategically important considering the current context in which new substances have to be developed or used to increase crop yield and quality or increase their resistance to biotic and abiotic stresses. Indeed, the biostimulatory activity highlighted by the biogenic ZnO-NPs developed in this work is worthy of consideration.Finally, given the value and numerous uses of the nanoparticle systems, a further possible application based on surface functionalisation should be considered. Biopolymeric systems can be obtained with metal oxide NPs functionalized with functional biopolymers . This tr