Patients with IgA nephropathy (IgAN) and persistent proteinuria are at risk of progression to kidney failure. Atacicept is a novel B-cell–targeted immunomodulator, shown to reduce immunoglobulin levels in patients with autoimmune diseases.JANUS (NCT02808429) was a phase II study that assessed the safety, pharmacodynamic effects, and efficacy of atacicept in patients with IgAN and proteinuria ≥1 g/d or 0.75 mg/mg on 24-hour UPCR despite maximal standard of care therapy.n = 5), atacicept 25 mg (n = 6), or atacicept 75 mg (n = 5) once weekly using subcutaneous injection. Twelve (75%) completed ≥48 weeks of treatment; 8 (50%) completed 72 weeks of treatment and the 24-week safety follow-up period. Fourteen patients reported treatment-emergent adverse events (TEAEs). Most TEAEs were mild or moderate in severity. Three patients  reported serious TEAEs, none of which were treatment related. Dose-dependent reductions in IgA, IgG, IgM, and galactose-deficient (Gd)-IgA1 with atacicept at week 24 were maintained to week 72. Early reduction in proteinuria was observed at week 24 with atacicept. Renal function progressively declined with placebo but remained stable under exposure to atacicept.A total of 16 patients were randomized 1:1:1 to placebo (Atacicept has an acceptable safety profile in patients with IgAN and is effective at reducing the levels of pathogenic factor Gd-IgA1, with potential improvements in proteinuria and renal function.  Key objectives were to evaluate the safety and tolerability, the pharmacodynamic effects on circulating Ig and Gd-IgA1, and the clinical response  of atacicept treatment for up to 72 weeks.JANUS was a randomized, double-blind, placebo-controlled phase II study of atacicept in patients with IgAN and persistent proteinuria . Eligible patients were randomized 1:1:1 to receive placebo, atacicept 25 mg, or atacicept 75 mg once weekly, administered by subcutaneous injections from prefilled 1 ml syringes. The planned treatment period was 72 weeks. Each patient entered a 24-week safety follow-up period following their last dose of treatment. See An external independent data monitoring committee monitored patient safety regularly. After at least 5 patients per arm had completed 12 weeks’ treatment, cumulative safety data was assessed. An interim efficacy analysis was performed after 16 patients completed 24 weeks’ treatment. Following this analysis, the sponsor decided that owing to unexpectedly slow enrollment, the study would be terminated once all enrolled patients had completed at least 48 weeks’ treatment and the safety follow-up period. This decision was not related to the clinical safety or efficacy of atacicept.All participants provided written informed consent before study initiation. Before commencement of the study at a given site, study protocol and consent forms were approved by the local independent ethics committee or institutional review board. The study was conducted in accordance with the principles of the Declaration of Helsinki and in compliance with the International Council for Harmonization E6 Guideline for Good Clinical Practice.Eligible patients were 18 years or older, with IgAN and persistent proteinuria despite being on a stable dose of ACEi and/or ARBs. Patients were required to initiate ACEi and/or ARB therapy at least 12 weeks before the screening visit, the dose of which must have been stable for at least 8 weeks before screening and considered optimal by the investigator. IgAN was confirmed by biopsy within the 60 months before screening, using the Oxford-MEST classification. UPCR ≥1 mg/mg by 24-hour urine collection during the screening period was required. Patients with UPCR ≥0.75 mg/mg at screening who had at least one documented UPCR ≥1 mg/mg within 12 months while on ACEi and/or ARB therapy were also eligible. The patients were required to have up-to-date vaccinations against pneumococcus and the influenza virus.2 for patients with kidney biopsy performed within 3 months before screening, eGFR <35 ml/min per 1.73 m2 for patients with kidney biopsy performed between 3 and 12 months before screening, and eGFR <45 ml/min per 1.73 m2 for patients with kidney biopsy performed between 12 and 60 months before screening. Further exclusion criteria were blood pressure higher than 140/90 mmHg, an active infection or high infectious risk, prior cyclophosphamide or concomitant immunosuppressant use, and prior use of B-cell–directed biological therapies such as rituximab.Patients with concomitant significant renal disease other than IgAN, including diabetic nephropathy, lupus nephritis, and Henoch Schönlein purpura, or with serum IgG levels <6 g/l were not eligible for the study. In addition, patients with >50% glomeruli with global glomerulosclerosis, or >50% cortical area involved by tubular atrophy or interstitial fibrosis were not eligible. Exclusion criteria also included those with estimated glomerular filtration rate (eGFR) of <25 ml/min per 1.73 mThe primary objective was to evaluate the safety and tolerability of atacicept in patients with IgAN and persistent proteinuria while on a stable dose of ACEi and/or ARB therapy. Safety was assessed based on the number of patients with TEAEs (MedDRA version 22.1), adverse events (AEs) leading to discontinuation, serious AEs, AEs leading to death, and AEs of special interest (AESIs). AESIs included cardiac failure, ischemic heart disease, cardiac arrhythmia, infections, hypersensitivity reactions , and injection-site reactions . Physical assessments, clinically significant vital signs, electrocardiograms, and laboratory assessments were also evaluated.Key secondary objectives were to evaluate the pharmacodynamic characteristics of atacicept, particularly the effect on circulating IgG, IgA, IgM, and Gd-IgA1 levels, as well as complement proteins C3 and C4. Blood samples were collected for analysis of these serum biomarkers at baseline and during treatment period.Other objectives included assessment of clinical efficacy of atacicept compared with placebo on proteinuria, assessed by 24-hour urine collection at baseline and weeks 24, 48 and 72 of the treatment period, and renal function as assessed by eGFR at baseline and during treatment period. eGFR was calculated as per the Chronic Kidney Disease Epidemiology Collaboration for all patients other than those in Japan, where eGFR was calculated using the Japan Association of Chronic Kidney Disease formula 2008. Exploratory objectives included changes in the levels of immune cell subsets, including total B cells, mature naïve B cells, and memory B cells, which were evaluated by flow cytometry. See This study was conducted between January 2017 and February 2020. A total of 47 patients at 17 centers were screened for participation in the study, including 1 center in Japan, 1 in the United Kingdom, and 15 in the USA. Of these 47 patients, 16 were considered eligible to participate in the study and were randomized; 5 patients were randomized to receive placebo, 6 to atacicept 25 mg and 5 to atacicept 75 mg .Figure 1Most (69%) patients completed at least 60 weeks of study treatment. The median  duration of treatment was 71.3  weeks for the placebo group, 52.6  weeks for the atacicept 25 mg, and 72.3  weeks for the atacicept 75 mg groups. The sponsor terminated the study early owing to slow enrollment, resulting in treatment discontinuation for 3 patients; 2 completed >60 weeks’ treatment and 1 completed >40 weeks’ treatment. All 3 patients completed 24 weeks of safety follow-up. A total of 5 patients discontinued treatment for other reasons .2) and all patients had high proteinuria (median UPCR 1.4–1.8 mg/mg by 24-hour urine collection) despite maximal supportive therapy.The mean (SD) age of the study population was 43 (11.1) years and 50% were female . The medA total of 14 (88%) patients experienced ≥1 TEAEs throughout the study; 60% of patients in the atacicept 75 mg group and 100% of patients in both the placebo and atacicept 25 mg groups reported TEAEs . All butn = 5), pruritus (n = 3), bruising (n = 3) and injection-site reactions (n = 3), and infections/infestations, including urinary tract infection (n = 3), and viral gastroenteritis (n = 2), were the most reported TEAEs. Infections were reported in all groups .Administration-site conditions, including erythema , injection-site reactions , injection-site pruritus  and injection-site bruising . Besides administration-site conditions, only 2 other treatment-related TEAEs were reported as follows: 1 case of herpes simplex in the placebo group and 1 case of pruritus in the atacicept 25 mg group.A total of 9 patients reported TEAEs that were considered treatment related. The proportion of patients who reported treatment-related TEAEs was higher in the atacicept 25 mg (83%) and 75 mg (60%) groups than the placebo (20%) group. Most treatment-related TEAEs (54 of 56 events) were local conditions including injection-site erythema  and 3 in the atacicept 25 mg group , none of which were considered treatment related. All serious AEs occurred during the treatment period except cervical spinal stenosis that occurred >4 months after last treatment of atacicept 25 mg. One patient (17%) in the atacicept 25 mg group discontinued treatment due to a TEAE of injection-site pruritus. No deaths were reported.n = 5, 83%) than the atacicept 75 mg group . The proportion of patients who reported AESIs of infections was 40% (n = 2), 83% (n = 5), and 20% (n = 20) in placebo, atacicept 25 mg, and atacicept 75 mg groups, respectively. The incidence of hypersensitivity-related TEAEs was lower with placebo  and atacicept 75 mg  than with atacicept 25 mg . Viral gastroenteritis was the only reported serious AESI of infection, reported by 1 patient who received atacicept 25 mg. No AESIs in the categories of cardiac failure, ischemic heart disease, cardiac arrhythmia, or demyelination were reported.All AESIs were mild or moderate. AESIs of injection-site reactions (≥1 injection-site symptom) were only reported by patients in the 2 atacicept groups, with higher incidence in the atacicept 25 mg group (No cases of severe hypogammaglobulinemia (IgG <3 g/l) were reported. Serum IgG <6 g/l was observed in 1 patient in the atacicept 25 mg group and 2 patients in the atacicept 75 mg group. First, 1 patient receiving atacicept 25 mg had IgG <6 g/l at weeks 12 and 24 of the safety follow-up period; IgG levels were within the normal range during the treatment period. Second, 1 patient receiving atacicept 75 mg had IgG <6 g/l from weeks 8 to 72 of the treatment period; IgG levels increased to within the normal range by week 12 of the safety follow-up visit. A third patient had IgG <6 g/l at weeks 24 and 32. This patient withdrew from the study at week 33 and their serum IgG levels returned to the normal range by the early termination visit. No safety concerns emerged following a review of vital signs, electrocardiograms, or clinical laboratory assessments.A consistent, dose-dependent reduction in serum IgA, IgG and IgM was observed with atacicept from baseline to week 24 . The greSimilarly, there was a clear dose-dependent reduction in median serum Gd-IgA1 levels from baseline to week 24 with atacicept 25 mg (−25%) and atacicept 75 mg (−60%) compared with placebo (2%), which was generally maintained over time . At weekSlight increases in serum C3 levels were observed with atacicept compared with placebo until week 24 and at week 48 a. At weeIn the atacicept groups, a decrease in mature naïve B cells was observed until week 72 . No pattAt week 24, a reduction in proteinuria, as assessed by 24-hour UPCR, was observed for both atacicept groups; median change from baseline was −24% with atacicept 25 mg and −25% with atacicept 75 mg, compared with an increase of 24% with placebo . This pe2), −8% at week 48  and −25% at week 72  ml/min per 1.73 m2, and 6.0  ml/min per 1.73 m2. Median change from baseline in eGFR with atacicept 75 mg was −4% at week 24, +9% at week 48 and −3% at week 72, with respective median  absolute changes of −2.0  ml/min per 1.73 m2, 3.0  ml/min per 1.73 m2, and −2.0  ml/min per 1.73 m2.There was a progressive decline in renal function assessed by eGFR in the placebo group; median change from baseline was −7% at week 24  and IgM (−60% vs −69%).Importantly, atacicept treatment also led to dose-dependent reductions in the critical pathogenic IgAN serum biomarker Gd-IgA1, which changed by −25% and −60% at week 24 with atacicept 25 mg and 75 mg, respectively, and these reductions were generally maintained to week 72. To date, no other immunomodulatory agent has shown the same magnitude of effect on reducing serum Gd-IgA1 levels in patients with IgAN.A clinically meaningful reduction in 24-hour proteinuria was observed at week 24 with atacicept, with continued improvement to week 72 with atacicept 25 mg. Despite a similar decrease in proteinuria at week 24 with atacicept 75 mg, this was not sustained over time. Proteinuria increased in 3 patients receiving atacicept at week 48, although these levels had reduced by week 72 in 2 of the patients. The proteinuria results in the atacicept 75 mg group are difficult to interpret given the small number of patients included and potential confounding factors . While there was a progressive decline in renal function by eGFR in the placebo group, both doses of atacicept resulted in stabilization of eGFR with improvements compared with placebo observed to week 72.Post hoc analyses showed correlation between reduced Gd-IgA1 and improvement in proteinuria with both atacicept doses at the patient level. Due to the robust reduction in Gd-IgA1 with atacicept 75 mg, which is considered clinically meaningful by the authors, and the overall trend for improvements in eGFR, these results provide important corroborative evidence for the potential benefit of atacicept for IgAN patients. Further investigation of additional atacicept doses that have a greater effect on pharmacodynamic parameters is warranted.While the atacicept 25 mg group showed better improvement in proteinuria, the atacicept 75 mg group had greater reductions in serum Gd-IgA1, an autoantigen that plays a central role in the pathogenesis of IgAN. The main limitation of this study was the small sample size; the results should therefore be interpreted with caution. The independent data monitoring committee provided recommendation to open enrollment for an atacicept 150 mg group, but this was not initiated due to the early termination of the trial. The early termination precluded longer-term assessments of the efficacy and safety of atacicept in patients with IgAN. A larger study of longer duration with inclusion of atacicept 150 mg is being conducted to confirm the findings reported here .In conclusion, in the phase II JANUS trial, atacicept demonstrated an acceptable safety profile in patients with IgAN and persistent proteinuria, with meaningful changes in 24-hour proteinuria, potential stabilization of renal function, and substantial reductions in the levels of Gd-IgA1 compared with placebo. Although undertaken in a small number of patients, these results suggest that the benefit–risk profile of atacicept is acceptable for patients with IgAN who are at risk of renal failure, providing early proof of concept that warrants further clinical investigation.Radica Alicic , Gerald Appel , Syed Babar , Jonathon Barratt , Diogo Belo , Yongen Chang , Scott Cohen , Claude Galphin , Gregory Greenwood , Nelson Kopyt , Sumit Kumar , Bradley Marder , Brad Rovin , Peter Santos , Meghan Sise , Yusuke Suzuki , James Tumlin JB is a consultant and Advisory Board/Study Steering Committee member for EMD Serono. JT is a consultant to and received grant funding from EMD Serono. YS is a consultant to Merck KGaA, Darmstadt, Germany, EMD Serono, Kyowa Kirin Ltd., and Novartis Pharmaceuticals, received research funding from EMD Serono, and has conducted collaborative studies with Visterra Inc., Moderna Inc., Retrophin Inc. and, Chinook Therapeutics. AK, AA and KP are employed by EMD Serono. HJ is employed by the healthcare business of Merck KGaA, Darmstadt, Germany; and HG is employed by Merck KGaA, Darmstadt, Germany. GA is a consultant to EMD Serono and conducted research studies with EMD Serono, Calliditas, and Retrophin Inc.


The cells were assembled with α-MoO3@CFC as the cathode and showed an outstanding Zn2+ storage capacity of 200.8 mAh g−1 at 200 mA g−1 current density. The capacity retention rate was 92.4 % after 100 cycles, along with an excellent cycling performance of 109.8 mAh g−1 following 500 cycles at 1000 mA g−1 current density. Subsequently, it was shown that CFC-loaded α-MoO3 cathode material possessed significantly improved electrochemical performance when compared to a cell constructed from commercial MoO3 using conventional slurry-based electrode methods. This work presents a novel yet simple method for preparing highly loaded and binder-free cathodic materials for aqueous ZIBs. The results suggest that the highly loaded cathode material with a high charge density may be potentially employed for future flexible device assembly and applications.Aqueous zinc-ion batteries (ZIBS) are becoming more popular as the use of energy storage devices grows, owing to advantages such as safety and an abundant zinc supply. In this study, molybdenum powder was loaded directly on carbon fiber cloth (CFC) via multi-arc ion plating to obtain Mo@CFC, which was then oxidatively heated in a muffle furnace for 20 min at 600 °C to produce high mass loading α-MoO It is still difficult to meet the demand for higher mass loading to achieve superior energy density and high capacity [Lithium-ion batteries have been widely used in consumer gadgets and automobiles as a result of their excellent energy density. However, the inherent disadvantages, including lack of safety, increased cost, and environmental concerns have limited their applications in grid-scale energy storage ,2,3. Aqud safety ,5. To dad safety ,7,8, vand safety ,10,11,12d safety ,14,15 hacapacity ,17. Thatcapacity ,19,20. C3) is theoretically promising as a high mass loading electrode material for high-performance ZIBs because of its unique layered structure and remarkable thermodynamic stability [3@CFC cathode with high mass loading and no binder via multi-arc ion plating and simple heat treatment is presented. This approach allows for a significant quantity of active material (about 12–15 mg cm−2) with a high binding strength to be placed on the CFC without the use of a conductive agent or binder, thereby improving the problems of active material shedding and the deterioration of battery performance. As a result, the prepared α-MoO3@CFC demonstrated an areal capacity of 2.61 mAh cm−2 at 200 mA g−1 current, and a higher energy density of 200.8 Wh kg−1 . This work may not only increase the ZIB capacity, but also facilitate the practical use of flexible wearable devices.In this regard, several studies have used the in situ growth of active materials on flexible substrates to create cathode materials for ZIBs. For example, a series of manganese-based and vanadium-based cathode materials with a high mass loading using different synthetic methods  have been studied ,22,23,24tability ,26. To mtability ,28. To mtability ,30. In t3@CFC. First, the molybdenum target  was placed in the multi-arc ion coating machine  along with the CFC, both of which had been sonicated with deionized water, acetone, and anhydrous ethanol. Following that, electrodeposition was initiated for 3 h, and the Mo@CFC was removed when the furnace temperature decreased to room temperature. The mass loading of Mo@CFC was about 8–10 mg cm−2. The mass ratio of Mo and CFC (about 11–13 mg cm−2) was about 0.62–0.91. Finally, Mo@CFC was oxidatively heated for 20 min in a muffle furnace at 600 °C to produce α-MoO3@CFC.A two-step procedure was used to fabricate the α-MoOCharacterization of the specimens was conducted using an X-ray diffractometer  and Cu Kα rays (λ = 1.54178Å) in the 3° to 80° range, and the morphology and structure were examined in detail using a scanning electron microscope (Nova Nano SEM 230) and a high-resolution transmission electron microscope .3@CFC was cut into small square pieces of 1 cm × 1 cm for the cathode, with active material mass loading of about 12–15 mg cm−2. A glass microfiber filter (GFF Whatman) was employed as a separator, and the electrolyte was a 2 M ZnSO4 solution. They were then assembled layer by layer and sealed with a fully automated sealer (MSK-110D) before being kept at room temperature for 24 h for electrochemical testing.The batteries utilized in this work for testing electrochemical performance were all coin cells, type CR2016, and all of the constructed ones were run in air. The anode was made from a zinc foil with a 12 mm diameter, whereas the manufactured α-MoO3 cells was fabricated by coating a slurry of commercial MoO3 powder , polyvinylidene difluoride (PVDF), and acetylene black, with an 8:1:1 mass ratio on the stainless-steel mesh, which was cut in a 12 mm diameter. The cathode, electrolyte, separator, and assembly procedures are the same as described above. Moreover, for the battery testing equipment, galvanostatic charge–discharge measurements (LAND CT2001A) were made between 0.4 and 1.4 V. Cyclic voltammetry (CV) was carried out at a scan rate of 1 mV s−1 using an electrochemical workstation . To conduct electrochemical impedance spectroscopy (EIS), a ZAHNER-IM6ex electrochemical workstation  was employed.The cathode for the commercial MoO3@CFC. Mo@CFC is directly produced by multi-arc ion plating. As a molybdenum source, six pure molybdenum targets (99.99% purity) are evenly distributed on both sides of the vacuum chamber, whereas the treated CFC is fixed as a carrier on a rotating rack located at the upper end of the vacuum chamber, allowing molybdenum to be uniformly plated on. The Mo@CFC is then placed within a quartz crucible and heated for 20 min at 600 °C, allowing Mo@CFC to fully oxidize to α-MoO3@CFC.3@CFC in 3 evenly covers the surface of the black pure CFC following 20 min of heating at 600 °C, forming α-MoO3@CFC with good flexibility.As depicted by the Mo@CFC and α-MoO3 are indexed well to orthorhombic α-MoO3 (JCPDS No.05-0508), with high crystallinity. In contrast, there is a diffraction peak attributed to Mo4O11 (JCPDS No. 05-0337), indicative of the existence of low-valence-state Mo after the oxidation treatment. The EDS result . With the increase in heating time, the oxidation of Mo is more complete. At 60 min, the characteristic peak of Mo4O11 disappears. However, with the extension of time, the CFC was destroyed by oxidation, and the mechanical properties of the samples weakened. Therefore, 20 min was determined as being the optimal preparation condition.The XRD patterns of α-MoO3@CFC are shown in 3@CFC with a one-dimensional rod-like shape are obtained, as presented in 3 is shown in 3. Moreover, the rod-like structure demonstrates the monocrystalline structure, as indicated by the SAED pattern , with an excellent energy density of 200.8 Wh kg−1.The electrochemical performances of ZIBs containing α-MoOnto MoO3 . A compa3@CFC-containing cathode at the 1st, 2nd, 5th, 50th, and 100th cycles, charged and discharged at 200 mA g−1. The initial discharge and charge capacities were equal to 409.3 and 288 mA h g−1, respectively. These values correspond to a Coulombic efficiency of 70.4%. The series of charge/discharge plateaus agree well with the CV curves shown in 3@CFC is significantly improved in comparison to that of commercial MoO3, as is evident in −1 at current densities ranging from 100 mA g−1 to 2000 mA g−1, respectively. The specific capacity increased to 308.2 mAh g−1 when the current density was returned to 100 mA g−1, and it remained steady in future cycles. The long-term cycling performance of α-MoO3@CFC at a current density of 1000 mA g−1 is shown in 3@CFC remained at 109.8 mAh g−1 following 500 cycles, whereas the commercial MoO3 had poor performance, with a rapid decline of the capacity. In addition, the ex situ SEM images of α-MoO3@CFC and commercial MoO3 after 20 cycles at a current density of 1000 mA g−1 are shown in 3@CFC is better.3@CFC electrode, electrochemical impedance spectroscopy (EIS) measurements are shown in 3@CFC and commercial MoO3 consist of semicircles at high frequencies and a straight line at lower frequencies. These regions correspond to the resistance of the electrode surface (Rf) and a charge-transfer resistance (Rct), respectively [3@CFC and commercial MoO3 electrodes before cycling showed that Rct values of the cathodes based on the α-MoO3@CFC and commercial MoO3 materials are equal to 43.73 and 82.09 Ω, respectively. These results confirmed that α-MoO3@CFC possessed better conductivity, as well as stronger ion and electron migration ability, than commercial MoO3. From a comparison of this work with some reported studies (high mass loading), in some respects, α-MoO3@CFC-based electrodes for ZIBs have possible application prospects  and binder-free cathode material, α-MoO3@CFC, prepared using multi-arc ion plating and simple heating. When operated at a current density of 200 mA g−1, the α-MoO3@CFC electrode has an electrochemical specific capacity of 200.8 mAh g−1 after 100 cycles. After 500 cycles at a current density of 1000 mA g−1, it still has a capacity of 109.8 mAh g−1. The exceptional mechanical characteristics and high areal capacity of 2.61 mAh cm−2 with an excellent energy density of 200.8 Wh kg−1 reveal considerable promise for flexible wearable and grid energy storage applications.In summary, ZIBs are fabricated with a high mass loading (12–15 mg cm


Proteins of the secretory pathway undergo glycosylation in the endoplasmic reticulum (ER) and the Golgi apparatus. Altered protein glycosylation can manifest in serious, sometimes fatal malfunctions. We recently showed that mutations in GDP-mannose pyrophosphorylase A (GMPPA) can cause a syndrome characterized by alacrima, achalasia, mental retardation, and myopathic alterations (AAMR syndrome). GMPPA acts as a feedback inhibitor of GDP-mannose pyrophosphorylase B (GMPPB), which provides GDP-mannose as a substrate for protein glycosylation. Loss of GMPPA thus enhances the incorporation of mannose into glycochains of various proteins, including α-dystroglycan (α-DG), a protein that links the extracellular matrix with the cytoskeleton. Here, we further characterized the consequences of loss of GMPPA for the secretory pathway. This includes a fragmentation of the Golgi apparatus, which comes along with a regulation of the abundance of several ER- and Golgi-resident proteins. We further show that the activity of the Golgi-associated endoprotease furin is reduced. Moreover, the fraction of α-DG, which is retained in the ER, is increased. Notably, WT cells cultured at a high mannose concentration display similar changes with increased retention of α-DG, altered structure of the Golgi apparatus, and a decrease in furin activity. In summary, our data underline the importance of a balanced mannose homeostasis for the secretory pathway. Glycosylation is the most common post-translational modification of proteins and lipids. It is relevant for the majority of plasma membranes and secreted proteins affecting their stability and conformation ,2,3. ThuGlycosylation starts in the cytoplasm with the generation of glycosyl donors, the activated form of sugars or sugar derivatives. This activation requires specific enzymes, which produce nucleotide-diphosphate-sugars using nucleotide-triphosphate as a substrate ,6. ActivAbnormal glycosylation of proteins can induce ER- and Golgi-stress and can finally manifest in serious, sometimes fatal malfunctions of different organ systems such as the brain and muscle . GMPPB-aMutations in proteins necessary for the structure of the ER and the Golgi apparatus can also lead to the aberrant glycosylation of proteins. Prominent examples are mutations in components of the conserved oligomeric complex (COG), which is a hetero-octameric peripheral membrane protein complex required for retrograde vesicular transport and glycoconjugate biosynthesis within the Golgi apparatus . MutatioAll animal experiments were approved by the Thüringer Landesamt für Lebensmittelsicherheit und Verbraucherschutz (TLLV) in Germany . Experiments were performed on WT and GMPPA KO litter mates on a C57BL/6 background in the 4th generation. GMPPA KO mice were generated as described previously . Mice weMice were sacrificed and organs removed. Muscles  were fixed in 4% paraformaldehyde (PFA) and washed with 1× phosphate buffered saline (PBS). After incubation in PBS with 0.25% Triton-X-100  overnight, fiber bundles were dissected. After blocking in 5% normal goat serum (NGS), fiber bundles were incubated with the following primary antibodies: rabbit anti-TGN38 1:250 , rabbit anti-GLG1 1:250 , and mouse anti-GM130 1:250  overnight. After washing with 1xPBS, fiber bundles were incubated with the corresponding secondary antibodies . Nuclei were stained with DAPI . Fiber bundles were washed with 1xPBS and mounted with Fluoromount-G . Images were taken with a Zeiss LSM880 Airyscan confocal microscope  with the Z-stack module and a 63× objective. Images were further analyzed with ImageJ. Colocalization analysis was performed with the Coloc2 module in ImageJ. Manderson overlap coefficients were used to quantify colocalization. Particle numbers were quantified in ImageJ using binary pictures and the plugin for particle analysis. To facilitate and accelerate image analysis, the ComDet v.0.5.5 plugin in ImageJ was used for colocalization analysis and particle number measurement. Since both approaches showed similar results, the ComDet plugin was used for the following analyses.Brains were cryo-sectioned into 8 µm thick sections. Immunofluorescence (IF) stainings were performed in Shandon chambers . Sections were fixed with 4% PFA and rinsed in phosphate-buffered saline (PBS). An amount of 0.25% Triton-X in 1xPBS was used to permeabilize cells. Blocking with 5% NGS was followed by incubations with primary and secondary antibodies as indicated above and mounted with Fluoromount-G . Images were taken with a Zeiss LSM880 Airyscan confocal microscope with the Z-stack module and a 63× objective. Images were further analyzed with ImageJ. Colocalization and particle numbers were analyzed using the ComDet v.0.5.5 plugin in ImageJ.MEFs were maintained in DMEM  and supplemented with 10% fetal bovine serum , penicillin (100 UI/mL), and streptomycin (100 mg/mL) .For α-DG-GFP localization experiments, cells were plated on coverslips. Twenty-four hours later, cells were transfected with 1 µg plasmid DNA/well encoding α-DG-GFP with lipofectamine 2000 reagent  according to the instructions of the manufacturer. Twenty-four hours post-transfection, cells were fixed with 4% PFA at room temperature, permeabilized with 0.25% Triton-X in 1xPBS, and blocked with 5% NGS. Immunofluorescence studies were performed as described above and sections were mounted with Fluoromount-G . For IF stainings of untransfected cells, cells were plated on coverslips for 2 days and then fixed with 4% PFA, permeabilized with 0.25% Triton-X in 1xPBS, and blocked with 5% NGS, and immunocytochemistry stainings were performed as described above. Images were taken with a Zeiss LSM880 Airyscan confocal microscope with the Z-stack module and a 63× objective.For IF experiments with supplementation of glucose or mannose, cells were plated in imaging plates . After 6 h, cells for α-DG-GFP localization experiments were transfected with 1 µg plasmid DNA/well encoding α-DG-GFP with lipofectamine reagent 2000  and medium was changed for all cells. Medium was supplemented with either 4.5 g/L glucose or with 4.5 g/L mannose. After 2 days, MEFs were fixed with 4% PFA at room temperature, permeabilized with 0.25% Triton-X in 1xPBS, and blocked with 5% NGS, and immunocytochemistry was performed as described above. The following primary antibodies were used: rabbit anti-TGN38 1:250 , rabbit anti-GLG1 1:250 , mouse anti-GM130 1:250 , mouse anti-PDI 1:500 , mouse anti-calnexin 1:500 , sheep anti-CLIMP63 1:500 , and rabbit anti-RTN4 1:500 . After washing, the appropriate secondary antibodies were used . After another washing round, cells were stained with DAPI , washed, and kept in 1XPBS  for image acquisition. Image acquisition was performed with a confocal high-content imaging microscope ImageXpress HT.ai  and a 40× objective. Images were further analyzed with ImageJ. Colocalization and particle numbers were analyzed using the ComDet v.0.5.5 plugin as well as the Coloc2 module in ImageJ see .g to remove nuclei and insoluble debris. The supernatant was stored at −80 °C. For protein concentration measurement, the Pierce BCA kit  was used according to the manufacturer’s instructions. Proteins were denatured at 90 °C for 5 min in laemmli sample buffer. After separation via SDS-polyacrylamide gel electrophoresis, proteins were transferred onto PVDF membranes . Membranes were blocked in 2% bovine serum albumin  in tris-buffered saline (TBS) buffer  supplemented with 0.1% Tween-20  for 1 h at room temperature. Membranes were incubated with primary antibodies at appropriate dilutions overnight at 4 °C: rabbit anti-GAPDH 1:1000 , rabbit GMPPA 1:500 , mouse anti-PDI 1:500 , mouse anti-pan cadherin 1:500 , sheep anti-dystroglycan 1:1000 , biotinylated lens culinaris lectin (LCH) 1:300 , and biotinylated concanavalin A lectin (Con A) 1:300 . Primary antibodies were detected with a horseradish peroxidase-conjugated secondary anti-rabbit antibody , anti-mouse antibody , anti-sheep antibody , and Streptavidin antibody , and the Super Signal Western Blot Enhancer Kit . The quantification of bands was done with ImageJ. All blots were repeated at least once. Coomassie blue staining of PVDF membranes was performed after protein detection. PVDF membranes were fixed for 3 min , stained in Coomassie blue solution , 45% EtOH (10% acetic acid) for 5 min, destained , rinsed in distilled H2O, and imaged [MEFs were cultured in DMEM  as indicated above. Cells were harvested with RIPA buffer. After, sonication homogenates were centrifuged at 16,900× d imaged .Supernatant was taken from confluent cells and frozen until further use. Sugar concentrations in the supernatant were measured with the D-mannose, D-fructose, D-glucose kit following the manufacturer’s instructions .g and stored at −80 °C. Cultured MEFs were harvested, washed with 1XPBS, and centrifuged at 1000× g for 5 min. Cell pellets were stored at −80 °C until further use. For measuring furin activity, brain tissue and cells were homogenized in 1XPBS (supplemented with protease inhibitors) and centrifuged  for 10 min at 4 °C and 10,000× g. Furin activity was measured in the supernatant according to the manufacturer´s instructions . Blood/serum samples were used directly.Mice were sacrificed and brain tissue was immediately frozen in liquid nitrogen and stored at −80 °C until further use. Blood was taken from unfasted mice and incubated on ice for 15 min. Samples were centrifuged for 10 min at 4 °C and 4000× g for 10 min. Supernatants were dried by speed vacuum, resuspended in 50 µL of methanol 50%, and analyzed by LC/MS, as described previously [MEFs were cultured after supplementation of either 4.5 g/L glucose or 4.5 g/L mannose for 10, 30 min, 4 h, or 20 h. For sample preparation, media were removed, cells were washed with cold water, quickly frozen in liquid nitrogen, and 250 µL of methanol/chloroform (9:1) was added to the plate. Cells were scraped from culture plates and centrifuged at 16,900× eviously .v/v) TritonX-100, complete protease inhibitor, and complete phosphatase inhibitor . Glycoproteins were eluted with 200 mM glycine buffer pH 2.5. After elution, 1 M Tris buffer pH 10.4 was added and samples were stored at −20 °C.For glycoprotein enrichment, 2 mg total protein from total brain or skeletal muscle lysates were incubated with concanavalin A (Con A) coupled agarose beads at 4 °C overnight, and washed with lysis buffer . Pellets were washed twice with 300 µL ice-cold 80% (v/v) acetone in water, then centrifuged at 20,800× g at 4 °C for 10 min. After removing the acetone, pellets were air-dried before addition of 25 µL of digestion buffer . Samples were resuspended with sonication as explained above; then, LysC  was added at a 1:100 (w/w) enzyme:protein ratio and digestion proceeded for 4 h at 37 °C under shaking . Samples were then diluted 1:1 with MilliQ water, and trypsin  was added at a 1:100 (w/w) enzyme:protein ratio. Samples were further digested overnight at 37 °C under shaking (650 rpm). The day after, digests were acidified by the addition of TFA to a final concentration of 10% (v/v), heated at 37 °C, and then desalted with Waters Oasis®® HLB µElution Plate 30 µm  under a soft vacuum following the manufacturer´s instruction. Briefly, the columns were conditioned with 3 × 100 µL solvent B (80% (v/v) acetonitrile; 0.05% (v/v) formic acid) and equilibrated with 3 × 100 µL solvent A (0.05% (v/v) formic acid in Milli-Q water). The samples were loaded, washed with 3 × 100 µL solvent A, and then eluted into 0.2 mL PCR tubes with 50 µL solvent B. The eluates were dried down using a speed vacuum centrifuge . Dried samples were stored at −20 °C until analysis.For proteomics analysis, samples were sonicated  for 10 cycles (30 s ON/60 s OFF) at high setting, at 20 °C, followed by boiling at 95 °C for 5 min. Reduction was followed by alkylation with iodoacetamide  for 30 min at room temperature in the dark. Protein amounts were estimated following an SDS-PAGE gel of 10 µL of each sample against an in-house cell lysate of known quantity. An amount of 30 µg of each sample was taken along for digestion. Proteins were precipitated overnight at −20 °C after the addition of 8× volume of ice-cold acetone. The following day, the samples were centrifuged at 20,800× m/z were acquired in profile mode in the Orbitrap with a resolution of 120,000 FWHM. The filling time was set at a maximum of 20 ms with an AGC target of 5 × 105 ions. DIA scans were acquired with 34 mass window segments of differing widths across the MS1 mass range. The HCD collision energy was set to 30%. MS/MS scan resolution in the Orbitrap was set to 30,000 FWHM with a fixed first mass of 200 m/z after accumulation of 1× 106 ions or after filling time of 70ms (whichever occurred first). Data were acquired in profile mode. For data acquisition and processing, Tune version 2.1 and Xcalibur 4.1 were employed.For data acquisition, approximatively 1 μg of reconstituted proteins were separated using a nanoAcquity UPLC  that was coupled online to the MS. Peptide mixtures were separated in trap/elute mode, using a trapping  and an analytical column . The outlet of the analytical column was coupled directly to an Orbitrap Fusion Lumos mass spectrometer  using the Proxeon nanospray source. Solvent A was water, 0.1% formic acid, and solvent B was acetonitrile, 0.1% formic acid. The samples were loaded with a constant flow of solvent A at 5 μL/min onto the trapping column. Trapping time was 6 min. Peptides were eluted via the analytical column with a constant flow of 300 nL/min. During the elution step, the percentage of solvent B increased in a nonlinear fashion from 0% to 40% in 90 min. Total runtime was 120 min, including cleanup and column re-equilibration. The peptides were introduced into the mass spectrometer via a Pico-Tip Emitter 360 µm OD × 20 µm ID; 10 µm tip (New Objective) and a spray voltage of 2.2 kV was applied. The capillary temperature was set at 300 °C. The RF lens was set to 30%. Full scan MS spectra with mass range 350–1650 DIA data were then uploaded and searched against the spectral library in Spectronaut . Relative quantification was performed in the software for each pairwise comparison using the replicates from each condition. The data (candidate table) and data reports were then exported as tables and further data analysis and visualization were performed with R-studio (version 0.99.902) using in-house pipelines and scripts. Data were then filtered for proteins that are known to be ER- and Golgi-resident and thus were assigned to the compartments “endoplasmic reticulum” and/or “Golgi”. These proteins are presented in The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE  partner g for 10 min at 4 °C. Pellets (nuclei fraction) were lysed in tris-buffered saline (TBS) supplemented with 0.5% SDS and sonicated. Supernatants were supplemented with NP40, TritonX-100, and digitonin until reaching a final concentration of 1% and then passed through injection needles of different sizes . Samples were centrifuged at 16,000× g at 4 °C for 1 h. Pellets (crude outer membrane fraction) were lysed in TBS + 0.5% SDS and sonicated. Supernatants were considered as cytoplasmic organelle fractions containing ER and Golgi. All fractions were supplemented with laemmli sample buffer and heated at 90 °C for 5 min. Then, samples were separated and immunoblotted as described above.MEFs were cultured in DMEM  as indicated above. Cells were seeded and transfected with 1 µg plasmid DNA/well encoding α-DG-GFP in 10 cm dishes with lipofectamine 3000 reagent  according to the instructions of the manufacturer. Medium was supplemented with either 4.5 g/L glucose or with 4.5 g/L mannose. After two days, cells were lysed in a homogenization buffer ) with an injection needle (21G) and a part was stored for total cell lysate fractions. The remaining lysates were centrifuged at 800× v/v). For contrast, 1% (w/v) uranyl acetate was added to 50% (v/v) acetone. The infiltration was carried out with epoxy resin. Processed biopsies were cut in 50 nm thick sections  and analyzed in a transmission electron microscope 900N  at 80 kV.Mice were perfused transcardially with 4% PFA and 2.5% glutaraldehyde in PBS. Skeletal muscle (gastrocnemius muscle) and the hippocampus were removed and postfixed. Afterwards, tissues were washed with 0.1 M cacodylate buffer pH 7.3 and postfixed in 1% osmium tetroxide/1% potassium ferrocyanide II in 0.1 M cacodylate buffer at 4 °C for 2 h. Tissues were dehydrated in the following accelerating acetone concentrations at 30%, 50%, 70%, 90%, 95%, and 100%  and individual data points with SEM are shown.For statistical analysis, raw data were analyzed for normal distribution with the Kolmogorov–Smirnov test or by graphical analysis using the Box-Plot and QQ-Plot in Graphpad prism 9. If appropriate, we either used 1-way ANOVA, 2-way ANOVA, or two-tailed Student’s t-tests. * indicates We used our recently established mouse model for AAMR syndrome  to studySince AAMR patients and mice show cognitive and motor impairment , we wondWe next assessed whether we could find similar alterations of the Golgi apparatus in mouse embryonic fibroblasts (MEFs) isolated from KO mice. In agreement with our in-vivo data, TGN38 positive particles and the overlap between GM130 and TGN38 positive compartments were decreased in KO MEFs K,L,O,P. Our findings were further strengthened by electron microscopy (EM) analysis of skeletal muscle and hippocampal neurons revealing enlarged secretory vesicles as well as enlarged Golgi cisternae, reduced Golgi stack numbers, and a fragmented trans-Golgi face Q–S.Taken together, disruption of GMPPA results in structural alterations of the Golgi apparatus.Because of the prominent structural alterations of the Golgi apparatus, we wondered whether the abundance of ER- and Golgi-resident glycoproteins might be changed in GMPPA KO mice. To this end, we enriched mannosylated proteins from skeletal muscle and brain protein lysates from WT and KO mice by Concanavalin A (Con A) pulldown. The efficiency of the enrichment was validated by immunoblot analysis showing a strong increase in the Con A lectin signal in enriched samples . SubsequWe also provide heatmaps for whole tissue lysates versus enriched samples for proteins found in the brain and skeletal muscle that were significantly altered between genotypes for the following processes: “Endoplasmic reticulum organization”, “Golgi organization”, “protease activity”, “protein maturation” and “protein processing”, and “vesicle mediated transport” B. NotablIn conclusion, Con A enrichment with subsequent mass spectrometry analysis shows that impaired glycosylation affects ER- and Golgi-resident proteins and a variety of cellular processes important for ER and Golgi structure, protein maturation, processing, and sorting. Because of the prominent alteration of the Golgi complex structure upon deletion of GMPPA, we wondered about possible consequences for its function. Besides glycosylation, protein cleavage is a major function of the Golgi apparatus and the TGN. Although furin also cycles between TGN and the cell surface, this endoprotease predominantly localizes to the TGN. Furin has a wide range of substrates, including e.g., pro-β-nerve growth factor (pro-β-NGF), pro-bone morphogenetic protein-4 (pro-BMP-4), and the insulin pro-receptor ,27. We mBecause of the prominent alteration of the Golgi complex structure upon deletion of GMPPA, we wondered about possible consequences for its function. Besides glycosylation, protein cleavage is a major function of the Golgi apparatus and the TGN. Although furin also cycles between TGN and the cell surface, this endoprotease predominantly localizes to the TGN. Furin has a wide range of substrates, including e.g., pro-β-nerve growth factor (pro-β-NGF), pro-bone morphogenetic protein-4 (pro-BMP-4), and the insulin pro-receptor ,27. We mIn our MS analysis, we identified several regulated ER-resident proteins in brain and skeletal muscle protein lysates of GMPPA KO mice, which raised the question of whether structural alterations of the Golgi apparatus in GMPPA KO mice come along with alterations of ER structure. To this end, we stained WT and GMPPA KO fibroblasts for phosphodiesterase (PDI), a luminal ER protein , and calTo get an idea about the trafficking of mannosylated proteins from the ER to the Golgi apparatus and because GMPPA KO mice show a reduced abundance of α-DG in the sarcolemma of skeletal muscle fibers , we tranWe recently showed that serum mannose concentrations are strongly increased in GMPPA KO mice , which iWe then overexpressed α-DG-GFP in MEFs and co-stained for the ER marker PDI or the Golgi marker GLG1. Notably, the overlap between α-DG-GFP with PDI was increased in MEFs cultured with mannose, while the overlap between α-DG-GFP with GLG1 was decreased. This suggests that the processing of α-DG-GFP in the ER is hampered by high mannose levels A–D. We aMutations in enzymes of the glycosylation machinery, which result in protein hypoglycosylation, can entail consequences for many cellular functions including protein processing in the ER and Golgi apparatus . WhetherUsually, the Golgi apparatus is composed of a series of flat, parallel, interconnected cisternae organized around the microtubule-organizing center in the perinuclear region. The Golgi apparatus is a highly dynamic structure, which can reversibly disassemble during mitosis, when the Golgi complex forms clusters of vesicles that disperse throughout the cytoplasm ,34. Our To obtain further clues about the role of the fragmentation of the TGN in GMPPA KO mice, we enriched mannosylated proteins, which were then identified and quantified by mass spectrometry. Notably, several ER- and Golgi-resident proteins important for protein folding and trafficking, glycosylation, and protein phosphorylation are regulated in GMPPA KO samples. Whole tissue lysates did not show a strong up- or down-regulation of proteins, while Con A enriched proteins showed robust alterations. Thus, these alterations rather reflect changes in the efficiency of proteins to be pulled down due to changes in their glycan chains or their interaction partners.Notably, the phosphofurin acidic cluster sorting protein1 (PACS1) was decreased in GMPPA KO tissues. PACS1, which is not glycosylated itself, plays a major role for the targeting of furin to the TGN ,45 and mDAG1) and are generated by cleavage of a common precursor in the endoplasmic reticulum [Because of these findings, we measured furin activity in serum as well as brain and MEF lysates from WT and GMPPA KO mice. Notably, its activity was decreased in GMPPA KO samples. This can have important consequences for the processing of its substrates, including α-DG. Alpha-DG is a highly glycosylated extracellular peripheral membrane protein noncovalently attached to the transmembrane protein β-DG. Both proteins are encoded by a single gene (eticulum . In the eticulum . α-DG bieticulum ,52.To assess the trafficking of a strongly mannosylated protein along the secretory pathway, we transfected WT and GMPPA KO MEFs with α-DG-GFP. Suggesting that the trafficking of α-DG is impaired, we found an increased retention of α-DG-GFP in the ER in the absence of GMPPA. Possibly, the altered structure of the Golgi apparatus impairs the trafficking of α-DG within the Golgi apparatus. Alternatively, the folding of α-DG-GFP may be impaired because of irregular glycosylation. In any case, this retention can partially explain the decreased abundance of α-DG in skeletal muscle fibers of AAMR patients and thus contribute to the pathogenesis of the myopathy associated with this disease. Since most glycoproteins are secreted and therefore undergo processing while trafficking through the ER and the Golgi apparatus , it is lWe recently showed that serum mannose concentrations are strongly increased in GMPPA KO mice, which was also evident in the supernatant of cultured MEFs isolated from GMPPA KO mice. Increased mannose levels in the supernatant may reflect increased release from glycans and/or increased generation of mannose from glucose. Although only ~2% of mannose entering the cell is used for glycosylation ,55, the Since we observed that dietary mannose restriction could at least in part prevent hypermannosylation and disease progression in GMPPA KO mice, we wondered whether mannose supplementation in WT MEFs could induce alterations as observed in GMPPA KO mice. Indeed, mannose induced a similar reorganization of the Golgi apparatus and the TGN in WT cells. Moreover, it decreased furin activity and promoted the retention of α-DG in the ER. It has been reported that external mannose is directly used for glycoprotein synthesis and our findings of elevated GDP-mannose levels in cells treated with mannose support this hypothesis ,55,56. WIt is tempting to speculate that dietary mannose supplementation may also have detrimental effects in-vivo. To our knowledge, however, there are no reports addressing mannose intake on cell metabolism.


The widespread environmental contaminant di-n-butyl phthalate (DBP) has been linked with reduced testosterone levels and adverse reproductive health outcomes in men. However, the underlying mechanisms of these anti-androgenic effects and the potential effects on other classes of steroid hormones remain to be elucidated. Here, we conducted mechanistic studies in human adrenocortical H295R cells exposed to 1–500 µM of DBP or its metabolite, mono-n-butyl phthalate (MBP), for 48 h. Quantification of steroid hormones in the cell medium by liquid chromatography-mass spectrometry revealed that both phthalates significantly decreased testosterone, androstenedione, corticosterone, and progesterone levels, in particular after dibutyryl-cyclic-AMP stimulation of steroidogenesis. Western blot analysis of key steroidogenic proteins showed that DBP induced a dose-dependent decrease of CYP11A1 and HSD3β2 levels, while MBP only significantly decreased CYP17A1 levels, indicating that the compounds affect early steps of the steroidogenesis differently. Both DBP and MBP exposure also lead to a dose-related decrease in HSD17β3, the enzyme which catalyzes the final step in the testosterone biosynthesis pathway, although these effects were not statistically significant. Interestingly, DBP increased the cortisol concentration, which may be due to the non-significant CYP11B1 increase in DBP-exposed cells. In contrast, MBP decreased cortisol concentration. Moreover, the analysis of superoxide generation and quantification of the protein oxidation marker nitrotyrosine demonstrated that DBP induced oxidative stress in H295R cells while MBP reduced protein nitrotyrosine levels. These findings confirm the anti-androgenic effects of DBP and MBP and reveal several differences in their toxicological mechanisms, with possible implications for future research on phthalate toxicity. Phthalates are synthetic chemicals found in numerous commercial products and materials, mainly as plasticizers . PhthalaWhile previous studies investigate the mechanism behind these anti-androgenic effects in vivo in mouse models  and in vThe H295R cells are a suitable model system to study the effects of environmental contaminants on steroidogenesis, including underlying mechanisms. These cells originate from the parent NCI-H295 cell line, which was established from adrenocortical carcinoma cells . H295R cThe aim of the current study was to examine and compare how DBP and its main metabolite, MBP, affect the steroid hormone biosynthesis, including key steroidogenic enzymes, and certain parameters of oxidative stress, using the human cell line H295R as an in vitro model. This study also examined if stimulating the steroidogenesis with dbcAMP affects how the cells respond to phthalate exposure.13C5, SIGMAFAST™ protease inhibitor tablets, 3--2,5-Diphenyltetrazolium Bromide (MTT), dimethyl sulfoxide (DMSO), and dbcAMP were all purchased from Sigma Aldrich . Dulbecco’s modified Eagle medium/HamF12 (DMEM/F12), FluoroBrite™ DMEM, Dulbecco’s Phosphate Buffered Saline (DPBS), and trypsin-EDTA were purchased from Gibco Life Technologies Europe . Insulin-Transferrin-Selenium (ITS+) premix and Nu-serum were purchased from Corning Discovery Labware, Inc. . Superblock™ blocking buffer was purchased from Thermo Scientific . Tween 20, NaCl and Tris-HCl, LC-MS grade water and methanol were purchased from Fisher . Ammonium fluoride was purchased from Honeywell Research Chemicals . Triton X-100 was purchased from VWR international . No-Stain™ Protein Labeling Reagent (Cat.no. A44717), dihydroethidium (DHE), cumene hydroperoxide, and Hoechst 33342 were purchased from Invitrogen, Life Technologies Corp. . TBS was purchased from Medicago AB . Sodium deoxycholate was purchased from Alfa Aesar ThermoFisher GmbH . Dry milk was purchased from Cell signaling Technology . The chemiluminescence ECL kit was purchased from Bio-Rad .DBP , MBP , analytical grade standards of testosterone, androstenedione, corticosterone, cortisol and progesterone, isotope-labeled testosterone-d3, corticosterone-d4 and progesterone-2 tissue culture flasks with 20 mL culture medium. The culture medium consisted of DMEM-F12 medium supplemented with 2.5% Nu-serum and 1% ITS+ premix. All incubations were done at 37 °C with 5% CO2.H295R cells were obtained from the American Type Culture Collection . The cells were cultured and maintained according to the OECD guideline 456. In brief, the cells were maintained as a monolayer in 75 cmCells were seeded in 96-well plates at a density of 50,000 cells/well in 200 µL culture medium. After 24 h incubation, the medium was replaced with new medium containing DBP or MBP added at concentrations of 0 , 1, 10, 100, or 500 µM. Forskolin (1 µM) and prochloraz (0.3 µM) were added as the positive and negative control, respectively, for the steroidogenesis assay. For each replicate, one plate was prepared with a standard culture medium and one plate with the addition of 0.1 mM dbcAMP to the medium to stimulate steroidogenesis. After 48 h exposure to the test compounds, the medium was collected and stored at −80 °C until analysis. The removed medium was then replaced with 100 µL/well of MTT (0.5 mg/mL) dissolved in DMEM-F12. After 45 min incubation at 37 °C, the MTT solution was removed, and the formazan generated was solubilized by adding 50 µL DMSO to each well and leaving the plate on a plate shaker at 500 rpm for 10 min. The absorbance of the dissolved formazan was measured at 570 nM using a SpectraMax i3 microplate reader .g for 15 min. The supernatant was filtered with a 96-well filter plate  by centrifugation at 3000× g for 10 min. The filtrate was then analyzed by liquid chromatography-mass spectrometry (LC-MS). Progesterone, corticosterone, androstenedione, and testosterone were quantified as previously described [13C5.The collected culture medium was thawed, diluted 1:2 with methanol and left on a plate shaker at 500 rpm for 20 min to precipitate proteins. The plate was then centrifuged at 2500× escribed , with thThe levels of nine key steroidogenic proteins were measured by western blot. Cells were cultured in 6-well plates at 500,000 cells/well. After attaching to the plate for 24 h, the cells were exposed to 10 and 100 µM DBP, 100 and 500 µM MBP, or solvent control (0.1% DMSO) dissolved in a culture medium with 0.1 mM dbcAMP. After 48 h exposure, the cells were washed with DPBS and trypsinized for 10 min. The detached cells were then collected in tubes and lysed with RIPA buffer . Protein concentration in the lysate was determined by the Lowry assay , and 20 2−) are highly reactive and have a short half-life, a shorter exposure time was selected based on pilot studies. Positive control was 1 h exposure to 25 µM cumene hydroperoxide. At the end of the exposure, the medium was changed to the staining solution, which consisted of 15 µM of the fluorogenic probe DHE that reacts strongly with ˙O2− and to a lesser extent with other ROS and NO, and 5 µg/ mL Hoechst 33342 dissolved in FluoroBrite DMEM, as normal DMEM-F12 has high autofluorescence that can interfere with the analysis. The cells were stained for 40 min, washed twice with DPBS, and then left in FluoroBrite medium for imaging. Images were acquired at 10× magnification from four sites per well using an ImageXpress Micro® Confocal High-Content Analysis System . ImageJ ver 1.53r  was used to count the number of DHE and Hoechst stained nuclei. Results are reported as the number of DHE-stained cells divided by the number of Hoechst-stained cells.Cells were seeded in black 96-well plates with a transparent bottom at a density of 25,000 cells/well, either in a normal culture medium or in a medium with 0.1 mM dbcAMP added, and cultured for 48 h. The cells were then exposed for 2 h to 1, 10, 100 or 500 µM MBP; 1, 10 or 100 µM DBP, or 0.1% DMSO (solvent control). Since reactive oxygen species (ROS) such as superoxide .Results are presented as mean ± standard deviation (SD) for each experiment. Differences compared to the control were analyzed by one-way analysis of variance (ANOVA) followed by Dunnet’s multiple comparison test for western blot results and by a one-sample p = 0.06) was observed at 500 µM DBP with 0.1 mM dbcAMP added to the cell medium, indicating that this concentration might have some cytotoxic effects. The same concentration without stimulation had less effect .The viability of the cells after 48 h phthalate exposure was determined by the MTT assay. No significant cell death was observed for any of the DBP A or MBP p = 0.12; MBP: 8.4% decrease, p = 0.005), but it was significant only for MBP (p = 0.0003) and 84% (p = 0.008) increase, respectively), while MBP induced a slight and non-significant decreasing trend  and testosterone  at 500 µM. While an overall decreasing trend could also be observed after MBP exposure, with a significant effect for testosterone  at 500 µM, a small but significant increase in androstenedione and testosterone levels (9% (p = 0.006) and 8% (p = 0.04) increase, respectively) were observed at 10 µM MBP , androstenedione , and testosterone  levels, while DBP exposure induced a significant decrease already at 100 µM for these hormones (17% (p = 0.04), 25% (p = 0.02) and 20% (p = 0.02) decrease, respectively; p = 0.04) and 56% (p = 0.02), respectively; p = 0.04; When the cells were exposed to the phthalates under stimulated conditions (0.1 mM dbcAMP), the trends were similar, but with overall larger effects for all the quantified hormones, except progesterone, where MBP still had no effect, but a significant decrease in hormone levels could now be observed at 500 µM DBP  and 46% (p = 0.004) decrease, respectively). The largest effect of MBP exposure was, in comparison, a less prominent decrease in testosterone at 500 µM MBP and 0.1 mM dbcAMP co-treatment .Both at basal conditions and when the steroidogenesis in the cells was stimulated with dbcAMP, DBP induced larger effects than MBP on all quantified hormones . After DTo examine the mechanisms behind the demonstrated changes in steroid hormone concentrations, the levels of nine key steroidogenic proteins were measured by western blot: steroidogenic acute regulator (StAR), cytochrome P450 (CYP) 11A1, hydroxysteroid dehydrogenase (HSD) 3β2, CYP17A1, CYP21A2, CYP11B2, CYP11B1, HSD11β2, and HSD17β3. Since the changes in hormone levels were mostly larger under stimulated conditions, these experiments were all conducted with 0.1 mM dbcAMP added to the medium. The highest DBP concentration included for this analysis was 100 µM, which was the lowest concentration that induced significantly altered steroid levels. Since MBP showed less potent effects on steroid levels, the 500 µM concentration was kept as the highest concentration for the western blot analysis of steroidogenic proteins.p = 0.02), and HSD3β2 (p = 0.04). A similar, but not statistically significant, decreasing trend could be observed for the levels of CYP17A1 (p = 0.45) and HSD17β3 (p = 0.28). DBP also induced a slight, but not significant, increase in CYP11B1 levels (p = 0.09). No changes or trends were observed for the remaining measured proteins (p = 0.02). A non-significant dose-related decreasing trend was observed for CYP11A1 (p = 0.23), HSD3β2 (p = 0.10), CYP11B2 (p = 0.20), HSD11β2 (p = 0.56) and HSD17β3 (p = 0.69). No trend was observed for StAR or CYP21A2 , which is produced by the pituitary. Previous studies have demonstrated that the H295R cells have a low response to ACTH and that forskolin or dbcAMP can be used instead to mimic this stimulation ,25. The StAR and CYP11B1 mRNA in the testis with less significant effects on the expression of the same genes in the adrenal gland [Epidemiological data has, in addition to the anti-androgenic effects, linked increased levels of phthalates in urine with increased concentrations of urinary cortisol levels . Interesal gland . Moreoveal gland . This hiOverall, the reduced testosterone levels observed in H295R cells after DBP or MBP exposure are consistent with our recent mouse study . The in To our knowledge, no previous studies have investigated oxidative stress in adrenal cells after DBP or MBP exposure. DBP exposure induced oxidative stress in the H295R cells, illustrated by a significant increase in superoxide generation, together with a small and non-significant increase in protein nitrotyrosine levels. This is in line with our mouse model, where a significant increase of the protein oxidation marker nitrotyrosine was observed one week after five weeks of oral exposure to DBP, even at the lower dose of 10 mg/kg/day . The difThis study confirms the anti-androgenic effects of DBP and MBP in human steroidogenically active cells, as the levels of two androgens, testosterone and androstenedione were reduced after exposure to the phthalates. While this study was done with adrenal cells, the compounds may likely induce similar effects in human Leydig cells, the main producer of testosterone in men , as the Although further studies are needed to fully elucidate the mechanism behind the observed effects on steroidogenesis, one possibility is that DBP/MBP interacts with different transcription factors to regulate the expression of steroidogenic enzymes. Activation of the cAMP/PKA pathway, for example, through ACTH stimulation, is the major signaling cascade regulating steroidogenesis , and it To conclude, this study provides a better mechanistic understanding of how DBP and its biologically active metabolite MBP affect steroid hormone biosynthesis in human cells. The findings revealed several differences in mechanism between these phthalates . The DBP


Actinidia valvata) is more tolerant to waterlogging than ‘Hayward’ (A. deliciosa) and is a potential resistant rootstock for kiwifruit production. Here, we focused on evaluating the performance of the waterlogging-sensitive kiwifruit scion cultivar ‘Zhongmi 2′ when grafted onto KR5 (referred to as ZM2/KR5) and Hayward (referred to as ZM2/HWD) rootstocks, respectively, under waterlogging stress. The results showed ‘Zhongmi 2′ performed much better when grafted onto KR5 than when grafted onto ‘Hayward’, exhibiting higher photosynthetic efficiency and reduced reactive oxygen species (ROS) damage. Furthermore, the roots of ZM2/KR5 plants showed greater root activity and energy supply, lower ROS damage, and more stable osmotic adjustment ability than the roots of ZM2/HWD plants under waterlogging stress. In addition, we detected the expression of six key genes involved in the kiwifruit waterlogging response mechanism, and these genes were remarkably induced in the ZM2/KR5 roots but not in the ZM2/HWD roots under waterlogging stress. Moreover, principal component analysis (PCA) further demonstrated the differences in the physiological responses of the ZM2/KR5 and ZM2/HWD plants under waterlogging stress. These results demonstrated that the KR5 rootstock can improve the waterlogging tolerance of grafted kiwi plants by regulating physiological and biochemical metabolism and molecular responses.Kiwifruit is commonly sensitive to waterlogging stress, and grafting onto a waterlogging-tolerant rootstock is an efficient strategy for enhancing the waterlogging tolerance of kiwifruit plants. KR5 ( Adverse environmental factors including salinity , alkalinCitrus sinensis × Poncirus trifoliata), the resistance of grafted plants to drought and heat stress combinations could be improved by modifying the scion antioxidant system [Plant tolerance to abiotic stress is a complex trait involving several environmental factors, and improving the tolerance of agricultural crops using modern breeding and biotechnological approaches has proven to be difficult . Graftint system . Moreovet system . Calogert system  also demActinidia spp.) is popular among consumers because of its rich vitamin content and unique taste [A. deliciosa as rootstocks, such as ‘Bruno’ and Hayward [A. deliciosa), ‘Qinmei’ (A. deliciosa), and some wild kiwifruit plants are widely used as rootstocks for kiwifruit production [A. chinensis and A. deliciosa are commonly considered to be sensitive to waterlogging stress [Kiwifruit  is more tolerant than ‘Hayward’ (A. deliciosa) to waterlogging stress [ADH1 (i1_LQ_K_c67155/f1p0/1459), ADH2 (i1_LQ_K_c38965/f1p0/1342), MnSOD1743 (i1_LQ_K_c14090/f1p1/1743), POD1591 (i1_HQ_K_c28263/f2p2/1591), ERF73 (i1_HQ_K_c88560/f4p0/1107), and ERF78 (i1_HQ_K_c68003/f14p0/1110) [A. deliciosa), a waterlogging-sensitive scion cultivar from the Zhengzhou Fruit Research Institute (ZFRI), Chinese Academy of Agricultural Sciences (CAAS) [Previously, our research demonstrated that the kiwifruit rootstock KR5  . In the s (CAAS) , onto KRp < 0.05), while the ZM2/KR5 plants presented stable root activity (Waterlogging damaged the growth of ‘Zhongmi 2′ scions grafted on ‘Hayward’ and KR5 rootstocks a. After activity c.p < 0.05). Although the Pn, Tr, Gs, and WUE of the ZM2/KR5 plant leaves were also inhibited under waterlogging stress, the reductions in these metrics were more pronounced and not significant in comparison to those of the control plants.As the ZM2/HWD plants died when exposed to being waterlogged for 8 d, the leaf net photosynthetic efficiency (Pn), transpiration rate (Tr), stomatal conductance (Gs), and water use efficiency (WUE) of the grafted plants were measured on days 0, 1, and 5 . The Pn,2−), hydrogen peroxide (H2O2), and malondialdehyde (MDA) contents continued to increase during the waterlogging treatment  activity, sucrose content, soluble sugar content, and proline content . ADH is ADH1 and ADH2 were significantly induced in the roots of the two grafted plants under waterlogging stress; however, the increase was greater in the ZM2/KR5 plants. Two antioxidant enzyme genes, MnSOD1743 and POD1591, were also induced significantly in the ZM2/KR5 roots, but no significant difference was observed in the ZM2/HWD roots. Similarly, ERF73 and ERF78, two ERF-VII members from A. valvata, were more strongly expressed under waterlogging stress in the ZM2/KR5 plants than in the sensitive ZM2/HWD plants.To further explore root molecular responses under waterlogging stress, we performed quantitative real-time PCR (qRT-PCR) to analyze the expression of key genes in the roots of the two scion–rootstock combinations . We foun2−, Leaf-H2O2, Leaf-MDA, Root-O2−, Root-H2O2 and Root-MDA content. Variances in the photosynthetic indices  were positively associated with PC2, which explained 22.60% of the total variance. Root activity, sucrose, soluble sugar, proline, and anaerobic fermentation enzyme ADH activity were negatively associated with PC1 and PC2. The short distance between the control groups showed that there was little difference between the two types of grafted plants before waterlogging, whereas after waterlogging treatment, the two types of plants could be separated based on PC1 or PC2. This indicates that rootstocks with different tolerances affected the response of grafted plants when exposed to waterlogging stress.PCA was performed to evaluate the overall effects of the two rootstocks with different waterlogging tolerances on the waterlogging tolerance of the scion–rootstock combinations . The firThe expansion of kiwifruit cultivation is restricted in waterlogged areas because of the low tolerance of this plant to waterlogging stress. When waterlogged, the root tips of plants were damaged initially because of oxygen deficiency, thereby affecting nutrient and water uptake, leading to plant wilting and even death in severe cases ,38. In tThe main pathway of gas exchange between plant leaves and the external environment is the stomata, and this process reflects the metabolic activities of plants , with stThe negative effects of waterlogging on roots mainly occur via (1) an energy crisis that causes anaerobic respiration, and (2) an imbalance between ROS accumulation and scavenging . At low When subjected to waterlogging stress, tolerant plants can change their intracellular water potential by rapidly accumulating osmotic regulatory substances, such as soluble sugar and proline, over a short period to deal with stress . We founADH genes that control ADH enzyme synthesis under waterlogging stress has been confirmed in many studies [ADH1 (i1_LQ_K_c67155/f1p0/1459) and ADH2 (i1_LQ_K_c38965/f1p0/1342) involved in anaerobic respiration under waterlogged conditions. We found that the expression of ADH1 and ADH2 in the ZM2/KR5 roots was higher than that in the ZM2/HWD roots. This was further verified by the higher ADH enzyme activity of the ZM2/KR5 roots. In addition, to maintain the balance between ROS accumulation and scavenging in adverse environments, plants have developed an antioxidant defense system comprising of SOD, POD, and CAT enzymes [MnSOD1743 (i1_LQ_K_c14090/f1p1/1743) and POD1591 (i1_HQ_K_c28263/f2p2/1591), was higher in the ZM2/KR5 roots than that in the ZM2/HWD roots. This finding further demonstrated the ROS scavenging ability of the ZM2/KR5 roots under waterlogging stress.An increase in the transcript abundance of the  studies ,47. Here enzymes ,49. BaseERF73 (i1_HQ_K_c88560/f4p0/1107) and ERF78 (i1_HQ_K_c68003/f14p0/1110), were remarkably induced in the ZM2/KR5 roots under waterlogging treatment, but they were almost absent in the roots of the ZM2/HWD plants. This is consistent with previous conclusions that indicated that ERF73 and ERF78 play crucial roles in the molecular-scale defense mechanism of plants under waterlogging stress [The ERF (Ethylene Responsive Factor) family plays an important role in the abiotic stress response, including drought , low temg stress .A. deliciosa, waterlogging sensitive) and KR5  were used as rootstocks, and the height of rootstock selected in this study was 15–20 cm, and the stem diameter was 0.6–0.8 cm. Then, A. deliciosa cultivar ‘Zhongmi’ 2 was grafted onto the two rootstocks, producing two scion–rootstock combinations Zhongmi 2/Hayward (ZM2/HWD) and Zhongmi 2/KR5 (ZM2/KR5). In total, thirty ZM2/HWD and thirty ZM2/KR5 potted plants were obtained through grafting.This study was carried out at the ZFRI, CAAS . To ensure the consistency of plant materials, two-year-old tissue culture plants of ‘Hayward’ .The survival rate of the two scion–rootstock combinations was determined manually after 8 d of waterlogging. Root activity was measured using the triphenyl tetrazolium chloride (TTC) method with some modifications, and expressed in mg/g/h . BrieflyThe net photosynthetic efficiency (Pn), transpiration rate (Tr), stomatal conductance (Gs), and water use efficiency (WUE) of six selected plants from each scion–rootstock combination were measured using a portable photosynthesis instrument CI-340  between 9:30 and 11:30 a.m. on days 0, 1, 5, and 8 of the waterlogging stress treatment. We marked the six selected grafted plants and performed continuous measurements during the experiment2− generation. Changes in the rate of O2− generation (nmol/g/min) were recorded using a spectrophotometer at 530 nm. H2O2 and MDA levels were determined according to the protocol of Hussain et al. [2O2 content was recorded using a spectrophotometer at 415 nm, and the results were expressed as µmol/g of fresh sample weight (FW). MDA content was measured using reactive substances of thiobarbituric acid (RSTBA), and recorded by subtracting at 600 nm from the absorbance value at 532 nm. The results were expressed as nmol/g of fresh sample weight.The method described by Huang et al.  was usedn et al. . H2O2 coADH (1 U) was defined as the amount of enzyme required to decompose 1 nmol of NADH per minute per mg protein . The suc®SYBR qPCR SuperMix Plus kit  using a LightCycler 480 II  on a 96-well plate. Each sample consisted of three technical replicates. The reaction mixture (20 μL) contained 10 μL of super mix, 0.8 μL of cDNA, 0.4 μL of each forward and reverse primers, and 8.4 μL of RNase-free water.Total RNA extraction from the grafted plant roots (control and treatment plants) was performed using a quick RNA isolation kit . RNA samples were stored at −80 °C until subsequent analyses. First strand cDNA was synthesized using a First Strand cDNA Synthesis Kit . Previous reported primers were used for qRT-PCR. qRT-PCR experiments were performed in a final volume of 20 µL with the NovoStartp ≤ 0.05). A heatmap was drawn using TBtools software v1.09861 , a toolkit developed for interactive analyses of big biological datasets [All experiments were performed in triplicate. Analysis of variance, mean comparisons, and data visualization were performed using OriginPro 2022 v 9.9.0.220 . Statistically significant differences were calculated using the LSD test (A. deliciosa) grafted onto waterlogging-tolerant rootstock KR5  performs better than the same cultivar grafted onto waterlogging-sensitive rootstock ‘Hayward’ (A. deliciosa) when exposed to waterlogging stress. Thus, as a rootstock, KR5 can maintain root activity, ensure energy supply, scavenge excessive ROS, and accumulate osmotic substances under waterlogging stress, thereby enhancing the tolerance of the scion to waterlogging stress. In addition, some key genes involved in the waterlogging tolerance mechanism increased remarkably in the ZM2/KR5 plants. PCA analysis further demonstrated the differences in the physiological responses of ZM2/HWD and ZM2/KR5 plants on their exposure to waterlogging stress. Based on our results, the use of more waterlogging-tolerant rootstocks could increase the environmental adaptation of kiwifruit scions, thereby improving performance under waterlogging stress. As such, the selection of stress-resistant rootstocks is important for kiwifruit production under adverse environmental conditions.This study demonstrated that scion cultivar ‘Zhongmi 2′ (