Body weight, serum parameters, histological analysis, and gene expression involved in lipid metabolism were quantified. Our results suggested that 100 μM of 3-HPP and 4-HPP inhibited lipid accumulation more significantly than parent RSV in an oleic acid-induced HepG2 cell line. Furthermore, 3-HPP, 4-HPP, and RSV effectively reduced liver weight and body weight, improved hepatic steatosis, and alleviated systemic inflammation in NAFLD mice. In addition, the results of quantitative real-time PCR showed that 3-HPP and 4-HPP altered the expression of cholesterol influx and efflux genes to a stronger extent than RSV. These results indicate that 3-HPP and 4-HPP are effective in regulating hepatic lipid metabolism.Resveratrol (RSV), a polyphenolic stilbene, has been widely studied for its protective effects against non-alcoholic fatty liver disease (NAFLD) by modulating intestinal microbiota. The microbial metabolites after RSV supplement would contribute to the bioeffects of RSV, while their impacts on NAFLD were unclear. Therefore, this study aimed to investigate the beneficial effects of the main microbial metabolites from RSV on lipid metabolism by combining in vitro and in vivo models. The mice were fed a high-fat diet and injected with RSV, 3-hydroxyphenyl propionic acid (3-HPP), and 4-HPP for 13 weeks ( Non-alcoholic fatty liver disease (NAFLD) is a chronic liver disease affecting 25% of adults and 3–10% of children globally . MoreoveResveratrol (RSV) is a polyphenolic stilbene, which is widely present in grapes, berries, and peanuts . PreviouGut microbiota, a virtual endocrine organ, can transform polyphenols into small molecular metabolites that increase the bioavailability of polyphenols . Recent Interestingly, except for resveratrol, procyanidins, epicatechin, and other flavanols can also be transformed into these phenolic acids by gut microbiota ,33,34. ITherefore, this work aimed to assess the efficacy of microbial phenolic acids after RSV supplementation on NAFLD using an oleic acid (OA)-induced cell model and an HFD-induced mice model. Our findings suggest that 3-HPP and 4-HPP can attenuate hepatic steatosis and systemic inflammation, and their effects were the same as those of RSV. Interestingly, 3-HPP and 4-HPP mainly influenced cholesterol transport, while RSV primarily affected FA oxidation and transport.RSV, 3-HBA, 3-HPP, 4-HBA, 4-HPA, and 4-HPP (purity > 95%) were purchased from Aladdin, Shanghai, China. The sodium salt of OA was obtained from J&K Scientific, Beijing, China. Phosphate buffered saline (PBS), polyethylene glycol 400, Cell Counting Kit-8 (CCK8), the BCA Protein Assay Kit, and Oil Red O (ORO) solution were obtained from Solarbio, Beijing, China.2O vapor pressure . When the cell confluence approached 70%, the cells could be passaged. Cultures between the 4th and 10th passages were used for different experiments.The human hepatocellular carcinoma cell line HepG2 was gifted by Professor Hongbo Hu . The cells were cultured in DMEM containing a 1% supplement of antibiotic solution  and 10% fetal bovine serum in an incubator with constant Hw/v) BSA solution to obtain the OA stock (10 mM/10% BSA). The stock was filtered and diluted with DMEM to obtain the OA working solution (1 mM OA/1% BSA). The NAFLD cell model was induced using the working solution, which was treated with HepG2 cells for 24 h. As a control, 1% BSA solution was used to treat cell cultures. HepG2 cells were then treated with RSV and phenolic acids and the OA working solution for 24 h. OA mother solution (100 mM) was prepared by dissolving sodium salt of OA powder in double distilled water at 70–75 °C. It was then combined with 15% . After 12–14 h, the cells were treated with various concentrations (25–200 μM) of RSV and phenolic acids in the absence and presence of the OA working solution for 24 h. The cells were then treated with 10% CCK8 reagent solution for 1 h in a dark environment. Following this, the absorbance was measured at 450 nm wavelength.HepG2 cells were seeded in 60 mm dishes. The cells were treated with 1% BSA solutions (C group), the OA working solution (OA group), the OA working solution with RSV (RSV group), and the OA working solution with phenolic acids (phenolic acids group) for 24 h. After these treatments, the cells were collected and then crushed with PBS by using an ultrasonic cell crusher . Then, the protein concentration of the crushed liquid was measured with the BCA Assay Kit to normalize the number of cells and the TG level in the cells was measured using the TG Assay Kit .v/v) ORO solution in the dark for 1 h. Lastly, the cells were sealed with pure water to observe and photograph under an inverted microscope  at 400× magnification. The results of ORO staining were semi-quantified using Image-Pro Plus 6.0.HepG2 cells were seeded in 35 mm dishes with slides. The cells were treated with 1% BSA solutions (C group), the OA working solution (OA group), the OA working solution with RSV (RSV group), and the OA working solution with phenolic acids (phenolic acids group) for 24 h. After these treatments, the cells were washed and fixed for 25–40 min. Following this, these were washed and stained with 60%  (No. AW70702202-4-1). A total of 30 4-week-old male C57BL/6J mice (16–18 g) were bought from Beijing Vital River Laboratory Animal Technology Co Ltd., China. After 7 days of adaptive feeding in a barrier environment , mice were kept in cages (three mice per cage) and classified into 5 groups with 6 mice in each group: the normal diet (ND), HFD, RSV, 3-HPP, and 4-HPP group.v/v) PEG solutions. The mice in the RSV, 3-HPP, and 4-HPP groups were intraperitoneally injected with the PEG solutions at a dose of 24 mg/kg BW twice a week. The mice of the ND and HFD groups were intraperitoneally injected with 20% PEG with saline.For 13 consecutive weeks, the ND group was supported with a control diet  and the other groups were fed HFD . The macronutrient composition of the two diets is shown in the 3 in size were fixed with 4% paraformaldehyde at a ratio of 10:1 for 24 h. The remaining samples were loaded into cryovials, placed in dry ice, and subsequently stored at −80 °C.On the 89th d, the mice were fasted for 16 h, weighed, and sacrificed. Following this, we collected their blood, white adipose tissue , and liver. The serum samples were acquired by centrifuging blood for 25 min at 3300 rpm at 4 °C. After weighing, a piece of epididymal fat and a piece of liver tissue that were approximately 1 cmw/v) glucose solution (2.0 g/kg BW). Blood samples were collected from the tail vein at several time points, and blood glucose levels were measured with glucometers . On the 82nd d, the mice were fasted for 16 h. Following this, the mice were intraperitoneally injected with 20%  were selected to assess the serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol (TC), TG, low-density lipoprotein cholesterol (LDL-c), HDL-c, and the hepatic TG and TC levels. The serum levels of insulin, interleukin-6 (IL-6), IL-8, and tumor necrosis factor-α (TNF-α) were measured using ELISA kits . Homeostatic model assessment for insulin resistance (HOMA-IR) was calculated according to a previous study . Fixed epididymal fat and liver were embedded with optimal cutting temperature compound and quickly frozen. When the specimen became white and hard, it was sectioned (8–10 μm) and dyed with hematoxylin and eosin (H&E) or ORO and hematoxylin. Staining results were visualized under an inverted microscope at 200× magnification and photographed using a digital camera. The results of H&E staining of liver tissues were scored on the basis of the NAFLD Activity Score (NAS) . Histologapdh expression and estimated with the 2−∆∆Ct method. Liver samples were pre-treated by liquid nitrogen milling and then total RNA was isolated. The concentration and purity of RNA were then assessed by detecting OD230, OD260, and OD280 using NanoDrop 2000 . High-purity RNA samples were converted into cDNA. Quantitative real-time polymerase chain reaction (qPCR) analysis was performed using the BioRad CFX96 System. The primer sequences  are listed in p < 0.05 was considered markedly different in accordance with Duncan’s test.Data are expressed as the means ± standard errors (SEMs). SPSS 20.0 was selected to compare statistically and GraphPad Prism 8 was used to show the results. All experiments were repeated 3 times in this work. One-way analysis of variance (ANOVA) was used to assess the significant difference among the 5 groups. The molecular structures of phenolic acids and RSV are shown in Five-week-old C57BL/6J mice were supported with an ND or HFD A. The miAfter 13 weeks, compared with ND, HFD significantly increased liver weight and liver index; however, 3-HPP, 4-HPP, and RSV supplementation reduced liver weight and liver index A,B. ThenGlucose homeostasis and inflammation have been reported to induce NAFLD . Comparecd36, fabp1, and slc27a5). Interestingly, only RSV supplementation downregulated the expression of cd36, illustrating that 3-HPP and 4-HPP had little influence on FA transport. Moreover, compared with ND, long-term HFD reduced the gene expressions of FA β oxidation . RSV, 3-HPP, and 4-HPP upregulated the expression of acadm, while only RSV upregulated the expression of cpt1a (abca1, abcg5, and abcg8, markers of cholesterol export, were downregulated in HFD mice. Whereas, the gene expression of cholesterol export, such as ldlr and scarb1, was not significantly influenced by HFD. RSV supplementation upregulated the expression of abca1 and abcg8, 3-HPP supplementation influenced the expression of abcg8 and scarb1, and 4-HPP administration upregulated the expression of abca1, abcg5, abcg8, and ldlr (The expression levels of genes in β oxidation of FA, FA transport, and cholesterol transport were assessed. In of cpt1a A. The exand ldlr C. These Increasing evidence has shown that RSV treatment has protective effects on NAFLD by modulating gut microbiota . IntriguIn the OA-induced HepG2 cell line, 3-HPP and 4-HPP were more effective in reducing lipid accumulation than RSV. By contrast, 3- HBA and 4-HBA did not affect lipid contents and 4-HPA had a weak lipid-lowering activity in HepG2 cells. This result suggests that the molecular structure of metabolites might be related to their effects on lipid accumulation and for the same benzene ring substitution, the length of the carboxyl chain had greater impacts on lipid-lowering activity than the position of the hydroxyl group. As a previous study indicated, the chemical structure of phenolic acids influences their antimicrobial potential .In NAFLD mice, intraperitoneal supplementation with 3-HPP and 4-HPP can alleviate hepatic steatosis, as indicated by liver weight, lipid contents in the liver and serum, and NAS. Surprisingly, the effects of 3-HPP and 4-HPP were the same as those of RSV. While DHR and LUN had little impact on body weight, adipose tissue mass and blood parameters in HFD-fed mice . Our fincd36 mRNA levels, while 3-HPP and 4-HPP did not influence the expression levels of marker genes of FA transport. Therefore, RSV had the ability to regulate FA transport, which was consistent with the effect of RSV supplementation with oral gavage [acadm mRNA levels and downregulated pparg mRNA levels. Moreover, RSV supplementation stimulated the expression of cpt1a, which was inhibited by HFD; this finding is consistent with previous findings [abca1 and abcg8, 3-HPP supplementation upregulated the expression of abcg8 and downregulated the expression of scarb1, and 4-HPP supplementation increased the expression of abca1, abcg5, abcg8, and ldlr. This finding indicates that the bioeffects of 3-HPP and 4-HPP on TC transport are stronger than RSV because RSV did not regulate genes of TG absorption while 3-HPP and 4-HPP did regulate. Importantly, RSV exhibited beneficial effects on lipid metabolism by influencing FA oxidation and transport, while 3-HPP and 4-HPP exhibited beneficial effects by influencing TC transport. After i.p, the majority of RSV and 3-HPP, 4-HPP were directly entered into the blood, while oral administration RSV was metabolized and entered systemic circulation together with its metabolites. Our findings help to illustrate the diverse biological activities of RSV oral administration. The liver is an essential organ for lipid metabolism. NAFLD primarily occurs as a result of gradually increased lipid accumulation. TG and TC are the major neutral lipids in the liver synthesized by FA, and hepatic steatosis results from an influx and outflux imbalance among these substances in hepatocytes . 3-HPP, l gavage . CPT1a, l gavage . Comparefindings . Scavengfindings . We founBesides hepatic steatosis, the development of NAFLD was highly associated with insulin resistance, chronic inflammation, and oxidative stress. We also found that HFD induced insulin resistance and systemic inflammation, which were reversed by 3-HPP, 4-HPP, and RSV supplementation. However, the effects of 3-HPP and 4-HPP on insulin resistance were weaker than those of RSV, which inspired us to find the real and active form in RSV decreasing insulin resistance. Finding this form would be of importance for preventing diabetes by RSV. In addition, 3-HPP and 4-HPP supplementation decreased systemic inflammation as well as RSV supplementation. Future studies could focus on antioxidant enzyme levels in the liver to assess the effects of 3-HPP and 4-HPP on oxidative stress. Moreover, the mechanisms by which 3-HPP and 4-HPP inhibit inflammation need to be explored in future studies.In conclusion, we found that 3-HPP and 4-HPP, gut microbiota-derived metabolites of RSV, can alleviate NAFLD in OA-induced HepG2 cells and HFD-fed mice by ameliorating hepatic steatosis and their bioeffects were equivalent to RSV at the same concentration. While the molecular mechanisms between the parent compound and microbial metabolites on regulating lipid metabolism are different, the former focuses on FA transport and the latter focuses on cholesterol transport. Our finding helps to explain the contradiction between low bioavailability and beneficial effects of RSV and other polyphenols. Furthermore, our study illustrates the individual difference of RSV bioactivity and provides further support for nutritional intervention of RSV.


Ilo/CAD TEVGs into the ovine carotid artery (n = 12) for 6 months. The primary patency was 50% . TEVGs were completely replaced with the vascular tissue, free from aneurysms, calcification, atherosclerosis and infection, completely endothelialised, and had clearly distinguishable medial and adventitial layers. Comparative proteomic profiling of TEVGs and contralateral carotid arteries found that TEVGs lacked contractile vascular smooth muscle cell markers, basement membrane components, and proteins mediating antioxidant defense, concurrently showing the protein signatures of upregulated protein synthesis, folding and assembly, enhanced energy metabolism, and macrophage-driven inflammation. Collectively, these results suggested a synchronised replacement of PCL with a newly formed vascular tissue but insufficient compliance of PCLIlo/CAD TEVGs, demanding their testing in the muscular artery position or stimulation of vascular smooth muscle cell specification after the implantation.Implementation of small-diameter tissue-engineered vascular grafts (TEVGs) into clinical practice is still delayed due to the frequent complications, including thrombosis, aneurysms, neointimal hyperplasia, calcification, atherosclerosis, and infection. Here, we conjugated a vasodilator/platelet inhibitor, iloprost, and an antimicrobial cationic amphiphilic drug, 1,5-bis- pentane tetrabromide, to the luminal surface of electrospun poly(ε-caprolactone) (PCL) TEVGs for preventing thrombosis and infection, additionally enveloped such TEVGs into the PCL sheath to preclude aneurysms, and implanted PCL In spite of significant efforts on developing biodegradable, durable, haemo- and biocompatible, and triple-resistant  small-diameter tissue-engineered vascular graft (TEVG), with compliance and elastance similar to the native arteries, current advances in this field are limited to the experimental prototypes showing at best 50% primary patency in the long term  ,3,4. TheThe most frequently used biodegradable polymer in vascular tissue engineering is poly(ε-caprolactone) (PCL), a synthetic compound which is sufficiently durable and elastic to withstand blood pressure until replacement of PCL with de novo vascular tissue ,17. AlthUnmodified PCL vascular grafts show insufficient haemocompatibility upon the implantation into sheep, a well-accepted “worst case model” in vascular tissue engineering which mimics: (1) pro-thrombotic conditions in patients with hereditary thrombophilia, cancer, autoimmune disorders, nephrotic and metabolic syndromes, obesity, prolonged immobilisation, and regular use of oral contraceptives; (2) pro-calcification conditions, i.e., advanced age, chronic kidney disease, diabetes mellitus, and osteopoenia/osteoporosis; (3) pro-aneurysmal conditions because of high blood velocity in elastic carotid artery (which is a common site of TEVG implantation in sheep) as compared to muscular arteries including coronary and internal thoracic arteries ,22. ClotAnother obstacle in the field is the dearth of unbiased mass spectrometry profiling in studies investigating vascular regeneration during TEVG degradation and synchronisation of these processes , as protHere, we showed that biodegradable tissue-engineered vascular grafts (TEVGs) modified with a vasodilator/platelet inhibitor, iloprost, and antimicrobial cationic amphiphilic drugs have non-inferior 6-month primary patency (50%) upon the implantation into the ovine carotid artery. To uncover the molecular events behind the TEVG remodeling, we, for the first time, performed label-free proteomic profiling of TEVGs and found them free of enzymatic degradation, thrombosis, aneurysms, calcification, atherosclerosis, or infection. Further, TEVGs demonstrated a complete endothelialization but lacked the basement membrane and hierarchical structure of the medial layer. In spite of a strong macrophage-driven inflammation, TEVGs fully retained their integrity, indicative of curbed proteolysis and controlled remodeling. Hence, we suggested that conjugation of cationic amphiphilic drugs and iloprost to the luminal surface endows TEVGs with perfect haemocompatibility and contributes to the physiological replacement of PCL with newly formed vascular tissue which, however, lacks regular elastic laminae and vascular smooth muscle cell layers, thereby posing insufficient compliance.Biodegradable TEVGs  were fabricated using electrospinning  from 12% PCL  dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol  solution. Electrospinning parameters were as follows: 22 kV voltage, 22G needle, 0.5 mL/h feed rate, 4 mm rotating drum diameter, 1000 rpm collector rotation speed, and 150 mm tip-to-collector distance. A metal pin with a 4 mm diameter was used as a collector. Upon the fabrication, TEVGs were coated with anti-aneurysmatic PCL  sheath by fused deposition modeling using an original 3D printer extruder and the following extrusion parameters: 1 rps roller speed, 1 mm/sec carriage speed, 0.5 mm extruder diameter, 0.5 mm/sec feed rate, and 160 °C feed temperature.−1 and at wavelengths ranging from 4000 to 500 cm−1 (FTIR).To increase haemocompatibility of TEVGs, we first immobilised a poly(vinylpyrrolidone) (PVP) hydrogel at their luminal surface by incubating TEVGs in 5% alcoholic solution of PVP  for 30 min, drying at room temperature and sterile conditions for 24 h, and irradiating in the argon atmosphere by a linear particle accelerator . As irradiation also sterilised TEVGs, subsequent procedures were conducted under sterile conditions. Then, TEVGs were cleansed from unconjugated polymer by washing in sterile water for 60 min. PVP conjugation was verified by Fourier-transform infrared spectroscopy  at a resolution of 4 cmAnti-thrombotic coating was created using iloprost  whilst antimicrobial effect was furnished with a cationic amphiphilic drug (CAD) 1,5-bis- pentane tetrabromide . The synthesis of the latter was conducted as previously described . CoatingFor both attached drugs  pentane tetrabromide), we assessed their conjugation with TEVGs and release kinetics. To investigate the controlled release of iloprost and 1,5-bis- pentane tetrabromide, TEVGs were incubated in the sterile phosphate-buffered saline  at 37 °C, and PBS was sequentially collected at 1, 5, 10, 15, 20, 30, 45, 60, 75 and 90 days.2 piece of each TEVG luminal surface  was analysed, and the spectra were obtained over the 620–4000 cm−1 region at a resolution of 4 cm−1 with a total of 64 scans. For the semi-quantitative analysis, the reflectance spectra (R) were converted to Kubelka–Munk equivalent absorbance units (F) according to the Equation (1), as the intensities of the absorption bands of many substances are proportional to their concentrations in the Kubelka–Munk model: Semi-quantitative detection of iloprost on TEVG luminal surface and time-resolved evaluation of its release into the milieu at all indicated time points was performed by Fourier-transform infrared spectroscopy  employing a zinc selenide attenuated total reflectance accessory . Around 1 cmBromine, an element appearing in TEVGs specifically upon the attachment of CAD 1,5-bis- pentane tetrabromide, was determined by energy-dispersive X-ray spectroscopy immediately after the fabrication and after storage at 4 °C for 1 day, at −20 °C for 1 day or 6 months, or after a 6 h incubation in PBS supplied with 0.05% Tween-20 at 37 °C. For energy-dispersive X-ray spectroscopy, dried samples were mounted on an aluminum stub using double-sided adhesive carbon tape, and sputter coated with carbon (60 nm thickness) using EM ACE 200 sputter coater . Energy-dispersive X-ray spectroscopy was conducted using a scanning electron microscope S-3400N  coupled with a high-resolution silicon drift detector  in a secondary electron mode at 20 kV accelerating voltage and high vacuum.The release profile of 1,5-bis- pentane tetrabromide into the milieu (PBS) was assessed by liquid chromatography–mass spectrometry  at 5 and 90 days.Ilo/CAD TEVGs  have been implanted for 6 months.The study was conducted according to the guidelines of the Declaration of Helsinki, and was approved by the Local Ethical Committee of the Research Institute for Complex Issues of Cardiovascular Diseases . Animal experiments were performed in accordance with the European Convention for the Protection of Vertebrate Animals  and Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes. For the implantation, we used female Edilbay sheep of 42–45 kg body weight which were received from the Animal Core Facility of the Research Institute for Complex Issues of Cardiovascular Diseases  and selected for the surgery by Doppler ultrasonography to identify those having carotid artery diameter of 4.0 ± 0.2 mm. Biodegradable PCLv/v), R-Pharm, Moscow, Russia) and intravenous infusion of atracurium besylate  with the constant control of heart rate, respiratory rate, blood pressure, and oxygen saturation. Through the whole time of the surgery, artificial ventilation parameters were as follows: 12–15/min respiratory rate, 7–9 mbar positive end-expiratory pressure (PEEP), 6–8 mL/kg respiratory volume, and 40–60% fraction of inspired oxygen (FiO2).Upon the premedication with xylazine  and atropine  and induction into the anaesthesia with propofol  and atracurium besylate , it was maintained by the endotracheal intubation and artificial ventilation with sevoflurane , we clamped the artery, excised a 4 cm segment, performed end-to-end implantation of a TEVG using the twisted seam , and conducted the wound closure . During the surgery, animals received 0.9% NaCl  and cefuroxime . After the wound closure, we injected an inhibitor of factor Xa enoxaparin sodium  and performed the extubation. Postoperative care included injections of cefuroxime , enoxaparin sodium , clopidogrel , and heparin sodium  during 5 days. Graft patency was assessed by Doppler ultrasonography immediately after the surgery and then at the following time points: 1 day, 3 days, 1 month, 3 months, and 6 months postoperation. At 6 months postoperation, sheep were sacrificed. Excised TEVGs and contralateral healthy carotid arteries were cut into four segments of equal length. The proximal segment was selected for the histological examination, two midgraft segments were used for the immunostaining and electron microscopy, and the distal segment was employed for the proteomic analysis.The proximal segment was fixed in two changes of 10% neutral phosphate buffered formalin  for 24 h at 4 °C, embedded into paraffin , and cut (8 µm sections) on a microtome  as previously described [The first midgraft segment was snap-frozen in the optimal cutting temperature medium  and cut on a cryostat  as described above. Before the immunostaining, sections (8 µm thickness) were fixed in 4% paraformaldehyde  for 10 min, permeabilised in Triton X-100  for 15 min, and blocked in 1% bovine serum albumin  for 1 h to prevent non-specific binding. Sections were then stained with rabbit anti-CD31  and mouse anti-α-SMA , rabbit anti-vWF , rabbit anti-collagen IV , rabbit anti-collagen III , rabbit anti-CD163 , or mouse anti-CD68  primary antibodies overnight at 4 °C. Next day, sections were further treated with goat anti-rabbit highly cross-adsorbed Alexa Fluor 488-conjugated  and donkey anti-mouse highly cross-adsorbed Alexa Fluor 555-conjugated  secondary antibodies for 1 h at room temperature. Autofluorescence was eliminated with Autofluorescence Eliminator Reagent  according to the manufacturer’s protocol. Counterstaining was performed with DAPI  for 30 min. At all stages, washing was conducted with 0.1% saline  solution of Tween-20 . Coverslips were mounted with ProLong Gold Antifade . Visualisation was performed by a confocal microscopy .After the electrospinning, TEVGs were sputter coated with gold and palladium  and visualised by scanning electron microscopy .4, 19110, Electron Microscopy Sciences, Hatfield, PA, USA), dehydrated in ascending ethanol series  and isopropanol , stained in 2% alcoholic uranyl acetate , impregnated with acetone : epoxy resin (1:1) and epoxy resin , embedded into epoxy resin, grinded, polished, counterstained with Reynolds’s lead citrate , sputter coated with carbon  and visualised by means of backscattered scanning electron microscopy  in accordance with an EM-BSEM (embedding and backscattered scanning electron microscopy) procedure as previously described [The second midgraft segment was fixed in 10% neutral phosphate buffered formalin , postfixed and stained in 1% phosphate buffered osmium tetroxide  for 10 min, supernatant was additionally centrifuged at 200,000× g  for 1 h to sediment insoluble ECM proteins. Quantification of total protein was conducted using BCA Protein Assay Kit  and Multiskan Sky microplate spectrophotometer  in accordance with the manufacturer’s protocol.Distal segments of TEVGs and intact contralateral carotid arteries were flushed with a physiological saline  and homogenised  in T-PER buffer  supplied with Halt protease and phosphatase inhibitor cocktail  according to the manufacturer’s protocol. Upon the initial centrifugation at 14,000× Upon the removal of RIPA buffer by acetone precipitation , protein pellet was resuspended in 8 mol/L urea  diluted in 50 mmol/L ammonium bicarbonate . The protein concentration was measured by Qubit 4 fluorometer  with QuDye Protein Quantification Kit  according to the manufacturer’s protocol. Protein samples (15 μg) were then incubated in 5 mmol/L dithiothreitol  for 1 h at 37 °C with the subsequent incubation in 15 mmol/L iodoacetamide for 30 min in the dark at room temperature . Next, the samples were diluted with 7 volumes of 50 mmol/L ammonium bicarbonate and incubated for 16 h at 37 °C with 200 ng of trypsin . The peptides were then frozen at −80 °C for 1 h and desalted with stage tips  according to the manufacturer’s protocol using methanol , acetonitrile , and 0.1% formic acid . Desalted peptides were dried in centrifuge concentrator  for 3 h and finally dissolved in 20 μL 0.1% formic acid for the further shotgun proteomics analysis.Approximately 500 ng of peptides were used for shotgun proteomics analysis by ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) with ion mobility in a TimsToF Pro mass spectrometer with a nanoElute UHPLC system . UHPLC was performed in the two-column separation mode with an Acclaim PepMap 5 mm Trap Cartridge (Thermo Fisher Scientific) and a Bruker Fifteen separation column  in a gradient mode with 400 nL/min flow rate and 40 °C. Phase A was water/0.1% formic acid, phase B was acetonitrile/0.1% formic acid . The gradient was from 2% to 30% phase B for 42 min, then to 95% phase B for 6 min with subsequent washing with 95% phase B for 6 min. Before each sample, the trap and separation columns were equilibrated with 10 and 4 column volumes, respectively. The CaptiveSpray ion source was used for electrospray ionization with 1600 V of capillary voltage, 3 L/min N2 flow, and 180 °C source temperature. The mass spectrometry acquisition was performed in DDA-PASEF mode with 0.5 s cycle in positive polarity with the fragmentation of ions with at least two charges in m/z range from 100 to 1700 and ion mobility range from 0.85 to 1.30 1/K0.Ovis aries) reference proteome UP000002356  and protein contaminants database CRAP (version of 4 March 2019). The search parameters were: parent mass error tolerance 10 ppm and fragment mass error tolerance 0.05 ppm, protein and peptide FDR < 1% and 0.1%, respectively, two possible missed cleavage sites, proteins with ≥ 2 unique peptides. Cysteine carbamidomethylation was set as a fixed modification. Methionine oxidation, N-terminal acetylation, asparagine and glutamine deamidation were set as variable modifications.Protein identification was performed in PEAKS Studio Xpro software  using sheep (https://github.com/ArseniyLobov/Proteomic-profiling-of-grafts-implanted-into-the-ovine-artery (accessed on 03 October 2022).The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE  partner p value ≤ 0.05. Bioinformatics analysis was performed using Gene Ontology [For the bioinformatic analysis, unique proteins were defined as those presented in ≥9 samples in each group  and ≤3 samples of another group. Differentially expressed proteins were defined as those with ≥4-fold higher or lower average peak area as compared to another experimental group as well as those with logarithmical fold change ≥ 1 and false discovery rate-corrected Ontology ,44, COMPOntology , UniProtOntology , ReactomOntology ,48, and Ontology ,50 databEqual amounts of protein (25 μg per sample) were mixed with NuPAGE lithium dodecyl sulfate sample buffer  at a 4:1 ratio and NuPAGE sample reducing agent  at a 10:1 ratio, denatured at 99 °C for 5 min, and then loaded on a 1.5 mm NuPAGE 4–12% Bis-Tris protein gel . The 1:1 mixture of Novex Sharp pre-stained protein standard  and MagicMark XP Western protein standard  was loaded as a molecular weight marker. Proteins were separated by the sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) at 150 V for 2 h using NuPAGE 2-(N-morpholino)ethanesulfonic acid SDS running buffer , NuPAGE Antioxidant , and XCell SureLock Mini-Cell vertical mini-protein gel electrophoresis system . Protein transfer was performed using polyvinylidene difluoride (PVDF) transfer stacks  and iBlot 2 Gel Transfer Device (Invitrogen) according to the manufacturer’s protocols using a standard transfer mode for 30–150 kDa proteins . PVDF membranes were then incubated in iBind Flex Solution  for 1 h to prevent non-specific binding.Blots were probed with mouse antibodies to CD68  and glyceraldehyde 3-phosphate dehydrogenase . Horseradish peroxidase-conjugated goat anti-mouse  secondary antibody was used at 1:1000 dilution. Incubation with the antibodies was performed using iBind Flex Solution Kit , iBind Flex Cards  and iBind Flex Western Device  during 3 h according to the manufacturer’s protocols. Chemiluminescent detection was performed using SuperSignal West Pico PLUS chemiluminescent substrate  and C-DiGit blot scanner  in a high-sensitivity mode (12-min scanning).p values ≤ 0.05 were regarded as statistically significant.Statistical analysis was performed using GraphPad Prism 8 . For descriptive statistics, data are presented as proportions, median, 25th and 75th percentiles, and range. Two independent groups were compared by the Mann–Whitney U-test. Proportions were compared by Pearson’s chi-squared test with Yates’s correction for continuity. The Ilo/CAD) revealed highly porous graft wall  analogue inhibiting platelet activation, aggregation, and adhesion and causing vasodilation through binding and activation of IP (prostacyclin or prostaglandin I2 receptor), EP2 (prostaglandin E2 receptor 2) and EP4 (prostaglandin E2 receptor 4) receptors. As iloprost also ameliorates endothelial dysfunction and hinders leukocyte adhesion to endothelial cells, it has been used to treat COVID-19-associated vasculopathy ,52. Seveaft wall a consistaft wall b. The ouaft wall c. Such saft wall c,d.−1 and C–O stretching bands at 1278 and 1054 cm−1 . The FTIR spectra of unmodified and PVP-modified TEVGs showed C=O stretching bands at 1724 cm054 cm−1 . In all ectively . In cont surface . As the Ilo/CAD TEVGs and in PBS solution at ascending time points of incubation at 37 °C. A gradual increase in the iloprost level on TEVG surface was observed during the first 20 days of incubation, followed by a decrement and end of iloprost release after 3 months  pentane tetrabromide to the luminal surface (Cationic amphiphilic drugs (CADs) have a characteristic bromine peak at 1.5 keV a. Energy surface . Storage surface . However surface .41H84Br3N4+ molecular ion (m/z 871.42256) or amphiphile molecular ion with phosphate as a counterion in PBS samples, testifying to the stable conjugation of 1,5-bis- pentane tetrabromide with the TEVG luminal surface  for 6 months, and all animals survived until the end of the follow-up. Doppler ultrasonography showed that primary patency was 83.3%  1 month postimplantation and 50.0%  3 and 6 months postimplantation, respectively. Six months postimplantation, gross examination demonstrated that the TEVGs fully retained their integrity and none of them suffered from aneurysms a. The luvasa vasorum, perivascular adipose cells, and fibroblasts (We then conducted a microscopic examination of ovine carotid arteries and TEVGs. Ovine carotid arteries consisted of: (1) dense endothelial cell (EC) monolayer attached to the collagen IV-enriched basement membrane; (2) thick medial layer with multiple lamellar units which included elastic laminae and vascular smooth muscle cells (VSMCs) embedded into the collagen III/IV extracellular matrix between them; (3) thick collagen-rich tunica adventitia . Similarroblasts . Hence, Six months postimplantation, TEVGs were completely resorbed and replaced with vascular tissue formed de novo . SimilarTo compare the molecular composition of the native carotid arteries and TEVGs 6 months postimplantation, we performed a label-free, pairwise proteomic profiling  by means of ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS). A proteomic approach was preferred over the RNA sequencing as TEVGs might contain physiologically significant proteins emerging from the circulation rather than produced in situ.Principal component analysis indicated a clear discrimination pattern between the native carotid arteries and TEVGs (71% explained variation), whereas the variation between the samples comprising each group was relatively low  Cellular Component  and UniPThe following analysis of GO Molecular Functions  and UniPFurther, bioinformatics analysis of differentially regulated biological processes employing GO Biological Process database revealed that actin cytoskeleton and basement membrane organization, actin filament depolymerization, stress fiber, myofibril, and focal adhesion assembly, integrin-mediated signaling pathway, muscle cell development, and muscle contraction were upregulated in the native carotid arteries . TEVGs sAnalysis of the Reactome database revealed concordant results, as the terms related to the integrin, syndecan, and laminin interactions, dermatan and chondroitin sulfate biosynthesis, fibronectin matrix formation, extracellular matrix proteoglycans, smooth muscle contraction, and elastic fiber formation were overrepresented in the native carotid arteries . TEVGs dA search of the Kyoto Encyclopedia of Genes and Genomes (KEGG) database also identified upregulated actin cytoskeleton regulation, focal adhesion, and VSMC contraction in the native carotid arteries as well as augmented lyso- and phagosomal activity and carbon/amino acid metabolism in TEVGs .Detailed analysis of unique and differentially expressed proteins identified an overrepresentation of the contractile proteins serving as VSMC markers (7/145), extracellular matrix proteins composing the basement membrane (16/145), and antioxidant defense proteins  in the native carotid arteries . StrikinExpression of lysosomal proteins (19/128) such as cathepsins , V-type proton ATPase subunits , and lysosome-associated membrane glycoproteins (LAMP1 and LAMP2) were restricted to or vastly increased in TEVGs indicative of chronic inflammation, as also evidenced by exclusive expression of a specific macrophage marker CD68 . Upregul+ cells) and proportions of anti-inflammatory and pro-fibrotic (M2) macrophages, notable for CD163 expression, did not differ significantly between the non-occluded and occluded TEVGs  and weaker antioxidant defense suggestive of a significant oxidative stress. Nevertheless, PCLIlo/CAD TEVGs did not show any signs or molecular signatures of thrombosis, calcification, atherosclerosis, or infection.Collectively, electron microscopy, immunostaining, and proteomic profiling demonstrated that the processes of polymer degradation and vascular tissue regeneration in PCLAlbeit, the development of commercially available off-the-shelf TEVG is highly relevant to the current demands of cardiac surgery, neurosurgery, and microsurgery and would significantly address a growing need in the increase in vascular reconstruction procedures, none of the TEVG prototypes has passed through the pre-clinical trials hitherto. Among the most frequent complications of TEVG implantation are thrombosis, aneurysms, neointimal hyperplasia, calcification, atherosclerosis, and infection ,5,6,7,8.Despite as many as 182 animal studies assessing the efficiency of TEVG implantation and 22 pre-clinical trials of various TEVGs (12 in the ovine and 10 in the porcine model) being carried out to-date , a vast Having considered protein categories overrepresented in native carotid arteries or TEVGs, we found VSMC markers, basement membrane components, and antioxidant defense proteins upregulated in the native carotid arteries and lysosomal markers, proteins responsible for the protein synthesis/folding, and glucose metabolism regulators overexpressed in TEVGs. Regarding the potential role of these categories in the long-term success of the TEVGs, VSMC contractile proteins mediate an adequate biomechanical response to the blood pressure changes through the controlled cyclic stretch, while basement membrane components contribute to the rapid and complete endothelialisation. Antioxidant defense proteins are probably in charge for the control of macrophage-driven inflammation, and their levels can be possibly corrected by an antioxidant-rich diet, such as the Mediterranean diet, in a clinical setting. Lysosomal enzymes and structural proteins (macrophage markers), components of the protein folding and synthesis machinery, and energy metabolism proteins are collectively responsible for the polymer degradation (requiring large amounts of cells enriched with acidic organelles and multiple enzymes to digest and recycle the polymer) and its replacement by the regenerative vascular tissue (demanding permanent protein supply to form the extracellular matrix), as both of these processes are extremely energy-consuming.As opposed to the bioprosthetic heart valves , TEVGs retained their integrity after a significant time postimplantation and we did not reveal the deposition of abundant serum proteins  or matrix metalloproteinases in the vascular tissue replacing the degrading PCL. In combination with active biosynthetic processes, evident by increased amounts of the proteins participating in the translation, protein folding and assembly, and energy metabolism, these molecular patterns testified to the synchronisation between PCL degradation and vascular regeneration. Further, TEVGs did not demonstrate any signs of the infection such as precipitation of complement components or neutrophil markers, instead showing informative signatures of chronic inflammation remodeling, namely structural lysosomal proteins (which are well-established macrophage markers) and cathepsins, known as lysosomal but not blood- or extracellular matrix-derived enzymes ,63,64. TIlo/CAD TEVGs included controlled replacement of the biodegradable polymer by de novo vascular tissue and freedom from a number of main complications accompanying the TEVG implantation . Although PCLIlo/CAD TEVGs did not have a clear basement membrane, it did not hinder endothelialisation, a mandatory pre-requisite for haemocompatibility and thromboresistance [Therefore, the advantages of our PCLsistance ,5,6,7,8,sistance . Yet, thsistance ,76 or posistance  sheath esistance . The lacsistance ,79,80. AThese results indicate two main issues remaining unresolved for the ultimate success in pre-clinical trials of bioresorbable TEVGs. The first, an inability to guide the assembly and formation of regular elastic fibers, is apparent but is probably not a major problem in the reconstruction of arteries with low shear stress . Even if the internal elastic lamina can be reproduced using certain composites, as was claimed in , it woulThe second problem—the lack of contractile VSMCs in the medial layer—seems to have a higher priority, as the recapitulation of VSMC differentiation in vivo remains challenging. Potential approaches to stimulate physiological, contractile VSMC specification of mesenchymal cells at cyclic stretch in the TEVG tunica media include dietary, vascular tissue engineering, and pharmacological interventions. First, a specific diet including recently defined factors of cardiovascular resilience such as calorie restriction, intermittent fasting, meal timing , antioxidant-rich diet, and increased physical activity ,82 mightWe propose that there is a correlation between the flow parameters  and VSMC specification, and regular measurements of the mentioned characteristics in TEVGs by means of various visualisation modalities can pave the way to our understanding of the transition between synthetic mesenchymal cells and contractile, fully functioning VSMCs. In other words, the key for the TEVG success currently lies within the medial layer. Possibly, TEVGs should be first tested in the position of muscular  but not elastic artery; the differences between the results of the TEVG implantation into artery or vein position have been convincingly described earlier .As shown in this study, unbiased transcriptomic or proteomic approaches are capable of gaining an insight into the vascular regeneration upon TEVG implantation and should be extensively employed in the pre-clinical trials of TEVGs to improve their design and testing settings. Further directions to apply high-throughput molecular characterisation in relation to TEVGs might include a detailed profiling of the EC monolayer, as its proper functioning is crucially important for providing acceptable primary patency ,90,91. T


Sulfonamides and their bioisosteres have drug-like properties well-known for decades and are suitable lead compounds for developing new pharmacological agent families for inhibiting carbonic anhydrases (CAs). CAs are a superfamily of metalloenzymes catalyzing the reversible reaction of CO2 hydration to HCO3− and H+, being present in most bacteria in multiple genetic families . These enzymes, acting as CO2 transducers, are promising drug targets because their activity influences microbe proliferation, biosynthetic pathways, and pathogen persistence in the host. In their natural or slightly modified scaffolds, sulfonamides/sulfamates/sulamides inhibit CAs in vitro and in vivo, in mouse models infected with antibiotic-resistant strains, confirming thus their role in contrasting bacterial antibiotic resistance.Resistance to antibiotic treatment developed by bacteria in humans and animals occurs when the microorganisms resist treatment with clinically approved antibiotics. Actions must be implemented to stop the further development of antibiotic resistance and the subsequent emergence of superbugs. Medication repurposing/repositioning is one strategy that can help find new antibiotics, as it speeds up drug development phases. Among them, the Zn Antibiotic resistance is a worldwide emergency that kills more people than HIV/AIDS and malaria combined . It killThus, to treat infections caused by those resistant bacteria, one strategy is to administer other antibiotics to which they are sensitive. However, they might also acquire resistance to the new class of antibiotics (multi-resistant organisms), and so switching to a new type of antibiotic is required until we arrive at bacteria resistant to all antibiotics (pan-resistant microorganisms) . The antStreptococcus pneumoniae, one of the six most hazardous antibiotic-resistant bacteria, exists such a vaccine); (iii) reduction of the use of antibiotics in non-human infection-treatment contexts, such as livestock farms; (iv) appropriate antibiotic use, as well as stopping their use for the management of viral infections; (v) maintenance of investments to make second-line antibiotics available, and the development of novel antibiotics and especially those with novel modes of action less susceptible to the onset of resistance, which can save lives [ESKAPE pathogens . Finally, the future of antibiotics is shaky because of questions about their effectiveness and safety [How do we intervene to stop antibiotic resistance? There is no shortage of strategies for dealing with the antibiotic resistance ,14,15,16ve lives ,14,15,16ve lives . Moreoved safety ,19.In this context, the drug repurposing or drug repositioning strategy offers the potential to “fast-track” the identification of novel antibiotics, since it speeds up the drug research process and reduces the time to market . FDA-app2) is a gas released into the atmosphere due to cellular respiration and oxidative metabolism, and all living creatures are responsible for its production [2 channels, controlled by CO2 levels inside the cell, make this transfer easier. However, CO2 is not merely a waste product; it can also trigger various cellular signaling pathways to increase bacteria virulence and pathogenicity [2, enhancing thus their chances of survival. This may be crucial because bacteria have to adapt to the relatively low CO2 levels of the outer atmosphere for the higher CO2 levels found inside most multicellular host organisms [2 levels rather than ambient CO2 levels to aid colonization or infection. Several such examples are Vibrio cholerae, which causes cholera, and produces enterotoxin as carbon dioxide levels rise, whereas bicarbonate produced by the CO2 hydration is the first positive effector of the primary V. cholerae virulence gene transcription activator (ToxT), responsible for the cholera virulence cascade [Pseudomonas aeruginosa, which can lead to infections in the blood, lungs (pneumonia), or other regions of the body following surgery, lives in vastly varying CO2 settings depending on whether or not it is colonizing a host [Carbon dioxide  [2 hydration/dehydration reaction is too slow at physiological pH values to fulfill the organism’s metabolic demands (kcat (hydration) = 0.15 s−1; kcat (dehydration) = 50.0 s−1) [Biomolecules in microbes related to COthe host ,27. In ts levels ,28. With0.0 s−1) .2 is strictly conserved across all CA-classes, illustrating the CA superfamily’s convergent evolution [2+, which is coordinated by three amino acid residues from the protein backbone [2+, such as Co2+, Cd2+, Fe2+, and Mn2+, can be coordinated by several CA-classes [Anabaena sp. [2 loss from bacteria by converting CO2 into bicarbonate, which is then transported inside the cytoplasm by bicarbonate transporters [2 and HCO3− equilibrium and regulating pH. Recently, β, γ, and ι-CAs with signal peptide at N-terminus have been found, attributing them a putative periplasmic localization and a physiological role similar to those mentioned above for the α-CAs [The classification system for CAs uses the Greek letters to represent the eight distinct families (or classes): α, β, γ, δ, ζ, η, θ, and ι ,31,32,33volution ,36,37,38backbone ,30,39. T-classes ,45,46,47aena sp. . The CA-aena sp. ,50,51,52aena sp. ,45,53,54aena sp. ; and twoaena sp. . A tandeaena sp. ,45,53,54aena sp. . Interesaena sp. ,58,59. Paena sp. ,36,37,38aena sp. ,29,33,49he α-CAs ,58,59.2 or HCO3−, pH homeostasis, secretion of electrolytes/toxins, and biosynthetic processes [2 concentrations is dependent on CA activity in bacteria such as Ralstonia eutropha (Gram-negative bacterium found in soil and water) and Escherichia coli (Gram-negative bacterium) [E. coli, the two β-CAs (CynT and CynT2) generate HCO3− to prevent bicarbonate depletion from cyanate breakdown and bacterial expansion at atmospheric CO2 concentrations, respectively [Mycobacterium tuberculosis [Helicobacter pylori [Vibrio cholerae [Brucella suis [Salmonella enterica [Pseudomonas aeruginosa [H. pylori, a Gram-negative, microaerophilic bacteria that colonizes the human stomach, are essential for the pathogen’s acid acclimation and, consequently, survival in the severe environment typical of this organ, with pH values as low as 1.5–2.0 [3 and CO2 are produced by the hydrolysis of urea [2 is then hydrated by β-CA, while the periplasmic α-CA hydrates the CO2 diffused in the periplasm. The produced ions (H+) by the CA-catalyzed reaction are used to form NH4+ by reacting with NH3+ in the periplasm and cytoplasm, which neutralizes the entering acid in the above environments [Vibrio cholerae, a Gram-negative bacterium already mentioned above, CAs are involved in the production of sodium bicarbonate, which stimulates the development of cholera toxin [V. cholerae employs CAs to colonize the host [Brucella suis, a non-motile Gram-negative coccobacillus, and Mycobacterium tuberculosis, a pathogenic bacterium that causes tuberculosis, were demonstrated to require functional CAs to proliferate [Salmonella enterica, the MIG5 gene, which encodes for a CA that is significantly expressed during bacterial infection, has been found [Pseudomonas aeruginosa reduced pathogenicity by decreasing calcium salt depositions [In the literature, many shreds of evidence support the opinion that the activity of CAs is connected to the survival of microbes because these enzymes are essential for supporting numerous physiological functions involving dissolved inorganic carbon, such as transport and supply of COrocesses ,33,60. Fcterium) ,62,63. Irculosis ,66,67,68r pylori ,70,71, Vcholerae , Brucelllla suis ,66,67,68enterica ,74,75, aruginosa . CAs enc 1.5–2.0 ,70,71. I of urea ,70,71. Tronments ,70,71. Ira toxin . It has the host . Again, liferate ,66,67,68en found ,74,75. Tositions .Because the many biochemical processes mentioned above involve the activity of bacterial CAs, their inhibition may reduce the pathogen’s survival and fitness. The good news is that CA inhibition suppresses bacterial growth differently from those demonstrated by traditional antibiotics, toward which the bacteria have developed or are developing antibiotic resistance. As reported in the scientific literature, many unique chemical classes of CA inhibitors (CAIs) exist . The CAI2+ ion binders, in particular, the sulfonamides and their bioisosteres, are considered the most promising compounds for the realization of novel antibacterials, avoiding antibiotic resistance [2-NH2, where R can be an aromatic, heterocyclic, aliphatic, or sugar scaffold, X = nothing, O or NH. Thus, sulfanilamide led to the discovery of the sulfa drugs and benzenesulfonamide CAIs of the type. They constitute an important class of drugs since they have drug-like properties well-known for decades and are suitable lead compounds for developing new pharmacological agent families for inhibiting CAs. Among them are the commercial derivatives 1–24 (AAZ-EPA (AAZ-EPA include acetazolamide (AAZ), methazolamide (MZA), ethoxzolamide (EZA), and dichlorophenamide (DCP) are classical, systemically working antiglaucoma CAIs. Dorzolamide (DZA) and brinzolamide (BRZ) are topically acting antiglaucoma agents. Benzolamide (BZA) is an orphan drug belonging to this class of pharmacological agents. Zonisamide (ZNS), sulthiame (SLT), and sulfamic acid ester topiramate (TPM) are widely used antiepileptic drugs. Sulpiride (SLP) and indisulam (IND) were also shown by our group to belong to this class of pharmacological agents, together with the COX2 selective inhibitors celecoxib (CLX) and valdecoxib (VLX). Saccharin (SAC) and the diuretic hydrochlorothiazide (HCT) are also known to act as CAIs. Famotidine (FAM) and epacadostat (EPA) are CAI sulfamide drugs clinically used respectively as a histamine H2 receptor antagonist and a selective indoleamine-2,3-dioxygenase 1 inhibitor. These inhibitors bind Zn (II) in a tetrahedral geometry, forming an extended network of hydrogen bonds with the enzyme amino acid residues, whereas the aromatic/heterocyclic portions of the inhibitor interact with the hydrophilic and hydrophobic residues found in the enzyme catalytic cavity, according to enzyme-inhibitor X-ray crystallographic data . Many of these studies were focused on bacterial CAs derived from pathogenic bacteria, such as ri, etc. ,83,84,85ri, etc. ,88,89,90ri, etc. . In thisEZA), an authorized diuretic and carbonic anhydrase inhibitor, kills Helicobacter pylori in vitro, suggesting it could be turned into an anti-H. pylori medication [For example, ethoxzolamide  and γ-CA (EcoCAγ), with a KI of 227 and 248 nM, respectively [2/HCO3− required for the bacterial metabolic need [The influence of the selective CA inhibitor re media . Carbon ectively ,95. AAZ lic need .AAZ could be used to design potent antienterococcal agents. The authors, modifying the AAZ scaffold, arrived at two leads possessing improved potency against clinical vancomycin-resistant enterococci (VRE) strains [AAZ showed a MIC of 2 μg/mL, while the two leads had a MIC = 0.007 μg/mL and 1 μg/mL, respectively. DZA, another classical CAIs, resulted in MIC values of 1–8 μg/mL against a panel of clinical VRE isolates [AAZ fared better than linezolid (a standard drug for VRE infections) when tested in two in vivo VRE mouse models –murine colonization–reduction and VRE septicemia [It has been reproposed the FDA-approved carbonic anhydrase drug  strains . The claisolates . Based oisolates ,98. In apticemia .AAZ inhibited the growth of the Gram-negative bacterium Neisseria gonorrhoeae both in the in vitro as well as in vivo mouse model of a gonococcal genital tract infection [Streptococcus mutans colonization in teeth treated with two potent bacterial CA sulfonamide inhibitors [Finally, nfection ,101. Recnfection  demonstrhibitors .It is also interesting to note that over the last few years, there has been a significant focus on developing non-classical CAIs for treating multiple diseases due to the prevalence of sulfonamide allergies among the general population as well as their use as potential new antibacterials . The cla2NH2), primary sulfamate (-OSO2NH2), and sulfamide (-NHSO2NH2), which are present in the structure of several clinically-approved drugs and an increasing number of investigational medicines, have been proven to affect the inhibition of the CAs encoded by pathogenic bacteria [I in the nanomolar range) of such CAs. These findings prompt scientists to consider the CAIs as a new approach to fight antibiotic resistance developed by bacteria versus the common FDA-approved antibiotics. Unlike common antibiotics, the CAIs impair the growth of pathogenic bacteria through a novel mechanism of action: perturbating/depleting the intracellular levels of CO2 and HCO3−, which are necessary to microbial biosynthetic pathways. In addition, the antibacterial growth due to the CA inhibition is strongly supported by the fact that the slow non-catalytic CO2 hydration/dehydration reaction is unable to restore these levels. These attractive facts led to the writing of fascinating manuscripts that discuss the repurposing of drugs such as ethoxzolamide (EZA), acetazolamide (AZA), and dorzolamide (DZA) as treatments for interfering with the life cycle of E. coli, Helicobacter pylori, Neisseria gonorrhoeae, and vancomycin-resistant enterococci.The well-known zinc-binding groups (ZBGs), such as the primary sulfonamide (-SObacteria ,79,80,81In conclusion, the licensed sulfonamides/sulfamates/sulfamides acting as CAIs exhibit antibacterial properties in their native or slightly modified scaffolds , hypothe


Pituitary corticotroph macroadenomas, which account for 7% to 23% of corticotroph adenomas, rarely present with apoplexy. This report aimed to describe a patient with a sparsely granulated corticotroph tumor (SGCT) presenting with apoplexy and remission of hypercortisolism.A 33-year-old male patient presented via ambulance with sudden onset of severe headache and nausea/vomiting. Physical examination revealed bitemporal hemianopsia, diplopia from right-sided third cranial nerve palsy, abdominal striae, facial plethora, and dorsal and supraclavicular fat pads. Magnetic resonance imaging demonstrated a 3.2-cm mass arising from the sella turcica with hemorrhage compressing the optic chiasm, extension into the sphenoid sinus and cavernous sinus. Initial investigations revealed a plasma cortisol level of 64.08  mcg/dL. He underwent emergent transsphenoidal surgery. Pathology was diagnostic of SGCT. Postoperatively, the following laboratory findings were found: (1) cortisol level, <1.8 ug/dL ; (2) adrenocorticotropic hormone level, 36 pg/mL ; (3) thyroid-stimulating hormone level, 0.07 uIU/mL ; (4) free thyroxine level, 1 ng/dL ; (5) luteinizing hormone level, <1 mIU/mL ; (6) follicle-stimulating hormone level, 1 mIU/mL ; and (7) testosterone level, 28.8 ng/dL , with ongoing requirement for hydrocortisone, levothyroxine, testosterone replacement, and continued follow-up.Corticotroph adenomas are divided into densely granulated, sparsely granulated, and Crooke cell tumors. Sparsely granulated pattern is associated with a larger tumor size and decreased remission rate after surgery.This report illustrates a rare case of hypercortisolism remission due to apoplexy of an SGCT with subsequent central adrenal insufficiency, hypothyroidism, and hypogonadism. This case report highlights the growing evidence that granulation pattern in corticotroph adenomas have clinical implications in disease presentation as well as prognosis.,The incidence of Cushing disease (CD) is estimated to be between 0.12 and 0.24 cases per 100 000 persons per year.A). He was transferred to a tertiary care center for neurosurgical management with endocrinology consultation postoperatively.A 33-year-old male patient who was otherwise healthy and not on any medications presented to a community hospital with sudden and severe headache accompanied by hypotension, nausea, vomiting, bitemporal hemianopsia, and diplopia. Computed tomography scan of the brain revealed a hyperattenuating 2.0 × 2.8 × 1.5-cm mass at the sella turcica with extension into the right cavernous sinus and encasement of the right internal carotid arteries A. He waIn retrospect, he reported a 3-year history of ongoing symptoms of hypercortisolism, including increased central obesity, dorsal and supraclavicular fat pads, facial plethora, abdominal purple striae, easy bruising, fatigue, decreased libido, and erectile dysfunction. Notably, at the time of presentation, he did not have a history of diabetes, hypertension, osteoporosis, fragility fractures, or proximal muscle weakness. He fathered 2 children previously. His physical examination was significant for Cushingoid facies, facial plethora, dorsal and supraclavicular fat pads, and central obesity with significant axillary and abdominal wide purple striae . NeuroloInitial laboratory data at the time of presentation to the hospital included an elevated plasma cortisol level , normal levels of thyroid-stimulating hormone (TSH)  and free thyroxine , evidence of central hypogonadism with a low total testosterone level , inappropriately normal levels of luteinizing hormone (LH)  and follicle-stimulating hormone (FSH) , low prolactin level , and normal insulin-like growth factor 1 (IGF-1) level . Adrenocorticotropic hormone (ACTH) was not drawn at the time of presentation.B and C).A pituitary gland dedicated magnetic resonance imaging (MRI) was performed to further characterize the mass, which redemonstrated a 1.9 × 3.2 × 2.4-cm heterogeneous mass at the sella turcica extending superiorly and flattening the optic chiasm, remodeling the floor of the sella, and bulging into the sphenoid sinus and extending laterally into the cavernous sinus with encasement of the right internal carotid artery. As per the radiologist’s diagnostic impression, this appearance was most in keeping with a pituitary macroadenoma with apoplexy B and C.A), t-box transcription factor nuclear immunolabeling (B), diffuse cytoplasmic CAM5.2 (low-molecular-weight cytokeratin) immunolabeling (C), and regional weak to moderate intense granular cytoplasmic ACTH immunostaining (D). The tumor was immunonegative for pituitary-specific positive transcription factor 1 and steroidogenic factor 1 transcription factors, growth hormone, prolactin, TSH, FSH, LH, estrogen receptor-α, and α-subunit. Crooke hyalinization was not identified in an adjacent compressed fragment of nonadenomatous anterior pituitary tissue. Ki-67 immunolabeling showed a 1.5% proliferative index (11 of 726 nuclei).The patient underwent urgent transsphenoidal surgery (TSS) and decompression with no acute complications. Pathologic examination of the pituitary adenoma showed features characteristic of a sparsely granulated corticotroph pituitary neuroendocrine tumor (adenoma),labeling B, diffulabeling C, and rstaining D. The tPostoperatively, he developed transient central diabetes insipidus requiring desmopressin but resolved on discharge. His postoperative cortisol was undetectable, and the following were noted: ACTH level, 36 pg/mL ; TSH level, 0.07 mIU/mL ; free thyroxine level, 1 ng/dL ; LH level, <1 mIU/mL ; FSH level, 1 mIU/mL ; and testosterone level, 28.8 ng/dL  . One monMicroadenomas account for the majority of corticotroph tumors, but 7% to 23% of patients are diagnosed with a macroadenoma.C).We also want to highlight the features of the preoperative radiographic findings that can provide valuable insight into the subsequent histology. Previous literature has shown that, on T2-weight MRI, silent corticotroph adenomas are strongly correlated with characteristic a multimicrocystic appearance, whereas nonfunctional gonadotroph macroadenomas are not correlated with this MRI finding.,The ability to distinguish between silent corticotroph macroadenoma and other macroadenomas is important for assessing rate of remission and recurrence risk. In 2017, the World Health Organization published updated classification for pituitary tumors.,,In multiple studies examining granulation pattern and tumor size, SGCTs were statistically larger.Similar to other cases of SGCT, the diagnosis was only arrived retrospective after pathologic confirmation.We report a case of a sparsely granulated corticotroph macroadenoma presenting with apoplexy leading to remission of hypercortisolism and development of central adrenal insufficiency, hypothyroidism, and hypogonadism requiring hormone replacement.The authors have no multiplicity of interest to disclose.


YUCCA, belonging to the class B flavin-dependent monooxygenases, catalyzes the rate-limiting step for endogenous auxin synthesis and is implicated in plant-growth regulation and stress response. Systematic analysis of the YUCCA gene family and its stress response benefits the dissection of regulation mechanisms and breeding applications. In this study, 12 YUCCA genes were identified from the mungbean (Vigna radiata L.) genome and were named based on their similarity to AtYUCCAs. Phylogenetic analysis revealed that the 12 VrYUCCAs could be divided into 4 subfamilies. The evidence from enzymatic assays in vitro and transgenetic Arabidopsis in vivo indicated that all the isolated VrYUCCAs had biological activity in response to IAA synthesis. Expression pattern analysis showed that functional redundancy and divergence existed in the VrYUCCA gene family. Four VrYUCCAs were expressed in most tissues, and five VrYUCCAs were specifically highly expressed in the floral organs. The response toward five stresses, namely, auxin , salinity, drought, high temperatures, and cold, was also investigated here. Five VrYUCCAs responded to IAA in the root, while only VrYUCCA8a was induced in the leaf. VrYUCCA2a, VrYUCCA6a, VrYUCCA8a, VrYUCCA8b, and VrYUCCA10 seemed to dominate under abiotic stresses, due to their sensitivity to the other four treatments. However, the response modes of the VrYUCCAs varied, indicating that they may regulate different stresses in distinct ways to finely adjust IAA content. The comprehensive analysis of the VrYUCCAs in this study lays a solid foundation for further investigation of VrYUCCA genes’ mechanisms and applications in breeding. Auxin/indole-3-acetic acid (IAA) is one of the major endogenous phytohormones, playing a critical role in regulating plant growth and development as well as the stress response , and it In plants, both tryptophan (Trp)-dependent and -independent pathways contribute to the auxin de novo synthesis, and the former is very well understood . There a(X)7ATGEN(X)5P), and ATG-containing motif2 (DxxxxATGY) [YUCCA belongs to class B flavin-dependent monooxygenases , the only class of FMOs found in plants based on the current status of available sequencing data . It has xxxATGY) ,14. AmonxxxATGY) . The FMOxxxATGY) .YUCCA family genes has been performed in more than 30 plants species, such as Arabidopis thaliana [Oryza sativa [Zea mays [Cucumis sativus [Glycine max [medicago truncatula [Populus trichocarpa [Lilium [malus domestica [Triticum aestivuml [Gossypium spp. [Brassica napus [YUCCA gene family and its potential roles in mungbean is limited. The biological functions of YUCCA genes have been demonstrated thoroughly in some species, especially in model plants. In Arabidopsis, the YUCCA(YUC) gene family includes 11 members with overlapping functions in plant development. In 2001, AtYUC1 was first identified from a dominant Arabidopsis mutant yucca involved in the production of IAA [YUCCA genes in Arabidopsis indicated that they played a vital role in developmental processes, including floral patterning and vascular formation  [AtYUC1,4,10,11) [AtYUC5) [AtYUC9) [AtYUC6 and AtYUC7 were reported to confer drought tolerance depending on distinct mechanisms [AtYUC7 could be induced by drought in an abscisic acid (ABA)-dependent manner, the auxin level elevation promoted lateral root growth to protect plants from drought [AtYUC8 and AtYUC9 were upregulated under high temperatures [PIF4) directly induced AtYUC8 expression to elevate endogenous free IAA levels, and the plant exhibited hypocotyl elongation [YUCCA genes have been identified, with a crucial role in crown root development  [OsNAL7/OsYUCCA8) [OsCOW1) [OsYUCCA4) [YUCCA genes were isolated from soybean, though with different naming rules [GmYUCCA2 (Glyma.03G208900) and GmYUCCA21 (Glyma.19G206200), identified in 2017 [GmYUC5-8-9a (Glyma.10G041800) and GmYUC5-8-9b (Glyma.13G128800) [YUCCA gene family. Overexpression of GmYUCCA5 (Glyma.04G070100) in Arabidopsis displayed downward curling of the leaf-blade margin, evident apical dominance, higher plant height, and shorter length of siliques [GmYUC2a (Glyma.08g038600) acted as a vital part in regulating both root growth and nodulation by modulating the auxin balance in soybean [To date, genome-wide identification of thaliana , Oryza sa sativa , Zea mayZea mays , Cucumis sativus , Glycinecine max , medicaguncatula , Populuschocarpa , Lilium  [Lilium , malus domestica , Triticuestivuml , Gossypiium spp. , and Braca napus . Howevern of IAA . Subsequ1,2,4,6) , embryog4,10,11) , hypocot[AtYUC5) , and the[AtYUC9) . AtYUC6 chanisms . AtYUC6 chanisms . While reratures . Under hsYUCCA1) , leaf mosYUCCA8) , maintai[OsCOW1) , and auxsYUCCA4) . Two resng rules ,20. Ther21 Glyma.G206200, 21 Glyma.G206200, YUC4-like protein regulated the chasmogamous flower trait in mungbean [YUCCA genes in developmental regulation. In this study, we systematically identified 12 YUCCA family genes (named as VrYUCCA) in the mungbean genome and investigated their gene structures, expression patterns, biological activity in vitro and in vivo, and potential roles under abiotic stresses. This research can provide valuable information about the functional explanation of the YUCCA gene family in mungbean, contributing to the application of these genes in mungbean genetic improvement.Mungbean is an important warm-season grain legume with rich nutrition and short duration, facilitating environmental sustainability . Our premungbean , indicatVrYUCCA genes were isolated from the mungbean genome , seedling true leaf (STL), compound leaf (CL), stem (St), young inflorescence (YI), flower bud (FB), opening flower (OF), 7-day pod (P7), and seed (Se), using real-time PCR , VrYUCCA6a (OF), VrYUCCA8b (OF), VrYUCCA8c (OF), and VrYUCCA11 (FB and OF). These results suggest that functional redundancy and divergence exist in the VrYUCCA gene family, and during different stages of growth or organs, distinct VrYUCCAs might play a major role.To explore the potential biological functions of time PCR . SeveralVrYUCCAs were overexpressed in Arabidopsis  and driven by the 35S strong promoter. The T1 generation of most VrYUCCAs transgenic plants showed typical auxin-related phenotypes , high salinity (200 mM NaCl), drought (15% PEG6000), heat (35 °C), and chilling (4 °C) for some time. The expression of VrYUCCA11 could not be detected well in the root and leaf, even under these stresses, due to its specific expression in flower organ. As shown in VrYUCCA2b, VrYUCCA6b, VrYUCCA8a, and VrYUCCA10 were inhibited markedly in the root by IAA, while VrYUCCA8b was induced. In the leaf, only the expression of VrYUCCA8a showed a highly significant increase.To investigate the response of VrYUCCA genes showed a visible increase and decrease under high-salinity treatment  was reported to be responsible for the mungbean chasmogamous mutant (CM) [VrYUCCAs during plant growth and development. In this study, we identified 12 VrYUCCAs in the mungbean genome, named based on their similarity to AtYUCCAs. Homologous genes of AtYUC1, AtYUC5, AtYUC7, and AtYUC9 were absent, but two each for AtYUC2, AtYUC4, and AtYUC6 and three for AtYUC8 were isolated in mungbean , excepts genome . Reportsant (CM) , indicatmungbean . This phVradi06g1650 was rpatterns , indicatYUCCA gene family. Subfamily I (YUC2–YUC6 group) and subfamily II (YUC1-YUC4 group) were reported to function in the inflorescence apex and flower in Arabidopsis, and AtYUC4 was specifically expressed in the floral meristems [VrYUCCA4b that had developmental defects in its flower organs [VrYUCCA4b (Vradi06g12650) seems to have a similar role in floral development, which is also highly expressed in young inflorescence  [YUCCA paralogous genes have functional redundancy to ensure normal plant development. Additionally, in Isatis indigotica Fort, IiYUCCA1, and IiYUCCA4 were also mainly detected in reproductive organs [Brassica napus, BnYUC4 was also highly expressed in early flower buds [YUC3-7-8-5-9 group) showed distinct expression patterns during root development in Arabidopsis [YUCs (YUC3-7-8-5-9), a multiple mutant (yucQ) could be rescued by expressing a YUCCA gene in subfamily III rather than in other subfamily members [VrYUCCA3 was detected in its seeds , which was assumed to be the most conserved group of YUCCAs in the plant kingdom [AtYUC10 and AtYUC11 mainly expressed in siliques and apices and were suggested to have overlapping functions with AtYUC1 and AtYUC4 during embryogenesis [VrYUCCA10 was the highest in all tissues , and transgenic Arabidopsis lines of 35S::CsYCU11 displayed defective pollens and reduced fertility [PpYUC11 increased during the late ripening stage in melting flesh peaches, implying its vital function during fruit ripening [YUCCA11 homologous genes increased in several Rosaceae species, such as apple (six YUCCA11) and white pear (seven YUCCA11). MdYUCCA11a, MdYUCCA11b, and MdYUCCA11d showed high expression in young fruit, the receptacle, and the pistil, respectively [YUCCA in other species, distinct VrYUCCA participated in specific biological processes with redundancy and differential functions.The 12 VrYUCCA proteins could be divided into four groups , possesseristems . We prevr organs . VrYUCCArescence . In Arabertility ; in mung mutant) . Thus, Ye organs . In Braswer buds . Thus, tbidopsis ,42. Inte members . At the ts seeds , implyinVradi06g150 seems  tissues , hintingand pods . In planripening . The numectively . Thus, lArabidopsis YUCCA1 protein could catalyze the N-hydroxylation of TAM to form N-hydroxy-TAM in vitro [VrYUCCAs transgenetic Arabidopsis lines all showed typical IAA over-accumulation defects , but the siliques showed apex density. Whether this line has special potential advantages during developmental processes or under stress need to be studied further.In this study, we verified 12 VrYUCCA proteins’ enzyme activity both in vivo and in vitro. The substrate of YUCCA was controversial. Initially, it was demonstrated that the in vitro . Meanwhiin vitro ,45. Subsin vitro ,46,47. H defects , indicatYUCCA expression. Here, five VrYUCCA genes responded to IAA stimulation, which mainly functions in the root. Notably, only VrYUCCA8a was induced in the leaf, but it was significantly inhibited in the root  in the promoter of YUCCA8 to activate its expression, which was deemed a conserved molecular mechanism [CsYUC8 and CsYUC9 were selectively upregulated after being exposed to 38 °C for 3 h [VrYUCCA8a and VrYUCCA8b were markedly induced by low temperatures rather than heat, similar to that in Populus [VrYUCCAs have a dual role, like AtYUC6 [Auxin, a central regulator involved in almost all aspects of the plant life cycle, helps plants to adaptive external stresses ,48. YUCCthe root , indicateatments ,22, indiresponse ,31. In treatment . VrYUCCAechanism . In cucue AtYUC6 , and traYUCCA gene family is relatively conservative among plants, and the homologous genes in different species tend to act similarly in their roles during plant growth and stress responses. The crucial role of YUCCAs in the auxin pathway is beyond doubt, so further in-depth research should be conducted to investigate its functional mechanism and application value.Overall, the http://www.plant.osakafu-u.ac.jp/~kagiana/gcorn/p/index18.html) (accessed on 1 September 2020) [YUCCA information in mungbean genome. Second, the amino acid sequences of the 11 Arabidopsis YUCCA (AtYUCs) were downloaded from TAIR (https://www.arabidopsis.org/index.jsp) (accessed on 1 September 2020) and then used as BlastP queries against mungbean genome database (http://plantgenomics.snu.ac.kr/mediawiki-1.21.3/index.php/Main_Page) (accessed on 1 September 2020) to reconfirm the VrYUCCAs. Then, the online tool SMART (http://smart.embl-heidelberg.de/) (accessed on 1 October 2020) was used to confirm whether the predicted VrYUCCA proteins had the FMO-like domain. Finally, the sequences information of VrYUCCAs was downloaded from NCBI (https://www.ncbi.nlm.nih.gov) (accessed on 1 October 2020). The online tool GSDS (http://gsds.gao-lab.org/) (accessed on 1 October 2020) was used to determine the gene structure. The online MEME suite (https://meme-suite.org/) (accessed on 1 October 2020) was used to identify the conserved motifs shared among VrYUCCA proteins, which were then visualized with TBtools, and the repeats logos were obtained through WEBLoGo (http://weblogo.threeplusone.com/create.cgi) (accessed on 1 October 2020). The theoretical pI (isoelectric point) and Mw (molecular weight) of VrYUCCAs were predicted using ExPASY (https://www.expasy.org/) (accessed on 1 October 2020). BaCelLo (http://gpcr.bio-comp.unibo.it/bacello/index.htm) (accessed on 1 October 2020) was used to predict the subcellular localization. Multiple sequence alignment of the identified VrYUCCA sequences was performed using MEGA-X and ClustalX2 and then visualized using ESPript 3.0 (https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi) (accessed on 1 October 2020). Multiple methods were used to identify mungbean YUCCAs. Firstly, Gcorn plant (er 2020) , a databArabidopsis (11), Oryza sativa (14), Glycine max (24), and Medicago truncatula (14) were downloaded from TAIR, RAP-DB (The Rice Annotation Project Database), NCBI, and phytozome (https://phytozome-next.jgi.doe.gov/) (accessed on 1 November 2020), respectively. The phylogenetic tree was constructed using MEGAX software with the neighbor-joining (NJ) method with 1000 bootstrap replicates. Additionally, it was optimized on the website iTOL (https://itol.embl.de/) (accessed on 1 November 2020).The amino acid sequences of YUCCA from The seedlings of mungbean cultivar ‘Sulyu 1’ were grown in the illumination incubator at 25 °C with 16 h light/8 h dark photoperiod. Adult mungbean was planted in experimental plots at Jiangsu Academy of Agricultural Sciences, Nanjing, China .VrYUCCA expression pattern analysis using real-time PCR.Seedling root, seedling true leaf, stem, compound leaf, young inflorescence, flower bud, opening flower, pod (7 d), and dry seed were sampled from mungbean seedlings (12 d) or adult plants. All the tissues were immediately frozen in liquid nitrogen, and each sample was collected independently three times for further VrYUCCA genes was then examined by real-time PCR.For IAA treatment, 12-day-old mungbean seedlings were soaked in liquid MS (Murashige and Skoog) medium with 1 μM IAA and liquid MS medium only (as the control) for 4 h. The treated roots and leaves were sampled separately after flushing with ddH2O and immediate freezing in liquid nitrogen. Treatment was repeated three times. The expression of w/v) polyethylene glycol (PEG) 6000 for 6 h and 24 h, respectively. The seedlings soaked in liquid MS medium served as the control, and the treated roots and leaves were sampled separately after flushing with ddH2O and immediate freezing in liquid nitrogen. Treatment was repeated three times. Then, the expression of VrYUCCA genes was analyzed using RNA-seq .For high-salinity and drought treatments, 12-day-old mungbean seedlings were soaked in liquid MS medium with 200 mM NaCl and 15% .For high- and low-temperature treatments, 12-day-old mungbean seedlings were placed in an incubator at 35 °C and 4 °C, respectively, for 24 h. The seedlings grown under 25 °C were taken as a control. Additionally, the treated leaves were sampled and immediately frozen in liquid nitrogen. The expression of ® RT6 cDNA Synthesis Kit Ver.2 . Real-time PCR was performed using ChamQ SYBR qPCR Master Mix  on an ABI prism 7500 real-time PCR System. Mungbean VrACTIN3 (Vradi03g00210) and Arabidopsis ACTIN2 (AT3G18780) were selected as reference genes to normalize the expression data. The 2−ΔCT or 2−ΔΔCT methods were used to analyze relative expression levels. The results were calculated from three biological replicates and three technical replicates. Statistical analysis and plot drawing were conducted using Excel and GraphPad Prism 5 software. Heat maps were drawing by TBtools after the FPKM value taken logarithm (LOG 2). The primers used in this study are listed in Total RNA was extracted using Polysaccharide polyphenol Plant total RNA Extraction Kit , and cDNAs were synthesized using GoldenstarVrYUCCA gene was ligated into a pMAL-c2x expression vector . Then, the recombinant VrYUCCA proteins were expressed in E. coli  and purified by dextrin Sepharose affinity chromatography , in accordance with the instructions described previously [The full-length CDS sequence of each eviously . The proThe enzymatic assays were conducted by referring to a previous study . A totalVrYUCCA gene was cloned into a pCAMBIA1305.1-GFP vector through XbaI and BamHI sites, to create a fusion construct under the control of the CaMV 35S promoter. The fusion vectors 35S::VrYUCCAs-GFP were transformed into Agrobacterium tumefaciens strain GV3101 and then were introduced into the Arabidopsis thaliana (Col-0) wild type (WT) using the floral dipping method described previously [Arabidopsis were screened on 1/2 MS culture media containing 15 mg/L hygromycin antibiotics. Phenotypic analysis was performed in the progeny-positive T1 lines.The CDS sequence of each eviously . The seeVrYUCCAs were systematically identified in mungbean, containing the six conserved motifs of the YUCCA gene family. The 12 VrYUCCA genes could be divided into four subfamilies according to the phylogenetic analysis. The VrYUCCAs showed different tissue expression patterns, and five had a highly specific transcript in the floral organs, including VrYUCCA4b, VrYUCCA6a, VrYUCCA8b, VrYUCCA8c, and VrYUCCA11. VrYUCCA2a, VrYUCCA6b, VrYUCCA8a, and VrYUCCA10 seemed to regulate the IAA content throughout the whole growing period, due to the constitutive expression pattern in all the tissues. The VrYUCCA proteins participated in the IPA pathway for IAA synthesis in vitro and exhibited veritable biological activity to the IAA product in vivo, which was certified by 35S::VrYUCCAs transgenetic Arabidopsis. Five YUCCAs in the root responded to IAA, but only VrYUCCA8a responded in the leaf. Regarding the other four stresses, VrYUCCA2a, VrYUCCA6a, VrYUCCA8a, VrYUCCA8b, and VrYUCCA10 might be mainly responsible for auxin regulation, due to their sensitivity to all four treatments used in this study.In this study, 12


This paper reports the powder X-ray diffraction patterns, argon isotherms at 87K, Brunauer–Emmett–Teller surface areas, pore size distributions, pore volumes,skeletal densities, and thermal gravimetric analyses for three NationalInstitute of Standards and Technology zeolitic reference materials, RM 8850(zeolite Y), RM 8851 (zeolite A), and RM 8852 (ZSM-5). They are well characterized, withreference values for chemical composition , loss on ignition (LOI), and loss on fusion (LOF), andinformational values for enthalpies of formation, particle size distributions,refractive indices, unit cell parameters, and mass variation with change in relativehumidity ⋅245.1H2O[1et al. [2] determined the formulasfor the zeolites from the reference values of the mass fraction percentagesfor the major components  and LOI and LOF values. Turner Zeolite Y was introduced as an acidic zeolitic catalyst for the cracking ofhydrocarbons in the 1960s ⋅209.4H2O⋅26.7H2O) , so it ireatment .The TGA plots of mass (%) as a function of temperature for the three as-is NISTreference zeolites are shown in et al. [vs.humidity for RM 8852 indicated its mass change is more sensitive to % RH (with up to1.3% mass change from 32% to 54% RH) than RM 8850 and RM 8851 [et al. [2/ZSM-5 isotherm at 20 °C after taking into account thesmall amount of mesoporosity and deammoniation of the ammonium ions duringpretreatment.molecular formula of the hydrated at 54% RH) forms of the zeolites (see Sec. 1):23.3%, 19.6%, and 6.7% mass water for RM 8850, RM 8851, and RM 8852, respectively.In the case of RM 8852, deammoniation may occur during activation , and the4% RH for [et al.  has sugg4This article reports additional characterization of the NIST zeolitic referencematerials RM 8850, RM 8851, and RM 8852. In particular, the powder X-ray diffractionpatterns, Ar isotherms at 87 K, surface areas, pore size distributions, porevolumes, skeletal densities, and thermal gravimetric analysis profiles of NIST RM8850, RM 8851, and RM 8852 are reported. Along with the Reports of Investigation,and previous reports, these data should be helpful to laboratories interested inusing the NIST zeolitic reference materials.


Among these, 27 novel associations were detected for AD, 34 for PD and 40 for MDD. Out of 18,781 genes with annotated variants within ±10 kb, 62 genes were enriched for associations with AD, 70 with PD and 125 with MDD (p < 2.7 × 10−6). Of these, seven genes were novel susceptibility loci for AD , two for PD  and two for MDD . The resulting network showed a significant excess of interactions (enrichment p = 1.0 × 10−16). The novel genes that were identified are involved in the organization of cytoskeletal architecture , telomere shortening (PIF1), the regulation of cellular aging  and neurodevelopment (EFNA3), thus, providing novel insights into the shared underlying biology of brain disorders and platelet parameters.Among candidate neurodegenerative/neuropsychiatric risk-predictive biomarkers, platelet count, mean platelet volume and platelet distribution width have been associated with the risk of major depressive disorder (MDD), Alzheimer’s disease (AD) and Parkinson’s disease (PD) through epidemiological and genomic studies, suggesting partial co-heritability. We exploited these relationships for a multi-trait association analysis, using publicly available summary statistics of genome-wide association studies (GWASs) of all traits reported above. Gene-based enrichment tests were carried out, as well as a network analysis of significantly enriched genes. We analyzed 4,540,326 single nucleotide polymorphisms shared among the analyzed GWASs, observing 149 genome-wide significant multi-trait LD-independent associations ( Platelets have represented, for decades, an interesting setting to investigate the biological underpinnings of neuropsychiatric and neurodegenerative disorders since they are considered “circulating mirrors of neurons” . Indeed,Epidemiological and genetic evidence also supports the existence of a bridge between platelet traits and neurodegenerative/neuropsychiatric disorders, such as Alzheimer’s disease (AD), Parkinson’s disease (PD) and major depressive disorder (MDD), which often suffer from a lack of risk-predictive biomarkers . Among cKALRN , encoding a PINK1 interactor previously implicated in schizophrenia, AD and PD [More recently, genomic studies investigated the genetic correlation-or single nucleotide polymorphism (SNP)-based co-heritability-of platelet traits and brain disorders, as well as potential causality links through Mendelian randomization (MR) approaches. In a large genome-wide association study (GWAS) of blood cell measures, Astle et al. performed a multivariable MR analysis on platelet parameters and MDD risk, which revealed no significant causal effect of the formers on the latter, although MPV and PDW showed marginally significant effects . Later oD and PD .Here, we aimed to identify novel genetic associations with AD, PD and MDD using a multi-trait association analysis (MTAG) approach , exploitp < 5.0 × 10−8; pairwise r2 < 0.6 in a 1 Mb window). Then, we selected novel genome-wide significant SNP associations with the analyzed disorders, defined as associations not detected as genome-wide significant in the original study, nor in previous GWASs of the same disorder, based on the GWAS catalog and literature search until 30 April 2022 (We used MTAG  to perforil 2022 .−6)  .To estimate protein–protein interactions (PPIs) among the genes enriched for associations, we used the search tool for the retrieval of interacting genes/proteins in db-STRING v11.5 . We analp < 5 × 10−8; pairwise r2 < 0.6; p = 2.36 × 10−180), 70 for PD  and 139 for MDD . Among these, we observed 27 novel associations with AD , 34 with PD  and 40 with MDD . Of these SNPs, seven were mapped in novel susceptibility genes for AD , two for PD (SLC26A1 and EFNA3) and two for MDD (HSPH1 and TRMT61A). The analysis of 18,781 genes tested in the gene-based enrichment analysis revealed 62, 70 and 125 genes with a significant enrichment of associations with AD, PD and MDD, surviving correction for multiple gene testing , 14 with PD  and 38 with MDD . None of these genes were overlapping across the three disorders. However, when we checked the overlap between each neurodegenerative/neuropsychiatric disorder and platelet parameters in terms of single variant associations, we found 24 SNPs associated with AD and 3 associated with PD, which were also associated with 1 or more platelet parameters. Of these, 12 AD- and 3 PD-associated SNPs represented novel associations never detected before  and three different platelet parameters , we analyzed 4,540,326 variants shared across the different source studies that passed QC (see Methods section). Given the main aim of the manuscript, we report in detail below the results of the associations with AD, PD and MDD, along with the overlap with platelet parameters in terms of single variant and the gene enrichment of associations. The full results of the MTAG analysis of platelet traits are reported in d before a,b. As fd before a–c.p = 1.0 × 10−16; 57 nodes, 57 edges vs. 2 expected, average node degree 2.00 and average local clustering coefficient 0.36), PD  and MDD    . Similarent 0.3) .negative regulation of amyloid precursor protein catabolic process  = 2.23(0.26); enrichment p after Bonferroni correction = 2.7 × 10−13) for AD, IgG binding  = 0.03(0.31); Pbonf = 0.0056) for PD and GABAergic synapse  = 0.74(0.13); Pbonf = 9.5 × 10−5) for MDD  term was APOE  [SNCA  [ZSCAN12  [ZSCAN12 encodes a Zinc finger and SCAN domain-containing protein involved in transcriptional regulation.We report the first multi-trait association analysis of structural platelet parameters routinely assessed in blood tests and three of the most common neurodegenerative/neuropsychiatric disorders, identifying novel candidate susceptibility genes for AD, PD and MDD. The most significant associations were detected in some of the most implicated genes in neurodegenerative/neuropsychiatric disorders, namely, with AD) , SNCA (awith PD)  and ZSCAith MDD) . APOE isith MDD) . Alpha sith MDD) ,44; moreith MDD) . ZSCAN12EPPK1, TTLL1, PACSIN2 and TPM4 play a role in the organization of cytoskeletal architecture, which has been identified as an important component in the development of neurodegenerative disorders [PIF1 prevents telomere elongation by inhibiting the action of telomerase, while ZNF689 and AZGP1P1 are transcription factors involved in cell viability and apoptosis, and both molecular functions can affect cellular aging and the development of age-related disorders such as AD and PD [TTLL1 [PACSIN2 [TPM4 [PIF1 [EFNA3, a novel gene resulting in an association with PD, encodes a member of the ephrin family, previously implicated in mediating developmental events, especially in the central nervous system [Still, MTAG analysis also revealed novel genes showing significant multi-trait associations, which, to our knowledge, were never associated with these disorders before. Among genes associated with AD, isorders ,47,48,49D and PD . MoreoveD [TTLL1 , PACSIN2[PACSIN2 ,40, TPM4N2 [TPM4  and PIF1M4 [PIF1  were pres system .A gene-based enrichment analysis also revealed novel genes associated with AD, PD and MDD. Interestingly, among these genes are several encode transcription factors that may be involved in development, maintenance and survival of neurons and olfactory receptors . Indeed,NDUFS2, NDUFAF2 and TOMM40L), cytoskeleton remodeling  and histone proteins . Indeed, NDUFS2 and NDUFAF2 encode for a subunit and for a chaperone involved in the assembly of complex I, located on the inner mitochondrial membrane, while TOMM40L is involved in mitochondrial transmembrane translocation. Several studies suggest that platelet mitochondrial dysfunction may be involved in neurodegenerative diseases such as AD and PD [Of note, we found several overlaps between genes and SNPs multi-trait associations with the brain disorders and platelet parameters analyzed. Among genes enriched for association, we identified clusters of genes encoding products involved in mitochondrial function  immunity [In particular, this highlighted some local networks of interests, such as the one among the histone proteins complex-patterns . Similarpatterns ,60, as wpatterns  and cardpatterns , while timmunity .Gene-set analysis revealed significant enrichments of GO terms involved in the regulation, formation and catabolic processes of amyloid beta and in the negative regulation of metalloendopeptidase activity for AD, supporting the hypothesis that metallopeptidases are implicated in the pathogenesis of several central nervous system diseases such as multiple sclerosis and AD . For PD,Overall, we provide insights into the shared underlying biology of these disorders and related platelet parameters, proposing novel molecular targets for the risk prediction and treatment of these disorders.The strengths of this study include the novelty of the analysis performed; indeed, to our knowledge, this represents the first attempt to identify the shared genomic underpinnings of platelet parameters and three of the most common neurodegenerative/neuropsychiatric disorders through a comprehensive approach, including not only multi-trait association analysis but also gene-/gene-set enrichment and molecular network analyses. Moreover, our analyses are based on large GWASs, with an important amount of genetic data, which confers robustness to our observations. Last, our focus on novel associations allowed identifying proteins and biological pathways, which should be functionally validated in the future.This study presents some limitations. First, currently only partial evidence of genetic correlation among the disorders and platelet parameters tested exists, which may have hampered the power of the analyses. Second, in multi-trait association approaches, association significance is often driven by the largest source GWAS involved in the MTAG analysis, which may have biased the analyses towards the largest studies. Still, this represents a useful approach to identifying the pleiotropic variants and genes influencing multiple traits and/or disorders, which were already proven successful with multiple correlated phenotypes . Third, However, although there are some limitations, these studies may reveal potential molecular targets for future treatments of three of the most common neurodegenerative/neuropsychiatric disorders.