Development of new treatment strategies for endometrial cancer that has become refractory or resistant to taxane/platinum is a critical need. The present study was a phase I/II study of gemcitabine, levofolinate, irinotecan, and 5-fluorouracil (5-FU) (GLIF) combination chemotherapy to determine optimal dosages, safety, and efficacy.Taxane/platinum-resistant or -refractory endometrial disease was defined as tumor progression within 6 months after a taxane/platinum-based regimen. Maximum tolerated dose was investigated by a 3 + 3-designed phase I study. The phase II study was conducted using the recommended doses determined in the phase I study.2, levofolinate 100 mg/m2, irinotecan 80 mg/m2, and 5-FU 1000 mg/m2. Thirty patients were enrolled, including the three patients who received GLIF therapy at the same dose as the recommended phase II dose in the phase I study. Two patients were excluded at this point due to study protocol violations, and the remaining 28 patients were included for analysis. Phase II revealed that the response and disease control rates were 7.1% (2/28) and 39.3% (11/28), respectively, and that the median PFS and OS were 3 months [95% confidence interval (CI) 3–7] and 12 months (95% CI 9–17), respectively. Febrile or grade 4 neutropenia was observed in 14% (4/28) of the cases. Grade 3 or 4 thrombocytopenia was not observed.The dosages recommended for the phase II trial were determined, in the phase I trial, to be: gemcitabine 800 mg/mWe found that GLIF combination chemotherapy is potentially a useful treatment option for endometrial cancers refractory or resistant to taxane/platinum-based chemotherapy. The incidence of endometrial carcinoma has been increasing. In most patients the tumor is still confined to the uterus at first diagnosis, and there is therefore a good prognosis. However, the prognosis for advanced or recurrent endometrial carcinoma becomes extremely poor . In partA combination chemotherapy using cisplatin/doxorubicin (AP) has long been the standard regimen for advanced or recurrent endometrial cancer . RecentlPrevious studies have shown that irinotecan and gemcitabine exhibit good response against advanced or recurrent endometrial cancer cases, respectively, although those cases were not yet resistant to taxane or platinum –7. In a This GOGO-EM2 study, approved by the institutional review board of Osaka University Hospital, was conducted from July 2011 to February 2016. Written informed consent was obtained from all patients.Patients with taxane/platinum-refractory or -resistant endometrial cancer were eligible for the study. Other eligibility criteria included: age ≥ 20 years, Eastern Cooperative Oncology Group performance status of 0–2, measurable or evaluable disease, a life expectancy ≥ 3 months, and adequate hematological, renal, and hepatic function. Taxane/platinum-resistant or -refractory disease was defined as progression during or within 6 months after a taxane/platinum-based regimen.The exclusion criteria included: patient with a *28 homozygous or *6 homozygous UGT1A1 gene polymorphism, hypersensitivity to chemotherapeutic drugs, interstitial pneumonia, ascites or pleural effusion that required drainage, active clinically significant inflammatory disease, duplicated cancer, symptomatic brain metastasis, uncontrolled diabetes mellitus, diarrhea, ileus, cardiac disease, edema, or patients whose circumstances did not permit them to complete the study.2 body surface area) over the course of 30 min, and irinotecan (80 mg/m2) over the course of 90 min, on days 1 and 15, respectively. Levofolinate (100 mg/m2 over 120 min) was administered intravenously on days 1, 2, 15, and 16. For 5-FU, an intravenous bolus of 400 mg/m2, followed by continuous infusion of 600 mg/m2 over the course of 480 min, was administered on days 1, 2, 15, and 16, respectively. This schedule was repeated every 4 weeks. Granulocyte colony-stimulating factor (G-CSF) was used to support hematopoiesis when grade 4 neutropenia, or grade 3 neutropenia with fever, was observed. A 3 + 3 study design was used for dose escalation. The first and second dose escalation indicated 800–1000 mg/m2 and 1200 mg/m2, respectively, for gemcitabine and 80–100 mg/m2 and 120 mg/m2, respectively, for irinotecan. The first and second dose reduction indicated 800–600 mg/m2 and 500 mg/m2, respectively, for gemcitabine, and 80–60 mg/m2 and 50 mg/m2, respectively, for irinotecan.The treatment schedule we used is shown in Fig. 3, (ii) the pretreatment platelet count was < 75,000/mm3, (iii) patients had diarrhea, of any grade, 24 h prior to chemotherapy, (iv) patients had fever ≥ 38 °C, (v) AST or ALT > 2.5 × the upper limit of normal (ULN) was observed, (vi) serum bilirubin > 1.5 × ULN was observed, (vii) pre-treatment serum creatinine level was elevated, or (viii) grade 3/4 non-hematologic toxicity occurred.Toxicity was assessed after every treatment cycle, graded according to the Common Terminology Criteria for Adverse Events (CTCAE), version 4.0 . Dose liFor the 3 + 3 design, the first three patients are treated by the starting dose level (Level 0). If no DLT occurs, the dose is escalated for the next cohort of three patients. If one DLT occurs, three additional patients are treated at the same level with dose escalation only if no additional DLT occurs. If two or three DLTs occur, the prior dose level is defined as MTD and recommended as the phase II dose.Eligibility and exclusion criteria were the same as in the phase I study. Any and all patients who received GLIF therapy at the same dose as the standard phase II dose in the phase I study were included in the analysis for the phase II study.2 body surface area) over the course of 30 min and irinotecan (80 mg/m2) over the course of 90 min on days 1 and 15, respectively. Levofolinate (100 mg/m2) over 120 min was administered intravenously on days 1, 2, 15, and 16. For 5-FU, an intravenous bolus of 400 mg/m2, followed by continuous 600 mg/m2 over the course of 480 min, was administered on days 1, 2, 15, and 16, respectively. This schedule was repeated every 4 weeks, until disease progression was confirmed by diagnostic imaging.Patients received intravenous gemcitabine  the pretreatment platelet count was < 75,000/mm3, (iii) patients had diarrhea of any grade 24 h prior to chemotherapy, (iv) patients had fever ≥ 38 °C, (v) AST or ALT > 2.5 × ULN was observed, (vi) serum bilirubin > 1.5 × ULN was observed, (vii) pre-treatment serum creatinine level was elevated, or (viii) grade 3/4 non-hematologic toxicity was observed. Patients who failed to recover adequate counts within a 2-week delay were to discontinue cytotoxic chemotherapy.Toxicity was assessed every treatment cycle and graded according to CTCAE. Subsequent cycles were delayed up to 2 weeks if (i) the pretreatment neutrophil count was < 1500 cells/mmDose adjustments were performed for hematological and other adverse events. A 20% dose reduction of gemcitabine, irinotecan, and 5-FU was done for grade 4 hematologic toxicity in the previous cycle, for diarrhea (grade 2 or worse), or for grade 3 or other non-hematologic toxicity. If these adverse events repeatedly occurred after level 1 dose reduction, another 20% dose reduction was done. The patient had to go off protocol if the following events occurred: (i) disease progression confirmed by a diagnostic imaging, (ii) hematologic/non-hematologic toxicity that violated drug administration criteria—despite a level 2 dose reduction, (iii) allergic reaction (grade 3 or worse), (iv) interstitial pneumonia, (v) hematologic/non-hematologic toxicity failed to recover within a 2-week delay, (vi) grade 4 non-hematologic toxicity, or (vii) patient’s voluntary withdrawal of participation in this study.The tumor response to treatment was evaluated by CT scan images after three chemotherapy cycles, according to RECIST guidelines, version1.1. Complete response (CR) was defined as disappearance of all target lesions, and partial response (PR) as at least a 30% decrease in the sum of diameters of target lesions, taking as reference the baseline sum diameters. Progressive disease (PD) was defined as at least a 20% increase in the sum of diameters of target lesions or the appearance of a new lesion, and stable disease (SD) as neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD. Those cases that had confirmed new lesions before completion of three cycles of chemotherapy were also defined as PD.Primary outcomes were response rate and toxicity. Secondary outcomes included progression-free survival (PFS) and overall survival (OS). PFS was defined as the interval from registration to the date of diseases progression—confirmed by diagnostic imaging. OS was defined as the interval from registration to the last follow-up or death. The median PFS and OS were estimated using the Kaplan–Meier method. Statistical analysis was conducted using JMP Pro 13 statistical software .2 for gemcitabine and 80 mg/m2 for irinotecan (Level 0). No patient encountered a DLT at these dose levels; therefore, the doses were escalated to 1000 mg/m2 for gemcitabine and 100 mg/m2 for irinotecan, respectively (Level 1). Two of the three patients encountered DLTs. Thus, Level 0 was considered to be the MTD. We proceeded with this as the recommended phase II dosage.During the phase I component of this study, assessable patients were enrolled to receive each dose level Table . InitialFrom November 2011 to July 2015, 30 patients were enrolled, including the 3 patients who received GLIF therapy at the same dose as the recommended phase II dose in the phase I study. Two patients were excluded at this point due to study protocol violations, and the remaining 28 patients were included for analysis. The characteristics of these 28 patients are summarized in Table In total, 28 patients received 143 cycles of GLIF. Their treatment response, after three cycles of chemotherapy, is shown in Table The results of hematologic and non-hematologic toxicities are shown in Table Advanced and recurrent endometrial cancers generally carry a poor prognosis. In particular, progression during treatment and shorter treatment-free intervals (TFIs) before progression are associated with poor prognosis. We were among the first to provide evidence that the majority of refractory or resistant diseases, if they progress during a first-line taxane/platinum chemotherapy (with or without anthracycline), or recur within 6 months of taxane/platinum, are non-responsive to the current regimens of second-line chemotherapy . We alsoAlthough multiple-agent chemotherapy regimens that include platinum- or taxane-based chemotherapeutic agents, such as carboplatin/paclitaxel (TC), cisplatin/doxorubicin (AP), cisplatin/doxorubicin/paclitaxel (TAP), or carboplatin/docetaxel (DC) are preferred for patients with recurrent endometrial cancer , 16, theThe efficacy of AP as a second-line regimen for recurrent endometrial cancer has been investigated previously . The medIn the present study, a GLIF combination chemotherapy, consisting of gemcitabine, levofolinate, irinotecan, and 5-FU, was found to be both safe and effective for taxane/platinum-resistant or -refractory endometrial disease. Response and disease control rates of 7.1% tended to be better than the 0% rate for patients with a TFI of < 6 months found in our previous retrospective study  of varioRecent phase-II studies have investigated the efficacy of molecular-targeted drugs for advanced/recurrent endometrial cancer , 19. TheIn conclusion, we have demonstrated that GLIF combination chemotherapy was potentially beneficial and could be a treatment option for patients with advanced or recurrent endometrial cancer that is not sensitive to taxane/platinum regimens.


MT mainly affected the production of C6 aromatic substances in the two varieties. In ‘Korla’, MT inhibited expression of PbHPL, and reduced hydroperoxide lyase (HPL) activity and content of hexanal and (E)-hex-2-enal. In contrast, MT inhibited activity of lipoxygenase (LOX), reduced expression of PbLOX1 and PbLOX2, promoted PbAAT gene expression, increased alcohol acyltransferase (AAT) activity, and increased propyl acetate, and hexyl acetate content in ‘Abbé Fetel’ that similarly led to the reduction in content of hexanal and (E)-hex-2-enal. Content of esters in ‘Abbé Fetel’ pear increased with increasing postharvest storage period. Although mechanisms differed between the two varieties, effects on aroma volatiles mediated by MT were driven by expression of genes encoding LOX, HPL, and AAT enzymes.Aroma affects the sensory quality of fruit and, consequently, consumer satisfaction. Melatonin (MT) is a plant growth regulator used to delay senescence in postharvest fruit during storage; however, its effect on aroma of pear fruit remains unclear. In this study, we assessed the effects of 0.1 mmol L N-acetyl-5-methoxytryptamine) is a nontoxic biological molecule produced by the pineal gland in animals and in different tissues in plants. Studies have identified a range of functions of MT in plants, including its scavenging of reactive oxygen species (ROS) that improves resistance to biotic and abiotic stresses, such as pathogen attack, extreme temperatures, salinity, drought, waterlogging, and low-sulfur [Melatonin (MT)  pathway [6 alcohols by alcohol dehydrogenase (ADH). Aroma esters are subsequently produced through alcohol acyltransferase (AAT) that catalyzes the final linkage of an acyl moiety and an alcohol. Although specific enzymes and genes involved in fatty acid-derived lactone (cyclic ester) formation have not been characterized [Pear varieties comprise two types: those that are ready to eat immediately upon harvest, where ethylene production has no effect on quality, and those that require ripening and softening triggered by an increase in respiration and ethylene production. ‘Korla’ pear is an example of the former. It is one of the most popular fruits in China  and is e pathway . LOX andcterized , studiescterized ,17.Here, we hypothesized that MT changes aroma components in pear fruit, because it inhibits the release of ethylene in postharvest fruit. We tested this hypothesis by analyzing the effects of MT on aroma components and indicators of the LOX pathway in two varieties of pear with contrasting ripening characteristics.There was no difference in the aroma maps of ‘Korla’ IAT and at PRC ; howeverAldehydes were the principal aroma components in ‘Korla’ pear , represeE)-hex-2-enal was similar to that of hexanal. MT increased ethyl ester content in ‘Korla’. IAT, MT had inhibited almost all volatile substances, but by the respiratory jump phase at PRC, content of propyl acetate, and hexyl ester had increased -hex-2-enal; ketones are 6-methylhept-5-en-2-one. Fifteen dominated in ‘Abbé Fetel’ pear IAT; among them, esters are ethyl acetate; propyl acetate; butyl acetate; 2-methylbutyl acetate; pentyl acetate; hexyl acetate; heptyl formate; alcohols are hex-2-yn-1-ol; 2-methylbutan-1-ol; hexan-1-ol; aldehydes are acetaldehyde; hexanal; 2-methylPent-4-enal; (E)-hex-2-enal; ketones is 6-methylhept-5-en-2-one. At PRC, content of four volatiles  were not detected -hex-2-enal, supporting previous research that similarly showed hexanal is the principal volatile in pear fruit [6 aldehydes and alcohols are characterized as green-note volatiles, and following treatment with MT, content of hexanal and (E)-hex-2-enal decreased in the two varieties. We showed that the fatty acid pathway produced C6 aldehydes and alcohols, indicated by linoleic and linolenic acids as precursors catalyzed by LOX. In ‘Korla’, MT increased content of linolenic and linoleic acids, but decreased content of hexanal. Although there was no difference shown in LOX activity, melatonin significantly downregulated HPL activity, indicating MT had reduced oxidation of the fatty acids that led to the lowered hexanal content. Although MT downregulated the PbLOX2 gene, there was no change in LOX activity in ‘Korla’ pear. HPL is known to catalyze the fatty acid hydroperoxide to generate hexanal and hexenal [PbHPL was downregulated IAT. These results indicate that MT regulates biosynthesis of aldehyde in ‘Korla’ pear by downregulating expression of HPL.When we analyzed the effect of MT on the aroma components in the two varieties of pear, we found they were dominated by hexanal and -hex-2-enal, reduced LOX activity, and downregulated PbLOX1 and PbLOX2 in ‘Abbé Fetel’ pear IAT, but there was no effect on content of linoleic and linolenic acids, HPL enzyme activity, or PbHPL gene expression. These results indicate that MT regulates content of (E)-hex-2-enal and hexanal by inhibiting LOX activity in ‘Abbé Fetel’ pear. Although MT effects on (E)-hex-2-enal and hexanal were consistent between ‘Korla’ and ‘Abbé Fetel’, the mechanisms differed, possibly due to variation in ethylene release driven by LOX activity that increases with ripening in ethylene-induced fruits; an increase in enzyme activity is a precursor to an increase in endogenous ethylene in climacteric fruits [In contrast, MT reduced content of , while higher ester content has been found in fruit treated with intermittent warming compared with 1-MCP treated fruit. Ethylene is positively related to ester content; however, in our study, while MT inhibited ethylene production, there was no effect on esters. The reason was that although melatonin inhibited ethylene production, it regulated the enzyme activity associated with aroma synthesis, activated activity of ADH and AAT, and promoted the conversion of alcohols to esters which ultimately increases content of esters in fruit. This is the main reason why the amount of aldehydes decreased after melatonin treatment, but the content of esters increased.We found that MT increased hexyl acetate content in ‘Abbé Fetel’ at PRC. ADH is a different class of green-note volatiles to Calcohols . With thd by AAT ,27, and d by AAT . Zhou etd by AAT  found th6 aldehydes, alcohols, and esters. In ‘Korla’, MT inhibited HPL activity and downregulated PbHPL expression to reduce content of hexanal and (E)-hex-2-enal, whereas in ‘Abbé Fetel’, MT inhibited LOX enzyme activity, downregulated expression of PbLOX1 and PbLOX2, and promoted activity of ADH and AAT that similarly reduced content of hexanal and (E)-hex-2-enal, and increased the content of propyl acetate, and hexyl acetate  and ‘Abbé Fetel’ (Pyrus communis L.) pear fruit were harvested from commercial orchards in Dali County, Shaanxi Province and Wugong County, Shaanxi Province, China, respectively, at commercial maturity on 3 September 2018. Treatments comprised immersion of 90 fruit of each variety in distilled water as the negative control (CK) or 100 μmol L−1 MT distilled water solution  at 25 °C for 12 h. ‘Korla’ fruit were treated immediately after harvest, whereas ‘Abbé Fetel’ fruit were initially stored at 4 °C and >95% relative humidity for 1 month, and treated subsequently. Following treatment, five replicates of 18 fruit per treatment were placed in sealed plastic bags, and stored at 25 °C. Treatment effects on all variables were measured immediately after treatment (IAT) and at the climacteric respiration peak of the CK fruit (PRC), unless stated otherwise. ‘Korla’ , and correction factor (CF) for each volatile standard are listed in Volatile compounds were determined using GC-MS based on solid-phase micro-extraction (SPME), according to a modified method reported by Zhang et al . We usedEthylene production from three replicates of 10 fruit per treatment was measured daily until the CK fruit had decayed, following the method described by Xie et al .g for 10 min. Supernatant was extracted, and residues were treated with 0.5 mL petroleum ether, before ultrasound extraction for 20 min and centrifugation at 10,000× g for 10 min. Two supernatants were combined and dried using N2, diluted to fixed volume (0.5 mL) with methanol, and then the solution was filtered into a sample bottle. The HPLC  liquid phase used a chromatographic column C18 , and the mobile phase conditions comprised 80:20 ratio of acetonitrile to 0.1% phosphoric acid, 204 nm wave length, and a column temperature of 30 °C.A sample of 0.5 g of fruit from each treatment was ground in a pre-cooled mortar with 1 mL of precooling reagent that comprised petroleum ether; the sample was then transferred to a new tube for ultrasound extraction for 40 min and centrifugation at 10,000× v/v) Triton X-100) was added, and then the mixture was centrifuged for 15 min at 4 °C and 15,000× g. The reaction system comprised 0.1 mM phosphate buffer (pH = 7.5), reaction substrate solution (8.6 mM linoleic acid and 0.25% (v/v) Tween-20 soluble in 0.1 mM phosphate buffer), and crude enzyme solution. Change in absorbance per min at OD280 was recorded 15 s after the addition of the enzyme solution, where each change in OD value was denoted as 1 U [LOX enzymes were extracted from 0.1 g of fruit tissue ground in liquid nitrogen using polyvinylpolypyrrolidone (PVPP), to which 1 mL of enzyme extract  glycerol, 4% PVPP, and 0.1mmol L−1 MPMSF (protease inhibitor). Then, samples were ground using a pre-chilled mortar and pestle, fully blended, and centrifuged at 15,000× g (4 °C) for 30 min. HPL activity was determined using a 3.5 mL reaction system, comprising 2 mL of analytical buffer , 0.75 mL of reaction substrate, 0.15 mL of 1.6 mmol L−1 NADH, 0.1 mL of ADH enzyme extract , and 0.5 mL of crude enzyme supernatant, and determined at a reaction temperature of 30 °C and wavelength of 340 nm. Sodium hydroperoxide linoleate was used as the substrate for determination of enzyme activity, and the reaction substrate comprised 10 mL of distilled water, 200 μL of 10 mmol L−1 sodium linoleate, 400 μL of LOX enzyme extract  that was heated in a water bath for 2 h at 30 °C. Change in absorbance per min at OD340 was recorded 15 s after addition of the enzyme solution, where each change in OD value was denoted as 1 U [HPL activity was determined following a modified version of the approach described by Vick , where 3d as 1 U . Enzyme 340 were recorded.The method of extraction of ADH enzymes was similar to that of LOX enzymes, where the reaction system comprised 0.15 mM NADH, 80 mM acetaldehyde, and enzyme extract. Changes in absorbance per min at ODg (4 °C) for 30 min. The reaction system, which comprised 0.5 mol·L−1 Tris-HCl buffer (pH = 7.0), 11.6 mmol·L−1 MgCl2, 0.3 mmol·L−1 acetyl-CoA, 10 mmol·L−1 butyl alcohol, and 0.6 mL of supernatant, was maintained at 35 °C for 15 min, before 150 μL of 20 mmol·L−1 dithio-bis-nitrobenzoic acid was added and kept at room temperature for 10 min. Then, AAT activity was measured at 412 nm 15 s after the enzyme solution had been added; changes in absorbance per 1 min at OD412 were recorded, where each change in OD value was denoted as 1 U .AAT acyltransferase activity was determined using a slightly modified version of the method described by EcheverrıÁ et al. , where 3Levels of expression of related genes were assessed as three biological replicates of 5 g composite samples of flesh taken from three fruit in each of the five replicates at the two time points (IAT and PRC); samples were frozen in liquid nitrogen and stored at −80 °C prior to analysis.Total RNA was purified using an RNAprep Pure Plant Kit , and RNA concentration was detected using UV spectrophotometry. Then, 1 mg of total RNA was reverse-transcribed to cDNA using a PrimeScript RTreagent kit with gDNA Eraser . Three replicates of qRT-PCR reactions were performed on an Icycler iQ5  with SYBR Premix Ex Taq II , according to the manufacturer’s instructions. Data were analyzed using iQ5 2.0 software  with the ddCT algorithm.https://www.ncbi.nlm.nih.gov/genome/12793) using BLAST searches of published sequences. For lipoxygenase genes, linoleate 13S-lipoxygenase 2-1 (XM_009356413.2) and linoleate 13S-lipoxygenase 2-1 (XM_009366759.2) were amplified and designated as PbLOX1 and PbLOX2, respectively; for fatty acid hydroperoxide lyase genes, fatty acid hydroperoxide lyase (XM_009348862.2) was amplified and designated as PbHPL; for alcohol dehydrogenase genes, alcohol dehydrogenase (XM_009362449.1), alcohol dehydrogenase-like 1 (XM_009350668.2), and alcohol dehydrogenase-like (XM_009374145.2) were amplified and designated as PbADH1, PbADH2, and PbADH3, respectively; and, for alcohol acyltransferase, methanol O-anthraniloyltransferase-like 1 (XM_018649523.1) was amplified and designated as PbAAT. Primers and references for actin, lipoxygenase genes, fatty acid hydroperoxide lyase genes, alcohol dehydrogenase genes, and alcohol acyltransferase related genes are listed in DNA coding sequences of candidate genes for RT-PCR were isolated from the pear genome (p < 0.05 in SPSS 23.0. Results are presented as means ± SDs of at least three replicate samples.Treatment differences were tested using the Student’s t test, at


Although all the members of this superfamily share a common conserved catalytic motif, the Nudix box, their substrate specificity lies in specific sequence traits, which give rise to different subfamilies. Among them, NADH pyrophosphatases or diphosphatases (NADDs) are poorly studied and nothing is known about their distribution. To address this, we designed a Prosite-compatible pattern to identify new NADDs sequences. In silico scanning of the UniProtKB database showed that 3% of Nudix proteins were NADDs and displayed 21 different domain architectures, the canonical architecture (NUDIX-like_zf-NADH-PPase_NUDIX) being the most abundant (53%). Interestingly, NADD fungal sequences were prominent among eukaryotes, and were distributed over several Classes, including Pezizomycetes. Unexpectedly, in this last fungal Class, NADDs were found to be present from the most common recent ancestor to Tuberaceae, following a molecular phylogeny distribution similar to that previously described using two thousand single concatenated genes. Finally, when truffle-forming ectomycorrhizal Tuber melanosporum NADD was biochemically characterized, it showed the highest NAD+/NADH catalytic efficiency ratio ever described.Nudix (for  They share the characteristic conserved sequence required for substrate catalysis named the Nudix box (GX5EX7REUXEEXGU), where U represents a bulky hydrophobic amino acid, usually Ile, Leu or Val5. Nucleotide sugars, diadenosine polyphosphates, nucleoside triphosphates, ADP-ribose, NADH and NAD+ are among their most common substrates6. In addition, some Nudix hydrolases have the ability to degrade protein-conjugated ADP-ribose, as is the case with human NUDT168. These compounds participate in crucial processes that require tight regulation. In fact, some Nudix hydrolases are overexpressed following cellular stress in order to recover homeostasis9.Nudix or NUDT (Nudix-type)3. Thus, a conserved proline was found 16 amino acids downstream the Nudix box for all members of the ADP-ribose pyrophosphatase subfamily, whereas an invariant tyrosine designated another subfamily, the diadenosine polyphosphate pyrophosphatases3. Finally, an array of eight conserved amino acids (SQPWPFPXS) was seen to be characteristic of NADH pyrophosphatases or diphosphatases (NADDs)10. The first member of this last family was biochemically characterized in Escherichia coli as the protein product of the nudC gene, formerly known as orf257, which catalyses the hydrolysis of NAD(H) to AMP and NMN(H)11. Several other NADH pyrophosphatases have been described in different organisms, such as mouse NUDT1312, human NUDT1213, and Saccharomyces cerevisiae and Caenorhabditis elegans NPY114. In addition, Arabidopsis thaliana present more than one NUDT representative, including NUDT1, NUDT2, NUDT6, NUDT7, NUDT10 and NUDT1917. However, AtNUDT1 activity was negligible at Mn2+ physiological levels18. AtNUDT2, 6, and 7 hydrolyse both ADP-ribose and NADH with almost equal catalytic efficiencies, whereas AtNUDT10 prefers ADP-ribose over NADH, and AtNUDT19, NADPH over NADH10. Of special interest are NADH pyrophosphatases from pathogenic organisms, such as those of Mycobacterium species , which have been found to be involved in the degradation of the active forms of anti-tuberculosis drugs, such as isoniazid (INH-NAD) and ethionamide (ETH-NAD)20. In addition, NADH pyrophosphatase plays a crucial role in the assimilation of exogenous NAD+ in Salmonella typhimurium21, whereas in Haemophilus influenzae, it has also been described as the enzyme responsible for the growth of the pathogen in NAD+ containing media22. Interestingly, E. coli NudC has also been recently related with the RNA-decapping process, since it was able to efficiently remove the NAD+ cap in different prokaryotic RNAs, hydrolysing the pyrophosphate bond to produce nicotinamide mononucleotide (NMN) and 5′-monophosphate RNA24.These enzymes can be divided into subfamilies depending on specific sequence traits that are involved in substrate recognition, as first stated by Bessman´s group when they studied four ADP-ribose pyrophosphatases23, giving rise to a new Prosite-compatible NADD pattern, which expands the previously published NADH diphosphatase sequence array. This in silico study provides the first phylogenetic distribution of NADDs, and also gives a picture of their domain architectures. The result revealed that NADH diphosphatases represent a small number of sequences (about 3%) of the Nudix superfamily. In addition, bacterial sequences are more abundant than those of eukaryotic organisms. However, among the latter, fungal sequences are profuse and widely distributed among different Classes, including Pezizomycetes, which produce relatively large fruiting bodies (apothecia) from epigeous, semi-hypogeous to hypogeous (truffles) origin25. Some of the last mentioned truffles are highly prized gastronomic delicacies, such as the black truffle of Périgord (Tuber melanosporum Vittad.)26. Of note, and totally unexpected, is the fact that the phylogenetic study made with known Pezizomycetes NADD sequences corroborated a previously reported molecular phylogeny carried out with several hundred conserved concatenated single-copy protein-coding genes27. In addition, the cloning and kinetic characterization of T. melanosporum NADD uncovers a new efficient biocatalyst with the highest NAD+/NADH catalytic efficiency ratio ever described.However, all the above studies have only provided a partial view of the NADH diphosphatase subfamily diversity. To address this, we have carried out an extensive bioinformatic analysis, taking into account other important amino acids recently described in the EcNudC structureE. coli NudC (aka EcNADD) structures   , in addition to Q192 and A241  [CD]x[CN]x[YF]Px(3)Px(2)IxGFx(4)Ex(7)REx(2)EExQ[PQ]W[PA]xPx[QLIMA]M was designed [CD]x[CN]x[YF]) and the conserved amino acids in the Nudix domain without β16 and α3 Ex(7)REx(2)EE) and part of the conserved eight-amino acid motif, known as NADD signature (Q[PQ]W[PA]xP)  Fig. , of whic124 Fig. . This laned Fig. . This paWhen this pattern was scanned against a Nudix protein sequence database composed of 234,112 sequences obtained using the Prosite pattern PS51462 (Nudix) in the UniprotKB database , 7479 sequences  were NADDs. Among them, 90% were from Bacteria, 9.4% from Eukaryota, 0.24% from Archaea and the rest from metagenomic sources. No viral sequences were obtained. In Archaea, they were basically found in the Methanomicrobia Class, whereas in Bacteria, they are almost equally distributed between Gammaproteobacteria (39%), Alphaproteobacteria (30%) and Actinobacteria (23%), with two Orders distinguished by their great number of sequences, Enterobacterales (1008) and Rhizobiales (809). In Eukaryota, the sequences corresponding to mammals (80), birds (35) and bony fishes (26), together with those of roundworms (31), are particularly numerous, although the largest number belongs to Fungi (478). Among them, Ascomycota is the most abundant (86.6%), followed by Basidiomycota (8.3%), Chytridiomycota (1.0%), Mucoromycota (1.8%), Zoopagomycota (1.8%), and one sequence from the fungal sp. No.14919 (0.2%)  (UniProtKB accession number: D5GP45), Burgundy truffle (T. aestivum Vittad.) (A0A292PI49), Piedmont white truffle (T. magnatum Pico) (A0A317SZB3) and whitish edible truffle (T. borchii Vittad.) (A0A2T6ZTD3).In addition, although NADDs are distributed among different fungal subphyla, Pezizomycotina (373) and Saccharomycotina (37) are clearly the most important in the retrieved sequences, followed by Agaricomycotina 25) , when the latter kingdom was considered alone, NADDs appeared to be more represented in Fungi (68%) than in the NUDIX (39%) and Peptidase S8 (38%) families, respectively  Table . The resly Table . This hily Table .Table 1C9. In addition, there are also some unusual domain architectures, one with an ADP ribosylation factor (ARF) domain that function as a regulator of vesicular traffic, three with an actin remodelling motif (NUDIX-like_NUDIX_Arf), one isochorismatase_NUDIX-like_NUDIX and one MULE_NUDIX-like_zf-NADH-PPase_NUDIX  Fig. , which iIX Table . The las+ (TrkA-N), or a putative transposase DNA-binding domain (OrfB-Zn-ribbon) with four conserved cysteines, which could play a similar role to zf-NADH-PPase domain  and its corresponding modelled structure were set as references. Circos representation shows that the information is basically concentrated in three main regions of the protein: residues 203–258 (Zn-binding domain), 292–314 (Nudix box) and 324–332 (NADD signature) (see outer histogram pMI and inner MI connection lines)  Server to Infer Coevolution (MISTIC) was used to predict positional correlations in the multiple sequence alignment (MSA) corresponding to NADDs sequences in order to guide the identification of structurally or functionally important positions in the fungal NADD family Fig. 30. T. mehttps://genome.jgi.doe.gov/mycocosm/home). A total of twenty-four sequences were found, including the five previously used from UniProtKB  are found together with the saprophytic cup fungus Peziza echinospora. The second clade shows six sub-clades each corresponding to members of the Ascodesmidaceae (1), Pyronemataceae (6), Sarcosomataceae (1), Sarcoscyphaceae (1), Discinaceae (1), Morchellaceae (3) and Tuberaceae (5) families. The clades obtained with NADD proteins was in accordance with the phylogram generated from maximum likelihood analysis of combined LSU, SSU, ITS, TEF and RPB2 sequence data from 40 pezizalian species25. The latter study also showed Ascobolaceae (1) and Pezizaceae in basal clade 1, Ascodesmidaceae and Pyronemataceae in clade 6, Sarcosomataceae and Sarcoscyphaceae in clade 5, and finally Discinaceae, Morchellaceae and Tuberaceae in clade 425. The above concordance with NADD distribution found for taxa of Pezizomycetes also agreed with the molecular phylogeny obtained using 2,093 concatenated conserved single-copy protein-coding genes from the eight Pezizomycetes27, in which NADD seems to be present in the most recent common ancestor (MRCA) of Pezizomycetes 470 ± 67 million years ago (Ma), starting from Ascobolaceae, and continuing present until Tuberaceae began diverging around 140 ± 10 Ma in the Early Cretaceous31. Interestingly, this MRCA seems to have the canonical NADD domain architecture (i.e. NUDIX-like_zf-NADH-PPase_NUDIX), since all the NADDs used in Fig. Choiromyces venosus NADD forms a distinct branch from Tuber spp. within Tuberaceae, and T. melanosporum from the rest of Tuber spp., as previously described in the molecular phylogeny27.In order to explore similar sequences in other Pezizomycetes, the fungal genomes described in the MycoCosm portal were also scanned (http://fungi.ensembl.org/index.html). However, this is not the case for the above used Tuber spp., whose genome organization reveals a common pattern in NADD flanking genes  (Perigord black truffle) was cloned into pET28a, transformed into E. coli Rosetta 2 and purified in two-steps, which combines an initial Ni2+ affinity chromatography step with a size exclusion in Superdex 200. The enzyme obtained was electrophoretically pure . In addition, Mn2+ gave rise to a 1.7-fold increase in activity at optimal conditions when compared with Mg2+  Fig.  from T. 14, data which are consistent with the general alkaline nature of most Nudix hydrolases described up to date5. In addition, at pH 9.0, TmNADD was also more stable than at any other pH values , which has an optimal temperature of 40 °C19. TmNADD displayed a half-life of about five hours at 37 °C, whereas at 20 °C it increased up to six hours , but with a clear preference for NADH. Thus, the obtained KM for NADH was 0.12 ± 0.01 mM, with a kcat of 10.7 ± 1.8 s−1 and a kcat/KM of 89.2 ± 19.8 mM−1 s−1, which is almost 10-fold higher than the catalytic efficiency shown towards NAD+ (kcat/KM of 9.3 ± 1.6 mM−1 s−1)  found in its sequence (GX5EX7REUXEEXGU) bind indispensable divalent cations, such as Mg2+ or Mn2+10. Their substrate specificity is associated with additional sequences to this box, giving rise to the several subfamilies3. Among them, NADH pyrophosphatases or diphosphatases have been poorly studied, especially in enzymes of fungal origin. Taking advantage of the large number of new sequences emanating from genomic sequencing projects and their translation into the corresponding public protein databases, this work provides a complete in silico analysis of this subfamily, finding that NADDs represent 3.2% of the Nudix proteins in the UniProtKB database. Bacterial sequences are the most abundant, followed by eukaryotic, archaeal and metagenomic, without any viral representative. Curiously, fungal NADDs, distributed among different subphyla, account for 6.4% of NADDs and 68% of eukaryotic sequences, the most relevant being Pezizomycotina and Saccharomycotina , fungal NADDs are also the most representative within structural cluster 3.90.79.10/3 (SC:3), where the nucleoside triphosphatases NudI, the ADP-ribose pyrophosphatases, the mitochondrial putative pre-mRNA cleavage factors, and the bifunctional NMN adenylyltransferases/Nudix hydrolases are also found. In fact, only one sequence from Nosema bombycis (strain CQ1 / CVCC 102059) (R0KWY1) was found in the nucleoside triphosphatase nudI sub-family when CATH sequences were retrieved from UniProt database.Nudix hydrolases are a vast and diverse family of proteins involved in the regulation of cellular responses and homeostasis, which are widely distributed in all kingdoms of lifeina Fig. , which iT. melanosporum). The sequences of these truffle NADDs show the canonical domain architecture (i.e. NUDIX-like_zf-NADH-PPase_NUDIX)  of Pezizomycetes, and has been maintained as an essential protein from Ascobolaceae to Tuberaceae31.NADDs are also found in Pezizomycetes basal groups, and in particular, in five economically important ectomycorrhizal truffle species, including the aromatic Périgord black truffle (IX) Fig. , which iIX) Fig.  and in tIX) Fig. . This ca9 accounts for 18.6% of the NADD domain architectures in eukaryotes, but only 0.42% in Fungi, in particular in the soil fungi Bifiguratus adelaidae (A0A261XXG3) and Syncephalastrum racemosum (A0A1X2H7S1). The latter fungus has occasionally been described as being the causative agent in toenail onychomycosis33. In addition, while the Ank-2 motif is found in 225,525 sequences with about 12,570 different Pfam domain architectures (http://pfam.xfam.org/family/Ank_2#tabview=tab0), the Ank-2_NUDIX-like_zf-NADH-PPase_NUDIX motif is only found in 88 sequences (http://pfam.xfam.org/family/zf-NADH-PPase#tabview=tab1) plus the two sequences found in this paper. This represents only 0.7% of the architectures where the Ank-2 motif is found. Its possible role associated with NADD is still unknown, although this Ank-2 domain is one of the most common protein-protein interaction platforms in nature. However, the presence of ankyrin domains  in NADDs is relatively frequent both in fungal and eukaryotes, but totally absent in bacteria and archaea.The bioinformatic study also showed for the first time a total of twenty-one different architectures for the NADDs used in this work Fig. , whereas2+ rather than Mg2+, with an optimal concentration (500 µM) higher than that described for hNUDT12 (50 µM) but similar to that of S. cerevisiae (300 µM)32. However, the optimal Mg2+ concentration was in the mM range (5–10 mM), similar to that described for E. coli, S. cerevisiae and C. elegans, but higher than that of hNUDT12 (0.4–2 mM)14. Its alkaline optimal pH was also a common feature of other NADDs, maintaining more than 50% activity for 6 hours in glycine buffer pH 9.0 at its optimum temperature (30–37 °C). Kinetically, TmNADD is 10 times more active towards NADH than NAD+. This ratio is lower than those of E. coli (120), S. cerevisiae (60), C. elegans (30) and even lower than that of hNUDT12 (20)14, meaning that the NAD+/NADH catalytic efficiency ratio is twice as high in TmNADD as in hNUDT12, making TmNADD the enzyme with the highest NAD+/NADH ratio ever described , was purchased from Genscript  and inserted into pET28a vector. The TmNADD-pET28a construct was transformed into Escherichia coli Rosetta2(DE3) and induced using 0.25 mM isopropyl-β-D-thiogalactoside (IPTG) for 16 h at 20 °C with constant shaking. The culture was centrifuged and the pellet resuspended in lysis buffer . Cells were disrupted using a Bead Beater homogenizer (Biospec). After ultracentrifugation , the supernatant was loaded at 4 °C onto a HiPrep IMAC 16/10 FF column  coupled to a FPLC chromatography system . The enzyme containing fractions were pooled, desalted and loaded into a Superdex 200 HiLoad 16/600 column (GE Lifesciences), obtaining an electrophoretically pure enzyme. TmNADD was stored at −20 °C with 10% glycerol.+ were carried out under standard NADD activity assay that have been described previously11. Reactions at 37 °C were prepared in 50 mM Tris-HCl pH 8.0 in the presence of 0.5 mM MnCl2, 5 mM NAD+ and 0.11 μM of TmNADD, unless otherwise stated. At regular intervals during the 15 min that the assay lasted, aliquots were taken, stopped at pH 3.0 with TFA , kept on ice for 10 min and centrifuged for 10 min at 12000 g. NAD+ conversion into NMN and AMP was followed by HPLC in a reverse phase C18 column  and a method involving buffer A  running at 1 mL min−1 in a gradient from 0 to 100% of buffer B  for 21 minutes. In these conditions, the reaction products NMN and AMP had a retention time of 5.1 and 13.1 minutes, respectively, and NAD+ had a retention time of 10.7 minutes. NAD+ diphosphatase activity was determined following the increase in the area of the AMP peak. Reactions towards NADH (5 mM) were carried out under the same conditions for 5 min, but using 0.02 μM of TmNADD. The reactions were stopped at regular intervals by the addition of 100 mM EDTA at pH 8.06. Under these conditions, the reaction products NMNH and AMP had retention times of 15.2 and 13.1 minutes, respectively, while that of NADH was 19.1 minutes. NADH diphosphatase activity was also determined following the increase in the area of the AMP peak.Reactions towards NADThe optimal pH of TmNADD was carried out by performing the standard activity assay in various buffers with different pH values: 6.0–10.0. The buffers used were as follows: sodium phosphate buffer (pH 6.0–7.5), Tris-HCl buffer , and glycine-NaOH buffer (pH 9.0–10.0). All buffers were at a concentration of 50 mM. Next, optimum temperature for TmNADD was determined using the standard NADD activity assay described above at different temperatures ranging from 20 to 60 °C.The effect of pH and temperature on enzyme stability were determined by incubating TmNADD at 37 °C at different pHs, or at pH 8.0 at different temperatures for increasing periods of time (0–360 min). The residual activity after the incubation period was measured in the standard reaction conditions.M) and maximal velocity (Vmax) were estimated using plots of initial rates vs. NADH or NAD+ concentrations, respectively. The reported values represent means ± SE of the fits of the curves to the Michaelis-Menten equation by non-linear regression based on triplicate experiments. The catalytic constant (kcat) and the catalytic efficiency (kcat /KM) were deduced from the obtained KM and Vmax values.The substrate specificity of TmNADD was determined at 37 °C and in 50 mM Tris-HCl pH 8.0 using various substrates. The Michaelis-Menten constant (Khttps://www.uniprot.org/), using the Prosite pattern PS51462 (NUDIX) as a query. Incomplete sequences and duplicate were removed, rendering the sequences used in the study. The NADD-like sequences were identified using the ScanProsite application (https://prosite.expasy.org/scanprosite/) and the pattern designed in this work. Sequences were aligned with the default parameters using MAFFT server  and displayed using ESPript 3.036. Subsequently, the tree was built on the same web server using the Neighbour-Joining (NJ) method, JTT substitution model, heterogeneity between ignored sites (α = ∞) and a bootstrap of 1000 replicates to increase the reliability of the tree obtained. Mutation correlation analysis was carried out with the retrieved fungal NADD sequences using MISTIC  web server (http://mistic.leloir.org.ar/)30. The domain architectures of retrieved sequences were obtained from the Pfam (https://pfam.xfam.org/) and visualized with iTOL (https://itol.embl.de/).Protein sequences were obtained from the UniProtKB database (Supplementary Information


Compounds 2, 7, 10, and 18 presented potent effects against Mycobacterium tuberculosis (57.2 µM ≤ MIC ≤ 70.9 µM). Hydrophilic effects caused by substituents on ring B increased antibacterial activity against Gram-positive species. Thus, log Po/w were calculated by using high-performance liquid chromatography-photodiode array detection (HPLC-PDA) analyses, and cinnamylideneacetophenones presented values ranging from 2.5 to 4.1. In addition, the effects of 3 and 4 were evaluated on pulmonary cells, indicating their moderate toxicity (46.3 µM ≤ IC50 ≤ 96.7 µM) when compared with doxorubicin. Bioactive compounds were subjected to in silico prediction of pharmacokinetic properties, and did not violate Lipinski’s and Veber’s rules, corroborating their potential bioavailability by an oral route.Cinnamaldehyde is a natural product with broad spectrum of antibacterial activity. In this work, it was used as a template for design and synthesis of a series of 17 cinnamylideneacetophenones. Phenolic compounds  Aliq3–6 and 9) exhibited potent antibacterial activity against Gram-positive species. Seven compounds  were able to inhibit M. tuberculosis growth. Preliminary SAR investigations suggested hydrophilicity is the central parameter for antibacterial and antitubercular activities. Furthermore, our results corroborated the potential of cinnamaldehyde as a privileged starting material and template for synthetic collection of hits; it did not violate Lipinski’s and Veber’s boundaries.In summary, a series of cinnamylideneacetophenones containing several substituents on ring B were designed and synthesized as part of our ongoing search for antibacterial and antitubercular compounds based on natural products. Five compounds (


Staphylococcus aureus (SA) infections cause high mortality and morbidity in humans. Being central to its pathogenesis, S. aureus thwarts the host defense by secreting a myriad of virulence factors, including bicomponent, pore-forming leukotoxins. While all vaccine development efforts that aimed at achieving opsonophagocytic killing have failed, targeting virulence by toxoid vaccines represents a novel approach to preventing mortality and morbidity that are caused by SA. The recently discovered leukotoxin LukAB kills human phagocytes and monocytes and it is present in all known S. aureus clinical isolates. While using a structure-guided approach, we generated a library of mutations that targeted functional domains within the LukAB heterodimer to identify attenuated toxoids as potential vaccine candidates. The mutants were evaluated based on expression, solubility, yield, biophysical properties, cytotoxicity, and immunogenicity, and several fully attenuated LukAB toxoids that were capable of eliciting high neutralizing antibody titers were identified. Rabbit polyclonal antibodies against the lead toxoid candidate provided potent neutralization of LukAB. While the neutralization of LukAB alone was not sufficient to fully suppress leukotoxicity in supernatants of S. aureus USA300 isolates, a combination of antibodies against LukAB, α-toxin, and Panton-Valentine leukocidin completely neutralized the cytotoxicity of these strains. These data strongly support the inclusion of LukAB toxoids in a multivalent toxoid vaccine for the prevention of S. aureus disease. Staphylococcus aureus, which is a pervasive human pathogen, is a leading cause of life-threatening community and hospital-acquired infections world-wide. This Gram-positive bacterium is often associated with a range of diseases, from mild skin and soft tissue infections to invasive bacteremia, septic arthritis, endocarditis, and osteomyelitis [S. aureus (MRSA) causes ~80,000 invasive infections and 11,000 deaths per year in the United States alone [myelitis . Moreovemyelitis ,3,4. MetS. aureus expresses a myriad of virulence factors, including cell surface attachment factors, capsular polysaccharides, enzymes, immune modulatory molecules, pore-forming toxins, and superantigens that aimed at establishing the infection or colonization as well as immune evasion [S. aureus produces single component alpha hemolysin (Hla or α-toxin) and bicomponent pore-forming toxins (BCPFTs), Panton-Valentine leukocidin , Leukocidin AB (LukAB), Leukocidin ED (LukED), and γ-hemolysins (HlgAB and HlgCB) [ evasion . Among td HlgCB) . While Hd HlgCB) , the BCPd HlgCB) . FollowiS. aureus (MRSA) strain circulating in the United States (US), revealed an octameric arrangement of four LukAB dimers with two unique interfaces 1 (intra-protomeric) and 2 (inter-protomeric), which primarily govern the cap and rim domains [Among the BCPFTs, LukAB is the most recently identified and it is one of the most potent members of the leukocidin family that kills human neutrophils, macrophages, monocytes, and dendritic cells ,10. Whil domains . Highly  domains ,12,13,14 domains . LukAB p domains ,12,13,16 domains . S. aureus infections as compared to healthy individuals or commercially available intravenous immunoglobulin (IVIG) [Highly virulent MRSA strains, like USA300, contribute to the clinical severity of SA infections by secreting a higher load of toxins to evade the innate immune system. Amongst other leukocidins, high titers to LukAB have been reported in acute and convalescent patients when compared to healthy controls . Addition (IVIG) . AltogetS. aureus toward neutrophils and monocytes.Here, iterative rounds of targeted single-amino acid and combination mutations within different functional domains of LukAB were designed in a structure-guided manner to identify the attenuated forms of LukAB. To this end, we first developed a novel production strategy that was based on co-expression from a single vector and a multistep purification process to generate tag-free, soluble dimers, and then functionally characterized them based on toxicity, thermostability, immunogenicity, and the ability to compete with cytotoxicity of wild type (WT) LukAB to identify several attenuated toxoids. Furthermore, we generated a polyclonal antibody against a selected attenuated LukAB mutant that demonstrated neutralizing activity towards supernatants from several laboratory and clinical strains, including USA300, which is responsible for the current CA-MRSA outbreak in the United States. Our findings further indicate that a multivalent toxoid vaccine targeting all the pore-forming toxins is needed to fully neutralize the cytotoxicity of different clinical strains of WT proteins ) as an adjuvant were examined for the induction of neutralizing antibodies. Groups of five female ICR (CD-1) mice were intramuscularly immunized (IM) with 10µg of LukAB mutants or LukABWT in 50µg of Al(OH)3 three times at two-week intervals and sera was collected 10 days after final immunization. Antibody-binding and -neutralizing titers were measured either for individual mice or from pooled sera for each mutant group by ELISA and toxin neutralization assays (TNA), respectively, as described in the Methods.The objective of this study was to generate attenuated forms of LukAB as the vaccine candidates. To this end, we used the available crystal structure of LukAB heterodimer (PDB: 5K59) and octamer  toxvailable , (3) thevailable D, and  (L61RLukBwt), reduced the toxicity by >30,000-fold as compared to WT (L61R/N/QLukBwt recorded melting temperatures (Tm) that were in the range of 51.3–52.2 °C, representing a positive thermal shift of 7.3–8 °C as compared to LukABWT (Tm = 44 °C)  and T28 of Panton-Valentine leukocidin (PVL) S subunit (LukS-PV) . N termi LukBD49 . We disr.027 nM) A,B. In ced to WT A. Additi= 44 °C) D. Howeve LukABWT . TherefoWT, the LukBE197A mutation modestly attenuated toxicity (~4.5-fold), while the other two mutations failed to attenuate cytolytic activity in PMNs with EC50s in the range of LukABwt  to introduce flexibility between residues D124 and G134 and to maintain structure stability. Of these mutants, only LukAwtLukBΔ125-G-133 could be expressed, although poorly, and demonstrated moderate 1218-fold attenuation with TNA titers similar to wild type  as LukAwtLukB127-SNGLS-133. While LukAwtLukB127-APGP-133 diminished the cytotoxicity by ~10-fold, the EC50 for LukAwtLukB127-SNGLS-133 toxicity was enhanced by ~100 fold  or replacing with the analogous sequence of HlgB (LukAwtLukBHlgB) (wtLukBΔ30aa due to the poor expression of LukBΔ30aa (data not shown), mutant LukAwtLukBHlgB was well expressed and it exhibited a cytotoxicity EC50 of >1000 nM, representing >30,000-fold attenuation  and HlgB suggested high structural similarity, except for the loops at the rim domain. Taking these observations together, we further investigated the role of the first 30 N terminal residues of LukB by either deleting them (LukAukBHlgB) . HlgB haenuation . The proenuation D and reltion  and/or varying pH 6–7.0 were implemented. Therefore, we sought to fractionate the proteins by reverse-phase (RP) HPLC. We first fractionated LukABWT, LukAD39ALukBR23E, and LukAwtLukBHlgB while using acetonitrile that resolved into two prominent peaks (WT exists as a dimer (~77 kDa). In contrast, LukS-PV (MW = 70.6 KDa) and LukF-PV (MW = 73.9 KDa) remained largely monomeric in the presence of glutaraldehyde. Mutants LukAD39ALukBR23E and LukAWTLukBHlgB are similar to LukABWT as dimers, indicating that they retain the heterodimeric structure that is important for eliciting neutralizing antibodies. Additionally, we also ran the poorly expressed LukAWTLukBΔ30aa mutant, in which the LukA component fails to associate with LukBΔ30AA, suggesting a previously unknown role for the N terminus of LukB in protomer-protomer interaction  of the mutants LukAD39ALukBR23E, LukAwtLukBHlgB, LukAwtLukBR23E, LukAwtLukB125-133_1G, LukAD39RLukBR23E, and LukAwtLukBK12/19/R23A, which are fully attenuated, or LukAD39ALukBwt, which retains toxicity, were incubated with PMNs along with a fixed concentration of 0.42 nM LukABWT. Relative toxicity of LukAB in the presence of various mutants was then plotted against the molar ratio of mutant to wild type . On the other hand, attenuated LukAWTLukB125-133_1G, having a deletion in the loop that is involved in pore formation, failed to reverse the lytic effect of LukAB, even at high concentrations, which suggested that this mutation can neither compete with receptor binding nor form mixed defective oligomers. As expected, LukAD39ALukBwt, a mutant that retains substantial toxicity, had no reverting effect on LukAB activity.The ability of mutants to bind to the cellular receptor CD11b would indicate an intact receptor binding site, which could be important for the induction of neutralizing antibodies. Therefore, we sought to determine whether the selected mutants compete with LukABild type A. ConsisD39ALukBR23E and LukAwtLukBHlgB, we tested the ability of LukAD39ALukBR23E for its ability to generate neutralizing antibodies in rabbits. The total IgG was purified from the sera of rabbits immunized with the LukAB mutant showing high and equal binding to both wild type and mutant LukAD39ALukBR23E , two PVL subunit toxoids LukSmut9 (αLukS) and LukFmut1 (αLukF), which we previously reported [H35LH48L (αHla). Besides three isolates of USA300, we also used a LukAB isogenic knock out of the USA300 strain SF8300 (SF8300ΔlukAB). As shown in Multiple pore-forming toxins, including Hla, PVL, LukED, HlgAB, HlgCB, and LukAB, mediate the cytotoxic activity of SA. While LukAB and PVL are most lytic to PMNs, this activity is shared by several other leukotoxins . We measreported , or the 50 0.04 nM, We next tested whether αLukAB also protects differentiated THP-1 cells against cytotoxic activity in the bacterial culture supernatants. For this, we examined the cell viability in presence of supernatants from SF8300 and SF8300ΔlukAB grown in TSB or BHI media to gauge the level of LukAB-mediated toxicity in differentiated THP-1 cells. The cytotoxicity of WT strain was 2.6-fold or 1.8-fold fold higher than SF8300ΔlukAB when grown in BHI or TSB, respectively C, which S. aureus have myopically focused on promoting the opsonophagocytic uptake of the bacteria and the subsequent killing by phagocytic cells, an approach that has been successful for several pathogens, such as S. pneumoniae, N. meningitidis, and H. influenzae B. However, these efforts have failed to deliver an effective vaccine for S. aureus and at least one of the experimental vaccines, Merck V710, led to increased mortality in vaccinated individuals who developed SA infection [The evolution and emergence of MRSA strains has become a major challenge to global health. Among the diverse virulence factors that were secreted by SA to subvert the encountering host defense system, α-toxin/Hla and the bicomponent leukotoxins, such as LukAB, HlgAB, HlgCB, LukED, and PVL, are the most potent, because they specifically target and kill innate immune cells and disrupt biological barriers by lysing epithelial and endothelial cells , as wellnfection , which snfection ,28,29. Wnfection . LukAB pnfection . HoweverAll of the bicomponent leukotoxins, except for LukAB, are secreted as S and F subunit monomers. Cell receptor binding is initiated by S subunit followed by the binding of F and oligomerization, which leads to pore formation . In contBadarau et al. solved the crystal structure of LukAB heterodimer in complex with a potent neutralizing antibody (ASN102) , as wellOf the electrostatic charges mediating protomer-protomer interactions (mediating heterodimer formation) and dimer-dimer interactions (mediating octamer formation), referred to here as interface 1 and 2, respectively, our data indicates that the residues within the salt bridges on interface 2 along with the bulky positively charged arginine and lysine residues at LukB N terminus are the key to mediating LukAB cytotoxicity. The electrostatic interactions that are part of the salt bridges within Interface 2, particularly participating LukB residues, which form a basic patch near the apical side of the cap domain, are crucial to cytotoxicity, as seen in this study. These residues have been previously shown to be important for LukAB octamerization and are known to be fully conserved between the LukAB sequence variants . In contMutations that were made to disrupt the buried hydrophobic pocket found within Interface 1, particularly L61, was found to be crucial to LukAB thermostability and attenuating toxicity; however, drastically hampering immunogenicity. As this residue is not surface exposed, the pocket near L61 is not an antigenic target, but it lowers immunogenicity, which is likely due to altered oligomeric status. The structures of LukAB and α-toxin show that L61 is nestled between β-strands that are part of the β-sandwich within the cap domain and the introduction of bulky mutations perturbs the hydrophobicity within this pocket affecting crucial protomer-protomer interactions that are sacred to stability, thereby reducing its ability to generate neutralizing antibodies. D39ALukBR23E that satisfied biochemical and biophysical characterization, in addition to showing complete attenuation in PMNs. It is interesting that the second-best candidate, LukAwtLukBHlgB mutant, which exhibited the complete attenuation in PMN lytic activity, retained some toxicity, albeit at high concentrations in the THP-1 cells, suggesting that the substituted HlgB residues may be responsible for the relapsed cytotoxic effects. Interestingly, both of these mutants were able to compete with WT LukAB toxicity at a low molar ratio of below 1, which indicated that the competition cannot be entirely due to receptor binding by the mutant. The reversal of LukAB toxicity by these mutants may relate to the formation of defective mixed oligomeric structures, an observation that warrants further investigation. Moreover, with this study, we can show that the identified vaccine candidate generates antibodies that can reverse the toxicity and show protection in PMNs, as well THP-1 monocytes.In our efforts, we have been successful in identifying a strong vaccine candidate in LukAPrevious studies showed the important role of LukAB in cell specific lysis of monocytes  and dendS. aureus. Our findings indicate that a multivalent approach targeting the related leukotoxins PVL, LukED, HlgAB, HlgCB, and the divergent LukAB, as well as the single component Hla is critical for protection against cytolytic activity of the most prevalent S. aureus strains. These data strongly support the development of a multivalent toxoid vaccine for S. aureus, which covers all major pore-forming toxins.In summary, in this study, we have developed at least two vaccine candidates for LukAB, which is an important virulence factor of NcoI-HindIII, and LukB was cloned into MCS2 using NdeI-XhoI restriction sites within the same vector. In another system, LukA and LukB were expressed while using two different pET-24a(+) plasmids within the same E. coli cell. We compared LukAB WT expression, yield, and toxicity from these two systems. All of the mutants were expressed while using the latter system, where LukA and LukB were expressed using separate plasmids within the same E. coli. Towards this end, LukA (WT or mutants) was cloned into pET-24a(+) with a Kanamycin resistant marker within NdeI-XhoI sites. Similarly, LukB (WT/mutants) was inserted within NdeI-XhoI sites, but we replaced the inherent Kanamycin resistant cassette in pET-24a(+) with Ampicillin. The origin of replication of pET-24a(+) was replaced by p15a resulting in a recombinant pET24a(+)AmpRp15a LukB vector to increase the plasmid compatibility. This LukB plasmid (WT/mutant) was transformed into BL21(DE3) (NEB) containing pET24a(+) LukA plasmid (WT/mutant) and the colonies were selected on LB plates with 50 ug/mL of Kanamycin (Kan50) and 100 ug/mL of Ampicillin (Amp100). The genes for LukAB (USA300_TCH1516) WT and mutants were codon optimized prior to transformation by GenScript®.General methods that are used for bacterial culture have been described previously in detail . In this study, we optimized LukAB expression while using two different vectors: pET Duet-1 (Novagen), where LukA was cloned into the multiple cloning site 1 (MCS1) while using E. coli that were grown at 37 °C in LB Kan50Amp100 were expanded to 0.5 L in a shaking incubator (225 rpm), until they reached an OD OD600 of 0.5. The cultures were immediately chilled on ice for 10 min. with periodic shaking and then induced with 0.3 mM IPTG  in a shaking incubator (225 rpm) overnight at 25 °C. The following day, the cells were harvested by centrifugation  and frozen at −80 °C. To lyse the cells, the pellet was resuspended in 3 mL of cell lysis buffer  per gram of cell paste and Hen egg lysozyme  at 1 mg/mL final concentration prior to incubation at 37 °C for 30 min. The partially lysed cells were then cooled in a wet/dry ice ethanol bath, followed by sonication while using a microtip . Lysis was confirmed by measuring the reduction of OD600 absorbance. Post lysis, the NaCl concentration was adjusted to 0.5 M and nucleic acid precipitation was carried out by the dropwise addition of 0.3–0.5% polyethyleneimine (PEI) while maintaining constant mixing. The PEI pellet was separated by centrifugation at 12,000 rpm in a Sorvall SS34 rotor and the supernatant containing the toxoid was subjected to ammonium sulphate (AmS04) precipitation. Towards this end, 0.472 g/mL of AmS04 powder was added to the PEI supernatant and then placed on a rotating mixer for 20 min. at room temperature. The resulting pellet that was obtained by centrifugation at 12,000× g for 30 min. at 4 °C was frozen at −80 °C until purification. Overnight cultures of 4 pellet was resuspended and buffer exchanged into 20 mM NaPi pH 6.5, 25 mM NaCl, 5% glycerol using a GE Healthcare PD10 desalting column. The mixture was then clarified by filtration using 0.8/0.2 µm Supor® low protein binding syringe filter . The toxoid from the resulting solution was purified while using a two-column purification approach. The first purification was carried over a 10 mL Poros 50 HS column using a 40-column volume (C.V.) gradient from 25 to 1000 mM NaCl in the phosphate buffer. The peak fractions were analyzed by standard SDS-PAGE analysis and accordingly pooled together for dialysis into 20 mM NaPi pH 6.8, 50 mM NaCl, 5% glycerol, the equilibration buffer for the second column—a 10 mL ceramic Hydroxyapatite (HTP) . The toxoid was eluted using a 40 C.V. gradient from 50 to 1000 mM NaCl in the phosphate buffer. The appropriate fractions were pooled together upon SDS-PAGE evaluation and then dialyzed into the final storage buffer . All purified LukAB proteins (MW = ~72 kDa) were concentrated while using Amicon 3K MWCO Ultra 15, filtered through 0.8/0.2 µm low protein binding membrane, and stored at −80 °C prior to use.The AmS0600 absorbance. The next day, culture supernatants were filtered through 0.2 µm filter to sterilize the supernatants. Sterility was confirmed by culturing 100 μL of the filtered supernatants on BHI or TSA agar plates overnight. 5 cells/mL. 1 × 107 cells were seeded in 30 mL and grown in culture media with 1.5% dimethyl sulfoxide (DMSO) for seven days to differentiate cells into neutrophils. CD11b expression using flow cytometry confirmed induction.The HL-60 cells  were cultured in RPMI 1640  supplemented with 16.4% heat inactivated fetal bovine serum (FBS), 4 mM L-glutamine, 82 U/mL each of penicillin, and streptomycin. Cells were passaged twice a week at a concentration of 6–8 × 10g  for 10 min. at 20 °C. Cells were washed and resuspended with phenol red-free RPMI 1640  supplemented with 2% FBS to a final concentration of 5 × 106 cells/mL. LukAB mutants (proteins) were serially diluted two-fold across 96-well plates (50 µL/well) and 100 µL of 5 × 106 cells/mL were added to each well. The plates were incubated at 37 °C, 5% CO2, 95% humidity for 3 h. After 3 h, either XTT  or CellTiter Glo (Promega) reagent was used to determine the cell viability and cytotoxicity [2, 95% humidity for 16–18 h. After incubation, the cells were pelleted by centrifugation at 3500 rpm 3565.9× g  for 3 min. The supernatants were transferred to 96-well ELISA plates and absorbance was read at 470 nm while using Spectramax 190 plate reader  and Softmax 5.4.5 software . When using CellTiter Glo, 50 μL of the reconstituted CellTiter Glo reagent was added to each well. The plate was shaken on an orbital shaker for 10–15 min., followed by measurement of luminescence (emission at 560 nm) using Cytation 5 imaging reader  and Gen5 2.09 software to determine cell viability. For the kinetic cytotoxicity studies, the replicates were incubated for 6 and 19 h, in addition to a 3-h incubation period. After incubation, the CellTiter Glo reagent was used to determine the cell viability.The induced HL-60 cells were harvested by centrifugation at 420× toxicity ,32. WhenDM, LukSmut1, LukFmut9, and LukAD39ABR23E toxoid as immunogens were generated by Genscript®  using >98% pure proteins as immunogens. Briefly, four New Zealand white rabbits were immunized per toxoid on day 0, 14, and 21 with 0.2 mg protein per rabbit, along with Freund’s Incomplete Adjuvant subcutaneously. The test bleeds and production bleeds were collected on day 21 and day 42. Hyperimmune sera were individually characterized for ELISA titer and TNA titers before pooling them together. The pooled serum was purified by Protein A affinity chromatography into total IgG and labeled, as follows: anti-Hla (IBT Cat: 1940-01 Rb pAb), anti-LukS- (IBT Cat: 1941-01 Rb pAb), anti-LukF-PV(IBT Cat: 1942-01 Rb pAb), and anti-LukAB (IBT Cat: 1944-02 Rb pAb). Full quality control (QC) were performed before use.Rabbit polyclonal antibody generation for anti-Hla6 cells/mL were added to each well. The plates were incubated at 37 °C, 5% CO2, 95% humidity for 3 h, followed by XTT or CellTiter Glo readout to determine the cell viability and neutralization [The serum samples were serially diluted two-fold in RPMI across 96-well plates (25 µL/well). Twenty-five microliters of 2.5 nM LukAB toxin was added to each well. 100 µL of induced cells were prepared, as described above, at 5 × 10lization ,32. 6 induced HL-60 cells/mL were added to each well. The plates were then incubated for 3 h at 37 °C, 5% CO2, 95% humidity, followed by CellTiter Glo readout determine cell viability and neutralization. Select LukAB mutants were serially diluted from a starting concentration of 12 nM in RPMI semi-log across 96-well plates (25 µL/well). Polyclonal αLukAB at 100 µg/mL or RPMI was added to each well (12.5 µL/well), followed by incubation at RT for 10 min. Following incubation, 12.5 µL of 5 nM LukAB WT and 100 µL of 5 × 10The proteins in the storage buffer  were mixed with 2X SYPRO orange dye  in a 96-well hard shell plate with clear bottom  and then placed into a thermal cycler, wherein the temperature scan rate was fixed at 0.5 °C/min over a range of 30–99 °C (21). The fluorescence intensities were plotted against temperature to get a sigmoidal curve and the melting temperatures (Tm) were calculated while using the first derivative. BSA (Pierce) was used as a control, which recorded a melting temperature of 66 ± 0 °C in 1 × PBS pH 7.4. WT and mutants at 50 ug/mL concentration were incubated with 0.25% glutaraldehyde  for 2 min. at 37 °C in a final volume of 100 μL in 20 mM HEPES pH 7.5, 50 mM NaCl. The reaction was stopped by adding 10 μL of 1M Tris, pH 8.0 and 4 × LDS sample buffer, followed by SDS-PAGE and Western blot analysis while using αLukAB and rabbit pAb αLukB-specific (IBT Cat: 0313-001). αLukS-PV and αLukF-PV were used as the negative controls. LukABv/v) in Line B with a flow rate of 1.00 mL/min over 60 min. and a column temperature of 27 °C.LukAB  was injected into an AdvanceBio RP-mAb diphenyl column  in a 1260 Infinity Quaternary instrument. A 30–90% gradient method was used consisting of 100% acetonitrile in Line A and 0.1% TFA  route prior to and 10 days after the third and final immunization.Female ICR (CD-1) mice, six weeks of age, were purchased from Envigo (US). The mice were maintained under pathogen-free conditions and fed laboratory chow and water ad libitum. All mouse work was conducted in accordance with protocols that were approved by institutional animal care and use committees (IACUC) of Integrated BioTherapeutics, where mouse studies were performed . The mice were intramuscularly immunized (IM) three times two weeks apart with 10 µg of LukAB mutant in 50 µg of Al(OH)Blood samples from mice were centrifuged in serum separator tubes and the serum samples were stored at −80 °C until further use in ELISA. Briefly, 96-well plates were coated with 300 ng/well of LukAB WT overnight at 4 °C. The plates were blocked with Starting Block (SB)  for one hour at room temperature (RT). Serum samples were diluted in a semi-log manner starting from 1:100 to 1:316,228 in a 96-well plate, using starting block buffer as the diluent. The plates were washed three times and sample dilutions were applied in 100 µL volume/well. The plates were incubated for 1h at RT and washed three times before applying the conjugate, goat anti-mouse IgG (H+L)- Horseradish Peroxidase (HRP) in SB. Plates were incubated for 1 h at RT, washed, as described above, and incubated with TMB  for 30 min. to detect HRP activity. Optical density at 650 nm was measured while using a Versamax™ plate reader . Data analysis for full dilution curves was performed using Softmax program and graphed in GraphPad Prism.+) up to 95.98% purity were purchased from BioIVT  and kept frozen in LiN2 until use. Acute monocytic leukemia THP-1 cells were purchased from ATCC (ATCC® TIB-202™) and were cultured at 4E5 cells/mL every 2–3 days at 37 °C + 5% CO2, as recommended in RPMI-1640, supplemented with 0.05 mM 2-mercaptoethanol and 10% FBS. To differentiate the THP-1 cells, 100 nM of Phorbol 12-myristate 13-acetate  was added to THP-1 media and seeded at 4E5 cells/mL. The cells were allowed to differentiate for three days and analyzed for extent of cell adherence and surface marker for differentiation  by flow cytometry. After three days, the media was replaced with non-PMA containing medium and allowed to rest for five days before use, feeding the cells with fresh THP-1 medium every two days. Purified human monocytes  for 10 min. The cells were then washed twice in 1% (w/v) RPMI+Cas  and pelleted by centrifugation. Proteins (12.5 µL) were serially diluted two-fold or in semi-log fashion, as indicated in 1% RPMI+ Cas and incubated with 75 µL cells (seeded at 1E5 cells/well in a 96-well plate) in a final volume of 125 µL for 1 h at 37 °C, 5% CO2, 95% humidity, followed by CellTiter Glo readout to determine the cell viability and % lysis. For experiments with supernatants from culture filtrates, the supernatants  or other toxins, such as α-toxin, LukSF, HlgAB, and HlgCB on differentiated THP-1 cells, the adherent cells were harvested by treatment with 1 × PBS + 0.05 mM EDTA at 37 °C for ~5 min. and immediately centrifuged at 420× 2+ and Mg2+, supplemented with 2% FBS (FACS buffer). For HL60 cells, CD11b-FITC  was added to each well at 1:40 dilution (50 µL) in FACS buffer and incubated at RT, being covered from light, for 15 min. For THP-1 cells, a combination of CD11b and CD14-APC/Cy7 stains  were used to monitor CD11b upregulation in addition to macrophage differentiation. The cells were washed twice with FACS buffer at 1400 rpm for 5 min. each and LIVE/DEAD Fixable Near-IR Dead Cell stain  was added to each well at a dilution of 1:500 (50 µL) and incubated on ice, covered from light, for 15 min. The cells were washed again and resuspended in 200 µL FACS buffer. Fluorescence measurements were acquired using either Guava flow cytometer  or they were acquired at a Symphony A3 . Data was analyzed with FlowJo software V10. Induction was considered to be successful if CD11b expression in viable cells was found to be ~70% or higher. HL60, DMSO-treated HL-60, THP-1, and PMA-treated THP-1 cells were seeded at 1.5E5 cells/well on a clear 96-well plate. The cells were washed with 200 µL/well of PBS without CaAn international patent application (WO2018232014A1) has been filed covering the composition of matter and method of used disclosed in this report.


However, the levels of pERK1/2, active β-catenin, and HES1 were not significantly altered. Therefore, we propose that as essential components of the extracellular microenvironment of MSCs, plasma Evs are taken up by MSCs and subsequently repress osteogenic differentiation through an AHSG-mediated decrease in pSmad1/5/8 levels. Our work identifies plasma Evs as novel regulators of MSC osteogenic differentiation.Extracellular vesicles (Evs) contain diverse functional proteins, mRNAs, miRNAs, and DNA fragments, are secreted by various types of cells, and play important roles in cellular communication. Here, we show for the first time that plasma Evs inhibited the osteogenic differentiation of mesenchymal stromal cells (MSCs) in vitro and the level of inhibition was positively correlated with the plasma Evs concentration. Plasma Evs downregulated the expression of markers such as osteocalcin (OCN), Runt-related transcription factor 2 (Runx2), and Osterix at mRNA levels required for osteogenic differentiation and reduced pSmad1/5/8 levels in MSCs. Furthermore, pSmad1/5/8 levels increased and MSCs underwent normal osteogenic differentiation after Evs-derived  Human bone tissue undergoes the dynamic process of bone reconstruction via continuous bone formation and absorption throughout life. MSCs are multipotent, nonhaematopoietic progenitor cells that are isolated from a variety of tissues , and theα2-HS glycoprotein (AHSG/fetuin-A), a liver secretory glycoprotein that is present at high levels in the plasma and extracellular matrix [β) receptor antagonists, sequence homology was found between TGFβ receptor type II and AHSG [β and with greater affinity to the TGFβ-related BMPs, thereby inhibiting angiosteosis and the osteogenesis differentiation of MSCs [Plasma Evs have been reported to contain r matrix , 13. AHSr matrix . Furtherand AHSG . AHSG bl of MSCs .In our previous study, plasma Evs inhibited the osteogenic differentiation of MSCs in vitro, but the mechanism was unknown. In the present study, the administration of AHSG in plasma Evs inhibited the osteogenic differentiation of MSCs in a dose-dependent manner. Levels of pSmad1/5/8 decreased as the concentration of Evs increased and exhibited a positive correlation with alkaline phosphatase (ALP) levels and alizarin red staining. We concluded that as essential components of the extracellular microenvironment of MSCs, plasma Evs are taken up by MSCs and subsequently repress osteogenic differentiation through an AHSG-mediated decrease in pSmad1/5/8 levels.6/mL) were cultured in low-glucose Dulbecco's modified Eagle's medium  containing 10% foetal bovine serum  at 37°C in a humidified air and 5% CO2 atmosphere. The medium was replenished every 2 days. MSCs that formed colonies within 10 to 14 days were harvested using 0.25% trypsin/ethylenediaminetetraacetic acid  [Adult bone marrow was extracted from the posterior superior iliac spine under sterile conditions after obtaining informed consent from healthy donors for the harvest of their tissues for research purposes. Bone marrow mononuclear cells were isolated by Ficoll density centrifugation. Mononuclear cells  . The expMSCs were digested and washed with phosphate-buffered saline . After incubation with specific antibodies, MSCs were washed again and resuspended in PBS. Flow cytometry was performed using a BD Biosciences Influx Cell Sorter . Human phycoerythrin- (PE-) conjugated anti-CD29, fluorescein isothiocyanate- (FITC-) conjugated anti-CD73, FITC-conjugated anti-CD105, FITC-conjugated anti-CD45, PE-conjugated anti-CD14, and PE-conjugated anti-HLA-DR antibodies were used for these experiments .5 cells/well) and cultured in osteogenic differentiation medium (OM) consisting of UltraCULTURE™ medium with 2% Ultroser™ G Evs-free serum, 0.1 μM dexamethasone, 10 mM β-glycerol phosphate, and 50 μM ascorbic acid . The medium was replaced every three days. The total culture duration was 14 days. Cells were stained with alizarin red S .MSCs were seeded into six-well plates (1 × 105 cells) were centrifuged at 600 ×g for 5 min in a 15 mL conical tubes and then cultured with UltraCULTURE™ medium supplemented with 2% Ultroser™ G Evs-free serum, 1% ITS-Premix (Corning), 50 mg/L ascorbic acid , 1 mM sodium pyruvate , 100 nM dexamethasone , and 10 ng/mL TGF-β3 . The medium was replaced every three days. The total culture duration was 21 days. Cells were stained with Alcian blue GX.MSCs (2.5 × 105 cells/well). UltraCULTURE™ medium was supplemented with 2% Ultroser™ G Evs-free serum, 1 μM dexamethasone, 10 μg/mL insulin, 0.5 mM 3-isobutyl-1-methylxanthine, and 0.2 mM indomethacin . After 3 days of induction, the medium was replaced with UltraCULTURE™ containing 10 μg/mL insulin for 1 day and then the medium was replaced with the initial induction medium. After three cycles of media changes, cells were cultured with UltraCULTURE™ containing 10 μg/mL insulin until the 14th day. Cells were stained with Oil Red O after paraformaldehyde fixation.MSCs were seeded into six-well plates  and centrifuged at 12,000 ×g for 45 min at 4°C, and the resulting supernatant was carefully transferred to a new ultracentrifuge tube and centrifuged at 110,000 ×g for 2 h at 4°C. Pellets were resuspended in 10 mL of PBS, and the resulting supernatant was filtered through a 0.22 μm filter . The flow-through was collected in a fresh ultracentrifuge tube, centrifuged at 110,000 ×g for 70 min at 4°C, and washed by centrifugation at 110,000 ×g for 70 min at 4°C. Evs were resuspended in PBS and stored at -80°C until use in subsequent studies. 12 samples from 3 batches of Evs were all assessed by transmission electron microscopy , Nanoparticle Tracking Analysis instrument , and immunoblotting.Fifty-four volunteers were recruited as donors of human plasma. Evs were isolated from human plasma samples using the procedure described by Thery et al. . BrieflyμL of PKH26 dye was added to 0.5 mL of Diluent C, and the mixture was incubated with the Evs solution for 4 min at room temperature. Two millilitres of 0.5% bovine serum albumin  in PBS was added to bind the excess dye. The labelled Evs were centrifuged at 110,000 ×g for 70 min at 4°C, and the Evs pellet was suspended in PBS and used in uptake experiments. PKH26-labelled Evs were cocultured with MSCs for the indicated times. Then, MSCs were fixed and stained with FITC-labelled phalloidin  and DAPI . Images were obtained using the confocal laser scanning microscope .For uptake studies, purified Evs were labelled with a PKH26 (Red) kit  using the previously reported protocols . Briefly4 cells/cm2 in growth medium (GM) consisting of UltraCULTURE™ medium  and 2% Ultroser™ G Evs-free serum . When the culture reached 80% confluence, the medium was replaced with OM consisting of UltraCULTURE™ medium with 2% Ultroser™ G Evs-free serum, 0.1 μM dexamethasone, 10 mM β-glycerol phosphate, and 50 μM ascorbic acid.MSCs were seeded in 12-well plates at a density of 1.5 × 10MSCs cultured in GM were designated the control group, MSCs cultured in OM formed the induction group, and MSCs cultured in OM with different concentrations of plasma Evs were designated the Ind+Evs group. The medium was replaced every 3 days for up to 14 days. For the neutralization study, the Ind+Evs group was treated with different antibodies.MSCs were fixed with 4% paraformaldehyde  and stained with 1% ARS (pH 4.3)  for 15 min. After 3 washes, the stained cells were observed under a microscope  and photomicrographs were captured. Cells were destained with 10% cetylpyridinium chloride monohydrate . A 200 mL aliquot was transferred to a 96-well plate, and the absorbance was measured at 562 nm using a Multimode Reader .ALP activity in MSCs was detected using an ALP activity kit . Briefly, MSCs were lysed in radioimmunoprecipitation assay lysis buffer  containing protease inhibitors . The lysate was centrifuged at 12,000 revolutions per minute for 30 min at 4°C, and the supernatant was incubated with reaction buffer at 37°C for 15 min. Colour development was terminated with stop solution, and the absorbance was measured at 405 nm. The protein concentration in the lysate was determined using a Pierce BCA protein assay kit . ALP activity in the supernatant is presented as units per gram of protein per 15 min.The ALP staining assay was performed using an ALP staining kit . Briefly, cells were fixed with a citrate-acetone-formaldehyde fixative solution and then incubated with the alkaline dye solution for 15 min in the dark. The histochemical detection of ALP was observed under a microscope, and photomicrographs were captured.(−ΔΔct) method and normalized to the housekeeping gene GAPDH. Primers were designed to amplify GAPDH , Runx2 , OCN , and Osterix .Total RNA was isolated from MSCs using a TRIzol reagent . The cDNA templates were synthesised using a PrimeScript RT reagent kit  according to the manufacturer's instructions. qPCR was performed on a Light Cycler 480 Real-Time PCR System  using SYBR Premix Ex Taq . Changes in the relative expression of each mRNA were assessed using the 2β-catenin , active β-catenin , HES1  TGFβ1  , CD63 , CD81 , TSG101 , ApoB , and AHSG , IgG1κ , BMP2  . Membranes were incubated with HRP-conjugated anti-mouse or anti-rabbit secondary antibodies  for 1 h at room temperature. Specific antibody-antigen complexes were detected using Immobilon Western Chemiluminescent HRP Substrate .MSCs were lysed, and proteins were quantified as described above. After boiling with sample loading buffer , equal amounts of protein were separated on a 10% polyacrylamide gel  and subsequently transferred to PVDF membranes . The PVDF membranes were incubated with the following primary antibodies overnight at 4°C: GAPDH , Smad1 , phosphorylated Smad1/5/8 , Smad2/3 , phosphorylated Smad2/3 , ERK1/2 , phosphorylated ERK1/2 , Evs were lysed, and proteins were quantified as described above. The LC-MS/MS analysis was performed by Guangzhou FitGene Biotechnology Co. Ltd., as previously described .The raw files were converted to Mascot generic format (.mgf) files using Proteome Discoverer 1.4  with default settings for an in-depth proteome analysis. Protein Pilot 5.0 software  was used for the in-depth proteome analysis and quantitative analysis of proteins with .mgf files as the input. The Paragon algorithm integrated in Protein Pilot 5.0 software was used to search the database.http://www.genome.jp/kegg/pathway.html) was employed to use the current knowledge of biochemical pathways and other types of molecular interactions to examine differentially accumulated proteins. Additionally, STRING 9.1 was used to explore the interaction network and functional relations among the differentially expressed proteins.Briefly, we chose the parameter “Thorough ID” mode with a 95% confidence interval. Only proteins with reasonable ratios across all channels were quantified to increase the confidence level –23. Finat-test was used for the analysis. Statistical analyses were conducted using SPSS software . A p value of less than 0.05 indicated a significant difference.Data are presented as mean ± standard errors (SEs). Depending on the type of data, one-way ANOVA or a Flow cytometry was used to identify the phenotypic surface markers of MSCs. All MSCs from the various donors were positive for CD29, CD73, and CD105 but negative for CD14, CD45, and HLA-DR, confirming a typical MSC phenotype . OsteogeNumerous characteristics of Evs were examined, including electron microscopic features, particle size, and surface markers. The transmission electron microscopy analysis of plasma Evs revealed the presence of spherical vesicles with a typical cup shape . We deteWe next examined whether plasma Evs were transferred to MSCs. After isolation from plasma samples, Evs were fluorescently labelled with PKH26 and incubated with MSCs. We confirmed the ability of MSCs to take up Evs as soon as 8 h after incubation .th day after osteogenic induction to investigate the inhibitory effects of plasma Evs. According to the ARS staining (8 particles/mL) and the level of inhibition was proportional to the concentration of plasma Evs. As the plasma Evs concentration decreased , the osteogenic differentiation and function of MSCs gradually recovered.ARS staining and quantification were performed on the 14staining  and quanstaining  results,th or 14th days that decreased thereafter , and Notch (HES1) signalling pathways. However, the levels of pSmad1/5/8 decreased as the concentration of Evs increased and were positively correlated with ALP activity and ARS staining. In order to distinguish the BMP/Smad and TGFβ/Smad signalling pathways, we also detected the protein expression levels of pSmad2/3. The results showed that pSmad2/3 levels were not significantly different among the groups  receptor antagonists, sequence homology was found between TGFβ receptor type II and AHSG [β and with greater affinity to the TGFβ-related BMPs, thereby inhibiting angiosteosis and the osteogenesis differentiation of MSCs [According to our experimental results, the inhibitory effect on osteogenic differentiation of Evs was reversed following the addition of anti-AHSG antibodies. The levels of pSmad1/5/8 were increased in the Ind+Evs 8+anti-AHSG group compared with the Ind+Evs 8 and Ind+Evs 8+isotypic group. Meanwhile, pERK1/2, active and AHSG . AHSG bl of MSCs . We specBased on our results, AHSG in plasma Evs may have potential in the treatment and prevention of some metabolic bone diseases. AHSG expression is decreased in patients with ankylosing spondylitis, which may be one reason for pathological osteogenesis . StrategIn addition to our discoveries, many issues remain to be addressed. First, although plasma Evs repressed MSC osteogenic differentiation, we failed to identify the specific cell type that produced these functional Evs, making the differences in signal transduction patterns between two cell types impossible to elucidate. Second, although plasma Evs are superior to Evs produced by a specific type of cells at mimicking the internal environment, they constitute only a small part of whole plasma and do not completely reflect and represent the complicated conditions in the extracellular matrix of the human body. Based on the results from our preliminary experiments, Evs-free plasma and complete human plasma promoted the osteogenic differentiation of MSCs (Supplementary In summary, our research identifies plasma Evs as novel regulators of MSC osteogenic differentiation. AHSG in plasma Evs inhibits the osteogenic differentiation of MSCs by decreasing pSmad1/5/8 levels.