There is a growing need for scaffold material with tissue-specific bioactivity for use in regenerative medicine, tissue engineering, and for surgical repair of structural defects. We developed a novel composite biomaterial by processing human cardiac extracellular matrix (ECM) into a hydrogel and combining it with cell-free amniotic membrane via a dry-coating procedure. Cardiac biocompatibility and immunogenicity were tested in vitro using human cardiac fibroblasts, epicardial progenitor cells, murine HL-1 cells, and human immune cells derived from buffy coat. Processing of the ECM preserved important matrix proteins as demonstrated by mass spectrometry. ECM coating did not alter the mechanical characteristics of decellularized amniotic membrane but did cause a clear increase in adhesion capacity, cell proliferation and viability. Activated monocytes secreted less pro-inflammatory cytokines, and both macrophage polarization towards the pro-inflammatory M1 type and T cell proliferation were prevented. We conclude that the incorporation of human cardiac ECM hydrogel shifts and enhances the bioactivity of decellularized amniotic membrane, facilitating its use in future cardiac applications. Cardiovascular disease remains the leading cause of death in industrialized societies and is on the rise in developing countries . For endAs a translationally competent biologic human scaffold system, we have previously shown that decellularized human amniotic membrane alone reduces post-infarct remodeling processes if applied epicardially . HoweverWe have previously succeeded in isolating Extracellular Matrix (ECM) from human myocardium (hcECM) with a more preserved protein composition compared to protocols reported by other groups , for exaHere, we designed a novel composite patch material, linking the essential biological characteristics of target organ ECM with a stable carrier material by combining hcECM hydrogel (hgECM) with the cell-free amniotic membrane and tested its cytoprotective and immunological properties.Processing into a hydrogel via Pepsin digestion may have a significant impact on ECM protein composition, greatly depending on the individual digestion and processing procedure ,17. We tA total of 113 proteins were identified in hgECM, of which 51 belong to the Extracellular Region Part  accordinFurthermore, several intracellular proteins were found that are involved in cell cycle, ECM regulation, or signaling pathway modifications such as acetyl-CoA acyltransferase 2, actin, actinin, different ATP synthases, complement component 3, desmin, chitinase, and heat shock 27 kDa protein 1. Thus, processing not only preserved a large number of ECM proteins but also cellular proteins relevant for cytoprotective support.Cell-free hcECM slices were processed into microparticles and homogenized into a hydrogel  to be usPulverization of the hcECM resulted in microparticles with essentially uniform size and shape a,b. Furt6 Pa in the human heart [The elastic modulus (E-modulus) is an important factor of a material that determines how a material elastically deforms when pressure is applied, such as the pressure placed on the walls of the heart by its movement. Once applied to the organ, the material must withstand the present stress, e.g., up to 0.5 × 10an heart . Uniaxia6 ± 1.7 × 106 Pa, 8.2 × 106 ± 1.5 × 106 Pa and 9.9 × 106 ± 1.7 × 106 Pa, respectively. Maximum stress  of the amniotic membrane (AM), the DeAM and the DeAM + E, to elucidate whether the processing of the AM affects the elastic behavior and stress resistance. Typical mechanical behavior of the membranes during a uniaxial pulling test was characterized by a toe region where the load was transferred to sample, a linear region where the E-modulus was obtained, and rupture once the maximum stress has been reached a. Graphim stress d for the2, serum and glucose deprivation) , human epicardial derived cells (EPDC), and murine cardiomyocyte-like cells (HL-1), both under normal culture conditions and in “simulated ischemia” (1% Oivation) .p = 0.07).On DeAM + E, HL-1 cells adhered better within the first 30 min, but no further increase in adhesion was observed during longer cultivation periods a. In conCulturing on DeAM + E reduced cell death for all tested cell types under normoxic conditions d–f. UndeThe cell growth rate was improved on DeAM + E for all cell types as seen by BrdU incorporation in HL-1 cells, EPDCs, and hCF, both under normoxia and in “simulated ischemia” j–o, high+-derived macrophage subpopulations , and monocytes as well as CD14ulations .Compared to standard culture plate conditions, cytokine secretion of naïve monocytes was affected by DeAM both with and without hgECM coating. IL-6 secretion was significantly decreased on both surfaces a but the+ monocytes once polarized towards the pro-inflammatory M1 type were unequivocally identifiable by a substantial increase in all measured cytokines IL-6, TNF-α and IL-10 (Macrophages derived from M-CSF stimulated CD14nd IL-10 g–i compand IL-10 i.The cytokine secretion profile of PBMCs demonstrated clearly that only the Phytohemagglutinin (PHA) stimulated PBMC (positive control) secreted significantly more IL-6, TNF-α and IL-10 j–l than Finally, we examined the impact of hgECM coating on macrophage polarization towards the M1 or M2 type by examining the magnitude of surface marker expression. In general, ECM has the potential to induce M2 polarization  and mighAfter macrophage polarization, the M1 macrophage polarization control group represents the pro-inflammatory positive control and displayed upregulated expression of the CD80 marker a. The an+ T cells as well as with a comparable proportion in the CD4+ and CD8+ T cell subpopulations  but rather trend to induce a polarization towards the anti-inflammatory M2-type. The kind of material has a sensitive effect on the macrophage polarization behavior. It was shown that an electrospun polymer could induce a polarization towards the pro-inflammatory M1-type ,27 whereWe primarily aim at using the DeAM + E composite material for sustained delivery of biologics  to the epicardial surface of the infarcted heart, since the epicardium is known to be highly involved in cardiac regeneration processes. In zebrafish and young mice, it has been shown that the epicardium can completely regenerate the infarcted myocardium ,40,41. TIn summary, we developed a cell-free human amniotic membrane/myocardial ECM composite scaffold material with favorable in vitro biological and mechanical properties that has the potential be used in large animal experiments and ultimately clinical studies. As a cell-free material, the production process can readily be scaled-up under GMP conditions. Further in vivo testing will reveal whether the DeAM + E material alone is able to significantly modify post-infarct remodeling processes. In any event, it may serve as a universal platform for epicardial delivery of a broad spectrum of cells and therapeutic agents.Left ventricular myocardium was collected from explanted hearts from patients who underwent heart transplantation for end-stage dilated cardiomyopathy. All patients were in New York Heart Association class II or IV, with a left ventricular ejection fraction <20%, and were not on mechanical circulatory support. Hepatitis B or C and HIV infection were ruled out preoperatively. The study protocol conforms to the ethical principles outlined in the Declaration of Helsinki. Patients provided informed consent for the use of the tissue for research purposes, and the process of tissue collection was approved by the Institutional Review Board and ethics committee of Charité—Universitätsmedizin Berlin .n = 17) were anonymously obtained after cesarean delivery of healthy and uncomplicated pregnancies from women who had given written informed consent for use of the placenta for research purposes.Human full term placentas  and placed in lysis buffer  for 1 h at room temperature, followed by incubation in 0.5% sodium dodecyl sulfate  solution for 4 h at room temperature with constant stirring. Next, the AM was washed in PBS three times at room temperature and finally overnight at 4 °C under constant agitation.Excess blood was removed by washing with Hank’s balanced salt solution  was mechanically peeled from the underlying chorion and washed several times in HBSS to remove blood. Subsequently, AM was washed with phosphate buffered saline without calcium and magnesium (PBSDecellularized AM (DeAM) was cut into appropriate pieces and lyophilized.w/v EDTA, pH 7.4; Carl-Roth) followed by SDS  incubation for 6 h under constant agitation at room temperature. Then, the slices were washed three times with PBS for 10 min to remove SDS, followed by an overnight washing period with Dulbecco’s phosphate buffered saline (DPBS) including 100 U/mL penicillin/streptomycin and nystatin to guarantee complete removal of SDS. Finally, the tissue was immersed in fetal bovine serum  for 3 h at 37 °C followed by three washes with DPBS/penicillin/streptomycin/nystatin for 10 min each. Prior to further processing and/or experimentation, cECM slices were stored at 4 °C for up to 2 weeks.Human myocardium was harvested under sterile conditions and stored for no longer than 48 h at 4 °C before sectioning. Human Cardiac Extracellular Matrix (hcECM) isolation and processing was performed as described . The myoTo process decellularized matrix, slices were snap-frozen in liquid nitrogen, lyophilized and pulverized using the Precellys ceramic Kit 1.4 mm  and the Minilys homogenizer (PEQLAB Biotechnologie) at a rate of six runs per 1 min at 4000 rpm. Resulting particles were resolved in distilled water and filtered using a 200 µm transfusion filter system to remove larger particles, lyophilized again for 48 h, and stored at −80 °C. For homogenization, 1 mg/mL pepsin from porcine gastric mucosa  was dissolved in 0.01 M HCl, and incubated with 10 mg/mL hcECM particles for 48 h under constant agitation. To inhibit the Pepsin activity, the hcECM solution was neutralized on ice and brought to physiological pH by adding pre-cooled 10× PBS and 0.1 M NaOH. Finally, the concentration was set to 8 mg/mL by dilution using 1× PBS and hcECM Hydrogel (hgECM) was stored at 4 °C for not longer than 24 h before use.2 hgECM solution and dried for 48 h at 37 °C in a non-humidified incubator. After two gentle washes with PBS, the coated membranes were used for experiments. DeAM and DeAM coated with hgECM (DeAM + E) were tested.With an 8 mm Biopsy-Punch , the DeAM was cut into discs and lyophilized. One disc of DeAM was placed into each well of a 48-well plate and held in place by rings cut from silicone tubing  providing an inner diameter equal to 96-well format, and washed with PBS. The samples were completely coated by adding 150 µL/cm2O)  followed by two final incubations in 100% Ethanol. Ethanol was removed by two incubations in Hexamethyldisilazane (Sigma-Aldrich) for 10 min each. Samples were air-dried overnight under a fume hood. Then, samples were placed on a stamp  with 12 mm PLANO Tab  and sputter coated with gold for 30 s . Finally, samples were imaged using the JCM 6000 benchtop SEM  in high vacuum mode at 10  kV.Samples were washed twice in PBS and fixed with 2.5% grade I glutaraldehyde (Sigma-Aldrich) for 30 min at room temperature. Subsequently, samples were washed again in PBS and dried using 5 min incubation steps in increasing Ethanol (Carl-Roth) concentration  using fixed clamps. Sample length was set to 1 cm, while thickness and width were individually measured using a micrometer (precision: ±0.001 µm) and a caliper (precision: ±0.01 mm) , respectively. Crosshead speed was set to 0.05 mm/s and pulling up to 0.9 strain. Elastic moduli were calculated as the slope of the most linear region presented on the stress over strain curve. Stress and strain were obtained in agreement with the initial cross sectional area and length of each sample. Maximum stress was obtained as the maximum load before sample rupture.Uniaxial pulling tests were performed in a wet state for the different groups under a BOSE testing bench (BOSE ElectroForceHomo sapiens (human) (20175 sequences); (ii) proteolytic enzyme: trypsin; (iii) maximum of accepted missed cleavages: 1, (iv) mass value: monoisotopic, (v) peptide mass tolerance 10 ppm; (vi) fragment mass tolerance: 0.05 Da; and (vii) variable modifications: oxidation. No fixed modifications were considered. Only proteins with scores corresponding to p < 0.05 were considered. The cut-off score for individual peptides was equivalent to p < 0.05 for each peptide as calculated by MASCOT (version number 2.2).The hgECM (140 µg) was resolved in 40 µL P-buffer (0.05 M Tris Base pH 7.5 (Sigma-Aldrich), 0.05 M potassium chloride , 0.11 M Chaps , 20% glycerine (Merck), PhosSTOP , cOmplete (Roche)) and sonicated on ice. Then, 200 µL UA  and 8 M Urea (Sigma-Aldrich)) were added and incubated for 10 min at room temperature. The solution was transferred into an amicon filter , followed by buffer exchange to trypsin solution,  and incubated at 37 °C overnight. Subsequently, ABC-buffer  was added and the solution centrifuged for 10 min at 14,000 rpm at room temperature. The flow-through was desalted using ZipTip C18 (Merck) pipet tips according the manufacturer’s protocol. Peptide samples were dried in a SpeedVac  concentrator and extracted afterwards in 20 µL 0.1% trifluoroacetic acid (TFA) for 15 min at room temperature. Analysis was performed using UPLC  ESI-QTOF-mass spectrometer . Mass spectra were evaluated using MASCOT software  automatically searching the SwissProt 51.9 database . MS/MS ion search was performed with the following set of parameters: (i) taxonomy: All cell types were negatively tested for mycoplasma contamination.2.Murine HL-1 cardiomyocytes (HL-1), immortalized using the simian virus SV40 T-antigen under the control of an atrial natriuretic factor (ANF) promotor , were geHuman cardiac fibroblasts  were cultured in DMEM (Gibco), 10% FBS (Biochrom) added and 100 U/mL penicillin, 100 µg/mL streptomycin (Gibco).Human immune cells  were cultured in VLE RPMI 1640 Medium (Biochrom), 10% hAB Serum (Sigma-Aldrich) 100 U/mL penicillin, 100 µg/mL streptomycin and 2 mM L-Glutamine (Gibco).Peripheral blood mononuclear cells (PMBCs) were isolated from Buffy Coat  using Biocoll (Biochrom) density gradient.+ Magnetic Cell Separation kit .Monocytes were isolated from PMBCs using the CD14Macrophages were differentiated from isolated monocytes by a 7 day incubation in culture medium with 50 ng/mL M-CSF (Miltenyi Biotec) and collected with a cell scraper.Adult human atrial samples (auricles) were obtained during cardiac surgery as redundant material anonymously collected as surgical waste under general informed consent. Epicardial Derived Cells (EPDC) were isolated from human heart auricles as described . Briefly2/5% CO2) for 5 h in a Binder CB150 incubator .Simulated ischemia cells were cultured on patch material or cell culture dishes for 24 h before being exposed to glucose/serum deprivation (glucose- and FBS-free DMEM (Thermo-Fisher)), with 100 U/mL penicillin/streptomycin (Gibco), 1% O2 were used. Cells were placed onto the scaffolds and incubated at 37 °C for 30 min, 60 min, or 120 min. Supernatants containing unbound cells were collected and cells were lysed with a final concentration of 1 °C% Triton-X100. Fluorescence intensity, representing the number of cells, was analyzed using the MithrasLB940 plate reader  following the manufacturer’s instructions. For determining adhesion rates, 125,000 HL-1 cells, or 20,000 hCF or EPDCs per cm2, were cultured onto the scaffolds or on standard cell culture conditions for controls. Cells were either cultured on standard culture conditions for 24 h or additionally exposed to simulated ischemia. Next, supernatants were analyzed for LDH release according to manufacturer’s instructions, and fluorescence was measured using the MithrasLB940 plate reader (Berthold Technologies). Results were normalized to normal cell culture conditions.Determination of cell necrosis was performed by measuring the release of Lactate Dehydrogenase (LDH) using the CytoTox-ONE™ Homogeneous Membrane Integrity Assay (Promega). Then, 125,000 HL-1 cells, or 20,000 hCF, or EPDCs per cm2SO4, and 100 µm of the solution was transferred to a fresh 96-well plate. Read out was performed using the SpectraMax 340PC384 .Cell growth was analyzed using the BrdU Cell Proliferation ELISA Kit (Roche). To determine cell growth, 62,500 HL-1 cells, or 20,000 hCFs or EPDCs per cm² were cultured onto the scaffolds or standard cell culture conditions as controls. Cells were cultured under standard conditions for 24 h at 37 °C in a humidified incubator. Subsequently, labelling reagent was added and incubated for 5 h at 37 °C under normoxia or simulated ischemia. Afterwards, cells were analyzed for BrdU-incorporation according to protocol. Finally, the reaction was stopped by adding 1 M HTo determine cytokine secretion, 100,000 monocytes were cultured onto patch materials and incubated for 24 h at 37 °C. For the positive control, medium was supplemented with 200 ng/mL Lipopolysaccharide (LPS). Supernatants were collected and cytokine secretion was detected using Human ELISA MAX™ Deluxe Kits (Thermo-Fisher) for IL-6, IL-10 and TNF-α following the manufacturer’s protocol.Cells were collected and washed once in buffer , 0.5% Sodium azide (Carl-Roth)). The supernatant was discarded completely and 50 µL staining solution in buffer containing all labeling antibodies was added and incubated for 30 min at 4 °C. Subsequently, the suspension was washed once with buffer and suspended in buffer containing 1% Paraformaldehyde  and analyzed the next day. The following antibodies were used for macrophage staining: CD163-Fitc , CD80-PE , CD16-PerCPCy5.5 , CD206-APC , CD14-APCCy7 , HLA-DR-PeCy7 , Life/Dead-V510 . Following antibodies were used for T cell staining in the proliferation setup: CD8-PE , CD4-APC , CD3-APCCy7 .7 cells in 1 mL assay buffer, washed twice in assay buffer and a labeled 200 µL CFSE working solution was added to 1.8 mL cell suspension in assay buffer. The labeled suspension was incubated for 15 min at room temperature. The reaction was stopped by adding medium followed by two washing steps with assay buffer. Finally, cells were suspended in the medium and used for the experiment. Then, 150,000 PBMCs were cultured on patch materials for 5 days. The positive control medium was supplemented with 5 µg/mL Phytohaemagglutinin (PHA). Subsequently, cells were harvested and analyzed for proliferation of CD3+, CD3CD4+ and CD3CD8+ T cell proliferation by flow cytometry . Briefly, PBMCs  were adjusted to 1 × 10To investigate macrophage polarization, 100,000 macrophages were cultured for 48 h on patch materials or cell culture dishes. To induce polarization towards M1, the culture medium was supplemented with 20 ng/mL INFy +100 ng/mL LPS. For M2a polarization, 20 ng/mL IL-4 and for M2c 20 ng/mL IL-10 was used.After 2 days of culture, supernatants were collected for detection of cytokine secretion via ELISA using Human ELISA MAX™ Deluxe Kits (Thermo-Fisher) for IL-6, IL-10 and TNF-α following the manufacturer’s protocol. Cells were harvested using Accutase ) and analyzed for polarization marker expression by flow cytometry  was used for data analysis and plotting. A p-value of p < 0.05 was considered significant.Data are shown as mean ± SEM. Comparisons passed normality and equal variance testing before the significance was tested. Differences between more than two groups were determined by one-way ANOVA with Bonferroni The source and composition of applied extracellular matrix is crucial for specific application. Here, we demonstrated the additional coating of cell-free amniotic membrane scaffolds with processed human cardiac extracellular matrix. The novel resulting patch material not only specifically supports the culture and interaction of cardiac cells such as cardiomyocytes, epicardial cells, and cardiac fibroblasts, but also exerts immunomodulatory effects on cell subsets relevant for cardiac regeneration and tissue engineering. The newly designed patch system may therefore enhance the therapeutic capacity of epicardial composite materials and pave the way for improved cardiac regeneration attempts.


The enediyne family of natural products has had a profound impact on modern chemistry, biology, and medicine, and yet only 11 enediynes have been structurally characterized to date. Here we report a genome survey of 3,400 actinomycetes, identifying 81 strains that harbor genes encoding the enediyne polyketide synthase cassettes that could be grouped into 28 distinct clades based on phylogenetic analysis. Genome sequencing of 31 representative strains confirmed that each clade harbors a distinct enediyne biosynthetic gene cluster. A genome neighborhood network allows prediction of new structural features and biosynthetic insights that could be exploited for enediyne discovery. We confirmed one clade as new C-1027 producers, with a significantly higher C-1027 titer than the original producer, and discovered a new family of enediyne natural products, the tiancimycins (TNMs), that exhibit potent cytotoxicity against a broad spectrum of cancer cell lines. Our results demonstrate the feasibility of rapid discovery of new enediynes from a large strain collection. Recent advances in microbial genomics clearly revealed that the biosynthetic potential of soil actinomycetes to produce enediynes is underappreciated. A great challenge is to develop innovative methods to discover new enediynes and produce them in sufficient quantities for chemical, biological, and clinical investigations. This work demonstrated the feasibility of rapid discovery of new enediynes from a large strain collection. The new C-1027 producers, with a significantly higher C-1027 titer than the original producer, will impact the practical supply of this important drug lead. The TNMs, with their extremely potent cytotoxicity against various cancer cells and their rapid and complete cancer cell killing characteristics, in comparison with the payloads used in FDA-approved antibody-drug conjugates (ADCs), are poised to be exploited as payload candidates for the next generation of anticancer ADCs. Follow-up studies on the other identified hits promise the discovery of new enediynes, radically expanding the chemical space for the enediyne family. Natural products offer unmatched chemical and structural diversity compared to any other small-molecule families. They continue to inspire novel chemistry, enzymology, and biology investigations and remain the best sources of drugs and drug leads . The ene3–The enediynes have had a profound impact on modern chemistry, biology, and medicine , 4, 7. A3–Streptomyces globisporus producer . By surveying 3,400 strains, we identified 81 potential producers, and genome sequencing of 31 representatives revealed at least 28 distinct enediyne biosynthetic gene clusters. We constructed an enediyne genome neighborhood network (GNN) to facilitate gene cluster annotation and to predict new structural features, thereby further streamlining the discovery of new enediyne natural products. To demonstrate the feasibility of our approach in rapidly discovering new enediynes from a large strain collection, we characterized a new C-1027 producer with a significantly higher C-1027 titer than the original producer , 10 and E3, E4, E5, E, and E10 (E3/E4/E5/E/E10) (20–Since the cloning of the first 9-membered C-1027)  and 10-m13–5/E/E10)   20–; no appa/E10) 20– and B. T/E10) 20– and C (5-1027  an/E10) 20–.E5/E or E/E10 , taxonomy, and geographic locations where the strains were isolated   . It is t to 69%) , suggestGenome sequencing of 31 representative hits from the 28 clades confirmed that they all contain an enediyne gene cluster and therefore are true enediyne producers . SignifiStreptomyces globisporus in 1993    (9). Mulnalysis) . The fac species  ensures For the other 27 clades, genome sequencing of representative hits unveiled gene clusters that are distinct from all enediyne biosynthetic gene clusters known to date , indicatE2 or R3 genes, respectively  are fundamentally different from the reported 10-membered enediyne biosynthetic gene clusters  from Streptomyces sp. strain CB03234 by genome sequencing , bu, buStrepuncialis , had nots genome  and inac SB18001 . HPLC anynthesis . We nextquencing . Remarkacassette , GNN anacassette  and may Streptomyces sp. strain CB03234, we first inactivated the tnmE gene in Streptomyces sp. strain CB03234 to afford the corresponding ΔtnmE mutant strain SB20001 . For example, TNM A is more potent than UCM, particularly against breast cancer cell lines  , a variall lines ; UCM is ll lines . Most imll lines  and C, tll lines , 46, supIn spite of their profound impact on modern chemistry, biology, and medicine, the enediyne family of natural products remains very small, with only 11 members structurally characterized to date. Recent advances in DNA sequencing and microbial genomics, however, clearly revealed that the biosynthetic potential of soil actinomycetes to produce enediynes is greatly underappreciated. A great challenge is that of developing innovative methods to discover new enediynes and producing them in sufficient quantities for chemical, biological, and clinical investigations. We recently reported a high-throughput real-time PCR method to prioritize strains for natural-product discovery. By adapting this method to identify strains that are highly likely to encode enediyne biosynthesis, followed by genome sequencing, bioinformatics analysis, genetic manipulation, and fermentation optimization, we have now demonstrated the feasibility of rapid discovery of new enediynes from a large strain collection. The new C-1027 producers, with a significantly higher C-1027 titer than the original producer, will impact the practical supply of the drug should it eventually be brought into clinical applications. The TNMs, with their extremely potent cytotoxicity against a broad spectrum of cancer cells and rapid and complete cell killing characteristics, in comparison with the payloads used in FDA-approved ADCs and ADCs in various stages of development, are poised to be exploited as payload candidates for the next generation of anticancer ADCs. Follow-up studies on the other hits that have already been identified in this study or on application of our strategy to other strain collections promise the discovery of new enediynes, radically expanding the chemical space for the enediyne family of natural products. Our results also support strain prioritization and genome mining for the discovery of other classes of natural products from ever-growing microbial strain collections. Together with other emerging strategies and technologies, these findings will inspire continued innovations in natural-product discovery.Primers, plasmids, and strains used and reported in this study are summarized in Genomic DNA (gDNA) was prepared from strains isolated from various unexplored and underexplored ecological niches , 47, 48.Real-time PCR was performed using an Applied Biosystems 7900HT Fast real-time PCR system. Preparation of gDNA arrays and application of the real-time PCR method to survey the 3,400 actinomycetes strains for the enediyne PKS gene cassette and to identify new enediyne producers followed the published protocol for strain prioritization with modifications . For furde novo assembly were done with CLC Genomics Workbench version 7.5.1 (CLC Bio.) using default settings. The resulting contigs were further extended and joined into a final scaffold by SSPACE version 2.0 (PRJNA293172) and S. uncialis DCA2648 (under BioProject PRJNA286672) reported in this study have been deposited in GenBank, with their accession numbers summarized in Genome sequencing of the representative enediyne producers was performed using an Illumina MiSeq sequencer (2 × 300 paired-end sequencing) at the Next Generation Sequencing and Microarray Core Facility, TSRI. Read quality filtering was performed using a tool developed in-house. Adapter trimming and sion 2.0  using alsion 2.0 . The draStreptomyces sp. strain CB02366, the ucm gene cluster from S. uncialis DCA2648, and the tnm gene cluster from Streptomyces sp. strain CB03234 were deposited in GenBank under accession numbers KU597647, KT762610, and KT716443, respectively. Accession numbers for the other new enediyne gene clusters are listed in Annotation of the new enediyne gene clusters and construction of the enediyne GNN were carried out as described previously , 6. AnnoE value corresponding to comparisons between proteins. A more detailed description is provided in The enediyne GNN was constructed and included (i) the 7 known 9-membered enediyne gene clusters , (ii) the 3 known 10-membered enediyne gene clusters , (iii) the newly characterized gene cluster for the 10-membered enediyne UCM, and (iv) the 31 new enediyne gene clusters  discovered in this study . CytoscaStreptomyces species was performed by gene replacement following literature procedures  mutant strains were cultured individually following previously reported procedures  following previously published procedures , and SB20002  mutant strains were cultured individually in 250-ml baffled flasks containing 50 ml of TSB liquid medium. After growth at 28°C and 250 rpm for 2 days, 5 ml of seed culture was inoculated into 250-ml baffled flasks containing 50 ml of the production medium . The resulting cultures were incubated at 28°C and 250 rpm for 7 days and individually harvested. Each culture was centrifuged, the supernatant was extracted with EtOAc, and the cell pellet was extracted with CH3COCH3. The combined extracts were concentrated in vacuum and dissolved in CH3OH for HPLC and HR-ESI-MS analysis  was isolated from 6 liters of fermentation culture of the ΔtnmH mutant strain SB20002. The structures of TNM A and C were established on the basis of extensive MS, CD, and NMR analysis  was isolated from 6 liters of fermentation culture of the analysis . For fur50s of TNMs against selected human cancer cell lines, including breast , melanoma (M14 and SK-MEL-5), non-small cell lung cancer (NCI-H226), and central nervous system (SF-295 and SF-539) cells, with UCM as a control, were determined by following standard protocols described previously (50 was determined by computerized curve fitting using GraphPad Prism (The ICad Prism . For comad Prism . FurtherStreptomyces sp. strain CB02366, the ucm gene cluster from S. uncialis DCA2648, and the tnm gene cluster from Streptomyces sp. strain CB03234 are KU597647, KT762610, and KT716443, respectively. Accession numbers for the draft genomes of the selected 31 hits, as well as the UCM producer S. uncialis DCA2648, are summarized in Accession numbers for the C-1027 gene cluster from Figure S1 Known enediynes and selected natural products as ADC payloads. Download Figure S1, PDF file, 0.5 MBFigure S2 A virtual survey of bacterial genomes for enediyne producers. Download Figure S2, PDF file, 0.5 MBFigure S3 Construction and confirmation of SB1036, SB18001, SB20001, and SB20002. Download Figure S3, PDF file, 0.2 MBFigure S4 1H and 13C NMR spectra of TNM A and TNM C. Download Figure S4, PDF file, 0.3 MBTable S1 Plasmids, strains, and primers used in this work.Table S1, PDF file, 0.1 MBTable S2 Taxonomy, geography, and accession numbers of sequenced genomes.Table S2, PDF file, 0.4 MBTable S3 S. uncialis DCA2648, and CB03234, respectively.Annotation of the C-1027, UCM, and TNM gene clusters from CB02366, Table S3, PDF file, 0.1 MBTable S4 Annotation of the 29 enediyne gene clusters from the representative hits.Table S4, PDF file, 0.4 MBTable S5 1H and 13C NMR data and other physicochemical data of TNM A and C.Table S5, PDF file, 0.3 MBText S1 Supplemental Experimental Procedures. Download Text S1, PDF file, 0.3 MB


Citrus sinensis) and “Sour pummelo” (Citrus grandis) were irrigated daily with a nutrient solution at a pH of 2.5, 3, 4, 5, or 6 for 9 months. Thereafter, the following responses were investigated: seedling growth; root, stem, and leaf concentrations of nutrient elements; leaf gas exchange, pigment concentration, ribulose-1,5-bisphosphate carboxylase/oxygenase activity and chlorophyll a fluorescence; relative water content, total soluble protein level, H2O2 production and electrolyte leakage in roots and leaves. This was done (a) to determine how low pH affects photosynthesis, related physiological parameters, and mineral nutrient profiles; and (b) to understand the mechanisms by which low pH may cause a decrease in leaf CO2 assimilation. The pH 2.5 greatly inhibited seedling growth, and many physiological parameters were altered only at pH 2.5; pH 3 slightly inhibited seedling growth; pH 4 had almost no influence on seedling growth; and seedling growth and many physiological parameters reached their maximum at pH 5. No seedlings died at any given pH. These results demonstrate that citrus survival is insensitive to low pH. H+-toxicity may directly damage citrus roots, thus affecting the uptake of mineral nutrients and water. H+-toxicity and a decreased uptake of nutrients  and water were likely responsible for the low pH-induced inhibition of growth. Leaf CO2 assimilation was inhibited only at pH 2.5. The combinations of an impaired photosynthetic electron transport chain, increased production of reactive oxygen species, and decreased uptake of nutrients and water might account for the pH 2.5-induced decrease in CO2 assimilation. Mottled bleached leaves only occurred in the pH 2.5-treated C. grandis seedlings. Furthermore, the pH 2.5-induced alterations of leaf CO2 assimilation, water-use efficiency, chlorophylls, polyphasic chlorophyll a fluorescence (OJIP) transients and many fluorescence parameters, root and leaf total soluble proteins, H2O2 production, and electrolyte leakage were all slightly greater in C. grandis than in C. sinensis seedlings. Hence, C. sinensis was slightly more tolerant to low pH than C. grandis. In conclusion, our findings provide novel insight into the causes of low pH-induced inhibition of seedling growth and leaf CO2 assimilation.Seedlings of “Xuegan” ( Acidic soils that limit crop growth and productivity are often observed all over the world, especially in the tropics and subtropics. Approximately 30% of the world's ice-free land is acidic, and approximately 12% of crops are cultivated on acidic soils  and a lack of nutrients—namely phosphorus (P), calcium (Ca), magnesium (Mg), potassium (K), and molybdenum (Mo)— and a reduced uptake of water . Malkanthi et al. . A pH 4.5 decreased the whole-root water conductivity in the H+-sensitive maize cultivar Adour 250, but it did not in the H+-tolerant maize cultivar BR 201 F  and red maple (Acer rubrum) ; they found that pH 3.0 decreased leaf photosynthesis, transpiration, and Chl level in the four clones as well as the leaf water-use efficiency (WUE) in the G4 leaves, but pH 3.0 did not affect WUE in the G9, G12, and G3 leaves. Zhang et al. (PSII), whereas it increased leaf non-photochemical quenching (NPQ); however, pH 3 had no effect upon leaf stomatal conductance, photochemical quenching (qP), and the maximum PSII efficiency of dark-adapted leaves (Fv/Fm), thus leading the authors to conclude that non-stomatal factors played a role in the low pH-induced inhibition of photosynthesis. Nonetheless, pH 4.0 did not influence spatial heterogeneity of Chl fluorescence, Fv/Fm, ΦPSII, and quantum yields of regulated (ΦNPQ) and nonregulated (ΦNO) energy dissipation in the leaves of Plantago algarbiensis and P. almogravensis  and decreased activities of superoxide dismutase (SOD) and catalase (CAT) in the roots of two rice cultivars . Martins et al.  found ths et al.  observed. et al.  reportedCitrus sinensis seedlings died within a few days at pH 2.0, but they were not killed for months at pH 2.5 and 3.0 though their growth was limited or negligible. Nevertheless, citrus do not thrive in trongly acidic soils, because serious problems may arise when the soil pH is 5.0 or lower  orchards in Pinghe, Zhangzhou, China had an average value of 4.34 and ranged from 3.26 to 6.22, with up to 90.0% of the orchard soils having a pH lower than 5.0. So far, however, only a handful of reports have empirically investigated the effects of low pH on citrus growth  leaves increased (decreased) with decreasing soil pH. The concentration of P and Ca in pummelo leaves decreased with decreasing soil pH  to determine how low pH affects gas exchange, related physiological parameters, and the mineral nutrient profiles in citrus seedlings; and (b) to understand the mechanisms by which low pH may lead to a decrease in leaf CO2 assimilation.The objectives of this work were (C. grandis) and “Xuegan” (C. sinensis) were germinated in plastic trays filled with clean river sand. Four weeks after germination, uniform seedlings that had a single stem were chosen and transplanted into 6-L terracotta pots (two seedlings per pot) containing clean river sand. Seedlings were grown in a greenhouse under a natural photoperiod at FAFU. One week after transporting, each pot was irrigated every other day with 500 mL of a nutrient solution containing 2.5 mM Ca(NO3)2, 2.5 mM KNO3, 1 mM MgSO4, 0.5 mM KH2PO4, 20 μM Fe-EDTA, 10 μM H3BO3, 2 μM MnCl2, 2 μM ZnSO4, 0.5 μM CuSO4, and 0.065 μM (NH4)6Mo7O24. Seven weeks after transplanting, each pot was fertilized daily until saturated with the same nutrient solution (approximately 500 mL), except that the pH of the nutrient solution was adjusted to 2.5, 3, 4, 5, or 6 with 1 M HCl. There were 20 replicates  per treatment in a completely randomized design. In this experiment, the pH 5 treatment served as the control because seedling growth and many physiological parameters reach their maximum at pH 5. Nine months after the pH treatment began, recent fully-expanded (approximately 7-week-old) leaves and approximately 5-mm-long white root apices were used for all measurements except that for root mineral element concentrations. After leaf gas exchange and Chl a fluorescence were measured, leaf disks (0.2826 cm2 in size) and approximately 5-mm-long white root apices from the same seedlings were harvested from randomly selected seedling at noon on a sunny day and immediately frozen in liquid N2, then stored at −80°C until they were used for the assays of ribulose-1,5-bisphosphate carbohylase/oxygenase (Rubisco), total soluble proteins, and pigments. The remaining seedlings that were not sampled were selected randomly to measure plant biomass, root and leaf relative water content (RWC), and electrolyte leakage, and the root, stem and leaf mineral element concentrations.This study was conducted at the Fujian Agriculture and Forestry University (FAFU) in Fuzhou, China. Seedling culture was performed according to Han et al.  and PengNine months after the pH treatment began, 10 seedlings per treatment from 10 pots were collected. The seedlings were divided into leaves, stems, and roots. Their DW was measured after being dried at 70°C for 48 h. Specific leaf weight was calculated as the ratio of leaf weight to leaf area.Leaf pigments were extracted with 80% (v/v) acetone. The Chl, Chl a and Chl b, and carotenoids (Car) in the extract were determined according to Lichtenthaler .2PO4-Na2HPO4 (pH 7.0) and 5% (v/v) insoluble polyvinylpyrrilodone (PVP), and assayed according to Bradford  from the same leaf or 20 approximately 5-mm-long white root apices taken at midday under full sun, were immediately transferred to a 50-mL tube filled with 15 mL of distilled water. The tubes were placed at room temperature in the dark for 24 h and the first electrical conductance (C1) was measured. Then the tubes were incubated in a boiling water bath for 15 min and the second electrical conductance (C2) was measured after being cooled. The electrolyte leakage was calculated as: electrolyte leakage (%) = (C1/C2) × 100.Root and leaf electrolyte leakage was assayed according to Han et al. . BrieflyRoot and leaf RWC were gravimetrically determined  were incubated in 2 mL of a 50 mM phosphate buffer (pH 7.0), 5 U horseradish GPX, and 0.05% (w/v) guaiacol for 2 h at room temperature in the dark. Then the absorbance was measured at 470 nm.Root and leaf Hn et al. . About 1Fibrous roots, the middle sections of stems, and approximately 7-week-old leaves (midribs and petioles removed) were collected and dried at 70°C for 48 h. Dried samples were ground in a mortar to pass through a 40-mesh sieve and stored for later analysis.3:H2O2 (5:2 v/v). P was determined colorimetrically as the blue molybdate-phosphate complexes according to Lu  in the roots, stems, and leaves. Element distributions in roots, stems, or leaves (%) were calculated as:  × 100.2 concentration under a controlled light intensity of 996–1004 μmol m−2 s−1 between 9:30 and 12:30 on a clear day. During all of these measurements, the leaf temperature and relative humidity were 30.0 ± 0.2°C and 64.5 ± 0.6%, respectively. Leaf Rubisco was extracted and assayed according to Chen et al.  at an ambient COn et al.  and Lin n et al. , respect−2 s−1, which was provided by an array of three light-emitting diodes (peak 650 nm) that were focused on the leaf surface to provide homogenous illumination over the exposed area of the leaf. All the measurements were performed on 3-h dark-adapted plants at room temperature.The polyphasic Chl a fluorescence (OJIP) transients were measured by a Handy Plant Efficiency Analyzer . The transient was induced by a saturating red light of approximately 3,400 μmol ms20μ, considered as the minimum fluorescence Fo), 50 μs (Fs50μ), 300 μs (Fs300μ), 2 ms , 30 ms , and P-step (considered as the maximum fluorescence Fm). The following parameters that refer to “time 0” (start of fluorescence induction) are: (a) fluorescence parameters derived from the extracted data, i.e., the maximum variable fluorescence Fv = Fm − Fo and the approximated initial slope (in ms−1) of the fluorescence transient V = f(t) [Mo = 4(Fs300μ−Fo)/(Fm−Fo)]; (b) the specific energy fluxes per reaction center (RC) for energy dissipation (DIo/RC) and absorption (ABS/RC); (c) the yields of the flux ratios, i.e., quantum yield for energy dissipation (φDo = DIo/ABS = Fo/Fm), maximum quantum yield of primary photochemistry (φPo = TRo/ABS = Fv/Fm), quantum yield for the reduction of the end acceptors of photosystem I (PSI) per photon absorbed (φRo = REo/ABS), and quantum yield for electron transport (φEo = ETo/ABS); (d) the overall grouping probability (P2G); and (e) the total performance index .The OJIP transients were analyzed according to the JIP test  and the maximum (Fm′) and minimum (Fo′) fluorescences were measured under natural light at midday in full sun. For this determination, the Fs was monitored to ensure it was stable before a reading was taken; the Fm′ was obtained by imposing a 1-s saturating flash of approximately 6,000 μmol m−2 s−1 at the leaf surface to reduce all the PSII centers. To measure the Fo′, a black cloth covered the leaf when a far-red light was switched on to rapidly oxidize the PSII centers by drawing electrons from PSII to PSI. The NPQ was calculated as: Fm/Fm′−1. The photochemical quenching coefficient, qP, was expressed as: (Fm′−Fs)/(Fm′−Fo′). The non-photochemical quenching coefficient, qNP, was defined as: (Fm−Fm′)/(Fm−Fo′). The ΦPSII was calculated as: (Fm′−Fs)/Fm′. The efficiency of excitation transfer to PSII RCs under natural light (Fm′/Fv′) was defined as: (Fm′−Fo′)/Fm′. Finally, the electron transport rate through PSII was estimated from (Fm′−Fs)/Fm′ × 0.5 × LA × photosynthetic photon flux (PPF), for which the PSI photochemistry was assumed equivalent to that of PSII . Both F2O2 production, RWC, electrolyte leakage, total soluble proteins, specific leaf weight, and mineral nutrients; and 7–15 replicates for the OJIP transients and the fluorescence parameters. The results are presented using the mean ± SE of 3–15 replicates. For a given dependent variable or parameter above, significant differences among the means of 10 treatment combinations were tested by a two (species) × five (pH levels) factorial ANOVA; the 10 means were compared on a pairwise basis by the Duncan's new multiple range test at P < 0.05. Linear and nonlinear regression was performed with the corresponding equations from SigmaPlot software .There were 10 replicates for plant biomass; three replicates for Rubisco; four replicates for gas exchange, pigments, HC. grandis seedlings treated with pH 2.5  H2O2 production was significantly higher in C. grandis than in C. sinensis at pH 2.5 , but similar between the citrus species at pH 3–6  leaves and stems increased as the pH increased from 2.5 to 4 (3), but it went unchanged with increasing pH. The root P level increased as the pH increased from 2.5 to 5, but it then kept stable with increasing pH. The K concentration in the C. sinensis leaves and stems and in the C. grandis leaves displayed little change in the range of pH 2.5–6; however, the K level in the C. sinensis roots and in the C. grandis stems and leaves was lower at pH 2.5 than at pH 3–6. Generally viewed, the Ca levels in the leaves, stems, and roots all increased as the pH increased from 2.5 to 4, after which they were relatively stable with increasing pH. The Mg level in the C. grandis leaves and stems and in the C. sinensis leaves decreased with decreasing pH, but the Mg level in the C. sinensis stems did not change in response to pH. The Mg level in the C. sinensis roots was reduced at pH 2.5, 3, and 4, but especially at pH 2.5 and 3, while its level in the C. grandis roots was elevated at pH 2.5 and pH 3, though especially at pH 3. Leaf and root S decreased with increasing pH, while the stem S level was higher at pH 2.5 than at the other pH treatments. Leaf P, K, Ca, and S, stem P, K, and S, and root P levels were all higher in C. sinensis than in C. grandis seedlings; or similar between the two citrus species at each given pH. Conversely, the leaf Mg, stem Ca and Mg, and root N, K, Ca, Mg, and S levels were all lower in C. sinensis than in C. grandis seedlings, or they were similar between the two citrus species at each given pH  stems increased as the pH increased from 2.5 to 3 (4), but it then kept relatively stable with increasing pH, though it decreased at pH 6. The root Fe concentration decreased with increasing pH. Leaf and stem Mn levels decreased with increasing pH. The root Mn level increased as the pH decreased from 6 to 3, then it decreased or went unchanged at pH 2.5. Leaf B concentration in the two citrus species was decreased only at pH 2.5. The B level in the C. sinensis (C. grandis) stems increased as the pH increased from 2.5 to 4 (3), but then it went unchanged with increasing pH, though it decreased at pH 6. Although the root B concentration increased as the pH increased from 2.5 to 5, it decreased at pH 6. The Cu level in the C. grandis leaves increased as the pH decreased from 6 to 4, after which it was little changed with decreasing pH; the Cu level in the C. sinensis leaves was highest at pH 5 and lowest at pH 6. Root Cu level in the two citrus species decreased as the pH increased from 2.5 to 4, but it then remained stable with increasing pH. The Zn level in the C. sinensis leaves and stems were lower at pH 5 and 6 than at pH 2.5, 3, and 4, while its level in the C. grandis leaves and stems were lower at pH 6 than at pH 2.5–5. The Zn level in the C. sinensis roots increased as the pH decreased from 6 to 3, but it then decreased at pH 2.5; the Zn level in C. grandis roots was highest at pH 3 and lowest at pH 6. Generally viewed, the leaf Fe, Mn, B and Cu, stem Fe, Mn, B, Cu and Zn, root Fe, B, Mn, and Zn concentrations all were higher in C. grandis than in C. sinensis, or they were similar between the two citrus species at each given pH. The exceptions to this generalization were that the Mn (Cu) level was higher in C. sinensis than in C. grandis leaves at pH 2.5 (5), and the Fe level was higher in C. sinensis than in C. grandis stems at pH 2.5. By contrast, the leaf Zn and root Cu concentrations were higher in the C. sinensis than in those of C. grandis, or they were similar between the two citrus species at pH 2.5–5, albeit leaf Zn lower was lower in the C. sinensis vs. C. grandis at pH 6 .Low pH very often affects the uptake of nutrients and water by plants  toxicity  increased with increasing leaf RWC . Thus, it is reasonable to assume that a low pH lowered the water uptake and induced water stress, thus inhibiting photosynthesis in the C. grandis and C. sinensis leaves.The leaf photosynthetic rate decreases with decreasing leaf RWC Lawlor, . Howevern Figure  was accon Figure , leaf Run Figure  and ETR,n Figure . Further Figures , Rubisco Figures , or ETR  Figures , and wit Figures ; leaf Ru2 assimilation decreased with increasing leaf N, P, Ca, or Mg concentrations  .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this study, we report how the cholera toxin (CT) A subunit (CTA), the enzyme moiety responsible for signaling alteration in host cells, enters the exosomal pathway, secretes extracellularly, transmits itself to a cell population. The first evidence for long-term transmission of CT’s toxic effect via extracellular vesicles was obtained in Chinese hamster ovary (CHO) cells. To follow the CT intracellular route towards exosome secretion, we used a novel strategy for generating metabolically-labeled fluorescent exosomes that can be counted by flow cytometry assay (FACS) and characterized. Our results clearly show the association of CT with exosomes, together with the heat shock protein 90 (HSP90) and Protein Disulfide Isomerase (PDI) molecules, proteins required for translocation of CTA across the ER membrane into the cytoplasm. Confocal microscopy showed direct internalization of CT containing fluorescent exo into CHO cells coupled with morphological changes in the recipient cells that are characteristic of CT action. Moreover, Me665 cells treated with CT-containing exosomes showed an increase in Adenosine 3’,5’-Cyclic Monophosphate (cAMP) level, reaching levels comparable to those seen in cells exposed directly to CT. Our results prompt the idea that CT can exploit an exosome-mediated cell communication pathway to extend its pathophysiological action beyond an initial host cell, into a multitude of cells. This finding could have implications for cholera disease pathogenesis and epidemiology. Vibrio cholerae which colonize the small intestine and secrete the Cholera Toxin (CT) protein [In the 21st century, cholera remains an epidemic or endemic disease in many parts of the world. It is caused by some serogroups of the bacterium  protein . The A sInternalization of CT depends on interaction of the CTB subunits of the toxin with GM1 gangliosides. GM1 gangliosides are typically concentrated in organized signaling centers such as lipid rafts and caveolae ,5,6. LocExtracellular secretion gives rise to a variety of vesicles (EV), including those strictly derived from MVBs and properly defined as exosomes. Exosomes (exo) are vesicles of 30–150 nm diameter that are secreted by cells into their environment. They are generated by inward budding of endosomal membranes to form multivesicular bodies (MVBs). Fusion of MVBs with the plasma membrane typically releases multiple exosomes ,9. An inBacillus anthracis virulence factor, is translocated into the lumen of endosomal intraluminal vesicles (ILVs). It persists in them for days, and can be transmitted to neighboring cells via exosomes [A few studies have examined the involvement of exo in toxin trafficking. The lethal factor (LF) of Anthrax toxin, a major exosomes . Trichosexosomes . In thisexosomes .We previously reported that Cav-1, a structural component of caveolae formation, is highly expressed in human metastatic melanoma cell lines, and is retrieved in isolated fractions of extracellular vesicles (EV) . Since c7 cells for both cell lines) were subjected to differential centrifugation to isolate EVs, and vesicle pellets were analyzed by western blot for CT presence. Both CTA and CTB subunits were present in EV pellets from both cell types, and were slightly more abundant in Me665 cells  [To better define the role of exosomes as a carrier of CT, we applied a method we have developed based on the production of metabolically labeled fluorescent exo (F-exo), by using the fatty acid analogue BODIPYic Acid) . Upon inEqual numbers of F-exo and F-exo CT were characterized by Western blot analysis for the presence of typical exo markers . To visualize the intracellular trafficking of CT, we first pulsed Me665 cells with BODIPY C16 for 5 h to metabolically label membrane sub-compartments. Cells were then treated with 12 nM CT for 20 min on ice to allow CT binding to the plasma membrane. The cells were then washed (T0) and incubated with fresh medium for a further 24 h at 37 °C (T24). Following fixation, cells were analyzed by confocal microscopy. As shown in To further characterize the subcellular localization of CT, we performed confocal microscopy of CT-treated cells (T24) using antibodies against BMP, a lipid whose occurrence is specific to the late endosomal MVB/lysosomal membrane tracking pathway , and TSGWith the aim of tracking F-exo CT biogenesis in Me665 cells, we pulsed cells with BODIPY C16 for 5 h to allow metabolic labeling of membrane compartments, including MVB before CT addition, for a further 2 h. Cells were then chased in fresh media for 1 h, before the media was removed and fresh media added for time intervals of up to 24 h. Exosome purification was performed on each of the conditioned media samples, to determine the kinetics of F-exo release into the extracellular medium A.From as early as 1 h, cells secrete a discrete population of F-exo that can be FACS counted.7 exo for each time point), were run on SDS-PAGE and immunoblotted with antibodies against CTA and CTB subunits. F-exo CT collected at 1 h and successively, after the addition of fresh medium, at 24 h , before the active CTA1 subunit reaches the cytosol and its final specific substrate . This laIn this study, we reported for the first time that CT can be propagated via EVs in a biologically active form, and that CT persists in cells long after treatment with the toxin. Accordingly, EVs collected from cells up to 72 h after their exposure to CT, still contain biologically active CT. Cells release different types of EVs, not all of endo/lysosomal origin, such as ectosomes that directly bud from the plasma membrane . To distFluorescent exo secreted in the extracellular medium can be purified and readily counted. It has also to be noted that the final purified vesicular pellet contains much less vesicles than those present in the initial medium. In fact, our labeling technique allowed us to determine that a considerable amount of fluorescent exosomes remain suspended or associated with different kind of debris and serum macromolecular components in the supernatant and in the low speed pellets. We show here that CTA is associated with a discrete population of fluorescent exo that express typical exo markers, indicating that CT is a bona fide component of exosomes . FurtherF-exo biogenesis studies allowed us to monitor CT secretion over time, highlighting an early release of F-exo containing CT (at 1 h). At later times (24 h), CT was still present in F-exo, albeit in lower amounts, showing that the bulk of CT associated with exo is released soon after toxin internalization. Interestingly, the host cell chaperone HSP90, an established exosomal marker , and proV. cholerae fast dynamic mechanism of intestinal infection relies on the effect of the cell-to-cell transmission of CT containing exo. In fact, in normal healthy adults, primary symptoms of cholera disease appear from half a day to five days after ingestion of at least 100 million bacteria, of which only few thousands survive the harsh acidic conditions of the stomach. Afterwards, only a small handful of bacteria penetrate the thick mucus wall to reach the V. cholerae natural setting, the small intestine, where they take hold and release CT [In conclusion, starting from the more general hypothesis which suggests that exosomes are vehicles that can influence cell homeostasis, we found that CT could exploit the exosome biogenesis pathway to spread its action from a single cell to an entire cell population. In light of our results, we may hypothesize that lease CT . We reasl-glutamine, penicillin (100 units/mL), and streptomycin (100 μg/mL)  at 37 °C in a humidified 5% CO2 atmosphere.The Human melanoma cell line, Me665/1 (Me665), stabilized from surgical specimen obtained from metastatic lesions at Istituto Nazionale Tumori  and CHO  cell line commercially available, were grown in Dulbecco’s modified Eagle’s medium (DMEM)  supplemented with 10% fetal bovine serum (FBS) , 5 mM Vibrio Cholerae 569 B, serotype Inaba, as described by [g for 20 min at 4 °C to pellet cells debris. Supernatants were transferred to new tubes and centrifuged in a SW41Ti rotor  at 10,000× g for 20 min at 4 °C, and finally ultracentrifuged at 100,000× g for 3 h. Pellets were washed in PBS and ultracentrifuged at the same speed for 3 h to further remove any residual CT still present in the exo preparation, since, along with vesicles, some free intact CT is also recycled to the extracellular milieu, and can be found in the supernatant after ultracentrifugation.For isolation of EVs, subconfluent monolayers of CHO and Me665 cells in exponential growth were incubated in DMEM supplemented with 0.3% FBS with or without 12 nM CT, and purified from culture filtrates of ribed by . After 2ribed by ,40, with4 cells/well), and DMEM supplemented with 0.3% FBS. Cells were incubated with or without 12 nM CT, or with EV and EV-CT (prepared as described above). After 6 h incubation, cells were inspected microscopically to analyze the morphological changes.Exponentially growing CHO cells were harvested, transferred to 12-well plates . The presence of GM1 ganglioside in cell lysates was assessed by the dot blot assay using horseradish peroxidase (HRP)-conjugated CTB subunit, and revealed with an ECL detection kit . Cell lysates (40 µg) were boiled and spotted onto a nitrocellulose membrane blocked using 5% Blotting Grade non-fat dry milk in a TBS-Tween (TBST) buffer (10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1% Tween 20) for 45 min at room temperature (RT), followed by incubation with HRP-CTB dissolved in TBST buffer (1:400) for 1 h at RT. The reactivity was detected using an ECL detection kit (Pierce). For Western Blot analysis, exosome preparations were lysed in Laemmli sample buffer, boiled, and loaded onto 10% or 14% SDS-PAGE gels under reducing or non-reducing conditions. Proteins were blotted onto a nitrocellulose membrane, and blocked using 5% Blotting Grade non-fat dry milk in TBS-Tween (TBST) buffer (10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1% Tween 20) for 1 h at RT followed by incubation with primary antibodies. The following primary antibodies were used: mouse anti-Cholera Toxin A (Clone 2H9)  1:500 and rabbit polyclonal Anti-Cholera toxin  1:1000; rabbit anti-Caveolin-1 (N-20)  1:1000; rabbit anti-Protein Disulfide Isomerase Polyclonal Antibody  1:1000; rabbit anti-HSP90 α/β (H-114) (Santa Cruz Biotechnology) 1:1000 dissolved in TBS-Tween (TBST) buffer for 1 h at RT; mouse anti-Alix (3A9) (Thermo Scientific) 1:1000, rabbit anti-CD63 (SBI) 1:1000, mouse anti-CD81 1:1000, mouse anti-TSG101  1:1000 dissolved in 0.25% Blotting Grade non-fat dry milk in TBST overnight at 4 °C. After washing with TBST, filters were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies  for 1 h at RT, and immunoreactivity was revealed by using an ECL detection kit (Pierce).Cell pellets were lysed in 20 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA with a Protease Inhibitor Cocktail  for 20 min on ice, and then centrifuged at 2000× 5) were incubated with 7 µM BODIPY FL C16  (BODIPY C16)  for 5 h at 37 °C in DMEM supplemented with 0.3% FBS. Excess probe was removed by washing cells twice with PBS. Subsequently, cells were incubated with or without 12 nM CT in DMEM supplemented with 0.3% FBS for 2 h at 37 °C, and washed twice with PBS to remove excess CT and further incubated for different times in complete media. Conditioned medium was centrifuged at 2000× g for 20 min to remove cells and cell debris. The pellet was discarded and the supernatant centrifuged at 10,000× g (10 K) to remove microvesicles. F-exo and F-exo CT were isolated by ultracentrifugation of the 10 K supernatant at 100,000× g (100 K) for 3 h, and the resulting pellet washed in PBS for 3 h at 100,000× g. All ultracentrifugation steps were performed at 4 °C using a SW41 Ti rotor . The final pellet was resuspended in PBS and quantified by FACS . To set the instrument, fluorescent beads (525/540 nm FL1) ranging in size from 0.1 to 0.5 µm were analyzed. Flow count fluorospheres, used to determine exosome number, were resuspended in PBS with F-exo and F-exo CT, and the instrument was set to fix the stopping gate on 2000 flow count fluorospheres. For further details, refer to [F-exo were purified and quantified as previously described . Brieflyrefer to . For F-eTM . The gradient was formed by layering 1 mL of 60% with 0.260 mL sample, 0.5 mL 40%, 0.5 mL 30% and 1.8 mL of 10% solutions on top in a 4.5 mL open top polyallomer tube . The gradient was then centrifuged for 18 h at 192,000× g at 4 °C (SW60 Ti rotor Beckman Coulter). Gradient fractions of 350 µL were collected from the top of the gradient and analyzed by FACS for exosome count. Fractions were loaded on a SDS-PAGE gel for Western blotting analysis using antibodies directed against exosome markers TSG101 and Alix. For CT detection, gradient fractions were TCA precipitated. The refractive index of each fraction was assessed with a refractometer , and the relative density was calculated using the linear relationship between refractive index (η) and the density (ρ) ρ = Aη − B.A discontinuous iodixanol gradient was used for further exosome purification, as described by several authors . SolutioTo analyze the intra/extra localization of CT, exo CT (16 µg) were incubated with or without 1 µg of chymotrypsin, in the absence or presence of 0.2% Triton X-100, at 37 °C for 3 h at pH 7.8. Digestion was stopped on ice with 5 µM phenylmethanesulfonyl fluoride (PMSF). Samples were then mixed with Laemmli buffer, heated at 95 °C for 5 min, and analyzed by SDS-PAGE and western blotting.5 cells/well) and incubated overnight at 37 °C in DMEM with 10% FBS to allow cell attachment. Plates were then washed with serum-free DMEM, and 1 mL of 1% FBS DMEM containing 0.5 mM 1-methyl-3-isobutylxantine  was added for 30 min. As control, IBMX treated cells were used. After incubation, 0.2 ng/mL of CT or a specific number (5.7 × 107) of F-exo and F-exo CT were added and plates were incubated at 37 °C for 4 h. Cells were washed in ice-cold PBS, and cAMP assay was performed, according to manufacturer’s instructions .Freshly trypsinized cells were transferred to 6-well plates (2 × 104) were cultured on 24-well plates containing coverslips until 60–70% of confluency. Cells were incubated at 37 °C with or without 7 µM BODIPY C16 for 4 h. 12 nM CT was added for 20 min on ice (T0), and cells were then incubated at 37 °C for 24 h (T24). Cells were fixed with 3 % paraformaldehyde (PFA) for 20 min at RT and permeabilized with 0.05% saponin. The primary antibodies used were: rabbit polyclonal anti-Cholera toxin (Sigma-Aldrich), mouse anti-TSG101 , and mouse anti-LBPA/BMP (6C4) . AlexaFluor647-conjugated and AlexaFluor488-conjugated goat anti mouse or anti rabbit (Life Technologies) was used as secondary antibody. Coverslips were mounted on the microscope slide with Vectashield antifade mounting medium containing DAPI . Images were taken by a FV1000 confocal microscope , using a (Olympus) planapo objective 60x oil A.N. 1.42. Excitation light was obtained by a Laser Dapi 408 nm for DAPI, an Argon Ion Laser (488 nm) for FITC (Alexa 488), and a Red Diode Laser (638 nm) for Alexa 647. DAPI emission was recorded from 415 to 485 nm, FITC emission was recorded from 495 to 550 nm, and Alexa 647 from 634 to 750 nm. Images recorded have an optical thickness of 0.3 mm.To analyze the intracellular localization of CT, Me665cells  . The amount of transferred exo was estimated, as previously described [4 cells were seeded on a sterilized coverslip and incubated with 2 × 108 F-exo, or F-exo CT for 4 h at 37 °C in DMEM with 0.1% FBS. At the end of incubation, the medium was removed, and the cells were washed with PBS and fixed with 3% PFA for analysis.To evaluate the transfer on target cells, F-exo were incubated with cells in a 24-well plate in duplicate in 1 mL DMEM with 0.1% FBS and kept for 4 h at 37 °C in COescribed . Briefly


Mesenchymal stem cells (MSCs) are known to both have powerful immunosuppressive properties and promote allograft tolerance. Determining the environmental oxygen tension and inflammatory conditions under which MSCs are optimally primed for this immunosuppressive function is essential to their utilization in promoting graft tolerance. Of particular interest is the mechanisms governing the interaction between MSCs and regulatory T cells (Tregs), which is relatively unknown. We performed our experiments utilizing rat bone marrow derived MSCs. We observed that priming MSCs in hypoxia promotes maintenance of stem-like characteristics, with greater expression of typical MSC cell-surface markers, increased proliferation, and maintenance of differentiation potential. Addition of autologous MSCs to CD4+/allogeneic endothelial cell (EC) co-culture increases regulatory T cell (Treg) proliferation, which is further enhanced when MSCs are primed in hypoxia. Furthermore, MSC-mediated Treg expansion does not require direct cell-cell contact. The expression of indolamine 2,3-dioxygenase, a mediator of MSC immunomodulation, increases when MSCs are primed in hypoxia, and inhibition of IDO significantly decreases the expansion of Tregs. Priming with inflammatory cytokines IFNγ and TNFα increases also expression of markers associated with MSC immunomodulatory function, but decreases MSC proliferation. The expression of IDO also increases when MSCs are primed with inflammatory cytokines. However, there is no increase in Treg expansion when MSCs are primed with IFNγ, suggesting an alternate mechanism for inflammatory-stimulated MSC immunomodulation. Overall, these results suggest that MSCs primed in hypoxia or inflammatory conditions are optimally primed for immunosuppressive function. These results provide a clearer picture of how to enhance MSC immunomodulation for clinical use. Mesenchymal stem cells (MSCs) are multipotent progenitor cells which have the potential to differentiate into osteocytes, adipocytes, and chondrocytes . In addiWhile these pathways have been relatively well delineated, the effect of MSCs on regulatory T cell (Treg) populations remains less well described. A handful of groups have described an increase in Treg expansion in response to MSC exposure –16. HoweFully defining the role of MSCs and their interaction with Tregs is of importance in the use of MSCs in prevention of acute rejection in transplantation. The immunosuppressive potential of MSCs has been demonstrated in several animal models, including skin grafts, solid-organ transplants, graft-versus-host disease, and most recently vascularized composite allotransplantation –27. HoweIn this study, we look at microenvironmental factors that can prime MSCs for optimal immunosuppressive function while maintaining stem-like characteristics. Because MSCs naturally reside in the bone marrow, which is hypoxic –30, primLewis rats were obtained from Charles River Laboratories  and maintained in the facilities at NYU School of Medicine, in compliance with IACUC policies and guidelines and approval for protocol 160702. Prior to harvesting bone marrow aspirates or spleens, rats were euthanized using carbon dioxide asphyxiation, followed by a bilateral pneumothorax.2, passaged at 80% confluency, and analyzed at passages 3 through 5. For mechanistic studies, MSCs were treated with 1-methyl-D-tryptophan (1-MT), a competitive inhibitor of indolamine 2,3-dioxygenase (IDO).Bone marrow derived-mesenchymal stem cells were obtained from male Lewis rats from femur, tibia, humerus and pelvis, as described . Cells wDoubling time was measured by counting trypsinized cells using a hemocytometer. Cells were trypsinized after reaching 80% confluency, at passages 1–5. Proliferation was determined using an MTT Cell Proliferation Assay Kit . Briefly, cells were seeded in a 96-well plate, cultured for 10 days, incubated with MTT for 4 hours, and measured spectrophotometrically.Migration was studied by modified transwell assays. MSCs were plated onto transwell inserts, with no cytokines or IFNγ and/or TNFα. SDF-1 was added to Mesencult in the lower chamber. After a 6 hour migration period, non-migrating cells were wiped from the top filter surface. An MTT assay, using the aforementioned kit, was used to determine relative cell numbers, as a measure of migration.MSCs at passages 3–5 were grown in either hypoxia or normoxia with adipogenic differentiation media of Mesencult + 0.25mM IBMX  + 1μM dexamethasone  + 1mM insulin  for 4 days, then only Mesencult + 1mM insulin for 10 days. Similar MSCs were also grown in osteogenic differentiation media of Mesencult + 10mM β-glycerophosphate  + 10nM dexamethasone + 100μM ascorbic acid  + 2mM L-glutamine  for 14 days. For chondrogenic differentiation, 200,000 cells were pelleted at 300g for 10 min, and grown in chondrogenic differentiation media of Mesencult basal media + 1% FBS + 100nM dexamethasone + 50μg/ml ascorbic acid + 40μg/ml L-proline  + 1% ITS supplement  + 1mM sodium pyruvate  + 10ng/ml TGFβ-3  + 100ng/ml BMP-2  for 14 days.Normoxic or hypoxic MSCs were stained for morphology with H&E at passage 3. Staining for adipogenic differentiation using Oil Red O and for osteogenic differentiation using Alizarin Red S also performed. Pellets were collected, fixed and processed into paraffin, then slides were stained for osteogenic differentiation with fast green and Safranin O. Images were collected using a Nikon DS-Fi1 camera  on an Olympus BX51 microscope  using NIS Elements software  and processed using Adobe Photoshop CC.RNA was isolated from cell pellets using an RNeasy Mini Kit . cDNA was generated using a high capacity cDNA synthesis kit, from 500ng mRNA . Real-time PCR was performed using SYBR Green Master mix . Primers were designed, and obtained from Life Technologies.3, and analyzed for presence of these cell surface markers using flow cytometer. 10,000 events were collected using a FACS Calibur cytometer , and data was analyzed using FlowJo software .MSCs were stained for 30 minutes at 4°C with anti-CD90-FITC, anti-CD29-PE, anti-CD11b-PerCP, anti-CD45-APC . Cells were washed and resuspended with 1xPBS +2% BSA + 0.1% NaNA kynurenine assay was performed using supernatants from normoxic or hypoxic MSCs. 50μl of supernatant was mixed with 50μl of 30% (w/v) trichloroacetic acid, incubated for 30 minutes at 50°C, centrifuged at 10,000g for 10 minutes and mixed in a 1:1 ratio with freshly prepared Ehrlich’s reagent  in a 96-well clear flat bottom plate. Kynurenine standard serial dilutions were also prepared and mixed with Ehrlich’s reagent in a 1:1 ratio. The plate remained at room temperature for 10 minutes, and was read on a plate reader at 490nm.7 cells. CD4+ cells were then magnetically sorted with MACS LS separator columns. CD4+ cells were co-cultured with passage 3 normoxic or hypoxic MSCs, and passage 2–3 allogeneic rat aortic ECs  in 24-well flat-bottom plates. CD4+ cells were plated 24 hours after MSCs and ECs in a MSC:EC:CD4+ cell ratio of 1:20:20. CD4+ cells were cultured with MSCs, ECs, or both. Additionally, each combination of cells was plated with transwell inserts to separate the different cell types. Cultures were maintained in a 1:1 ratio of Mesencult and Endothelial Cell Growth Medium  with rhTGFβ (20ng/ml), rhIL-2 (300U/ml) , and anti-CD3 (2.5μg/ml)  for 48 hours in hypoxic conditions. Cells were stained with anti-CD4 and anti-FoxP3 antibodies  using a FoxP3 Staining Buffer Kit . Flow cytometry for CD4 and FoxP3 was performed and analyzed as above, for CD4+ cells that stained FoxP3+ using a ZE5 Cell Analyzer .Spleens were excised from Lewis rats, and CD4+ T cells were isolated using magnetic cell sorting , according to manufacturer’s protocol. Briefly, splenocytes were resuspended in 1xPBS + 1%BSA, and labeled with 10ul anti-rat CD4 microbeads/10xg and 4°C, cells were resuspended to 105 cells/200μL in cold 2%FBS/PBS. Using a Cytospin 4 , 200μL of cell suspension was loaded and centrifuged at 1000rpm (113xg) for 3 minutes. Slides were immediately fixed in 4% paraformaldehyde, followed by permeabilization with 0.1% Triton-X-100. Cells were stained for CD4 and FoxP3, and photographed using a Zeiss Observer microsope (Germany).CD4+ splenocytes from co-cultures were collected in chilled 1.5mL Eppendorf tubes and counted. Following a brief centrifugation at 300All data were analyzed using Graphpad Prism . All experiments were performed with at least n = 6, and analyzed using Students’ t-tests and ANOVA for multiple pairwise comparisons. A p-value of < 0.05 determined statistical significance.We first sought to ensure that growing MSCs under hypoxia conditions maintains stemness, by positive staining for CD90 and CD29, and negative staining for CD45 and CD11b, which are known positive and negative markers of MSCs . Other pIn investigating the effect of hypoxic culture on MSC proliferation, we found that MSCs cultured in hypoxia, vs normoxia, demonstrate an over 2-fold increase in relative proliferation  . HypoxicFinally, we looked at expression of chemokines known to facilitate MSC immunosuppression, CXCL10 and CXCL9 . InteresWe then studied whether hypoxic culture affected differentiation potential by growing MSCs in either adipogenic, osteogenic, or chondrogenic differentiation media. We found that MSCs grown under hypoxic conditions in osteogenic differentiation media showed greater staining of calcium deposits with alizarin red . HypoxicThese data suggest that growth in hypoxia maintains stem-like properties, including the potential for tri-lineage differentiation, and increases proliferation, all properties of MSCs that are involved in exertion of their immunomodulatory functions.2 on MSC stemness functions, we next investigated the effect of growth in a near anoxic environment of 0.5% O2. When MSCs were cultured in near anoxia, we found an increase in the percent of MSCs expressing CD90, although expression of CD29 remained relatively unchanged. Overall, there was a significant increase in the percentage of CD90+/CD29+/CD45-/CD11b- MSCs (97.9%), indicating that stemness markers are further preserved at very low oxygen tensions  .To determine whether this interaction among hypoxic MSCs, ECs and CD4+ cells requires direct contact, we used transwell inserts in our co-cultures to physically separate the different cell populations. First, we cultured CD4+ cells in an insert overlaying only MSCs, and found significant reduction in FoxP3+ cells, in contrast to the original co-culture conditions . Then, wAfter determining that hypoxic enhances MSC immunomodulation via Treg stimulation, we then investigated the role of IDO in this interaction. Because IDO has been shown to mediate aspects of MSC immunosuppression , 37, we After confirming that hypoxic MSCs indeed have a greater IDO expression, we wanted to determine whether IDO mediated the effect of MSCs on Treg proliferation. We inhibited the activity of IDO by culturing hypoxic MSCs with the competitive inhibitor 1-MT, and found that proliferation of Tregs significantly decreased . In fact, inhibition of IDO almost negated the effects of the addition of MSCs on Treg proliferation .We then investigated the effect of inflammatory conditions on MSC immunomodulation and stem-like characteristics. MSCs exposed to any combination of cytokines demonstrated decreased proliferation, but was significant only when IFNγ was included  . HoweverNormoxic MSCs exposed to almost every combination of cytokines show significant decrease in markers of differentiation in lpl  and alp . Hypoxic MSCs also show significant decrease with exposure to any combination of cytokines, in lpl  and alp   . CompariExposure of normoxic MSCs to IFNγ or IFNγ/TNFα results in significant increase in CXCL10  and CXCL9 . There was no increase when exposed to TNFα alone. A similar increase was seen in hypoxic MSCs  . The magAdditionally, exposure to inflammatory conditions in both normoxia and hypoxia increases the expression of iNOS, a vasoactive molecule. This vasodilation allows for the trafficking of lymphocytes to MSCs by chemokines   . OverallAfter determining that inflammatory exposures increase markers of immunomodulation in MSCs, we turned to look at the role of inflammatory conditions in IDO expression and Treg stimulation. When exposed to IFNγ for 3 days, expression of IDO significantly increased. In normoxia, IDO expression increased 17.8±5.6 times when exposed to IFNγ/TNFα (p<0.0001) and increased 4.9±3.3 times with IFNγ only, although this difference was not significant. In hypoxia, IDO expression increased 5.9±3.6 times with IFNγ (p<0.05) and 15.1±5.7 times with IFNγ/TNFα (p<0.001). IFNγ/TNFα increased IDO significantly more than IFNγ alone, in both normoxia (p<0.05) and hypoxia (p<0.01). When MSCs were exposed to TNFα alone, IDO expression was unchanged .Longer exposure to either IFNγ or IFNγ/TNFα for 7 days increased IDO expression to a significantly greater level. In normoxia, IDO expression increased 127.2±48.9-fold with IFNγ exposure, and 1711.1±12024.5-fold with IFNγ/TNFα (p<0.0001 for both). In hypoxia, IDO expression increased 6338.1±598.8-fold with IFNγ, and 8601.3±1245.0 –fold with IFNγ/TNFα (p<0.0001 for both). Again no change was seen with TNFα alone .However, we did find a negative impact of longer exposure to cytokines on MSC morphology. After 3 days of exposure, MSCs still maintain their spindle-like characteristics. After 7 days, MSCs begin to look wider and flatter . Our finGiven the significant increase in IDO expression by MSCs when exposed to inflammatory conditions, we wondered if this would impact the ability of MSCs to stimulate Treg proliferation. Surprisingly, exposure of MSCs to IFNγ had no impact on the percentage of Tregs, as compared to culture with unexposed MSCs, in both normoxia  and hypoxia  .MSC-mediated immunosuppression provides a powerful potential tool for decreasing the incidence and severity of acute rejection of transplanted allografts ,4,7,39. 2 is sufficient to prime MSCs for an immunomodulatory phenotype.We found that 5% hypoxic conditions, similar to oxygen tension in the native bone marrow environment –29, favoWe found that stem-like characteristics are maintained in hypoxia. This is of special importance as any potential therapy using MSCs hinges on MSCs being able to maintain their stem-like state. Our findings that a greater percentage of MSCs express typical stem cell markers in hypoxia, and that hypoxic MSCs phenotypically look more spindle-like, a typical characteristic of MSCs, support this point. Although we considered the possibility that increased proliferation under hypoxic conditions may lead to loss of stem-like properties of MSC, our observations indicate that this is not the case–despite increased proliferation, hypoxic MSCs maintain stemness characteristics better than those grown under normoxic conditions. Our results are supported by other studies showing the maintenance of stemness of despite proliferation is typical of bone marrow-derived MSCs ,41.Additionally, at the range of passages studied, hypoxic MSCs retain a greater tri-lineage differentiation potential than normoxic MSCs. Although some studies in the literature question the potential of hypoxic MSCs to differentiate ,43, otheTo investigate the effect of hypoxia on MSC immunosuppressive potential, we looked at CXCL9 and CXCL10, chemokines that recruit helper and effector T cells to the site of rejection ,47, therMuch of our study focused on the interactions of MSCs and Tregs. Although the effects of MSCs on cytotoxic and helper T cells have been relatively well established –13, the The increase in Treg proliferation even when MSCs are physically separated from ECs and T cells demonstrates that a soluble factor mediates MSC immunomodulation. This is consistent with reports of MSC mediated immunosuppression via exosomal functions ,50,51. FWe found that IDO is a key mediator of both hypoxia-stimulated and IFNγ-stimulated immunosuppression. The role of IDO has been debated in the literature. We found only one study that investigated the role of IDO in MSCs under hypoxic conditions, which showed no change in IDO expression in hypoxia . HoweverMigration of MSCs, an important aspect of immunomodulation, was increased following exposure to inflammatory cytokines, which is in agreement with previous studies ,54. AlthOur extensive in vitro analyses give us greater insight into conditions that would optimally prime MSCs for immunosuppression. There are obvious potential applications of priming MSCs for an immunomodulatory phenotype to clinical use. We have shown the efficacy of these processes in rats, and our model can be easily adapted to human allografts in future studies.S1 Raw Data(PDF)Click here for additional data file.


The metabolic pathway analysis was performed with MetaMapp and drawn by CytoScape. Results show that the combined DON and ZEN treatment has an obvious “antagonistic effect” in serum and liver tissue metabolic profiling of mice. The blood biochemical indexes, like alkaline phosphatase, alanine transaminase, and albumin (ALB)/globulin (GLO), reveal a moderated trend in the combined DON + ZEN treatment group, which is consistent with histopathological examination. The metabolic pathway analysis demonstrated that the combined DON and ZEN treatment could down-regulate the valine, leucine and isoleucine biosynthesis, glycine, serine and threonine metabolism, and O-glycosyl compounds related glucose metabolism in liver tissue. The metabolic profiling in serum confirmed the finding that the combined DON and ZEN treatment has an “antagonistic effect” on liver metabolism of mice.Metabolic profiling in liver and serum of mice was studied for the combined toxic effects of deoxynivalenol (DON) and zearalenone (ZEN), through gas chromatography mass spectrum. The spectrum of serum and liver sample of mice, treated with individual 2 mg/kg DON, 20 mg/kg ZEN, and the combined DON + ZEN with final concentration 2 mg/kg DON and 20 mg/kg ZEN for 21 days, were deconvoluted, aligned and identified with MS DIAL. The data matrix was processed with univariate analysis and multivariate analysis for selection of metabolites with variable importance for the projection (VIP) > 1,  During environmental mycotoxin exposure, humans and animals most often encounter mixtures of toxins, rather than single toxins, such as deoxynivalenol (DON) and zearalenone (ZEN) . DeoxyniFusarium species as well as the metabolites zearalanone, α-zearalanol (α-ZOL) and β-zearalanol (β-ZOL). Both α- and β-ZOL exert harmful heath effect via their strong estrogenic activities, resulting in decreased fertility, increased fetal resorption, and changes in the weight of endocrine glands and serum hormone levels 2012-0002) approved all experimental procedures on 7 April 2016, project ID: JN.No20160322-20160701 . A totalg at 4 °C for 15 min. 100 μL serum was extracted with 1 mL extraction solution  [g at 4 °C for 15 min. Finally, 300 μL metabolites solution supernatant (liver or serum) was transferred to a new 2 mL GC/MS glass vial. Quality control: An equal volume of 10 μL (based on the number of samples) was taken from each sample into the 2 mL GC-MS glass vial as a mixed sample [v/v) added into each sample before being sealed again and incubated at 70 °C for an hour. Finally, the mix wells were waiting for GC-MS analysis [Freeze-dried liver powder (20 mg) was extracted with 1 mL of 50% cold acetonitrile (ACN) by a homogenizer, five times operated at 5500 rpm for 20 s with 30 s intervals . The homs 3:3:2) . The samd sample . Followianalysis . Blood b−1 and the gas flow rate through the column was 20 mL·min−1. The initial temperature was kept at 70 °C for 1 min, then raised to 280 °C at a rate of 6 °C·min−1, and maintained for 5 min at 280 °C. The injection, transfer line and ion source temperatures were 280 °C, 280 °C, and 250 °C, respectively. The energy was −70 eV in electron impact mode. The mass spectrometry data were acquired in full-scan mode with the m/z range of 50–600 at a rate of 20 spectra per second after a solvent delay of 366 s.GC-MS analysis was performed using a Shimadzu QP2010 Ultra gas chromatograph system coupled with mass spectrometer. The system utilized an Rxi-5Sil MS column . A 1 μL aliquot of the sample solution was injected into a splitless injector. Helium was used as the carrier gas, the front inlet purge flow was 3 mL·minm/z tolerance was 0.5 Da, the EI similarity cut-off was 70%, and the identification score cut-off was 70%. In the alignment parameters setting process, the retention time tolerance was 0.075 min, and retention time factor was 0.5.Raw data was converted to “mzXML” format with the GCMS PostRun from Shimadzu company, and then converted to “abf” format with the ABF converter. The MS DIAL with Fiehn library were used for raw peaks exaction (files of “abf” format), the data baseline filtering and calibration of the baseline, peak alignment, deconvolution analysis, peak identification and integration of the peak height . The exat-test.Statistical data analysis was performed using Simca-P 14+, which was used for the principal component analysis (PCA) and orthogonal projection to latent structures-discriminant analysis (OPLS-DA). Heatmap analysis was performed with Metaboanalyst 3.0 , a web-based tool for pathway analysis and visualization metabolomics. The pathway mapping was analyzed with MetaMapp, and drawn by CytoScape 3.4.0.  GraphPad Prism , R i386 3.3.1, and Origin were used for the statistical analysis, including one-way ANOVA test (Dunnett’s multiple comparison test) and


Staphylococcus aureus (MRSA) in cattle and buffalo and to study their antibiotic resistance pattern.This study was conducted to determine the prevalence of methicillin-resistant S. aureus-specific 16S rRNA by polymerase chain reaction (PCR). The isolates were further analyzed for the presence of mec A gene by PCR. The antimicrobial susceptibility profiling was performed by disc diffusion method.A total of 136 samples  from cattle and buffalo were collected. MRSA was identified by conventional bacterial culture techniques which were further confirmed by amplification of mec A gene in 36.4% and 58% MRSA isolates from cattle and buffalo, respectively. Antimicrobial susceptibility test found MRSA resistant to penicillin and oxytetracycline (88% each), cefoxitin (75%), cotrimoxazole (62%), and amoxyclav (50%). 100% sensitivity was observed against ciprofloxacin, amikacin, chloramphenicol, and gentamicin. Three (16.7%) MRSA isolates from buffalo were found resistant to vancomycin.The prevalence of MRSA in the current study was 28.57% and 34.28% in cattle nasal and skin swab, respectively, with an overall prevalence of 31.43% MRSA among cattle. Buffalo nasal and skin sample showed MRSA prevalence of 54.55% and 39.4%, respectively, with 46.9% overall prevalence. PCR could detect S. aureus (VRSA) in the current study indicates the emergence of VRSA in animal population which may be transmitted to the human beings working in close contact to the animals.Cattle and buffalo were identified as a potential carrier of MRSA in Bihar (India). The isolation of vancomycin-resistant  Staphylococcus aureus is an opportunistic pathogen of human and animal [S. aureus and first isolation of penicillin-resistant S. aureus was made in 1942 from clinical cases [S. aureus (MRSA) appeared in the year 1961 which later on became a serious nosocomial infection worldwide [mecA) gene and other antibiotic resistance determinants [d animal  which usd animal . Alexandal cases . It was orldwide  into therminants , leavingData regarding the prevalence of MRSA in India have been reported from various regions -11. HoweHence, this study was planned for isolation, identification and molecular detection of methicillin resistance in staphylococci and their antibiotic resistance pattern from bovines.S. aureus-specific polymerase chain reaction (PCR) targeting 16S rRNA using the primer sequence as F-5’ GTAGGTGGCCAAGCGTTATCC 3’ and R-5’ CGCACATCAGCGTCAG 3’ [2), 5 mM of deoxynucleotide triphosphates (dNTPs), 20 pmol of forward and reverse primers, 1 U Taq DNA polymerase, 2 µl of DNA template per reaction and final volume was adjusted to 25 µl with nuclease free water. The cycling conditions were optimized with initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 50°C for 1 min, and extension at 72°C for 1 min with the final extension at 72°C for 10 min.Skin and nasal swab samples 136) from apparently healthy cattle and buffalo were collected from Institutional Livestock Farm Complex, Bihar Veterinary College (BVC), Patna, and from the patients coming to teaching veterinary clinical complex of BVC, Patna. The sample comprised skin and anterior nostril swab of cattle (35 each) and buffalo (33 each). The skin samples were collected from the axillary region of the animals. The samples were inoculated in tryptone soya broth (TSB) with 10% sodium chloride salt (TSB-S) (Hi-media) and incubated at 37°C for 24 h. The samples which showed turbidity in TSB-S broth were streaked on mannitol salt agar (Hi-media) with 6 mg/L oxacillin (O-MS agar) and incubated for 24 h at 37°C. Bacterial growth with mannitol fermentation was observed for the presence of round-shaped, typical golden, yellow, or pale color colonies of oxacillin-resistant staphylococci. The presumptive isolates were further confirmed by Gram’s-staining (Hi-media) and catalase test . The tes6 from apmecA gene by PCR. The PCR assay was standardized for amplification of mecA gene by forward (5’ GTA GAA ATG ACT GAA CGT CCG ATAA 3’) and reverse (5’ CCAATTCC ACATTGT TTCG GTC TAA 3’) primer [2), 0.5 µl of dNTP mixture (10 mM each), 2 µl (10 pmol/µl) of forward and reverse primers, 1 µl (1 unit) Taq DNA polymerase, 5 µl of bacterial lysate and final volume was adjusted with nuclease free water. The bacterial lysate was prepared by boiling and snap chilling [The confirmed isolates of MRSA were further screened for the presence of ) primer  with somchilling  from oveThe amplified products were analyzed by agarose gel electrophoresis using 1.5% LE agarose  on tris acetate-ethylenediaminetetraacetic acid buffer containing ethidium bromide (0.5 µg/ml). Gels were visualized and photographed under gel documentation system (Biorad).viz., ciprofloxacin (5 µg), amoxiclav (30 µg), ofloxacin (5 µg), amikacin (30 µg), cefoxitin (30 µg), ceftriaxone (30 µg), chloramphenicol (30 µg), clindamycin (2 µg), penicillin (10 µg), gentamicin (10 µg), oxytetracycline (30 µg), tetracycline (30 µg), cotrimoxazole (25 µg), and vancomycin (30 µg). The interpretation of results was made according to CLSI guidelines [Antibiogram study of MRSA isolates was performed by disc diffusion method  using a idelines .S. aureus-specific 16S rRNA (228 bp) gene  gene . The prebp) gene .mecA gene has been used for specific and rapid identification of MRSA among the oxacillin-resistant S. aureus isolates. The test detected mecA gene  and amoxyclav (50%) whereas no resistance was observed against ciprofloxacin, amikacin, chloramphenicol, gentamicin, and vancomycin , i.e., PBP2a is a useful molecular marker of β-lactam resistance in Staphylococci [mecA gene have been used in the present study for specific identification of MRSA among the oxacillin-resistant S. aureus isolates [mecA gene from cattle and buffalo, respectively. Similar findings of mecA gene detection in cattle nasal carriage have been reported by Alzohairy [mecA gene amplification by PCR among MRSA isolates in the current study may be due to penicillinase activity of S. aureus [mecA gene homologue, mecALGA251 called mecC gene in the isolates [The findings of this study revealed that prevalence of MRSA in buffalo (46.9%) was higher in comparison to cattle (31.43%) which corroborate with earlier findings on isolation of MRSA from nasal or skin samples of cattle ,18. Howeylococci ,21. Hencisolates ,23. The lzohairy . In contlzohairy . The low. aureus  or due tisolates  which comecA positive isolates of buffalo origin. It revealed that 88% MRSA isolates of cattle origin, having mecA gene were resistant to penicillin and oxytetracycline whereas 75% were resistant to cefoxitin followed by cotrimoxazole (62%) and amoxyclav (50%) whereas no resistance was observed against ciprofloxacin, amikacin, chloramphenicol, gentamicin, and vancomycin  in MRSA isolates of cattle, whereas 75% were resistant to cefoxitin, (62%) cotrimoxazole and (50%) amoxyclav. No resistance was observed against ciprofloxacin, amikacin, chloramphenicol, gentamicin and vancomycin. Similar pattern of antimicrobial resistance for penicillin, cotrimoxazole, cefoxitin, ciprofloxacin, amikacin, and chloramphenicol was observed in ncomycin . This an8% in MRS [et al. , and Cha [et al.  have theS. aureus (VRSA) in the current study indicates the emergence of VRSA in animal population which may have a serious implication in human infection.The current finding also revealed resistance against vancomycin, in 16.7% of MRSA isolates from buffalo which is considered as the drug of choice for treatment of MRSA infections . AlthougThe present finding concludes that cattle and buffalo may act as a potential carrier of MRSA which may act as a risk factor for human’s infection who are in direct contact with live animals .PK was the mentor and project leader. AK and Anjay were responsible for project design and performed most of the work. PK and MK were responsible for experimental work and support. All authors read and approved the final manuscript.