This previously unrecognized methane cycling is not discernible from geochemical profiles due to high net methane consumption. High-throughput sequencing and Catalyzed Reporter Deposition-Fluorescence in situ Hybridization (CARD-FISH) results suggested that anaerobic methane-oxidizing archaea (ANME)-2 and -3 coupled to sulfate-reducing bacteria (SRB) carried out SO42–-AOM, and alternative ANME-2 and -3 solely or coupled to iron-reducing bacteria (IRB) might participate in Fe-AOM in sulfate-depleted environments. This finding suggested that ANME could alter AOM metabolic pathways according to geochemical changes. Furthermore, the majority of methylotrophic methanogens belonged to Methanimicrococcus, and hydrogenotrophic and acetoclastic methanogens were likely inhibited by sulfate or iron respiration. Fe-AOM and methylotrophic methanogenesis are overlooked potential sources and sinks of methane in methane seep ecosystems, thus influencing methane budgets and even the global carbon budget in the ocean.Methane seeps are widespread seafloor ecosystems shaped by complex physicochemical-biological interactions over geological timescales, and seep microbiomes play a vital role in global biogeochemical cycling of key elements on Earth. However, the mechanisms underlying the coexistence of methane-cycling microbial communities remain largely elusive. Here, high-resolution sediment incubation experiments revealed a cryptic methane cycle in the South China Sea (SCS) methane seep ecosystem, showing the coexistence of sulfate (SO Methane seeps are methane-dependent chemosynthetic ecosystems  that occMethanococcoides/ANME-3 (Methanoperedens nitroreducens)  coupled with sulfate reduction . Howevers/ANME-3 , ANME-2as/ANME-3 , ANME-2deducens) , Candidareducens , and Mettivorans  might betivorans . It is sIn addition, in methane seep ecosystems, acetoclastic and hydrogenotrophic methanogenesis are often considered the dominant methanogenic processes below the Sulfate-methane transition zone (SMTZ) . ThermodThen, it is becoming clear that methylotrophic methanogenesis plays a major role in marine sediments, especially surficial sediments . MethyloThe northern continental slope of the South China Sea (SCS) has various sites of methane seepage, covering a wide range of water depths (200–3000 m). The Jiaolong and Haima methane seeps are the two still-active seep sites in SCS. Previous studies on the seeps in SCS have led to many significant advancements including new insight into the methane seep structure , mineralIn this study, the Jiaolong methane seep was chosen as our research object. In 2018, a remotely operated underwater vehicle, The Remotely Operated Platform for Ocean Science (ROPOS), found a new methane seepage site in this area. Combining high-throughput sequencing, CARD-FISH, and enrichment culture methods with pore water biogeochemistry, we investigated the microbially driven metabolic processes of methane production and consumption. The coexistence of sulfate- or iron-dependent AOM and methylotrophic methanogenesis was found, and the potential rates of these processes were assessed. These results are of great importance to the understanding of the biogeochemical processes in global methane seep ecosystems.During the R/V Tan Kah Kee 1083 expedition (April–May 2018) in the northern SCS, a remotely operated underwater vehicle  was used to search for new undisturbed active methane seeps at a water depth of 900–1,200 m. A new active methane seep was found and named Jiaolong F3 site . Two push cores with a length of 32 cm were retrieved beneath the black microbial mats by ROPOS . One pus2+).Before sampling, holes were drilled at both sides of the biogeochemistry push cores to collect porewater and methane gas. Holes in one side were drilled at 2 cm intervals, in the other at 2.5 cm, and sealed with diffusion-tight tape. Porewater was extracted using Rhizons  at 2 cm intervals . Cut-off2CO3/0.8 mM NaHCO3, flow: 1 ml/min) . For measurement of dissolved iron, 1 ml of pore water was acidified with ultrapure HCl and assessed using a 797 VA Computrace as previously described .The pore water sample used for analysis of DIC was quickly transferred into glass vials (2 ml), leaving no headspace for concentration measurements and stored at 4°C. In the laboratory, pore water was measured using a CN analyzer multi N\C 3100  . For anaex situ measurement of the methane concentration, 3 ml of wet sediment sample was removed from the push core at an interval of 2.5 cm with a cut-off syringe and extruded into a vial (20 ml) containing 6 ml of sodium hydroxide (2.5% w/w). The vial was closed immediately with a butyl rubber stopper, sealed with an aluminum crimp, and stored upside down at 4°C until measurement by gas chromatography . Purified amplicons were pooled in equimolar amounts and paired-end sequenced (2 × 300) on an Illumina MiSeq platform . Raw fastq files were demultiplexed, quality filtered by Trimmomatic, and merged by FLASH. Operational taxonomic units (OTUs) were clustered with a 97% similarity cutoff using UPARSE (version7.1), and chimeric sequences were identified and removed using UCHIME. Taxonomy assignment was performed using the SILVA 16S rRNA database (version 132). The distance-based maximum likelihood was used for phylogenetic analysis. Bootstrap analysis was performed using 1000 replications. Chao1 and Shannon–Weaver diversity indices and rarefaction curves were calculated by MOTHUR. Principal coordinates analysis (PCoA) was computed using PAST. Sequencing data are stored on the NCBI Sequence Read Archive .Environmental DNA was extracted using a modified SDS-based extraction method . The fing for 10 min) using PBS at 4°C and stored in ethanol/PBS buffer (1:1) at −20°C for further processing. After that, 100 μl of fixed sediments were diluted with 900 μl ethanol/PBS buffer (1:1) and dispersed using ultrasound. Then, 20 μl of dispersed sediments were diluted in 20 ml of Milli Q filtered water. The suspension sediments were filtered on polycarbonate filters, and 0.1% low melting point agarose was dripped onto the filters and dried at 46°C in an incubator. The microbes were permeabilized using 15 μg/ml proteinase K. Then, 3% H2O2 was used to inactivate the endogenous peroxidases.The CARD-FISH protocol was based on a previous study . The pro2O2, 1 × PBS [pH7.4], 0.1% (w/v) blocking reagent) and 1 μl of Alexa488 labeled tyramides . The probes were incubated at 46°C in amplification solution for at least 30 min in the dark.For hybridization, filters were placed in a tube and mixed with 500 μl hybridization solution , 0.9 M NaCl and formamide], and 1 μl of probe working solution  . MicroorTM, Thermo Fisher, United States).For second hybridizations, the first probe-label peroxidase was inactivated by incubating the filter sections in 0.01 M HCl for 10 min at room temperature and washing the sections with 50 ml of Milli Q water. Then, the CARD-FISH protocol was repeated two times with the same filter sections by using different probes. The second hybridization used Alexa 647 labeled tyramides  and mounted with ProLong Gold Antifade reagent . Cell counting was performed using ImageJ.The push core (30 cm length) stored at 4°C was processed for microbial AOM and methanogenesis activity. We determined which substrates were available for AOM and methanogenesis in the Jiaolong methane seep. Push cores were positioned vertically, and the sediment was pushed out of the liner using a plastic plunger. From the center of the push core, samples were taken and incubated for determination of AOM and methanogenesis activity.2, and reduced with Na2S ⋅ 9H2O (0.5 g/L) and L-cysteine (0.5 g/L). The bottles were filled with 10% methane and incubated for 6 months at 4°C in the dark  as a response to AOM activity. Initial DIC concentrations in the medium and those at time-points DIC were determined.Methods for determining potential rates of AOM have been published previously . For meathe dark . Then, t2/CO2 80/20 atmosphere + molybdate (20 mM), (3) acetate (20 mM) + molybdate (20 mM), and (4) methanol (20 mM) + molybdate (20 mM). Molybdate was used as an enzymatic inhibitor for sulfate reduction  and L-cysteine (0.5 g/L). Then, the sediments were incubated for 6 months at 4°C in the dark. Methane production was measured by sampling the headspace of the serum vials by syringe and analyzing with GC-FID , using manual injection of 100 μl of headspace gas : 23 g NaCl, 0.0136 g KHsolution , and 1 msolution .The geochemistry results showed that the concentrations of sulfate, methane, and DIC changed rapidly in the depth range of 0–6 cm . The conThe vertical distribution of the microbial community was investigated in sediments from different depths . In totaSulfurovum (60.0179%), Methyloprofundus (11.1006%), and Sulfurimonas (3%). In the sediments at depths of 4–28 cm, the dominant groups belonged to SRB, including SEEP-SRB1 (12–28%), and Unclassified Desulfobulbaceae (8–20%) (Shewanella (0.01–0.07%), Pseudomonas (0.01–0.1%), Desulfuromonas (0.11-0.92%), Geobacter (0.004–0.02%), and so on  , 4. The nd so on .Nitrosopumilus (70.65%) and ANME-2a/b (18.84%). As the depth of sediment increased, Nitrosopumilus rapidly disappeared, while ANME-2a/b became more abundant, peaking (44.5%) at the depth of 8-10 cm, and then decreased. ANME-3 appeared at the depth of 4–6 cm, and its abundance increased with depth. ANME-2a/b was replaced by ANME-3 at the depth of 20–22 cm. ANME-2c appeared mainly in sediments at depths of 4–6 cm  (Methanosphaera (0.0014–0.0024%), Methanomethylovorans (0.0014–0.0076%), Methanosalsum (0.0014–0.0024%), Methanolobus (0.0178–0.0855%), Methanohalophilus (0.31–3.83%), Methanococcoides (0.09–2.34%), Methermicoccaceae (0.004–0.05%), and Methanomassiliicoccus (0.0051–0.0136%)  , 4. Furt0.0136%) , 4.8 cells g–1 (3.25 × 107 cells g–1 for archaea and 9.7 × 107 cells g–1 for bacteria) with bacteria as the dominant type. The numbers of SRB and ANME were below the detection limit. At the depth of 2–6 cm, the number of cells increased sharply, reaching 3.0 × 109 cells g–1 at a depth of 4 cm (2.0 × 109 cells g–1 for bacteria and 8.0 × 108 cells g–1 for archaea). Below 6 cm, the total number of cells decreased with increasing depth, reaching 1.0 × 107 cells g–1 at the bottom layer (1.1 × 106 cells g–1 for bacteria and 5.8 × 106 cells g–1 for archaea).As shown in 8 cells g–1 for SRB and 1.4 × 108 cells g–1 for ANME). As the depth increased, the number of SRB gradually decreased to 1.0 × 106 cells g–1. The number of ANMEs was maintained at 9.0 × 107 cells g–1 at depths of 6–12 cm and gradually decreased to 4.1 × 106 cells g–1 at the bottom. Notably, there was no significant change in the abundance of ANME at depths of 6–12 cm, whereas the number of SRB decreased rapidly , and the net rate of SO42–-AOM increased to its maximum of 900.20 nmol cm–3 day–1 at a depth of 8–10 cm, below which the rate decreased, reaching 580.79 nmol cm–3 day–1 at a depth of 28–30 cm.AOM activities was assessed as shown in –3 day–1. The highest rate of Fe-AOM was 37.99 nmol cm–3 day–1 at a depth of 12–14 cm where the concentration of ferrous iron was the highest (26.60 μM). Furthermore, no obvious increase in nitrite/nitrate-dependent AOM activity had been detected. These results suggested the potential existence of Fe-AOM below the SMTZ in the Jiaolong methane seep.In other experiments, sulfate reduction was inhibited with the addition of molybdate (20 mM), and methanogenesis was also inhibited by 20 mM BES. When ferrihydrite was supplied, the net Fe-AOM activity appeared below the SMTZ at a depth of 8–10 cm with its rate reaching 30.00 nmol cm–3 day–1. The rate peaked at 0.17 nmol cm–3 day–1 at a depth of 16–18 cm, and then decreased to 0.10 nmol cm–3 day–1 at a depth of 28–30 cm .Sequencing data showed the coexistence of methanogenic archaea and ANME in the sediments, suggesting the cooccurrence of methanogenesis and AOM. Hence, an enrichment experiment was carried out to test our hypothesis. All cultures were incubated at 4°C for 6 months. The data showed that there was no methanogenic potential in the surface sediments. The efflux of methane appeared at the depth of 4–6 cm, with its rate reaching 0.02 nmol cm28–30 cm . Further6–2.0 × 108 cells g–1 for archaea,1.1 × 106–2.0 × 109 cells g–1 for bacteria) than other methane seeps in the SCS, such as the Haima methane seep   (5–106 cells g–1)   , the GMGacteria) , and theCR data) .Sulfurovum, Methyloprofundus, Sulfurimonas, Nitrosopumilus, and ANME-2a/b. In contrast, in the deepest sediments, the dominant sequence cluster belonged mainly to ANME-2, ANME-3, and Methanimicrococcus. The microbial diversity obtained in this study obviously differed from those in previous studies at methane seeps in the northern SCS. In June 2013, researchers found that in the Jiaolong methane seep, the majority of the microbial inhabitants at the surface layers (0–6 cm) were Sulfurimonas, Sulfurovum, and ANME-1, while SEEP-SRB1, ANME-1, and ANME-2 dominated the deepest layers (8–14 cm). The percentage of ANME-3 was the lowest in all layers, and SEEP-SRB3 and SEEP-SRB4 were not detected   , Candidareducens , or Methtivorans  might beMethylomirabilis oxyfera, Methylobacter, Methylosarcina, Methylomonas, and Methylococcus) were not detected. Under the condition that 20 mM BES was added to inhibit methanogens archaea in the incubation experiments, the Fe-AOM activity appeared below SMTZ. These evidences excluded the potential participation of methanogenic archaea and methanotrophic bacteria in the process of Fe-AOM. CARD-FISH data showed that there was no significant change in the number of ANME at the depth of 6–12 cm, while the number of SRB decreased rapidly, and some ANME-2 and ANME-3 were not coupled with SRB , Geobacter (0.004–0.02%), Pseudomonas (0.01–0.1%), and Desulfuromonas (0.11–0.92%). Interestingly, the abundance of IRB was relatively higher in the 12–14 cm depth. To date, two potential ways of Fe-AOM were described in previous studies. (1) ANME oxidizes methane and transfer electrons directly to soluble metal ions or complexes, or solid metal oxides  ANMl oxides . It is wMethylomirabilis oxyfera, and nitrate-dependent AOM is performed by the ANME-2d  (–3 d–1) (–3 d–1)   , Aarhus (–3 d–1) , 2018, a(–3 d–1) . This pr3 day–1)  in shallIn summary, the incubation experiments revealed the coexistence of sulfate-driven AOM, iron-driven AOM, and methylotrophic methanogenesis in Jiaolong methane seep sediments of the northern SCS where terrigenous sediments rich in iron oxide are imported in large quantities. Fe-AOM and methylotrophic methanogenesis are overlooked potential sources and sinks of methane in SCS methane cycle. Globally, large amounts of iron [∼730 Tg/year] from rivers are transported to ocean continental margins , and metThe datasets generated for this study can be found in the NCBI, PRJNA574743 and PRJNA574745.HL, QY, and HZ designed the research and wrote the manuscript. HL performed the experiments and analyzed the data. All authors commented on the manuscript.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.


Also, DM caused significant myocardial damage and fibrosis; elevation of myocardial MDA; NE with upregulation of myocardial caspase-3, TNF-α, TGF-β, and TH; and significant decrease in serum insulin and myocardial GSH and CAT (p < 0.05). Administration of either GLP1 analog or SGLT2i caused a significant improvement in all studied parameters (p < 0.05). Conclusion: We concluded that both GLP1 and SGLT2i exhibited cardioprotective effects against DCM in T2DM, with the upper hand for SGLT2i. This might be due to attenuation of fibrosis, oxidative stress, apoptosis (caspase-3), sympathetic nerve activity, and inflammatory cytokines (TNF-α and TGF-β).The present study investigated the possible cardioprotective effects of GLP1 and SGLT2i against diabetic cardiomyopathy (DCM) in type 2 diabetic rats and the possible underlying mechanisms. Methods: Thirty-two male Sprague Dawley rats were randomly subdivided into 4 equal groups: (a) control group, (b) DM group, type 2 diabetic rats with saline daily for 4 weeks, (c) DM + GLP1, as DM group with GLP1 analogue (liraglutide) at a dose of 75 µg/kg for 4 weeks, and (d) DM + SGLT2i as DM group with SGLT2 inhibitor (dapagliflozin) at a dose of 1 mg/kg for 4 weeks. By the end of treatment (4 weeks), serum blood glucose, homeostasis model assessment insulin resistance (HOMA-IR), insulin, and cardiac enzymes  were measured. Also, the cardiac histopathology, myocardial oxidative stress markers , glutathione (GSH) and CAT) and norepinephrine (NE), myocardial fibrosis, the expression of caspase-3, TGF-β, TNF-α, and tyrosine hydroxylase (TH) in myocardial tissues were measured. Results: T2DM caused significant increase in serum glucose, HOMA-IR, serum CK-MB, and LDH ( Diabetes mellitus (DM) is a systemic metabolic disease, which is characterized by chronic hyperglycemia due to lack of insulin secretion, insulin action, or both ,2. In 201c (HbA1c) [A novel class of medication called sodium/glucose cotransporter 2 inhibitors (SGLT2i) are used nowadays in the treatment of T2DM. Several experimental studies demonstrated the beneficial cardiovascular effects of SGLT2i independent of their anti-hyperglycemic actions ,12,13,14 (HbA1c) . In the Thirty-two male Sprague Dawley rats, aged 10–12 weeks, weighing 190 ± 10 g, were housed in the medical experimental research center (MERC) at Mansoura Faculty of Medicine in separate cages under controlled environmental conditions (12 h light/dark cycle and temperature 24 °C). Rats had a free access to water. All protocols of the present study were approved by the institutional review board (IRB) of Mansoura Faculty of Medicine .Normal control group—including normal nondiabetic rats that received saline 0.5 mL via gastric gavage and 0.5 mL saline via subcutaneous (S.C.) injection;Diabetes Mellitus (DM) group—including T2DM rats that received saline 0.5 mL via gastric gavage and 0.5 mL via S.C. saline injection;DM + GLP1 group—including T2DM rats that received GLP1 analogue (liraglutide) at a dose of 75 µg/kg dissolved in 0.5 mL saline via S.C. injection and 0.5 mL saline via gastric gavage once daily for 4 weeks ;DM + SGLT2i group—including T2DM rats that received SGLT2 inhibitor (dapagliflozin) at a dose of 1 mg/kg dissolved in 0.5 mL saline via oral gastric gavage and 0.5 mL via S.C. saline injection daily for 4 weeks .Rats were randomly subdivided into 4 equal groups (8 rats each) as follows:The details of induction of Type 2 DM in rats was mentioned in our previous study . BrieflyFasting blood sugar and insulin were measured using commercially available kits  according to the manufacturer’s instructions. The homeostasis model assessment (HOMA) insulin resistance (IR) index was calculated from fasting insulin and fasting blood glucose using Mathew’s formula .The serum levels of creatine kinase (CK)-MB and lactate dehydrogenase (LDH) were measured using commercially available kits  according to the manufacturer’s instructions.A piece of left ventricle (about 50–100 mg) was dissected and homogenized in cold buffer  using a mortar and pestle. The tissue homogenate was centrifuged at 4000 rpm for 15 min at 4 °C. Then, the supernatant was collected and stored at −20 °C until time of biochemical assay. The concentrations of malondialdehyde (MDA) and reduced glutathione (GSH) and the catalase enzyme activity were measured in the supernatant using a colorimetric method according to the manufacturer’s instructions .A piece of left ventricle (50–100 mg) was homogenized in 1 mL of Trizol to obtain total RNA according to the manufacturer’s instructions . Reverse transcription was done using 1 µg total RNA and a cDNA kit (high-capacity cDNA archive kit). The sequence of the primers of the tested genes were TNF-α: TNFα (295 bp) forward 5′-TACTGAACTTCGGGGTGATTGGTCC-3′, reverse 5′-CAGCCTTGTCCCTTGAAGAGAACC-3′; and GAPDH forward: 5′-TATCGGACGCCTGGTTAC-3′, reverse: 5′-CTGTGCCGTTGAACTTGC-3′. The details of PCR reaction and data analysis for the expression of nrf2 were mentioned in our previous work .The left ventricle of the heart was dissected and fixed in 10% neutral buffered formalin, then heart tissues were embedded in paraffin and sectioned at 3 μm thickness and stained using hematoxylin and eosin (H&E). Slides were examined for signs of cardiomyopathy, which included deranged myocardial cells, uneven cytoplasm distribution, edema, inflammatory cells infiltrate, hypertrophy of muscle fibers, rupture of myocardial fibers and irregular nuclei , using lThe tissue section was deparaffinized, rehydrated, washed, immersed in 3% hydrogen peroxide and then digested with pepsin for antigen retrieval. After the blocking of unspecific binding by serum, the section was incubated with primary antibodies of anti-caspase-3 , dilution 1:1000; mouse monoclonal antibody to TGF-β, dilution 3:1000 (DAKO); and TH  at 4 °C overnight. Diaminobenzidine/peroxidase substrate was used to produce a brown-colored signal. The section was counterstained, dehydrated, cleared and coverslipped. Phosphate buffered saline (PBS) was used to replace primary antibody, and adjacent sections were used as negative control. TH was quantified as the percent of myocardial area  occupied by positive staining  for each left ventricular area by using image J software).p < 0.05 was considered significant.Data processing and analysis was done by SPSS  version 17 . One-way ANOVA with Tukey’s post hoc test was used. p < 0.05). On the other hand, the levels of blood glucose, HOMA, LDH and CK-MB were significantly attenuated in DM + SGLT2i and DM + GLP1 groups compared to the DM group, while insulin was significantly increased in treated groups compared to the DM group (p ˂ 0.05). Moreover, there were no statistically significant differences between DM + SGLT2i and GLP1 groups in these parameters, except blood glucose, which was significantly lower in the DM + SGLT2i group than the DM + GLPT1 group  and a significant decrease in treated groups (DM + SGLT2i and DM + GLP1 groups) compared to that of the DM group (p ˂ 0.01). Also, MDA levels were significantly lower in the DM + SGLT2i group than DM + GLP1 group (p ˂ 0.001) with significant increase in their levels in treated groups (DM + SGLT2i and DM + GLP1 groups) compared to the DM group (p ˂ 0.01). There was no statistically significant difference in the CAT activity between DM + SGLT2i and DM + GLP1 groups, while the concentration of GSH was significantly higher in DM + SGLT2i than that in the DM + GLP1 group . This elevation in TNF-α expression was significantly attenuated in treated groups (DM + SGLT2i and DM + GLP1 groups) compared to the DM group (p < 0.01). Moreover, the DM + SGLT2i group showed significant reduction in TNF-α expression compared to that of the DM + GLP1 group (p < 0.01)  .H&E histopathological examination revealed normal myocardial architecture with regular arrangement of myocardial fibers and nuclei in the normal control group A, while Masson trichrome examination of the heart tissues for fibrosis revealed that no collagen fibers and fibrosis in heart specimens were obtained from the normal control group A, while p < 0.001). This elevation was significantly attenuated in DM + SGLT2i and DM + GLP1 groups compared to the DM group (p < 0.01). Also, the level of NE and the density of TH were significantly lower in the DM + SGLT2i group than that in the DM + GLP1 group (p < 0.01)  A,B. The  < 0.01) C. Hearts < 0.01) D. Also,  < 0.01) E,F.p ≤ 0.001). In addition, its expression was significantly attenuated in treated groups (DM + SGLT2i and DM + GLP1) compared to that of the DM group (p ≤ 0.01). Moreover, its expression was significantly lower in the DM + SGLT2i group compared to that of the DM + GLP1 group (p ≤ 0.01)  A. Heart  ≤ 0.01) B. Hearts ≤ 0.01) C. On the ≤ 0.01) D, while  ≤ 0.01) E.p ≤ 0.001). In addition, its expression was significantly attenuated in treated groups (DM + SGLT2i and DM + GLP1) compared to that of the DM group (p ≤ 0.01). Moreover, its expression was significantly lower in the DM + SGLT2i group compared to that of the DM + GLP1 group (p ≤ 0.01)  A. Heart  ≤ 0.01) B, while  ≤ 0.01) C. On the ≤ 0.01) D, while  ≤ 0.01) E.The main findings of the present study include the following: a) T2DM was characterized by significant elevation of blood glucose and cardiac enzymes, insulin resistance, disturbed myocardial morphology with myocardial fibrosis and oxidative stress. Also, b) T2DM was associated with up-regulation of apoptotic markers (caspase-3) and inflammatory cytokines (TGF-β) and increased sympathetic nerve density with higher myocardial NE, and c) pretreatment with either SGLT2i or GLP1 significantly improved the studied parameters, and SGLT2i offered a more protective effect than GLP1.In the present study, a well-known rat model of T2DM (high-fat diet for one month followed by a single low dose of STZ) was adopted. The present study found high blood glucose with relative insulin deficiency and high HOMA-IR in the DM group, indicating T2DM development. Previous studies reported similar findings ,22,23. LThe present study demonstrated significant cardiac damage in T2DM, as evidenced by the rise in the levels of cardiac enzymes (CK-MB and LDH) and by the morphological changes such as myocardial atrophy, degeneration, necrosis and inflammatory cell infiltrates observed in hearts of the DM group. The rise in cardiac enzymes in serum might be due to loss of the integrity of cardiomyocytes and release of cytosolic cardiac enzymes. Similar findings were demonstrated by other researchers ,26,27. MPathogenesis of DCM is a complex and multifactorial process in which persistent hyperglycemia and insulin resistance lead to myocardial oxidative stress, inflammation, activation of the renin angiotensin aldosterone system (RAAS), autonomic neuropathy and myocardial apoptotic cell deaths. These molecular changes promote development of cardiac hypertrophy and fibrosis . Activat2+ transporters, matrix metalloproteinases and oxidative stress. Also, they reported that liraglutide (GLP1) attenuated oxidative stress and fibrosis in cardiac tissues in mice treated with a high-fat diet.Oxidative stress plays an important role in the pathophysiology of diabetic cardiomyopathy (DCM). Hyperglycemia leads to excessive generation of free oxygen radicals, which promote the development of myocardial apoptosis and fibrosis. We found, in the current work, enhanced myocardial oxidative stress in diabetic rats as evidenced by a significant rise in MDA (marker of lipid peroxidation) and with significant decreases in CAT activity and the concentration of GSH in the hearts of the DM group. Yang et al.  and WilsApoptotic cell damage is another mechanism for DCM, in which elevated blood sugar in DM promotes the release of mitochondrial cytochrome C to the cytosol, resulting in activation of caspase-3 and apoptotic cell death of cardiomyocytes . Also, pMyocardial fibrosis and inflammation are key features in DCM ,40. The An in vitro study by Dorecka and colleagues  demonstrAlso, in the present study we investigated the effects of GLP1 and SGLT2i on the sympathetic nerve fiber density and myocardial NE content. The current showed significant increase in the density and number of sympathetic fibers in diabetic hearts, as evidenced by the significant increase in TH density in the hearts of type 2 diabetic rats with higher levels of NE in the myocardium, suggesting sympathetic overactivity in heart tissues in early DM. In line with these findings, Otake et al.  demonstrT2DM might be associated with DCM, which is characterized by myocardial fibrosis and which might be due to enhanced oxidative stress, upregulation of inflammatory cytokines  and apoptotic proteins (caspase-3) and increased activity of myocardial sympathetic nerve fibers. Treatment of diabetic rats with either SGLT2i (dapagliflozin) or GLP1 (liraglutide) protected the hearts from DCM, and SGLT2i offered more cardioprotective effects than GLP1. The cardioprotective effects of both agents might be due to inhibition of oxidative stress, the apoptotic process, the inflammatory process, fibrosis, the sympathetic nerve fiber activity and myocardial NE.


ABM300 was characterized in vitro  and in vivo  to assess the effects of the compound in dysregulated behaviors within the transgenic models. In vitro, ABM300 increased CB1R agonist binding but acted as an inhibitor of CB1R agonist induced signaling, including β-arrestin2 translocation, ERK phosphorylation and cAMP inhibition. In vivo, ABM300 did not elicit anxiogenic-like or cannabimimetic effects, but it decreased novelty-induced hyperactivity, exaggerated stereotypy, and vertical exploration in both transgenic models of hyperdopaminergia, as well as normalizing PPI in DATKO mice. The data demonstrate for the first time that a CB1R allosteric modulator ameliorates the behavioral deficits in two models of increased dopamine, warranting further investigation as a potential therapeutic target in psychiatry.The endocannabinoid system (eCBs) encompasses the endocannabinoids, their synthetic and degradative enzymes, and cannabinoid (CB) receptors. The eCBs mediates inhibition of neurotransmitter release and acts as a major homeostatic system. Many aspects of the eCBs are altered in a number of psychiatric disorders including schizophrenia, which is characterized by dysregulation of dopaminergic signaling. The GluN1-Knockdown (GluN1KD) and Dopamine Transporter Knockout (DATKO) mice are models of hyperdopaminergia, which display abnormal psychosis-related behaviors, including hyperlocomotion and changes in pre-pulse inhibition (PPI). Here, we investigate the ability of a novel CB However, antipsychotics are associated with extrapyramidal side effects, sedation, metabolic syndrome, and weight gain  CB1R agonist binding but functionally acts as an inhibitor of CB1R agonist-mediated signaling CP55,940 . Non-specific binding was defined in the presence of 10 µM WIN55,212-2.Binding assays in hCBhttps://www.eurofinsdiscoveryservices.com).The PathHunter® β-Arrestin assay was conducted by Eurofins Pharma Discovery Services  according to the manufacturer’s protocol, in hCB1R CHO cells plated at a density of 40,000 cells/well (96-well). ABM300 IC50 values were determined in the presence of increasing concentrations of ABM300 at the EC80 for CP55,940 (40 nM). Results are presented as the percent stimulation of ERK1/2 phosphorylation by CP55,940 alone.CB1R CHO cells were seeded at a density of 40,000 cells/well (white 96-well), and after 24 h were serum-starved in the presence of 0.1% Bovine Serum Albumin for 60 min prior to pretreatment for 30 min with increasing concentrations of ABM300 (10−10–10−5 M) and a final 30 min incubation with 40 nM CP55,940 and 5 μM forskolin. The cAMP BRET experiments were performed as previously described CP55,940 to hCB1R CHO  and an α value of 4.33 ± 0.79 (logα 0.622 ± 0.08) (Supplementary Table In line with our previous studies using a related compound (Org275) , we findCHO Fig.  with an 1R assay, CP55,940-stimulated β-arrestin recruitment with an EC50 value of 5.37 nM (pIC50 8.28 ± 0.06) and an Emax of 104.3 ± 0.90%. In the presence of the EC80 of CP55,940 (10 nM), ABM300 produced a concentration-related reduction in β-arrestin recruitment with an IC50 value of 49.7 nM (pIC50 7.31 ± 0.02)  Fig. .1R CHO cells. In the presence of vehicle, CP55,940 induced ERK1/2 phosphorylation with an EC50 of 12.1 nM (pEC50 8.14 ± 0.23) , to 40.3 ± 5.57% and 14.7 ± 5.22%, respectively  of AEA from 100.3 ± 0.20% to 80.23 ± 14.4% and 31.88 ± 6.09%, respectively  of CP55,940, ABM300 produced a concentration-related reduction in ERK1/2 phosphorylation with an IC50 value of 47.0 nM (pIC50 7.38 ± 0.12) . At CP55,940 EC80 (40 nM), ABM300 blocked this inhibition with an IC50 value of 379 nM (pIC50 6.45 ± 0.08)  Fig. . At concely Fig. . ABM300 ely Fig. . Additioely Fig. . In the 12) Fig. . CP55,9408) Fig. .We further characterized the real-time kinetic effect of ABM300 using a cAMP BRET sensor assay in HEK293 cells. Similar to previous observations with Org275 , ABM300 2, Dopamine D1 and D2, NMDA, 5HT1A, 5HT1B, 5HT2A, 5HT2B, and 5HT3 . Previous reports demonstrated adverse effects observed with CB1R orthosteric agonists/inverse agonists [p = 0.3335), whereas rimonabant significantly reduced open arm time compared to vehicle (p = 0.0059).We confirmed that ABM300 (10 mg/kg) did not have agonist activity via the cannabinoid-induced tetrad, compared to THC (10 mg/kg). In all four tetrad measures , and GluN1KD mice responded to ABM300 (p < 0.0001) when compared to vehicle. Significant effects of genotype were observed for stereotypy and vertical exploration . ABM300 significantly reduced exaggerated stereotypic activity . Sensorimotor gating deficits, along with acoustic startle response, did not respond to ABM300 or the atypical antipsychotic olanzapine  , indicating that ABM300 has genotype-specific effects on the exacerbated hyperactivity endophenotype in DATKO. There was an effect of genotype on exaggerated stereotypic movements and mania-like rearing behavior , which ABM300 had a beneficial effect in decreasing  . Neither genotype, nor treatment with ABM300, had an effect on the acoustic startle response in the DATKO model .The DATKO mouse, another model of hyperdopaminergia, displays hyperactivity, increased stereotypy and vertical exploration, with an impairment in sensorimotor gating , 31. Simly) Fig. . Furtherly) Fig. , with a p = 0.007). Furthermore, olanzapine, but not ABM300, enhanced escape responding during CAR  and an interaction between drug treatment and testing day (p = 0.034). Lastly, neither ABM300 nor olanzapine affected escape failures  [1R agonist-mediated β-arrestin recruitment , ERK phosphorylation , and cAMP inhibition .Our molecular docking data indicate that ABM300 binds to the recently elucidated binding site for the original allosteric modulator Org275 . The Orgund CB1R , which mo Org275 , 15, whe ± 0.79) , but act1R allosteric modulators such as Org275 and PSNCBAM-1 [1Rs can significantly differ in various neuronal cell types; CB1R on GABAergic interneurons have significantly higher agonist affinity than those found on glutamatergic terminals, but the coupling efficacy of glutamatergic CB1R is significantly higher [1R signaling in vivo and, potentially, selectively modulates the endocannabinoid system in specific neurotransmitter system pathways to ameliorate the dysregulated behaviors observed in these mice.As previously described , the uniSNCBAM-1 . This may higher , 46. Thi1R to the soma, while cholesterol, which binds to the same site as ABM300 and acts as a positive modulator, allows for the enrichment of CB1R at the axon [1R membrane localization by CB1R allosterics. The consequences of this in a complex neuronal network, and pathological states, are yet to be fully elucidated.Studies have demonstrated that administration of compounds that increase endocannabinoid levels modulate a number of schizophrenia-like responses in mice . Studiesthe axon . Thus, i2 receptor [To assess psychosis-like behaviors, and therapeutic efficacy of ABM300, we focused on two main behavioral categories: exploratory behavior (hyperactivity) and sensorimotor gating (disruption in PPI). The literature related to psychosis-like behaviors in genetically modified mouse models has classically focused on the same two categories . Althougreceptor . Thereforeceptor . Meanwhireceptor .1R antagonism and loss of function may enhance some forms of learning and memory, and it is possible that CB1R-negative allosteric modulators may be pro-cognitive in preclinical schizophrenic models [Here, we show that ABM300 restores dysregulated dopamine-mediated exploratory activity in both genetic models: decreasing exaggerated hyperactivity, stereotypy and rearing. Furthermore, ABM300 rescues PPI deficits in the DATKO model. Since psychosis symptomology is never present alone in a disease state such as schizophrenia, it would be intriguing to investigate the effects of ABM300 in other symptomatic domains, such as cognition. CBc models , 53.We saw no effect of the compound in CAR in rat even though olanzapine produced significant suppression of CAR as has been shown in previous studies . CAR is 1R in the in vivo effect of ABM300 in genetic models. Directly implicating CB1R is challenging. One approach would be to create a double knockout of CB1R-/- and DAT-/- or GluN1-/-. However, given the crucial role of the CB1R in dopaminergic and glutamatergic signaling, there is a strong likelihood that the double knockouts could have a novel phenotype (such as seizures) or that previous phenotypes would be exacerbated [1R has been shown to have effects on dopamine signaling [1R orthosteric antagonist would block the effects of ABM300, thereby implicating CB1R. However, there is also doubt as to whether an orthosteric CB1R antagonist would block the effects of a negative allosteric inhibitor (antagonist blocking inhibitor); it is conceivable that a synergistic effect might be observed. Taken together, these limitations highlight the complexity of directly implicating CB1R in the mechanism of action of ABM300 in vivo in these genetic models. We plan to make this the subject of future investigations involving a variety of in vivo and ex vivo approaches. The pharmacological profiling and assessment of the potential for off-target interactions of ABM300 in binding screens suggest that the compound is CB1R-selective. Taken together, the potent inhibitory effect of the compound on CB1R signaling at nM concentrations in vitro and the favorable PK and brain penetration data, there is substantial support for the hypothesis that the CB1R mediates the effects of ABM300 in vivo in the genetic mouse models of hyperdopaminergia. It is also notable that the present study only included acute administration of one dose of ABM300. Future experiments will focus on various dose regimens, chronic dosing, development of tolerance and the effects of ABM300 in combination with an antipsychotic.It is important to note that we do not have direct evidence for the involvement of the CBcerbated , 57. Furignaling , 59. Ano1R allosteric modulator, with a unique pharmacological profile, effectively ameliorates certain behavioral deficits in two distinct models of increased dopamine. These data highlight that the allosteric-binding pocket on the CB1R warrants further investigation as a potentially important therapeutic target in psychiatry.The data presented here offer the first evidence that acute administration of a CBThe authors declare the following financial and biomedical conflict of interests: Ruth A. Ross, Catharine A. Mielnik, Amy J. Ramsey, Iain R. Greig, Laurent A. Trembleau, Mostafa H. Abdelrahman are co-inventors on a patent application related to ABM300 and structural analogs. Kim S. Sugamori, David B. Finlay, Hayley H.A. Thorpe, Matthieu Schapira, Nirunthan Sivananthan, Chun Kit Li, Vincent M. Lam, Sean Harrington, W. McIntyre Burnham, Jibran Y. Khokhar, Ali Salahpour, Michelle Glass reported no biomedical financial interests or potential conflicts of interest. W. McIntyre Burnham received Δ9- (THC) as a gift from MedReleaf. The authors would like to gratefully acknowledge Wendy Horsfall for mouse colony maintenance. The work was funded by grants to RAR from CIHR , CIHR funding to AJR (MOP119298) and CIHR funding to AS (PJT-15619).Mielniketal2020_Supplemental Material


Aspergillus oryzae, resulting in the production of 22 DDPs, including 15 new analogues. We also demonstrate the advantage of expanding the diversity of DDPs to probe various bioactive molecules through a wide range of biological evaluations.A synthetic biology method based on heterologous biosynthesis coupled with genome mining is a promising approach for increasing the opportunities to rationally access natural product with novel structures and biological activities through total biosynthesis and combinatorial biosynthesis. Here, we demonstrate the advantage of the synthetic biology method to explore biological activity-related chemical space through the comprehensive heterologous biosynthesis of fungal decalin-containing diterpenoid pyrones (DDPs). Genome mining reveals putative DDP biosynthetic gene clusters distributed in five fungal genera. In addition, we design extended DDP pathways by combinatorial biosynthesis. In total, ten DDP pathways, including five native pathways, four extended pathways and one shunt pathway, are heterologously reconstituted in a genetically tractable heterologous host,  Aspergillus oryzae.Combining genome mining and heterologous expression in a genetically tractable host can lead to bioactive natural products discovery and production. Here, the authors employ this strategy for new decalin-containing diterpenoid pyrenes production by expressing native, extended, and shunt pathways in  During secondary metabolite biosynthesis in living organisms, natural products are formed in vivo through multi-step enzymatic reactions4. In other words, natural product structures are constructed and tailored in repeated interaction with proteins in cells. This biosynthetic manner provides natural product with drug-advantageous properties such as potential protein interactivity, water solubility, and membrane permeability5. For this reason, the natural product chemical space is highly relevant to biological space6. Therefore, the discovery of natural products with novel structures and biological activities is an important challenge in the pharmaceutical field. Natural products with valuable biological activities are recognized to contain biologically relevant privileged structures,8 whose analogues and congeners serve as excellent guides to discover not only more potent bioactive compounds but also various additional biological activities10. Thus, expanding the chemical space around biologically important natural products may accelerate drug discovery research11. Chemical synthesis, including divergent and diverted total syntheses, is a powerful strategy to produce bioactive natural product analogues and congeners13. A late-stage modification strategy is also a beneficial tool to generate natural product derivatives, such as diversity-oriented synthesis using natural products and their intermediate starting points17. However, the structural complexity and limited availability of natural products remain obstacles to synthesizing a large collection of natural products and their structural analogues in sufficient amounts. Thus, the development of a method that enables rapid and rational access to the chemical space around bioactive natural products and a reliable supply is required for drug discovery.Natural products are historically unparalleled successful sources for drug discovery18. It is also well known that fungi possess extraordinary biosynthetic gene clusters that may encode highly diverse natural product biosynthetic pathways, and biosynthetic gene information has been accumulating at a rapidly accelerating rate in the past a decade because of genome sequencing innovation19. However, most biosynthetic pathways distributed in fungal genomes have not been linked with structural information20. This implies the presence of a large number of novel natural products that may be produced via those untapped pathways. A synthetic biology method based on heterologous biosynthesis coupled with genome mining is a promising approach to translate enormous amounts of biosynthetic gene information to richly diverse natural products. Genome mining based on biosynthetic studies has enabled rapid access to biosynthetic pathways not only for a target natural product and its related analogues but also for unexplored natural products. Heterologous biosynthesis has enabled to access compounds encoded by biosynthetic pathways found by genome mining and a number of total biosynthesis of natural products and discovering novel natural products have been reported28. In addition, combinatorial biosynthesis approach is powerful tool to generate non-natural analogues, which may be a great advantage to construct pharmaceutical beneficial screening library32. Thus, we apply the synthetic biology approach for rationally expanding the chemical space around a target natural product as follows , a type of meroterpenoid natural products composed of a diterpenoid-derived decalin ring system linked to pyrone biosynthesized via polyketide pathwayssubA–F) in an entomopathogenic fungus, Metarhizium robertsii, has been estimated  aggregation in nucleation phase, paralysing activity against adult Drosophila and suppressing insect innate immune signal transduction. Most these biological activities firstly have been found in DDPs in this study.Here, we demonstrate the advantage of the synthetic biology approach based on heterologous and combinatorial biosynthesis coupled with genome mining for constructing a biologically relevant DDP-focused library composed of a variety of DDPs. Although biosynthetic studies on DDPs are limited, a putative biosynthetic gene cluster and pathway for subglutinols -dependent epoxidase (FMOep) and terpene cyclase (TC) were found in five fungal genomes, Fusarium graminearum PH-1 (dpfgABCDEGHIJK), Macrophomina phaseolina MS6 (dpmpABCDEGHIJ) Colletotrichum higginsianum IMI349063 (dpchABCDEFGH), Metarhizium anisopliae E6 (dpmaABCDEF), and Arthrinium sacchari (dpasABCDEF), which we previously isolated from a spider  are highly conserved in each cluster, suggesting that all the pathways share the biosynthetic pathway for a common intermediate 4. The differences in the genes at the tailoring steps in each pathway may diversify DDP biosynthesis. The three biosynthetic gene clusters in F. graminearum (dpfg cluster), M. phaseolina (dpmp cluster) and C. higginsianum (dpch cluster) contain two types of short chain dehydrogenase reductase (SDR) genes, dpxxG (SDR1) and dpxxH (SDR2). Each of the dpxxG and dpxxH genes can be recognized as orthologous because of their high similarity ; that is, each set of SDRs, dpfgGH, dpmpGH and dpchGH, may provide the same product from 4. Both the dpfg and dpmp clusters include an orthologous methyltransferase gene, dpxxI (MT1), which it is absent in the dpch cluster, indicating that the dpch pathway is probably divided from the dpfg and dpmp pathways after SDR modification steps and that a remaining dpchF (FMO) would lead to the end product in the dpch pathway. Both the dpmp and dpfg clusters contain a P450 gene, dpmpJ and dpfgJ, respectively, but they show low similarity to each other , suggesting that the dpfg and dpmp pathways may have branched after the MT1 modification step. Subsequently, dpmpJ and dpfgJK afford the final products in each pathway. However, the dpas and dpma gene clusters possess only a modifying enzyme, FMO dpasF and FAD-dependent BBE domain-containing oxidoreductase (BBE) dpmaF, respectively. Therefore, both pathways are likely branched at the initial tailoring stage, leading to the final products. As a result, we predicted the treelike native DDP pathways, as depicted in Fig. dpch and dpma pathways may produce higginsianin A40 and subglutinol A46, respectively. However, the orphan dpfg, dpmp and dpas pathways may provide new DDP analogues.To find biosynthetic gene clusters that may encode DDP pathways, we performed genome mining of the public databases and our original gene resources by using SubA (NR-PKS) as a query. As a result, five candidate gene clusters with A. oryzae NSAR1 according to the hypothetical biosynthetic pathways , which, as expected, produced prenylated (C20) α-pyrone 2   and B  as previously suggested in the sub cluster46  as well as subglutinols A  and B  , while AO-dpasABCDE-dpmpGH gave higginsianin B    producing transformants as a platform. The introduction of dpmpI and dpfgI into the platforms yielded AO-dpasABCDE-dpmpGHI and AO-dpasABCDE-dpfgGHI, both of which produced a new intermediate 11 . Moreover, dpmpIJ and dpfgIJK were introduced to construct AO-dpasABCDE-dpmpGHIJ and AO-dpasABCDE-dpfgGHIJK. The transformant expressing the whole dpmp gene cluster, AO-dpasABCDE-dpmpGHIJ, afforded a new DDP with a C-26 primary alcohol on the γ-pyrone moiety   and 14  with a highly oxidized γ-pyrone moiety including a methyl ester  and higginsianin A (10), respectively, and the orphan dpas, dpmp and dpfg pathways provided new DDP analogues.We then aimed to reconstitute the s48 Fig. . We also−1) Fig. . We reco−1) Fig. . On the ter Fig. , like coA. oryzae heterologous expression system. In summary, DpmaF and DpfgJ strictly recognized the C-8 configuration, while DpasF, DpchF, DpmpI, DpfgI and DpmpJ showed tolerant substrate selectivity, which became an advantage for the generating diversity in combinatorial biosynthesis. Through the experiments, we found and reconstituted a shunt DDP pathway in A. oryzae, which afforded viridoxin A hydrolysate 1533 (42 mg L−1) and a new DDP analogue 16 (6 mg L−1)  enables the generation of four types of C5 unit moieties through its multifunctional oxidative ability, and we designed two extended pathways by adding dpasF to the dpmp and dpfg pathways. In addition, MT1 (dpmpI) was connected to the dpas and dpch pathways. Thus, we designed and reconstituted the four extended pathways in A. oryzae , as expected, produced four new analogues, 17 (5 mg L−1), 18 (3 mg L−1), 19 (16 mg L−1), and 20 (5 mg L−1), due to the promiscuity of DpmpJ  afforded only the new analogue 21 (6 mg L−1), the most modified compound in this study because DpfgJ (P450) strictly recognizes C-8 stereochemistry , as expected, produced O-methylated higginsianin A , and the fourth transformant (dpas pathway + dpmpI) provided 23 (23 mg L−1), 24 (7 mg L−1) and 25 (3 mg L−1) , 18  mg L−1, pJ Figs. . AnotherL−1), 18  mg L−1, ry Figs. . In thisL−1), 18  mg L−1, 1) Figs. . As expeA. oryzae and produced 22 DDPs, including 15 new compounds. Among these 22 compounds, 11 compounds came from native pathways, 2 compounds were biosynthesized through shunt pathways, and 9 compounds were produced via extended pathways  was determined by the modified Mosher’s method49 -positive cells55. We selected compounds 5, 11, 12, 17, 18, 20, 22 and 23 according to dose-dependent cytotoxicity against the bulk of MCF-7 cells for the mammosphere formation assay , which are a small sub-population in tumour bulk identified in most cancer cells and clinical samplesion Fig.  more efflls Fig. . The moslls Fig. . Thus, fDrosophila assay system for cytotoxicity, inhibitory activity of innate immune signalling and insecticidal activity57. The cytotoxicity of the DDPs was evaluated using two cell lines, embryonic macrophage-derived DL1 cells60 and larval blood cell-derived l(2)mbn cells61, and their IC50 values are listed in Supplementary Table 5 unit  showed potent antiproliferative activity, and they affected DL1 cell lines more potently. Notably, subglutinol A (5) and 21 strongly inhibited cell growth in the DL-1 cell line (IC50 = 12 nM (5) and 15 nM (21)).We screened the DDPs on the 57. No compound showed a remarkable effect on the Toll pathway, while 21 inhibited the IMD pathway with an IC50 of 0.27 ± 0.01 µM  is one of the virulence factors of entomopathogenic Metarhizium fungi against insects. Thus, we successfully found unique biological activity against Drosophila in 5 and 21, which may potentially be developed as insecticides.We next tested the ability of the DDPs to affect the innate immune pathways, namely, the Toll and immune deficiency (IMD) pathways, which are the front line of defence against infection by microorganisms µM Fig. . This reila Fig. . The res62 using AZT as positive control (EC50 = 29.7 nM and CC50 > 10000 nM). Some of compounds, such as 5, 10, 11, 12, 14, 17, 18, 20, 23 and 24, exerted specific anti-HIV activity (EC50 < 200 nM) without affecting other viruses . Other compounds also showed moderate to substantial activity but the effective concentrations were high; therefore, their toxicity made it difficult to clearly distinguish their anti-HIV effect.We examined the inhibitory activities of DDPs against various species of bacteria (both gram-negative and gram-positive bacteria), fungi and viruses. No DDPs showed significant anti-bacterial or anti-fungal activities. In contrast, preliminary screening of the DDPs for anti-viral agents with hepatitis B virus (HBV), measles virus (MV), Epstein-Barr virus (EBV), herpes simplex virus (HSV) and human immunodeficiency virus-1 (HIV-1) indicated their selective inhibition of HIV. We further investigated the anti-HIV activity of the DDPs by the MAGI assay63. A nucleation-dependent polymerization model that is composed of nucleation and elongation phases is generally accepted as the aggregation mechanism of Aβ4264. In the nucleation phase, the monomer of Aβ42 gradually forms low-molecular-weight oligomers , which cause synaptotoxicity and memory loss65.The aggregation of the 42-mer-amyloid β (Aβ42) is involved in the pathogenesis of Alzheimer’s disease (AD)12, 13, 14, 20, and 21, and tested them again  assay, which is a conventional and useful method for quantifying Aβ aggregates . The possible peaks of Aβ42–21 adducts were detected, and then the mass envelopes at +7, +6, +5, and +4 charge distribution corresponded to the Michael adduct based on the loss of 32 Da  using E22P, M35ox-Aβ9-35 as a toxic conformer surrogate67. Based on a large number of fragmented b ions and y ions, Lys16 in Aβ42 (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA) seemed to be one of the target amino acid residues of 21 , potent inhibitory activity of an insect innate immune system, the IMD pathway (21), potent paralysing activity against adult Drosophila (5), selectively inhibited HIV proliferation (20), and Aβ aggregation in the nucleation phase through trapping lysine residue on highly modified γ-pyrone motif (13 and 21). Notably, compounds produced via extended pathways indeed showed unique biological activities. Thus, this study showcases the capability of combinatorial synthetic biology in acceleration of drug discovery.We then screened the DDP-focused library with various biological activity assays. DDPs produced in this study shared same skeleton and showed similar antiproliferative activities against cancer cells and inhibition of mitochondrial complex III each other. However, interestingly, small structure differences in each DDP gave unique functions of markedly reducing CSC-like populations in MCF-7 bulk cells  and RP-18 F254S (Merck). Column chromatography and flash chromatography were carried out on silica gel 60 N  and silica gel 60 N , respectively. NMR spectra were recorded on a Burker AVANCE III spectrometer. Chemical shifts for 1H and 13C NMR are given in parts per million (δ) relative to tetramethylsilane (δH 0.00) and residual solvent signals (δC 77.0) for CDCl3 as internal standard. Reverse phase HPLC analysis was performed on HITACHI LaChrom Elite series equipped with L-2130 pump, L-2200 autosampler, L-2455 Diode Array Detector, and D-2000 system manager. LC-MS analysis was performed on HITACHI Chromaster series equipped with 5110 pump, 5430 Diode Array Detector, 5610 MS Detector, MSD system manager. High-resolution mass spectra were measured on a Thermo Fischer Scientific Exactive Mass spectrometer. UV spectra were recorded on a JASCO-V-730 spectrophotometer. IR spectra were recorded on JASCO-FT/IR-4200 spectrometer. Optical rotations were recorded on JASCO-P-1030.Polymerase chain reaction (PCR) was performed using a TaKaRa PCR Thermal Cycler Dice® Gradient (Takara Bio) and Thermal Cycler LifeECO (Nippon Genetics). Oligonucleotide primers for PCR were purchased from Hokkaido System Science Co., Ltd.  and listed in Supplementary Table A. sacchari Kumo-3 was obtained in previous study . Genome mining was performed by Protein BLAST search against A. sacchari Kumo-3 genome sequence and the NCBI non-redundant database and 2ndFind program (http://biosyn.nih.go.jp/2ndfind/). The results of bioinformatics analyses are shown in Supplementary Tables Draft genome sequence of Arthrinium sacchari (strain Kumo-3) was isolated from a spider previously69. Genomic DNA of Fusarium graminearum 50218 was obtained from the Medical Mycology Research Center, Chiba University . Macrophomina phaseolina NBRC 7317 and Metarhizium anisopliae NBRC 103233 were obtained from the Biological Resource Center, National Institute of Technology and Evaluation . Colletotrichum higginsianum MAFF 305635 was obtained from the Genetic Resource Center, National Agriculture and Food Research Organization .Escherichia coli DH5a  was used for cloning experiments. Aspergillus oryzae NSAR147, a quadruple auxotrophic mutant  was used as the host for fungal expression.A. sacchari Kumo-3, M. phaseolina NBRC 7317, M. anisopliae NBRC 103233, and C. higginsianum MAFF 305635 were cultivated on Potato dextrose agar (PDA) (2.4% Potato dextrose broth (Difco), 1.5% agar). The spores and mycelium from the plate were inoculated into 60 mL of Potato dextrose medium (2.4% Potato dextrose broth). After several days cultivation at 30 °C , the mycelia of each strain were collected by filtration, washed with water, and frozen at −80 °C. The frozen mycelium was ground to fine powder, suspended in TE buffer (pH 8.0), and equal volume of lytic buffer  was added. After incubation at room temperature for 5 min, the supernatant was extracted with phenol: chloroform: isoamyl alcohol (25:24:1) solution (pH 7.9). After ethanol precipitation, the isolated DNA was dissolved in TE buffer (pH 8.0) and stored at −20 °C before use.E. coli DH5α  following standard techniques and recombinant plasmids were extracted with GenEluteTM Plasmid Miniprep Kit (Sigma-Aldrich) from E. coli cells.PCR was performed with PrimeSTAR® Max DNA Polymerase (Takara Bio) unless otherwise noted. PCR products and linearized plasmids were purified with QIAEX® II Gel Extraction Kit (QIAGEN). DNA cloning experiments were performed using dpfg, dpmp, dpch, dpas, and dpma clusters were amplified by PCR from genomic DNA of F. graminearum 50218, M. phaseolina NBRC 7317, C. higginsianum MAFF 305635, A. sacchari Kumo-3, and M. anisopliae NBRC 103233 as templates by using gene specific primers listed in Supplementary Table Asp718 or NotI) of the pUARA2, pUADEA2, pUSCA2, or pUPTRA2   method as follows: A. oryzae host strains were cultivated on agar plate  or Casamino acid , 0.01% adenine (for adenine auxotrophic strains)). After 20 hour cultivation at 30 °C , mycelium was collected by filtration and washed with water. The mycelium was incubated with 10 mL protoplasting buffer , 1 mg mL−1 Cellulase Onozuka R-10 (SERVA Electrophoresis GmbH), 0.8 M NaCl, 10 mM phosphate buffer (pH 6.0)). After 3-h incubation at 30 °C, undigested mycelia was removed by filtration with Miracloth (Merck), and the protoplasts were collected by centrifugation at 1300 × g for 5 min. The protoplasts were washed with 0.8 M NaCl, resuspended in solution I ) to obtain 2.0 × 108 cells mL−1. 0.2 Volumes of solution II ) was added with gentle mixing, and then 200 µL of this protoplast suspension was combined with 10–20 µg of plasmid DNA (<20 µL). After 40 min incubation on ice, 1 mL of solution II was added to the aliquot with gentle mixing. After another 15 min of incubation at room temperature, 20 mL of solution I was added to the mixture and the protoplasts were collected by centrifugation at 2000 rpm for 5 min. The protoplasts were resuspended in 600 µL of solution I, and the suspension was combined with molten soft-top agar  supplemented with appropriate nutrients (Supplementary Table Transformation of uffer pH .0). Afteuffer pH .0). Afteuffer pH .0). AfteA. oryzae NSAR1 using expression plasmids containing the aforementioned genes to construct all the transformants expressed the native DDP pathways  buffer for 10 min. The genomic DNA solution was diluted with water to prepare the PCR template at appropriate concentration.We obtained about four to ten transformants per one-transformation experiments. Thus, we checked gene insertion of all the transformant by PCR. Target gene integration into the 18 Packed Column  with acetonitrile and water containing 0.01% trifluoroacetic acid  at a flow rate of 1.0 mL min−1. The LC-MS analysis was performed on COSMOSIL 5C18 Packed Column  with acetonitrile containing 0.1% formic acid and water containing 0.1% formic acid  at a flow rate of 1.0 mL min−1 using positive mode electro spray ionization.All the transformant that pass the gene integration check were cultivated and analysed their metabolite profiles by reverse phase HPLC and LC-MS analyses. Each transformant was inoculated into 60 mL of CPS medium , 0.3% meat peptone , 0.3% soy peptone, 2% soluble starch , 1% maltose monohydrate , 0.01 % adenine (for adenine auxotrophic strains)) or 1/2 CPS medium , 0.15% meat peptone , 0.15% soy peptone, 1% soluble starch , 0.5% maltose monohydrate , 0.01 % adenine (for adenine auxotrophic strains). About 1 week (6–10 days) after cultivation at 30 °C, 150 rpm, the culture media and mycelium were separated by filtration. The culture media (3 mL) was extracted with EtOAc (2 mL), and the organic layer was concentrated in vacuo. The residue was then dissolved in 150 µL of MeOH to prepare sample for reverse phase HPLC and LC-MS analysis. The freeze-dried mycelium (20 mg) was extracted with MeOH (1 mL), and the extract was concentrated in vacuo. The residue was then dissolved in 100 µL of MeOH to prepare sample for HPLC and LC-MS analysis. For the HPLC and LC-MS analysis, 10 µL of the samples were used. The HPLC analysis was performed on COSMOSIL 5CA. oryzae transformant was cultivated in 1/2 CPS medium , 0.15% meat peptone , 0.15% soy peptone, 1% soluble starch , 0.5% maltose monohydrate , 0.01% adenine (for adenine auxotrophic strains) for 1 week at 30 °C, and the culture media and mycelium were separated by filtration. The culture media was extracted with EtOAc two times, and the organic layer were combined and concentrated in vacuo to give crude extract. The freeze-dried mycelium was extracted with EtOAc (20% MeOH), and the extract was concentrated in vacuo. The mycelia extract was then dissolved in MeOH (0.1% water), washed with hexane three times, concentrated in vacuo to give crude extract. Each of the crude extract of culture and mycelium was fractionated by silica gel column chromatography, and subjected to further purification by flash chromatography and preparative TLC (SiO2 or ODS). All the DDP structures were fully determined by spectral analyses , anti-HIV assay  to prepare 5 mM solutions. The Each DDP solution (5 mM) was used for antiproliferative effects on cancer cell lines (Supplementary Notes Further information on research design is available in the Supplementary InformationPeer ReviewReporting SummaryDescription of Additional Supplementary FilesSupplementary Data 1


For years, moderate hypothermia (32 °C) has been proposed as an unorthodox therapy for liver injuries, with proven hepatoprotective potential. Yet, limited mechanistic understanding has largely denied its acceptance over conventional pharmaceuticals for hepatoprotection. Today, facing a high prevalence of acetaminophen-induced liver injury (AILI) which accounts for the highest incidence of acute liver failure, hypothermia was evaluated as a potential therapy to combat AILI. For which, transforming growth factor-α transgenic mouse hepatocytes (TAMH) were subjected to concomitant 5 mM acetaminophen toxicity and moderate hypothermic conditioning for 24 h. Thereafter, its impact on mitophagy, mitochondrial biogenesis, glutathione homeostasis and c-Jun N-terminal kinase (JNK) signaling pathways were investigated. In the presence of AILI, hypothermia displayed simultaneous mitophagy and mitochondrial biogenesis to conserve functional mitochondria. Furthermore, antioxidant response was apparent with higher glutathione recycling and repressed JNK activation. These effects were, however, unremarkable with hypothermia alone without liver injury. This may suggest an adaptive response of hypothermia only to the injured sites, rendering it favorable as a potential targeted therapy. In fact, its cytoprotective effects were displayed in other DILI of similar pathology as acetaminophen i.e., valproate- and diclofenac-induced liver injury and this further corroborates the mechanistic findings of hypothermic actions on AILI. The discovery of acetaminophen (APAP), as an analgesic and antipyretic agent, can be dated back to the 1960s . Yet, itToday, the broad understanding on the toxico-pathological mechanism of AILI involves two key aspects—mitochondrial dysfunction and oxidative stress. These cellular perturbations are initiated by an accumulation of the reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI), following the metabolism of APAP in the liver . While NCurrently, the only approved treatment for AILI is via the administration of N-acetyl cysteine (NAC) . Being sNotwithstanding these intriguing findings, hypothermic conditioning today remains an unorthodox therapy, in contrast to targeted pharmaceutics. On hindsight, the reservations may stem from its unexplored mechanism in liver injury. Unlike ischemic injuries where hypothermia exhibits proven antioxidant and promitochondrial properties ,19,20,21v/v) of insulin-selenium-transferrin supplement  at 37 °C in a humidified atmosphere containing 5% CO2, based on Wu et al. [v/v) fetal bovine serum  at 37 °C in a humidified atmosphere containing 5% CO2. APAP  was prepared in PBS at 40 mM and diluted with fresh media into 5 mM; diclofenac sodium  was prepared in dimethyl sulfoxide at 100 mM and diluted with fresh media into 2.5 mM while sodium valproate  was prepared in ultrapure water at 100 mM and diluted with serum-free media into 10 mM. Anti-AMPKα, anti-phospho-AMPKα (Thr172), anti-p70S6K, anti-phospho-p70S6K (Thr389), anti-ULK1, anti-phospho-ULK1 (Ser757), anti-phospho-ULK1 (Ser317), anti-Beclin-1, anti-phospho-Beclin-1(Ser93), anti-JNK, anti-phospho-JNK (Thr183/Tyr185) and anti-GAPDH antibodies were purchased from Cell Signaling ; anti-LC3B, anti-VDAC1, anti-3-nitrotyrosine and anti-PGC-1α antibodies were purchased from Abcam , while anti-β-actin, anti-TOM20 and anti-TFAM were obtained from Santa Cruz Biotechnology . The horseradish peroxidase (HRP)-conjugated antirabbit and antimouse secondary antibodies were purchased from Cell Signaling . All other reagents were obtained from Sigma-Aldrich  or Thermo Scientific .Transforming growth factor-α transgenic mouse hepatocytes, TAMH,  were cultured in Dulbecco’s modified eagle medium: nutrient mixture F-12  supplemented with 10 mM nicotinamide , 100 nM dexamethasone , 2 µg/mL of gentamicin  and 0.1% , for 2 h. Thereafter, cells were treated with APAP at 32 °C for 24 h, together with a concomitant CQ treatment for 24 h. On the other hand, to demonstrate the concomitant occurrence of mitophagy and mitochondrial biogenesis, TAMH was treated with 250 nM MitoTracker® Red CM-H2XRos  for 30 min at 37 °C prior to APAP and hypothermic treatment for 24 h. Following on, cells were fixed with 4% (w/v) paraformaldehyde  for 15 min and permeabilized with 0.1% (v/v) Triton X-100  for 3 min at room temperature. Next, blocking of non-specific binding sites was performed with 2% (w/v) bovine serum albumin (BSA) in PBS for an hour at room temperature before incubating with primary antibodies diluted in 1:200 with 2% BSA (w/v) in PBS for another hour at room temperature. Cells were then washed thrice with PBS and incubated with Alexa Fluor 488 and/or Alexa Fluor 594  secondary antibodies for an hour at room temperature. This was followed by 3 PBS washes, where cells were incubated with Hoechst 33342 dye  during the second wash. Finally, coverslips were mounted with mounting medium  and viewed with an Olympus Fluoview FV10i confocal microscope .1 × 105 cells/well were seeded on 6-well plates for 24 h prior to APAP treatment at 32 °C for 24 h. Thereafter, cells were incubated with 250 nM tetramethylrhodamine methyl ester (TMRM)  at 37 °C for 30 min. Following on, cells were trypsinized and resuspended in PBS. Mitochondrial membrane potential (ΔΨM) was measured by flow cytometry using cytoFLEX  based on 20,000 events. Cells treated with 10 µM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP)  for 2 h at 37 °C were used as positive control and the relative levels of ΔΨM, in percentage, were determined by normalizing against the negative control i.e., APAP-free treatment with continuous 37 °C incubation. A total of 3 biological replicates were performed.2 × 10g for 10 min. The supernatant was then collected and further centrifuged at 10,000× g for 20 min. Here, the supernatant was collected as the cytosolic fraction while the resultant pellet forms the mitochondrial fraction. Finally, the pellet was resuspended in cell lysis buffer comprising of the abovementioned protease inhibitor cocktail, together with 1% (v/v) octylphenoxypolyethoxyethanol, 0.5% (w/v) sodium deoxycholate and 0.1% (w/v) sodium dodecyl sulfate (SDS) diluted in PBS. All chemicals stated were obtained from Sigma-Aldrich .Cells were seeded on 10 cm dish to reach 90% confluence prior to APAP treatment at 32 °C for 24 h and the isolation of mitochondrial and cytosolic fraction was performed as previously described [5 cells/well were seeded on 6-well plates for 24 h prior to APAP treatment at 32 °C for 24 h. Following hypothermic and APAP treatment, cell samples were lysed in cell lysis buffer stated in the previous section. 20 μg of protein samples were separated via 8–15% (v/v) SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes . Following on, all membranes were blocked with 5% (w/v) BSA in tris-buffered saline containing 0.1% (v/v) Tween-20 (TBS-T) for an hour with the exception of assay with anti-3-nitrotyrosine antibodies, which was blocked with 5% skim milk in TBS-T for 3 h. Thereafter, all membranes were incubated overnight at 4 °C with primary antibodies diluted in 1:1000, except anti-β-actin and anti-GAPDH antibody which were diluted in 1:10,000. Next, all membranes were washed with TBS-T before incubating with HRP-conjugated secondary antibodies, at 1:10,000 dilution, for an hour. The protein bands were then visualized with chemiluminescence image analyzer  using western lightning plus-ECL reagent . β-actin was used as the housekeeping protein for most blots while GAPDH and VDAC1 were used as the housekeeping protein for cytosolic and mitochondrial fractions respectively in samples involving subcellular fractionation.2 × 105 cells/well were seeded on 6-well plates for 24 h prior to APAP treatment at 32 °C for 24 h. Following hypothermia and APAP treatment, total RNA was extracted using RNeasy mini kit  while total DNA was extracted using DNeasy blood and tissue kit . Thereafter, RNA and DNA content were quantified with NanoDrop 1000 UV/Vis spectrophotometer  and cDNA was synthesized from 1 µg of total RNA using qScript cDNA SuperMix  based on manufacturer’s recommendations. The list of primers  used are shown in 2 × 104 cells/well were seeded on 96-well plates for 24 h prior to APAP treatment at 32 °C for 24 h. Thereafter, to determine the relative hepatic GSH content in TAMH, both live and dead cells were harvested and glutathione cell-based detection kit  was used according to manufacturer’s instructions. By using monochlorobimane as the GSH substrate, it reacted with GSH and the resultant fluorescence intensity was measured at 380 nm excitation/480 nm emission wavelength on a microplate reader . Relative changes, by percentage, in the GSH content were determined by normalizing against the negative control i.e., APAP-free treatment with continuous 37 °C incubation. A total of 3 biological replicates was performed.1.5 × 104 cells/well were seeded on 96-well plates for 24 h prior to APAP treatment at 32 °C for 24 h. Next, both the live and dead cells were harvested and the GSH/GSSG ratio detection assay kit  was used according to manufacturer’s instructions. With the use of thiol green fluorophore, the fluorescence intensity representing the total glutathione and reduced GSH were measured at 490 nm excitation/520 nm emission wavelength on a microplate reader. Thereafter, the level of GSSG was calculated and GSH/GSSG ratio was determined. All samples were run in duplicates and 3 biological replicates were performed.To further examine the extent of reduced (GSH) and oxidized glutathione (GSSG) levels, 1.5 × 104 cells/well were seeded on 96-well white plates for 24 h prior to diclofenac or sodium valproate treatment at 32 °C for 24 h. Thereafter, CellTiter-Glo luminescent cell viability assay  was used according to manufacturer’s instructions. Briefly, CellTiter-Glo reagent was added in the same volume as the culture medium in each well after hypothermic and drug treatment. Following on, the plates were placed on an orbital shaker at room temperature for 10 min before measuring luminescence intensity with a microplate reader. The relative percentage changes in luminescence reading were determined by normalizing against the negative control i.e., drug-free treatment with continuous 37 °C incubation. All samples were run in triplicates and 3 biological replicates were performed.1 × 105 cells/well were seeded on 6-well plates for 24 h prior to diclofenac or sodium valproate treatment at 32 °C for 24 h. Following on, both live and dead cells were harvested and stained with 10 μg/mL propidium iodide , diluted in PBS. The percentage of dead cells were determined with flow cytometry based on 20,000 events and a total of 3 biological replicates was performed.2 × 10t-test was performed for all statistical analysis and differences between groups were considered statistically significant for p < 0.05.All data were expressed as mean ± SD of 3 biological replicates. Statistical analysis was conducted using GraphPad Prism for Windows (version 7.00) . Unpaired Student’s To combat AIHI, the removal of damaged mitochondria by autophagy has been frequently reported ,27. SincIn relation to AIHI, the phenomenon of autophagic flux was further explored in its role in removing damaged mitochondria, otherwise known as mitophagy. To do so, one of the mitochondrial proteins within the translocases of outer membrane (TOMs) complex i.e., TOM20 was used as a surrogate marker for mitochondria. With immunofluorescence imaging, the apparent overlay between TOM20 and the microtubules associated with the autophagosome membrane i.e., LC3B, illustrated an internalization of mitochondria within the autophagosome prior to its degradation, following hypothermia E. SimilaBeyond mitophagy, the investigation proceeded to explore mitochondrial biogenesis in TAMH in order to provide a more holistic understanding of its overall health following hypothermia in AIHI. Accordingly, an increased protein expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), the master regulator of mitochondrial biogenesis ,30, was ® Red CM-H2XRos to track the live mitochondria preceding APAP and/or hypothermic treatment in TAMH. Thereafter, TOM20 was used as the surrogate marker of mitochondria to track the total mitochondria levels at the end of APAP toxicity for 24 h. The overlay of these fluorescent signals would, therefore, represent the mitochondria of cells which survived APAP toxicity while the fluorescent signal from TOM20 alone, without overlay, would suggest new mitochondria biosynthesis. As such, the changes in these signals would provide an indication of the dynamic changes in mitochondrial count following APAP and/or hypothermic treatment. Based on the immunofluorescence imaging, there was a reduced intensity of MitoTracker® Red and a reduced overlay between MitoTracker® Red and TOM20 after hypothermic conditioning in AIHI. For the former, it depicted a reduced level of initial mitochondria that survived the APAP and/or hypothermic treatment; for the latter, a higher amount of TOM20 signals alone represented de novo synthesis of mitochondria in the sample after APAP and/or hypothermic treatment. In other words, these clearly illustrate a concomitant manifestation of mitophagy and mitochondrial biogenesis, respectively  of similar pathophysiology involving mitochondrial dysfunction and oxidative stress ,35. IndeThe concept of therapeutic hypothermia for clinical interventions has a longstanding history of more than a century . AmongstHerein, we employed mouse hepatocytes, TAMH, for a detailed in vitro assessment of hypothermic mechanisms in AIHI. This is in continuation from our past work, which demonstrated hepatoprotection with hypothermia in both TAMH and L-02 cell lines . While TSecondly, moderate hypothermia could enhance mitochondrial biogenesis in AIHI, concomitant with mitophagy . While pNext, moderate hypothermia could allay oxidative stress by blocking GSH depletion and impeding JNK signaling pathway . In thisAccompanying GSH restoration, JNK activation was also disrupted with hypothermia in AIHI . With ren ≥ 4, or an in vivo study to cross-validate the observed outcomes. Nonetheless, in the current study, all these may, essentially, serve to rationalize and instill confidence for hypothermia as an unconventional, yet feasible and effective therapy against AILI, and even for other DILIs of similar pathology.Altogether, we have scrutinized the biochemical role of hypothermia in major pathological features encompassing AIHI. This includes the impact of temperature downregulation on mitochondrial dysfunction as well as oxidative stress-mediated pathways . Notably®  has enabled controlled regional cooling of subcutaneous fats to achieve body shaping effects [In summary, we hope to transform the hepatoprotective capacity of hypothermia to a clinically actionable therapy for DILI. Hitherto, the therapeutic potential of hypothermia has been most recognized for its neuroprotection in cardiac arrest and neonatal encephalopathy ,71. For  effects . This co effects .


Exogen phenolic compounds were associated with the modification of the content of endogenous polyphenolic and the induction of the production of the putative auxin-a, adenosine, cellulose and 1,26-hexacosanediol-diferulate. We suggest that in EC of avocado, there is an enhanced phenylpropanoid metabolism for the production of the building blocks of lignin and flavonoid compounds having a role in cell wall reinforcement for tolerating stress response. Data are available at ProteomeXchange with the identifier PXD019705.Somatic embryogenesis (SE) is a valuable model for understanding the mechanism of plant embryogenesis and a tool for the mass production of plants. However, establishing SE in avocado has been complicated due to the very low efficiency of embryo induction and plant regeneration. To understand the molecular foundation of the SE induction and development in avocado, we compared embryogenic (EC) and non-embryogenic (NEC) cultures of two avocado varieties using proteomic and metabolomic approaches. Although Criollo and Hass EC exhibited similarities in the proteome and metabolome profile, in general, we observed a more active phenylpropanoid pathway in EC than NEC. This pathway is associated with the tolerance of stress responses, probably through the reinforcement of the cell wall and flavonoid production. We could corroborate that particular polyphenolics compounds, including  Persea americana Mill.) is a crop of great economic importance. However, avocado production is threatened by different agents, mainly fungal phytopathogens, including Colletotrichum species, Phytophthora cinnamomi, and Euwallacea kuroshio  phenol, caffeic acid, and chlorogenic acid, inhibit SE [p-CA after 28 days, has been associated with the repression of SE induction in Larix leptolepis, Coffea canephora and Daucus carota [p-CA and t-FA may comprise redirection of the metabolic flow from the synthesis of polyphenolics to the production of the building blocks of lignin and flavonoid compounds having a role in cell wall reinforcement and ROS scavenging, respectively. The general reduction in endogenous content of polyphenolics in EC treated with p-CA and t-FA during the first 12 h supports our assumption , 4-hydroxy-benzoic acid (99-96-7), caffeic acid (6034 S), 4-coumaric acid (6031 A), ferulic acid (6077 A), quercetin 3, 4-di-O-glucoside (1347 S), quercetin 3-D-O-galactoside (1027 S) standards were purchased from Extrasynthese (https://www.extrasynthese.com/). All reagents used for proteomic analysis were purchased from Sigma-Aldrich , except as otherwise specified in the corresponding section.Solvents used for the extraction and analysis of phytochemicals  were LC-MS grade . Authentic standards for mangiferin, (+)-catechin, quercetin-3-D-galactoside, quercetin, gallic acid, (-)-epicatechin and quercetin-3-glucoside  were used. Kaempferol-3-−1, thiamine HCl 4 mg L−1, myo-inositol 100 mg L−1 and picloram 0.41 µM; this medium is named MSP [−1  (https://caissonlabs.com/), and autoclaving at 1.5 kg cm−2 at 121 °C for 15 min. Under aseptic conditions, aliquots of media (20 mL) were dispensed onto sterile plastic Petri dishes (100 × 15 mm). The cultures were incubated in darkness at 25 ± 1 °C. The explants were subcultured every two weeks on the same culture medium over seven months.Immature fruits 5–10 mm) of avocado varieties “Hass” and “Criollo” were collected and ECs of both varieties were established from immature zygotic embryos . A small mm of av−1 of protease inhibitor cocktail . The mixtures were homogenized with a tissue homogenizer  and centrifuged at 10,000× g for 45 min at 25 °C. Afterwards, the supernatants were recovered and kept at −80 °C for future proteomic analysis. The protein assay was carried out with the BCA™ Assay Kit  using bovine serum albumin (BSA) as a standard.Proteins were obtained from 0.5 g of frozen NEC and EC collected from seven-month-old cultures and after two weeks of subculturing. The in vitro cultures were first ground with liquid nitrogen in a mortar with a pestle. The powder was suspended in three volumes of phosphate buffer 100 mM (pH 7.0) containing SDS 2%, NaCl 150 mM, and 20 µL gThe general procedure of proteomic analysis, including protein digestion and isobaric labeling, has been published elsewhere . Detailew/w) trypsin protein ratio for 16 h at 37 °C. Afterwards, additional freshly made trypsin was added in 1:60 (w/w) trypsin protein ratio for 4 h at 37 °C. Then, peptides were labeled with TMT6-plex reagents according to the manufacturer’s instructions . The labels 126, 127 and 128 were used for NECs, while labels 129, 130 and 131 were used for ECs. Then, samples were pooled and fractionated using strong cation exchange (SCX) cartridges . Fractions were desalted with C18 cartridges and dried using a CentriVap .We started with 100 µg of protein by reducing it with tris (2-carboxyethyl) phosphine  and alkylating with iodoacetamide . Then, proteins were digested with trypsin  at a 1:30  at a flow rate (FR) of 3 µL min−1, and fractionated on an EASY spray C18 RSLC column  adapted to a nanoLC . A 100 min gradient was used with an FR of 300 nL min−1 and two solvents (solvent A: 0.1% formic acid in water and solvent B: 0.1% formic acid in 90% acetonitrile). The gradient was set as follows: 10 min solvent A, 7–20% solvent B for 25 min, 20% solvent B for 15 min, 20–25% solvent B for 15 min, 25–95% solvent B for 20 min, and 8 min solvent A. Full MS scans in the Orbitrap analyzer  were carried out with: 120,000 of resolution (FWHM), scan range 350–1500 m/z, AGC of 2.0e5, maximum injection time of 50 ms, intensity threshold of 5 × 103, dynamic exclusion 1 at 70 s, and 10 ppm mass tolerance. For MS2 analysis, the 20 most abundant MS1s were isolated with charge states set to 2–7. A precursor selection mass range of 400–1200 m/z was used, with a precursor ion exclusion width range of 18 to 5 m/z, and an isobaric tag loss TMT. MS3 spectra were acquired using synchronous precursor selection (SPS) with ten isolation notches, as previously described [Each reconstituted sample (5 µL) was injected into a nanoviper CN-terminal/lysine residues (+229.163 Da). Dynamic modifications comprised methionine oxidation (+15.995 Da) and deamidation in asparagine/glutamine (+0.984 Da). We used for the TMT6-plex quantification method ± 10 ppm mass tolerance, highest confidence centroid, and a precursor co-isolation filter of 45%. Protein identification was carried out with tolerances of ±10 ppm and ±0.6 Da. Peptide hits were filtered for a maximum of 1% FDR using the Percolator algorithm. Functional annotation of proteins was carried out by Blast2Go software (https://www.blast2go.com/), and gene ontology (GO) enrichment was carried out by David bioinformatic source (https://david.ncifcrf.gov/) using Arabidopsis homologs. We used the REVIGO web server (http://revigo.irb.hr/) for GO clustering and visual representation of biological processes . The subsequent search was carried out with SEQUEST HT, MASCOT , and AMANDA against the avocado proteins databases. Parameters in the search included full-tryptic protease specificity, two missed cleavage allowed. Static modifications covered carbamidomethylation of cysteine (+57.021 Da) and TMT 6-plex rocesses .g for 15 min at 4 °C. The supernatant was split off for further analysis. Three biological replicates were analyzed for NEC and EC.Fifty milligrams of EC and NEC samples were lyophilized in a freeze dryer  and suspended in 1 mL of methanol containing 0.1% formic acid and then homogenized with a tissue homogenizer  in a 1.5 mL centrifuge tube. Afterwards, the samples were placed in an ultrasonic bath  for 45 min at 4 °C. Then, the mixtures were centrifuged at 3000× Phenolics were identified and quantified using a UPLC system  coupled to the QqQ mass spectrometer  with a dynamic multiple reaction monitoring (dMRM) method for the searching for up to 60 compounds, as previously described by our research group . In addihttp://www.leicamicrosystems.com).Fresh tissue samples of CE and NEC of Hass and Criollo varieties were observed in a light stereoscope Leica S6D (Schweiz) with a 63× objective. The images were processed using LAS V4.12 software  and observations were carried out in an FEI-Quanta250 FEG microscope (Czech Republic), operated at 5 Kv acceleration voltage.The samples from 7-month-old EC and NECs were fixed in glutaraldehyde 2.5% (pH 7.2) for 12 h. After fixation, the cultures were rinsed thrice in phosphate buffer (pH 7.2) and dehydrated in an ethanol series for 60 min each . After ethanol dehydration, the samples were dried in a critical point drier  using liquid COhttp://www.leicamicrosystems.com) and mounted on copper grids and studied with a JEM 1400 Plus Transmission Electron Microscope .The samples were fixed overnight with 0.1 M ‘Sorensens’s buffer (pH 7.2) containing paraformaldehyde 2%, glutaraldehyde 1% and sucrose 0.8%. Afterwards, the samples were rinsed with the same buffer and post-fixed with OsO4 1% for 2 h. Thereafter, samples were dehydrated with different concentrations of ethanol for 10 min each . Then the samples were embedded in ‘Spurr’s resin  and polymerized at 60 °C for 24 h. Afterward, thin cross-sections of 70 nm were obtained with an Ultracut ultramicrotome   were fixed in 4% paraformaldehyde in phosphate-buffered saline, 0.2 M . Samples were stained with 10 µL of calcofluor white  for 10 min and rinsed in PBS for 5 min. Images were acquired with a Leica TCS-SP8+STED microscope  using a plan-apochromat 63x  objective. Calcofluor-stained samples  were recorded in the grey channel . Deconvolution images were processed with SVI Hygens professional software v.18.04.1 .p-hydroxybenzoic acid, p-coumaric acid (1 µM) or trans-ferulic acid for 28 d. At the same time, Hass EC was cultured for the same time in MSP medium containing either pCA 10 µM or tFA 100 µM. We sampled EC for 0, 6, 12, 24, 336 (14 d) and 672 h (28 d). The number of somatic globular embryos was visually recorded at 14 and 28 d of each treatment. The experiment consisted of five repetitions per treatment.Criollo EC was cultured in Petri dishes (100 × 15 mm) containing 25 mL of MSP medium supplements with PAL1, AT2G37040), chalcone-flavanone isomerases  and flavanone 3-hydroxylase  were chosen. The primers were designed using Primer Express Software . Primer or probe sequences were selected to be complementary to an exon-exon junction, to ensure amplification from the cDNA template and not from genomic DNA. The transcript abundance of the genes in this study was analyzed by quantitative real-time PCR (qRT-PCR) with the StepOne™ Real-Time PCR System  using TaqMan assays. The gene expression values were normalized to Rubisco (accession No. AY337727) expression. The RT-PCR reaction was performed under the following conditions: 50 °C for 2 min, 10 min at 95 °C, followed by 40 cycles at 95 °C each 10 s, 60 °C for 1 min for denaturation, primer alignment, and amplification, respectively. Finally, qRT-PCR data were analyzed by the 2− ΔΔCt method [p < 0.05), followed by Tukey’s honestly significant test calculated at 5% levels of probability using the Statistical Software R®.Total RNA was extracted using “Mini Kit Plant RNeasy” following the manufacturer’s instructions . Sequence-specific primers were designed for each gene. Arabidopsis genes homologs in avocado, including phenylalanine ammonia-lyase 1 .Protein abundances were quantile, and log2 normalized. A linear model for microarrays data  approachOur proteomic-metabolomic approach provides insight into the improvement of SE in avocado. Our findings highlight the critical features of EC, including an active PP metabolism and cell wall fortification that might be linked with molecular features associated with embryogenic competence. Both ECs and NEC experience stress due to culture conditions, proteins associated with oxidation-reduction processes, and other stress response mechanisms were, however, present in a higher proportion in EC than NEC. This suggests that the ability to respond to stress is a hallmark of SE establishment in avocado. Our obtained evidence indicates that specific polyphenolics can induce the proliferation of embryos, which could be useful for the obtention of starting biological material during the establishment of an efficient pipeline for the improvement of embryo maturation/germination in the particular case of avocado SE.


The 16S rRNA genes of “Candidatus Brocadia”, “Candidatus Anammoxoglobus”, and five new deduced anammox bacterial phylotypes were detected in the shallow groundwater samples. Diverse anammox bacteria were broadly distributed in the shallow groundwater aquifer of the Kathmandu Valley.The abundance and diversity of anaerobic ammonium oxidation (anammox) bacteria were assessed in 152 groundwater samples in the Kathmandu Valley, Nepal. Anammox bacterial 16S rRNA genes were detected in 54% (37/68) of samples collected in the dry season at 1.6×10 In the wet season (August and September), shallow groundwater samples (n=84) were collected in the gravel subsurface (n=29) and clay subsurface (n=55) (Escherichia coli strain DH5α using a pMD19 T-vector system (Takara Bio). A PCR-Restriction Fragment Length Polymorphism (RFLP) analysis and clone library analysis were performed as previously reported . The DDBJ/EMBL/GenBank accession numbers of the 16S rRNA gene sequences of clones are LC553643 to LC553674.We selected the Kathmandu Valley (area 664 kme (n=55)  and S2. e (n=55) , a clay e (n=55) , a grave5 and 7.6×106 copies L–1 in groundwater in the gravel subsurface and between 1.6×105 and 8.8×106 copies L–1 in those in the clay subsurface. In the wet season, the 16S rRNA genes of anammox bacteria were detected in 69% (20/29) of shallow groundwater samples in the gravel subsurface and in 55% (30/55) of those in the clay subsurface, with 1.2×105 to 1.2×107 copies L–1 and between 4.3×104 and 3.1×106 copies L–1 in groundwater samples in gravel and clay subsurfaces, respectively (–1) was as high as that in other freshwater samples , while the Anammoxoglobus group consisted of “Candidatus Anammoxoglobus” and RFLP18 (five clones). Cluster 1 comprised 16 RFLP groups including 66 clones. Clusters 2 and 3 each consisted of a single RFLP group, RFLP23 (46 clones) and RFLP24 (1 clone), respectively. Cluster 4 comprised three RFLP groups (13 clones), Cluster 5 consisted of six RFLP groups (seven clones), and Cluster 6 had two RFLP groups (two clones). “Ca. Brocadia” is recognized as a major anammox group in non-saline environments, such as freshwater sediments, freshwater, soils, and groundwater, while “Ca. Anammoxoglobus” has also been frequently detected in non-saline environments, including soil, freshwater, estuaries, and groundwater  in groundwater in more detail.Based on the results of the PCR-RFLP and clone library analyses for the anammox bacterial 16S rRNA gene, 143 clones were divided into 32 RFLP groups . A phylo seasons . These rIn conclusion, various anammox bacteria were widely distributed throughout the shallow groundwater basin in the Kathmandu Valley during the wet and dry seasons. Therefore, anammox bacteria may contribute to N removal in this groundwater basin.Microbes Environ 36: ME20143.Nakano, M., Kamei, T., Man Shakya, B., Nakamura, T., Tanaka, Y., Haramoto, E., et al. (2021) Distribution and Community Composition of Anammox Bacteria in Shallow Groundwater of the Kathmandu Valley, Nepal. https://doi.org/10.1264/jsme2.ME20143Supplementary Material