Broussonetia papyrifera L. (B. papyrifera) is a good feeding material with high crude protein, crude fat, and low crude fiber, which is widely distributed in China. In this study, 12 Dorper ♂×Hu ♀  crossbred weaned male lambs were seleted into four groups based on the feed that ratio of the B. papyrifera fermented feed in the total mixed diet , to character the lambs’ longissimus dorsi (LD) fatty acids, morphology and transcriptome. Results showed that the muscle fiber’s diameter and area were the smallest in the 100% group. The highest content of beneficial fatty acids and the lowest content of harmful fatty acids in group 18%. RNA-seq identified 443 differentially expressed genes (DEGs) in the LD of lambs from 4 groups. Among these genes, 169 (38.1%) were up-regulated and 274 (61.9%) were down-regulated. The DEGs were mostly enriched in in fatty acid metabolism, arginine and proline metabolism, and PPAR signaling pathways. Our results provide knowledge to understand effect of different ratios of B. papyrifera fermented feed on sheep meat quality traits, also a basis for understanding of the molecular regulation mechanism of B. papyrifera fermented feed affecting on sheep meat quality.To date, utilization of feed grains is increasing, which competes for human food. It is imperative to develop and utilize unconventional feed materials.  Broussonetia papyrifera L. (B. papyrifera), is a broad-leaved woody specie with a deciduous, dioecious, and dichogamous plant . The plant B. papyrifera has strong stress resistance, can grow normally at arid hillside, valley, and roadside. Studies have been indicated thet it has multiple function, such as the role of manufacturing paper with its inner bark , its leaves are ideal for feeding animals  and it can also be used in the pharmaceutical industry . It is found that B. papyrifera silage also reduce the ruminal biohydrogenation  and increase the poly-unsaturated fatty acids (PUFA) concentration in the milk .industry . DomestiB. papyrifera in China, widely distributes in basins of Yellow river, Yangtze river, Pearl river and Mingjiang river. B. papyrifera is a kind of nutrient-rich woody feed. It contains high crude protein, crude ash, crude fat, phosphorus, and suitable crude fiber content. It has the potential to alleviate the lack of protein feed in China and its dependence on foreign sources. It was reported that the crude protein of B. papyrifera had high degradation rate in the rumen of cows. Therefore, this kind of unconventional woody forage was considered to be of great development value . Also the B. papyrifera fruits polysaccharides have antioxidant and antibacterial activities . There is report about the radix of B. papyrifera that had the greatest antinociceptive and anti-inflammatory effects when different parts of the plant were compared as treatment for chemical-induced pain and inflammation in rodents .There are about 300,000 hectares of tivities . Many oftivities . AntioxiLin et al., 2014). Due to its high protein content, rich nutrient content, and low fat and cholesterol content, mutton has a growing demand for mutton. The main indicators for comprehensively evaluating individual animal meat production performance and meat nutritional value include meat production, meat quality, muscle nutrition and fatty acid content. The important indicators for evaluating meat quality are tenderness, marble pattern, meat color, etc.  staining was measured to observe the longissimus dorsi (LD) phenotype, to study the feeding effect of B. papyrifera fermented feed; then, the transcriptome data was alanyzed, to screen differential genes that relate to B. papyrifera fermented feed. Conjoint analysis explored the influence of B. papyrifera fermented feed on the quality of lamb meat, and screened out the appropriate addition ratio.With the improvement of people’s living standards, consumers’ demand for livestock and poultry products has undergone great changes. People not only demand good taste and flavor, but also demand rich nutrition and benefit human health  fed that ratio of the B. papyrifera fermented feed in the total mixed diet  for 60 days. HE staining and analysis of fatty acid content of LD muscle, a suitable ratio of fermented feed for B. papyrifera was initially selected. Then conduct transcriptomics analysis to screen the key genes that affect meat quality and predict the biological functions of these genes in fatty acid synthesis and metabolism. Finally, combining the phenotypic data and the transcriptome results, explore the effect of B. papyrifera fermented feed on the quality of lamb meat, and select the appropriate addition ratio , TRIpure Reagent , CHCl3 (Beyotime), NaCl , 14%BF3-CH3OH , Eosin Staining Solution (Beyotime), Hematoxylin Staining Solution (Beyotime), Evo M-MLV RT Kit with gDNA Clean for qPCR (Accurate Biotechnology (Hunan) Co., Ltd), YBR Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology (Hunan) Co., Ltd).NaOH , CHExperimental animals were reviewed and approved by the Animal Committee of Gansu Agricultural University (GSAU-2019-76).B. papyrifera fermented feed in the total mixed diet . There is transition period of 7 days, a pre-test period of 7 days and a formal period of 60 days. Refer to NRC (2007) standard for 20 kg lamb with a daily weight gain of 200g/d. The diet formula was shown in 12 crossbred weaned male lambs Dorper ♂ × Hu ♀ were selected, which with same weight (20 kg/lamb), similar ages and good health. All fed in Gansu Zhongtian Sheep Industry Co., Ltd. . Randomly divided into four groups (three lambs per group) based on the feed that ratio of the 3/CH3OH  as described by Liu , 2015) with some modification. Extracted lipids (approximately 10 mg) were combined with BHT and 2.0 mL NaOH-MeOH solution (0.1 mol/L), saponify at 60 °C for 60 min. After the solution was clear and transparent, test the tube under 4 °C running water cool to room temperature; then add 2.0 mL of 14% BF3-CH3OH solution, methylate at 60 °C for 15 min, and cool the test tube to RT under 4 °C of tap water; add three mL of n-hexane, one mL of 25% NaCl solution and six mL of distilled water was shaken vigorously, centrifuged at 6,000 r/min for 10 min, the upper layer of FAME was collected, the lower layer solution was repeatedly extracted once with n-hexane, combined FAME, added an appropriate amount of anhydrous sodium sulfate, and all were sucked out with a disposable needle, and the organic phase was filtered. The membrane is placed at the front of the needle tube for filtration, concentrated to dryness under the protection of nitrogen, the residue is dissolved in n-hexane and fixed to a 2.0 mL volumetric flask, stored at −20 °C for future use, until GC detection.Frozen samples were thawed 12 h prior to analyse at 4 °C, and weighed 5g LD muscle on an electronic balance. In order to accurately and quickly extract the lipids in the LD, total lipids were extracted with CHCl−1, The flow rate of gas (Hydrogen) was 40 mL min−1, The flow rate of air was 400mL min−1. Split injection, split ratio 60:1, injection volume 1.0 μL. Injector temperature was set as 260 °C. The initial oven temperature was programmed at 140 °C and maintained for 5 min, then increased to 200 °C at 2 °C min−1, increased to 230 °C at 6  °C/min and keep 20 min.Derivatized methyl esters of fatty acids were separated and quantified by gas chromatograph . The capillary column was a 100 m ×0.25 mm ×0.2 µm . The carrier gas (Nitrogen) flow rate was 1.5 mL minAsuka et al., 2017). Then was observed under a microscope, and photographed using ImageView.Section of LD of was stained with H&E as described previously  and Agilent 2100 (Agilent). The total RNA of the aforementioned samples was used to construct an RNA-Seq library, which were sequenced in parallel on Illumina HiSeq 2000 system by Biomarker Technologies .Mortazavi et al., 2008). Differential expression analyses of genes among four groups were implemented using the Cuffdiff  . GAPDH was used as a reference gene in quantitative analysis. Using Evo M-MLV RT Kit with gDNA Clean for qPCR to reverse transcribe RNA into cDNA, and using SYBR Green Premix Pro Taq HS qPCR Kit and run on the Roche LightCycler96. The relative expression was calculated using the 2–ΔΔCt method  was performed for nine randomly selected DEGs , the least significant difference (LSD with Fischer’s) method in one-way analysis of variance (ANOVA). The results were expressed as mean ±standard error. B. papyrifera fermented feed, including 4 odd saturated fatty acids and 7 even saturated fatty acids. Among the even saturated fatty acids, butyric acid (C4:0), capric acid (C10:0) palmitic acid (C6:0) and stearic acid (C18:0)were not significantly different among the four groups (P > 0.05). The content of lauric acid(C12:0), myristic acid (C14:0) and arachidic acid (C20:0) in the 100% group was significantly higher than the other three groups(P < 0.05). Among the odd saturated fatty acids, tridecanoic acid (C13:0) and tricosanoic acid (C23:0) were not significantly different among the four groups (P > 0.05). The content of pentadecanoic acid (C15:0) and heptadecanoic acid (C17:0) group 100% was significantly higher than other groups , palmitoleic acid (C16:1), nervonic acid (C24: 1) and trans linoleic acid (C18:2n6t) (P > 0.05). Among the unsaturated fatty acids, the content of Cis-10-pentadecenoic acid (C15:1) and cis-10-heptadecenoic acid (C17:1) and were gradually increased with the addition of B. papyrifera fermented feed. Among the polyunsaturated fatty acids, the content of four fatty acids also increased with the addition of mulberry fermented feed, including linoleic acid (C18:2n6c), γ-linolenic acid (C18:3n6), alpha-linolenic acid (C18:3n3), cis-8, 11,14-eicosatrienoic acid (C20:3n6) and DHA (C22:6n3)  .P > 0.05), but its content increased with the amount of addition, the content of n3 in the group 100% was significantly higher than other groups (P < 0.05). Both n6/n3 and P/S can be used to measure the nutritional value of meat. In this study, P/S was the highest in the 100% group, while n6/n3 was the highest in the group 18% , while the difference between 6% and 18 was not significant (P > 0.05), but 18% was less than 6%, indicating that with the increased in the amount of B. papyrifera fermented feed the muscle fiber’s area and diameter was decreased.HE staining of the LD of lamb in different groups, the cross-section staining results showed that the muscle fibers were polygonal, all arranged tightly, the muscle bundle gap was large, and the intramuscular membrane was obvious –1D. The B. papyrifera fermented feed on the meat quality of fattening lamb, transcription level changes in the LD were analyzed using the Illumina HiSeq 2000 platform, and prepared to construct cDNA libraries for RNA-seq. As shown in In view of the influence of different proportions of B. papyrifera in the three groups  to 98 (100%), and the down-regulated DEGs increased from 52 (6%) to 171 (100%) , 104 (38 up-regulated and 66 down-regulated) and 198 (84 up-regulated and 141 down-regulated) DEGs belonged to 6%,18% and 100%, respectively. The 6 DEGs (2 up-regulated and 4 down-regulated) were commonly regulated by effecting of fermented feed from e groups . A total1 (100%) . Hierarc1 (100%) . The resB. fermenta. While the genes in clusters 1 and 5 were up-regulated from 0% to 18%, and gradually down-regulated within 100%. This result indicates that those genes were transiently promoted by B. fermenta. Most of them participated in pathways such as “glucagon signaling pathway”, “PPAR signaling pathway” and “fat digestion and absorption”. The genes belonging to clusters 2, 7 and 8 were continuously down-regulated. Transcription is inhibited by B. fermented feed, most of which are rich in “MAPK signaling pathway” and “cAMP signaling pathway” pathways. Cluster 3 contains fewer genes (31), and the expression levels of these genes are significantly up-regulated with the addition of fermented feed from B. papyrifera, most of which are rich in the “AMPK signaling pathway” and “Circadian rhythm” pathways.According to the expression profiles, 443 DEGs were classified into 8 clusters by co-expression clustering . A totalAnnotated genes were divided into three major functional categories: biological processes (BP), cellular components (CC) and molecular functions(MF). It is obvious that there were more functional terms for BP and relatively few transcripts for CC and MF. Compared with down-regulated transcripts, detoxification (GO: 0098754), synapse (0045202), synapse part (0044456) antioxidant activity (GO:0016209) terms were peculiar in up-regulated DEGs; nucleoid (0009295) was unique in upregulation GO terms .B. fermenta. fermented feed. Top20 of KEGG enrichment showed that up-regulated DEGs were highlighted in “fatty acid degradation (ko00071)”, “circadian rhythm(ko04710)”, “tryptophan metabolism (ko00380)”, “arginine and proline metabolism (ko00330)”, “regulation of lipolysis in adipocytes (ko04923)”, “glycerolipid metabolism (ko00561)” and “cGMP-PKG signaling pathway (ko04022)”. Moreover, “pertussis (ko05133)”, “proteoglycans in cancer (ko05205)”, “complement and coagulation cascades (ko04610)”, “ECM-receptor interaction (ko04512)”, “pathways in cancer (ko05200)”and “Rap1 signaling pathway (ko04015)” annotated the most down-regulated DEGs , ACADVL (degree = 9), ECI1 (degree = 9), ECHS1 (degree = 9) were the top four hub genes.The PPI network of DEGs related to meat quality in the LD muscle of lamb were presented in Bidanel et al., 1991).In this study, the diameter and area of muscle fibers decreased with the increase of the ratio of B. papyrifera fermented feed . PUFA have been proven to prevent and treat cardiovascular diseases, and also have important physiological effects such as delaying aging and anti-cancer. MUFA plays an important role in lowering cholesterol . In the present study, PUFA were higher in other three groups than that in control group, and it might due to the antimicrobial activities in the diet supplemented with B. papyrifera fermented feed , which affects the biohydrogenation of unsaturated fatty acids by ruminant rumen microorganisms, and also dietary antioxidant activity might increase PUFA content in meat (α-linolenic acid(C18:3n3) and harmful fatty acids such as lauric acid(C12:0), myristic acid(C14:0) and stearic acid(C18:0) in the 4 groups, it showed that the 18% group has a higher content of beneficial fatty acids and a lower count of harmful fatty acid, which would be more beneficial for the consumer’s health.Lauric acid (12:0), myristic acid (14:0) and palmitic acid (16:0) in long-chain SFA are closely related to cholesterol and can harden human blood vessels, which was the reason to cause cardiovascular disease and arteriosclerosis, there are some epidemiology showing that stearic acid (C18:0) is related to coronary heart disease . In thislesterol . The n3 Refer to , as it i in meat  . CombinB. papyrifera fermented feed mainly improves the intramuscular fat content of fattening lamb through nutritional regulation, thereby affecting meat quality performance. By analyzing the KEGG pathway of differentially expressed genes, it can be seen that 4 DEGs  are enriched in the PPAR signaling pathway . AQP7, a water/glycerol transporting protein, regulates adipocyte glycerol efflux and influences lipid and glucose homeostasis . PLIN5 is expressed on both lipid droplets and mitochondria, and may participate in the interaction between lipid droplets and mitochondria . Muscle-specific overexpression of PLIN5 increases the storage of lipid droplets in muscle cells and also increases the rate of oxidative gene expression and metabolism  that were significantly enriched in the fatty acid metabolism signaling pathway . ACADVL mainly catalyzes the oxidation of fatty acids to form C 2-C 3 double bonds, and usually catalyzes C 16-acetyl COA or even longer chain fatty acids . The long-chain fatty acyl COA is transported to the mitochondria, firstly binds to VLCAD, and interacts with the mitochondrial trifunctional protein. After 2-3 cycles of oxidation, the medium-chain fatty acyl CoA is formed (ECI1 was an important mitochondrial enzyme involved in the β oxidation of unsaturated fatty acids, which was mainly involved in the metabolic process of fatty acids (ECI1 increased with the addition of B. papyrifera fermented feed, reaching the highest in the 18% group, but decreased in the 100% group. The expression level of ACAVDL and ECHS1 rose to the highest after 6% B. papyrifera fermented feed, and then decreased with the increase of the added amount (Refer to B. papyrifera fermented feed would increase the synthesis of long-chain fatty acids, while excessive addition of B. papyrifera fermented feed would increase fatty acid metabolism and reduce fatty acid content in the body, thereby affecting the fat deposition on meat quality in the LD of lamb.By analyzing the KEGG pathway of differentially expressed genes, it could be seen that there were four genes (Refer to . ECHS1 is formed . The proty acids . In thisRefer to . It showB. papyrifera in animal breeding, they have focused on its antioxidant activity, rumen hydrogenation and its effect on fatty acids. There is no report on the specific influence mechanism of B. papyrifera fermented feed on the meat quality traits of animal. To our best knowledge, this is the first report about the effect of adding different B. papyrifera fermented feed on the meat quality traits and changes of the longissimus dorsal transcriptome of fattening lamb. First, the fatty acid content and muscle fiber morphology of the longissimus dorsal were studied. It was found that after adding B. papyrifera fermented feed, the muscle fiber’s diameter of the muscle became smaller, the tenderness became more tender, and the content of polyunsaturated fatty acid was the highest in the group18%. Finally, a total of 443 DEGs were identified in the four groups. We screened some DEGs that related to synthesis of fatty acids, most of which were up-regulated in the 6% and 18% groups, and down-regulated in the 100% group. Our results show that proper addition of Pichia fermented feed can help the synthesis of long-chain unsaturated fatty acids and the deposition of intramuscular fat. Therefore, it is recommended to add 18% groups, B. papyrifera fermented feed to lamb feed to improve meat quality traits.Although there are many researches on 10.7717/peerj.11295/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.11295/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.11295/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.11295/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.11295/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.11295/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.11295/supp-7Supplemental Information 7Click here for additional data file.10.7717/peerj.11295/supp-8Supplemental Information 8Click here for additional data file.


Mycobacterium smegmatis in apoprotein and cofactor-bound forms. Based on structural analysis and mutagenesis, we identified several catalytically critical, positively charged residues at a putative RNA-binding site. We found that mutation of any of these sites is sufficient to abolish methylation activity, whereas the corresponding RNA-binding affinity of Erm38 remains unchanged. The methylation reaction thus appears to require a precise ensemble of amino acids to accurately position the RNA substrate, such that the target nucleotide can be methylated. In addition, we computationally constructed a model of Erm38 in complex with a 32-mer RNA substrate. This model shows the RNA substrate stably bound to Erm38 by a patch of positively charged residues. Furthermore, a π-π stacking interaction between a key aromatic residue of Erm38 and a target adenine of the RNA substrate forms a critical interaction needed for methylation. Taken together, these data provide valuable insights into Erm–RNA interactions, which will aid subsequent structure-based drug design efforts.Erythromycin resistance methyltransferases (Erms) confer resistance to macrolide, lincosamide, and streptogramin antibiotics in Gram-positive bacteria and mycobacteria. Although structural information for ErmAM, ErmC, and ErmE exists from Gram-positive bacteria, little is known about the Erms in mycobacteria, as there are limited biochemical data and no structures available. Here, we present crystal structures of Erm38 from  One major mechanism of AMR in Gram-positive and mycobacterial pathogens affects three broad classes of front-line antibiotics — macrolides, lincosamides, and streptogramins (MLS) — and is conferred by a family of horizontally and vertically transmitted genes that encode the so-called erythromycin resistance methyltransferases (Erms)  in virtually every clinically important bacterial pathogen represents a true crisis with major societal and economic impact  (https:/s (Erms) , 4, 5, 6s (Erms) . Horizonbscessus , 9, 10. B, and C , which germ genes, such as erm37 in M. tuberculosis, erm38 in Mycobacterium smegmatis, and erm41 in M. abscessus, as well as ermE of Saccharopolyspora erythraea, are genomically encoded, whereas the erm genes of Gram-positive bacteria, including ermA and ermC of methicillin-resistant S. aureus and ermB of vancomycin-resistant enterococci, are found on highly mobile plasmids involved in horizontal gene transfer. In terms of size, Erms A, B, and C share similar molecular weights (∼30 kDa), whereas Erm37 and Erm41 are smaller (∼20 kDa) because of the lack of a C-terminal domain  (1QAO) (6NVM)  , ErmC (P) (1QAO) , 16, and) (6NVM) . However) (6NVM) , and Ermfections . Here, wEscherichia coli resulted in a heterogenous population of partially degraded proteins with molecular weights ranging from 30 to 45 kDa, in spite of length variations , with analysis using the Database of Disordered Protein Predictions (riations . The comriations . The higriations  that difA). Generally, Erm38 shares highly a similar structural fold with all published Erm structures and KsgA, a 16S rRNA methyltransferase (B) reveals that both structures are highly similar with RMSD of 2.3 Å for 237 superimposed alpha carbon atoms, despite sharing only 22% sequence identity. Compared to ErmC, the N-terminal region of Erm38 has a longer α1 helix and longer loops connecting α3–β4 and α5–α6. In the C-terminal helical domain, Erm38 possesses a longer α8 helix but shorter α9 and α10 helices, along with an extra 310 helix-labeled η5.Erm38 features at its N-terminal catalytic domain the classic Rossmann fold, an α/β sandwich that contains the SAM cofactor-binding site and a helical C-terminal domain A. Generansferase . A compansferase B revealsB). This was expected given the high chemical homology between SAM and sinefungin. The SAM-binding pocket of Erm38 was also compared with ErmC (A and C). Despite sharing only 22% sequence identity, residues at the SAM-binding pocket are highly conserved between Erm38 and ErmC proteins with minor differences found at V62/I60, A41/S39, and F93/I85, whereas residues F15, G40, G42, E61, D92, N108, and P110 are identical for the two enzymes. Particularly, G40, G42, E61, D92, and P110 of Erm38 are strictly conserved among Erms A, B, C, E, 37, 38, and 41  to cause full MLS . As these solvent-exposed basic residues are likely to be involved in binding the RNA substrate, we produced an alanine mutant for each site and compared the enzymatic activities of the mutant and WT enzymes. R31A was selected as a negative control based on the fact that it is distant from the SAM- and RNA-binding sites, and we concluded that it does not participate in any intramolecular interaction needed to maintain the fold of the protein. E61K is known to reduce methylation activity by disrupting the SAM-binding site . Surprisingly, the RNA-binding profiles for WT and mutants were similar, with an interaction model was best represented by 2:1 heterogeneous binding and two dissociation constants (KD)  . The micE. coli 23S rRNA) lying in the vicinity of F111 of Erm38, an aromatic residue known to interact with the adenine to facilitate methylation  simulation to further refine the binding interface. 3FTF). The Erm38–RNA complex is stable in the 10-ns simulation, whereas this alternate model lacks the π-π stacking interaction and it does not interact with R140 of Erm38, which was shown to be important according to our mutagenesis data  that resulted in complete loss of erythromycin resistance, whereas mutations R112A and K133A in ErmC  reduced erythromycin resistance. These three amino acid positions are highly conserved across all seven Erms compared in this study . This rules out X-ray crystallography, cryo-EM, and solution-state NMR techniques for structure determination. Solid-state NMR offers a solution to analyze the precipitate, whereas the 40 kDa size of the Erm38–RNA complex poses a problem for NMR resolution.et al.  was produced by the NTU Protein Production Platform (Singapore). The truncated Erm38 is referred to throughout as Erm38. Site-directed mutagenesis was performed on Erm38 to introduce the single mutations R31A, R119A, R140A, R141A, R142A, and K166A, using primers listed in E. coli BL21(DE3)-T1R Rosetta strain, Erm38 proteins were expressed by growing in LB broth containing 34 μg/ml chloramphenicol and 50 μg/ml kanamycin at 37 °C, with expression induced with 0.2 mM IPTG for 18 h at 16 °C. Cell pellets were lysed by sonication in 50 mM Na Hepes pH7.5, 500 mM NaCl, 10% glycerol, 0.5 mM DTT, and 10 mM Imidazole. After removal of cell debris by centrifugation at 16,000g, the supernatant was sterile filtered and subjected to a series of purification steps. For WT Erm38, high-purity protein was critical for crystallization studies, which necessitated a three-step purification involving immobilized metal affinity chromatography (IMAC), cation-exchange chromatography, and size-exclusion chromatography  on Intelli-plates 96-3 (Art Robbins Instruments) , 27, 28.3FTF and 3FUV as search models. Several rounds of manual model building were then performed using program COOT  were determined by plotting the SAH concentration as a function of RNA concentration tested and the data were fitted by nonlinear regression with GraphPad Prism version 9 .All assays were performed in 10 μl total volume in a 384-well plate . Erm reaction kinetics were quantified using the MTase-Glo methyltransferase assay kit (Promega), which detects the SAH product of SAM demethylation in a coupled luminescence reaction , 40 that1 mM DTT , 42. To d values for each Erm38 protein, a reference sensor with loaded RNA but no protein was subtracted from the data before fitting. Curve fitting using a 2:1 interaction model was used to measure the dissociation constant (KD) for WT and mutants of Erm38.The binding affinities of WT, R119A, and R130A Erm38 proteins with the 32-mer RNA substrate were measured by biolayer interferometry using an Octet RED96e (ForteBio), with binding data acquired (kinetics mode) and analyzed using built in software. Biosensors were hydrated in phosphate-buffered saline with Tween (PBST) buffer  for 20 min at ambient temperature preceding data acquisition, and experiments were performed at 25 °C. Erm38 proteins were diluted in PBST buffer. A biotinylated 32-mer RNA substrate was synthesized (Sigma-Aldrich) with biotin appended at the 3′-end of the oligo. The biotinylated RNA was diluted to 100 nM and immobilized on a streptavidin-coated biosensor with a signal threshold of 0.2 nm. A baseline level was established for 60 s before Erm38 proteins were exposed to RNA-loaded biosensors for 120 s and dissociated in PBST buffer for 120 s. To determine KE. coli numbering) pointing outwards, which makes this RNA model primed to interact with the methylation site of Erm. Docking of Erm38 and 32-mer RNA was performed using HDOCK 2, where k is 1 kcal mol−1 Å−2 and xref is the initial atom coordinates. Finally, 20 ns production simulations were performed without constraints. The simulation was performed under the NPT ensemble assuming the CHARMM36 force field , 7F8B (Erm38 complexed with SAM), and 7F8C (Erm38 complexed with SFG).This article contains The authors declare that they have no conflicts of interest with the contents of this article.


RNA sequencing reveals primary myocyte-specific gene-regulatory niche effects for β-hydroxybutyrate but do not support a general signaling of β-hydroxybutyrate role in other primary cells or during cellular differentiation in vitro. Various forms of fasting and ketogenic diet have shown promise in (pre-)clinical studies to normalize body weight, improve metabolic health, and protect against disease. Recent studies suggest that β-hydroxybutyrate (βOHB), a fasting-characteristic ketone body, potentially acts as a signaling molecule mediating its beneficial effects via histone deacetylase inhibition. Here, we have investigated whether βOHB, in comparison to the well-established histone deacetylase inhibitor butyrate, influences cellular differentiation and gene expression. In various cell lines and primary cell types, millimolar concentrations of βOHB did not alter differentiation in vitro, as determined by gene expression and histological assessment, whereas equimolar concentrations of butyrate consistently impaired differentiation. RNA sequencing revealed that unlike butyrate, βOHB minimally impacted gene expression in primary adipocytes, macrophages, and hepatocytes. However, in myocytes, βOHB up-regulated genes involved in the TCA cycle and oxidative phosphorylation, while down-regulating genes belonging to cytokine and chemokine signal transduction. Overall, our data do not support the notion that βOHB serves as a powerful signaling molecule regulating gene expression but suggest that βOHB may act as a niche signaling molecule in myocytes. Prevalence rates for obesity are spiraling out of control in many communities across the world. Inasmuch as obesity is a major risk factor for many chronic diseases, including type 2 diabetes, cardiovascular disease, and certain types of cancer , effectiInterestingly, it has been suggested that intermittent fasting may confer cardiometabolic health benefits independently of caloric restriction and concomitant weight loss , 8. A nuFOXO3a, Catalase, and MnSOD , and Fabp4, whereas rosiglitazone significantly up-regulated these genes. In line with the lack of effect of βOHB on 3T3-L1 differentiation, βOHB had a minor impact on the expression of Slc2a4 (Glut4) and no impact on the expression of Adipoq or Fabp4  or class II (MHCII). Expression of Myh3, Myh7, and Myh8, representing MHCI, was unchanged between βOHB and control-treated myoblasts. Expression of Myh1, Myh2, and Myh4, representing MHCII, was also unchanged between βOHB and control , Slc16a7 (Mct2), and Slc16a6 (Mct7), which are responsible for the transport of βOHB and butyrate , of which 50–52% were down-regulated .Next, we studied the similarity in gene regulation by butyrate among the different cell types. Hierarchical biclustering of all significantly regulated genes per condition showed marked similarity in the response to butyrate. Furthermore, Venn diagrams for the butyrate-treated cells revealed that a large fraction of the significantly regulated genes were shared in all cell types, confirming the similarity in gene regulation by butyrate. Indeed, 18%  of all significantly up-regulated genes were up-regulated in every cell type. Similarly, 15%  of all significantly down-regulated genes were down-regulated in every cell type . Heat maTo examine the similarity in gene regulation by butyrate across the different cell types at the level of pathways, we performed gene set enrichment analysis (GSEA) using the top 100 up- and down-regulated genes according to the T-statistic. The overlap in significantly regulated pathways (FDR q < 0.1) are shown in a Venn diagram, revealing a high overlap for butyrate-induced and repressed pathways among the four cell types. 20 out of 61 pathways were induced in at least two cell types, while the four pathways  were induced in all four cell types . InteresP < 0.001. qRT-PCR analysis for Clec4a1 and Apoc2 confirmed their down-regulation by βOHB , which was TCA cycle (not shown). The top 40 list of most highly up-regulated genes presents a diverse set of genes involved in cell cycle progression, tissue and cell remodeling as well as gene regulation . Many of the down-regulated pathways were related to immunity and inflammation . EnrichrGene set enrichment analysis negatively enriched pathways by βOHB treatment in primary myocytes.Table S3 2Fc values for all genes significantly down-regulated by βOHB in a correlation plot and determined the number of genes that fell within a fold-change ratio of 0.75× to 1.25×. Approximately 40–50% of all βOHB DEGs and of genes commonly regulated by βOHB and butyrate fell within this artificial cutoff, indicating that a substantial number of genes regulated by βOHB were regulated by butyrate to a similar extent  to influence cellular differentiation and for the first time performed whole genome expression analysis in primary adipocytes, macrophages, myocytes, and hepatocytes comparing βOHB side-by-side with the well-established HDAC inhibitor butyrate. At physiologically relevant plasma concentrations of βOHB as measured after fasting or ketogenic diet, βOHB did not affect the differentiation of 3T3-L1 adipocytes, C2C12 myotubes, THP-1 macrophages, primary mouse adipocytes, and primary human monocytes. Furthermore, acute βOHB treatment minimally influenced gene expression in primary adipocytes, macrophages, and hepatocytes but altered the expression of a substantial number of genes in primary myocytes. The results from βOHB are in stark contrast to the consistent inhibition of differentiation by butyrate in 3T3-L1, C2C12, THP-1, primary mouse adipocytes, and primary human monocytes, and the profound and consistent gene expression changes caused by butyrate in the various primary cells. Together, these data do not support the notion that βOHB serves as a potent signaling molecule regulating gene expression in adipocytes, macrophages, and hepatocytes. The suppressive effect of βOHB in myocytes on the expression of genes involved in immunity merits further study.Interest in ketones has surged in the recent years. Illustrated by the sheer abundance of reviews and perspective articles on the potential benefits of ketosis, βOHB is considered as a potential mediator of the putative fasting-related health benefits , 22, 23.Sirpa, Clec4a1, Fcgr3, Cd93, Syk, Ms4a6c, Hck, C1qc, Btk, C1qb, and Ccr1. Considering that the Mct transporter expression profile is similar among the primary cells, it is unclear why βOHB only exerted these effects in myocytes and not, for example, in macrophages. Nevertheless, one could speculate that the down-regulation of immune-related pathways in muscle cells by βOHB may be part of a broader mechanism to suppress immunity during starvation. Indeed, it is well recognized that starvation presents a trade-off between, on the one hand, saving energy to prolong survival and, on the other hand, investing a sufficient amount of energy to maintain immune defenses. It can be hypothesized that βOHB may serve as a signaling molecule that mediates the suppressive effect of starvation on specific immune-related processes -(–)-3-hydroxybutyric acid sodium salt from Sigma-Aldrich (#298360). Butyrate was Sodium butyrate from Sigma-Aldrich (#303410).3T3-L1 fibroblasts were maintained in DMEM supplemented with 10% newborn calf serum and 1% penicillin/streptomycin (P/S) . Experiments were performed in six-well plates. For Oil red O staining, cells were differentiated using the standard protocol. 2 d post-confluence, cells were switched to DMEM supplemented with 10% FBS, 1% P/S, 0.5 mM isobutylmethylxanthine, 1 μM dexamethasone, and 5 μg/ml insulin for 2 d in the presence of either 8 mM βOHB or 8 mM Butyrate. After 2 d, the cells were switched to DMEM supplemented with 10% FBS, 1% P/S, 5 μg/ml insulin, and the tested compounds for another 2 d. Then cells were maintained in normal DMEM medium (2–3 d), in the presence of the tested compounds until Oil red O staining on Day 10. Oil red O staining was performed following standard protocols. For qRT-PCR experiments, the cells were differentiated using the mild protocol, which allows for more sensitive assessment of compounds promoting the differentiation process at Day 4 of differentiation . 2 d posC2C12 skeletal muscle cells were cultured in DMEM supplemented with 20% FBS  and induced to differentiate with DMEM supplemented with 2% horse serum (HS)  upon reaching confluence in the presence of either 5 mM βOHB or 5 mM Butyrate. DM was renewed every other day. Myotube formation was complete  by Day 5.THP-1 cells were cultured in RPMI 1640 + heat-inactivated FBS and 1% P/S. Differentiation to macrophages was induced with 62.5 ng/ml phorbol 12-myristate 13-acetate  for 24 h in the presence of either 8 mM βOHB or butyrate.Human primary monocytes were isolated from buffycoat blood (Sanquin) using the Miltenyi magnet system (CD14 positive selection) and differentiated with 50 ng/ml macrophage colony-stimulating factor (M-CSF) for 7 d. 2 mM butyrate and 2 mM βOHB were supplied to the differentiation medium from Day 0. Cells were collected at Day 7 for qRT-PCR. Cells were cultured in the RPMI medium and supplemented with 10% FCS, 1% P/S, and 1% GlutaMAX .2.Microscopic pictures were taken and cells were subsequently frozen for RNA isolation. All cells were cultured at 37°C with 5% CO2, 1.5 mg/ml collagenase type II , 10% FBS, 0.5% BSA, and 15 mM Hepes) for 1 h at 37°C, with occasional vortexing. Cells were filtered through a 100-μm cell strainer . Subsequently, the cell suspension was centrifuged at 500g for 10 min and the pellet was resuspended in erythrocyte lysis buffer . Upon incubation for 2 min at room temperature, the cells were centrifuged at 500g for 5 min and the pelleted cells were resuspended in DMEM containing 10% FBS and 1% PS (DMEM/FBS/PS) and plated. Upon confluence, the cells were differentiated according to the protocol as described previously  supplemented with 1% Penicillin/Streptomycin (PS) and 1% BSA (Sigma-Aldrich). Material was minced finely with scissors and digested in collagenase-containing medium  in 6–8 d in DMEM/FBS/PS supplemented with 20% L929-conditioned medium (L929). After 6–8 d, non-adherent cells were removed, and adherent cells were washed and plated in 12-well plates in DMEM/FBS/PS + 10% L929. After 24 h, medium was switched to 2% L929 in DMEM/FBS/PS overnight. Cells were treated the following day.2 incubator at 37°C. Confluence was reached latest after 5 d in culture, upon which cells were trypsinized (0.25% trypsin), filtered with 70-μm filters, centrifuged at 300g for 5 min, and then seeded on an uncoated T150 flask for 45 min to get rid of fibroblasts. Subsequently, myoblasts were seeded in PM at 150,000 cells/ml onto Matrigel-coated 12-well plates cells. Upon reaching confluence, differentiation was induced by switching to differentiation medium (DM: Ham’s F-10 Nutrient Mix supplemented with 5% HS and 1% PS). DM was replaced every other day. Myotubes fully differentiated by Day 5 of differentiation in DM. The medium was renewed every other day.Myoblasts from hindlimb muscle of WT-C57Bl/6 male mice were isolated as previously described . In brie2. After 4 h of attachment, cells were washed with PBS and allowed to rest in dexamethasone-free medium overnight before treatment.Primary hepatocytes were isolated from C57BL/6NHsd male mice via collagenase perfusion as described previously , 63, 64.Primary cells were treated for 6 h with 5 mM βOHB or Butyrate, with PBS as control. Adipocytes and Macrophages were treated in DMEM/FCS/PS. Myotubes were treated in DM. Hepatocytes were treated in Williams E medium. Cells were washed with PBS once and stored in −80°C until RNA was isolated.Total RNA from all cell culture samples was extracted using TRIzol reagent (Thermo Fisher Scientific) and purified using the QIAGEN RNeasy Mini kit (QIAGEN) according to the manufacturer’s instructions. RNA concentration was measured with a NanoDrop 1000 spectrometer and RNA integrity was determined using an Agilent 2100 Bioanalyzer with RNA 6000 microchips (Agilent Technologies). Library construction and RNA sequencing on BGISEQ-500 were conducted at Beijing Genomics Institute (BGI) for pair-end 150 bp runs. At BGI, genomic DNA was removed with two digestions using amplification grade DNAse I (Invitrogen). The RNA was sheared and reverse transcribed using random primers to obtain cDNA, which was used for library construction. The library quality was determined by using Bioanalyzer 2100 (Agilent Technologies). Then, the library was used for sequencing with the sequencing platform BGISEQ-500 (BGI). All the generated raw sequencing reads were filtered, by removing reads with adaptors, reads with more than 10% of unknown bases, and low quality reads. Clean reads were then obtained and stored as FASTQ format.Salmon (tximport (version 1.16.1) (limma (version 3.44.3)  , scaled  1.16.1) . Differe 3.44.3)  using th 3.44.3) , based o 3.44.3) . Counts pipeline . Differepipeline , 72. GenP‐value cutoff) is used, GSEA is an unbiased approach. A GSEA score is computed based on all genes in gene set, which boosts the signal-to-noise ratio and allows to detect affected biological processes that are due to only subtle changes in expression of individual genes. This GSEA score called normalized enrichment score can be considered as a proxy for gene set activity. Gene sets were retrieved from the expert-curated KEGG pathway database  according to the manufacturer’s protocol using 250 ng RNA for in vitro studies. Quantitative PCR amplifications were carried out on a CFX 384 Bio-Rad thermal cycler (Bio-Rad) using SensiMix PCR reagents . Gene expression values were normalized to one of the housekeeping genes and analyzed using delta ΔΔCt method. Primer sequences of genes are provided in Table S4.Primers for qPCR.Table S4 am to 6:00 pm).Animals for primary cell experiments were all housed at the Centre for Small Animals, which is part of the Centralized Facilities for Animal Research at Wageningen University and Research (CARUS) and were approved by the Local Animal Ethics Committee of Wageningen University . Mice were maintained at 21°C, on rodent chow and kept on a regular day–night cycle . Mice were fed ad libitum with regular rodent chow.P-values < 0.05 were considered statistically significant.Statistical analysis of the transcriptomics data was performed as described in the previous paragraph. Data are presented as mean ± SD. GSE179023.The RNAseq data from publication have been deposited to the GEO database and assigned the accession number:


Dianthus broteri as a model, we compared the ecophysiological behaviour of colchicine-induced neotetraploids (4xNeo) to diploids (2x) and naturally occurring tetraploids (4xNat). Leaf gas-exchange and chlorophyll fluorescence analyses were performed in order to asses to what extent post-polyploidization evolutionary processes have affected 4xNat. Genomic diploidization and phenotypic novelty were evident. Distinct patterns of variation revealed that post-polyploidization processes altered the phenotypic shifts directly mediated by genome doubling. The photosynthetic phenotype was affected in several ways but the main effect was phenotypic diploidization (i.e. 2x and 4xNat were closer to each other than to 4xNeo). Overall, our results show the potential benefits of considering experimentally synthetized versus naturally established polyploids when exploring the role of polyploidization in promoting functional divergence.Whole-genome duplication and post-polyploidization genome downsizing play key roles in the evolution of land plants; however, the impact of genomic diploidization on functional traits still remains poorly understood. Using  Dianthus broteri demonstrates that ‘phenotypic diploidization’ occurs in functional traits, which can lead to the creative role of polyploidization being underestimated.Colchicine-induced polyploidization in  This Iberian taxon encompasses the largest known autopolyploid complex within the genus (n = 2x = 30), tetraploid (2n = 4x = 60), hexaploid (2n = 6x = 90), and dodecaploid (2n = 12x = 180) monocytotypic populations. This complex seems to have evolved recently (0.9–2.1 Mya) following a rapid cytotypic adaptive divergence in concert with an extensive geographical disjunction and an ecological niche differentiation within an aridity gradient, with the higher cytotypes (6x and 12x) inhabiting more restricted and extreme habitats . The ploidy level and DNA content of this population have previously been characterized by flow cytometry together with chromosome counts (D. broteri diploids (–2 s–1). Germination was recorded every 2 d for 21 d, and the seedlings obtained were sown in individual 2.5-l pots filled with a mixture of a commercial organic substrate (Gramoflor) and perlite (3:1) and placed in a greenhouse with controlled temperature of 21–25 ºC, 40–60 % relative humidity (RH), and natural daylight ranging from 200–1200 μmol m−2 s−1 photosynthetic photon flux density (PPFD) incident on leaves during the day. The pots were adequately irrigated with tap water and seedling survival was checked weekly for 90 d after sowing . In addition, diploid and tetraploid plants from natural populations spanning the whole range of distribution of this species (Seeds were collected in 2018 from a diploid population (15 plants) of  species  were alsx, 4xNeo, and 4xNat) were randomly selected 15 d prior to analysis and placed in a controlled-environment chamber  with a 14/10 h photoperiod (300 μmol m−2 s−1) at 25/18 °C and 40–60 % RH. This period under controlled conditions was to avoid interference by possible border effects within the greenhouse.For the ecophysiological characterization, 10 plants per group (i.e. 2D. broteri (D. broteri seedlings was used for nuclear suspensions together with Pisum sativum L. cv. ‘Ctirad’ as the genome size control (2C nuclear DNA = 9.09 pg). All the FC measurements showed high quality (>5000 nuclei per peak and CV <5 %) and peak means were determined through manual gating using the Kaluza Analysis 2.1 software (Beckman Coulter). DNA content (2C) was estimated for the main FC peak  of the higher-ploidy-level cells. According to these results, each plant was classified as diploid (2n=2x), neotetraploid (2n=4xNeo), diploid-tetraploid mixoploid (2n=2x+4x), or tetraploid-octoploid mixoploid (2n=4x+8x). The mixoploids were discarded for further analysis. Monoploid genome size (1Cx) was estimated as the amount of 2C nuclear DNA divided by the ploidy level. In addition, 1Cx data for the naturally occurring tetraploids (4xNat) were obtained from The DNA content and ploidy level of the seedlings were estimated by flow cytometry (FC) following a specific protocol for xNeo plants, and randomly chosen plants for 2x and 4xNat). Epidermal impressions of the region near the leaf tip were taken on the adaxial surface of the youngest fully expanded leaf using Germolene New Skin Liquid Plaster  and examined under a Carl-Zeiss Axiophot photomicroscope equipped with a Sony DXC-390P Exwave HAD camera. The resulting images were analysed using ImageJ v.1.51j8, and the mean stomatal density together with mean lengths and widths were determined on the basis of 10 images per plant, at 10× magnification for density and 40× for the dimensions , stomatal conductance (gs), and intercellular CO2 concentration (Ci) were recorded at a PPFD of 1000 µmol m−2 s−1 , vapour pressure deficit of 2.0–3.0 kPa, ambient CO2 concentration (Ca) of 400 µmol CO2 mol−1, leaf temperature of 25±2 °C, and 50±5% RH. In addition, leaves were dark-adapted for 10 min for measurement of dark respiration (Rd).A LI-COR LI-6400 infrared gas analyser in an open system equipped with a Li-6400-02B LED light source was used for instantaneous gas-exchange measurements and for constructing gm) and maximum carboxylation activity of Rubisco  by the curve-fitting method (A/Ci responses were measured once the leaves had reached a steady-state under the conditions described above (~20 min), at which point Ca was decreased in the following steps: 400, 350, 300, 250, 200, 150, 100, and 50 µmol mol−1. The chamber conditions were then restored to their initial levels and Ca was increased in the following steps to complete the curve: 500, 750, 1000, 1250, 1500, 1750, and 2000 µmol mol−1. At each step, gas exchange was allowed to equilibrate to avoid significant variations in Rubisco activity . The maximum quantum efficiency of PSII (Fv/Fm) was determined by using a 0.8 s saturating light beam with an intensity of 8000 μmol m−1 s−1 (a) were obtained via OJIP tests implemented in the pre-programmed protocols of the FluorPen FP100 (n=10). Fv/Fm, absorbed energy flux (ABS/CS), trapped energy flux (TR/CS), electron transport energy flux (ET/CS), and dissipated energy flux (DI/CS) per leaf cross-section, together with performance index (PI) derived from OJIP were calculated according to max) was also obtained using the pre-programmed rapid light curve (RLC) protocol (n=6), which consisted of the exposure of dark-adapted leaves (30 min) to increasing light levels  and recording the quantum yield at each step.Modulated Chlorophyll http://www.R-project.org/). The effects of the colchicine treatments (concentration and duration as main factors) on seed germination and seedling survival (dependent variables) were tested by fitting generalized linear models (GLMs) using a binomial error structure and the ‘logit’ link function for each dependent variable. The effectiveness of the colchicine treatments in producing viable polyploids was examined using the Fisher’s exact test. One-way ANOVA and post hoc Tukey’s tests were used for detecting differences between the cytotypes in the 1Cx DNA contents and the ecophysiological parameters derived from the OJIP tests and RLC protocol.Data analyses were carried out using R v.3.6.3 . The frequency of polyploidy induction was significantly higher as the dose (i.e. concentration and incubation time) of colchicine increased  of which 74 were diploids (the 21 controls and 53 treated plants), 11 were tetraploids, and nine were mixoploids  was about twice that of the diploids (1.82±0.03 pg), whilst the natural tetraploids showed a mean genome size of 3.47±0.13 pg. Accordingly, the monoploid DNA amount (1Cx) was significantly dependent on cytotype  and 4xNeo (0.900±0.017 pg) were compared (P>0.05), 4xNat showed a significantly lower 1Cx DNA value than either of them (P<0.05), varying from 0.77–0.94 pg.The nuclear DNA amount of the synthetic 4F2,30=25.79, P<0.001) and width , as well as density . Whilst length, width, and density were indistinguishable between 2x and 4xNat (P>0.05), varying between 29–39 μm, 19–28 μm, and 66–154 mm−2, respectively, the synthetic neotetraploids showed significantly longer  and wider  stomata than either of them, but had a lower density . Notably, the synthetic tetraploids showed an increased variation in stomatal length , but a more consistent density .Stomatal traits were affected differently by polyploidization . ComparixNat did not always display the same trends than 4xNeo. Analysis of leaf gas exchange showed that the synthetic neopolyploids had a significant increase in net photosynthetic rate , stomatal conductance , and mesophyll conductance  in comparison to the diploids and natural tetraploids, which showed similar values to each other , which had similar values. The 4xNeo plants also showed ~2-fold greater gm than 2x (P<0.05), whilst 4xNat showed intermediate values between those of 2x and 4xNeo. Although the results showed broadly similar patterns, no significant differences were found among the cytotypes for the other photosynthetic parameters that were measured, namely intercellular CO2 concentration (Ci), maximum rate of Rubisco activity , and the rate of dark respiration (Rd) (P>0.05).Photosynthetic performance was also affected by polyploidization, but 4ch other . AN and max; F2,20=5.64, P<0.05) and maximum quantum efficiency of PSII . In both cases, higher values were found in 4xNeo compared to 2x, whilst vales in 4xNat were intermediate  than 2x , whilst 4xNat showed a 30.8% increase in ABS/CS compared to 2x (P<0.01). No significant difference was found between 4xNat and 4xNeo (P>0.05). The same post-WGD divergence was also observed for the trapped energy flux per cross-section , but there were no significant differences among the cytotypes , with mean values of 1717, 1960, and 2065 for 2x, 4xNeo, and 4xNat, respectively ,with 4xNeo showing ~0.3-fold lower dissipation values than 2x and 4xNat , resulting in larger stomata with greater aperture area available for gas exchange  in the natural tetraploids. Variation in cell size (related to the loss of DNA content) will play a crucial role in optimizing gas exchange, potential carbon gain, and water use after WGD  of D. broteri could have also triggered adaptation to harsh environments , dark respiration (Rd), CO2 concentration in the substomatal cavities (Ci), or the electron transport energy flux per leaf cross-section (ET/CS) (x and 12x) of the D. broteri complex, suggesting a photoprotective adaptation to their stressful ecological niches  in maximum carboxylation rate of Rubisco ( (ET/CS) , so the gs and gm; xNat. In agreement with our results (i.e. 4xNeo having higher conductances than 2x), neopolyploids frequently display increased gs and gm. , which correlates with gs and gm (among other parameters), showed higher values for 4xNeo whilst it did not differ between 2x and 4xNat  and maximum quantum efficiency of PSII (Fv/Fm), and we also found that WGD per se decreased the dissipated energy flux per leaf cross-section (DI/CS). These results indicated that much of the energy absorbed by the 4xNeo plants would have been transformed in their photosystems and directed to the photochemical pathway , our design allowed us to distinguish them  would have the potential to facilitate their establishment through the exploitation of new niches, and would thus explain the wider ecological range of the tetraploids (Our study has highlighted the relative importance of WGD In summary, the results of our study not only suggest that many important plant functional traits are directly affected by polyploidization, but they also reveal that the process of phenotypic diploidization can lead to the creative role of polyploidization being underestimated. Thus, phenotypic novelty in neopolyploids might be constrained by regulatory mechanisms related to physiology as well as by other evolutionary processes related to environmental pressures.The following supplementary data are available at Fig. S1. Flow cytometry analyses of the plants arising from colchicine-treated seeds.2 concentration, maximum carboxylation rate of Rubisco, dark respiration, and electron transport energy flux per leaf cross-section in the different cytotypes..Fig. S2. Intercellular COerab179_suppl_Supplementary_Figures_S1-S2Click here for additional data file.


MBT27, via application of metabolic analysis and molecular networking. Actinomycins L1 and L2 are diastereomers, and the structure of actinomycin L2 was resolved using NMR and single crystal X-ray crystallography. Actinomycin L is formed via spirolinkage of anthranilamide to the 4-oxoproline moiety of actinomycin X2, prior to the condensation of the actinomycin halves. Such a structural feature has not previously been identified in naturally occurring actinomycins. Adding anthranilamide to cultures of the actinomycin X2 producer Streptomyces antibioticus, which has the same biosynthetic gene cluster as Streptomyces sp. MBT27, resulted in the production of actinomycin L. This supports a biosynthetic pathway whereby actinomycin L is produced from two distinct metabolic routes, namely those for actinomycin X2 and for anthranilamide. Actinomycins L1 and L2 showed significant antimicrobial activity against Gram-positive bacteria. Our work shows how new molecules can still be identified even in the oldest of natural product families.Streptomycetes are major producers of bioactive natural products, including the majority of the naturally produced antibiotics. While much of the low-hanging fruit has been discovered, it is predicted that less than 5% of the chemical space of natural products has been mined. Here, we describe the discovery of the novel actinomycins L Actinobacteria have been the source for the majority of the antibiotics in use today4. Of the Actinobacteria, members of the genus Streptomyces produce over half of all currently characterized antibiotics3. Genome sequencing revealed that Actinobacteria have much more biosynthetic potential to produce bioactive molecules than originally anticipated, with even the model organisms harbouring many so-called cryptic or silent biosynthetic gene clusters (BGCs) that specify yet unknown compounds7. Triggering the expression of silent BGCs by genetic and cultivation-based techniques should facilitate unlocking this yet unexplored chemical diversity, allowing the discovery of novel molecules9. This strategy relies on altering the regulatory networks of the producing organism in response to fluctuating culturing conditions, such as carbon, nitrogen or phosphate concentration12. Manipulation of fermentation conditions of promising producer strains, known as “one strain many compounds” (OSMAC) approach, is effective in enhancing secondary metabolites production14. Novel secondary metabolites have been discovered via modification of cultivation parameters, including nutrients16, and addition of chemical elicitors18.Considering the emerging crisis of antibiotic resistance that spreads among bacterial pathogens and increasing incidence of cancer, the search for new, efficient and less toxic drugs remains a priority19. Supervised statistical methods are able to classify a response like a biological activity, and to determine the most discriminant metabolite(s) related to such response20. Moreover, simultaneous dereplication of differentially expressed compounds is implemented into the drug-discovery pipelines in order to avoid rediscovery of already known compounds21. MS-based metabolomics provides important information on the distribution of the metabolites that are present in complex mixtures, but the identification of their structures is complicated. For this purpose, the Global Natural Products Social Molecular Networking (GNPS) platform was developed, applying both molecular networking and automated searches of tandem mass spectrometry (MS/MS) fragmentation spectra against spectral libraries, to identify structural relationships between metabolites23. This greatly facilitates the annotation and dereplication of known molecules.Metabolic profiling of crude extracts obtained under different growth conditions represents a challenging analytical task since these mixtures are composed of hundreds of natural products. Therefore, metabolomics, particularly those based on mass spectrometry (MS), became more and more valuable and greatly increased the efficiency of such screenings24. Actinomycins are produced by various Streptomyces strains and are composed of a chromophore group and two pentapeptide chains with a variable composition of amino acids25  assembly line, and actinomycins are formed through oxidative condensation of two 3-hydroxy-4-methylanthranilic acid (4-MHA) pentapeptide lactones (PPLs)27.Actinomycin is a DNA-targeting antibiotic and anticancer compound discovered in 1940 by Waksman & Woodruff, and in fact the first antibiotic that was isolated from an actinobacteriums25 Fig. . Actinom1 and L2, from the extracts of Streptomyces sp. MBT27. Multivariate data analysis combined with molecular networking indicated that the antimicrobial activity of the extracts correlated with novel actinomycins L1 and L2 and known actinomycins D, X0β and X2. NMR and single crystal X-ray crystallography revealed that an anthranilamide moiety was linked through a spiro-center to a proline residue in the structure of actinomycins L1 and L2. Such a structural feature has not previously been identified in naturally occurring actinomycins.In this work we report the discovery of new actinomycin analogues, actinomycin LStreptomyces sp. MBT27 is a gifted natural product producer that was isolated from Qinling mountains in China, with potent antibacterial activity against various MDR (multi-drug resistant) bacteria28. We previously showed that the strain among others produces the novel quinazolinones A and B29. To investigate the antibiotic activity of Streptomyces sp. MBT27 the strain was fermented in minimal medium (MM) with either of the following carbon sources (percentages in w/v): 1% of both mannitol and glycerol, 1% mannitol, 2% mannitol, 1% glycerol, 2% glycerol, 1% glucose, 2% glucose, 1% fructose, 1% arabinose, or 1% N-acetylglucosamine (GlcNAc). Supernatants of Streptomyces sp. MBT27 cultures were extracted with ethyl acetate and bioactivity assays were performed against Bacillus subtilis 168. Interestingly, the carbon sources had a huge effect on the antimicrobial activity  indicated that the model has a good reliability and ability of prediction. A permutation test was performed (n = 100) and the resulting R2Y and Q2Y values were significantly lower , which indicated that there was no overfitting in the model30 . The first two PCs accounted for 37% and 16%, respectively, of the total data variation. PCA analysis failed to show significant metabolic separation in relation to the observed bioactivity , m/z 1255.6353 (8.53 min), m/z 1277.6175 (8.52 min), m/z 1271.651 (7.56 min) and m/z 1293.612 (7.56 min)  to the known actinomycins, suggesting that the molecule was a novel actinomycin. Statistical analysis showed that the extracts with stronger antimicrobial activity contained higher concentrations of actinomycins X2, X0β, D and the new compound, in comparison with the less active ones  molecular networkingStreptomyces sp. MBT27 followed by bioactivity guided fractionation. The purification process resulted in the isolation of two compounds (1) and (2), with the same mass .To allow identification and structural analysis of the likely novel actinomycin analogue, we performed large-scale fermentation of ss Figs. , S5. Thess Figs. –S12. Basues Fig. . The pro2) was crystallized successfully. Single-crystal X-ray diffraction confirmed the structure obtained for (1) based on NMR, and established the absolute configuration to be 2′S, 2″S, 4′R, 4″R, 10′R, 12′S, 12″S, 18′S, 18″S, 23′R, 23″R by anomalous-dispersion effects in diffraction measurements on the crystal , X2, and X0β detected in the extracts of Streptomyces sp. MBT27 are members of the actinomycin X complex. Recently it was shown that actinomycins X0β and X2 are formed through the sequential oxidation of the γ-prolyl carbon by the cytochrome P450 enzyme saAcmM35. Based on its structure, actinomycin L is most likely formed through an aminalization reaction between the two amino groups of anthranilamide and the γ‐keto group on the proline residue of actinomycin X2. Accordingly, its production should be arrested when one of the precursors is not available. Interestingly, Streptomyces sp. MBT27 produced actinomycin L in very low amounts when grown with fructose (1% w/v) as the sole carbon source. Moreover, ANOVA statistical analysis showed that anthranilamide was produced in equally low amounts under the same growth conditions  was added to cultures of Streptomyces sp. MBT27 and of S. antibioticus IMRU 3720. 4-MHB is a structural analogue of 4-MHA that replaces 4-MHA as a starter unit in the nonribosomal assembly of the actinomycin halves34. When 4-MHB replaces 4-MHA, 4-MHB containing PPLs accumulate, because they cannot react with each other to give a phenoxazinone ring, as is the case with 4-MHA PPLs36. LC–MS analysis of the 4-MHB-supplemented extracts showed the appearance of the previously reported 4-MHB-containing pentapeptide lactones PPL 1, PPL 0, and PPL 2, and new PPL, designated as PPL 3 , which is formed by methylation of 3-hydroxykynurenine (3-HK)26. Our work surprisingly revealed a novel structure within the extensively studied actinomycin family, namely that of actinomycin L, which arises via attachment of an anthranilamide moiety to the γ-carbon of one of the proline residues through aminal formation. ANOVA statistical analysis proved that production of anthranilamide is the limiting factor in the biosynthesis of actinomycin L. Feeding experiments with anthranilamide suggested that actinomycin L is formed through the spontaneous reaction of anthranilamide with the 4-oxoproline site of actinomycin X2 prior to the condensation of the two 4-MHA PPLs into actinomycin L. To the best of our knowledge, the attachment of anthranilamide to a 4-oxoproline moiety is a novel observation.Actinomycin was the first antibiotic identified in ActinobacteriaStreptomyces sp. MBT27 harbors the same genes as that of S. antibioticus, with high homology between the genes, which strongly suggests that the modification of actinomycin X2 to actinomycin L is not encoded by the BGC itself. Indeed, we anticipate that anthranilamide is derived from anthranilic acid in Streptomyces sp. MBT27, whereby anthranilic acid in turn is biosynthesized through the shikimate pathway40. Anthranilic acid is a commonly produced primary metabolite in Streptomyces, while anthranilamide is less common43. The actinomycin X2 producer S. antibioticus IMRU 3720 fails to convert anthranilic acid into anthranilamide, which explains why actinomycin L was also not detected in the extracts. However, actinomycin L was produced when we fed cultures of S. antibioticus IMRU 3720 with additional anthranilamide, which is fully in line with our proposed biosynthetic pathway. Thus, actinomycin L is an example of a natural product that requires the joining of two separate metabolic pathways, and this is a concept that needs more attention. After all, scientists rely increasingly on heterologous expression and synthetic biology approaches44, and these will likely fail if genes are required that do not fall within the main BGC.The actinomycin BGC of Streptomyces spp. is strongly influenced by the carbon source, whereby the preferred carbon source varies from strain to strain49. D-galactose favors actinomycin production in Streptomyces antibioticus over arabinose, xylose, glucose, fructose and rhamnose45, while glycerol was the optimal carbon source for actinomycin production by S. antibioticus Tü 6040 and S. antibioticus SR15.447. In the case of Streptomyces sp. MBT27, growth on MM with glycerol, GlcNAc, fructose and glucose  as sole carbon sources were the best carbon sources to promote the production of actinomycins. However, increasing the glucose concentration to 2% blocked the production of actinomycins. Glucose was previously reported to repress the transcription of the gene for hydroxykynureninase, which is involved in the formation of the main actinomycin precursor 4-MHA45. Importantly, in our experiments the carbon source not only promoted the overall production levels, but also contributed to the chemical diversity of the actinomycins, including the production of actinomycin L. This coincided with the production of anthranilamide, an essential substrate to form this novel actinomycin variant.The production of actinomycins by 50. What is important to note is that this also applies to well-known families of molecules and in extensively studied model organisms. Examples are the highly rearranged cryptic polyketide lugdunomycin that belongs to the family of angucyclines51, the new glycopeptide corbomycin with a novel mode of action52, as well as the discovery of coelimycin53 and a novel branch of the actinorhodin biosynthetic pathway54 in the model organism Streptomyces coelicolor. The discovery of actinomycin L provides another interesting example that we have not yet exhausted the known part of the chemical space. Indeed, the isolation of these novel actinomycins underlines that the biosynthetic potential of Actinobacteria still has major surprises in store, and that we can expect that new molecules can be discovered even within extensively studied microbes and compound classes.In the twenty-first century, genome mining and renewed drug discovery efforts have revealed that Actinobacteria may produce many more molecules than was expected55. NMR spectra were recorded on a Bruker Ascend 850 MHz NMR spectrometer (Bruker BioSpin GmbH). Data was analyzed using MestReNova 14 software . The structures of molecules were drawn using ChemDraw Professional version 16.0 (Perkin-Elmer Informatics). HPLC purification was performed on Waters preparative HPLC system as described29. All solvents and chemicals were of HPLC or LC–MS grade, depending on the experiment.Optical rotation, FT-IR and UV were measured as previously describedStreptomyces sp. MBT27 was obtained from the Leiden University strain collection and had previously been isolated from the Qingling Mountains, Shanxi province, China28. Cultures were grown in triplicate in 100 mL Erlenmeyer flasks containing 30 mL of liquid minimal medium (MM)56, supplemented with various carbon sources, and inoculated with 10 µL of 109/mL spore suspension. The carbon sources (percentages in w/v) were: 1% mannitol + 1% glycerol, 1% mannitol, 2% mannitol, 1% glycerol, 2% glycerol, 1% glucose, 2% glucose, 1% fructose, 1% arabinose or 1% N-acetylglucosamine (GlcNAc). The cultures were incubated in a rotary shaker at 30 °C and 220 rpm for seven days. Following fermentation, culture supernatants were extracted with ethyl acetate (EtOAc) and evaporated under reduced pressure. In the series of feeding experiments Streptomyces sp. MBT27, S. antibioticus IMRU 3720 and S. chrysomallus ATCC11523 were fermented in MM supplemented with 1% fructose and 0.7 mM anthranilamide. For the directed biosynthesis of non-natural actinomycin X halves Streptomyces sp. MBT27 and S. antibioticus IMRU 3720 were grown in MM with 1% w/v fructose, 0.7 mM anthranilamide and 0.7 mM 3-hydroxy-4-methylbenzoic acid (4-MHB) and extracted with EtOAc.Streptomyces sp. MBT27 was grown in YEME at 30 °C and 220 rpm for 48 h. DNA was extracted from Streptomyces sp. MBT27 as described56. DNA quality was verified by agarose gel electrophoresis. PacBio sequencing and assembly was performed by Novogene (UK). Generally, library was prepared using SMRTbell template prep kit  according to manufacturer instructions. Sequencing was then performed using PacBio Sequel platform in continuous long reads mode. Assembly was done using Flye (version 2.8.1)57. Biosynthetic gene clusters (BGCs) in this genome were annotated using AntiSMASH 6.037. The actinomycin BGC was then extracted and compared with the same cluster from S. antibioticus IMRU 3720 using clinker version 0.0.20) with default settings58.29. The fractions eluted with n-hexane–acetone (1:1) was subjected to a SunFire C18 column  eluted with a H2O–MeOH gradient of 50–100% in 20 min, at a flow rate of 15 mL/min. The fraction containing the actinomycins was collected and further purified on semi-preparative SunFire C18 column , run at 3 mL/min, and eluted using a H2O–MeOH gradient of 70–100% in 20 min, to yield actinomycins L1 , L2 , X2 , X0β , and D .Large-scale fermentation, extraction end fractionation were performed as previously described1 (1): red amorphous powder; [α] D25-38 ; UV (MeOH) λmax (log ε) 211 (1.957), 312 (0.124), 427.5 (0.152), 438 (0.151) nm; IR νmax 3301, 2963, 2921, 2859, 1740, 1662, 1644, 1585, 1521, 1465, 1405, 1300, 1262, 1191, 1097 cm−1; 1H and 13C NMR data, see Table m/z 1387.6681 [M + H]+ .Actinomycin L2 (2): red amorphous powder; [α] D25 -54 ; UV (MeOH) λmax (log ε) 226 (2.189), 364 (0.186) nm, 426.5 (0.367) nm; IR νmax 3308, 2943, 2929, 2831, 1748, 1662, 1644, 1585, 1448, 1406, 1302, 1262, 1191, 1113 cm−1; HRESIMS (positive mode) m/z 1387.6674 [M + H]+ . The X-ray diffraction experiment on a crystal grown from MeOH further confirmed the structure and determined the absolute configuration of compound 2 , vancomycin-sensitive Enterococcus. faecium , and vancomycin-resistant Enterococcus faecium VanB , as described60. Each compound was serially diluted in DMSO with a dilution factor of 2 to test 10 concentrations starting at 128 μg/mL in all the antimicrobial assays. The MIC was defined as the lowest concentration of compound that inhibited ≥ 95% of the growth of a microorganism after overnight incubation. The Genedata Screener software  was used to process and analyze the data and to calculate the RZ’ factor in the assay that was between 0.90 and 0.98 supporting its robustness.The antimicrobial activity of the compounds was tested in liquid inhibition assays against seven pathogens including Gram-negative and Gram-positive bacteria  equipped with an electrospray ionization source (ESI) as described29.For LC–MS analyses, extracts were dissolved in MeOH to a final concentration of 1 mg/mL, and 1 μL was injected into Waters Acquity UPLC system coupled to Agilent 6530 QTOF MS. Samples were analyzed according to the protocol that was previously published62 and spectral networks were assembled using Global Natural Product Social molecular networking (GNPS) (https://gnps.ucsd.edu) as described23. Briefly, the precursor ion mass tolerance was set to 2.0 Da and a MS/MS fragment ion tolerance of 0.5 Da, while the minimum cosine score was set to 0.7. The data were clustered using MSCluster with a minimum cluster size of three spectra. The spectra in the network were also searched against GNPS spectral libraries. A minimum score of 0.7 was set for spectral library search, with at least six fragment peaks matching. Cytoscape 3.7.2 was used for visualization of the generated molecular networks32. In Cytoscape the detected chemical space is displayed as nodes and edges . The edge thickness was set to represent the cosine score, with thicker lines indicating higher similarity between nodes. LC–MS/MS data were deposited in the MassIVE Public GNPS data set (MSV000085106). The molecular networking job in GNPS can be found at https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=0c9153470404488d8927289139f875d3. The annotated MS/MS spectra were deposited in the GNPS spectral library for actinomycin L1 (CCMSLIB00005718892) and L2 (CCMSLIB00005718891).MS/MS raw data were converted to a 32-bit mzXML file using MSConvert (ProteoWizard)63 and processed as previously described29. The aligned peak list was exported as a comma-separated file for statistical analysis. Statistical analysis was performed using MetaboAnalyst64, where log transformation and pareto scaling was initially applied to the data. The normalized data were subjected to principal components analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). The quality of the models was evaluated with the relevant R2 and Q2. To identify the difference in intensity of a single mass feature among multiple growth conditions, one-way ANOVA was performed, followed by a post hoc Tukey’s honest significant difference (HSD) test.Prior to statistical analysis, mzXML files, which were converted using Shimadzu LabSolutions Postrun Analysis, were imported into Mzmine 2.31Supplementary Information.


The objectives of this research were to determine the risk extension and microcystin-LR levels, and to identify the environmental factors that trigger the toxic cyanobacteria growth and microcystin-LR occurrence in 24 water reservoirs in eastern Cuba. Samplings were performed in the early morning hours, with in situ determination and physicochemical analysis carried out in the laboratory. Microcystin-LR were determined in water and within the cells (intracellular toxins) using UPLC–MS analysis after solid phase extraction. The reservoirs studied were found to be affected by eutrophication, with high levels of TN:TP ratio and phytoplankton cell concentrations, high water temperatures and low transparency, which cause collateral effect such as cyanobacterial bloom and microcystin-LR occurrence. In Hatillo, Chalóns, Parada, Mícara, Baraguá, Cautillo, La Yaya, Guisa and Jaibo reservoirs, concentrations of MC-LR higher than the WHO limits for drinking water (1 µg·L The increasing eutrophication of aquatic environments caused by industrial development favors the massive growth of microorganisms such as cyanobacteria ; these sMoreover, the current changing climate could potentially favor toxic cyanobacteria growth ,9,10. ThIt is crucial to note that water coverage is one of the most important indicators for human development . Water rIn Cuba, even though irrigation is identified as the main use for water reservoirs, the drinking water supply has the most important and complex use because it needs water treatment availability and management in order to comply with safe water quality standards . Water iIn Cuban legislation, there are gaps concerning cyanobacteria and cyanotoxin occurrence and its potential impacts. Even though cyanotoxin levels in water reservoirs need to be analyzed, there is some evidence of potentially toxic cyanobacteria occurrence ,18. TherMicrocystis, Oscillatoria and Anabaena toxic species in water reservoirs has frequently been reported [Microcystis dominance being remarkable. Evidence obtained in reservoirs of Santiago de Cuba province describes in the early studies the occurrence of Microcystis viridis, Aphanothece minutissima, Oscillatoria chalybea, O. limosa, O. tenuissima, Anabaena torulosa, Planktothrix sp., Lyngbya sp. and Synechococcus sp., in three water reservoirs . The occurrence of some potentially toxic species were afterwards described in other reservoirs, and the Microcystis genus was the best represented, with six species: M. aeruginosa, M. comperei, M. flos-aquae, M. panniformis, M. viridis and M. wesenbergii [Globally, the presence of reported ,22,23,24enbergii . In ordeMoreover, the factors related to cyanotoxin occurrence and its social impact are not yet completely understood ,18 sinceThe main activities in the studied catchment areas were forestry (68%) and agriculture (30%). Other activities such as industry and mining are less important (2%). The highest agriculture use in the catchment area was identified in Bio (90%) and La Yaya reservoirs (70%), while the highest forestry use (80%) was identified in Paso Malo and Charco Mono. Deforested areas were present in all the studied reservoirs, Chalóns reservoir being the highest (30%), while Cautillo, Hatillo and Bio reservoirs showed deforestation of only 5% .−1. The lowest conductivity was measured in Gota Blanca (445 µS·cm−1), while the highest value was observed in Parada reservoir (1174 µS·cm−1)  . The disa concentration average was 53.5 µg·L−1, the maximum values were obtained in Chalóns (127.4 µg·L−1), and the lowest concentration was in Mícara (19.3 µg·L−1) , and the maximum transparency was measured in Gota Blanca (1.8 m) . The chl µg·L−1) .−1, with a minimum concentration in Gilbert (0.3 mg·L−1) and a maximum in Mícara (2.5 mg·L−1). The TN values were higher than 1.5 mg·L−1 in Parada, La Yaya, Cautillo and Gibara reservoirs , while Cautillo reservoir has the highest values (0.2 mg·L−1)  in the studied reservoirs was 1.0 mg·Lservoirs . The ave mg·L−1) . In rega4 cell·mL−1. The highest phytoplankton counting was recorded in Parada reservoir (26 × 104 cell·mL−1), and the lowest in Gota Blanca (2.0 × 104 cell·mL−1). Specifically, the cyanobacteria average concentration was 3.4 × 104 cells·mL−1 with maximum densities in Mícara (19 × 104 cell·mL−1), and the lowest in Moa reservoir (0.28 × 104 cells·mL−1). Chalóns (9.8 × 104 cells·mL−1), Hatillo (9.4 × 104 cells·mL−1) and Baraguá (8.6 × 104 cells·mL−1) reservoirs had concentrations of cyanobacteria cells near the 105 cells·mL−1 . Chlorophyta and Heterokontophyta divisions had 23 and 19% cyanobacteria, respectively. The Bacillariophyta were not well represented; they comprised only 2% of the species. Twenty-three species of potentially toxic cyanobacteria were identified, which represents 61% of the total cyanobacteria species. The list of cyanobacteria species and potentially toxigenic species is presented in Synechocystis aquatilis and Aphanocapsa sp.; both were present in 15 reservoirs (62.5%), followed by Planktothrix sp. and Merismopedia punctata (54%); while Romeria simplex was still present in 50% of the reservoirs studied , La Yaya (60.5 µg·L−1) and Mícara reservoirs (55.8 µg·L−1). In general, water samples exceeded the World Health Organization (WHO) MC-LR limits for drinking water supply (1 µg·L−1) in nine reservoirs (29%), Cautillo and La Yaya reservoirs being significantly higher (p < 0.05). The methodological accuracy to the MC-LR quantification was assessed through a calibration curve carried out in UPLC–MS, with a linear relationship in the calibration concentration range. Highest values of microcystin-LR  were detThe analysis revealed a negative correlation between air temperature and DO (r = −0.51) and transparency (r = −0.41) . Furthera (r = −0.41), corroborating the usefulness of these parameters to evaluate water quality and ecosystem trophic status [In addition, Secchi disk had a negative correlation with water temperature (r = −0.42) and with chlorophyll c status ,37, whicc status .a (r = −0.45)  and cyanobacteria cell concentration (r = 0.53). Finally, conductivity has negative correlation with cyanobacteria cell concentration (r = −4.46) and chlorophyll = −0.45) . AccordiThere are several reports on environmental factor influences regarding the occurrence of cyanobacteria and cyanotoxins around the world. Some of these studies were conducted in the tropical and subtropical regions of Asia ,39,40,41The eastern part of Cuba has environmental conditions which might favor the cyanobacteria proliferation, including the nutrient enrichment of aquatic systems, warm temperatures and calm stable water conditions. In all the reservoirs studied, an aquaculture was developed, with an important contribution to the nutrient enrichment , water ea and phycocyanin (PC). The first axis shows 32.77% of total variance and the second axis shows 20.92%, with 53.69% of the total variance explained.PCA analysis revealed that component 1 is strongly influenced by water and air temperature, TP, phytoplankton and cyanobacteria concentration cells. Component 2 is positively related with TN and TN:TP ratio, and negatively with pigments  . During −1 , all of them containing cyanobacteria blooms. The former confirms the results of previous reports, which explained that DO diminishes during cyanobacteria blooms [Parada reservoir presented the lowest DO concentration, but other reservoirs have DO values within the range 5 to 6 mg·La blooms .Consequently, the deteriorating quality of water in reservoirs accelerates the ecosystem damage and water functions, which endangers the use of water resources ,56, prod3− is the main available carbon source, which favors cyanobacteria growth [The low transparency measured in the reservoirs indicates poor water quality, which reveals that high turbidity is most probably associated with cyanobacteria abundance . Anothera growth ,60.a was higher than 100 µg·L−1 in Camazán and Chalóns reservoirs, which show the worst conditions concerning the trophic status in recent studies [a is considered a good indicator of trophic conditions in water reservoirs, positively correlated with poor water quality [a [a concentration.Concentration of chlorophyll  studies . Althoug quality , the preality [a . This wa−1, which is considered another indicator of eutrophy [All the reservoirs studied were found to be eutrophic or hypertrophic when the TP or TN concentrations were analyzed. Additionally, all the reservoirs had phytoplankton concentrations higher than 1500 cell·mLeutrophy ,28,63. T−1, advisable for drinking waters [Sixteen reservoirs showed TN:TP atomic ratios higher than 20, especially Cautillo, Guisa, Hatillo, Jaibo, La Yaya, Mícara and Parada reservoirs, with MC-LR concentrations at the upper OMS guideline limit value of 1.0 μg·Lg waters . Though g waters , the res−1: Chalóns reservoir showed the lowest TN:TP atomic ratio (7), but its cyanobacteria concentration, mainly due to Synechocystis aquatilis bloom, was 9.8 × 104 cyanobacteria cells·mL−1; similar behavior was found in Baraguá reservoir (26 × 104 cyanobacteria cells·mL−1) with a TN:TP atomic ratio (11), associated with a bloom involving Cylindrospermopsis curvispora and Oscillatoria sp. This result points to the need for risk management related to cyanobacteria growth and cyanotoxin production, and suggests that low TN:TP should not favor microcystin-producing cyanobacteria [Two reservoirs showed TN:TP ratios lower than 20, and MC-LR concentrations, both intracellular and in water samples higher than 1 µg·Lbacteria .TN:TP atomic ratio analysis is useful as an indicator of cyanobacteria risk, according to different authors, but this analysis needs to consider other variables, such as phytoplankton and cyanobacteria cell concentrations, diversity and bloom occurrence .The highest values of MC-LR in water were detected in Hatillo, Baraguá, Chalóns, Mícara and Cautillo reservoirs, with high TP concentrations; the other two reservoirs, Parada and Mícara, showed high TN values. In those reservoirs less enriched with nitrogen and phosphorus, such as Los Plátanos and Clotilde, MC-LR were not detected in water. This means that TN and TP are important variables to consider into the analysis and management of cyanotoxin risk. Additionally, it is important to note that TP was involved in TSI analyses.−1 were considered eutrophic by different authors [4 cell·mL−1) was measured in Gota Blanca reservoir; concentrations higher than 15 × 104 cell·mL−1 were measured in Guisa, La Yaya, Hatillo, Mícara and Parada reservoirs.Another important aspect in this research is the variation of the phytoplankton concentration. Reservoirs with phytoplankton concentrations higher than 1500 cell·mL authors ,28,63. R4 cell·mL−1); Hatillo, Chalóns and Baraguá had cyanobacteria concentrations higher than 7 × 104 cell·mL−1. Regarding cyanobacteria percentage, besides Mícara reservoir (100%), Baraguá and Chalóns reservoirs are highlights, with more than 80% of cyanobacteria. The cyanobacteria percentage was higher than 40% of the total phytoplankton in 70% of the reservoirs. The Mícara reservoir is a special case with a Microcystis species blooms, which imply the need to develop more studies in relation to environmental variable fluctuation, the seasonal changes, sediment features, and stratification profiles.Concerning cyanobacteria, maximum cell densities were recorded in Mícara  and TSIaChl and TSIP, as well as eutrophic level had between 20 and 40% of normalized importance.Water temperature is widely reported as an influencing factor in the growth and development of cyanobacteria, and the production of cyanotoxins. High temperatures would not be the cause of cyanobacteria blooms, but they would be associated with other phenomena such as thermic stratification and changes in the depth of the mix zone, which can favor the development of cyanobacteria with gas vesicles . ChorophThis study shows that the combination of high-water temperature, high TN:TP ratios and low transparency, even within a pH range inside the guideline values (6.5–8.5) ,58, can −1).The climatic conditions in eastern Cuba favor the occurrence of cyanobacteria and cyanotoxin in the reservoirs studied. These are affected by eutrophication, evidenced by high levels of TN:TP ratios and phytoplankton cell concentrations, high water temperatures and low transparency, which cause collateral effects such as cyanobacterial bloom and microcystin-LR occurrence. The most affected reservoirs were Hatillo, Chalóns, Parada, Mícara, Baraguá, Cautillo, La Yaya, Guisa and Jaibo, with MC-LR concentrations higher than the drinking water WHO limits  . Twenty-All the reservoirs were sampled randomly in rainy and dry seasons, in order to evaluate the risk extension of the phenomena. A unique sampling was established during the morning in each reservoir during the first stage of this study, in order to analyze the influence of physicochemical and environmental factors, and the occurrence of toxic cyanobacteria growth and microcystin-LR, presenting evidence for the risk characterization, including the extension and microcystin levels during the sampling.Catchment areas were identified using the geographic information system , and SAGA software. Elevation data were defined by with the Digital Elevation Model (DEM) provided by the Shuttle Radar Topography Mission (SRTM) with 90-m resolution . Channel2, which corresponds to 55% of the total area of eastern Cuba ; 19 of the studied reservoirs belong to the three main watersheds of this region [The total catchment area of all reservoirs included in this study is 14,182 kms region : Toa-May2 and it is one of the most affected by human activities, containing about 37% of all water resources in the entire country [The Cauto watershed catchment includes three provinces . This watershed is the biggest of Cuba with an area of 8969 km country . The Toa country .Samples were collected in the early morning hours (8:00–9:00 am) at the water surface layer (0.5 m depth) of the reservoirs. Three locations were established in each reservoir, considering at least one point of the national monitoring water quality program, but other points were located near the reservoir tapping, and/or in relation to foam or the occurrence of cyanobacteria blooms .−1) and dissolved oxygen (DO) (mg·L−1), were measured in situ with a multi-parameter water quality meter . Transparency (m) was determined using a 20 cm Secchi-disc. To the in situ chlorophyll measurements, an AquafluorTM handheld fluorometer  was used, previously calibrated with chlorophyll a standard  (Chl a) correction factor was used, considering the resulting calibration curve equation, which was prepared from serial dilutions of a standard Chl a solution in methanol 90% (10 to 1000 μg·L−1) [Water temperature (°C), pH, conductivity (µS·cm μg·L−1) .The Cuban Norms 1021:2014 and 25:1999 established to assess the water quality of reservoirs for drinking water supply and fishing interest ,75 were −1 [Total nitrogen (TN) and total phosphorus (TP) were determined after persulfate oxidation to nitrate  and orth−1 .The phytoplankton samples collected at the water surface were preserved with Lugol 10% solution . Each saCyanobacteria counting was performed using a Neubauer hemocytometer chamber and a binocular microscope Olympus , and percentage of cyanobacteria cell concentration in relation to the total phytoplankton concentration was calculated. Taxonomical identification was performed for species classification, when it was possible, following the expert criteria ,82,83,84® glass microfiber filters Grade GF/F 25 mm, then, the filters with biomass were frozen at −20 °C [Microcystin-LR determination was performed for both intracellular and extracellular level, using water and filtered samples. Water samples (100 mL) were filtered through Whatmant −20 °C . Consequt −20 °C . This so® HPLC grade): water mix (3:6). The analyte-adsorbed column was washed sequentially with 6 mL water and 10 mL of MilliQ water: methanol 20% (v/v), dried at low-pressure. Microcystins then were eluted with 10 mL of MilliQ water: methanol 80% (v/v) [For MC-LR purification, the extracts were centrifuged  and the supernatant was purified through an Empore™ SPE Cartridge C18, which was previously conditioned with methanol (ROTISOLV0% (v/v) . The elu0% (v/v) ,89,90.The MC-LR was also extracted from filtered water samples (100 mL) through a purification process as described above.−1; ≥95%, HPLC). Serial dilutions were applied to produce standard solutions with concentrations ranging from 1 μg·L−1 to 100 μg·L−1. The calibration curve was based on the peak area in the chromatogram, measured with the software Empower 3.0 against the standard concentrations of microcystin-LR.Working standards for the calibration curve to quantified MC-LR were prepared using a stock solution standard from Enzo Life Sciences . The separation was achieved via an ACQUITY BEH-C18 column  and BEH VanGuard Pre-column  from Waters . Methanol , acetonitrile  and formic acid  were used. Milli-Q water was obtained using a Millipore purification system.−1 initially with 80% (A) and 20% (B) until minute 3, changing the proportion from minute 3 to 8 to 35% (A) and 65% (B) and keeping it until minute 10. Finally, it was changed to 80% (A) and 20% (B) until minute 13 [Columns were kept at 35 °C during analysis. The injection volume was 20 µL. The mobile phase A was Milli-Q water + 0.1% formic acid  and mobile phase B was acetonitrile + 0.1% formic acid . The gradient elution was performed as a variation  with a finute 13 .Detection and quantification of microcystins were performed in ACQUITY QDa Performance MS Detector  equipped with an electrospray ionization source (ESI), capillary voltage of 0.8 kV, using selected ion recording (SIR) with mass 995.60 Da for MC-LR determination. The source off-set was 15 V and the temperature was 400 °C ,90.Validation of the method consisted of defining the linearity of the limit of detection (LOD) and limit of quantification (LOQ). Linearity was assessed by the relationship between the signal (peak area) and the analyte concentration expressed by linear regression of 7 different standard concentrations .The determination of LOD and LOQ was based on the standard deviation of the response of blank samples injected, and the slope (m) of the calibration curve. LOD was calculated as 3.3 times the standard deviation (S) of the signal-to-noise ratio (urve (m) ,93,94,95−1 of microcystin-LR (Enzo Life Sciences) to water samples (100 mL), and to filters containing phytoplankton biomass. The same methodology was used in both cases. The SPE purification and analysis using UPLC–MS were performed in triplicate.Microcystin-LR recoveries were obtained by spiking 10 μg·Lp < 0.05), and principal component analysis (PCA), were conducted using R studio (Version 1.2.5019). The data were normalized using Ln (water temperature), Log10 , Ln (1/X) in microcystin-LR (intracellular and extracellular), inverse (conductivity) and square root of arcsine (transparency) transformation. Graphs were produced with SigmaPlot Version (11.0.0.77) software.Mean, variance , standard deviation, relative error and correlation tests between variables  were considered in water and in biomass.A study of neural networks was conducted using IBM SPSS Version 19.0 and Sigmaplot Version (11.0.0.77) software to analyze the normalized importance of the factors that condition the occurrence of microcystins, using 60% training, 30% test and 10% reserve. Standardized methods were used considering 2 factors (season and trophic state index), 7 covariates (phytoplankton, water temperature, TN:TP ratio, chlorophyll