Saccharomyces cerevisiae is widely employed as a cell factory for the production of biofuels. However, product toxicity has hindered improvements in biofuel production. Here, we engineer the actin cytoskeleton in S. cerevisiae to increase both the cell growth and production of n-butanol and medium-chain fatty acids. Actin cable tortuosity is regulated using an n-butanol responsive promoter-based autonomous bidirectional signal conditioner in S. cerevisiae. The budding index is increased by 14.0%, resulting in the highest n-butanol titer of 1674.3 mg L−1. Moreover, actin patch density is fine-tuned using a medium-chain fatty acid responsive promoter-based autonomous bidirectional signal conditioner. The intracellular pH is stabilized at 6.4, yielding the highest medium-chain fatty acids titer of 692.3 mg L−1 in yeast extract peptone dextrose medium. Engineering the actin cytoskeleton in S. cerevisiae can efficiently alleviate biofuels toxicity and enhance biofuels production. Saccharomyces cerevisiae.Product toxicity is one of the factors that hinder biofuel production. Here, the authors engineer the actin cytoskeleton to increase cell growth and production of n-butanol and medium-chain fatty acids in  Saccharomyces cerevisiae is a workhorse for the production of large amounts of biofuels, such as ethanol, n-butanol, isobutanol, and fatty acids from various feedstocks1. Owing to its high tolerance to harsh fermentation conditions and relative ease of genetic manipulation2, S. cerevisiae has been engineered to produce biofuels, such as short-chain alcohols and medium-chain fatty acids (MCFAs)4. For example, S. cerevisiae can produce 1.05 g L−1 n-butanol and 1.39 g L−1 MCFAs after the engineering of its native 2-keto-acid pathway5 and fatty acid synthases6, respectively. However, there are limits on further increases in titer, yield, and productivity of biofuels because the elevated hydrophobicity and lipophilicity of biofuels make them toxic to yeast3.S. cerevisiae. In evolutionary engineering, generating and taking advantage of plasticity of the genome is key to obtaining robust microbial cell factories, which can then be refined by adaptive laboratory evolution7 and artificial synthetic evolution8. Adaptive laboratory evolution has been widely applied to increase the stress tolerance of yeast to biofuels, such as MCFAs6, n-hexanol9, isobutanol10, and isoprenol11. Moreover, the rapid development of DNA synthesis methodologies8, multiplex automated genome evolution12, trackable multiplex recombineering13, recombinase-mediated evolution14, and CRISPR-mediated genomic editing technology15 have allowed direct evolution of microbial genomes as a means of augmenting tolerance to biofuels. For instance, after applying a random recombination strategy to assemble multiple stress defense circuits, including heat shock proteins, ubiquitins, antioxidant proteins, and stress-related transcriptional factors, ethanol toxicity to S. cerevisiae was alleviated, and ethanol production was increased by 6.9% on an industrial scale16. In transcriptional factor engineering, the goal is to regulate a group of genes. This can be achieved by engineering specific transcription factors17 and/or global transcription processes18. Global transcriptional regulators, such as sigma factors18, mediator complex19, and histone-like nucleoid structuring factor20, have been widely used to enhance biofuel tolerance. For example, engineering sigma factors (RpoD) by random mutagenesis successfully augmented ethanol tolerance in Zymomonas mobilis and increased ethanol production by 1.8-fold compared to the control21. In efflux pump engineering, the purpose is to relieve cellular toxicity by promoting the export of noxious compounds from the cell. Five families of efflux pumps, including the ATP-binding cassette (ABC) family22, the multidrug and toxin extrusion family23, the major facilitator superfamily24, the small multidrug resistance family25, and resistance nodulation division efflux pump family25, have been engineered to export the toxic biofuels to alleviate the impact on growth. For example, to reduce the cellular toxicity of alkane, the heterogeneous membrane ABC transporters ABC2 and ABC3 were introduced into S. cerevisiae, leading to enhanced alkane tolerance and a 30-fold increase in the export of undecane26. Together, these three strategies are efficient in improving the stress tolerance of cellular factories to biofuels; however, they are laborious and limited by the complexity of the stress tolerance mechanism. To further increase stress tolerance, the ways in which biofuels cause damage to the cell need to be elucidated. Knowing these damage patterns will facilitate development of a rational and targeted strategy.Various strategies, such as evolution engineering, transcriptional factor engineering, and efflux pump engineering, have been attempted to further increase the titer, yield, and productivity of biofuels in In this work, a rational strategy to improve biofuel tolerance is implemented by targeting the pattern of damage induced by n-butanol and MCFAs. Accordingly, n-butanol stress tolerance and production are enhanced by shortening actin cable tortuosity using an n-butanol responsive promoter-based autonomous bidirectional signal conditioner. MCFA stress tolerance and production are also elevated by increasing the actin patch density using an MCFA responsive promoter-based autonomous bidirectional signal conditioner. We demonstrate that this strategy has widespread potential to increase stress tolerance and improve the production of biofuels in yeast.600) was decreased by 53.9%  n-butanol, cell growth at the population level as measured by cell density  was decreased by 48.4%  dropped from 7.0 to 5.4  n-butanol, respectively. The bud6 overexpression strain showed a 6.1% decrease in budding index under the same conditions. Therefore, spa2 and cdc42 were selected as targets to improve the budding index. Furthermore, simultaneous deletion of spa2 and overexpression of cdc42 in strains W303-1A, W303-1B, BY4741, BY4742, Cen.PK2-1C, and thTAM in YNB medium with 0.8% (v/v) n-butanol was found to increase the OD600 by 88.3%, 79.6%, 53.9%, 104.0%, 106.7%, and 108.6%, respectively , and the second group included three deleted genes . As shown in Fig. pal1, syp1, sla2, and ent2 led to increases in actin patch density of 57.1%, 110.7%, 152.9%, and 18.8%, respectively, whereas overexpression of ent1 led to a decrease of 8.1%. On the contrary, the deletion of sla1 and clc1 resulted in increases in actin patch density of 57.1% and 129.9%, respectively, whereas deletion of ede1 resulted in a decrease of 4.3% . The aggregation was found only in the syp1-overexpressing strain, in which pHi was decreased, but not the clc1Δ strain or the sla2-overexpressing strain  and patches (clc1Δsla2OE) resulted in 13.1% and 12.7% decreases in the OD600 values compared with the control strain  containing an activation unit, a repression unit, and a biofuel signal converter was constructed Fig. .Fig. 4CoPRD), a hybrid LexO-cup1 promoter comprising a LexA operator (LexO), a cup1 core promoter sequence, and a GFP reporter gene. LexA-Ste12PRD can be recruited to LexO-cup1 to allow for transcriptional activation of the GFP reporter. In the absence of α-factor, the yeast mating pheromone response pathway is inactivated, which prevents the activation of LexA-Ste12PRD and the expression of GFP. Conversely, in the presence of the α-factor, LexA-Ste12PRD is activated, which induces the cup1 promoter and finally activates GFP expression  and the core promoters of LexO-cup1 were changed. As a result, GFP fluorescence intensity increased 12.7-fold and 16.3-fold following treatment with 0.8% (v/v) n-butanol and 0.2 mM decanoic acid, respectively  n-butanol triggered a 6.2-fold increase in GFP fluorescence intensity driven by the activation unit and a 4.8-fold decrease in mKate2 fluorescence intensity driven by the repression unit compared to the control Fig. , d. The rol Fig. , d. The 600) in strain LH001-B were increased by 1.2- and 1.7-fold, respectively  was 56.2% higher than that of the control strain  n-butanol treatment, compared to the control strain, the budding index and cell density   the repair phase, in which ABSC-butanol is self-induced by n-butanol, leading to the repression of spa2 expression by Gal80 and the activation of cdc42 expression by the tdh3 promoter.cum Fig. . Strain nol Fig. . Comparenol Fig. , (2) a 2nol Fig. , (3) a 400) Fig. , and (4)00) Fig. . These r00) Fig.  as follocdc42 and spa2, respectively. As shown in Fig. spa2 was activated during the first 50 h of n-butanol fermentation but was repressed thereafter, whereas cdc42 was increasingly expressed between 50 and 120 h  and the fraction of viable cells was 59.6% and 26.8% higher than that of strain LH002-B1, respectively  and a 42.4% decrease in the fraction of viable cells, compared to the corresponding values of the control strain LH003-M0  and the fraction of viable cells of strain LH003-M5 were 36.0% and 43.8% higher compared to those of strains LH003-M1 and LH003-M0, respectively ely Fig. . Consequely Fig. . These vi, respectively; (2) the actin cytoskeleton was engineered to improve the budding index and stabilize the pHi; (3) an autonomous bidirectional signal conditioner was constructed to manipulate the gene expression of the actin cytoskeleton upon accumulation of the biofuels. Based on these strategies, the production of n-butanol and MCFAs significantly increased to 1674.3 mg L−1 and 692.3 mg L−1, respectively. These results demonstrate that engineering the actin cytoskeleton can improve the performance of industrial strains.In this study, the actin cytoskeleton was engineered to improve cell growth and enhance the production of n-butanol and MCFAs  direct alleviation of the toxic effect of biofuels and (2) less disruption to the normal cell division cycle. Moreover, pHi is vital for maintaining the enzymatic activity of metabolic enzymes and partly determines cellular homeostasis52. Strategies to maintain pHi have focused on engineering weak acid stress response transcription factors53  and engineering membrane transporters6 . However, these two strategies might not directly stabilize pHi homeostasis, because toxic compounds may not be exported out of the cells selectively54. Here, the actin patch was engineered to improve the endocytic process and ensure correct formation of the vacuole, which is crucial for pHi homeostasis in yeast55; consequently, the pHi was normalized in an MCFA producing strain. The engineering of actin patch engineering leads to fewer side effects than engineering transcription factors39 and a lower growth burden than engineering transporters to alleviate the pHi defects22.Engineering the actin cytoskeleton could improve the budding index and stabilize the pH56 and modular engieering57) are not sufficient to enable microbial cells to adapt to the stochastic metabolic perturbations and a polytropic fermentation environment58. Therefore, autonomous decision-making genetic circuits may be a better solution to adjust microbial cells to intracellular or extracellular changes59. These genetic circuits consist of a sensing module, a processing module, and an output module, with the former two modules deciding the utility and efficiency. (i) The sensing module: the biosensor senses the intracellular  or extracellular signals . A series of genetic circuits have been developed based on these biosensors; a typical genetic circuit depends on a biosensor that detects the level of intermediates in a pathway. For example, a dynamic sensor-regulator system (DSRS), based on the fatty acid-sensing protein FadR, was developed to manage the trade-off between gene expression and fatty acid ethyl ester synthesis63. As a result, the DSRS substantially improved the stability of biodiesel-producing strains and increased the titer of fatty acid ethyl ester to 1.5 g L−1. Similar to the DSRS, the ABSC system was based on an n-butanol or MCFA responsive promoter; thus, it could autonomously and temporally regulate the gene expression without adding any small-molecule inducers  and could be used to control the biosynthetic pathway through changing the corresponding product as required. (ii) The processing module: the convertor, such as a nucleic acid- or protein-based state machine65, Boolean logic gate67, and CRISPRi system68, are key components of the processing module that act to convert the upstream signal into cellular behaviors. In the ABSC system, the endogenous yeast pheromone response pathway was used as the convertor, which could convert signals from biofuel accumulation into endogenous ones to regulate the expression of the corresponding genes. Through this convertor, the ABSC system could maintain cellular homeostasis69 while avoiding the problem of expression of genes competing for cellular resources  and providing high compatibility for different conditions.Finally, an ABSC was constructed that could autonomously and temporally regulate the gene expression of the actin cytoskeleton upon the accumulation of biofuels. To construct an efficient microbial cell factory, static strategies  adh1, adh2, adh3, adh4, and adh5  were knocked out. Then, the hbd encoding 3-hydroxybutyryl-CoA dehydrogenase, crt encoding 3-hydroxybutyryl-CoA dehydratase, adhE2 encoding alcohol dehydrogenase, and ter encoding trans-enoyl-CoA reductase from Clostridium acetobutylicum, and eutE encoding butyraldehyde dehydrogenase from E. coli were codon-optimized to S. cerevisiae and synthesized by Genewiz. After that, these genes consisted of the heterologous n-butanol synthesis cassettes, and then they were integrated into the genomic ura3-1 locus of S. cerevisiae W303-1A. Additionally, coaA and adhEA267T/E568K/R577S from E. coli were integrated into genomic adh6 and sfa1 locus, respectively. Finally, the native promoter of fms1, erg10, ald2, and pan6 were replaced into adh1, hxt7, fba1, and gpm1, respectively. For n-butanol production in a shake flask, yeast nitrogen base (YNB) medium (6.7 g L−1 yeast nitrogen base without amino acids and 20 g L−1 glucose) was used as a regular culture for each yeast strain. The seed cultures were inoculated in the YNB medium until reaching the exponential phase. After washing by sterilized double distilled water two times, the seed cultures were inoculated aerobically into a 100 mL flasks with 50 mL fermentation medium71 2SO4, 20 mM KH2PO4, supplemented with 0.171 mM uracil, 0.439 mM leucine, 0.093 mM tryptophan, 0.124 mM histidine, 0.134 mM methionine, 0.08 mM adenine and 20 g L−1 glucose) with a starting optical density at 600 nm (OD600) of 0.3 for fermenting 120 h under 200 r.p.m at 30 °C.For constructing the n-butanol synthesis pathway in S. cerevisiae Cen. PK2-1c, the gene pox1 (encoding fatty-acyl coenzyme A oxidase), faa1, and faa4 (encoding long-chain fatty acyl-CoA synthetase) were knockout simultaneously. Then, Fas2-expressing cassettes [tdh3promoter-Scfas2-fba1terminator] were integrated into chromosome loci X-2. Moreover, acyl-CoA thioesterase from Yersinia pestis (tesB), ATP:citrate lyase from Mus musculus (acl), cytosolic NADP+-dependent malic enzyme from Rhodosporidium toruloides (me) and citrate synthase from R. toruloides (cit) were were codon-optimized and synthesized by Genewiz. acl, me, and cit expression cassette were integrated into hfd1 locus, tef1p-acc1(S659A & 1137-1170 → GGGG)-adh1t was integrated into gpd1 locus, and tesB expression cassette was integrated into eeb1 locus. At last, the native promoter of pyc1, idp2, and yhm2 was replaced by pyk1, adh1, and gpd1, respectively. For medium-chain fatty acids (MCFA) production, the pre-cultures were inoculated in modified YPD medium  until reaching the exponential phase under 200 r.p.m at 30 °C. After washing by sterilized double distilled water two times, the seed cultures were inoculated into 250 mL with 50 mL YPD (containing 200 μg mL−1 geneticin and nourseothricin) with an initial OD600 of 0.1 for fermenting 60 h under 200 r.p.m at 30 °C.For constructing MCFA synthesis pathway in R, leu2) and yCPlac33 . In MCFAs producing strain, to adapt the ABSC system in YPD medium, the auxotrophic marker of yEPlac181 and yCPlac33 were replaced to KanMX4 and NatMX6, and the plasmids were named yEPlac182 and yCPlac34, respectively. Geneticin (200 μg mL−1) and nourseothricin (200 μg mL−1) were added to maintain the expression of plasmids.For autonomous and temporal control of actin cytoskeleton in n-butanol and MCFA producing strain, Gibson assembly and molecular cloning techniques were applied to construct the ABSC related plasmids. In n-butanol producing strain, the ABSC system were constructed based on plasmid yEPlac181  and incubated for another 10 h. After that, the cells were collected, centrifuged, and then resuspended with phosphate-buffered saline (PBS) containing propidium iodide. The cells were dyed at room temperature for 15 min under dark conditions. During the measurement of cell growth, two steps were implemented to prevent evaporation of n-butanol. (1) For analyzing the spot assay, the YNB agar plates contained n-butanol were wrapped with parafilm and taped. (2) For PI stain and cell density analysis, the yeast cells were grown on glass tube with rubber cap and the syringe needle was applied to collect the samples.For analyzing the spot assay of 600 corresponds to a cell dry weight (DCW) of 0.213 g L−1. Finally, the cell viability was analyzed by the BD FACSArica III cytometer. And the data were analyzed by FlowJo software (FlowJo-V10).Moreover, in this study, one unit of ODg for 5 min, and then the precipitate was by phosphate-buffered saline at pH 7.4 for three times. After that, the supernatant was discarded, and then the cells were fixated by adding 2.5% glutaraldehyde at room temperature for 4 h. Later, the cells were washed with phosphate-buffered saline three times. At last, the cells were prepared by the FESEM . For time-lapse microscopy, yeast cells were cultivated at 30 °C for 8–10 h, then cells were diluted to an optical density OD600 = 0.2 in YPD medium, grown for 6 h to mid-log phase. After that, the yeast cells were collected by centrifugation at 5000 g for 5 min, and then the cells were diluted to OD600 = 1. Then, 2.5 μL diluted yeast cell culture was placed on a YPD plate in a microscope cavity slide. Finally, the cells were in suit incubated at 30 °C and this time was set as zero-time. Microscopy images were taken using a Nikon ECLIPSE 80i microscope equipped with a ×100 oil-immersion objective.For field emission scanning electron microscope (FESEM) assay, the yeast cells were collected by centrifugation at 5000 To detect the yeast budding index, the yeast cells were incubated on a YNB medium supplemented with needed amino acids for 10 h at 30 °C. Then, the yeast cells were treated with or without 0.8% (v/v) n-butanol treatment for 12 h at 30 °C. After washing by sterilized double distilled water three times, the cell was suspended by YNB medium supplemented with needed amino acids and 20 μM alpha-factor  for synchronizing the cell cycle in the G1 phase. After washing by sterilized double distilled water three times, the treated cells were released into a pre-warmed YNB medium. The released cells were collected every 10 min and then spread on YNB solid plates supplemented with needed amino acid. Lastly, the colony-forming unit (CFU) was calculated to analyze the budding index of each strain. For applying Cdc10-GFP for analyzing relative budding index, the genomic Cdc10 was fused with GFP by using the CRISPR-Cas9 system, and then the cells were observed by fluorescence microscope.For FM4-64 labeling, yeast cells were grown to the mid-log phase, and then cells were stained with FM4-64 for 20 min on ice. After that, cells strain with FM4-64 were released to 30 °C. Finally, the cells were observed at intervals of up to 2 min. For alpha-factor labeling assay, the yeast cells were grown to the mid-log phase and placed on ice for 15 min. After that, the yeast cells were mixed with 2 μM Cy5-α-factor. After 1 h on ice, the yeast cells were washed three times by ice-cold PBS and then resuspended by room temperature PBS to detect the internalization of α-factor by FACS.600 = 0.6 in YPD medium, and then the cells were fixed directly by adding formaldehyde into the medium to a final concentration of 3.7% for 10 min. After that, the cells were centrifuged for 5 min at 5000 g. And, the cells were fixed again by PBS containing 3.7% formaldehyde. After washing by PBS three times, the cells were stained with Alexa Fluor 488-phalloidin at room temperature for 1 h. Finally, the cells were imaged by Leica TCS SP8, and then the ratio of cable length to the distance between cable endpoint (cable tortuosity)33 were analyzed by Image J . For analyzing actin patch density, the genomic Abp1 was fused with GFP and the cells were imaged by Leica TCS SP8, and the cell area and patch amount were analyzed by Image J, respectively.Yeast cells were grown to OD72.410 nm and Fi470 nm is the mean fluorescence intensities of cells that expressing pHluorin-based pH probes, and Fi410 nm background and Fi470 nm background is mean fluorescence intensities of cells without pHluorin73.max and RpHmin are the limited value for the ratio at extremely acidic (pH 5.0) and alkaline (pH 8.5), respectively. The constant pKa was analyzed by in situ calibration curve72.The dual excitation of pHluorin was tested by a blue solid-state laser that emits 50 mW of light at 488 nm and a violet diode laser that emits 100 mW of light at 405 nm. In each trial, 20,000 cells were analyzed, and the data were analyzed with FlowJo software. The pHluorin fluorescence was emitted at 510 nm and excited at 410 and 470 nm, respectively. The mean fluorescence intensities of each cell after excitation at 410 nm and 470 nm were recorded and correlated with pHi by using Eqs. 2−1 Na2HPO4, 0.32 g L−1 NaH2PO4, and 0.9 g L−1 NaCl), and then repeat centrifuge-resuspend steps two times. The collected cells were incubated at 37 °C for 30 min with buffered solutions that contained 20 μM nigericin, 150 mM KCl, and 50 mM buffering agents . After treating the cells with 10 μM nigericin, the cells were incubated on ice for 30 min to equilibrate the intracellular pH with external pH. Then, the mean fluorescence intensities of cells were analyzed through flow cytometry, and plotted the calibration curve between pHi and mean fluorescence intensities. Finally, the pKa was determined from the calibration curve. The measurement of intracellular pH is consistent with previously published studies75 in both normal and MCFA treated conditions.For analyzing in situ calibration curve, The yeast cells were incubated on the ice for 5 min, then centrifuged and resuspended by ice-cold PBS buffer .GFP or mKate2 expression Eqs. –6 in ABS600 of 0.6 at 30 °C. The synthetic alpha-factor  was added into the cultures for synchronizing the cell cycle in the G1 phase, then the mixture was incubated at 30 °C for 2 h. After that, the cells were washed twice by pre-warmed YPD medium, and the synchronized cells were released in pre-warmed YPD medium with or without 0.8 % (v/v) n-butanol. The samples were collected every 15 min, and the cells were then fixed in 70% ethanol for 10 min and kept at 4 °C for further processing. After centrifugation, the pellets were resuspended with ice-cold 50 mM sodium citrate solution containing 0.1 mg mL−1 Rnase A and then grown overnight under 30 °C. Finally, after centrifugation, the pellets were suspended with a 50 mM sodium citrate solution containing propidium iodide, and then the mixture was prepared in a FACS tube for analysis by BD FACSArica III cytometer. And the data were analyzed by FlowJo software (FlowJo-V10).To analyze the cell cycle, the yeast cells were incubated in a YPD medium to ODTM III cytometer  was used to measure the fluorescence of each sample. GFP, Annexin V, and NBD-lipids were detected in the FITC channel , whereas mKate2 and PI stain were detected in the PE-Cy5-A Red channel . 10,000 cells were analyzed for each sample. All data were analyzed by FlowJos software (FlowJo-V10).The yeast cells were washed twice by PBS buffer and resuspended to an OD600 of 0.1. A BD FACSAricaact1, encoding the β-actin, was used as a loading control to normalize the gene expression.The yeast cells were cultivated to the mid-log phase and harvested. After washing by DEPC-treated water, total RNA was extracted using the MiniBEST universal RNA extraction kit , and cDNA was synthesized by PrimeScript II first-strand cDNA synthesis kit . After that, the quantitative analysis of the mRNA level of each sample was performed by using TB Green qPCR Master Mix  in Bio-Rad CFX96 Touch . In this study, the For measuring the fluorescence intensity, the yeast cells were cultured on a YNB medium supplemented with the needed amino acid for 10 h at 30 °C. The fluorescence of cell culture was measured by a SpectraMax M3 microplate reader . The detection of GFP fluorescence intensity was set at 480 ± 10 nm of excitation wavelength and 515 ± 10 nm of emission wavelength. And the detection of mKate2 fluorescence intensity was set at 588 ± 10 nm of excitation wavelength and 645 ± 10 nm of emission wavelength.For n-butanol quantification, the fermentation broth was centrifuged at 8000 g for 10 min, and the supernatant was collected. Then the n-butanol concentration was determined by gas chromatography-mass spectroscopy (GC-MS) an Agilent 7890 A GC system and HP-INNOWAX column. For the extraction of medium-chain fatty acids (MCFAs), the fermentation broth was centrifuged at 8000 g for 10 min, and the supernatant was collected. Then, the mixture of hexane: chloroform  was used for extracting the MCFAs. After that, the organic layer was collected and evaporated through nitrogen. To measure the concentration of MCFAs, samples were resolved in a mixture of methanol: sulfuric acid: chloroform  to heat at 70 °C for 1 h. Subsequently, the Fatty acid methyl esters (FAMEs) were extracted by hexane. Finally, the FA methyl esters were measured GC-MS on an Agilent 7890 A GC system and a Rxi-5Sil column , following the method of an initial temperature of 35 °C held for 1 min, 6 °C/min to 200 °C, 30 °C/min to 270 °C, held for 1 min.t-test by SPSS statistics software (SPSS R24.0.0.0). The data analysis and graphing were performed by Origin 2019 64 bit. All experiments in this study were performed in three biological repeats.The significance of groups of data was determined by a two-tailed Student’s Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Reporting Summary


SDG) gene family, the full-length coding sequence (CDS) of CsSDG36 was cloned from the tea cultivar ‘Fuding Dabaicha’. Bioinformatics analysis showed that the open reading frame (ORF) of the CsSDG36 gene was 3138 bp, encoding 1045 amino acids and containing the conserved structural domains of PWWP, PHD, SET and PostSET. The CsSDG36 protein showed a close relationship to AtATX4 of the TRX subfamily, with a molecular weight of 118,249.89 Da, and a theoretical isoelectric point of 8.87, belonging to a hydrophilic protein without a transmembrane domain, probably located on the nucleus. The expression of CsSDG36 was not detected in the wild type, while it was clearly detected in the over-expression lines of Arabidopsis. Compared with the wild type, the over-expression lines exhibited lower hyperosmotic resistance by accelerating plant water loss, increasing reactive oxygen species (ROS) pressure, and increasing leaf stomatal density. RNA-seq analysis suggested that the CsSDG36 overexpression caused the differential expression of genes related to chromatin assembly, microtubule assembly, and leaf stomatal development pathways. qRT-PCR analysis revealed the significant down-regulation of stomatal development-related genes , EPF2, TCX3, CHAL, TMM, SPCH, ERL1, and EPFL9) in the overexpression lines. This study provides a novel sight on the function of histone methyltransferase CsSDG36 under drought stress.Histone methylation plays an important regulatory role in the drought response of many plants, but its regulatory mechanism in the drought response of the tea plant remains poorly understood. Here, drought stress was shown to induce lower relative water content and significantly downregulate the methylations of histone H3K4 in the tea plant. Based on our previous analysis of the SET Domain Group ( Arabidopsis  was used for hyperosmotic stress treatment. Seedlings were pre-grown for 2 months in a basic solution ((NH4)2SO4 (0.25 mM), NH4NO3 (0.5 mM), KH2PO4 (0.5 mM), K2SO4 (0.2 mM), Ca(NO3)2 (0.5 mM), MgSO4 (0.8 mM), Al2(SO4)3 (0.08 mM), NaFeEDTA (35.0 μM), H3BO3 (23.0 μM), MnSO4 (4.5 μM), CuSO4 (0.15 μM), ZnSO4 (1.0 μM) and Na2MoO4 (0.05 μM))  × 100%, where M0: fresh weight of rosette leaves; M1: weight of rosette leaves after soaking in water for 24 h; M2: weight of rosette leaves after drying at 80 °C for 24 h. The results were obtained from three biological experiments with three technical repetitions.For physiological analysis, Arabidopsis leaves and roots were ground in liquid nitrogen and dissolved in 1×PBS buffer (pH = 7.8). After centrifugation, the supernatant was collected to detect the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT).The SOD activity was determined by NBT photoreduction as reported by Beauchamp and Fridovich (1971) with slight modifications. Briefly, the enzyme solution (0.05 mL) was added to 2.95 mL reaction solution , 0.1 mmol/L EDTA, 0.22 mol/L methionine and 33 mol/L riboflavin) for photochemical reduction reaction under 4000 lx rays for 20 min, and the OD value was obtained at 560 nm. SOD activity = [(A560CK − A560e) × V]/(A560CK × 0.5 × DW × Vt), where A560CK is the absorption value of the control substance at 560 nm; A560E, the absorption value at 560 nm; V, the total volume of crude extract; DW, the sample dry weight; and Vt, the volume of crude extract used for measurement. The results were obtained from three biological experiments with three technical repetitions.2O2 after cooling. The light absorption value was determined at 470 nm. POD activity = (ΔA470 × Vt)/(0.01 × t × Vs × DW), where ΔA470 is differences in the absorption value at 470 nm; V, total volume of crude extract; DW, sample dry weight; Vt, volume of crude extract used for measurement; and T, reaction time. The results were obtained from three biological experiments with three technical repetitions.The POD activity was determined by guaiacol assay as reported by Gajewska and Skl Odowska (2007). The enzyme solution (0.05 mL) was mixed with the reaction solution (50 mL 0.05 mol /L PBS (pH = 6.0) and 28 μL guaiacol), followed by heating to dissolve and adding 19 μL 30% H2O2 method as described by Okuda (1991). Briefly, the enzyme solution (0.1 mL) was added to 1.9 mL PBS (pH = 7.0) and 1 mL 0.1 mol/L H2O2 to determine the absorbance value at 240 nm. The CAT activity = (ΔA240 × Vt)/(DW 0.01 × Vs), where ΔA240 is differences in the absorption value at 240 nm; V, total volume of crude extract; DW, sample dry weight; Vt, volume of crude extract used for measurement; and T, reaction time. The results were obtained from three biological experiments with three technical repetitions.The CAT activity was determined by the HThe malondialdehyde (MDA) content was determined by spectrophotometry described by Chen et al. (2021). Briefly, the MDA in the sample was extracted with trichloroacetic acid, reacted with thiobarbituric acid (TBA) to form a pink compound, and its absorbance value at 532 nm was determined and compared with the standard. The MDA content = c × V/m where c is the MDA concentration calculated from the absorbance value at 532 nm and standard curve; V, volume of sample solution; and DW, sample dry weight. The results were obtained from three biological experiments with three technical repetitions.Arabidopsis thaliana were sampled every day. Finally, the untreated and treated Arabidopsis thaliana leaves were fixed with 1% glutaraldehyde fixative solution, and chlorinated trichloroacetaldehyde was used to facilitate the observation of leaf stomata. Under a full dynamic normal fluorescence microscope, the stomatal number in the leaf lower epidermis was observed and the number per unit area was counted. A minimum of 10 fields of view were counted for each plant line, and the stomatal length-width ratio was counted for at least 50 stomatal in each sample. The results were obtained from three biological experiments with three technical repetitions.The stomatal number was analyzed using the nutrient solution culture method as reported by Conn et al. (2013). Briefly, homozygous Arabidopsis seeds were disinfected and seeded on agar, which was placed on an opaque 0.5 L box filled with nutrient solution. After 14 days of growth, the seedlings were transferred to an 8 L sealed container filled with nutrient solution and cultured for 6 days, followed by 2 days of treatment with 10% PEG 6000. The leaves of Arabidopsis thaliana strains, the leaf and root samples of Arabidopsis thaliana overexpression line 2 and wild type were collected for RNA isolation using the rapid RNA isolation kit  and the quality of the RNA was tested. The qualified RNA samples were embedded in dry ice and sent to Tianjin Novogene Co., LTD. Tianjin For the library construction process, RNA samples were pooled, respectively, according to the effective concentration and the amount of target data for RNA-seq. After statistics, quality control, assembly analysis and functional annotation for the raw transcriptome data, the obtained transcripts were subjected to differential gene screening, GO enrichment and KEGG pathway analysis. The results were obtained from three biological experiments.In order to study the gene expression dynamics of overexpressed GADPH as the reference gene, using the primers shown in −Δ CT = 2−(CT target gene − CT reference gene). The results were obtained from three biological experiments with three technical repetitions.Total plant RNA was extracted using the “rapid RNA isolation kit” , and reverse transcription was performed using the TRUEscript RT Kit (+gDNA Eraser) reagent from Beijing Adlai Biotechnology Co., Ltd., Beijing, China. Agarose gel electrophoresis was used to detect the PCR products. The qRT-PCR reaction was performed using the “2× SYBR Green qPCR Mix (with 100 × ROX)” reagent from Beijing Adlet Biotechnology Co., Ltd., Beijing, China. The PCR cycle was performed with a three-step method, with t-test was used to determine the significant difference between the two samples. The results were considered statistically significant at p < 0.05.All the data and error bars were calculated using three independent experiments. The


Indocyanine green was repurposed as iron theranostics for simultaneous detection and treatment of iron-overload disorders. 3+ in vitro and in vivo. The high sensitivity and specificity of ICG-Fe interaction facilitated MR images with a marked correlation between T1 signal intensity ratio (T1SIR) changes and Fe3+ concentration in rodent models and humans. On the basis of these findings, a rational design for coordination-driven self-assembly ICG-Lecithin (ICG/Leci) was proposed to determine Fe3+. The enhancement of photoacoustic signal at 890 nm with increasing Fe3+ concentration showed an over 600% higher linear slope than that of T1SIR changes in animal models. ICG/Leci also promoted a 100% increase in iron depletion in the liver compared with deferoxamine. The high MR sensitivity and superior photoacoustic contrast, combined with enhanced iron depletion, demonstrate that ICG/Leci is a promising theranostic agent for simultaneous detection and treatment of iron-overload disorders.Precise and noninvasive theranostic methods to quantify and deplete focal iron are of crucial importance for iron-overload disorders. Here, we developed an indocyanine green (ICG)–based imaging platform to reveal Fe Iron is an essential element for a variety of metabolic processes in most living systems  capabilities offering better treatment planning and assessing therapeutic outcomes. Compared with free ICG and deferoxamine (DFO), one of the world’s most prescribed iron depletion drugs, ICG/Leci could more significantly increase iron excretion and reduce serum ferritin levels in vivo. DFO nephrotoxicity featuring the infiltration of inflammatory cells in the kidneys was found in the DFO group, but the good safety of ICG/Leci was demonstrated here. Overall, this ICG-based system could potentially address the critical need for iron deposition theranostics in the clinic.It is believed that ICG molecules assembled with short- and long-range order can display optical properties distinct from the individually dispersed monomers (−1 (fig. S1). When ferric chloride was added to ICG, several aggregation modes were investigated with density functional theory (DFT) calculations  at −36.4 kcal mol−1 for ICG-Fe3+. Thus, the addition of ferric chloride to ICG can promote aggregation. To further confirm the aggregation, the binding affinity of Fe3+ for ICG was detected by isothermal titration calorimetry (ITC) with a dissociation constant (Kd) of 0.64 μM. ΔG was −35.33 kcal mol−1 for the first stage, Kd was 6.91 μM, and ΔG was −29.44 kcal mol−1 for the second stage (fig. S3A). We also measured Kd with 6520.49 μM and ΔG at −12.47 kcal mol−1 for Fe3+-DFO (fig. S3B), which indicated that ICG has a much higher affinity than DFO  to Fe3+ under this condition.The interaction energy, calculated by Gaussian09, between ICG molecules is −20.2 kcal molr1 value of Fe3+, which was approximately 50-fold lower than that of free Fe3+ under the MRI scanner  (fig. S4A) was independent of the added ICG (fig. S4B). The r1 value of Fe3+/a-ferritin hybrid decreased with a significant value of 2.7 mM−1 subtracted from (Fe3+ + a-ferritin) to (Fe3+ + a-ferritin)/ICG . In contrast, normative T2* values of (Fe3+ + a-ferritin) and (Fe3+ + a-ferritin)/ICG showed no significant difference  probably due to the strong susceptibility artifact under high field strength /ICG . The r1 fference . Furtherstrength . This in3+ or ICG-Fe3+ injection . Then, two different iron-overload phenotype knockout mice, Hfe−/− mice and Hjv−/− mice , Hfe−/−, and Hjv−/− mice at 4 hours after intravenous injection of ICG. T1SIM /T1SIL  for mice treated with ICG. At 0-, 1-, and 4-hour time points after treatment with ICG, the signal from Fe3+ in the liver of WT mice showed only subtle changes from 0, −3.61, to 1.74%. However, the signal changes in the Hfe−/− and Hjv−/− mice showed more apparent changes of 19.19 and 29.97% at 4 hours, respectively. Linear regression analysis showed that mice treated with ICG had an iron concentration–dependent increase  with a regression line of y = 0.03207x − 1.314 in T1SIM/T1SIL change at 4 hours  mice with hepatic iron overload (−1), T1SIM/T1SIL of the liver tissues in HBV-Tg mice showed a 14.91% change of the relative T1SI at 4 hours versus 0 hours (−1 tested by atomic absorption spectrometry was in good agreement with that from the MRI measurement (−1) before surgical removal of the tumor , the T1 signal response in case 1 showed a −0.5% change at 1 hour versus 0 hour, while there was a 14.71% change in case 2 (−1(mild positive Prussian blue staining) and 677.54 ± 146.31 μg g−1 (moderate positive Prussian blue staining), respectively  0 hours . Moreovesurement . Note thhe tumor . Relativn case 2 , and the3+ concentrations exhibited an increasing peak at 875 nm over time . However, this process is time-consuming and requires a high Fe3+ concentration (>3 μM) (fig. S8J). To further promote the aggregation and surmount monomerization challenges from circulating albumin in vivo, Leci was introduced to control the orientation of ICG molecules and thus contribute to the formation of J-aggregation (−1 after Leci was added into ICG (fig. S9), which was significantly higher than when ferric chloride was added to ICG/Leci (Kd with 34.49 μM after Fe3+ was dropped into ICG/Leci (fig. S11B); high selectivity for Fe3+ over Zn2+, Cu2+, Mg2+, Ca2+, and Gd3+ was demonstrated . However, no noticeable signals at 890 nm were seen in the control groups without adding Fe3+ as shown in fig. S13. Consequently, the PA intensity at 890 nm gradually increased upon the addition of Fe+3 (3+ (fig. S14B). Fe3+/ICG/Leci retained low fluorescence intensity, which might be primarily due to solid π-π stacking between ICG and the inner Leci shell mediated by Fe3+. This interaction contributes to the maximum PA intensity at 890 nm. In the presence of ICG/Leci, we also observed a significant decrease in the value of r1 of Fe3+ -2,5-diphenyltetrazolium bromide) and (Cell Counting Kit-8) (CCK8) assays. Fencreased . As showT1SI changes were observed when treated with different concentrations of ICG or ICG/Leci . However, there was a significant reduction of fluorescence intensity in the ICG/Leci group when cells were preincubated with ferric ammonium citrate (FAC) . In contrast to the free FAC group, few ICG/Leci . NoticeaICG/Leci . The T1S−/− mice was recorded by fluorescence at 672 nm. Figure S17 (A and B) shows that the liver fluorescence intensity in the ICG/Leci group was much higher than that of the ICG group in the WT mice, but it decreased more quickly in the Hfe−/− mice with time. It is believed that this is due to the increasing π-π stacking among ICG molecules under activation of excess Fe3+. Figure S18 (A and B) shows the co-registered ultrasound + PA images of Hjv−/− mice from 0-, 1-, 4-, 8-, 12-, to 24-hour time points after intravenous injection with ICG and ICG/Leci. The PA signal at 890 nm gradually increased. After ICG injection, little PA signal change could be observed. Encouraged by that, more experiments were conducted in WT and Hfe−/− mice with a moderate iron concentration in the liver. The cross-sectional 3D images were reconstructed from the PA signals at 4 hours as shown in −/− and Hjv−/− mice, which is consistent with the distribution of iron surrounding the vessels among the liver parenchyma as seen in Prussian blue images . The optical contrast of vessels in the 3D images remained clear in WT mice originating from Hb and HbO2. The PAI signal intensity at 890 nm in Hfe−/− and Hjv−/− mice increased with time  .Before MRI/PAI, the biodistribution of ICG and ICG/Leci in the livers of WT and Hfeith time . It was  − 34.87 , with a − 1.314) . In addiTo verify the potential clinical application of ICG/Leci as a PAI contrast agent for iron deposited in the liver, we next screened tissue specimens from the iron-overload patients confirmed by liver biopsy measurements. No PA signal was observed before ICG/Leci staining (fig. S20A). On the contrary, the PA signal was positive when the iron-deposited section was stained , left. T−/−, and Hjv/− mice. T1 signal from the WT, Hfe−/−, and Hjv−/−mice at 0 and 8 hours after intravenous injection of ICG/Leci. The relative changes in T1SIM/T1SIL signals were 0.2, 18.73, and 29.54% for WT, Hfe−/−, and Hjv−/−, respectively , Western blot, and immunohistochemistry. The iron content is shown in The in vivo toxicity of ICG/Leci was also characterized by histochemical and blood biochemical analyses. Both the Fenton reaction caused by deposited iron and administration of DFO would lead to renal injury. This helped ensure inflammatory cell infiltration . Conversr1 value  are vital for developing sufficiently sensitive probes for LIC clinical detection. Repurposing approved drugs for LIC detection is a critical resource for potential probes. The initial goal of this study is to determine the ferric ion chelation capability of ICG in situ for signal amplification to decrease the detection limit in LIC mouse models and human subjects; here, high-performance MR measurements could be calibrated via atomic absorption spectrometry and biopsy. This strategy fits with the mechanism of water exchange restrictions around Fe ions influenced via the hydrophobic group of ICG, which leads to a shorter spin-lattice relaxation time and a lower 3+) in a short time. This indicates a good linear relation between T1SIR  and LIC. However, the different types of iron influence R2 and R2* relaxometry  in different ways that result in an inaccurate reflection of LIC  and produce a detectable PA signal after chelating FeTo the best of our knowledge, no previous work has introduced a probe for simultaneous iron quantification and depletion in vivo. The ICG/Leci formulas with dual-modality imaging can not only accurately diagnose LIC for better treatment planning but also simultaneously exhibit much higher iron depletion efficacy than DFO following image-guided assessment of therapeutic outcomes. Our results further suggest that polymeric ICG could more significantly increase iron excretion and reduce kidney failure in mice than most extensively used DFO. Notably, DFO or other FDA-approved iron chelators face unfavorable pharmacokinetics and significant toxicity, although they are macromolecules with a long circulation half-life  with 100 μM ICG. Thus, the 2.5 mg kg−1 dose of ICG could completely satisfy the reaction concentration of chelatable iron pool in mouse liver. Considering these two points, 2.5 mg kg−1 ICG satisfies security and validity in mice. More accurate calculations can be introduced to future dosage designs in mice or further pilot clinical studies. The second is ion selection. There are many kinds of metal ions  in the liver . Lecithin was purchased from Sangon Biotech (Shanghai) Co. Ltd. MTT and 4′,6-diamidino-2-phenylindole (DAPI) were obtained from Sigma-Aldrich Co. Ltd. . Dulbecco’s modified Eagle’s medium and fetal bovine serum were purchased from Hyclone Inc. All other chemicals were purchased from Sigma-Aldrich in analytical grade and used without further purification.All calculations were carried out using Gaussian09. Optimization of closed shell singlet spin states was performed with restricted-B3LYP (RB3LYP), and the high spin states were performed using unrestricted-B3LYP (UB3LYP). A mixed basis set 6-31G(d) for the normal atoms and Stuttgart Dresden (SDD) for Fe were used for geometry optimizations and frequency analysis. Computed structures were illustrated using CYLView.3+ binding with ICG or ICG/Leci at 20°C. Hepes buffer , which mimicked acid environments in lysosomes, containing CH3OH, CHCl3, and n-hexane (15:6:1), was mixed and allowed to stand for 24 hours. ICG (2 mM), DFO (2 mM), and ICG/Leci (2 mM:4 mM) were injected 19 times (2 μl per time) into Fe3+ (0.1 mM). The data were repeated twice and analyzed by OriginPro 6s software.A MicroCal ITC 200 microcalorimeter  was used to measure the Fe3 were dissolved in HCl (pH 4.5). The changes in fluorescence, absorbance, and optoacoustic signal of ICG and ICG/Leci upon addition of various concentrations of Fe3+ were recorded after different rounds of mixing. An ultraviolet-visible (UV-vis) system was applied to detect the absorbance changes from 200 to 1000 nm. Fluorescence production was recorded at an excitation wavelength of 680 nm. The photoacoustic signal was acquired at 890 nm .ICG, lecithin, and FeCl3+, Fe3+/Leci, Fe3+/ICG, Fe3+/ICG/Leci, ferritin, a-ferritin, ferritin/ICG, and a-ferritin/ICG at final Fe3+ concentrations  were dispersed in 2% agarose and recorded with an MRI scanner. The signal intensity was evaluated by analyzing the region of interest (ROI): ICG—0, 0.0156, 0.0313, 0.0625, 0.125, 0.25, and 0.5 mM; lecithin—0, 0.0313, 0.0625, 0.125, 0.25, 0.5, and 1 mM.The MRI experiment was performed on a 1.5-, 3-, and 9.4-T MRI scanner. Different concentrations of Fe3+ , or Fe3+  following ICG . The MRI experiment was performed under a 9.4-T MRI scanner with the same parameters as above.Animal experiments were approved by the Animal Care and Use Committee of Xiamen University. WT 129S1/SvTmJSlac and c57 mice were obtained from Beijing Vital River Laboratory Animal Technology Co. Ltd. The isolation of liver tissue was performed in 129S1/SvTmJSlac mice after being anesthetized with 1% isoflurane. Liver tissues were subsequently collected from the abdomen and cut into small pieces and then injected with ICG , Fe5 cells per well at 37°C under 5% CO2 before they were incubated with Fe3+ at final Fe3+ concentrations of 0, 5, 10, 20, 50, 100, 200, and 500 μM at 37°C for 4 hours in a CO2 incubator. The cells were then incubated with ICG/Leci  for another 20 hours. Next, the medium was replaced with culture medium, and 10 μl of MTT solution  was added to each well. The mixture was then incubated for 4 hours. The MTT solution was carefully removed, and 150 μl of dimethyl sulfoxide was added for 10 min to solubilize the violet formazan crystals. Thereafter, the absorbance of each well was measured using a Thermo Fisher Scientific Multiskan MK3 ELISA reader (Thermo Fisher Scientific) at 570 nm. As for CCK8 assays, 10 μl of CCK solution was added to each well at the end of 20-hour incubation time and further incubated for another 2 hours, and the absorbance was recorded at 450 nm.Primary hepatocytes were harvested from the livers of mice and seeded into six-well plates at a density of 5 × 103+ were added to primary hepatocytes harvested as above. These cells were allowed to adhere to glass culture dishes. Four hours later, ICG/Leci  was added and incubated for another 2 hours. Next, DAPI was incubated with cells for 15 min (room temperature). The dishes were washed with PBS three times and prepared for fluorescence imaging. Similarly, 1 × 107 cells cultured with Fe3+ and ICG/Leci were detached from dishes, washed three times, and fixed into a cell ball with 1% paraformaldehyde for 1 hour. The cell balls were then subjected to MRI, fluorescence, and PAI.Various concentrations of Fe3+ and ICG/Leci were lysed using radioimmunoprecipitation assay lysis buffer, and the total protein was loaded on a 12% SDS–polyacrylamide gel electrophoresis. The samples were then transferred to nitrocellulose membranes. After blocking with 5% bovine serum albumin, the material was incubated overnight with a rabbit anti-human H-ferritin primary antibody at 1:1000 dilution , rabbit anti-mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) at 1:10,000 dilution , rabbit anti-mouse TfR1 at 1:1000 dilution , rabbit anti-human ferroportin at 1:1000 dilution , and anti-rabbit immunoglobulin G secondary antibodies at 1:2500 dilution ; the samples were detected with the ECL System (Pierce).Mouse primary hepatocytes cultured with Fe−/−, Hjv−/−, and HBV-Tg mice were maintained in the animal facility of State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics with a 12-hour light/12-hour dark cycle at ~18° to 23°C and ~50% humidity. All animals received humane care, and all experiments were conducted in compliance with institutional and national guidelines and supervised by staff from the Xiamen University. Mice (8 weeks old) received ICG and ICG/Leci (ICG dose: 2.5 mg kg−1) via tail vein and were anesthetized with 1% isoflurane. Before the MRI/PAI, real-time fluorescence imaging (Carestream FX Pro) was used to curve the biodistribution of ICG and ICG/Leci in livers at 672 nm.The HfeT1-weighted MR images from the abdomen with the following parameters: repetition time (TR), 1000 ms; echo time (TE), 8.5 ms; flip angle, 180°; number of excitations, 4; matrix size, 256 × 256; field of view (FOV), 4 × 4 cm2; slice number, 13; and slice thickness, 1 mm. For PAI, a Vevo LAZR system with consistent parameters  was also conducted after intravenous injection of ICG or ICG/Leci (ICG dose: 2.5 mg/kg). Imaging was done at t = 0, 1, 4, 8, 12, and 24 hours. Signal intensities of both images were measured in a defined ROI, and the signal-to-noise ratio values were calculated using different formulas.MRI used a 9.4-T magnetic field micro-MR scanner (Bruker) to record −1) via cubital vein injection. MRI measurements were conducted using an 18-element Tim coil at a field strength of 3.0 T . Liver T1 MRI measurement was conducted using T1-weighted VIBE (Volume Interpolated Breath-Hold Examination) sequences with the following parameters: TR, 3.97 ms; TE, 1.29 ms; and FOV, 308 × 380 cm2. The muscle-to-liver T1 ratio was calculated in eight ROIs of two slices per time point. The eight ROIs were placed in the eight segments of the liver. The study was approved by the Institutional Review Board (IRB) of Xiang’an Hospital of Xiamen University (IRB no. XAHLL2020004) and registered at ClinicalTrials.gov (ID: ChiCTR2000035648). The subjects signed and gave written informed consent.Three subjects  were recruited. After having blood drawn to detect liver function, serum ferritin, and transferrin saturation, the subjects received an iodine/ICG allergy test and were treated with ICG , and ICG/Leci  were tail vein–injected into 8-week-old Hfe−/− mice (n = 5 per group) with a single dose. Another five mice were intravenously treated with equivalent doses of DFO as a positive control group. After the injection, these mice were put into metabolic cages with a regular diet. Excreted urine and feces were collected at different time points  and digested by a mixture of HNO3 and H2O2 (1:3). The iron concentration of these samples was detected by ICP-MS to calculate the urinary and feces iron excretion.PBS (250 μl), ICG . ICP-MS was used to detect the iron concentration. Part of the liver was fixed by paraformaldehyde and stained with Prussian blue. The remaining organs, including the liver, spleen, kidneys, heart, lungs, and brain, were fixed and stained with H&E.The serum ferritin efficacy study for these blood samples was measured by ELISA conducted with an automatic biochemical analyzer . Organs  were then harvested from sacrificed mice. The weighed part of the liver and spleen was also digested with a mixture of HNO


Porcine epidemic diarrhea virus (PEDV) is a dangerous virus causing large piglet losses. PEDV spread rapidly between pig farms and caused the death of up to 90% of infected piglets. Current vaccines are only partially effective in providing immunity to suckling due to the rapid dissemination and ongoing evolution of PEDV.In this study, the complete genome of a PEDV strain in Vietnam 2018 (IBT/VN/2018 strain) has been sequenced. The nucleotide sequence of each fragment was assembled to build a continuous complete sequence using the DNASTAR program. The complete nucleotide sequences and amino acid sequences of S, N, and ORF3 genes were aligned and analyzed to detect the mutations.766P > L766 in the epitope SS6; two in the S0subdomain (135DN136 > 135SI136 and N144> D144); two in subdomain SHR1 at aa 1009L > M1009 and 1089S > L1089; one at aa 1279P > S1279 in subdomain SHR2 of the S protein; two at aa 364N > I364 and 378N > S378 in the N protein; four at aa 25L > S25, 70I > V70, 107C > F107, and 168D > N168 in the ORF3 protein. We identified two insertions (at aa 59NQGV62 and aa 145N) and one deletion (at aa 168DI169) in S protein. Remarkable, eight amino acid substitutions  were found in SA subdomain. Besides, N- and O-glycosylation analysis of S, N, and ORF3 protein reveals three known sites  and three novel sites  in the IBT/VN/2018 strain compared with the vaccine strains. Taken together, the results showed that mutations in the S, N, and ORF3 genes can affect receptor specificity, viral pathogenicity, and the ability to evade the host immune system of the IBT/VN/2018 strain. Our results highlight the importance of molecular characterization of field strains of PEDV for the development of an effective vaccine to control PEDV infections in Vietnam.The full-length genome was determined with 28,031 nucleotides in length which consisted of the 5′UTR, ORF1ab, S protein, ORF3, E protein, M protein, N protein, and 3′UTR region. The phylogenetic analysis showed that the IBT/VN/2018 strain was highly virulent belonged to the G2b subgroup along with the Northern American and Asian S-INDEL strains. Multiple sequence alignment of deduced amino acids revealed numerous mutations in the S, N, and ORF3 regions including one substitution  Coronaviridae family in Nidovirales order (genus Alphacoronavirus) (. The genome of PEDV is approximately 28 kb in length. It comprises a 5′ untranslated region (5′ UTR), a 3′ UTR with a polyadenylated tail, and seven open reading frames  encoding four structural proteins , two nonstructural proteins, and one accessory protein ORF3 (Porcine epidemic diarrhea (PED) characterized by watery diarrhea, vomiting, and severe dehydration in suckling piglets and led to 50%–90% mortality among susceptible piglets . The disnavirus) . The genein ORF3 .The S protein (a glycoprotein) contains a specific receptor binding site that is an antigenic target for neutralizing antibodies and relates to its pathogenicity and immunogenicity . This prcampaign for piglets has been conducted since 2011 for PEDV control, diarrhea disease still exists in many provinces and causes serious damage to the livestock industry in recent years in Vietnam. The low effectiveness of the vaccines could be due to the genetic differences between vaccines and field epidemic strains, emphasizing the necessity of novel vaccines against new viral strains. In Vietnam, PEDV caused an outbreak for the first time in 2009 and then occurred again in 2013 . SeveralThe PED-specific PCR-positive fecal samples were collected from the piglets that died of diarrhea in 2018, in Hung Yen province (in North of Vietnam), transported to the laboratory, and stored at −20 °C until used. Total RNA was extracted using Trizol reagent  according to the manufacturer’s instructions and suspended in DEPC-treated water  and stored at −80 °C until use.The first-strand cDNA synthesis was performed using the Maxima Reverse Transcriptase Kit  following the manufacturer’s protocols. The complete genome sequence of the PEDV strain was amplified by 20 pairs of primers  designedPhylogenetic trees were constructed based on the full-length genome and S nucleotide sequences by the neighbor-joining method using MEGA 7 software  of the VThe complete nucleotide sequences and amino acid sequences of S, N, and ORF3 genes were aligned and compared by the BioEdit software (version 7.0.9.0)  to detecThe glycosylation analysis was performed to detect the glycosylation sites in the S, N, and ORF3 proteins using the glycosylation prediction software . For homMT198679. The full-length genome was 28,031 nucleotide (nt) in length, excluding the 3′ poly-A. It consisted of a 5′ UTR region with 292 nt in length; an ORF1ab region with 20,248 nt; a S protein with 4,158 nt ; an ORF3 region with 675 nt (224 aa); an E protein with 231 nt (76 aa); a M protein with 681 nt (226 aa); a N protein with 1,326 nt (441 aa); and a 3′ UTR region with 229 nt. Full-length genome analysis indicated that IBT/VN/2018 strain shared nucleotide similarity ranged from 95.7%–99.3% in comparison with other PEDV strains (KR610993) isolated from Thailand in 2014 and the highest identity was 99.3% compared with the CH Hubei strain (KY928065) isolated from China in 2016.The complete genome of the IBT/VN/2018 strain was determined and deposited in GenBank under accession number  strains . The lowThe phylogenetic tree constructed based on the full-length genome sequence of 37 PEDV references indicated that the PEDV strains could be divided into two major groups G1 and G2 . Group 1The phylogenetic tree based on the S protein sequences also showed that all the strains could be divided into two groups . Group 1751YSNIGVCK758 (SS2), 766LSQSGQVKI774(SS6), and 1370GPRLQPY1376 (2C10). In the epitope SS6, substitution 766P > L766 was identified in IBT/VN/2018, HUA PED45 and HUA PED47 strains isolated in Vietnam only while it was not detected in other strains investigated.The S gene sequence of the IBT/VN/2018 strain was 4,158 nt in length and encoded a protein consisting of 1,385 aa. The genetic identity of the nucleotide sequence and amino acid sequence of the S gene of the Vietnamese strain was 92.2%–98.1% and 91.1%–98.4% compared to the strains from other countries, respectively . The hig0 subdomain of the IBT/VN/2018 strain, two substitutions (135DN136 > 135SI136 and 144N > D144) were found when compared to the vaccine strains   at aa positions: 294I > M294, 318A > S318, 335V > I335, 361A > T361, 497R > T497, 501SH502 > 501IY502, 506I > T506, 682V > I682, and 777P > L777. The IBT/VN/2018 strain also had two insertions (at aa position 59NQGV62 and 145N) and one deletion (168DI169) in S protein when compared with the CV777/CN/KT323979, AJ1102/CN/JX188454, SM98/Korea/KJ857455, and DR13/Korea/JQ023162 strains , S2 , or subdomains as described by Q023162) . In the  strains .857V > A857, 1009L > M1009, 1089S > L1089, 1207T > D1207, 1221F > Y1221, 1229S > G1229, 1251D > E1251, and 1279P > S1279 were detected in comparison to the CV777/CN/KT323979, AJ1102/CN/JX188454, SM98/Korea/KJ857455, and DR13/Korea/JQ023162 strains. Among them, two substitutions were found in subdomain SHR1 at aa position 1009L > M1009 and 1089S > L1089, and one substitution at aa 1279P > S1279 in subdomain SHR2.The S2 domain presents the typical structural features of class I fusion proteins, including a hydrophobic fusion peptide , two heptad repeat regions , and a C-terminal transmembrane domain . In the S2 domain of IBT/VN/2018 strain, eight substitutions at aa positions 364V > I364 and 378N > S378 were only detected in the IBT/VN/2018 strain  isolated in a severe outbreak in North Vietnam 2018 has been sequenced and analyzed. The genome sequence analysis indicated that the IBT/VN/2018 strain carried specific structural characteristics of members of the  strains . The PED strains  and the  strains  showed t strains . This re strains .766P > L766 was also found in the IBT/VN/2018 strain in the epitope SS6. These changes may lead to the ineffectiveness of the drugs and vaccines.The S protein composes of 1,383 to 1,386 amino acids consisting of the S1 subunit (aa 1–789) and the S2 subunit  , dependi144N > D144, 318A > S318, 335V > I335, 501SH502 > 501IY502, 682V > I682, 1009L > M1009, and 1089S > L1089  and some strains from China , Korea (Korea/2010/GU937797 and Korea/2011/KC879280), and Thailand (Thailand/2011/KC764959). Remarkably, we found seven substitutions that were only found in the IBT/VN/2018 strain in comparison to other PEDV strains including  > L1089 . The res > L1089 . These r > L1089 . However0 also known as the N-terminal region is a functional receptor for the porcine epidemic diarrhea virus. PEDV uses the N-terminal region as the major receptor for cell entry  into the target cell membrane, then the fusion protein combined with HR1 and HR2 regions form a stable structure. This process took the virus’s transmembrane domain accessed and fused it with the membrane of the host cell   , S3 sugg0 domain does not remarkably affect its overall 3D structure  were detected in the ORF3, N, and S proteins of the IBT/VN/2018 strain when compared with the vaccine strains. Among that three novel glycosylation sites  were detected in the IBT/VN/2018 strain. As a consequence, the amino acid substitutions in the S, N and ORF3 proteins may help the IBT/VN/2018 strain evade the host immune system and probably change the virulence of the virus.It has been known that glycosylation plays an important role receptor binding, virus entry, protein proteolysis, and protein transport, thereby altering the virulence and immune evasion of virus . The gly294I > M294, 318A > S318, 335V > I335, 361A > T361, 497R > T497, 501SH502 > 501IY502, 506I > T506, 682V > I682, and 777P > L777) were found in SA subdomain in the Vietnamese strain compared with the vaccine strains. In addition to these substitutions, three novel N- and O-glycosylation sites  were detected in the S protein of IBT/VN/2018 strain. The continual mutations in these genes may have generated a novel antigenic strain and help the virus escape from the host immune response induced by the vaccine. Our results highlight the importance of molecular characterization of PEDV strains circulating in Vietnam and provide a molecular potential for the development of an effective vaccine to control PEDV infections of pigs in Vietnam.Our study showed that the IBT/VN/2018 strain belonged to the G2b subgroup that along with the Northern American and Asian S-INDEL strains and are considered as a highly virulent group. Remarkably, eight amino acid substitutions , deletion (at aa position 168DI169), and the substitutions at aa 135DN136 > 135SI136, 497R > T497, 506I > T506, 857V > A857, 1221F > Y1221, and 1279P > S1279 in S protein of IBT/VN/2018 strain when comparison with the vaccine strains CV777/CN, SM98/Korea and DR13/Korea. The red arrow pointed the substitutions at aa 144N > D144, 294I > M294, 318A > S318, 335V > I335, 361A > T361, 501SH502 > 501IY502, 682L > F682, 777P >  L777, 1009L > M1009, 1089S > L1089, 1207T > D1207, 1229S > G1229, and 1251D > E1251 in S protein of IBT/VN/2018 strain when comparison with the vaccine strains AJ1102/CN, CV777/CN, SM98/Korea and DR13/Korea.The blue arrow pointed the insertions  and some of PEDV strains (from China and Thailand) compared to the vaccine strains CV777/CN and DR13/Korea. The green arrow pointed the changes at aa positions 364V > I364 and 378N > S378 between IBT/VN/2018 strain and other strains.The blue arrow pointed the changes at aa positions Click here for additional data file.10.7717/peerj.12329/supp-3Supplemental Information 325L > S25, 70I > V70, 107C > F107, and 168D > N168 that found in PEDV-VN  strains and GD1/CN/2011/JX647847, GDA/CN/2012/JX112709, CH Hubei/CN/2016/KY928065, and CBR1/Thailand/2014/KR610993 strains compared to other strains.There were four substitutions at aa positions Click here for additional data file.10.7717/peerj.12329/supp-4Supplemental Information 4No changes were observed in the 3D structure of the S protein of IBT/VN/2018 at mutated sites p.P766L, p.L1009M, and p.S1089L as compared to 6U7K strain.Click here for additional data file.10.7717/peerj.12329/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.12329/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.12329/supp-7Supplemental Information 7Click here for additional data file.10.7717/peerj.12329/supp-8Supplemental Information 8Click here for additional data file.


The surficial hydrothermal sediments of Guaymas Basin harbor complex microbial communities where oxidative and reductive nitrogen, sulfur, and carbon-cycling populations and processes overlap and coexist. Here, we resolve microbial community profiles in hydrothermal sediment cores of Guaymas Basin on a scale of 2 millimeters, using Denaturing Gradient Gel Electrophoresis (DGGE) to visualize the rapid downcore changes among dominant bacteria and archaea. DGGE analysis of bacterial 16S rRNA gene amplicons identified free-living and syntrophic deltaproteobacterial sulfate-reducing bacteria, fermentative Cytophagales, members of the Chloroflexi (Thermoflexia), Aminicenantes, and uncultured sediment clades. The DGGE pattern indicates a gradually changing downcore community structure where small changes on a 2-millimeter scale accumulate to significantly changing populations within the top 4 cm sediment layer. Functional gene DGGE analyses identified anaerobic methane-oxidizing archaea (ANME) based on methyl-coenzyme M reductase genes, and members of the Betaproteobacteria and Thaumarchaeota based on bacterial and archaeal ammonia monooxygenase genes, respectively. The co-existence and overlapping habitat range of aerobic, nitrifying, sulfate-reducing and fermentative bacteria and archaea, including thermophiles, in the surficial sediments is consistent with dynamic redox and thermal gradients that sustain highly complex microbial communities in the hydrothermal sediments of Guaymas Basin. Beggiatoaceae and aerobic ammonia-oxidizing Thaumarchaeota  to separate PCR-amplified 16S rRNA and functional gene sequences of dominant phylotypes. Essentially a molecular fingerprinting method, DGGE separates PCR amplicons into different phylotypes with distinct migration patterns in denaturing gels that can be visually matched side by side, and probed or excised and sequenced for closer identification . The seqAlvin dive 4868 on December 19, 2016, at the Mat Mound Massif in the southern trough of Guaymas Basin [27°00.40’N/111°24.57’W] . In the sampling area, hydrothermal sediments were overgrown with microbial mats, and occurred interlaced with hydrothermal mineral precipitates that provided holdfasts for Riftia tube worms. Thermal gradient measurements with the Alvin heatflow probe showed that in situ temperatures at the sampling location, starting at bottom water temperature of 3°C right above the sediment surface, reached 60°C at 10 cm depth, 100°C at 20 cm depth, 112°C at 30 cm depth and 115°C at 40 and 50 cm depth . In core 4868-7, a total of 28 cm dark-brown sediment was recovered; gas voids appeared below 3.8 cm. The sediment changed from very soft, non-consolidated to fine-grained, more consolidated sediment at 12.7 cm, still containing gas voids. The overlying orange Beggiatoaceae mat was collected, and the sediment core (inner diameter 6.25 cm) was frozen at −80°C immediately after shipboard recovery, and transported frozen to the Institute for the Chemistry and Biology of the Marine Environment (ICBM) at Oldenburg University, Oldenburg, Germany.Two hydrothermal sediment cores (No. 4868-7 and 4868-10) were collected at a depth of 2003 m during The parallel core from the same site (4868-10) was used for rhizon sampling of porewater  as described previously . The oveBeggiatoaceae filaments that were left behind after lifting off the mat and freezing the core.The microbiology core was gradually thawed at room temperature and sliced into approx. 2 mm intervals, by pushing the sediment upwards with a custom-designed piston. The highly fluid sediment was pushed to the rim of the core liner, and a 2 mm interval was siphoned off with 1 ml pipet tips [tips cut off for a wider opening] and collected for DNA extraction, before pushing the remaining sediment core again to the rim of the core liner and sampling the next layer; sediment granules and inhomogeneities prohibited sampling on a finer scale. The procedure was repeated until the upper 4 cm of the core were collected in approx. 2 millimeter intervals; deeper layers were collected in 4 millimeter intervals. The highly fluid sediment surface was dominated by supernatant water and possibly porewater that had frozen out of the core and diluted the sediment; this mixed surface layer was siphoned off while the piston was gently pushed up in two intervals . The sediment in the proper sense started at 3 mm depth; this cutoff value was chosen for the DGGE analyses. The sediment contained small clumps and carbonate concretions that interfered with exact 2 mm slicing; therefore the depth of each sediment sample layer was checked and recalculated based on sample volume: 0.29 – 0.48 cm, 0.48 – 0.66 cm, 0.66 – 0.82 cm, 0.82 – 1.02 cm, 1.02 – 1.21 cm, 1.21 – 1.43 cm, 1.43 – 1.62 cm, 1.62 – 1.85 cm, 1.85 – 2.08 cm, 2.08 – 2.28 cm, 2.28 – 2.44 cm, 2.44 – 2.64 cm, 2.64 – 2.85 cm, 2.85 – 3.04 cm, 3.04 – 3.25 cm, 3.25 – 3.45 cm, 3.45 – 3.63 cm, 3.63 – 3.83 cm, 3.83 – 4.02 cm, 4.02 – 4.20 cm. The depth values are rounded to full or half millimeters in the DGGE figures. The upper two sediment layers contained some orange-colored DNA was extracted from each sediment sample using the DNeasy power soil kit , following the manufacturer’s instructions, except 0.5 g instead of 0.25 g of sample were used. DNA extracts were quantified using NanoDrop . For PCRCGC CCG CCG CGC CCC GCGCCC GGC CCG CCG CCC CCG CCC CCC TAC GGG AGG CAG CAG-3′), and the reverse primer 907R (5′-CCG TCA ATT CCT TTG AGT TT-3′), slightly modified versions of a previously published bacterial 16S rRNA gene primer pair (CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCC CCG CCT TCA TTG CRT AGT TWG GRT AGT T-3′) as a reverse primer with the GC-clamp (2 (25 mM), 1 μl BSA (10 mg/ml), 1 μl MolTaq (1 U/μl), 2 × 1 μl of the respective primers (10 pmol/μl) and 1 μl of DNA template.A 550 bp segment of bacterial 16S rRNA genes was amplified using the forward primer GC-357f extended with the underlined GC-clamp , with the following parameters for amplification of 16S rRNA gene fragments: 96°C initial hold for 4 min to activate the Taq polymerase , followed by 37 cycles of amplification, with each cycle consisting of 30 s of denaturation at 96°C, 45 s of annealing at 57°C, and 1 min extension at 72°C, and concluded with a final extension step of 10 min at 72°C. Amplification conditions for mcrA genes were identical, except the annealing temperature was adjusted to 56°C, and the amplification was performed by 30 cycles. PCR conditions for archaeal amoA gene amplification were adjusted to an initial hold at 94°C for 5 min, and 46 cycles consisting of denaturation at 94°C for 45 s, annealing at 53°C for 1 min, and extension at 72°C for 1 min . In the DGGE gel, 30 μl of each PCR sample plus 5 μl of buffer were loaded into the central gel slots, and 15 μl of marker and 5 μl of loading buffer were loaded on both sides of the gel. DGGE was performed with PCR products loaded onto 6% polyacrylamide gels in 1× TAE buffer  with a denaturing gradient from 40 to 70% (with 100% denaturant corresponding to 7 M urea and 40% formamide) at 100 V and 60°C for 20 h for bacterial 16S rRNA genes and mcrA genes. The denaturant gradient and running time were adjusted to 15–55% and 15 h for archaeal amoA genes, and to 30–60% and 20 h for bacterial amoA genes. After the gradient gel had been poured with a gradient mixer, it was topped with a denaturant-free gel portion that surrounded the teeth of the DGGE comb (forming the PCR product loading slots) to facilitate the start of migration of the PCR products through the DGGE gel. We note that DGGE analyses of archaeal 16S rRNA gene fragments were attempted repeatedly, but did not result in clearly discernible DGGE patterns or defined DGGE bands, and are therefore not included in this study.® PCR Purification kit . Then, 5 μl of PCR product were mixed with 5 μl of forward primer (5 pmol/μl), and submitted to GATC Biotech AG, Konstanz, Germany, for sequencing.After the DGGE runs, the gel bands were stained using SybrGold  for 1–2 h after completion of electrophoresis. The band patterns were analyzed by cluster analysis using the software package GelComparII  on DGGE gel images taken under UV-light as described . Small gEach DGGE band sequence was checked against related sequences of cultured and uncultured bacteria and archaea in GenBank using BLASTN . PhylogeBeggiatoaceae mats , without claims of having chosen a particularly representative site, its characteristics are generally consistent with previous surveys of Guaymas Basin hydrothermal sediments, where microbial abundance and activity are strongly concentrated toward the top few centimeters. Surficial sediments immediately below the seawater-sediment interface show the highest degree of microbial activity, as shown by peak rates of sulfate reduction (In this case study, the results of DNA extraction, PCR amplification and DGGE demonstrated that DNA of sufficient quantity or quality for this type of analysis was limited to the surficial centimeters of this Guaymas Basin core, approx. 2–4 cm depth depending on the gene target. The microbial populations that are profiled by DGGE are sandwiched into this narrow surface layer, although the thermal regime at the time of measurement would have permitted thermophilic or hyperthermophilic microbial life until at least 20 cm depth with of 100°C . Even theduction  and aceteduction , by rapieduction , and by eduction , concenteduction , and dileduction . In thiseduction . To summin situ temperatures of 100°C; interpolating temperatures for 4 cm depth, the depth extent of DGGE analysis, would yield ca. 25–30°C (The thermal regime at the time of measurement would have permitted thermophilic or hyperthermophilic microbial life until at least 20 cm depth with  25–30°C . Even co 25–30°C , 2016 or 25–30°C . Close e 25–30°C  suggestiDesulfatiglans lineage that can perform complete oxidation of aromatic hydrocarbons (Beggiatoaceae in the upper sediment layers (Beggiatoaceae mats in Guaymas Basin (The DGGE analysis shows the coexistence of microbial populations with aerobic and anaerobic metabolisms in the surficial sediments: aerobic ammonia-oxidizing Betaproteobacteria and marine Thaumarchaeota ; sulfateocarbons ; thermopocarbons ; syntropocarbons ; microaeocarbons , and micocarbons . Orange t layers  perform t layers , potentit layers . The depas Basin . DGGE an4 to 105 sequences per sample (see in situ by Alvin’s bottom-facing camera (In contrast to high-throughput sequencing approaches that yield 10g camera . This isg camera . Possiblg camera .Beggiatoaceae mats that require simultaneous availability of electron acceptors and donors by turbulent mixing of seawater and vent fluids (The spatial overlap of microorganisms with aerobic ammonia-oxidizing and anaerobic sulfate-reducing and methane-oxidizing microbial communities indicates fluctuating redox conditions in mat-covered surficial sediments, and is consistent with previous observations of a dynamic temperature regime in Guaymas Basin hydrothermal sediments ; it is at fluids . Overlapt fluids . Usuallyt fluids . Recent t fluids . The envMW775652 – MW775666 for 16S rRNA gene amplicons, MW980147 – MW980167 for bacterial amoA gene amplicons, MW911582 – MW911595 for archaeal amoA gene amplicons, and MW980168 – MW980174 for mcrA gene amplicons. Geochemical data are available from the Biological and Chemical Oceanography Data Management Office at the Woods Hole Oceanographic Institution under Project Number 474317 and can be found under Sequences are available from GenBank under accession numbers Alvin dive 4868. GW performed the porewater geochemical analyses. AT led the expedition, conceptualized the experiments, performed the phylogenetic analyses, and wrote the manuscript. All authors contributed to the article and approved the submitted version.BE supervised the DGGE experiments, guided undergraduate student assistants TN, SK, KS, and HT through the experimental procedure and together with BH submitted the DGGE band sequences to GenBank. RP collected the sediment cores and measured the thermal gradients during The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Escherichia coli (STEC) are among common foodborne bacterial pathogens and healthy livestock are the main source of this bacterium. Severe diseases attribute to two types of cytotoxin Stx1 and Stx2, which are also called Shiga toxin (Stx). Infection of humans with STEC may result in Acute diarrhea with or without bleeding, hemorrhagic colitis (HC) and the hemolytic uremic syndrome (HUS). As antibiotic resistance is increasingly being reported among STEC isolates obtained from livestock and patients worldwide, in this study the pattern of antibiotic resistance in clinical isolates was determined.Shiga toxin‐producing Stool samples were collected from patients with diarrhea. All samples were cultured and identified by biochemical and molecular tests. Antimicrobial susceptibility test and assessment of extended-spectrum β-lactamase (ESBL)-related genes were conducted. Moreover, phylogenetic groups were analyzed using quadruplex PCR, and DNA analysis assessed multi-locus sequence types (MLST).E. coli samples, 174 were identified as STEC by PCR. Antimicrobial susceptibility test results showed that, 99.4%, 96% and 93.1% of isolates were susceptible to imipenem/ertapenem, piperacillin–tazobactam and amikacin, respectively. The highest resistance was towards ampicillin (68.4%), followed by trimethoprim–sulfamethoxazole (59.8%), and tetracycline (57.5%). A total of 106 (60.9%) isolates were multidrug resistance (MDR) and 40.8% of isolates were determined to be extended spectrum β-lactamase producers. In 94.4% of isolates, genes responsible for ESBL production could be detected, and blaTEM was the most prevalent, followed by blaCTX-M9. Furthermore, phylogenetic grouping revealed that majority of STEC strains belonged to Group C, followed by Groups E, B2 and A. MLST unveiled diverse ST types.Out of 340 A periodical surveillance studies and thorough understanding of antibiotic resistant profiles in STEC isolates could help select effective antibiotic treatment for patients and develop strategies to effectively manage food contamination and human infections. Escherichia coli (STEC), including O157:H7 and non-O157 serotypes, are among common foodborne pathogens. Outbreaks of several human gastrointestinal diseases, including acute diarrhea with or without bleeding, hemorrhagic colitis (HC) and the hemolytic uremic syndrome (HUS), are caused by STEC [eae), that is involved in creating attaching and effacing (A/E) lesions [Shiga toxin‐producing  by STEC . The eld by STEC . Healthy by STEC , 3. Huma lesions , 3. In c lesions .Enterobacteriaceae. Their common spread mechanism is by horizontal gene transfer and can be bacterial chromosome and plasmid mediated [Enterobacteriaceae [In recent years, due to extensive application of various antibiotics (such as β-lactams) against infections, high levels of antibiotic resistance and extended-spectrum beta-lactamase (ESBL)-producing bacteria are being detected . Extendemediated . The maieriaceae . The plaeriaceae , which lEscherichia coli clinical strains have been classified into several phylogenetic groups , based on the combination of certain genes and a DNA fragment  [E. coli strains usually belong to groups A and B1 [E. coli clinical strains provides information about the relation of strains and disease and frequency of their occurrence in the environment. But compared to Uropathogenic E. coli, few studies have determined phylogenetic groups of diarrheagenic E. coli. Also the Clermont triplex PCR method, which is only capable of determining phylogroups A, B1, B2, and D, has been used more extensively. Therefore, as disposition of phylogroups for diarrheagenic E. coli is still unclear, STEC could belong to any of the phylogroups [spE4.C2) . EvaluatspE4.C2) . The comA and B1 , whereasA and B1 . Phylogelogroups .As antibiotic resistance is increasingly being reported among STEC isolates obtained from livestock and patients worldwide, and death is specifically associated to antibiotic‐resistant STEC strains, in this study the pattern of antibiotic resistance, as well as ESBL production and phylogenetic groups associated with clinical isolates, was determined.E. coli. Five colonies from each of the confirmed isolates’ MacConkey agar plate tested by PCR to identify the presence or absence of stx1A, stx2A, and eae genes [Escherichia coli strain O157/H7 was used as a positive control. Non-pathogenic E. coli strain DH5α was used as negative control to monitor PCR contamination. All E. coli isolates were submitted to DNA extraction by boiling method as follows: 1 ml from overnight cultures of each colony was centrifuged at 12000 rpm for 2 min. The supernatant was discarded, and the pellet was washed with 1 ml of normal saline by centrifugation at 12000 rpm for 1 min. The pellet was resuspended in 200 μl DNase/RNase free distilled water (ThermoScientific) and subjected to boiling at 100 °C for 10 min. After centrifugation at 14000 rpm for 2 min, 50 μl of supernatant was collected to be used as template. The detection of virulence genes were carried out under the following PCR conditions: 95 °C for 3 min, 30 cycles of 94 °C for 1 min, 60 °C for 45 s, 72 °C for 45 s, and a final extension at 72 °C for 5 min.During 1-year period 2014), diarrhea patients’ stool samples inoculated on MacConkey agar were collected from different reference hospitals. Following standard microbiological techniques , 340 samae genes , 14. Esc, diarrheEscherichia coli ATCC 25922 was used for quality control. Detection of ESBLs was performed by combined disc assay [Antimicrobial susceptibility tests were performed on Mueller–Hinton agar  using commercial antimicrobial discs , based on standard disk diffusion method and according to the guidelines of the clinical and laboratory standards institute (CLSI) . The antsc assay , using cblaCTX-M9, blaSHV, blaOXA and blaTEM using primer sequences presented in Table 2 2 mM (Ampliqon), 9 μl of DNase/RNase free distilled water (ThermoScientific), 1 μl of 10 pM for reverse and forward primers, and 2 μl of DNA template.PCR was performed to detect β-lactamase genes All STEC strains were appointed to a phylogenetic group  based on Clermont’s quadruplex multiplex PCR scheme . GenomicE. coli pathogenic isolates were selected to perform the test. Pasteur MLST system was carried out by amplifying eight house-keeping genes  from E. coli chromosomal DNA. The amplified products were purified and subjected to Sanger dideoxy DNA sequencing . To determine sequence types (STs), the sequences of these genes were then compared with known alleles at each locus, which are available from Institute Pasteur’s MLST website, at www.pasteur.fr/mlst.Although MLST is one of the choice methods for typing of epidemiologically important strains, but as it is a costly and time-consuming technique, only a fraction of the P-values < 0.05 were considered statistically significant. Descriptive statistics such as frequency and percentage were also used to indicate categorical data.Comparison of proportions and graphic were performed with IBM SPSS 19.0 software and Microsoft Excel . Pearson Chi-square test was used to determine the difference in resistance between ESBL positive and ESBL negative isolates. A two-sided E. coli isolates were tested for stx1A, stx2A and eae genes using PCR; from which STEC’s virulence genes were detected in 174 (51.2%) of diarrhea cases. Frequency of virulence factors among confirmed samples was as follows; stx1 171 (98.3%), stx2 10 (5.7%), eae 16 (9.2%), and five different virulence profiles were observed i.e., 148 (85.0%) isolates showed only stx1 gene, 15 (8.6%) were positive for stx1 and eae, 7 (4.0%) for stx1 and stx2, 3 (1.7%) for stx2 and only one of the isolates was positive for all 3 genes. Moreover, the positive cases comprised of 97 males and 77 females with male: female ratio of 1.26:1; from which 44 male and 27 female belonged to children under 6 years, 27 male and 25 female belonged to 10–30 year olds, and the rest of the isolates were from patients older than 30 years , 36 (33.9%) and 27 (25.5%) belonged to 3, 4 and 5 classes of antibiotics, respectively. The isolates resistance pattern ranged from 3 to 7 antibiotics and showed 20 different profiles that are shown in Table blaTEM gene was more common with frequency of 60 (84.5%), followed by blaCTX-M9 gene 49 (69.0%), blaSHV gene 35 (49.3%), and blaOXA gene 28 (39.4%). Molecular evaluation of ESBL-producing isolates indicates 4, 3, 2 and 1 gene patterns . The highest co-existence rate of β-lactamase producing genes belonged to blaCTX-M9/blaTEM, followed by blaOXA/blaTEM, blaCTX-M9/blaSHV, blaCTX-M9/blaOXA and blaSHV/blaTEM . The Molecular Patterns are demonstrated in Table Four genes responsible for β-lactamase production have been investigated by PCR from 71 ESBL positive phenotypes. The results showed 67 (94.4%) isolates contained at least 1 of these ESBL encoding genes. The of 60 84.%, followThe 174 STEC isolates were assigned into different phylogroups according to Clermont’s quadruplex scheme Fig. .Fig. 1PhAccording to virulence genes, ESBL genes and resistance profile, total of 25 isolates; 18 STEC and 7 EPEC (previously published data), were analyzed using Pasteur MLST scheme. Examining gene sequences of the strains and assigning specific alleles for each locus resulted in the identification of diverse sequence types (STs) Table . The tesE. coli isolates in this study, 174 strains were identified as STEC among which prevalence of stx1 and stx2 genes was 98.3% and 5.7%, respectively. Although in general infection of humans by stx2a may cause more severe clinical outcomes [Of the 340 outcomes , a recenoutcomes . The curoutcomes , 23.Overuse of antibiotics with and/or without prescription is responsible for emergence of multidrug resistance among clinical isolates, which is a serious public health issue and can create therapeutic difficulties for patients. The findings of this study show high levels of resistance to ampicillin, trimethoprim–sulfamethoxazole and tetracycline among STEC, which is in agreement with previous findings in Japan, China, South Africa, Iran, Mexico and Michigan –29. AlsoblaTEM was more common, followed by blaCTX-M9, blaSHV and blaOXA. In respect to higher prevalence of blaTEM, our findings are similar with a study from India [blaCTX-M was more common [The presence of ESBL-producing isolates in clinical samples is worrisome in both developed and developing countries. Rapid expansion of ESBL positive isolates highly affects the activity of broad-spectrum antibiotics, creating major therapeutic difficulties . Also, tom India , wherease common , 31.E. coli clinical strains provides information about the frequency of occurrence in the environment. In this study, we determined the phylogenetic group of 174 STEC strains isolated from the stool of diarrheal patients and showed that the majority of STEC strains belonged to 1 of 5 phylogroups; C, E, B2, A and B1, while ESBL positive and MDR strains represented more affiliation with Group E. Furlan et al. [E. coli [Phylogenetic analysis of n et al.  assessedn et al.  reported[E. coli . These dE. coli population structure using the sequences of eight housekeeping genes, and MLST results indicated a highly diverse sequence types (STs), which are in accordance with other findings [In current study, we also used Pasteur MLST scheme to analyze the diarrheagenic findings , 38.Escherichia coli isolates in this study have demonstrated high levels of resistance to various antimicrobial agents. The emergences of ESBL-producing isolates were detected, which poses a serious public health challenges. Our results also revealed worrying increase in the prevalence of MDR among ESBL-producing STEC, especially those affiliated to phylogroup E. These findings emphasize the health risks and difficulties that could be encountered in eradicating STEC infections. Thus more research should be directed towards periodical surveillance studies to screen/identify patients that are carriers of ESBL-producing bacteria, in order to develop an effective antibiotic therapy guideline. Moreover, to prevent the further spread of MDR isolates, empirical treatments and overuse of antibiotics by patients should be discouraged.