Glycine max. RNA-seq transcriptome data from different tissues in public databases revealed tissue-specific and differential expression profiles of the GmCBSDUFs, and qPCR analysis revealed that certain groups of soybean CBSDUFs are likely involved in specific stress responses. In addition, GmCBSDUF3 transgenic Arabidopsis was subjected to phenotypic analysis under NaCl, PEG, and ABA stress treatments. The overexpression of GmCBSDUF3 could enhance tolerance to drought and salt stress in Arabidopsis. This study presents a first comprehensive look at soybean CBSDUF proteins and provides valuable resources for functionally elucidating this protein subgroup within the CBS domain-containing protein family.Cystathionine-β-synthase (CBS) domain-containing proteins (CDCPs) constitute a large family in plants, and members of this family have been implicated in a variety of biological processes. However, the precise functions and the underlying mechanisms of most members of this family in plants remain to be elucidated. CBSDUF proteins belong to the CDCP superfamily, which contains one domain of unknown function (DUF21) and an N terminus that is adjacent to two intracellular CBS domains. In this study, a comprehensive genome database analysis of soybean was performed to investigate the role(s) of these CBSDUFs and to explore their nomenclature, classification, chromosomal distribution, exon–intron organization, protein structure, and phylogenetic relationships; the analysis identified a total of 18 putative CBSDUF genes. Using specific protein domains and phylogenetic analysis, the CBSDUF gene family was subdivided into eight groups. The soybean CBSDUF genes showed an uneven distribution on 12 chromosomes of The online version of this article (10.1007/s10528-020-09991-w) contains supplementary material, which is available to authorized users. Archaebacterium Methanococcus jannaschii  in most of these proteins, which may indicate their probable functions. Based on whether they have additional domain(s), these proteins were further classified into different subclasses: CBSX, CBSCLC, CBSSIS, CBSPPR, CBSIMPDH, CBSCBS, CBSCBSPB and CBSDUF. These subclasses possess various functions, including cytoplasmic targeting, subcellular localization of chloride channels (CLC), protein–protein interaction, protein regulation, sensing of cellular energy status, and maintenance of intracellular ion gradients  (PF01595) and an N terminus that is adjacent to two intracellular CBS domains. This transmembrane region has no known function. Many of the sequences in this family are annotated as hemolysins because of their similarity to Q54318 (HLYC_BRAHO), which does not contain this domain. Therefore, the functions of DUF21 are still unknown. DUF21 often exists together with CBS domains and plays important roles in plant growth and development. The characteristics of the CBSDUFs in this subgroup are not yet clear. In our previous study, we identified CDCPs in soybean, but there was no detailed analysis of the CBSDUF subgroup. We found that overexpression of soybean GmCBS21, which belongs to the CBSDUF subgroup, possesses a novel function to improve low nitrogen tolerance in https://phytozome.jgi.doe.gov/pz/portal.html#). If more than one transcript existed, the primary transcript was selected as a representative. Using the same approach, 8, 10, 10, 4, 9, 4, and 4 putative CBSDUF members were identified from common bean (Phaseolus vulgaris), M. truncatula, Lotus japonicus, sorghum, Arabidopsis, rice, and maize, respectively. Table Eighteen putative GmCBSDUF members were found in the NCBI database and used as queries to conduct BLAST searches against the public genome database  were identified, with sequence identities ranging from 60.47 to 99.26%.To some extent, functional information can be derived from structural similarity. Knowledge of the structure is often essential for interpreting functional data. GmCBSDUF protein structures are shown in Fig. S1. It is clear that GmCBSDUF proteins have a highly conserved hydrophobicity profile, with one hydrophobic segment located at the N terminus. SMART allows the identification and annotation of genetically mobile domains and the analysis of domain architectures. The results are shown in Fig. Bradyrhizobium japonicum inoculation (HAI), in mock-inoculated root hairs at 24 HAI, and in stripped roots at 48 HAI were also constructed  was sprayed on soybean seedlings growing on B&D agar medium. The results showed that inoculation with B. japonicum significantly increased the expression of GmCBSDUF8/9, but not other GmCBSDUFs, in root hairs. Therefore, we suspect that GmCBSDUF8/9 may be required for bacterial recognition, nodulation, and nitrogen fixation.Based on the publicly available soybean RNA-Seq data  genome database (Phytozome 12) provides high-resolution gene expression data for a diverse set of 17 soybean GeneAtlas tissue samples, such as flower (open and unopened), lateral root (standard), leaf , nodule (symbiotic condition), root tip (standard), root , shoot tip (standard), stem (standard), and 9 soybean normal tissue samples . These data were also analyzed and represented as heat maps . Expression analyses of all GmCBSDUF genes revealed that the different members have different tissue-specific expression. Among all 18 analyzed genes, GmCBSDUF5 showed the highest level of constitutive expression in all tissues, followed by GmCBSDUF3, GmCBSDUF2, and GmCBSDUF12. This high level of constitutive expression indicates a significant role in all these soybean tissues . A cluster of genes showed low levels of expression in all tissues. They are GmCBSDUF8/9/11/13. GmCBSDUF16 is highly expressed only in root nodules, but its expression is very low in symbiotic conditions. These results are basically consistent with the results in Fig. Furthermore, the soybean (https://www.phytozome.net/soybean), the promoter regions located 2 kb upstream of the translation start codons of the GmCBSDUF genes were analyzed using the PlantCARE promoter analysis program (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/). Multiple elements were identified, and the stress and hormone signaling-related sites are shown in Table Based on the soybean genome database  after stress treatment. This may indicate that these genes play a major role in nitrogen assimilation. Future studies are needed to demonstrate the functional roles of genes responsive to low N stress in relation to N metabolism.2O2, ABA, and salinity stress were examined. The raw expression values for the genes are shown in Table S2.As described in Table Two-week-old soybean seedlings were exposed to cold stress at 4 °C for 0, 0.5, 5, or 12 h, and the expression of GmCBSDUFs was detected. The results revealed that cold stress altered the expression of GmCBSDUFs, which could be grouped into 3 categories. As indicated in Fig. Figure 2O2 on the transcription of GmCBSDUFs in the roots of soybean seedlings. It is clear that H2O2 treatment (1) increased the transcript levels of GmCBSDUF6/7/8/10/11/12/16, (2) decreased the transcript levels of GmCBSDUF2/3/14/15/17/18, and (3) did not change the transcript levels of other GmCBSDUFs.Figure Figure Figure CBSDUFs may be involved in multiple stress responses in plants. As described above, when induced with some stresses, the expression of GmCBSDUF genes is significantly altered. Our previous study found that GmCBSDUF3 could improve plant nitrogen use efficiency. Therefore, we chose GmCBSDUF3 for further functional exploration. Two homozygous constitutively overexpressing Arabidopsis lines (GmCBSDUF3-1 and GmCBSDUF3-2) with higher GmCBSDUF3 expression were selected for phenotypic analysis under NaCl, PEG, and ABA stress treatments. As shown in Fig. Although some CDCPs, such as IMPDH  and L. japonicus genome database (https://www.kazusa.or.jp/lotus/). Sequences with an E value < 1.0 were selected for further analysis. A search with the keywords PF00571 for the CBS domain and PF01595 for the DUF21 domain was conducted for putative soybean CBSDUFs by searching ontologies against the Phytozome (v12.0) database (https://www.phytozome.net). If more than one transcript existed, the primary transcript was selected as a representative.The known DUF21 and CBS domain-containing protein sequences from soybean, Arabidopsis, common bean, https://gsds.cbi.pku.edu.cn/) 2·4H2O, 2.5 mM KNO3, 0.5 mM NH4NO3, 0.5 mM KH2PO4, 1 mM MgSO4·7H2O, 0.05 mM Fe-EDTA, 0.005 mM KI, 0.1 mM H3BO3, 0.1 mM MnSO4·H2O, 0.03 mM ZnSO4·7H2O, 0.0001 mM CuSO4·5H2O, 0.001 mM Na2MO4·2H2O, 0.0001 mM CoCl2·6H2O) or low nitrogen solution (0.2 mM Ca(NO3)2·4H2O, 1.8 mM CaCl2·2H2O, 0.25 mM KNO3, 1.125 mM K2SO4, 0.05 mM NH4NO3, 0.5 mM KH2PO4, 1 mM MgSO4·7H2O, 0.05 mM Fe-EDTA, 0.005 mM KI, 0.1 mM H3BO3, 0.1 mM MnSO4·H2O, 0.03 mM ZnSO4·7H2O, 0.0001 mM CuSO4·5H2O, 0.001 mM Na2MO4·2H2O, 0.0001 mM CoCl2·6H2O) at 25 °C in a chamber with a 12-h light and 12-h dark photoperiod. All treatments were performed over a continuous time course . Roots, stems, and leaves from control and stress-treated plants (five plants were collected as mixed samples at each time point) were collected as samples in three biological replicates for RNA preparation, and the samples were quickly frozen in liquid nitrogen and stored at − 80 °C until use.For low nitrogen treatment, seeds of a low N-tolerant soybean variety (Pohuang) were germinated. After 7 days, the seedlings were grown hydroponically in half-strength modified Hoagland solution until the first trifoliate leaf was fully developed and then grown in normal nitrogen solution  stresses were applied to 2-week-old soybean seedlings. For salt stress, the roots of seedlings were dipped into solutions of 200 mM NaCl. For dehydration, the root systems of whole plants were placed onto filter paper with 70% humidity at room temperature for induction of a rapid drought treatment  and treated with DNase I (Invitrogen) to avoid genomic DNA contamination. First-strand cDNA was synthesized using Superscript II reverse transcriptase (Invitrogen). Gene-specific primers were designed according to gene sequences using Primer 5.0 software (Table S1). The quantitative RT-PCR was performed with a CFX96TM real-time system (Bio-Rad) in a 20 μl system containing 2 μl of a tenfold diluted cDNA, 10 μl of 2 × SYBR green real-time PCR master mix (Takara), and 1 μl each of 10 μM forward and reverse primers. β-actin was used as the internal control. Statistical analyses were performed using the Agrobacterium tumefaciens strain GV3101 and then transformed into Arabidopsis (Columbia) via the floral dip method. The transgenic plants were screened on MS medium with 100 mg/L hygromycin and confirmed by PCR analyses. The expression levels of GmCBSDUF3 in transgenic plants were determined by qPCR .The full-length coding sequence (the primers 5′ ATGGCGGCAGAGATACCG 3′ and 5′ CTATTGATTCCTTAGTGACTCACT 3′.) of GmCBSDUF3 was TA cloned into the plant expression vector pCXSN. The recombinant construct containing the 35S::GmCBSDUF3  cassette was introduced into Seeds of transgenic overexpressing Arabidopsis and WT plants were grown on 10 × 10 cm MS agar plates. They were routinely kept for 2 days in darkness at 4 °C to break dormancy and transferred in a light growth chamber under a day/night 16/8 h cycle at 23 °C. For stress treatment, the seeds of transgenic lines or WT were kept on MS media supplemented with 50 mM NaCl, 2% PEG, or 1.5 μM ABA. Each treatment contained three independent replicates.Supplementary file1 (TIF 531 kb)—Figure S1. Transmembrane topology analysis of soybean CBSDUF proteins. The protein transmembrane topology was predicted by using TMHMM Server v2.0. The predicted transmembrane helixes are shown as red peaks.Supplementary file2 (TIF 132 kb)—Figure S2. Generation and RT-PCR testing of GmCBSDUF3 lines (A) GmCBSDUF3 overexpression plasmid profile; (B) relative expression levels of GmCBSDUF3 lines.Supplementary file3 (TIF 98 kb)—Figure S3. Tissue-specific expression patterns of GmCBSDUF genes. The figure shows the gene expression patterns of GmCBSDUF genes in 17 soybean GeneAtlas tissue samples, including flower (open and unopened), lateral root (standard), leaf , nodule (symbiotic condition), root tip (standard), root , shoot tip (standard), stem (standard) and 9 soybeannormal tissue samples . The data were obtained from the soybean genome database Phytozome 12.Supplementary file4 (TIF 572 kb)—Figure S4. Expression of soybean GmCBSDUF genes in response to low nitrogen stresses.A: Leaves, B: stems, C: roots.Data were obtained by real-time PCR normalized against the reference gene ACT11 and are shown as apercentage of expression in the control at 0 h. Blue columns represent the expression under normalnitrogen conditions, and red columns represent the expression under low nitrogen conditions. GmCBSDUF13, which was not expressed in soybean roots, stems and leaves under normal conditions,was not induced under low nitrogen stress and is not shown in this figure.Supplementary file5 (XLSX 10 kb)—Table S1. Primer listSupplementary file6 (XLSX 15 kb)—Table S2. Responses of 18 soybean CBSDUF genes to abiotic stresses by qRT-PCR with the 2-ΔΔCT methodBelow is the link to the electronic supplementary material.


V1.3 is a subtype of the voltage-gated sodium channel family. It has been implicated in the pathogenesis of neuropathic pain, although the contribution of this channel to neuronal excitability is not well understood. Tf2, a β-scorpion toxin previously identified from the venom of Tityus fasciolatus, has been reported to selectively activate NaV1.3. Here, we describe the activity of synthetic Tf2 and assess its suitability as a pharmacological probe for NaV1.3. As described for the native toxin, synthetic Tf2 (1 µM) caused early channel opening, decreased the peak current, and shifted the voltage dependence of NaV1.3 activation in the hyperpolarizing direction by −11.3 mV, with no activity at NaV1.1, NaV1.2, and NaV1.4-NaV1.8. Additional activity was found at NaV1.9, tested using the hNav1.9_C4 chimera, where Tf2 (1 µM) shifted the voltage dependence of activation by −6.3 mV. In an attempt to convert Tf2 into an NaV1.3 inhibitor, we synthetized the analogue Tf2[S14R], a mutation previously described to remove the excitatory activity of related β-scorpion toxins. Indeed, Tf2[S14R](10 µM) had reduced excitatory activity at NaV1.3, although it still caused a small −5.8 mV shift in the voltage dependence of activation. Intraplantar injection of Tf2 (1 µM) in mice caused spontaneous flinching and swelling, which was not reduced by the NaV1.1/1.3 inhibitor ICA-121431 nor in NaV1.9-/- mice, suggesting off-target activity. In addition, despite a loss of excitatory activity, intraplantar injection of Tf2[S14R](10 µM) still caused swelling, providing strong evidence that Tf2 has additional off-target activity at one or more non-neuronal targets. Therefore, due to activity at NaV1.9 and other yet to be identified target(s), the use of Tf2 as a selective pharmacological probe may be limited.Na Peptides were purified by preparative reverse-phase (RP) HPLC on a Shimadzu Prominence system . HPLC fractions containing the desired peptide (judged by HPLC/MS) of similar purity were pooled, lyophilized, and stored at −20 °C.Tf2 was synthesized in two segments consisting of Tf2(1–26)-α-thioester and Tf2(27–62), which were linked by native chemical ligation. Tf2(1–26)-α-thioester was synthesized via Boc chemistry on trityl-associated mercaptopropionic acid lysine resin to yield the C-terminal α-thioester upon hydrogen fluoride  cleavage. Tf2(27–62) was synthesized using Fmoc chemistry on Rink amide resin to yield the C-terminally amidated peptide segment after trifluoroacetic acid  cleavage. The cleaved crude peptides were precipitated and washed twice with cold diethyl ether. The peptides were then dissolved in 50% acetonitrile, 0.1% TFA : 4031.7 Da, observed MW: 4031.2 ± 0.5 Da.Tf2(1–26)-α-thioester: KEGYAMDHEGCKFSCFIRPSGFCDGY-[COS-CH2-CH2-CO]-Lys, expected MW (assuming average isotope composition): 3164.5 Da, observed MW: 3164.4 ± 0.5 Da. Tf2(27–62): CKTHLKASSGYCAWPACYCYGVPSNIKVWDYATNKC-NH2HPO4, 40 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and 50 mM 4-mercaptophenylacetic acid (MPAA), pH 7.25. The solution was stirred under nitrogen for 16 h and the reaction progress was monitored by HPLC-UV. The reduced full-length Tf2 (1–62) ligation product was isolated via RP-HPLC on an Agilent Zorbax 300SB, C3 column . Yield: 42.5 mg; expected MW (assuming average isotope composition): 6963.0 Da, observed MW: 6962.8 ± 1.0 Da.Native chemical ligation was done as previously described . BrieflyFor oxidative folding of Tf2, 1.7 μmol (15 mg) of reduced Tf2 were dissolved in 3.75 mL of 6 M guanidine hydrochloride. Folding was initiated by diluting the peptide solution with 150 mL of folding buffer  and stirring for 72 h at 4 °C. Oxidized Tf2 was then purified by RP-HPLC on a Agilent Zorbax 300SB, C3 column . Yield: 2.2 mg. Calculated MW (assuming most abundant isotope composition): 6949.03 Da, observed MW: 6949.12 ± 0.20 Da. All reagents were obtained from Sigma-Aldrich  unless otherwise stated.V1.1–1.7/β1  were maintained in minimum essential media (MEM) supplemented with 10% fetal bovine serum (FBS) and 2 mM l-glutamine, along with the selection antibiotics blasticidin, geneticin, and zeocin as recommended by the manufacturer. Chinese hamster ovary (CHO) cells expressing hNaV1.8/β3 via a tetracycline inducible system  were maintained in MEM, 10% FBS, and 2 mM l-glutamine. Tetracycline (1 µg mL−1) was added to culture media 48–72 h prior to assay to induce hNaV1.8 expression. Cells were passaged every 3–4 days after reaching 70–80% confluence with TrypLE Express  and grown in an incubator at 37 °C with 5% CO2.Human embryonic kidney (HEK) 293 cells that constitutively express the human VGSC channel α subunits NaV1.9_C4 plasmid  were maintained in high glucose Dulbecco’s Modified Eagle Medium  with a glucose level of 4.5 g/L and supplemented with 10% FBS . ND7/23 cells were transiently transfected with 2.75 µg hNa3 vector ), 0.25 μV1.1–1.7, we utilized the FLIPRTetra fluorescent imaging plate reader . Cells were plated in 384-well black-walled plates at a density of 10,000–15,000 per well in normal growth media 24–48 h before membrane potential experiments. Cells were loaded with red membrane potential dye (Molecular Devices) diluted in physiological salt solution  and incubated for 30 min at 37 °C as per the manufacturer’s instructions. All synthetic toxins were diluted in PSS and 0.1% bovine serum albumin (BSA) to avoid adsorption to plastic surfaces. Changes in membrane potential (excitation/emission 510–545 nm/565–625 nm) were recorded each second for 5 min after the addition of toxin and the area under the curve (AUC) was computed using ScreenWorks .To rapidly assess the activity of Tf2 and Tf2[S14R] at hNaV1.3, hNaV1.7, and hNaV1.8 cell lines were performed using the QPatch-16 automated electrophysiology platform . This set-up utilizes 16-well planar chips plates  with a standard resistance of 2 ± 0.02 mΩ. Cells were expanded in T-175 flasks and maintained in 37 °C and 5% CO2 48–72 h prior to assay. On the day of experiment, cells were harvested with TrypLE Express and resuspended in DMEM with 25 mM HEPES, 100 U/mL penicillin-streptomycin, and 0.04 mg/mL trypsin inhibitor from Glycine max (soybean) and stirred for 30–60 min prior to use.Whole cell patch-clamp experiments on hNa2 (2), MgCl2 (1), HEPES (10), TEA-Cl (20), and glucose (10) containing 0.1% BSA. The pH was adjusted to 7.4 with NaOH and osmolarity adjusted to 305 mOsm with sucrose. The intracellular solution (ICS) consisted of (in mM): CsF (140), EGTA/CsOH (1/5), HEPES (10), and NaCl (10) adjusted to pH 7.4 with CsOH and 320 mOsm with sucrose. The cell positioning pressure was set to −60 mbar, minimum seal resistance 0.1 GΩ, holding pressure −20 mbar, and currents were filtered at 25 kHz . A standard P/4 leak subtraction protocol was included, and as such, leak subtracted and non-leak subtracted currents were acquired in parallel.The external solution (ECS) for all automated VGSC recordings contained (in mM): NaCl (140), KCl (4), CaClV1.3 and hNaV1.7 or +10 mV for hNaV1.8 to assess the voltage dependence of fast inactivation. Peak current values were normalized to a buffer control. Toxins were diluted in ECS with 0.1% BSA and incubated for 5 min prior to recording.After obtaining the stable whole-cell configuration, voltage clamp experiments proceeded as follows from a holding potential of −90 mV. All protocols included a brief 15 ms) pre-conditioning pulse to 0 mV followed by a 120 ms recovery to allow voltage-sensor trapping by β-scorpion toxins i of 100–200 DRG cells per experiment was monitored in parallel using a Nikon Ti-E Deconvolution inverted microscope , equipped with a Lumencor Spectra LED Lightsource . Images were acquired at 10x objective at 1 frame/s . For each experiment, baseline fluorescence was monitored for 30 s. At 30 s, 60 s, and 180 s, assay solution was replaced with fresh assay solution (as a negative control), Tf2 (1 µM in assay solution), and KCl (30 mM in assay solution), respectively. Experiments with TTX and Tf2[S14R] were performed in the same way. Cells responding to KCl and/or Tf2 were considered neuronal and grouped according to size: Large (>600 µm2), medium (300–600 µm2), and small diameter (<300 µm2). Experiments involving the use of mouse tissue were approved by The University of Queensland Animal Ethics Committee. Data were derived from 2–3 independent experiments, and plotted using GraphPad Prism 8. Where differences were tested, unpaired t-tests were used.Primary cultures of mouse DRG neurons were prepared as previously described ,17. BrieV1.9-deficient (NaV1.9-/-) mice aged 6–8 weeks (~25g) on a C57BL/6J background. Homozygous Nav1.9 knockout mice on a C57BL/6J background were generated using CRISPR/Cas9 technology by the Queensland Facility for Advanced Genome Editing . In brief, two sgRNAs were designed to target exon 2 of the Scn11a gene. The deletion of exon 2 generated a frame shift mutation resulting in the introduction of stop codons in exon 3. The guide sgRNAs were microinjected together with Cas9 mRNA into fertilized eggs collected from C57BL/6J mice and transferred into pseudopregnant surrogate CD1 female mice. Offspring born to the foster mothers were genotyped by PCR (see below) and DNA sequencing. One F0 male mouse was bred to C57BL/6J female mice to generate F1 heterozygous mice, and F1 heterozygous mice were inbred to obtain homozygous mice. Genomic DNA was extracted from ear clippings and mice were genotyped by PCR using the following primers (forward: 5′-GCTGCTCAGACACTCACAGT-3′) and (reverse: 5′ATTCTGCCACCAGAGACTGC-3′) by the AEGRC genotyping and sequencing facility (The University of Queensland). Animals were housed in groups of 3 or 4 per cage, under 12-h light-dark cycles, and had standard rodent chow and water ad libitum. Age-matched controls were used for studies involving knockout animals. All behavioral assessment was performed by a blinded observer unaware of the genotype and/or treatments received.For behavioral assessment, we used adult male wildtype or homozygous NaEthical approval for in vivo experiments was obtained from The University of Queensland Animal Ethics Committee prior to experimentation. All animals experiments were conducted in accordance with local and national regulations, including the International Association for the Study of Pain Guidelines for the Use of Animals in Research in agreement with the Animal Care and Protection Regulation Qld (2012), and the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, 8th edition (2013) .w/v BSA and administered either alone or in combination by intraplantar (i.pl) injection into the left hind paw in a volume of 40 µL under light isoflurane (3 %) anesthesia. Mice were then placed individually into polyvinyl boxes (10 × 10 × 10 cm) and recorded by video for 20 min post-injection. The number of spontaneous pain behaviors, including licks, shakes, and flinches, were counted later by a blinded observer. Once spontaneous pain behavior had ceased, mechanical thresholds were assessed using an electronic von Frey apparatus , heat thresholds were assessed using the thermal probe test , and weight bearing was assessed using the Catwalk XT  as previously described  (10 µM), or ICA-121431 (500 nM) were diluted in saline with 0.1% escribed ,19. To qIn a separate cohort of animals, paw thickness was measured along the distal–proximal axis at the metatarsal level using a digital vernier caliper  10 min after i.pl. injection of Tf2 (1 µM) or Tf2[S14R] (10 µM).p < 0.05 and was determined by t-test or one-way ANOVA with Dunnett’s post-test, as indicated. In case of deactivation, multiple t-tests (one per row) were used without assuming equal standard deviation and with the Holm–Sidak method for correcting p-values for multiple comparisons. Concentration–response curves were fitted with a four-parameter Hill equation with a variable Hill coefficient. Conductance–voltage relationships for activation were calculated for each voltage step using rev is the reversal potential. Resulting data were fitted using a Boltzmann equation: GNa =Na is the voltage-dependent sodium conductance, GNa,max is the maximal sodium conductance, Vm is the membrane potential, V1/2 is the membrane potential at half-maximal activation, and k is the slope factor. Current–voltage relationships for inactivation were fitted using a Boltzmann equation: INa = Na is the voltage-dependent sodium current, INa,max is the maximal sodium current, Vm is the membrane potential, V1/2 is the half-maximal inactivation, and k is the slope factor. The time course of current decay at deactivating voltages over 10 ms was fitted by the double-exponential equation,0 is the current amplitude at steady state, amp1 and amp2 are the amplitude coefficient for the fast and slow time constants, and τ1 and τ2 are the fast and slow time constants. Data are expressed as the mean ± standard error of the mean (SEM).Data were plotted and analyzed by GraphPad Prism, versions 8.2.0 and 8.4.2. Statistical significance was defined as We used chemical synthesis to prepare multi-milligram quantities of Tf2. This toxin is a relatively large (62 amino acid residues) and post-translationally modified protein, whose 3-D structure is stabilized by four disulfide crosslinks. The target polypeptide chain was split into two segments of manageable size (26 and 36 residues), which were assembled individually by solid-phase peptide synthesis and purified. The full-length polypeptide chain was obtained in good yield by native chemical ligation of the two segments. Folding of the 62 amino acid peptide chain and oxidation of the eight cysteines required extensive optimization and allowed the isolation of synthetic Tf2 in acceptable yields and excellent purity for further functional studies. Functional equivalence with venom-derived Tf2 was established through activity testing (see below) as well as partial assignment of NMR data, which are in agreement with the expected cysteine-stabilized αβ fold that is characteristic for this class of scorpion toxins .V1.1–1.7 expressed heterologously in HEK293 cells. Synthetic Tf2 potently activated hNaV1.3, with an EC50 of 213 ± 57 nM  at 1 µM caused a decrease in the peak current and a hyperpolarizing shift (Δ −11.3 mV) in the voltage dependence of activation , or the voltage dependence of fast inactivation  or deactivation  or E15 (in Cn2 and Css4) with an arginine can uncouple the ‘excitatory’ activity from the ‘inhibitory’ activity, producing β-scorpion toxin mutants that inhibit the peak current without causing early channel opening ,20,21. Wal assay A. In patV1.3 is expressed at low levels in the adult rodent peripheral nervous system [2+ concentration ([Ca2+]i) in a subset of neurons (p < 0.05) in the presence of 1 µM TTX (2+ influx in mouse DRG neurons is dependent on the expression of TTX-sensitive VGSCs. At the same concentration (1 µM), Tf2[S14R] was not significantly different to cells treated with the negative control (p = 0.71), indicating that this analogue lacks the capacity to activate DRG neurons, consistent with its reduced excitatory activity of NaV1.3.It has been reported that Nas system . Applica neurons A,B. Smal neurons C. Activa1 µM TTX D, indicaV1.3 and in DRG neurons seen in vitro  Tf2; however, it had no effect on Tf2-induced spontaneous flinching  had no effect on Tf2-induced spontaneous flinching  and swelling  controls, NaV1.9-/- mice did not exhibit a significant reduction in spontaneous pain behaviors  or paw swelling  after injection of Tf2  Tf2 in mice caused a rapid induction of spontaneous pain behaviors, including flinching, licking, and shaking of the injected hind paw, that slowly subsided over 20 min A,B. In cswelling C, which n of Tf2 E,F. OnceV1.1–1.9) constitutively expressed in mammalian cells, on the calcium influx in primary sensory DRG neurons, and on nociceptive behaviors in mice in vivo, with the overall aim to investigate its suitability as a selective NaV1.3 pharmacological probe. Modern chemical peptide synthesis strategies applied to α- and β-long chain scorpion toxins have allowed the production of these molecules on the multi-milligram scale and have allowed structural and functional studies that are difficult to perform with the small quantities usually isolated from scorpion venom , where we mutated the serine at position 14 to a positively charged arginine. In the homologous β-scorpion toxin Ts1, mutation of S14 to an arginine resulted in a toxin that could still bind to NaV1.4 but no longer shifted the voltage dependence of activation  required application at a concentration 200 times higher than the native to inhibit NaV1.6 in vivo , which lost activity at NaV1.3, also lost excitatory activity in DRG neurons suggests that the pharmacophore of Tf2 at this other neuronal target(s) overlaps at least partially with that of NaV1.3. In addition, the observation that Tf2[S14R], which lost excitatory activity in DRG neurons, still caused swelling in vivo suggests that Tf2-induced inflammation is mediated by an additional non-neuronal target. Scorpion venom- or toxin-induced inflammatory responses have been described previously and have been linked to the activation of innate immune cells via interaction with pattern recognition receptors  cause inflammation in vivo could be explored further to understand how scorpion toxin envenomation affects inflammatory processes in humans.In summary, Tf2 does not appear to be a suitable tool to study the effects of Nav1.3 channel activation in sensory neurons. This is due to additional activity at Na


Peganum harmala  seed oil and its antifungal properties against 10 fungal species. Seed oils of six populations were analyzed using high performance liquid chromatography (HPLC) and gas chromatograph/mass spectrometry (GC-MS). The HPLC analysis indicated that P. harmala seed oil exhibited a very high level of tocopherol contents, with values in the range of 2385.66–2722.68 mg/100 g. The most abundant tocopherol isomer was δ-tocopherol (90.39%), followed by γ-tocopherol (8.08%) and α-tocopherol (1.14%). We discovered for the first time the presence of tocotrenols in P. harmala seed oils of the six populations studied. The GC-MS analyses revealed that linoleic acid was the main fatty acid (65.17%), followed by oleic acid (23.12%), palmitic acid (5.36%) and stearic acid (3.08%). We also studied the antifungal activity of seed oil of the Medenine (MD) population on ten fungal pathogens. The antifungal effects differed among pathogens and depended on oil concentrations. Seed oil of the MD population caused a significant decrease in mycelial growth of all fungi tested, with values ranging 31.50–82.11%, except for Alternaria sp., which showed no inhibition. The antifungal activity against the 10 selected fungi can be explained by the richness in tocols of the extracted oil and make P. harmala a promising crop for biological control. Furthermore, the importance of fatty acids and the wide geographic spread in Tunisia of this species make this crop a potential source of renewable energy.This investigation included the chemical analysis of  Peganum harmala Linn, also known as wild rue, harmel or esphand, is a perennial plant species belonging to the Nitrariaceae family . The Nitrariaceae is a widespread family including 27 genera and 285 species subdivided into five subfamilies Melaleuca5), which was resistant against all concentrations , (4) and (3), respectively.The MICs ranged within 3.9–125.0 µL/mL (dilutions 1/8 to 1/256). The antifungal activity indicated that seed oil inhibited the growth of all tested fungi at all dilutions, except for  at 80 °C and finally stored at −20 °C for further analysis. The oil content was expressed as the lipid content percentage on seed powder. All chemicals and reagents used were of analytical grade and were obtained from Merck , Sigma-Aldrich , Acros Organics  and Fisher Scientific Co. .The mature seeds collected from fruits were dried and then milled in a K/IKA-WERKE M20 grinder. The seed oil was extracted as follows: 80 g of ground seeds were placed in a cellulose paper cone mixed with 250 mL of P. harmala seed oils were analyzed by high performance liquid chromatography (HPLC) according to the ISO 9936 standard. The HPLC system was equipped with a Shimadzu LC-20AT pump , a Shimadzu RF 20A fluorescence detector  and a Rheodyne 7725i manual injector . The tocols were separated on a normal phase column  at ambient temperature and with mobile phase flow level of 0.5 mL/min. The mobile phase was a 99.5:0.5 (v/v) n-hexane/isopropanol mixture. The data were analyzed using the Shimadzu LC solution software Version 2.53. Of seed oil, 0.5 g was diluted with 50 mL of hexane, and 20 µL of this was injected into the column. A mixture of standard tocol isomers  was dissolved in hexane at 2 μg/mL each and used for identification and quantification of the HPLC peaks. The amount of tocols in extracts was calculated as mg tocols/100 g oil.The tocols present in The fatty acid methyl esters (FAME) composition was determined by converting the oil to fatty acidmethyl esters by addition of 1 mL of n-hexane to 40 mg of oil followed by 200 µL of sodium methoxide (2 M). The mixture is heated in the bath at 50 °C for a few seconds followed by adding 200 µL HCl (2 N). The top layer (1 µL) was injected onto a GC  equipped with a flame ionization detector (FID) and a polarcapillary column  to obtain individual peaks of fatty acid methyl esters. The detector temperature was 275 °C and the column temperature was 150 °C held for min and increased at the rate of 15 °C/min to 200 °C and the rate of 2 °C/min to 250 °C and held for 4min. The run time was 45 min. The fatty acid methyl esters peaks were identified comparing their retention times with individual standard FAME  and analyzed with the Agilent Technologies Chemstation A09.01 Software. The relative percentage of the fatty acid was calculated on the basis of the peak area of a fatty acid species to the total peak area of all the fatty acids in the oil sample. Fatty acid methyl esters peak identification was confirmed by GC-MS (NIST 2002 database) operating under similar conditions as used for the GC-FID.Rhizoctonia solani (1); Macrophomina phaseolina (2); Pythium sp.1 (3); Pythium sp. 2 (4); Alternaria sp. (5); Colletotrichum sp. (6); Monosporascus cannonballus (7); Fusarium solani f.sp. cucurbitae (8); Fusarium oxysporum f.sp. melonis (9); and Fusarium oxysporum f.sp. niveum (10)) were identified and conserved in the Laboratory of Phytopathology of the High Institute of Agronomy of Chott-Mariem in Tunisia. Before testing antifungal activity of seed oil, fungal isolates (one for each species) were grown on Potato Dextrose Agar media for 7–10 days.Ten fungal isolates  and diluted up to 50 mL with distilled water. The serial dilution method (1/2n) from the prepared stock solution was used to prepare nine different concentrations: D1, 500 µL oil/500 µL DMSO; D2, 250 µL oil/750 µL DMSO; D3, 125 µL oil/875 µL DMSO; D4, 62.5 µL oil/937.5 µL DMSO; D5, 31.25 µL oil/968.75 µL DMSO; D6, 15.62 µL oil/984.38 µL DMSO; D7, 7.81 µL oil/992.19 µL DMSO; D8, 3.9 µL oil/996.1 µL DMSO; and D9, 1.95 µL oil/998.05 µL DMSO. The negative control was DMSO (1%). Mycelia disks (0.7-mm diameter), from five-day-old cultures, of the isolates used were deposited in the center of the plates. Mycelium growth was measured after 48, 72 and 96 h of incubation in darkness at 25 °C. Three replicates were used for each concentration. Each measure was compared with the control cultures under the same conditions. The percent inhibition of growth diameter (IP) was calculated accordingly to the following formula:The p < 0.05 according to Tukey’s test.A one-way analysis of variance (ANOVA) procedure was applied, using Minitab Statistical Software Release 14 . Significance differences between reported means were established at P. harmala populations contained very high levels of tocols (2722.68 mg/100 g), with δ-T the most abundant isomer (2441.78 mg/100 g) followed by γ-T (205.46 mg/100 g). We noted also the presence of tocotrienols for the first time in P. harmala. The preponderance of the two components (δ- and γ-T) explained the pertinent antifungal activity against the selected fungi and suggests that P. harmala has promise as an alternative approach for biological control of other microorganisms. In addition, the high levels in fatty acids in the P. harmala seeds, compared with other plants cited in the literature, revealed the importance of this plant as a new potential source of biofuel and for further medicinal applications. This idea is supported by the wide geographic spread of P. harmala in Tunisia and the adaptability of this species to various climactic conditions.All seed oils of


Inflammation is the main pathophysiology of dry eye, characterized by tear film instability and hyperosmolarity. The aim of this study was to investigate the association of inflammation and cellular autophagy using an in vitro dry eye model with primary cultured human corneal epithelial cells (HCECs). Primary HCECs cultured with fresh limbal explants from donors were switched to a hyperosmotic medium (450 mOsM) by adding sodium chloride into the culture medium. We observed the stimulated inflammatory mediators, TNF-α, IL-1β, IL-6 and IL-8, as well as the increased expression of autophagy related genes, Ulk1, Beclin1, Atg5 and LC3B, as evaluated by RT-qPCR and ELISA. The immunofluorescent staining of LC3B and Western blotting revealed the activated autophagosome formation and autophagic flux, as evidenced by the increased LC3B autophagic cells with activated Beclin1, Atg5, Atg7 and LC3B proteins, and the decreased levels of P62 protein in HCECs. Interestingly, the autophagy activation was later at 24 h than inflammation induced at 4 h in HCECs exposed to 450 mOsM. Furthermore, application of rapamycin enhanced autophagy activation also reduced the inflammatory mediators and restored cell viability in HCECs exposed to the hyperosmotic medium. Our findings for the first time demonstrate that the autophagy activation is a late phase response to hyperosmotic stress, and is enhanced by rapamycin, which protects HCECs by suppressing inflammation and promoting cells survival, suggesting a new therapeutic potential to treat dry eye diseases. Dry eye is a common disease that affects up to 50% of the population worldwide . It is cAutophagy is a highly conservative self-degradation pathway , which iHowever, the pathophysiological role of autophagy in regulating inflammation of the dry eye disease has not been well elucidated. Byun and colleagues observed that in Sjögren’s syndrome dry eye, the levels of ATG5 and LC3B II/I were significantly higher in tears and conjunctival epithelium, but decreased after one month of treatment with topical corticosteroid . These fp < 0.05, compared with 312 mOsM). Therefore, 500 mOsM was excluded for the subsequent study. Both the mRNA expression and protein production of pro-inflammatory cytokines TNF-α (Primary HCECs cultured in the isosmolar medium (312 mOsM), were switched to the hyperosmolar medium  with the addition of 44, 69 or 94 mM of NaCl, respectively. As evaluated by MTT cell viability assay A, HCECs es TNF-α B,C, IL-1es TNF-α D,E and Ies TNF-α F,G, as wes TNF-α H,I were n = 3, n = 3, TNF-α mRNA expression by HCECs was sharply stimulated to 2.17 ± 1.11 and 4.82 ± 1.89 fold respectively n = 3, B as the n = 3, n = 3, IL-1β mRNA expression increased to 2.32 ± 0.98 fold n = 3, D, accompn = 3, n = 3, IL-6 mRNA expression increased to 2.71 ± 0.63 fold when exposed to the 450 mOsM medium n = 3, F. Meanwhn = 3, n = 3, The mRNA expression of chemokine IL-8 was raised to 2.37 ± 0.57 fold n = 3, H in the n = 3, Autophagy begins with activation of Ulk1 in the initiation phase, which stimulates Beclin1 in the nucleation phase to regulate and generate formation of the nascent autophagosome in the elongation phase whereby cytosolic components are enclosed into an isolation membrane. We found that the expression of Ulk1 A in the n = 3, n = 3, LC3B is a marker commonly associated with autophagy activation with autophagosome formation. LC3B mRNA levels increased moderately to 1.82 ± 0.43 but dramatically to 6.39 ± 1.17 fold, respectively, in response to hyperosmolar medium with 400 and 450mOsM D. The imp < 0.05, control vs. 24 or 48 h, n = 3). Interestingly, the production of p62 protein, also known as SQSTM1, decreased significantly in 24–48 h, indicating the autophagic flux enhanced by hyperosmolarity  vs. Rapa (+) at 24 h (87.9 ± 3.0% vs. 91.8 ± 2.1%), 36 h (90.3 ± 2.9% vs. 95.0 ± 2.8%), or 48 h (88.2 ± 2.0% vs. 93.2 ± 1.8%), p < 0.05; Meanwhile, the release of inflammatory cytokines was suppressed by rapamycin, evidenced by much lower levels of TNF-α (43.17 ± 12.64 pg/mL), IL-1β (40.11 ± 11.06 pg/mL), IL-6 (4.38 ± 0.84 ng/mL) and IL-8 (6.13 ± 0.79 ng/mL) protein levels in the medium supernatants C–F. InteHyperosmolarity and inflammation are the key pathogenesis in dry eye disease, as acknowledged by TFOS DEWS II . As a rePrevious studies, including ours, have confirmed that hyperosmolarity can stimulate an inflammatory reaction via different pro-inflammatory cytokines and chemokine ,34,35,36Our study further observed the increase of mRNA expression levels of Ulk1 in initiation phase, Beclin1 in nucleation phase, as well as Atg5 and LC3B in elongation phase during autophagosome formation, which reveal the autophagy activation, an instinctive response to stress . The proThe formation of autophagosome, detected by fluorescence of LC3B, not only prompts autophagic activation, but also prompts autophagy dysregulation, such as slowed lysosomal degradation or inefficient fusion due to a lack of lysosomal fusion. Considering this, it is essential to monitor the autophagy flux, which is the degradative process that the autophagosome fuses with the lysosome and subsequently breaks down. The autophagy marker LC3B and substrates such as P62/SQSTM1 protein are the common autophagy reporters . P62/SQSThe crosstalk between inflammation and autophagy has recently become an interesting topic, although the mechanism is not fully elucidated ,24,40,41In this study, we interestingly found that hyperosmolarity triggered autophagy activation later than inflammation in primary HCECs. Different from the inflammation factors activated at 4 h, autophagy is a late response to hyperosmotic stress in HCECs at 24 h. After autophagosome formation in HCECs exposed to hyperosmolarity, evidenced by the increases of Beclin1, Atg5, Atg7 and LC3B, as well as the decrease of P62, a few upward blips in cell viability were observed, accompanied by a moderate drawdown of TNF-α, IL-1β, IL-6 as well as IL-8. Autophagy modulates inflammation through influencing the inflammatory cells survival conditions and altering secretion of inflammatory cytokines ,44, by wTo confirm the protective role of autophagy activation, we further performed the experiments using an autophagy inducer rapamycin. We observed that rapamycin did not show its protective effect on HCECs in hyperosmolarity within 4–16 h. It is supposed that the activation of autophagy is still a late response by HCECs with rapamycin. The autophagy activation was significantly enhanced after 24 h, when rapamycin may accelerate its effect, and then suppress the secretion of inflammatory cytokines and chemokine. The cell viability reduced by hyperosmolarity was also restored by rapamycin. The findings established a link between the autophagy activation by rapamycin and the restoration of cell homeostasis. Further investigation is needed to explore the potential regulation mechanism of autophagy in the pathogenesis of dry eye at different stages. Thorough illustration of the mechanism of autophagy and its interaction with other pathological events, such as inflammatory response and oxidative stress ,34,45,46Fresh human corneal tissues not suitable for transplant were provided by the Lions Eye Bank of Texas . The corneal tissues were obtained from donors within 72 h after death (age of donors ranged from 18–65 years). Primary HCECs were cultured from donors’ limbus explants in a supplemented hormonal epidermal medium (SHEM) containing 5% FBS as our previous publications . FourteeTM 2000 spectrophotometer . Then, the first strand cDNA was synthesized by reverse transcription using 2.0 μg of the total RNA extraction and Ready-To-Go You-Prime First-Strand Beads . As per our previous described methods [® Gene Expression Assays  were: TNF-α (Hs00174128_m1), IL-1β (Hs01555413_m1), IL-6 (Hs00174131_m1), IL-8 (Hs00174103_m1), as well as GAPDH (Hs99999905_m1) as an internal control. The results were analyzed by the comparative threshold cycle method and normalized by GAPDH as an internal control.Total RNA extraction was conducted with the RNeasy Plus Mini Kit , quantified with a NanoDrop methods , RT-qPCR® M200 PRO multimode microplate readers . Double-sandwich ELISA for human TNF-α, IL-1β, IL-6 and IL-8  was conducted to determine the protein concentration of these mediators in the conditioned medium with different treatments . Absorba® TGX Stain-Free™ Protein Gel  and transferred electrophoretically to 0.2 μm PVDF membranes . The membranes were blocked with 5% nonfat milk in TTBS for 1 h at room temperature and incubated with primary antibodies against human Beclin1, ATG5, ATG7, LC3B, P62/SQSTM1 , or β-Actin  overnight at 4 °C. After five washes with TTBS for 5 min each, the membranes were incubated with HRP conjugated Goat anti-Rabbit IgG (H+L) Secondary Antibody  or Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody  for 1 h at room temperature. The signals were detected with a Clarity™ Western ECL Substrate , and the images were obtained by a ChemiDoc™ MP Imaging System . As described in our previous publication , equal a® 488-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) secondary antibody  was applied, and 4′,6-diamidino-2-phenylindole  was used for nuclear counterstaining. As a negative control, secondary antibody was applied alone and compared to isotype goat IgG. The staining was then photographed with Nikon A1 Confocal Laser Microscope System  and processed with Image J software . Primary HCECs, cultured on 8-chamber slides, were fixed with methanol at –20 °C for 5 min. Indirect immunofluorescent staining was conducted as our previously described . Primary4 cells/ml onto 96-well plates with 200 μL/well of SHEM overnight, and treated for 24 h or different time periods  in isosmolar (312 mOsM) or hyperosmolar medium (450 mOsM), with 1 h prior co-incubation with rapamycin  or not. The proliferative activity of the cells was quantitatively measured by a Cell Growth Determination Kit, MTT based . The optical density (OD) of absorbance at 570 nm was read by Infinite® M200 PRO multimode microplate readers  [Primary HCECs were seeded at 2 × 10 medium 4 mOsM, wit-test was used to compare two groups. One-way ANOVA test was used to compare three or more groups, followed by Dunnett’s post-hoc test to compare within groups. p < 0.05 was considered statistically significant. Student’s Our findings for the first time demonstrate that the autophagy activation is a late response to hyperosmotic stress after triggering inflammation in HCECs, which protects HCECs by reducing the inflammatory mediators and promote sell survival. These protective effects are further strengthened when the autophagy activation is enhanced by rapamycin in HCECs exposed to hyperosmolarity, an in vitro dry eye model, suggesting a new therapeutic potential to treat dry eye diseases. Further investigations are needed to explore the mechanisms of autophagy regulation underlying in the pathogenesis of dry eye diseases.


By using a GPER antagonist G15, we demonstrated that the GPER mediates the action of estrogen. Together, the data from this in vitro study demonstrate that estrogen can regulate LDLR levels mainly through GPER activation, which prevents PCSK9-dependent LDLR degradation in HepG2 cells.Plasma levels of PCSK9 are significantly higher in postmenopausal women. Pharmacologically increased estrogen levels have been shown to lower PCSK9 and LDL-C levels in animals and humans. The action of estrogen suggests that it has the ability to prevent PCSK9-mediated LDLR degradation in liver cells. However, little is known about how estrogen alters PCSK9-mediated LDLR degradation. Here, we report that 17β-estradiol (βE2) reduces PCSK9-mediated LDLR degradation by a mechanism that involves activation of the G protein-coupled estrogen receptor (GPER). In cultured HepG2 cells, βE2 prevented the internalization of PCSK9, which subsequently lead to PCSK9-mediated LDLR degradation. The altered LDLR levels also resulted in an increase in LDL uptake that was not observed in the absence of PCSK9. In addition, we showed that clathrin was rapidly increased in the presence of PCSK9, and this increase was blocked by βE2 incubation, suggesting rapid recruitment of clathrin in HepG2 cells. PLCγ activation and intracellular Ca Estrogen plays a role in lipid metabolism in both physiological and pharmacological contexts. Because of the benefits of estrogen, as documented in the past decade, premenopausal women have experience delayed first-time myocardial infarction –3 and haIt has been shown that the G protein-coupled estrogen receptor (GPER) mediates the rapid effect of estrogen via a non-genomic effect , 32, 41.2+ signaling, which alters the distribution of clathrin, thereby inhibiting PCSK9-mediated degradation of LDLR. These data may help understand the molecular mechanisms underlying the physiological effects of estrogens.It has recently been reported that the GPER is a receptor in the plasma membrane that can be translocated to the endoplasmic reticulum (ER) . In HepGTM FL LDL (L3483), Alexa Fluor® 488 protein labeling kit (A10235), Alexa Fluor® 488-conjugated goat anti-mouse IgG (A11001) and Alexa Fluor® 488-conjugated goat anti-rabbit IgG (A11008) were all purchased from Thermo Fisher . A cAMP parameter assay kit (KGE002B) was purchased from R&D . The PrimeScript™ II 1st Strand cDNA synthesis kit (6210A) and TB GreenTM Premix Ex Taq II (RR420B) were purchased from TaKaRa .Recombinant human PCSK9  was purchased from Genlocus . βE2 (E2758-1G) was purchased from Sigma-Aldrich . The GPER antagonist G15 (1161002-05-6) was purchased from Cayman Chemical . The PLC inhibitor U73122 (U6756-5 MG) was purchased from Sigma-Aldrich . Anti-LDLR antibody (sc-373830) was ordered from Santa Cruz . Anti-phospho-PLCγ (Tyr783) polyclonal antibody (2821) and anti-PLCγ rabbit antibody (2822) were purchased from Cell Signaling . Anti-clathrin rabbit polyclonal antibody (ab59710) was purchased from Abcam . The BODIPYHepG2 cells  were maintained at 37°C in phenol red-free DMEM supplemented with 10% fetal bovine serum, 100 IU/mL penicillin, and 100 μg/mL streptomycin and allowed to grow until 85% confluent prior to the experiment.® 488 from the protein labeling kit (AF−PCSK9). To determine whether βE2 blocked the internalization of PCSK9, HepG2 cells were seeded into 48-well plates at 4 × 104 cells per well in growth medium. After serum deprivation for 16 h, the cells were incubated with 25 μg/mL AF−PCSK9 for 0.5–6 h and treated with 0.01–10 μM βE2 for 2 h in the presence of AF−PCSK9 (25 μg/mL). For all assays, the cells were pretreated with 1 μM G15 for 15 min prior to the addition of βE2 to block GPER action. After a series of wash steps with PBS, internalized AF−PCSK9 was directly observed under an inverted fluorescence microscope, and the fluorescence intensity of AF−PCSK9 in isopropyl alcohol was detected by a SpectraMax M5 reader and reported in RFUs.For the internalization assay, rhPCSK9 was labeled with an Alexa Fluor2-cell imaging dish  at 6 × 104 cells per well in growth medium and allowed to adhere to the plate for 24 h. After serum deprivation for 16 h, the cells were treated with or without 0.01–1 μM βE2 for 6 h in the presence or absence of rhPCSK9 (25 μg/mL). The expression level of LDLR was detected by western blotting and immunofluorescence.To investigate whether βE2 prevents PCSK9-mediated LDLR degradation, HepG2 cells were seeded into 6.2 cm5 cells per well in growth medium and allowed to adhere to the plate for 24 h. After serum deprivation for 16 h, the cells were incubated with or without 0.01–1 μM βE2 for 6 h in the presence of rhPCSK9 (25 μg/mL). Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. cDNA was prepared from 1 μg RNA using a PrimeScript™ II 1st strand cDNA synthesis kit according to the manufacturer's protocol (TaKaRa). Real-time quantitative PCR was performed to detect the expression of LDLR mRNA in a CFX96 PCR system (Bio-Rad) with 1 μL of cDNA diluted 25-fold in 12.5 μL of the reaction mixture with TB Green Premix Ex Taq II and 0.1 μM forward and reverse primers for LDLR and β-actin. Specific primers for LDLR and β-actin were used: for LDLR, forward, 5′-GTTATTCAGGGAGAACGGCT-3′ and reversse, 5′-GAGTCAACCCAGTAGAGGC-3′; and for β-actin, forward, 5′-CTGGGCATGGAGTCCTGTG-3′, and reverse, 5′-ATCTTCATTGTGCTGGGTG-3′. The primers were obtained from GENEWIZ . We set amplification parameters: 95°C for 30 s; 40 cycles of 95°C for 5 s; and 56°C for 30 s.For detection of LDLR mRNA, HepG2 cells were seeded into 6-well plates at 1 × 10® FL kit. HepG2 cells were seeded into 48-well plates at 4 × 104 cells per well in growth medium and allowed to adhere to the plate for 24 h. After serum deprivation for 16 h, the cells were treated with or without 0.01–10 μM βE2 for 4 h in the presence of rhPCSK9 (25 μg/mL). For the assay, the cells were pretreated with 1 μM G15 for 15 min prior to the addition of βE2 to block GPER action. BODIPYTM FL LDL (6 μg/ml) was added for 3 h, and then the cells were then washed with PBS. Isopropyl alcohol was added to measure the uptake of BODIPYTM FL LDL. The plates were read with a SpectraMax M5 reader .LDL uptake was measured using a BODIPY2-cell imaging dish  at 6 × 104 cells per well in growth medium and allowed to adhere to the plate for 24 h. After serum deprivation for 16 h, the cells were incubated with 25 μg/mL rhPCSK9 for 0–2 h and treated with 0.01–1 μM βE2 for 2 h in the presence of rhPCSK9 (25 μg/mL). For all assays, the cells were pretreated with 1 μM G15 for 15 min prior to the addition of βE2 to block GPER action. After a series of wash steps with PBS, clathrin was detected by immunofluorescence with the corresponding antibodies.Clathrin was also measured for the distribution assay. HepG2 cells were seeded into 6.2 cm4 cells per well and allowed to adhere to the plate for 24 h. After serum deprivation for 16 h, the cells were washed with Ca2+-free HBSS  and then loaded with 5 μM Fluo-4 AM for 20 min. The cells were washed again with Ca2+-free HBSS. Each cell imaging dish was placed in a laser scanning confocal microscope . First, the fluorescence intensity images were collected every 15 s for 5 min, and 0.1 μM E2 was then added and fluorescence was monitored continuously for 15 min. For the assay, the cells were treated with 1 μM G15 and 1 μM U73122 for 15 min prior to the addition of E2 to block GPER and PLCγ action. Data were quantified and analyzed using ZEN 2010 software. The background was subtracted, and the fluorescence was expressed relative to values at the beginning of the experiment.Calcium mobilization was detected by Fluo-4 AM . HepG2 cells were seeded in a cell imaging dish at 6 × 105 cells with 5 mL growth medium. After serum deprivation for 16 h, the cells were treated with or without 0.1 μM βE2 for 5 min in the presence of rhPCSK9 (25 μg/mL). For the assay, the cells were pretreated with 1 μM G15 for 15 min prior to the addition of βE2 to block GPER action. After treated with 0.1 μM βE2 for 5 min or 0.01–1 μM βE2 for 6 h, the cells were washed twice with cold PBS and lysed with 1 × RIPA buffer  supplemented with a protease inhibitor PMSF (1 mM) to obtain the total protein. An equal amount of total protein from each lysate was loaded and separated on 4%−10% gels for SDS-PAGE and electroblotted to polyvinylidene fluoride (PVDF) membranes. The primary antibodies used included anti-LDLR mouse monoclonal antibody (1:150 diluted in PBS), anti-pPLCγ (Tyr783) rabbit antibody (1:500 diluted in PBS), and anti-PLCγ rabbit antibody (1:500 diluted in PBS). The secondary HRP-conjugated antibody was goat anti-mouse IgG (1:4000 diluted in PBS) and goat anti-rabbit IgG (1:4000 diluted in PBS). Western blots were measured for chemiluminescence (Thermo Fisher), detected by ImageQuant LAS 500 , and analyzed by Image Plus 6.0 analysis software.HepG2 cells were seeded into T25-cell culture bottles at 5 × 10® 488-conjugated goat anti-mouse IgG (1:250 diluted in PBS) and Fluor® 488-conjugated goat anti-rabbit IgG (1:250 diluted in PBS) at room temperature for 1 h. Excess antibody was removed by washing the cells with PBS, and the cells were stained with 4′6-diamidino-2-phenylindole (DAPI) and then viewed with a confocal microscope .Experiments were performed in cell imaging dishes as described in the methods section LDLR Protein Assay and Clathrin Assay. HepG2 cells were washed three times with PBS and fixed with a 4% paraformaldehyde solution for 20 min. The fixed cells were permeabilized with PBS containing 0.3% Triton X-100 for 3 min at 4°C and washed three times with PBS containing 0.5% BSA. The cells were incubated with 3% BSA in PBS for 30 min at 37°C and then incubated with primary antibodies: anti-LDLR mouse monoclonal antibody (1:100 diluted in PBS) and anti-clathrin rabbit polyclonal antibody (1:500 diluted in PBS) at 4°C overnight. Antibodies were stained with Alexa FluorP < 0.05.Data are presented as the mean ± SEM. Differences among groups were tested by SPSS 17.0 software with one-way ANOVAs followed by an LSD or Duncan test. Comparisons were considered to be statistically significant if AF−PCSK9) in HepG2 cells expressing LDLR. The fluorescence signals were detectable in the HepG2 cells at 30 min and peaked at 2 and 4 h after AF−PCSK9 (25 μg/mL) was added to the cells. The localization of AF−PCSK9 was initially at the cell surface, and it was later localized throughout the cytoplasm  was added. In contrast, βE2 treatment decreased the cytoplasmic distribution of AF−PCSK9 and increased the distribution of AF−PCSK9 at the cell surface  pretreatment of cells  pathway in other cell types . The actocytosis , 46. In ependent , and cAM2+ release, which in turn alters clathrin distribution or influences the clathrin trafficking pathway to affect the internalization of the PCSK9-LDLR complex. Another possible explanation is that the GPER is localized to the endoplasmic reticulum (eticulum , where teticulum . The coaeticulum . InvestiIn summary, our results highlight the important role of estrogen in regulating LDLR levels through GPER activation and being associated with clathrin trafficking pathway. Therefore, estrogen may have a role in modulating the levels of total blood cholesterol and LDL-C in hyperchlesterolemic women.All datasets generated for this study are included in the article/supplementary material.WF designed and planned the study and performed the experiments. X-PG and L-MY designed and planned the study and wrote the manuscript. SZ, Y-PD, and W-JZ performed the experiments and analyzed the data.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.


Chlamydomonas reinhardtii with microcystin-LR (MC-LR) to determine its allelopathic effects. Results showed that MC-LR markedly suppressed C. reinhardtii cell viability. Comparative proteomic and physiological analyses revealed that MC-LR significantly up-regulated protein abundance of antioxidants ascorbate peroxidase (APX) and catalase (CAT) at the beginning stage of exposure. This was accompanied by an over-accumulation of hydrogen peroxide (H2O2), suggesting that MC-LR suppresses cell viability via oxidative damage. Furthermore, we found that MCs induced desulfhydrase (DES) activity for hydrogen sulfide (H2S) generation at the beginning stage. Additional H2S donors reactivated antioxidant enzyme activity, which reduced H2O2 accumulation and ultimately enhanced C. reinhardtii tolerance to MC-LR damage. This effect could be reserved by inhibiting H2S biosynthesis. Simultaneously, we found that H2S also suppressed MC-LR-induced cell autophagy, and thus attenuated the toxic effects of MC-LR. Our findings suggest that oxidative bursts may be the main reason for the allelopathic effects of MC-LR on C. reinhardtii viability and that H2S signaling may enhance C. reinhardtii tolerance to MC-LR through the activation of antioxidant enzyme activity and suppression of cell autophagy.Cyanobacterial blooms have become more frequent and serious in recent years. Not only do massive blooms cause environmental pollution and nutrient eutrophication, but they also produce microcystins (MCs), a group of toxic cycloheptapeptides, which threaten aquatic ecosystem and human health. As such, clarifying the allelopathic interactions between cyanobacteria and other algae is critical to better understand the driving factors of blooms. To date, however, such studies remain largely insufficient. Here, we treated model alga  Microcystis aeruginosa is the most common bloom-forming species due to its wide distribution, which has reached up to 70% of the global cyanobacteria biomass, and has strong ability to produce microcystin (MC) toxins. MCs belong to a group of cycloheptapeptide inhibitors of intracellular protein phosphatases 1&2A (PP1/PP2A). These toxins have been found to have adverse effects on the liver, small intestine, colon, brain, kidney, lung, heart, and reproductive system, and also exhibit potential cancer-promoting activity , a small gaseous molecule, is involved in multiple physiological functions in mammals, such as blood vessel relaxation, neurotransmission, insulin signaling, and angiogenesis lyase protein family and are mainly responsible for the generation of H2S in Arabidopsis  and triggering oxidative stress . Oxidatihrocytes . In planhrocytes . Hydrogeogenesis . H2S is l stress . In planpyruvate . Desulfhbidopsis . Previoumulation . H2S is utophagy . Our preutophagy . It is rutophagy , suggestC. reinhardtii with the cyanobacterial toxin MC-LR. Using comparative proteomics and physiological experiments, we found that MC-LR induced the differential expression of several proteins, including APX and CAT, induced the over-accumulation of ROS, mainly hydrogen peroxide (H2O2), and increased the activities of APX and CAT at the early stage of exposure. We also found that MC-LR induced DES activity for the release of H2S. Interestingly, additional H2S also enhanced the activity of APX and CAT to efficiently scavenge ROS production, and thus ultimately improved the tolerance of C. reinhardtii to MC-LR. H2S signaling also suppressed MC-LR-induced cell autophagy and thus reduced toxic damage in C. reinhardtii. Our findings revealed a novel mechanism by which H2S is involved in the allelopathic interaction between cyanobacteria and other algae.To better understand the allelopathic interactions between cyanobacteria and other algae, we treated unicellular C. reinhardtii wild-type strain was purchased from the Freshwater Algae Culture Collection Center at the Institute of Hydrobiology (Chinese Academy of Sciences (CAS), Wuhan, China). Cells were cultured under continuous light at 23°C in Tris-acetate-phosphate (TAP) medium. MC-LR was purchased from the Sigma Chemical Company . For MC-LR treatment, C. reinhardtii cells in the stationary phase (106 cells/ml) were treated with different concentrations of MC-LR. At different time points, cells were removed for further analysis.The 2S artifical donors NAHS or GYY, and H2S scavenger hypotaturine (HY) was used for investigating H2S function as previous report  were incubated with 0.1% (w/v) Evans blue for 5 min, then washed once with 50 ml of TAP medium, and re-suspended in an equal volume of TAP medium. Viability was determined by counting the colored cells in a hemocytometer, with 10 random counts performed. Control tests of viability were performed with suspension cultures fixed in 70% ethanol or in FAA . The photosynthesis Fv/Fm ratio was measured as reported  was used as a marker of cell death . In briereported .g for 15 min at 4°C. The resulting pellet was collected, frozen in liquid N2, and well ground using a mortar and pestle, keeping the sample submerged in liquid N2. Ice-cold protein extraction buffer (10 ml) containing KCl (100 mM), Triton X-100 (1% v/v), β-mercaptoethanol (1% v/v), L-ascorbic acid (50 mM), and phenylmethanesulfonyl fluoride (1 mM) in Tris-HCl buffer  was used and powdered, and the suspension was mixed with an equal volume of Tris-phenol (pH 8.0) and vortexed thoroughly. The upper phenolic phase was obtained after centrifugation at 12,000g for 30 min at 4°C and mixed with five volumes of ammonium acetate (10 mM) in methanol, with the mixture kept at −20°C overnight. The protein pellet was then obtained after centrifugation at 12,000g for 15 min at 4°C, followed by washing in cold acetone containing 0.1% β-mercaptoethanol and drying for storage at −80°C. To dissolve the protein pellet, Tris–HCl lysis buffer  containing urea (7 M), EDTA (2 mM), thiourea (2 M), CHAPS (4% v/v), and PMSF (1 mM) was added at a final ratio of 10 mg/ml, with the solution sonicated at 200 W for 15 min to accelerate dissolution, then centrifuged for 15 min at 12,000g in a cold room. The pellet was discarded, and the supernatant was transferred to another tube. After this, 10 mM DTT was added to reduce the disulfide bond, iodoacetamide  was added under dark conditions to covalently block cysteine, and five volumes of cold acetone were added under −20°C for 2 h to the pellet protein, which was then dried and re-dissolved in 500 μl TEAB  and centrifuged for 15 min at 12,000g in a cold room. The protein concentration was quantified by the Bradford method  at a ratio of 30:1 (protein/trypsin) for 16 h at room temperature. The digested peptides were dried using vacuum centrifugation, and then dissolved in TEAB buffer (0.5 M) as recommended by the protocols for the 8-plex iTRAQ reagent . One unit of iTRAQ reagent 113 to 117 isobaric tags in 24 μl of isopropanol was used to label the sample for 2 h under room temperature. After labeling, the peptide mixture was pooled and dried using vacuum centrifugation, then re-suspended in 4 ml of dissolution buffer containing 25 mM NaH2PO4 and 25% acetonitrile (pH 2.7). The sample was separated by an Ultremex strong cationic exchange column (4.6 × 250 mm) in a Shimadzu LC-20AB HPLC Pump system . Sample fractions were eluted with a linear gradient flow at 1 ml min−1 and an elution program of 5% buffer B  for 7 min, 5–60% gradient buffer B for 20 min, 60–100% buffer B for 2 min, and maintenance with 100% buffer B. The elution was divided into 20 fractions by monitoring absorbance at 214 nm, and then desalted using a C-18 column. The desalted fraction was dissolved in buffer C containing 5% ACN and 0.1% formic acid at a ratio of 0.5 µg µl−1 and centrifuged at 20,000g for 10 min at 4°C. For high-performance liquid chromatography (HPLC)-mass spectrometry (LC-MS-MS), 5 μl of the supernatant was loaded by the sampler onto a Shimadzu LC-20 AD nanoHPLC  with a C18 column (200 µm inner diameter). The peptide was eluted with a linear gradient program, with a washing program of 5% buffer D  for 5 min, 3–35% buffer D for 35 min, 60–80% buffer D for 2min, and maintenance in 80% buffer D. Mass spectrometry following HPLC was performed using a TripleTOF 5600 system  equipped with a Nanospray III source, and raw data were obtained under ion spray voltage at 2.5 kV, 30 psi N gas, 15 psi nebulize gas, and heater temperature at 150°C. The instrument was operated in information dependent acquisition (IDA) mode, with mass range between 100–2 400 m/z and detection resolution over 30,000 (FWHM) in the Orbitrap analyzer set for TOF-MS scans. The 30 most intense ions above a threshold of 120 cps with a 2+ to 5+ charge state were selected for fragmentation. A sweeping collision energy at 35 ± 5 eV coupled with iTRAQ adjusted rolling collision energy was used for all precursor ions during collision-induced dissociation.Extracted protein (100 µg) from C. reinhardtii proteome database (http://plants.ensembl.org). Peptide mass and fragment mass tolerance values were 10 ppm and 0.1 Da, respectively, and one missing cleavage in the trypsin digestion was allowed. For all searching, oxidation of methionine residues and pyroglutamate formation of N-terminal glutamine residues were set as the variable modifications, and carbamidomethyl formation of cysteine residues and iTRAQ labeling of lysine sites were set as the fixed modifications. Only peptides with significance scores over 20 at a 99% confidence interval and false discovery rate (FDR) of less than 1.5% were selected for quantification. Cutoffs of 1.5- or 0.6-fold with a p-value of less than 0.05 were set to indicate significantly differential protein expression.The raw MS/MS data files were converted to Mascot generic files by Proteome Discoverer 1.2 and searching was performed using the in situ detection of H2O2, 3, 3’-diaminobenzidine (DAB) staining was performed as described previously . The reaction was terminated at 6–7 h post-inoculation when a brown precipitate became visible in the cell after microscope observation. The cells were then fixed for 15 min in ethanol:acetic acid:glycerol (3:1:1) and destained with acetone for 12 h. The cells were observed by light microscopy under bright field at 100× magnification. The H2O2 levels were determined using a Synergy 2 Multi-mode Reader  equipped for excitation in the range of 530–560 nm and fluorescence emission at 590 nm. The H2O2 concentration was calculated using a H2O2 standard curve.For eviously . In brieg for 15 min at 4°C. The resulting pellet was quickly homogenized in liquid nitrogen and extracted with 5 ml of extraction buffer  for 10 min. The homogenates were filtered through two layers of cheesecloth and centrifuged at 4°C and 15,000g for 15 min. The supernatants were desalted on a Sephadex G-50 column and used to measure enzymatic antioxidant activities. Total soluble protein concentration of the supernatants was determined by the Bradford method. The activities of APX and CAT were determined as described previously . Enzyme activity was quantified using the molar extinction coefficient for ascorbate (2.8 mM− 1 cm−1) and results were expressed in μmol min−1 gFW−1 , taking into consideration that 2 mol of ascorbate is required for reduction of 1 mol of H2O2. Results from four independent replicates were analyzed .The activity of APX was determined based on the oxidation of ascorbate. The reaction solution (3 ml) contained 0.1mM sodium acetate buffer (pH 5.4), 1 M EDTA, 0.8 mM H2O2. The reaction solution (1.5 ml) contained 35 mM phosphate buffer (pH 7.0), 15 mM H2O2, and 100 μl of extracted solution. The reaction was initiated by adding the enzyme solution. Changes in absorbance at 240 nm were read every 10 s for 90 s using a spectrophotometer. Enzyme activity was quantified using the molar extinction coefficient for H2O2 (36 M−1 cm−1) and results were expressed in μmol min−1 gFW− 1. Results from three to eight independent replicates were analyzed .The activity of CAT was determined based on the oxidation of H2S was absorbed in a zinc acetate trap. The trap consisted of a small glass tube containing 3 ml of 0.5% (w/v) zinc acetate fixed to the bottom of the reaction bottle. After 30-min reaction, 0.3 ml of 20 mM dimethyl-p-phenylenediamine was dissolved in 7.2 mM HCl and added to the trap. This was followed by injection of 0.3 ml of 30 mM ferric ammonium sulfate in 1.2 ml of HCl. After incubation for 15 min at room temperature, the amount of H2S in the zinc acetate trap was determined colorimetrically at 667 nm. A calibration curve was established by NaHS according to the above method, and H2S content in C. reinhardtii was expressed as nmol g−1 fresh weight.After MC-LR treatment, the cells (50 ml) were harvested and ground to a fine powder with liquid nitrogen and extracted in 10 ml of phosphate-buffered saline  containing 0.1 mM EDTA and 0.2 mM ascorbic acid. The homogenate was mixed in a test tube containing 100 mM PBS (pH 7.4), 10 mM L-Cys, and 2 mM phosphopyridoxal at room temperature, and the released H3 dissolved in 1.2 N HCl and 100 ml of 20 mM N,N-dimethyl-p-phenylenediamine dihydrochloride dissolved in 7.2 N HCl. The formation of methylene blue was determined at 670 nm, and enzyme activity was calculated using the extinction coefficient of 15 × 106 cm2 mol−1 . First-strand cDNA synthesis and qRT-PCR analysis were performed as described previously  were obtained from AgriSera , and diluted 1:3,000 for anti-tubulin and 1:1,000 for ATG8.Immunoblotting was performed as described previously . Total pC. reinhardtii, we first examined the dose effect of MC-LR on C. reinhardtii biomass. The C. reinhardtii cells were cultured in TAP medium for 3 d, followed by treatment with different concentrations of MC-LR for 7 d. After this, we checked cell viability and biomass. As shown in C. reinhardtii, accompanied by low cell death, as determined by Evans blue staining. Once the concentration of MC-LR was over 100 nM, cell biomass was significantly reduced by 40% and cell death dramatically increased, reaching over 80% at 300 nM MC-LR. Thus, we selected 100 nM MC-LR for further study. As shown in C. reinhardtii.To determine the toxic effects of MC-LR on the growth of C. reinhardtii response to MC-LR, we performed comparative iTRAQ proteomics to monitor the dynamic protein changes after 100 nM MC-LR treatment. Samples were collected at 1, 3, and 5 d for iTRAQ labeling. Samples without MC-LR exposure were used as the control. Each iTRAQ experiment was replicated three times. Protein prediction after data collection was conducted using Mascot software and the public C. reinhardtii protein database. Protein abundance was divided into three groups: group 1 (1 d of MC-LR treatment/untreated control), group 2 (3 d of MC-LR treatment/untreated control), and group 3 (5 d of MC-LR treatment/untreated control), with a fold-change cut off of >1.5-fold for significantly increased expression and <0.6-fold for significantly decreased expression. In total, we detected 68 proteins with a significant change in expression in response to MC-LR treatment (p <0.05) (To understand the mechanism of the p <0.05) Table 1.C. reinhardtii cell viability through different strategies.We also conducted hierarchical cluster analysis to categorize the proteins that showed differential expression profiling during MC-LR toxicity stress , H2S biosynthesis enzyme inhibitor aminooxyacetic acid (AOA) for 24 h, and then compared H2O2 accumulation. As shown in 2O2 accumulation, whereas addition of AOA and HY aggravated MC-LR-induced H2O2 accumulation. Furthermore, we measured the activity of APX and CAT after MC-LR and different chemical treatment. As shown in 2S signaling protects C. reinhardtii from the damage induced by MC-LR through the up-regulation of APX and CAT activity to reduce H2O2 over-accumulation.As shown above, MC-LR induced the over-accumulation of ROS in enge ROS , we reas2S signaling suppresses cell autophagy lyase and cysteine synthase increased at the beginning stage of MC-LR treatment. These proteins are both associated with the biosynthesis of H in vivo . In addil stress , suggest2O2, are strongly induced by environmental stress to damage plants. As a result, plants have evolved a defense mechanism to avoid ROS-induced damage, e.g., enhancing antioxidant enzyme activity to scavenge ROS accumulation  MC-LR treatment. It is possible that the reason why MC-LR caused an increase in ROS is because MC-LR did not efficiently sustain high levels of enzyme activity to successfully scavenge the over-accumulation of ROS. We demonstrated that H2S signaling activated antioxidant enzyme activity, thus it is possible that H2S enhanced the tolerance to C. reinhardtii against MC by activating antioxidant enzymes to scavenge ROS toxicity. We treated C. reinhardtii with MC-LR and additional H2S donors and found that the addition of NaHS and GYY improved cell viability, including an increase in cell density and Fv/Fm ratio. In contrast, inhibiting H2S biosynthesis with AOA and HY aggravated MC-LR toxicity on C. reinhardtii cell viability. Thus, these findings confirm the protective function of H2S against MC damage. We also found that the addition of H2S donors reactivated APX and CAT activity compared to that after MC-LR treatment alone, whereas addition of H2S biosynthesis inhibitors further suppressed enzyme activity. Therefore, we propose that H2S enhances the tolerance of C. reinhardtii cells to MC exposure by up-regulating and sustaining high levels of antioxidant enzyme activity to scavenge MC-LR-induced ROS.ROS, mainly Hmulation . Our proC. reinhardtii cell viability through ROS-induced cell autophagy. As H2S signaling negatively regulates cell autophagy , National Natural Science Foundation of China (41766006), and Six Talent Peaks Project in Jiangsu Province.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.


The transverse relaxation time (T2) of water molecules differ between tissues. Because the molecular motion of water is significantly affected by macro molecules, tissue containing fibrous polymers such as collagen has shorter T2 values. Patients with Duchenne Muscular Dystrophy (DMD) develop myocardial fibrosis in the late stage of the disease. The goal of this study was to determine the association of myocardial T2 distribution to the severity of DMD.1 = 6 ms, TE2 = 34 ms) DMD patients were grouped according to the ejection fraction (EF) and the circumferential strain (εcc) as: A: EF ≥ 56%, εcc ≥ 12%, mean age = 10.5 yrs, B: EF ≥ 56%, εcc ≤ 12%, mean age = 17.5 yrs, C: EF ≤ 56%, mean age = 17.5 yrs. T2 values were plotted for each subject as a histogram. The normalized mean histograms from each group were compared by the Full Width of Half Maximum (FWHM).Twenty six DMD patients and eight normal subjects were studied. T2 maps of the left ventricle were generated using a black blood dual spin echo method.  higher in group B (0.42 ± 0.06) compared to Group A (0.54 ± 0.08). Regression analysis show moderate association between FWHM/T2mean and |Ecc| in group A and B. (Pearson correlation coefficient r = .51).The distribution of T2 in the LV of the DMD subjects were remarkably heterogeneous compared to that of normal subjects. These characteristics may suggest early signs of diffused collagen accumulation in the LV in DMD.


De novo synthesis of sphingolipids in mammalian cells is inhibited by the family of three ORMDL proteins . However, the cell and tissue-specific functions of ORMDL proteins in mast cell signaling are poorly understood. This study aimed to determine cross-talk of ORMDL2 and ORMDL3 proteins in IgE-mediated responses. To this end, we prepared mice with whole-body knockout (KO) of Ormdl2 and/or Ormdl3 genes and studied their role in mast cell-dependent activation events in vitro and in vivo. We found that the absence of ORMDL3 in bone marrow-derived mast cells (BMMCs) increased the levels of cellular sphingolipids. Such an increase was further raised by simultaneous ORMDL2 deficiency, which alone had no effect on sphingolipid levels. Cells with double ORMDL2 and ORMDL3 KO exhibited increased intracellular levels of sphingosine-1-phosphate (S1P). Furthermore, we found that concurrent ORMDL2 and ORMDL3 deficiency increased IκB-α phosphorylation, degranulation, and production of IL-4, IL-6, and TNF-α cytokines in antigen-activated mast cells. Interestingly, the chemotaxis towards antigen was increased in all mutant cell types analyzed. Experiments in vivo showed that passive cutaneous anaphylaxis (PCA), which is initiated by mast cell activation, was increased only in ORMDL2,3 double KO mice, supporting our in vitro observations with mast cells. On the other hand, ORMDL3 KO and ORMDL2,3 double KO mice showed faster recovery from passive systemic anaphylaxis, which could be mediated by increased levels of blood S1P presented in such mice. Our findings demonstrate that Ormdl2 deficiency potentiates the ORMDL3-dependent changes in mast cell signaling.The systemic anaphylactic reaction is a life-threatening allergic response initiated by activated mast cells. Sphingolipids are an essential player in the development and attenuation of this response.  Subsequently, as a consequence of an enzymatic cascade, ceramide is formed. The ceramide serves as a central base to synthesize more complex glycosphingolipids  induammation , and 3) ammation . In contammation . The sphammation , 15 and ammation , 16.ORMDL3 gene resides, have been associated in genome-wide association studies with the childhood onset asthma and increased ORMDL3 mRNA levels  in chromosome 17q12-q21, where the human A levels . RecentlA levels . In vivoresponse . Furtherresponse .Previous experiments suggested that besides ORMDL3, other members of the ORMDL family have critical biological roles. Thus, in HeLa and HEK293 cells, only silencing of all three members of the ORMDL family increased the ceramide levels , 23. FurOrmdl1 and Ormdl2 genes, but their involvement in the FcϵRI activation events was not determined  with reduced or enhanced levels of ORMDL3 and found that ORMDL3 is a negative regulator of high-affinity IgE receptor (FcϵRI) signaling . We alsotermined . Mast ceesponses . Besidesesponses . These responses , 27. S1Pesponses . SIPR2 iesponses . The FcϵIn this study, we focused on the role of ORMDL family members 2 and 3 in mast cell signaling. We prepared whole-body ORMDL2 KO and ORMDL3 KO mice and mice with combined ORMDL2,3 deficiency , and then we studied the properties of their mast cells and mast cell-dependent immune responses. The combined data suggest that ORMDL2 together with ORMDL3 co-ensure the proper mast cell functions.Antibodies specific for HPRT (sc-376938), IκB-α (sc-371), p38 (sc-535), p-p38 , and SPTLC1 (sc-374143) were obtained from Santa Cruz Biotechnology. Antibodies specific for p-IκB-α  and p-SYK  were purchased from Cell Signaling Technology. Antibody against SPTLC2 (ab23696) was obtained from Abcam. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (sc-2005) and HRP-conjugated-goat anti-rabbit IgG (sc-2004) were from Santa Cruz Biotechnology or Jackson ImmunoResearch Lab. . Anti-mouse FcϵRI α chain-fluorescein isothiocyanate (FITC) conjugate (Cat. No. 11-5898) and anti-mouse c-KIT (CD117)-allophycocyanin (APC) conjugate (Cat. No. 17-1171) were obtained from eBiosciences. PE-conjugated hamster anti-mouse CD29 -specific antibody was purchased from BD Biosciences. APC/Fire™ 750-conjugated anti-human/mouse β7 integrin (Cat. No. 321224)-specific antibody was purchased from BioLegend. IgE monoclonal antibody (mAb) specific for 2,4,6-trinitrophenol (TNP), clone IGEL b4 1 was produced in our laboratory from hybridoma cells obtained from Dr. A. K. Rudolph , 31. SpeOrmdl2 and Ormdl3 were designed using the gRNA selection tool CRISPOR  was generated using pX330 vector , 35 cycles , and final elongation step for 3 min at 72°C using Phusion High-Fidelity DNA Polymerase and the corresponding buffer, and pX330-U6-Chimeric_BB-CBh-hSpCas9  to final concentrations of 100 ng/μl for Cas9 mRNA and 50 ng/μl for gRNAs prior to microinjection into the male pronuclei of C57BL/6 one-cell embryos. The offspring were screened by PCR-based Heteroduplex Mobility shift Assay (HMA) with primers targeting ORMDL2 loci 5′-ATTCAGGGATTACAAGCATGAGC-3′ and 5′-GTAGGAAGATATACATTGCCTGG-3′; or ORMDL3 loci 5′-GGCAATTGATTGCTGGTCACC-3′ and 5′-GGGAGACATGGACGATGTGC-3′, respectively. The screening PCR was performed under the following conditions: initial denaturation for 5 min, 95°C, followed by 34 cycles: 30 s, 95°C; 30 s, 59°C; 1 min, 72°C. The final elongation step lasted 3 min at 72°C. For PCR we used DreamTaq™ Hot Start Green DNA polymerase and the corresponding buffer (Thermo Scientific). The PCR products were subjected to HMA using 5% native polyacrylamide gel electrophoresis (PAGE) and separated for 45 to 60 min with a constant electric potential of 80 V. Positive founders were identified by slower migrating bands as previously described (Guide (g)RNAs targeting  CRISPOR . Ormdl2-0 vector . Ormdl3 -hSpCas9  as a tem® Total RNA kit (OMEGA bio-tek). M-MLV reverse transcriptase (Invitrogen) with random hexamer primers (Invitrogen) was used according to the manufacturer’s instructions to produce single-stranded cDNA. Real-time PCR amplification was performed in 10-μl reaction volumes of qPCR mix containing 1 M 1,2-propanediol, 0.2 M trehalose and SYBR green 1 , and 4% WEHI-3 culture supernatant as a source of IL-3 and 4% culture supernatant from Chinese hamster ovary cells transfected with the cDNA encoding murine stem cell factor (SCF), a kind gift of Dr. P. Dubreuil; INSERM, Marseille, France). BMMCs were grown for 6 to 8 weeks, tested for the presence of surface c-Kit (CD117) and FcϵRI, and then used within 2 months for the experiments.BMMCs were isolated after washing the bone marrow of WT, ORMDL2 KO, ORMDL3 KO, and ORMDL2,3 dKO mice with 10 ml of culture medium. The cells were cultured in RPMI-1640 medium  supplemented with antibiotics , 71 μM 2-mercaptoethanol (2-ME), MEM non-essential amino acids, 0.7 mM sodium pyruvate, 2.5 mM PDMCs were isolated after washing the peritoneum of WT, ORMDL2 KO, ORMDL3 KO and ORMDL2,3 dKO mice with 15 ml of culture medium. The cells were cultured as mentioned above. PDMCs were grown for 4 weeks and subsequently tested for sphingolipids.2, 5.6 mM glucose, 20 mM HEPES, pH 7.4) supplemented with 0.1% essential fatty acid-free and globulin-free bovine serum albumin . The cells were sensitized with TNP-specific mouse IgE, clone IGEL b4 1 , followed by solubilizing them in hot sodium dodecyl sulfate-(SDS)-PAGE sample buffer , sonicat6) were collected, washed in BSS-BSA buffer and then in distilled water of chromatography grade (Sigma-Aldrich). Each cell pellet was suspended in 400 µl of distilled water and sonicated three times for 5 s. One part of the sonicate was saved to detect proteins by immunoblotting. Concentration of the proteins was determined by the Bradford assay (Thermo Fisher Scientific). Samples containing 40 µg of proteins were placed into glass vials containing internal standards . Then, 1 ml of 2:1 chloroform/methanol solution was added. Samples were shaken horizontally for 1 h at 22°C followed by filtration through hydrophobic Millex LH 0.45 µm filters (Merck Millipore).BMMCs or PDMCs (6 × 106) were collected, washed in BSS-BSA buffer and then in distilled water of chromatography grade (Sigma-Aldrich). Each pellet was suspended in 200 µl of MilliQ water and homogenized by sonication for 10 s in a cup horn. Extraction of S1P was performed from 100 µl of cell homogenate by the slightly modified method of Aerts et al. (g. The upper phase (supernatant) was transferred to another 5 ml vial, 300 µl of chloroform and 450 µl of MilliQ water were added and 10 min vortex mixing followed. Liquid phases were separated by 5 min centrifugation at 2500g. The upper phase was transferred to a new 5 ml vial. The lower phase was reextracted by 1200 µl methanol/water  and 10 min vortex mixing followed by 5 min centrifugation at 2500g. Both upper phases were combined and evaporated under the stream of nitrogen. The extract was re-suspended in 500 µl of MilliQ water by vortex-mixing and 5 min sonication in sonication bath. S1P was then extracted by 500 µl of water-saturated 1-butanol  and 10 min vortex mixing followed by 5 min centrifugation at 2500g. Upper phase was transferred to new glass vial. Remaining lower phase was repeatedly extracted by 500 µl of water saturated 1-butanol and vortex mixing. The upper phase was separated by centrifugation and combined with the first one. Combined butanol extracts were evaporated under the stream of nitrogen. The final extract was reconstituted in 300 µl methanol and filtered through hydrophobic Millex LH 0.45 µm filters.BMMCs . Isocratic elution was done in a mobile phase consisting of methanol (Biosolve) with 5 mM of ammonium acetate (LC-MS grade). The elution program for isocratic HPLC separation was as follows: flow rate 100 μl/min for 5 min. Mass spectra were measured in an AB/MDS SCIEX API 4000 tandem mass spectrometer (SCIEX: AB Sciex LLC) equipped with an ESI source and coupled to an Agilent HPLC 1290 series (Agilent Technologies). Samples were reconstituted in 300 µl of methanol with 5 mM ammonium acetate and 20 µl was introduced by the autosampler. The Analyst 1.6.3 software  was used to operate the instruments and process the data.via generating the [M+H+]+ precursor ions. The product ion used for ceramide was 264.4 m/z corresponding to C18:1 sphingoid base, whereas the sphingosine product ions in m/z were 268.3 , 282.3 (C18:1), 284.3 (C18:0), 310.3 (C20:1) and 312.3 (C20:1) and S1Ps product ions in m/z were 250.4 , 264.4 (C18:1), 284.4 (C18:0), 292.4 (C20:1) and 312.4 (C20:0). Delays between mass transitions were set to 5 ms with Q1 and Q3 operating at the unit mass resolution. The settling time was set to 500 ms for ceramides and sphingosines and no settling time was used for S1Ps. The curtain gas pressure was 20 psi. Nitrogen was used as the collision gas with the pressure set at 10 psi. The capillary spray voltage was 5.5 kV. The temperature of the nebulizing gas (nitrogen) was 200°C. The ion source gas pressure was adjusted to 20 psi for source gas 1 and 50 psi for source gas 2. The interface heater was turned on during the analysis. The ion optics settings for ceramide molecular species measurements were 50 V for the declustering potential and 10 V for the entrance potential. The collision energy was set to 36 V with a collision cell exit potential of 5 V. The dwell time for each ion transition was set to 100 ms. For sphingoid base molecular species, the ion optics setting was as follows: declustering potential was set to 40 V and entrance potential was 10 V. The collision energy was set to 17 V with a collision cell exit potential of 5.5 V. The dwell time for each ion transition was set to 300 ms. The ion optics settings for S1P molecular species measurements were 215 V for the declustering potential and 11 V for the entrance potential. The collision energy was set to 25 V with a collision cell exit potential of 5 V. The dwell time for each ion transition was set to 1000 ms.Ceramides, sphingosines, and S1Ps were analyzed by selected reaction monitoring (SRM) in the positive ion mode Concentrations were calculated using a known amount of internal standards 12.5 ng for C17:0, d18:1 ceramide (MW = 551.6), and C17:1 sphingosine (MW = 285.4) and 100 ng for C17:1 S1P (MW = 365.4) added to the samples. Final concentrations were expressed as pmol of lipids per mg of proteins, volume of serum and cell counts. Molecular species (isoform) profiles were constructed using the measured signals of individual species and expressed as pmol of lipids per mg of the protein .2+ response, cells were loaded with Fura-2-AM and probenecid to a final concentration of 1 µM and 2.5 mM, respectively. After 20 min incubation at 37°C, the cells were washed in BSS-BSA/probenecid and immediately before measurement centrifuged at 805g at 20°C for 5 min, followed by resuspending in BSS-BSA at concentration 5 × 105/ml. Aliquots of 100 µl were inserted into wells of a white 96-well plate. The cells were activated after 40 s with 100 µl of BSS-BSA supplemented with TNP-BSA (1 μg/ml). Changes in concentrations of intracellular Ca2+ ([Ca2+]i) were determined by spectrofluorometry using an Infinite 200M plate reader (TECAN) with excitation wavelengths at 340 and 380 nm and with constant emission at 510 nm. The activator was added manually after 30 s of basal Ca2+ measurements. Data are shown as the ratio em510exc340/em510exc380. β-glucuronidase release was performed as described elsewhere  in 120 μl of chemotaxis medium (RPMI-1640 supplemented with 20 mM HEPES and 1% BSA) were added into the upper well, and chemoattractant  in 0.6 ml of the chemotactic medium was added to the lower compartment. Cells migrating into the lower wells during the 8-h incubation period  were counted in 50 μl aliquots using an Accuri C6 flow cytometer (BD Biosciences).Chemotaxis responses were assayed in 24-well transwell chambers  with 8 µm-pore-size polycarbonate filters in the upper wells. TNP-specific IgE-sensitized BMMCs (0.3 ×106) were then activated or not with antigen  for 5 min. After centrifugation the cells were lysed on ice in 0.8 ml lysis buffer  supplemented with 1% Brij-96. The gradient was formed by adding 0.5 ml of 80% sucrose stock solution to the bottom of the polyallomer tube  followed by 1.5 ml of 40% sucrose containing the cell lysate, 2 ml of 30% sucrose, and 1 ml of 10% sucrose. After 4 h centrifugation at 4°C and 210,000g, using an SW55 Ti rotor, nine fractions were collected from the top of the gradient, and individual fractions were size-fractionated by SDS-PAGE followed by immunoblotting with PY20-HRP conjugate or LYN-specific antibody. The position of phosphorylated PAG, LYN, LAT1, and LAT2 is known from our previous studies  or β7 integrin analysis were stained separately with PE-CD29-specific or APC/FireTM 750-β7 integrin-specific 200-fold-diluted antibodies. After washing with PBS, the cells were measured by flow cytometry. Data were acquired with BD Symphony. Mast cell populations positive for FcϵRI, c-Kit, β1 integrin, and β7 integrin were determined and further processed using FlowJo software (TreeStar).6) were activated or not with 1 μg/ml of TNP-BSA for 5 h. Then, the cells were centrifuged and the supernatants (25 μl) were used for cytokine quantification using the bead-based immunoassay  according to the manufacturer’s instructions. Data were acquired using a BD Symphony flow cytometer equipped with 488 and 637 nm lasers. The data were analyzed using LEGENDplex™ data analysis software. IL-2, IL-12p70, IL-17A, and IFN-γ were not detected in the supernatants of non-activated or activated mast cells.IgE-sensitized BMMCs  were intravenously challenged with antigen . Two hours later, the mice were killed and the ears removed for measurement of the amount of extravasated dye. Formamide (1 ml) was then added to each ear, which was then homogenized with the T-25 ULTRATURRAX Digital High-Speed Homogenizer Systems (IKA) and incubated at 80°C for 2 h. The samples were centrifuged at 14,000in vivo research instruments system. Monitoring of body temperature was initiated 2 weeks after acclimatization of mice  in the experimental room. Female mice aged 11 to 16 weeks were sensitized by intraperitoneal injection with TNP-specific IgE (45 µg in 400 µl of PBS) and 18 h later, anaphylaxis was induced by tail vein injection of antigen . The body temperature was recorded at 10-min intervals for at least 90 min after antigen challenge.The body temperature was measured with an accuracy of 0.1°C using a rectal thermometer from BIOSEB g and snap frozen in nitrogen. The assessment of histamine in the plasma was done within one month of plasma collection using an EIA kit (IM2015) according to the manufacturer’s instructions (BeckmanCoulter). Total histamine was determined from 5 × 105 BMMCs according to the manufacturer’s instructions.PSA was induced in mice  as described above, but blood was collected 90 s upon antigen injection by facial vein collection into chilled plastic tubes coated with EDTA. Plasma was centrifuged for 15 min at 4°C, 1000t-test for comparison of two groups. For comparison of more than three groups in the experiment, one-way or two-way ANOVA with Tukey’s or Dunnett’s post hoc comparison was used. Multiple comparisons were performed within the group. Calculated P values of less than 0.05 were considered significant. For each analysis, the data met the assumptions of the statistical test. Data are expressed as mean ± s.e.m. All statistical analyses were performed using GraphPad Prism version 7.3.Statistical analysis was carried out using the Student’s unpaired two-tailed Ormdl3. A founder with 5-nucleotide deletion at position 49-53 downstream of ATG was selected. The presumable non-sense transcript starts from the original ORMDL3 initiating ATG codon and terminates after 345 nucleotides  in ORMDL2,3 dKO mast cells  was observed 50 min after antigen administration, followed by a slow recovery, which was not completed 90 min after the challenge with antigen . We therde novo biosynthesis pathway  when compared with WT, ORMDL2 KO, and ORMDL3 KO BMMCs. The mature PDMCs exhibited comparable profiles of total sphingosines and ceramides, suggesting that this phenotype is not constrained on BMMCs. These data support the concept of functional excess of ORMDL proteins relative to SPT , β-glucuronidase release , chemotaxis , IκB-α phosphorylation , secretion of IL-4, IL-6, and TNF-α , and PCA study  corroborated our previous results showing that ORMDL3 negatively regulates mast cell signaling , Ag-dependent Caignaling . In the ignaling , 47–49. ignaling . Phosphoignaling , 49. SPHignaling , 49. Theignaling . FcϵRI-sin vitro and in vivo . MM was supported in part by the Faculty of Science, Charles University, Prague. LDe was supported in part by the First Faculty of Medicine, Charles University, Prague.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.