Cell viability was detected using CCK-8 assays. Wound healing and Transwell assays were performed to detect cell motility. ROS production was determined by immunofluorescence using DCFH-DA probes. Western blotting and RT-qPCR were carried out to measure gene expression levels. Inflammation responses were determined by measuring inflammatory cytokines. Immunohistochemistry staining was conducted to measure α2-adrenergic receptor levels in tissue samples. DEX alleviated the H2O2-induced cytotoxicity, attenuated the promoting effects of H2O2 on cell malignancy, and protected VSMCs against H2O2-induced oxidative damage and inflammation response. DEX regulated the GSK-3β/MKP-1/NRF2 pathway via the α2AR. DEX alleviates the inflammatory responses and oxidative damage of VSMCs by regulating the GSK-3β/MKP-1/NRF2 pathway via the α2AR in IA.Vascular smooth muscle cell (VSMC) phenotypic modulation regulates the initiation and progression of intracranial aneurysm (IA). Dexmedetomidine (DEX) is suggested to play neuroprotective roles in patients with craniocerebral injury. Therefore, we investigated the biological functions of DEX and its mechanisms against IA formation and progression in the current study. The rat primary VSMCs were isolated from Sprague–Dawley rats. IA and superficial temporal artery (STA) tissue samples were obtained from patients with IA. Flow cytometry was conducted to identify the characteristics of isolated VSMCs. Hydrogen peroxide (HThe online version contains supplementary material available at 10.1186/s40360-022-00607-0. Intracranial aneurysm (IA) is a regional bulging on intracranial artery characterized by asymptomatic lesion . RuptureVSMCs, a major cell type in vessel walls, is well-known for their multifunctional properties, including maintaining the vessel integrity, regulating blood pressure, and redistributing blood flow , 10. VSM2-adrenergic receptor (α2AR) agonist, exhibits sympatholytic, sedative, amnestic and analgesic properties [Dexmedetomidine (DEX), a highly selective αoperties , 15. DEXoperties , 17. A poperties . In patioperties . DEX plaoperties . In an ioperties . Furtheroperties , 23. HowMultiple proteins participate in the regulation of the antioxidant response, including glycogen synthase kinase 3β (GSK-3β), mitogen-activated protein kinase phosphatase-1 (MKP-1), and the nuclear factor erythroid 2-related factor 2 (NRF2) , 25. GSK2O2), an inducer of oxidative stress, is involved in apoptosis of VSMCs [2O2 has been used to establish an in vitro model of IA via inducing the apoptosis of VSMCs [2O2-induced cellular model of IA. We hypothesized that DEX might prevent progression of rupture of IA. We believed that our study would provide theoretical reference for IA treatment.Hydrogen peroxide . All participates were over 16 years of age. The study protocols were also based on the ethical principles of the Declaration of Helsinki for medical research involving human subjects. 80 patients with IA participated in this study. IA tissue samples were obtained during the surgery, and superficial temporal artery (STA) vascular tissue samples were collected as controls. All samples were fixed in 10% formaldehyde  and embedded in paraffin.https://arriveguidelines.org). Rat brain VSMCs were separated from the Circle of Willis. The brain vessels were collected under sterile conditions followed by phosphate buffer solution  washing. After washing, excess fat and fascia were removed. Subsequently, the vessels were placed in enzyme solution containing 0.2% collagenase II and 1 mg/ml soybean trypsin inhibitor for 20 min at 37℃. For VSMCs isolation, the blood vessels were cut off along the long axis and the intima was tore off. Next, the vessels were cut into pieces (1 mm3) and 0.125% trypsin was added. After incubation for 15 min at 37℃, DMEM  supplemented with 10% fetal bovine serum  was added. Then the digested VSMCs were collected by centrifugation and the supernatant was removed. The collected cells were cultured in DMEM supplemented with 10% FBS, 100U/ml penicillin, and 100 mg/ml streptomycin at 37℃ with 5% CO2 in a humidified condition. After 3 days of primary culture, the cells grew adhering to the wall. After 2 weeks, the cells were observed to be spindle shaped using a light microscope . VSMCs at passages 3 to 8 were used for subsequent experiments.Sprague–Dawley rats  were purchased from Hunan SJA Laboratory Animal CO., LTD . All experimental procedures were based on the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and approved by the Ethics Committee of the First Hospital of Hebei Medical University . All methods were conducted in accordance with the ARRIVE guidelines  and S10 calcium-binding protein B  were added. All antibodies were used at the manufacturer’s recommended dilutions and cellular concentrations. Subsequently, the signals of labelled cells were detected by flow cytometry and the data were analyzed by FlowJo software .The SMC differentiation markers and stem cell markers using flow cytometry. The cells were cultured in a Fluorescence-Activated Cell Sorting (FACS) buffer containing 2% FBS and PBS. After fixation and permeabilization, SMC-specific markers myosin heavy chain 11 , calponin 1 , Smoothelin2  and stem cell markers CD44 , stem cell antigen-1  and DEX by conducting CCK-8 assays. The VSMCs were first exposed to different concentrations  of DEX for 120 min. The treated cells seeded in the 96-well plates (5 × 103 cells/well) until 80% confluence. Then 10 μl of CCK-8 reagent  was added into the culture medium. Another 2 h was given the cells for incubation at 37℃. Absorbance at 450 nm was measured using a microscope . To identify optimal concentration of H2O2, the cells were exposed to different concentrations  of H2O2 for 12 h, and then CCK-8 assays were conducted as described above. To identify optimal concentration of DEX, we pretreated VSMC with different concentrations  DEX for 2 h before exposure to 1 mM/ml H2O2 for 12 h. Then 10 μl of CCK-8 reagent was added, and the optimal concentration of DEX was obtained based on cell viability. The optimal concentration of DEX and H2O2 was 1 μM and 1 mM/ml, respectively. Then the VSMCs were divided into the control group (DMEM), the DEX group (1 μM DEX), H2O2 group (1 mM/ml H2O2), H2O2 + NS group , H2O2 + DEX group (1 mM/ml H2O2 + 1 μM DEX), the H2O2 + DEX + BRL  group.The first step of the study was to find out the optimal concentration of hydrogen peroxide (H5 cells/well) to measure the intracellular ROS levels. After treatment as described above, PBS washing was conducted. The cells were then incubated at 37℃ with 10 μM DCFH-DA  for 1 h. Next, the VSMCs were washed three times using the serum-free DMEM and suspended in PBS. A fluorescence microscope  was used to visualize the fluorescence intensity of DCF at 488 nm (excitation) and 525 nm (emission).The VSMCs cells were seeded into 6-well plates (1 × 104 cells/well) and incubated at 37℃ with 5% CO2 for 24 h. Until the cells reached 90% confluence, a pipette tip (10 μl) was used to make scratches on the cells. The cells were then washed using PBS and subsequently cultured at 37℃ with 5% CO2 for 24 h. The wound width was imaged using an AxioCam camera  at 0 and 24 h. ImageJ software was used to evaluate the remaining area [The cells were seeded into 24-well plates  and stained with crystal violet. Number of migrated cells were measured using an inverted microscope (Leica). Six fields were randomly selected, and the average was calculated .The proteins were collected using RIPA lysis buffer (Sigma-Aldrich) containing protease inhibitor. Cell lysates were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes (PVDF). After blocking with 5% skimmed milk, the membranes were incubated overnight with primary antibodies against SM22α , αSMA , α2AR , p-GSK-3β , GSK-3β , MKP-1 , NRF2  and GAPDH  at 4℃. Then the membranes were washed three times. After that, the membranes were incubated with secondary antibodies for 2 h at room temperature. After being probed with enhanced chemiluminescence (Yeasen Biotechnology), the proteins were visualized using an image analysis system . The protein intensity was evaluated by ImageJ software.−△△Ct, and expressed as ratio of GAPDH. The primer sequences were listed in Table IA and STA tissue samples were stored at -80℃ and placed in pre-cooled lysis buffer (Sigma-Aldrich) after stirring with an electric homogenate machine. After centrifugation at 1200 rpm for 30 min, the supernatant was collected. TRIzol (Sigma-Aldrich) was used for total RNA isolation from lysed tissues and VSMCs. Reverse transcription into cDNA was done using the Reverse Transcription Kit (Sigma-Aldrich). RT-qPCR was performed on the ABI 7300 system . The relative level was calculated using 2Cellular supernatants were collected after 1000 g centrifugation for 5 min. Interleukin-1β, IL-6 and tumor necrosis factor (TNF-α) ELISA kits  were applied to measure the levels of inflammatory cytokines according to the kit instructions.The paraffin-embedded IA and STA tissue sections were cut into 5 μM and then placed on poly-L-lysine coated slides. For immunohistochemistry, the slides were incubated with primary antibody against α2AR  overnight at 4℃. After washing, the slides were incubated with secondary antibody. The slides were visualized using a DAB plus chromogen (Thermo Fisher Scientific). Under 400 × magnification, the pictures were taken in 5 random fields.t test and one way analysis of variance followed by Tukey’s post hoc test were used to compare differences. p < 0.05 was considered statistically significant.Each experiment was performed in triplicate. Statistical analyses were performed using the GraphPad Prism 6.0 software . Data were described as the mean ± standard deviation. Student’s After 2 weeks, the cells were observed to be spindle shaped as shown in Fig. 2O2 on the viability of VSMCs. Compared with that in the control group, the viability of VSMCs was reduced with increasing concentration of H2O2, as CCK-8 revealed  reversed the inhibitory effects of DEX . We found that HDEX Fig. A-C. As RDEX Fig. D-F. Finaent Fig. G-I. In s2O2-induced VSMC injury. DEX promoted the activation of GSK-3β/MKP-1/NRF2 pathway via the α2AR to increase the SM22α and αSMA levels and inhibit oxidative stress, leading to the conversion of VSMCs from synthetic type into contractile phenotype, thereby suppressing the proliferation, migration, and invasion of VSMCs.Compared with that in STA, the level of α2AR was downregulated in IAs, as shown by RT-qPCR Fig. A. The reThe rupture of IA leads to SAH, causing severe mortality and morbidity . Inflamm2O2 on cell viability and restored the promotion in cell migration and invasion. SM22 α and αSMA are the markers of contractile phenotype. Herein, DEX reversed the inhibitory effects of H2O2 on SM22 α and αSMA levels. NQO-1 and SOD1 are antioxidant enzymes, and TNF-α, IL-6 and MCP-1 participate in inflammation initiation [As a highly selective α2AR agonist, DEX works in the intensive care unit as an anesthetic adjunct for patient sedation and in the operating room settings for general anesthesia . Accumulitiation . GCLC liitiation , 48. In DEX is reported to exert neuroprotective efficacy in patients with IA . The undIn summary, this study demonstrated the protective roles of DEX against H2O2-induced cell proliferation, invasion, migration, oxidative damage, and inflammation responses, leading to the conversion of VSMCs from synthetic type to contraction type by via the α2AR/GSK-3β/MKP-1/NRF2 axis. To be honest, there are some limitations in this study. First, in vivo experiments and more comprehensive studies are needed in the subsequent studies. Second, the studies on α2AR are inadequate. Despite these limitations, we believed that our study would provide a theoretical basis for IA treatment.Additional file 1.


In only two years, the coronavirus disease 2019 (COVID-19) pandemic has had a devastating effect on public health all over the world and caused irreparable economic damage across all countries. Due to the limited therapeutic management of COVID-19 and the lack of tailor-made antiviral agents, finding new methods to combat this viral illness is now a priority. Herein, we report on a specific oligonucleotide-based RNA inhibitor targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It displayed remarkable spontaneous cellular uptake, >94% efficiency in reducing RNA-dependent RNA polymerase (RdRp) RNA levels in transfected lung cell lines, and >98% efficiency in reducing SARS-CoV-2 RNA levels in samples from patients hospitalized with COVID-19 following a single application. Since the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019, the number of globally confirmed cases according to World Health Organization statistics exceeded 400 million across 222 countries by February 2022, with confirmed deaths of more than 5.7 million patients ,4,5,6. TSARS-CoV-2 is an enveloped positive-sense unsegmented single-stranded RNA virus, meaning that the base sequence of the RNA is recognized and treated as a later messenger RNA in the host’s cell. With a length of about 30 kb, the genome of SARS-CoV-2 is the largest RNA genome of all known RNA viruses. The replication of SARS-CoV-2 is operated by a set of non-structural proteins (nsps) that assemble into a multi-subunit polymerase complex. Among them, nsp12 is the catalytic subunit with RNA-dependent RNA polymerase (RdRp) activity ,8. RdRp Therapeutic oligonucleotides have attracted great interest due to their potency and potential for changing the therapeutic landscape of many pathological conditions, including those of viral origin. Targeting conserved SARS-CoV-2 RNA sequences essential for viral replication offers a rational approach to inhibit viral infection and thereby halt disease progression ,9. The sThis proprietary platform, now called ESiNAR-X, served as basis for the development of a highly specific oligonucleotide-based lead pharmacological modality against SARS-CoV-2, namely ASC1R. It was designed to target the RNA region encoding the nsp12 catalytic subunit with RdRp activity. The present study demonstrates the antiviral potential of ASC1R in terms of its ability to reduce levels of RdRp RNA in transfected lung cell lines by more than 94% and levels of SARS-CoV-2 RNA in patient samples by more than 98% within 24 h after a single application.12)GCAGTTGTGGCATCTCCT-biotin. The high-performance liquid chromatography-purified compound was stored at −20 °C in either lyophilized form or dissolved in nuclease-free water.ASC1R, a 37mer phosphorothioate DNA with an internal poly(ethylene glycol) (PEG) linker, was synthesized by Selecta Biotech  using an automated H-28 DNA synthesizer . The oligonucleotide was labeled with Cyanine 5 (Cy5) and biotin at the 5’ and 3’ ends, respectively, to generate 5’-Cy5-CAAATGTTAAAAACACTAT for cloning purposes.A 119 nt single-stranded DNA (5’-CCCA^AGCTTGTGAAATGGTCATGTGTGGCGGTTCACTATATGTTAAACCAGGTGGAACCTCATCAGGAGATGCCACAACTGCTTATGCTAATAGTGTTTTTAACATTTGG^AATTCCG) corresponding to the target RdRp region, including overhang sequences from a 5’ Primers for the amplification of the 119 nt DNA sequence for cloning purposes  and primers and probe for the quantification of the RdRp region  were purchased from Metabion International AG .A CE IVD gb SARS-CoV-2 Multiplex Kit for RT-qPCR analysis of the E gene was purchased from Generi Biotech .An Hs01060665_g1 Assay Kit for RT-qPCR analysis of the b-actin housekeeping gene was purchased from Thermo Fisher Scientific .12 molecules each) and incubated in a hybridization buffer comprising 0.001% sodium dodecyl sulfate , 0.1 M NaCl , and 1 mM EDTA  for 30 min at 37 °C. The resultant heteroduplexes, control ASC1R (1012 molecules), and RdRp sequence (1013 molecules) were then loaded onto a 6% polyacrylamide gel and subjected to electrophoresis at 110 V . Afterwards, nucleic acids were stained with GelRed Nucleic Acid Stain  for 20 min and visualized at 590 nm, and Cy5-labeled ASC1R was visualized at 680 nm, both using a ChemiDoc MP instrument .ASC1R and the 119 nt target RdRp sequence were mixed in an equimolar ratio (10HindIII and EcoRI (Thermo Fisher Scientific) according to the manufacturer’s instructions. Products were separated on a 1.5% agarose gel , excised, and purified using a QIAEX II Gel Extraction Kit (Qiagen). Ligation was performed overnight at 4 °C using T4 DNA ligase  at a 1:3 molar ratio of vector to insert.The 119 nt target RdRp sequence was amplified using high-fidelity DNA polymerase and Phusion Plus PCR Master Mix  to ensure high accuracy during DNA synthesis, and amplicons were purified using a QIAquick PCR Purification Kit . Purified amplicons (1 µg) and 5 µg of pcDNA 3.1 vector  were cleaved using FastDigest Escherichia coli cells  were transformed with the vector according to the manufacturer’s instructions. Briefly, 10 ng of plasmid DNA was carefully mixed with cells and incubated on ice for 30 min. Cells were heat shocked at 42 °C for 30 s and chilled on ice for 5 min. After the addition of 950 µL of SOC medium , the mixture was shaken at 200 rpm for 60 min at 37 °C, and the cells were diluted and plated on selective LB agar  plates containing 100 µg/mL ampicillin . After incubation overnight at 37 °C, selected colonies were inoculated into 3 mL of fresh LB medium containing 100 µg/mL ampicillin and incubated overnight at 37 °C with shaking at 200 rpm. The vector construct with the RdRp sequence was verified by Sanger sequencing. Plasmids for subsequent transfection of lung cells were purified using a PureLink HiPure Plasmid Miniprep Kit .NEB 5-alpha competent 2. Before experiments, the amount of FBS was gradually decreased to 2% to slow cell growth and avoid contact inhibition.A primary cell line of human embryo lung cells (HEL299) and stable adenocarcinomic human alveolar basal epithelial cells (A549) were used in this study. HEL299 and A549 cells were maintained in Minimum Essential Medium  and low-glucose Dulbecco’s Modified Eagle’s Medium  supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin  at 37 °C with 5% CO5 cells/well. Cells were transfected using ViaFect  according to the manufacturer’s instructions. Specifically, the transfection reagent and plasmid were pre-mixed at a ratio of 1.5:1 v/w and incubated for 15 min at room temperature. The transfection complex was then added to cells and incubated for 24 h at 37 °C with 5% CO2. HEL299 cells, as a primary cell line, were used for experiments immediately after transfection due to their limited proliferative capacity [HEL299 and A549 cells were seeded 1 day before transfection in 48-well plates in 200 µL MEM and low-glucose DMEM supplemented with 2% FBS and 1% P/S at a density of 1 × 10capacity . Transfe4 cells/slide, exposed to 0.005–10 µM of Cy5-labeled ASC1R, and incubated at 37 °C and 5% CO2 for 24 h. For the quantification of cellular uptake, transfected HEL299 and A549 cells (seeded in 48-well plates in 200 µL of media at a density of 5 × 104 cells/well) were exposed to 0.005–10 µM of Cy5-labeled ASC1R and incubated at 37 °C and 5% CO2 for 24 h. To analyze the efficacy of ASC1R, transfected HEL299 and A549 cells (seeded in 48-well plates in 200 µL of media at a density of 5 × 104 cells/well) were exposed to 5 µM of Cy5-labeled ASC1R and incubated at 37 °C and 5% CO2 for 24 h. Non-treated cells served as controls and were incubated in parallel.For confocal microscopy, transfected HEL299 and A549 cells were seeded onto glass slides at a density of 2 × 10After treatment, cells on glass slides were washed with phosphate-buffered saline  to remove extracellular ASC1R, and a fresh medium without Phenol Red was added. Cell membranes were stained with FM 4-64FX membrane stain  and visualized at 744 nm. Intracellular ASC1R was visualized at 680 nm using a Leica TCS SP8 AOBS confocal microscope .g, and washed with PBS to remove extracellular ASC1R. Cells (5 × 104) were lysed using 40 µL of lysis buffer containing RIPA  and 10% SDS at 4 °C for 30 min, and then briefly sonicated using a Sonopuls Mini 20 instrument . A 10 µL sample of cell lysate was loaded onto a 6% polyacrylamide gel and subjected to electrophoresis . ASC1R was visualized at 680 nm, and the amount of ASC1R was normalized against a standard and expressed as the number of ASC1R molecules per single cell.After treatment, cells were trypsinized, pelleted at 200 × Twenty-six biological specimens were obtained from patients diagnosed with COVID-19 using nasopharyngeal swabs collected in 1.5 mL microcentrifuge tubes containing 1 mL of MEM supplemented with 2% FBS. The study was approved by the Ethics Committee of the National Cancer Institute in Bratislava, Slovakia, and written informed consent was provided by all patients. All procedures were performed in compliance with relevant laws and institutional guidelines and in accordance with the ethical standards of the Declaration of Helsinki.g, resuspended in 400 µL of MEM supplemented with 2% FBS, and divided into two equal aliquots (200 µL each). One aliquot was exposed to 5 µM of Cy5-labeled ASC1R and incubated at 37 °C and 5% CO2 for 24 h, while the other served as a non-treated control and was incubated under the same conditions in parallel.Swabs were squeezed into microcentrifuge tubes, vigorously vortexed, removed from the tubes, and properly disposed. Cells were pelleted at 300 × g, and washed with PBS to remove extracellular ASC1R. Treated patient samples were pelleted at 300× g and washed with PBS. RNA was isolated using RNAGEM  following the manufacturer’s instructions. Non-treated parallels were processed identically.After treatment, HEL299 and A549 cells were trypsinized, pelleted at 200× -ΔΔCt method.Real-time RT-PCR was carried out in a final volume of 20 µL containing 2 µL of RNA using AmpliTune 1-step RT-qPCR Probe Mix according to the instructions for users. Amplification was carried out on an AriaMX automated PCR instrument . Briefly, RNA was reverse-transcribed at 50 °C for 10 min, whereas subsequent cDNA amplification involved the initial activation of DNA polymerase at 95 °C for 3 min and 40 cycles of denaturation (95 °C for 15 s) and annealing/elongation (58 °C for 30 s). All data were normalized against b-actin. Data from treated cells were also normalized against controls and analyzed by the 2The fundamental requirement for the therapeutic action of any oligonucleotide-based modality is its ability to bind to the target RNA. ASC1R has an unconventional design with an internal linker between the recognition sequences of the inhibitor ; hence, As shown in One of the major problems hindering the smooth translation of therapeutic oligonucleotides into clinical practice is their unsatisfactory cellular uptake . For thiTransfected lung cells treated with 5 µM ASC1R and non-treated controls were incubated in parallel for 24 h. The efficacy of ASC1R was evaluated as the percentage difference between the amounts of expressed RdRp RNA in treated and non-treated cells post-incubation. Raw Ct values for non-treated HEL299 and A549 parallels were 18.7, 19.6, and 20.0 and 20.5, 24.5, and 27.8, respectively. As shown in N = 26; Q25–Q75 range = 22.0–28.8). As shown in Twenty-six nasopharyngeal samples from patients diagnosed with COVID-19 were used in this study. Each sample was handled, treated, and evaluated independently. Equal aliquots of non-treated and ASC1R-treated cells derived from each patient sample were incubated in parallel for 24 h. Similarly to transfected cells, the efficacy of ASC1R was evaluated as the percentage difference in the amount of SARS-CoV-2 RNA post-incubation. The median raw Ct values for non-treated parallels after a 24 h incubation was 24.7 (The persistent coronavirus pandemic and the high mortality associated with COVID-19 demand urgent solutions. In this study, we reported the antiviral potential of a tailor-made oligonucleotide-based inhibitor targeting SARS-CoV-2, which is capable of spontaneously penetrating the cells, binding to viral RNA, and inducing its enzymatic degradation with a remarkable > 98% efficacy after a single application. The therapeutic potential of ASC1R demonstrated herein could translate into substantial clinical benefits for patients with COVID-19. Furthermore, in the context of infectious diseases, our results provide implications for the research and development of analogous antivirals for other diseases of viral origin. The findings could help to meet the global challenge of developing new and safe treatment modalities.


Two new organic-inorganic hybrid rare-earth double perovskites exhibit interesting properties such as photoluminescence, magnetism, and structural phase transition. 4CsSm(NO3)8 (1) (R3HQ = (R)-(-)-3-quinuclidinol) and (R3HQ)4CsEu(NO3)8 (2) were synthesized and characterized. Compounds 1 and 2 exhibit obvious phase transitions at 379 and 375 K, respectively, confirmed by differential scanning calorimetry (DSC) and variable temperature powder X-ray diffraction. The rapid switching between high- and low-dielectric states makes it a typical dielectric material with a switchable dielectric constant for thermal stimulus response. Furthermore, 1 and 2 show attractive photoluminescence and paramagnetic behavior, and the fluorescence quantum yield of 2 reached 14.6%. These results show that compounds 1 and 2 can be used as excellent candidates for multifunctional intelligent materials, which also provides a new way for development of multifunctional materials.Two new organic–inorganic hybrid double perovskites (R3HQ) The compounds not only undergo a reversible structural phase transition at about 379 K for 1 and 375 K for 2 but the phase temperature is also increased by about 30 K compared to the reported (R3HQ)4RbSm(NO3)8 and (R3HQ)4RbSm(NO3)8. Interestingly, 1 and 2 exhibit photoluminescence under UV excitation, and the quantum yield of 2 reached 14.6%. In addition, the direct current (DC) magnetic susceptibility of compounds shows obvious paramagnetic signals. The successful construction of the compounds provides a new idea for further enriching the rare-earth double perovskite system -(-)-3-quinuclidinol) aqueous solution was obtained by adding R3HQ and HNO3 solution (65–68%) to an aqueous solution in a molar ratio of 1:1. Then, high-quality colorless transparent block crystals were obtained by slowly evaporating the mixed aqueous solution of Sm(NO3)3/Eu(NO3)3, CsNO3, and R3HQNO3 in a molar ratio of 1:1:2 at room temperature for about 2 weeks. Anal. (%) Calc. for C28H56CsSmN12O28: C, 25.25, H, 4.23, N, 13.94. Found: C, 26.03, H, 4.37, N, 13.01. Anal. (%) Calc. for C28H56CsEuN12O28: C, 25.30, H, 4.19, N, 13.17. Found: C, 26.00, H, 4.36, N, 12.99.All reagents were purchased commercially and used as received without any further purification. First, an unseparated R3HQNO−1 heating/cooling rate by heating and cooling the crystalline samples. The real part ε′ of the complex permittivity which is related to the temperature was performed using a TH2828A variable temperature dielectric measuring instrument in the temperature range of about 345–390 K. The variable temperature powder X-ray diffraction data were measured on a Rigaku D/MAX 2000 PC X-ray diffraction system with Cu-Kα radiation in the 2θ range of 5°–50° with a step size of 0.02° and in the temperature range 298–403 K. Photoluminescence was recorded on an Edinburgh FL980 UV/V/NIR fluorescence spectrometer. The quantum yield was measured by using an Edinburgh FLS 1000 UV/V/NIR fluorescence spectrometer. The single-crystal X-ray diffraction data were collected on a Rigaku Synergy apparatus with graphite-monochromate Mo-Kα radiation at 278 K for 1 and 301 K for 2, respectively. Thermogravimetric analysis was performed on NETZSCH STA 449F3 in the temperature range of about 298–1070 K.DSC measurements were performed with a NETZSCH differential scanning calorimeter 214 Polyma in the temperature range 300–390 K under atmospheric pressure with a 20 K min1 crystallized in the tetragonal chiral space group P4322 (no. 95) at 278 K with cell parameter a = 10.1274(1) Å, b = 10.1274(1) Å, c = 46.2252(11) Å, and Z = 4. The crystallographic asymmetric unit of 1 comprises Sm and Cs sharing a nitrate and each connecting a complete nitrate and an incomplete nitrate, and two separate organic cations, the complete molecules being generated by inversion symmetry. Both Sm and Cs are coordinated with six NO3− as bridging ligands to chelate into an alternately arranged corner-sharing octahedral structure to form a framework of inorganic metal layers, and the double layer of R3HQ organic cations occupied the space between the inorganic layers  of at 298 K . It can at 298 K . Therefo 1 and 2  remain u1 and 2 were investigated by DSC under nitrogen and atmospheric pressure with a heating/cooling rate of 20 K min⁻1. The DSC curve 4RbSm(NO3)8 and (R3HQ)4RbSm(NO3)8, the phase transition temperature is increased by about 30 K. Its average temperature is taken and calculated, and the change in entropy and enthalpy is 17.44 kJ mol⁻1 and 46.0254 J mol⁻1 K⁻1, respectively. Similarly, compound 2 showed corresponding thermal anomaly peaks at 375 and 359 K , where R is the gas constant and N is the ration of the number of respective geometrically distinguishable orientations in high- and low-temperature phases  is an effective thermodynamic method used for determining whether crystals undergo phase transition resulting from temperature induction. The phase transitions of SC curve  of 1 disnd 359 K . The chae phases , the valechanism . Combini of the temperature-dependent dielectric constant of 1 was investigated in the temperature range of 348–390 K and the frequency of 100 kHz. The dielectric constant shows a trend of slow increase in the range of about 348–365 K, and a trend of slow decrease in the temperature range of about 369–395 K , which leads to the population of excited states. The ground state energy level is split into seven 7FJ states caused by spin-orbit coupling. Hence, J is no longer 0, it can change from 0 to 6. Therefore, the value of χmT gradually decreases and approaches 0 with the decrease in temperature. For 2, SmIII is the same as EuIII, the value of χmT decreases gradually with the decrease in temperature, which is a paramagnetic signal.The direct current (DC) magnetic susceptibility of compounds  samples . In geneIn summary, we obtained two organic–inorganic hybrid rare-earth double perovskites, which adopt a Ruddlesden–Popper (RP)-type perovskite structure. The reversible dielectric phase transition and switchable dielectric constant between high- and low-dielectric states of the compounds are verified by measuring temperature-dependence of the real part of the dielectric constant using differential scanning calorimetry (DSC) and variable temperature PXRD. Simultaneously, the compounds also have significant photoluminescence and paramagnetic behavior. Therefore, the compounds have potential application in the field of stimulus-responsive multifunctional intelligent materials, and the successful synthesis of the two compounds further enriches the rare-earth two-dimensional perovskite family.


We investigated the possibility of single-step genome editing in small ruminants by CRISPR-Cas9 zygote electroporation. We targeted SOCS2 and PDX1 in sheep embryos and OTX2 in goat embryos, utilizing a dual sgRNA approach. Gene editing efficiency was compared between microinjection and three different electroporation settings performed at four different times of embryo development. Electroporation of sheep zygotes 6 h after fertilization with settings that included short high-voltage (poring) and long low-voltage (transfer) pulses was efficient at producing SOCS2 knock-out blastocysts. The mutation rate after CRISPR/Cas9 electroporation was 95.6% ± 8%, including 95.4% ± 9% biallelic mutations; which compared favorably to 82.3% ± 8% and 25% ± 10%, respectively, when using microinjection. We also successfully disrupted the PDX1 gene in sheep and the OTX2 gene in goat embryos. The biallelic mutation rate was 81 ± 5% for PDX1 and 85% ± 6% for OTX2. In conclusion, using single-step CRISPR-Cas9 zygote electroporation, we successfully introduced biallelic deletions in the genome of small ruminant embryos. Small ruminants are important animals in agricultural systems and biomedical research. Their size, anatomy, and gestation period make them appropriate for sustainable food production, human diseases investigations, and xenotransplantation research . The CRIThere are two general approaches to producing genome-edited animals, the single-step approach and the two-step approach. In the single-step approach, editing occurs directly in zygotes, which are then transferred to surrogates to generate a gene-edited animal. In the two-step approach, gene edits are introduced into cultured cells, and then gene-edited cells are used to create an animal by somatic cell nuclear transfer (SCNT)/cloning. In general, because of the technical difficulty and the low efficiency of SCNT/cloning , the sinWhen producing genome-edited animals in a single-step approach, the CRISPR-Cas9 system is delivered directly to the zygotic cytoplasm. Microinjection is an effective method for delivering genome editing reagents to a zygote. However, microinjection requires expensive equipment and intensive training. In addition, the microinjection process is time-consuming, and results can be inconsistent, especially when processing a large number of zygotes. Moreover, mosaicism has been repeatedly reported in animals developed from microinjection of engineered endonucleases to zygotes, hindering the benefit of the single-step CRISPR-Cas9 strategy . The altThis study investigated the efficiency of electroporation as a delivery method of CRISPR-Cas9 components to the sheep and goat zygotes. We targeted two different loci in the ovine genome: SOCS2  on chromosome 3 and PDX1  on ovine chromosome 10, and one locus in the caprine genome: OTX2 (orthodenticle homeobox 2) on chromosome 10. SOCS2 regulates somatic growth by inhibiting the transcription pathways induced by GH, EGF, and IGF-1 . PDX1 isTo optimize gene editing in sheep zygotes, CRISPR/Cas9 reagents targeting sheep SOCS2 were delivered to sheep oocytes/zygotes using different parameters. Sheep oocytes/zygotes were electroporated using one of three different settings . Settingp < 0.01) reduced blastocyst development in an electroporation parameter-dependent manner, with setting 3 showing the lowest developmental rate, 9.3% ± 4% (7/75), compared to 36% ± 4% (27/75) in the control. Conversely, microinjection of MII oocytes had no significant effect on the day 7 development rate. The blastocyst percentages were 16% ± 4% (12/75), 14.6% ± 2% (11/75) and 30.7% ± 2% (23/75) for electroporation setting 1, electroporation setting 2 and microinjection (p > 0.05) effect on day 7 development rate. The blastocyst percentage was 33.3% ± 4% (25/75) in the control group, 30.6% ± 3% (23/75) in electroporation setting 1 group, 30.6% ± 4% (23/75) in electroporation setting 2 group, 25.3% ± 2% (19/75) in electroporation setting 3 group, and 32% ± 1% (24/75) in microinjection group (p > 0.05) effect on blastocyst rate except for the electroporation setting 3 group. When the electroporation was performed 8 h after fertilization, the blastocyst percentages were 37.3% ± 2% (28/75), 34.6% ± 2% (26/75), 34.6% ± 2% (26/75), and 30.6% ± 2% (23/75) in the control, electroporation setting 1, 2, and 3 groups, respectively (Day 7 blastocyst percentages are summarized in njection A. Notablon group B. Similaectively C. While ectively D.p < 0.01) to electroporation setting 1  (p < 0.01) than in setting 1 , when the delivery of the CRISPR-Cas9 system was performed 6 h after fertilization , setting 3 , and microinjection  were superior  A. Likewilization B.p < 0.01) than in setting 1  higher when MII oocytes were electroporated using settings 2  and 3 , compared to setting  1 and microinjection   A. At 6 h%; 8/21) B. Past 6%; 8/21) C further%; 8/21) D.To assess the efficiency of electroporation-based single-step genome editing targeting an alternative locus, we electroporated dual sgRNA CRISPR/Cas9 reagents targeting PDX1. Electroporation, 6 h after fertilization, using setting 2, successfully deleted 208 bp of PDX1 sequence in 37 out of 45 electroporated sheep zygotes (82.2% ± 8%), with 30 out of 37 mutated embryos (81% ± 5%) having a biallelic mutation. The blastocyst rate was 30% ± 2% (45/150) in the electroporated group compared to 35.5% ± 4% (53/150) in the control .To assess if electroporation of CRISPR/Cas9 would also work for goat zygotes, we targeted OTX2 with a dual sgRNA approach. A long deletion of 1.1 kb was successfully introduced in the OTX2 locus of the goat genome by electroporation of zygotes 6 h after fertilization using setting 2. The blastocyst rate was 27.3% ± 3% (41/150) compared to 32.7% ± 3% (49/150) in the control. The mutation percentage was 80.5% ± 5% (33/41), with a biallelic mutation rate of 85% ± 6% (28/33) .Ovis aries) and goat (Capra hircus), using electroporation to deliver the CRISPR-Cas9 system reagents. Different electroporation settings were used at different times of embryo development to optimize the creation of biallelic mutant (knock-out) embryos. Electroporating sheep zygotes 6 h after in vitro fertilization using a setting consisting of poring and transfer pulses (setting 2) was superior to microinjection in producing more biallelic mutant blastocysts. The mutation percentage increased from 83.3% ± 8% (20/24) to 95.6% ± 8% (22/23)  B.During microinjection, a single opening is usually made in the plasma membrane, and the CRISPR-Cas9 RNPs are placed close to the plasma membrane. In electroporation, the poring electric pulses induce millions of small (~1 nm) resealable pores in the plasma membranes  for a tiBiallelic mutation rate decreased when the time of CRISPR-Cas9 RNPs delivery was delayed in microinjection from 38% ± 10% (8/21) (MII oocytes) to 25% ± 10% 5/20) (6 h zygotes) and in electroporation from 100% ± 0% (11/11) in MII oocytes to 95.4% ± 9% (21/22) in 6 h zygotes, 76% ± 12% (19/25) in 8-h zygotes and 56% ± 12% (14/25) in 10-h zygotes. The RNPs delivery time is thus of critical importance for genome editing of zygotes. To reduce the chances of mosaicism, DNA editing must be completed before the first DNA replication, which occurs ahead of the first embryonic cleavage. In mice, electroporation of the zygotes 12 h after fertilization increased the rate of mosaicism [ (6 h zygAn experienced technician could process up to 500 zygotes in one hour by electroporation (from stripping the zygotes to the culture of the electroporated embryo) compared to 40–50 zygotes that could be processed by microinjection. Moreover, electroporation maintains consistency across embryos in the dose of RNPs delivered, which is harder to control in microinjection, especially when processing large numbers of zygotes.p < 0.01) the developmental rate from 30.7% ± 7% in microinjection to 14.6% ± 2% and 9.3% ± 4% in settings 2 and 3 of electroporation, respectively. Electroporation changes the structure and the components of the plasma membrane. Besides inducing resealable pores in the membrane lipid bilayers, electroporation increases lipid peroxidation [Electroporation of the MII oocyte with settings 2 and 3  producedxidation  and can xidation , which mMicroinjection and electroporation using settings 2 and 3 were superior to electroporation setting 1 . The effThe decrease in blastocyst rate and mutation percentage of setting 3 when compared to setting 2 could be associated with a higher number of pulses in setting 3 (6 bipolar pulses of 30 volts for 3 ms). Additionally, short-length pulses can disturb DNA transmembrane transport  and may The high efficiency of CRISPR/Cas9 RNP electroporation for introducing a biallelic mutation in sheep embryos was additionally confirmed by targeting the PDX1 gene with the optimized conditions. The mutation rate was 82.2% ± 2%, with a biallelic mutation rate of 81% ± 5%. In a previous study, the same sgRNAs produced 46% and 20% biallelic mutations when microinjected into sheep MII oocytes and zygotes, respectively .Finally, electroporation efficiency for delivering the CRISPR-Cas9 system was further confirmed by targeting goat OTX2 using a dual gRNA approach resulting in a long deletion of 1126 bp in 33 out of 41 zygotes (80.5% ± 5%) with 28 embryos (85% ± 6%) presenting biallelic mutations. These results confirm the high efficiency of both the CRISPR-Cas9 and electroporation to introduce long deletions.To the best of our knowledge, this is the first report of a successful introduction of the CRISPR-Cas9 system into the intact zygote of sheep and goats by electroporation to generate knock-out blastocysts.We designed three experiments to examine the efficiency of electroporation in delivering the CRISPR-Cas9 into the small ruminants’ zygotes, each with three replicates. In experiment 1, we targeted sheep SOCS2 locus and tested three different electroporation settings at four different developmental times: MII oocytes and zygotes at 6, 8, and 10 h after fertilization. We compared the efficiency of gene editing with microinjection at two times: MII oocytes and 6–8 h after fertilization. In experiment 2, we selected the most efficient electroporation conditions to target the sheep PDX1 locus to confirm the electroporation efficiency at another sheep locus. In experiment 3, we targeted the goat OTX2 locus to confirm the electroporation efficiency in targeting another small ruminant species.http://www.e-crisp.org, accessed on 28 August 2019) and  and were ordered from Synthego . The selected sgRNAs fulfilled stringent criteria of efficiency and specificity. For ovine SOCS2 locus, two sgRNAs were selected to target the beginning and the end of exon 1  and transported to the laboratory in warm saline. COCs (1–8 mm) were aspirated by using a 21 G butterfly needle using vacuum pressure. All work was performed in a warm and disinfected workstation.Fresh ram semen was collected before in vitro fertilization and extended using Andromed  at a 1:1 ratio. Frozen goat semen was provided by B & D Genetics.2, 5% CO2, and 90% N2.All work was performed in a warm and disinfected workstation. All media were pre-warmed before use. Incubation work was performed at 38.5 °C in a humified environment at 5% OSuitable-quality COCs with several cumulus cells and homogeneous cytoplasm layers were selected for in vitro maturation. First, COCs were washed five times with warm washing media and then transferred in groups of 50 to 400 µL of maturation medium  and incubated for 22–24 h.g for 5 min with 2 mL of semen preparation media . Sperm concentration was adjusted with fertilization media to 2 × 106/mL and added to COCs. The final fertilization drop volume was 500 µL. When the microinjection or electroporation was performed at the MII oocyte, the cumulus was stripped, and the stripped oocytes were incubated with sperms in the presence of 10 COCs.The expanded COCs were washed three times with warm and pre-equilibrated fertilization media  and transferred in groups of 50 to 400 µL of fertilization media. Semen was washed twice by centrifugation at 328× Fertilized COCs were stripped by vortexing for 3 min in SOF-Hepes media, washed five times, and transferred in a group of 50 to 500 µL of culture media , covered with 400 µL of mineral oil, and incubated for seven days.All work of RNA was carried out in a disinfected and RNA-free workstation at room temperature. Before each delivery session, the sgRNA was mixed with Cas9 protein  at a 1:2 concentration ratio and incubated on ice for 10 min. The final concentration of each sgRNA was 40 ng/µL.Microinjection was performed in a micromanipulation station that consisted of micromanipulators  attached to an inverted microscope  and two hydraulic oil microinjectors . Denuded zygotes and MII oocytes were washed three times with SOF-Hepes and transferred in a group of 25 to 50 µL drops of SOF-Hepes covered with mineral oil. To confirm getting into the cytoplasm, a zygote or MII oocyte was secured by a holding pipette (150–180 µm outside diameter), and the zona pellucida was drilled by a laser system , followed by aspirating part of the cytoplasm through the injection needle (5–7 µm) via applying an appropriate negative pressure . FinallyElectroporation was performed using NEPA21 Super Electroporator and Nepa Electroporation Cuvettes 1 mm gap (EC-001) . All work was performed in a disinfected and RNA-free workstation. Denuded MII oocytes/zygotes were washed three times with SOF-Hepes, three times with warm Opti-MEM reduced serum medium (Thermo Fisher Scientific #31985062), then moved to a new drop of Opti-MEM and mixed with RNPs to bring the volume to 20 µL and loaded to the bottom of the cuvette. The electroporation process was conducted in one of three different settings . After ehttps://ice.synthego.com/, accessed on 28 August 2019) to estimate the editing efficiency. The samples were classified as either wild-type or mutant (We used Sanger sequencing and Tracking of Indels by Decomposition (TIDE) to evaluate the editing efficiency . Brieflyr mutant . The wilp < 0.05, a Tukey range test was employed to compare the means.The percentages of blastocysts, mutation, and biallelic mutations were analyzed by one-way ANOVA. When there was a significant difference at


The search for novel antimicrobial agents is crucial as antibiotic-resistant pathogens continue to emerge, rendering the available antibiotics no longer effective. Likewise, new anti-cancer drugs are needed to combat the emergence of multi-drug resistant tumors. Marine environments are wealthy sources for natural products. Additionally, extreme marine environments are interesting niches to search for bioactive natural compounds. In the current study, a fosmid library of metagenomic DNA isolated from Atlantis II Deep Lower Convective Layer (ATII LCL), was functionally screened for antibacterial activity as well as anticancer effects.Bacillus Cc6 strain, namely clones 102-5A and 88-1G and they were further tested against eleven other challenging strains, including six safe relatives of ESKAPE pathogens , a safe relative to Mycobacterium tuberculosis and four resistant clinical isolates. Clone 88-1G resulted in clear zones of inhibition against eight bacterial strains, while clone 102-5A resulted in zones of inhibition against five bacterial strains. The whole cell lysates of clone 88-1G showed 15% inhibition of Mtb ClpP protease -Mycobacterium tuberculosis drug target-, while whole cell lysates of clone 102-5A showed 19% inhibition of Mtb ClpP protease. Whole cell lysates from the selected clones exhibited anticancer effects against MCF-7 breast cancer cells (cell viability at 50% v/v was 46.2% ± 9.9 for 88-1G clone and 38% ± 7 for 102-5A clone), U2OS osteosarcoma cells (cell viability at 50% v/v was 64.6% ± 12.3 for 88-1G clone and 28.3% ± 1.7 for 102-5A clone) and 1BR hTERT human fibroblast cells (cell viability at 50% v/v was 74.4% ± 5.6 for 88-1G clone and 57.6% ± 8.9 for 102-5A clone). Sequencing of 102-5A and 88-1G clones, and further annotation detected putative proteases and putative biosynthetic genes in clones 102-5A and 88-1G, respectively.Two clones exhibited antibacterial effects against the marine Mycobacterium tuberculosis. Thus, searching the microbial dark matter of extreme environments is a promising approach to identify new molecules with pharmaceutical potential use.The ATII LCL metagenome hosts putative peptidases and biosynthetic genes that confer antibiotic and anti-cancer effects. The tested clones exhibited promising antibacterial activities against safe relative strains to ESKAPE pathogens and The online version contains supplementary material available at 10.1186/s12934-022-01835-z. Antibiotic resistance is a major global health challenge, which necessitates the search for new antimicrobial agents . AdditioSpecialized metabolites are produced by a subset of organisms, and the enzymes producing them are encoded by biosynthetic gene clusters (BGCs) in the genome of the producing organism –6. CurreAmong the marine ecological niches that harbor a huge microbial diversity are environments with extreme conditions , 19. TheEarlier, we have screened the metagenomic fosmid library that harbored prokaryotic environmental DNA from the Atlantis II Red Sea brine pool LCL water samples (ATII) , 24. In The aim of this work was to screen the ATII LCL metagenomic fosmid library by a functional assay for anti-bacterial effects. Two positive clones were then selected to assess their antibacterial effects against clinically relevant bacterial strains. Furthermore, cytotoxic effects of the extracts from the two clones were determined against breast cancer and bone cancer cells in addition to non-cancerous fibroblasts. Lastly, the selected clone inserts were sequenced, and their putative protein-coding genes were annotated. The present two clones harbored individual genes which resulted in the observed effects.Bacillus Cc6. The antibacterial overlay assay was also conducted against eleven additional challenging strains  and EPI300 control. The 102-5A fosmid extract was slightly more potent and demonstrated an inhibition activity equivalent to 19% that was also statistically significant when compared to the negative controls  and sequenced in an Illumina MiSeq platform. After trimming the vector and After the vector and host sequences were trimmed, the number of PEGs (protein-encoding genes) detected by RAST (Rapid Annotations using Subsystems Technology) in each assembly was 21 and 22 in inserts 102-5A and 88-1G, respectively  a putative CopG family transcriptional regulator [Natronococcus occultus], (3) a putative serine protease AprX [Bradyrhizobium erythrophlei] and (4) a putative glycosyltransferase [Candidatus Micrarchaeota archaeon CG1_02_49_24].Clone 88-1G insert harbored the following PEGs Fig. , Table 3Nocardia pseudobrasiliensis], (2) peptidase S8 [Streptomyces sp. CB02613] and (3) cobalt-precorrin 5A hydrolase [Hyphomicrobium facile].Annotation of clone 102-5A insert revealed two putative peptidases and a putative hydrolase, with the best hits being Fig. , Table 3Bacillus Cc6 marine strain, which is a Gram-positive marine bacterial strain  and Acinetobacter (Acinetobacter AB2) clinical isolates were obtained from hospitalized patients in Egypt. Clone 88-1G inhibited the growth of Acinetobacter AB2 strain, MRSA-ZC1, and MRSA-ZC2. Additionally, clone 88-1G resulted in an inhibition zone with the acid-fast bacteria Mycobacterium smegmatis. This result was corroborated with the results of the Mtb ClpP protease assay [M. tuberculosis target enzyme.Clone 88-1G exhibited antibacterial activity against se assay , 31, as Bacillus Cc6 marine strain , when compared to the 88-1G clone, which exhibited a cell viability of 46.2% ± 9.9   of Atlantis II Red Sea brine pool (21° 20.72’ N and 38° 04.59’ E) was previously collected in the 2010 KAUST/WHOI/HCMR expedition . ProkaryE. coli CBAA11 strain producing the antibacterial tambjamine was used as the positive control strain [Bacillus Cc6 strain which is a marine Bacillus strain associated with the Australian marine sponge Cymbastela concentrica  [Bacillus Cc6 strain was used as a challenging strain because it was isolated from a marine environment and because it was reported in a functional screening assay utilizing the same vector CC2 as used in our study [E. coli CBAA11 strain was chosen as a positive control because it produces the antibacterial tambjamine and was reported to result in clear zones against Bacillus Cc6 [E.coli CBAA11) and a negative control (EPI300).An antimicrobial overlay assay was used to test for clones with anti-bacterial activity similar to that reported in the literature , 46. E. l strain , 34. Theh Wales) , 34. Bacur study , 34. E. llus Cc6 , 34. AllBacillus Cc6 strain. The following day, the plates were incubated overnight at 25 °C and an overnight culture of Bacillus CC6 in LB/chloramphenicol was prepared and incubated at 37 °C with shaking. The following day, the starter culture was used to inoculate 100 ml LB/chloramphenicol of Bacillus CC6 till the OD reached 0.5. Then Bacillus CC6 was poured on the plates using top agar at 1:1000 dilution. The overlaid plates were finally incubated overnight at 25 °C and observed carefully for clear zones in the top layer [First the clones were plated on LB/chloramphenicol/arabinose overnight at 37 °C. Chloramphenicol was not added to the other strains, other than op layer . Clones E. coli bearing the eDNA from the positive clones -previously supplemented with auto-induction solution and chloramphenicol- were centrifuged at 3,500 rpm for 10 min. Afterwards, the cell pellets were resuspended in 0.01 M Tris–HCl pH 7. The extracts were then sonicated on ice at 20% maximal amplitude for 370 s . Finally, the extracts were sterile-filtered with 0.2 µm membrane filters (Corning) [Whole-cell extracts were prepared from the clones namely 102-5A and 88-1G, using a method similar to that reported in the literature . OvernigCorning) . Two neg2 at a seeding density of 104 cells/well.Three cell lines were used for the cytotoxicity assay, a human breast adenocarcinoma cell line (MCF-7) , an oste4 cells/well and left overnight to adhere to the bottom of the 96-well plates . The old media was discarded the following day and the media containing the different v/v% concentrations of the extracts was added . The cells were exposed for 48 h to the extracts before assessing the cell viability.The initial seeding density was adjusted to 10The extracts were incubated for 48 h with MCF-7, U2OS and 1BR hTERT cell lines with different v/v% concentrations  . Cell viSeveral negative controls were used in the experiments. Cells with no cell lysates added were used as the first negative control cells. Cells exposed to the buffer only with no cell lysates were used to test the buffer effect. Additionally, cells exposed to the cell lysates of EPI300 strain were used to test the effect of the strain.595 control) were supplemented with complete media. The wells used for measuring A595 blank were only supplemented with media and no cells. Absorbance was finally measured at 595 nm (A595), using a SPECTROstar Nano microplate reader . Cell viability was calculated as follows:All the negative control cells  [Prior to annotation, the vector sequences—pCC2FOS™- were trimmed from the resulting scaffolds. ch 2017) . Each PEch 2017) . The PEGM. tuberculosis that is composed of subunits P1 and P2 is a proven drug target against M. tuberculosis. Mtb ClpP assay was used to determine the inhibition of the peptidase activity of Mtb ClpP1P2. The cleavage of the labeled peptide substrate generates fluorescence at 460 nm (excitation at 355 nm). The assay was performed in a F-bottom, black plate, clear bottom 96- well plate with a final reaction volume of 30 µl, as per the manufacturer's instructions . The assay buffer and Mtb ClP1P2 were mixed. For the negative control, DMSO was used, and for the positive control, the Mtb ClP1P2 was boiled at 95 °C for 10 min .Statistical analysis was conducted by one-way ANOVA and Post-hoc Tukey test to calculate the lts Fig. . R progrR program was also used to generate Fig. Additional file 1: Figure S1. The anti-bacterial overlay assay results of 88-1G fosmid clone, Figure S2. The anti-bacterial overlay assay results of 102-5A fosmid clone, Figure S3. Negative control for the overlay assay. Figure S4. The alignment of the PFAM hits of the selected PEGs in 88_1G and 102_5A clones. Table S1. Annotation of all PEGs after filtering and trimming of the putative orphan biosynthetic gene clusters on 88-1G clone insert, Table S2. Annotation of all PEGs after filtering and trimming of the putative orphan biosynthetic gene clusters on 102-5A clone insert.


Morphological changes as well as markers of oxidative stress, including thiobarbituric acid reactive substance (TBARS) levels, the oxidation of protein sulfhydryl (-SH) groups, methemoglobin formation (% MetHb), apoptotic cells, a reduced glutathione/oxidized glutathione ratio, Band 3 protein (B3p) content, and anion exchange capability through B3p were analyzed in RBCs treated with HgCl2 with or without 10 μM HT and/or HVA pre-treatment for 15 min. Our data show that 10 µM HT and/or HVA pre-incubation impaired both acanthocytes formation, due to 10 µM HgCl2, and mercury-induced oxidative stress injury and, moreover, restored the endogenous antioxidant system. Interestingly, HgCl2 treatment was associated with a decrease in the rate constant for SO42− uptake through B3p as well as MetHb formation. Both alterations were attenuated by pre-treatment with HT and/or HVA. These findings provide mechanistic insights into benefits deriving from the use of naturally occurring polyphenols against oxidative stress induced by HgCl2 on RBCs. Thus, dietary supplementation with polyphenols might be useful in populations exposed to HgCl2 poisoning.Mercury is a toxic heavy metal widely dispersed in the natural environment. Mercury exposure induces an increase in oxidative stress in red blood cells (RBCs) through the production of reactive species and alteration of the endogenous antioxidant defense system. Recently, among various natural antioxidants, the polyphenols from extra-virgin olive oil (EVOO), an important element of the Mediterranean diet, have generated growing interest. Here, we examined the potential protective effects of hydroxytyrosol (HT) and/or homovanillyl alcohol (HVA) on an oxidative stress model represented by human RBCs treated with HgCl Human exposure to heavy metals has increased dramatically in the last five decades due to exponential increase in their use in various industries and products ,2. Among2 and CO2 between lungs and tissues, these circulatory cells are equipped with efficient endogenous anti-oxidative systems that make them mobile free radical scavengers providing antioxidant protection, not only to RBCs themselves but also to other tissues and organs in the body  L cells × 10−2 by means of a standard curve previously obtained by precipitating known SO42− concentrations. The rate constant of SO42− uptake (min−1) was derived from the following equation: Ct = C∞ (1 − e−rt) + C0, where Ct, C∞, and C0 indicate the intracellular SO42− concentrations measured at times t, ∞, and 0, respectively, with e representing the Neper number (2.7182818), r indicating the rate constant accounting for the process velocity, and t being the specific time at which the SO42− concentration was measured. The rate constant is the inverse of the time needed to reach ~63% of the total SO42− intracellular concentration [42−] L cells × 10−2 reported in figures represents the SO42− micromolar concentration internalized by 5 mL RBCs suspended at 3% hematocrit.An SOlsewhere ,51,52,53activity ,55, was 2 (4 h at 37 °C) or pre-incubated with 10 µM HT and/or HVA , were centrifuged  to replace the supernatant with SO42− medium. The rate constant of SO42− uptake was then determined as described for the control condition.Samples (3% hematocrit), which were left untreated or exposed to 10 µM HgCl2 (4 h at 37 °C) or pre-incubated with 10 µM HT and/or HVA , were centrifuged  and resuspended in isotonic solution. After treatments, the content of GSH was measured by Cayman’s GSH assay kit using an enzymatic recycling method with glutathione reductase. This assay is based on the oxidation of GSH by Ellman’s reagent DTNB, which produces oxidized glutathione (GSSG) and 3-thio-2-nitro-benzoic acid (TNB), absorbing at a wavelength of 412 nm. The amount of GSSG was calculated by the following formula: 1/2 GSSG = GSH total-GSH reduced. Results are expressed as a GSH/ GSSG ratio.GSH levels were assayed according to Teti and collaborators , with slp < 0.05; (n) corresponds to the number of separate measurements.All data are expressed as arithmetic mean ± standard error of the mean. For statistical analysis and graphics, GraphPad Prism  and Excel  software were used. Data normality was verified with the D’Agostino and Pearson Omnibus normality test. Significant differences between mean values were determined by one-way analysis of variance (ANOVA), followed by Bonferroni’s multiple comparison post-test or ANOVA with Dunnett’s post-test, as appropriate. Statistically significant differences were assumed at 2 induced the morphological alteration of RBCs with 44.3% of acanthocytes (RBCs with surface blebs) detected by scanning electron microscopy analysis (SEM). However, in samples pre-treated for 15 min at 25 °C with 10 μM HT and/or HVA and then treated with 10 μM HgCl2, the percentage of morphologically altered cells was reduced to 3.5% and 2.9%, respectively (As depicted in ectively .2 with or without pre-exposure to 10 µM HT and/or HVA for 15 min at 25 °C. 2). The evaluation of ROS species was carried out by flow cytometry in RBCs left untreated or, alternatively, exposed to 10 µM HgCl2 showed a significant increase in ROS levels compared to the control samples. After 30 min, the levels of ROS increased by 50% in the 10 µM HgCl2-treated samples and remained unchanged over time. In 2 failed to significantly increase ROS levels, with these being unchanged compared to control values  for 1 h were significantly higher with respect to those of RBCs left untreated (control). Moreover, the TBARS levels of RBCs treated with 10 µM HgCl2 for 4 h were significantly higher than those of RBCs left untreated (control). Importantly, in RBCs pre-treated with 10 µM HT and/or HVA and then exposed to 10 µM HgCl2, TBARS levels were significantly reduced compared to those measured in 10 µM HgCl2-treated RBCs. It is of note that olive oil polyphenols alone did not significantly affect TBARS levels.The measurement of Thiobarbituric acid reactive substances (TBARSs) in RBCs is reported in 2 for 4 h with or without HT and/or HVA pre-treatment. As expected, exposure to NEM led to a significant reduction in the sulfhydryl group content. Sulfhydryl groups in 10 µM HgCl2-treated RBCs were also significantly reduced with respect to the control. Importantly, pre-treatment with 10 µM HT and/or HVA significantly restored sulfhydryl group total content in 10 µM HgCl2-treated RBCs (ted RBCs b. Olive ted RBCs b.2 (4 h at 37 °C) with or without pre-treatment with 10 µM HT and/or HVA (1 h at 25 °C), or, alternatively, treated with the well-known MetHb-forming agent NaNO2 (4 mM for 1 h at 25 °C). Methemoglobin levels measured after incubation with NaNO2 were significantly higher than those detected in RBCs left untreated (control). In parallel, MetHb levels measured following exposure to 10 µM HgCl2 were significantly higher than those measured in the control (left untreated). Pre-exposure to 10 µM HT and/or HVA significantly reduced the MetHb levels in 10 µM HgCl2-treated RBCs towards values that did not differ from control values. Olive oil polyphenols alone did not significantly affect the %MetHb.2 (4 h at 37 °C) with or without pre-treatment with 10 µM HT and/or HVA (1 h at 25 °C). Regarding the percentage of RBCs in eryptosis, significant differences were detected after treatment with 10 µM HgCl2 for 4 h. However, pre-treatment with 10 µM HT and/or HVA significantly reduced eryptosis levels in 10 µM HgCl2-treated RBCs. Olive oil polyphenols alone did not significantly affect eryptosis.2 (4 h at 37 °C) with or without pre-treatment with 10 µM HT and/or HVA (1 h at 25 °C). Band 3 protein levels after treatments were not significantly different with respect to those determined in control RBCs.42− uptake as a function of time in RBCs left untreated (control) and in RBCs treated with 10 µM HgCl2 (4 h at 37 °C) with or without pre-treatment with 10 µM HT and/or HVA (1 h at 25°C). In control conditions, SO42− uptake progressively increased and reached equilibrium within 45 min (rate constant of SO42− uptake = 0.058 ± 0.001 min−1). Red blood cells treated with 10 µM HT and/or HVA alone showed a rate constant of SO42− uptake that was not significantly different with respect to the control. Conversely, the rate constant value (0.043 ± 0.001 min−1) in RBCs treated with 10 µM HgCl2 was significantly lower than in the control (*** p < 0.001). In RBCs pre-incubated with 10 µM HT (1 h at 25 °C) and then treated with 10 µM HgCl2 (1 h at 25 °C), the rate constant (0.058 ± 0.001 min−1) was significantly higher than that of RBCs treated with 10 µM HgCl2 (0.043 ± 0.001 min−1) while not being significantly different with respect to the control (2 (4 h at 37 °C), the rate constant (0.062 ± 0.001 min−1) was significantly different with respect to that of RBCs treated with 10 µM HgCl2 (0.043 ± 0.001 min−1)  was significantly lower than what was determined in both control and treated RBCs  . SO42− u control , while i control . In DIDS2 for 4 h at 37 °C with or without pre-treatment with 10 µM HT and/or HVA (15 min at 25 °C). The GSH/GSSG ratio measured after incubation with 10 µM HgCl2 was significantly lower than that detected in control RBCs. This effect can be associated with an increased GSSG abundance and/or decreased GSH concentration, both of which are indicative of an increase in cellular oxidative stress.2-induced oxidative stress model in human RBCs were investigated. With regard to the first aspect, the susceptibility of RBCs to 10 µM HgCl2 exposure was verified by scanning electron microscopy (SEM), revealing dramatic changes in the RBC shape. Such a finding is in line with what was already reported by Lim and collaborators [2. In the present case, the typical biconcave shape was lost in a significant number of cells displaying surface blebs . Indeed, the percentage of acanthocytes was higher (44.3%) than that detected in untreated cells (3%). However, pre-treatment with 10 µM HT and/or HVA polyphenol extracts prevented the morphological changes  on morphology and function on an HgClborators  when the changes c,d, with changes . These d2 has also been associated with eryptosis [2 exposure induced PS translocation to the external surface of the RBC membranes after 4 h of treatment , were completely restored by 10 µM Q pre-treatment (1 h). Similarly, the oxidation of protein sulfhydryl groups was totally restored after pre-treatment (1 h) with Q in RBCs treated with 20 mM H2O2 (1 h). Additionally, the protective effects of freeze-dried Açaì extract—the main chemical components present in the Açaí pulp are polyphenols—in a D-Galactose-induced aging model in human RBCs have been also reported [The biological effects of olive oil are related to its chemical matrix, which includes numerous phytochemicals components, such as flavonoids . These mreported . 2-induced oxidative damage resulted in the oxidation of the ferrous iron of hemoglobin to a ferric form given methemoglobin (MetHb), which is inactive as an oxygen transporter in the blood [2 for 4 h increased the levels of MetHb in RBCs  [Another major property of RBC oxidation is the clustering and/or the breakdown of B3p and the binding of oxidized hemoglobin (MetHb) to high affinity sites on B3p. Band 3 protein is an integral membrane protein that accounts for approximately 25% of the RBC membrane. It has several crucial functions and is composed by two rather different domains of a similar size . In RBCshe blood . To bett in RBCs c. These ife span . Interesoduction d, which on (2O2) . 2 toxicity is its impact on membrane transport systems. The Band 3 protein is mainly involved in the chloride–bicarbonate exchange. Firstly, the content of B3p was determined. No change in the protein content was detected under these experimental conditions  concentrations induced oxidative stress and provoked a decrease in the rate constant for SO42− uptake via B3p [42− uptake as well as the reduction in B3p expression levels observed following treatment with H2O2 [2O2 doses (10 µM for 30 min) [2O2 exposure is most likely linked to the formation of oxidizing hemoglobin after oxidative stress. Therefore, the different effect on the SO42− transport kinetics observed in a former study is not surprising. Indeed, in other models of oxidative stress obtained by exposing RBCs to high concentrations of D-Galactose and D-Glucose, the acceleration of the anion exchange through B3p, instead of a slowing, was detected [The evidence that B3p exhibits modifications in the rate constant for SO via B3p . Howeverith H2O2 . Moreove 30 min) . This prdetected ,32. It idetected ,80. 2 toxicity is correlated with a high affinity of HgCl2 for sulfhydryl groups that can severely alter enzyme activities, including Glutathione [2 ions react with sulfhydryl groups of cysteine residue, thus depleting intracellular thiols [2 treatment reduced the GSH/GSSG ratio. However, the pre-incubation of RBCs with 10 µM HT and/or HVA completely restored the redox balance (+-induced oxidative injury [2 increases the production of hydrogen peroxide and GSH depletion in renal mitochondria, thus causing oxidative damage in cell membranes, resulting in hemolysis.Finally, we directed our focus towards an assay of the endogenous antioxidant system. The underlying mechanism of HgCltathione . The HgCr thiols . A decre balance . To suppe injury . Glutathe injury . Its depe injury  suggests2 induced increased oxidative stress in human RBCs. Indeed, the damaged RBCs may quickly be removed from circulation, thus leading to the shortened lifespan of RBCs, resulting in anemia. However, pre-treatment with HT and/or HVA avoided the formation of acanthocytes observed after exposure to HgCl2 prevented HgCl2-induced oxidative stress damage, including ROS production, lipid peroxidation, and sulfhydryl group oxidation of the total proteins on the plasma membrane. Moreover, HgCl2 exposure was associated with a reduction in the rate constant of SO42− uptake through B3p as well as MetHb production. Both alterations were attenuated by pre-treatment with HT and/or HVA. Importantly, pre-incubation with both polyphenols restored the GSH/GSSG ratio, resulting in the improvement of the endogenous antioxidant system.In summary, exposure to HgCl2-induced oxidative stress damage on a cellular level. Additionally, the results obtained here confirm that the measurement of the B3p anion exchange capability remains a suitable tool for monitoring the impact of oxidative stress on RBC homeostasis. Considering the involvement of oxidative stress damage in HgCl2-induced poisoning, new biomarkers with both diagnostic and monitoring potential are needed. Blood can be obtained from patients with minimally invasive procedures, with it reflecting the physiological status of peripheral tissues, and therefore it might represent a convenient source of biomarkers. In this light, future investigations are needed to clarify the signaling underlying the protective activity of HT and/or HVA on the interaction between B3p and cytoskeletal proteins such as ankyrin and spectrin and their potential post-translation modifications. These results are a step towards a better understanding of the biochemical mode of mercury-induced toxicity and cell damage. This will allow for appropriate methods to overcome the harmful effects of this extremely dangerous metal and widespread environmental pollutant to be devised.In conclusion, the present investigation elucidates benefits deriving from the use of naturally occurring polyphenols against HgCl


Our present data demonstrate that BEO inhibits cholinergically- and non cholinergically-mediated contractions in rat isolated gut and that linalool is the most active component. These results suggest that the phytocomplex might be useful in the treatment of spastic disorders in ENS mainly characterized by the presence of pain; incidentally, irritable bowel syndrome (IBS) is a painful condition in which a role for neurotransmitter dysfunction has been envisaged. More investigation is required for clinical translation of the present data.Preclinical data indicate that bergamot essential oil (BEO) can modulate the synaptic functions within the central nervous system (CNS). Particularly, several data shows that essential oil is endowed with reproducible analgesic and anxiolytic effects that may derived from the ability to modulate the excitatory and inhibitory neurotransmission in the CNS. Although there are differences in the functional complexity of the enteric nervous system (ENS), it is likely that the phytocomplex has biological properties in gut superimposable to those showed in the CNS. Accordingly, the aim of this study was to investigate ex-vivo the effect of bergamot essential oil and its main constituents on the contractile activity of rat isolated colon, jejunum and ileum induced by different muscle stimulants such as acetylcholine (10 Citrus bergamia Risso & et Poiteau, syn.: Citrus aurantium L. subsp. bergamia (Risso et Poiteau), an aromatic citrus fruit belonging to genus Citrus, family Rutaceae, is worldwide used to extract the essential oil from the peel by a cold-pressing procedure [2+-dependent mechanism, when low concentrations of BEO were used, and a non-Ca2+-dependent carrier mechanism at high concentrations of phytocomplex [A [Bergamot  and the CNS both contain integrative neural circuitry and similarities between them . It is kIn this study, using isolated organ bath technique, the effects of BEO were characterized on cholinergically- and non-cholinergically-mediated contractions induced respectively by acetylcholine (ACh) and potassium chloride (KCl) on different tissues  of the enteric system of rat. It was also investigated the activity of single oxygenated components of the volatile fraction of BEO such as limonene, α-pinene, linalyl-acetate and linalool reported to contribute in the pharmacological properties of the essential oil ,15,20,21v/v). ACh and ATR  were dissolved in distilled water. BEO and its constituents were dissolved in dimethylsulphoxide (DMSO)  and its amount was less than 1% of the tissue bath volume.Atropine (ATR), ACh, (R)-(+)-limonene, α-pinene, linalyl acetate and (-)-linalool were purchased from Sigma-Aldrich . The crude bergamot essential oil was kindly provided by “Capua Company1880 S.r.l.”, Campo Calabro . Chromatographic analysis of BEO reports: (R)-(+)-limonene ~48.6%, α-pinene ~3.2%, linalyl acetate ~23.6%, (-)-linalool ~5.5% of the total volume ; . Animal procedures were conducted under approved guidelines by the University Animal Welfare Committee (OPBA) of the Department of Pharmacy, Health and Nutritional Sciences of University of Calabria   and in accordance with the Council Directive (86/609/EEC) and D.Lgs 26/2014 in order to apply the principles of reduction and refinement. Experiments were performed on adult male rats (250–300 g), obtained from Charles River Laboratories . Animals were housed in 36 × 18.5 × 24 cm clear polyethylene cages in a room with controlled temperature (22 ± 1 °C) and under a light-dark schedule (lights on 7 a.m. to 7 p.m.) for at least 7 days before being used. Food and water were freely available. The rats were sacrificed by exposure to 4% isoflurane air followed by cervical dislocation.4 2.5; NaHCO3 25; KH2P04 1.2; CaCl2 2.5; glucose 11.1 . The segments, cleaned from adjacent and connective tissue were cut into full thickness strips along the longitudinal (15 mm long and 5 mm wide) and were vertically suspended in two channel organ bath containing 40 mL of oxygenated (95% O2 and 5% CO2) Krebs solution at 37 °C. Before use, the preparations were allowed to equilibrate for 45 min. Then, contractions were evoked by exogenously applied ACh (10−6 M) and KCl (80 mM); the latter substances were administrated and left in the tissue baths for 5 min to obtain reproducible responses to use as control. After obtaining of three reproducible contractions, BEO (2.5 × 10−5 to 2.5 × 10−3% v/v), linalool (2.5 × 10−6 M to 2.5 × 10−4 M), limonene (2.5 × 10−6 M to 2.5 × 10−3 M), linalyl acetate (2.5 × 10−6 M to 2.5 × 10−3 M) and α-pinene (2.5 × 10−6 M to 2.5 × 10−3 M)  were added to the bath 5 min before ACh. The distal end of each segment was tied to an organ holder and the proximal end was secured with a silk thread to an isometric force transducer . Mechanical activity was digitized on an A/D converter, visualized, recorded and analyzed on a personal computer using the Data Capsule-Evo system .Following the opening of the abdominal cavity, whole segments of jejunum, ileum and colon were excised and the content gently flushed out with Krebs solution consisting of (mM): NaCI 119; KCI 4.5; MgS0n = 3–6 in all experiments. GraphPad PRISM 7.0 for Windows  was used for statistical analysis and graphics production. Effects were considered statistically significant if they had a null hypothesis probability lower than 5% (p ˂ 0.05). Statistical significance of any difference between unpaired data was determined using Student’s test (t-test).Data are expressed as the mean ± S.E.M, −6 M) elicited a contraction (indicated as 100%) in rat isolated colon, jejunum and ileum that was completely inhibited by pre-treatment with ATR (10−6 M) for 5 min , 2.5 × 10−4%, (v/v) and 2.5 × 10−3%, (v/v), respectively. In jejunum, values of 96.4 ± 3.6%, 60.2 ± 5.5% and 1.0 ± 1.0% were measured with increased volumes of phytocomplex. Highest effects were observed in ileum where cholinergic contraction was reduced by BEO to 83.2 ± 6.2%, 58.9 ± 6.5% and 0.75 ± 0.7%, respectively  to 74.2 ± 13.2%, 93.3 ± 3.5% and 93.5 ± 1%, respectively. Lower concentrations of the compounds failed to affect muscarinic contraction . On the other hand, linalyl acetate induced a concentration-dependent (2.5 × 10−5–2.5 × 10−3 M) decrease of ACh contraction to 94.6 ± 2.9%, 81.8 ± 9.1% and 5.0 ± 2.9% respectively. Similarly, linalool reduced, in a concentration-dependent manner (2.5 × 10−6–2.5 × 10−4 M), the contraction induced by ACh to 93.5 ± 5.9%, 67.7 ± 6.4% and 0.17 ± 0.17%, respectively  only at the highest concentration (2.5 × 10ectively .−3 M) reaching the values of 93.5 ± 3.4% and 92.8 ± 3.6%, respectively. Conversely, linalyl acetate (2.5 × 10−5–2.5 × 10−3 M) and linalool (2.5 × 10−6–2.5 × 10−4 M) induced a concentration-dependent decrease of cholinergic contraction. Particularly, linalyl acetate induced a reduction to 88.4 ± 3.2%, 75.4 ± 6.4% and 67.7 ± 8.0% and linalool to 84.7 ± 2.3%, 52.1 ± 3.4% and 0.6 ± 0.6%, respectively  for 5 min suggesting that muscarinic receptors were not involved (Administration of KCl (80 mM) to the bath elicited a contraction (indicated as 100%) in rat isolated colon, jejunum and ileum that was not modified by pre-treatment with ATR ) induced a concentration-dependent decrease of KCl contraction and, similarly to what was observed with cholinergic contractions, its most active constituent was linalool. For the sake of brevity, we only report the effect of linalool  while linalool induced a reduction to 64.3 ± 8.5% and 2.94 ± 2.8% at the concentrations of 2.5 × 10−5–2.5 × 10−4 M, respectively  .The results obtained show that contractions induced by ACh in colon, jejunum or ileum of rat are inhibited by BEO in a concentration-dependent manner and abolished by the highest volume tested. Similarly, BEO decreased KCl contractions in gut tissues, suggesting that phytocomplex counteracts both cholinergically- and non cholinergically-mediated contractions. The ability of BEO to inhibit non cholinergically-mediated contractions in isolated gut supports recent data by Straface and colleagues (2020)  and is fCitrus spp on gut smooth muscles reviewed by Heghes and colleagues (2019) [Citrus aurantifolia demonstrated a spasmolytic activity with a progressive reduction in amplitude of contractions and muscle tone in isolated rabbit jejunum preparation [Citrus sinensis on rat ileum [Incidentally, our results indicate that some of the compounds included in the volatile fraction contribute to spasmolytic effect of phytocomplex and that linalool is the most efficacious and potent constituent of BEO to counteract gut contractions elicited by the different spasmogenic agents. Particularly, linalool induces a dose-dependent reduction of the cholinergic contraction in all gut tissues investigated, while limonene and α-pinene elicit a relaxant effect only at the highest concentration used and linalyl acetate shows a concentration-dependent effect only in jejunum and ileum. Moreover, the monoterpene linalool is active at concentrations ten times less than those used with the other compounds investigated. The results obtained by testing BEO constituents show that linalool is also the most effective to counteract the KCl-evoked contraction. Our results support data on the antispasmodic activity of different essential oils from s (2019) . Particuparation . More reat ileum . Interesat ileum .+, Ca2+ and K+ intracellular levels [+ ions is fundamental for initiating the genesis of an action potential, which in turn determines an intracellular increase of Ca2+ ions to guarantee the fusion of synaptic vesicles on the presynaptic membrane. It follows that a reduction in the intracellular levels of Na+ reduces the possibility of generating action potential and consequently release of neurotransmitters, including acetylcholine [+-channels in central neurons of Caucasotachea atrolabiata [+ current other than other ions currents in olfactory receptor cells of rat [+-channels and blocks the membrane voltage-gated Ca2+ channels, producing endothelium-independent vasorelaxation given by hyperpolarization and by inhibition of Ca2+ influx, fundamental in the interaction between actin and myosin filaments [The ability of BEO and linalool to interfere with the contractions elicited by different spasmogenic compounds seems to suggest that their effect may be due to the modulation of intracellular processes common to different stimuli rather than a receptor-type mechanism and supports data by Straface and colleagues . Particur levels . In nervlcholine . Incidenolabiata . Moreoves of rat . In mousilaments . Althougilaments . Interesilaments .All these data suggest that antispasmodic activity of bergamot essential oil underlies the involvement of complex mechanisms and deserve further investigation.2+ influx by actions on the L-type Ca2+ channels [Overall, our results confirm that BEO is endowed with reproducible antispasmodic effects, mainly through linalool, and it could be helpful in the complementary treatment of intestinal disorder related to excessive smooth muscle contractility. Particularly, the phytocomplex might be useful in the treatment of irritable bowel syndrome (IBS), a chronic, functional gastrointestinal syndrome characterized by relapsing abdominal pain and altered bowel habits, with either predominant symptoms of diarrhoea and constipation . Currentchannels .IBS is a painful condition in which a role for neurotransmitter dysfunction has been envisaged and like all functional gastrointestinal disorders, it is a disorder of brain–gut interactions ,36. PainIn this context, BEO, endowed with reproducible antispasmodic, analgesic and anxiolytic effects, might be really beneficial in the treatment of IBS. However, more investigation is required for clinical translation of the present data, as it occurred for clinical translation of engineered BEO in pain underlying agitation in dementia ,43,44.These results show that BEO is endowed with reproducible antispasmodic effects on colon, jejunum and ileum of rat and that linalool is the most active constituent. Although the mechanism of action deserves further investigation, the whole essential oil and linalool could be beneficial in the complementary treatment of painful intestinal disorder related to excessive smooth muscle contractility such as IBS.


Staphylococcus aureus. Changes in the cell membrane fluidity of methicillin-resistant S. aureus were found to be a major component of the mechanism of synergistic antibiotic activity of Ricini Semen extract and its constituent fatty acids. In our model, changes in cellular membrane fluidity disrupted the normal function of bacterial signaling membrane proteins BlaR1 and MecR1, which are known to detect oxacillin, and resulted in the incomplete expression of penicillin-binding proteins 2a and β-lactamase. Utilizing the mechanism presented in this study presents the possibility of developing a method for treating antibiotic-resistant bacteria using traditional antibiotics with plant-based compounds.Resistant bacteria are emerging as a critical problem in the treatment of bacterial infections by neutralizing antibiotic activity. The development of new traditional mechanisms of antibiotics is not the optimal solution. A more reasonable approach may be to use relatively safe, plant-based compounds in combination with conventional antibiotics in an effort to increase their efficacy or restore their activity against resistant bacteria. We present our study of mixing Ricini Semen extract, or its constituent fatty acids, with oxacillin and testing the effects of each on the growth of methicillin-resistant  Staphylococcus aureus was the third most common species of pathogens found in blood infections detected during hospitalization, accounting for 16.6%, of which more than half were methicillin-resistant S. aureus (MRSA) [S. aureus is a skin bacterium that destroys the skin barrier and stimulates immunity, causing various skin diseases, including atopic dermatitis [S. aureus are both well-known and clinically important antibiotic-resistant bacteria [One of the most critical threats to human health is the emergence of antibiotic-resistant bacteria ,2. In 20s (MRSA) . S. aurermatitis ,6. MRSA bacteria .MRSA presents a clinical challenge to multidrug resistance (MDR) as well as resistance to methicillin . MRSA isSynergistic agents that increase the antibiotic activity of traditional antibiotics are one of the most promising options for successfully treating resistant bacterial infections . For synRicinus communis, the castor oil plant, is a species of flowering plant that produces an oily seed, Ricini Semen [S. aureus and Escherichia coli [ni Semen . Castor ni Semen ,22,23. Ahia coli ,25. The S. aureus [S. aureus when treated at 20 µg/mL [Ricinoleic acid, an unsaturated omega-9 fatty acid containing a hydroxyl group, accounts for approximately 85% of castor oil ,27,28. R. aureus . Palmiti. aureus . Palmiti. aureus . Stearic. aureus . Stearic20 µg/mL . Linolei20 µg/mL .Staphylococcus spp. and other Gram-positive bacteria [S. aureus is that bacterial cellular membrane sensor proteins, MecR1/BlaR1, detect the antibiotic and deactivate the repressor proteins MecI/BlaI [mecR1-mecI system, a blaR1-blaI system with a high degree of homology with mecR1-mecI, takes over and serves to regulate the expression of mecA [Oxacillin is a β-lactam antibiotic and a derivative of penicillin with high antibiotic activity against bacteria ,34. The ecI/BlaI . The relecI/BlaI ,36,37. I of mecA .mecA is present in a mobile genetic element, staphylococcal cassette chromosome mec (SCCmec) [mec types are classified [mec type II, and 50.1% were SCCmec type IV [(SCCmec) . Currentassified . Accordi type IV .mec types of MRSA. This study also provides data regarding the possible biological mechanisms of Ricini Semen that inhibit MRSA growth when used in combination with oxacillin.Extracts having synergistic antibiotic activity with oxacillin against MRSA were identified within the 397-edible plant extract library (data not shown). Among them, we found that Ricini Semen extract and its chemical components, ricinoleic acid, palmitic acid, and stearic acid, conferred synergistic activity with oxacillin against two of the SCCmec type of the MRSA CCARM 3807 and MRSA CCARM 3820 strains used in this study, we verified the presence of SCCmec in each strain , is significantly upregulated [PBP2a, a penicillin-binding protein synthesized from . aureus . MRSA CCne model ,58, a siegulated . mec type IV element with no mecI and a truncated mecR1 [mecA and blaZ [blaZ for PC1 and mecA for PBP2a were both significantly upregulated [The MRSA CCARM 3820 had an SCCed mecR1 . Becauseand blaZ ,59. Oxacegulated .mecA, mecR1, and blaR1 by Ricini Semen extract and ricinoleic, palmitic, and stearic acids were analyzed using RT-PCR  and plated on Tryptic Soy Agar (TSA) plates made by adding 1.5% Bacto-Agar  to TSB.w/v) of sodium chloride  to distilled water. All solutions were sterilized at 121 °C for 20 min. The strains were cultured at 37 °C with a shaking speed of 250 rpm for liquid cultures.Saline was prepared by adding 0.85%  was dissolved in distilled water. Ricinoleic acid , palmitic acid , stearic acid , linoleic acid , and oleic acid  were dissolved in ethanol . Laurdan  was purchased from Invitrogen (ThermoFisher Scientific Korea). Phosphate-buffered saline  was purchased from Sigma-Aldrich Inc.Ricini Semen ethanol extract was prepared according to a previous study  with sommec type of MRSA strain was confirmed by polymerase chain reaction (PCR) according to a previous study [mecA, α2 (5′-TAAAGGCATCAATGCACAAACACT-3′) and βc (5′-ATTGCCTTGATAATAGCCTCT-3′) for ccrA2-ccrB2, mA7 (5′-ATATACCAAACCCGACAACTACA-3′) and mI6 (5′-CATAACTTCCCATTCTGCAGATG-3′) for mecA-mecI, and mA7 and IS7 (5′-ATGCTTAATGATAGCATCCGAATG-3′) for mecA-IS1272. Genomic DNA was extracted using the MiniBEST Bacteria Genomic DNA Extraction Kit Ver.3.0 . The PCR reaction mixture consisted of 0.8 µL of chromosomal DNA, 0.8 µL of dNTP (each 10 mM), 4 µL of ×10 pfu buffer, 0.4 µL of forward primer (50 pmol/µL), 0.4 µL of reverse primer (50 pmol/µL), 0.4 µL of pfu polymerase , and 33.2 µL of distilled water. PCR conditions for mecA and ccrA2-ccrB were one cycle of initial denaturation at 94 °C for 2 min, 30 cycles of denaturation at 94 °C for 2 min, annealing at 57 °C for 1 min and extension at 72 °C for 2 min, and one cycle of final elongation at 72 °C for 2 min [The SCCus study . The primecA-mecI and mecA-IS1272, all PCR conditions were the same as those described above, except the annealing temperature was increased from 57 °C to 60 °C [For to 60 °C . The PCRMRSA CCARM 3807 and MRSA CCARM 3820 were plated on TSB agar. The plates were then incubated at 37 °C for 24 h, after which the bacteria were used to inoculate 5 mL of TSB and precultured at 37 °C for 24 h with 250 rpm continuous shaking.600 = 0.05 of the final cell densities were subcultured into another 5 mL of TSB medium to which each chemical or extract was added. These cultures were incubated at 37 °C for 24 h with 250 rpm continuous shaking, after which the cell density by Abs600 was measured. Each experiment was repeated twice over three experiments total for each set of conditions. The minimum concentration at which cells did not grow in all three cultures was calculated as the minimum inhibitory concentration (MIC).The precultured cells with Absw/v) sodium chloride. After centrifugation at 699× g for 1 min and subsequent filtration with syringeless filters 0.20 µm , the hexane layer was analyzed.The Ricini Semen extract was analyzed by gas chromatography for the quantification of each component, including linoleic acid, oleic acid, palmitic acid, ricinoleic acid, and stearic acid. The samples were pretreated according to the protocol used in a previous study  with few2, the injector temperature was set at 270 °C, the detector temperature was set at 280 °C, the H2 flow rate was set at 30 mL/min, and the airflow rate was set at 300 mL/min [The fatty acids in the Ricini Semen extract were analyzed by gas chromatography  with a flame ionization detector and with the use of a column . The oven conditions were held at 120 °C for 1 min, after which the temperature was increased at a rate of 10 °C per minute to 240 °C, and this temperature was maintained for 10 min. The gas flow was set at 1.8 mL/min, and 1 µL of each sample was analyzed in split mode 1:50. The mobile phase gas was N0 mL/min .600 = 0.05. The concentrations of compounds and extracts used to measure the cell growth curve are specified in corresponding sections. The cultures were incubated at 37 °C for 24 h with 250 rpm of continuous shaking. The concentration of bacterial cells was measured by absorbance at 600 nm at hourly intervals. The experiment was repeated three times, and the mean and standard deviations were calculated.Bacterial cells were cultured as described in the section “Measuring the minimal inhibition concentration of the MRSA strains” up to the preculture step. The precultured cells were inoculated into 20 mL aliquots of TSB medium to which each chemical or extract was added. This culture was then tested so that the bacterial concentration at AbsColony-forming units (CFU) were measured to estimate the number of living bacterial cells after culture. In the previous growth curve experiment, a 24-h culture sample was diluted with saline and spread on TSA plates. After incubation at 37 °C for 24 h, the number of bacterial colonies on the TSA plates was counted. The experiment was repeated three times, and the mean and standard deviations were calculated.Control − CFUSample)/CFUControl × 100. CFUControl is the CFU value of cell culture without any testing extract or compound. Compared with the relative growth inhibition of each sample, if the relative growth inhibition by mixing two samples was more than doubled, it was designated as synergistic activity.The synergistic antibiotic activity was determined by using the CFU value instead of the absorbance of the culture medium in a previous paper . The relThe cell culture and material treatment conditions for this portion of our study are the same as in our growth curve experiment. After the main culture for 3 h, cells were broken by TissueLyser LT  , and RNAA total of 3 µL of the purified RNA template, 1 µL of forward primer (5 pmol/µL), 1 µL of reverse primer (5 pmol/µL), 1 µL of enzyme mix from the TOPreal One-step RT qPCR Kit (SYBR Green with low ROX) , 10 µL of reaction mix from the TOPreal One-step RT qPCR Kit, and 4 µL of water (RNase free) were mixed, and the mRNA concentration was analyzed using QuantStudio5 (ThermoFisher Scientific Korea) in preparation for the real-time polymerase chain reaction (RT-PCR).mecA forward (5′-GTTAGATTGGGATCATAGCGTCATT-3′) [mecA reverse (5′-TGCCTAATCTCATATGTGTTCCTGTAT-3′) [mecR1 forward (5′-AAGCACCGTTACTATCTGCACA-3′) [mecR1 reverse (5′-GAGTAATTTTGGTCGAATGCC-3′) [blaR1 forward (5′-CATGACAATGAAGTAGAAGC-3′) [blaR1 reverse (5′-CTTATGATTCCATGACATACG-3′) [mecA were initial denaturation at 95 °C for 10 min followed by 40 cycles of denaturation at 95 °C for 15 sec, annealing at 60 °C for 1 min, and extension at 72 °C for 30 sec. Initial denaturation for blaR1 was performed at 95 °C for 10 min, followed by 45 cycles of denaturation at 95 °C for 30 sec, annealing at 51 °C for 30 sec), and extension at 72 °C for 30 sec. Initial denaturation of mecR1 was completed at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 10 sec, annealing at 60 °C for 15 sec, and extension at 72 °C for 20 sec. The specificity of the PCR products was confirmed by the performance of a melting curve analysis between 60 °C and 95 °C. This experiment was performed in duplicate.The primer sequences were 16S rRNA forward (5′-TCCGGAATTATTGGGCGTAA-3′) , 16S rRNCATT-3′) ,67, mecAGTAT-3′) ,67, mecRCACA-3′) , mecR1 rTGCC-3′) , blaR1 fAAGC-3′) , and blaTACG-3′) . The cDNI435 − I490)/(I435 + I490). The experiment was performed independently in triplicate, and the mean value and standard deviation value were calculated.Bacterial cellular membrane fluidity was measured according to the protocol presented in a previous study, with few modifications . The bacS. aureus is changed by Ricini Semen extract and its constituent fatty acids. The altered fluidity in the proposed model diminishes the ability of membrane proteins BlaR1 and MecR1 to detect oxacillin in the SCCmec element, conferring resistance to oxacillin. Interfering with the degradation of BlaI and MecI by inactivating BlaR1 and MecR1, respectively, may prevent the expression of β-lactamase (BlaZ) and penicillin-binding proteins 2a (PBP2a), which are resistance mechanisms induced by oxacillin. Ricini Semen extract and its constituent fatty acids are suggested to enhance the activity of β-lactam antibiotics and restore their therapeutic activity on MRSA infections.Ricini Semen extract and its constituent fatty acids synergistically enhance the antibiotic activity of oxacillin. The cellular membrane fluidity of