Experimental colitis can persist as a chronic disease, accompanied with an underlying risk of development into colorectal cancer. Metastasis-associated protein 1 (MTA1), as a chromatin modifier, exerts notable association with multiple diseases, including colitis. The current study aims to investigate the mechanism of MTA1/HIF1A/AQP4 axis in experimental colitis in mice. First, experimental colitis mouse models were established using dextran sulfate sodium (DSS) and in vitro colonic epithelial cells FHC inflammation models were with lipopolysaccharide (LPS) for determination of MTA1 and HIF1A expressions. It was found that MTA1 and HIF1A were both highly-expressed in experimental colitis samples. Results of dual-luciferase reporter gene assay and ChIP assay further revealed that MTA1 activated HIF1A, and subsequently induced AQP4 transcription to up-regulate AQP4 in experimental colitis. Following loss- and gain-function, the effects of MTA1/HIF1A/AQP4 axis on apoptosis and viability of colon epithelial cells were detected by a combination of TUNEL staining and flow cytometry, and CCK-8 assay. It was observed that silencing of MAT1 in the FHC and NCM460 cells reduced IL-1β and TNF-α expressions induced by LPS. Meanwhile, AQP4 promoted LPS-induced inflammation, and exacerbated apoptosis of colon epithelial cells and augmented experimental colitis development in mice. In vivo experiments further verified that TGN-020 treatment effectively alleviated DSS-induced experimental colitis in mice and diminished apoptosis of colon epithelial cells. Altogether, MTA1 may promote AQP4 transcription by activating HIF1A, thus exacerbating DSS-induced experimental colitis in mice, which provides a novel direction for the treatment of experimental colitis. Acute and chronic colitis are episodic, inflammatory conditions that affect millions of people across the globe . Moreove2 was previously uncovered to elevate the levels of aquaporin 4 (AQP4) in human placenta [Metastasis-associated protein 1 (MTA1) is well-established as a chromatin modifier with notable involvement in the prognoses of numerous malignancies . What’s placenta . Aquaporplacenta . As prevplacenta . In lighAfter experimental colitis was induced in mice with 2% DSS, results illustrated a notable reduction in weight of mice Fig. , accompaWe analyzed the experimental colitis gene expression dataset GSE53835 and found that MTA1 was highly-expressed in experimental colitis Fig. . mRNA anNext, we adopted sh-MTA1#1 and sh-MTA1#2 to silence MTA1 in the human normal colon epithelial FHC cells. The two independent short hairpin RNAs (shRNAs) achieved good silencing efficiency, while MTA1#1 exhibited superior silencing efficiency, and thus was adopted for subsequent experimentation Fig. . FollowiFurthermore, analyses of GSE53835 indicated that HIF1A was overexpressed Fig. , while cWe subsequently analyzed interaction network of AQP4 using GeneCards database, and identified 25 interacting genes Fig. . With KOIn addition, LPS treatment up-regulated AQP4 mRNA and protein levels Fig. . ThereafExperimental mice presented with increased DSS-induced apoptosis Fig. . SubsequFollowing DSS treatment on TGN-020-treated mice, TGN-020 alleviated weight loss and colon shortening of mice after DSS treatment Fig. . MeanwhiInitial experimentation illustrated that MTA1 was highly-expressed in DSS-induced experimental colitis mouse models. Despite the lack of research regarding the role of MTA1 in experimental colitis, a number of studies have documented the involvement of MTA1 in colorectal cancer and inflammatory responses. For instance, MTA1, as a direct target of miR-421, was previously illustrated to manipulate the cell progression of colorectal cancer . Moreovevia up-regulating HIF1A. Similarly, numerous studies have come across interaction between MTA1 and HIF1A under various conditions. One particular study identified that up-regulation of MTA1 elevated HIF1A in trophoblasts under hypoxic conditions, and could further serve as mediator of trophoblast function as well as differentiation in an early pregnancy [In addition, MTA1 facilitated experimental colitis regnancy . Furtherregnancy . What’s regnancy . Moreoveregnancy . The Huaregnancy , 27. Bairegnancy . Strikinregnancy . Meanwhiregnancy .Furthermore, another vital discovery was that HIF1A induced transcription of AQP4, which exacerbated the habitual occurrence of experimental colitis. Thanks to the hard-done work of our peers, the role of AQP4 in experimental colitis is well-established. For instance, AQP4 was previously identified as a significantly up-regulated gene in experimental colitis . MoreoveIn light of the aforementioned findings, MTA1 can facilitate the binding of HIF1A in the promoter region of AQP4, which augments expression of AQP4, and thus promotes epithelial cell inflammation and apoptosis in experimental colitis Fig. .Fig. 7ThThe current study was conducted with the approval of the Ethics committee of the Northern Jiangsu People’s Hospital. Animal experimentation protocols were in compliance with the Guidelines for the care and use of laboratory animals issued by the National Institutes of Health.www.atcc.org). In addition, human colonic epithelial cell line NCM460 was obtained from CGMCC. The obtained HEK293T and NCM460 cells were then cultured in DMEM, and the FHC cells were cultured with RPMI-1640 (Gibco) at 37 °C with 5% CO2 in air.Human embryonic kidney cells HEK293T and human normal colonic epithelial cells FHC cells were procured from the ATCC , shMTA1#1, shMTA1#2, shAQP4#1, shAQP4#2, shHIF1A#1, shHIF1A#2. The AQP4 overexpression plasmid was obtained from Addgene .Afterward, FHC and NCM460 cells were treated with 1 μg/mL LPS  for 24 h to induce an in vitro inflammatory response .7 infectious particles/mL virus. For further amplification, the obtained virus supernatant was adopted to infect the cells in the 100-mm culture disk, and the virus was isolated for subsequent infection of HEK293T cells in a 150-mm culture disk to prepare an appropriate concentration of the virus. Next, 15% CsCl and 40% CsCl were added to a Beckman centrifuge tube to prepare the CsCl gradient solution. Afterward, the virus supernatant enriched with virus particles was added to the CsCl gradient solution. Ultracentrifugation was then performed at 30000 rpm at 4 °C for 16 h. Two bands were identified after centrifugation. The band presenting with the higher position and weaker color was regarded the primarily empty adenovirus shells, which did not have the ability to infect; the required band with lower position and brighter color containing live virus particles was isolated with a 16 needle. Dialysis was subsequently performed in tris-buffered saline (TBS) for 1 h, followed by two regimens of dialysis in TBS containing 10% glycerol, for 1 h each time. The purified adenovirus was aliquoted into Eppendorf tubes. In the Eppendorf biophotometer, 1 μg viral protein was found to be equivalent to 4 × 109 virus particles. Finally, the collected samples were stored at −80 °C.Prior to transfection, the target plasmid was treated with Pac I . Subsequently, plasmids treated with 6 μg Pac I were transfected into HEK293T cells with PEI or other transfection reagents. After 8 h, 4 mL DMEM  was added. The cells were then scraped from the bottle and transferred into a 50-mL conical tube 7 to 10 days after transfection. Following centrifugation, the cells were resuspended with 2.0 mL PBS. Afterward, the cells were preserved in liquid nitrogen, dissolved in a 37 °C water bath, and shaken vigorously. The preceding steps were performed 4 times. Later, the HEK293T cells were placed in a 60-mm culture disk with 50–70% cell confluence, and the virus-contained supernatant was added at a volume ratio of 30–50%. Significant cell lysing or CPE was evident 2–3 days after infection. When 1/3rd to 1/2 of the cells were free and floating, the virus was isolated 3–5 days after infection. The presence of recombinant adenovirus was subsequently validated by western blot analysis or by the addition of 5 μL viral supernatant with 10 μL PCR-grade Protease K, digestion at 55 °C for 1 h, boiling for 5 min, centrifugation, and PCR (1–2 mL). Viral supernatant was collected with a minimum concentration of 1 × 10Forty female BALB/c mice (aged 5–6 weeks) were provided by Shanghai SLAC Laboratory Animal Co., Ltd. . After 5 days of acclimatization, the mice were fed with (1) normal water, (2) 2% DSS solution, (3) dimethyl sulfoxide (DMSO), (4) DMSO + 2% DSS (fed with DMSO containing 2% DSS), and (5) TGN-020 + 2% DSS  once daily for a period of 7 days, following which the alternations in the weight of mice were continuously observed and recorded. After 30 days, the mice were euthanized and the colon tissues were harvested for subsequent analyses. The investigator was blinded to the group allocation during the experiment and/or when assessing the outcomes.First, 2 cm severe inflammation colon tissue samples were fixed with 10% formaldehyde solution for 24 h, paraffin-embedded and sectioned (5 μm). After dewaxing with xylene, the sections were dehydrated with gradient alcohol, and then immersed in hematoxylin solution for 5 min, in 1% hydrochloric acid alcohol for 30 s, and in 0.5% eosin solution for 5 min, and finally dehydrated with gradient alcohol. Afterward, the sections were cleared, sealed, and observed under a microscope .Two centimeters of severe inflammatory colon tissue samples were fixed in 10% formalin solution, paraffin-embedded, and sectioned (5 μm). Immunohistochemistry was performed as previously described . After c−△△Ct method.Total RNA content was extracted with instructions of TRIzol  kit, followed by determination of RNA concentration. Reverse transcription was performed using cDNA reverse transcription kits . Fluorescent quantitative PCR was carried out with a fluorescence quantitative PCR instrument . The primers were synthesized by Sangon Biotechnology   was added for ultrasonic lysing. Subsequently, bicinchoninic acid protein quantitative kits (Boster) were adopted to determine protein concentration. Next, the proteins were separated by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane. After blocking, diluted primary antibodies against AQP4 , HIF1A , MTA1 , and GAPDH  were subsequently used to probe the membrane at 4 °C overnight. The following day, the membrane was re-probed with the HRP-labeled secondary antibody  for 1 h. Enhanced chemiluminescence working solution  was added for culture for 1 min. Later, the membrane was placed in the Bio-rad automatic imager for exposure. Image Lab software was applied to quantify the gray levels.Following restriction endonuclease digestion, the AQP4 fragment containing HIF1A WT or MUT binding sites was inserted into the pGL3 vector reporter plasmid with the help of T4 DNA ligase to obtain pGL3-AQP4-WT or pGL3-AQP4-MUT. HIF1A overexpression vector or control vector was, respectively, co-transfected in cells for 36 h. Subsequently, the growth medium was completely aspirated, followed by the addition of 1 × passive lysis buffer for complete lysis. A dual-luciferase reporter system  and luciferase substrate luminescent detector were adopted for detection. All steps were carried out in strict accordance with the product manual and the recommended procedures in the operation manual.The cells were fixed with 1% formaldehyde at 37 °C for 10 min, and then rinsed twice with cold phosphate containing several protease inhibitors . Subsequently, the cells were incubated in pH 8.1 lysis buffer comprising of 1% SDS, 10 mM ethylene diamine tetraacetic acid (EDTA), and 50 mM Tris-HCl for 10 min, after which the genomic DNA was sliced using ultrasound. Following 10-min centrifugation of lysate at 13,000 r/min and 4 °C, the supernatant was diluted with a variety of reagents, including 0.01% SDS, 1% Triton X-100, 2 mM EDTA, 16.7 mM Tris-HCl, 167 mM NaCl (pH 8.1) and protease inhibitor. Anti-HIF1A  antibody was then supplemented to the lysate and cultured overnight at 4 °C. The following day, DNA fragments were recovered and PCR amplification was performed with the specific primers to detect the degree of enrichment of AQP4 promoter fragment binding to HIF1A.In strict accordance with the manufacturer’s instructions, the DeadEnd Colorimetric TUNEL System (Promega) was employed to evaluate the degree of apoptosis of colonic mucosal epithelial cells. Briefly, the DAB chromogenic solution was added to the samples for 3–6 min to facilitate color development. Following hematoxylin counterstaining, the cell apoptosis was observed under an optical microscope.3 cells/well. In compliance with the provided instructions, the CCK-8 reagent  was adopted to measure the cell survival rate at 48 h. The optical density (OD) value was documented with an ELISA instrument  to calculate the relative cell survival rate.FHC cells were seeded in 96-well plates, at 1 × 105 treated FHC cells were attained. Apoptosis was subsequently detected using an Annexin V-fluorescein isothiocyanate/propidium iodide (PI) apoptosis detection kit , and the stained cells were identified with a flow cytometer. Annexin V-positive and PI-negative cells were regarded as apoptotic cells. The apoptosis rate was calculated using FlowJo software .Briefly, 1 × 10t test, and those among multiple groups by one-way ANOVA or repeated measures of ANOVA, followed by Tukey’s post hoc tests. p < 0.05 was regarded as statistically significant.Data analyses were performed using SPSS 21.0. Measurement data were summarized as mean ± standard deviation. Data between two groups were compared by unpaired Figrue S1Figrue S2Figrue S3Figrue S4Figrue S5Supplementary Figure LegendsSupplementary Tablesauthor-contribution-form


Chiliadenus montanus  Brullo. (Asteraceae), a medicinal plant known for producing volatile organic compounds (VOCs). Callus induction was achieved successfully by culturing leaf explants on full-strength Murashige and Skoog medium (MS) supplemented with 2.2 µM 2, 4-dichlorophenoxy acetic acid  and 6.9 µM kinetin (Kin). Successful direct shoot regeneration was achieved using nodal explants cultured onto half-strength MS media supplemented with 1.4 μM Gibberellic Acid (GA3) and 4.4 μM 6-Benzylaminopurine (BAP). Indirect microshoots were successfully regenerated using callus cultured on MS media supplemented with 8.8 μM BAP, 2.2 μM Zeatin, and 1.4 μM GA3 followed by culturing on MS media supplemented with 8.8 μM BAP and 0.5 μM naphthalene acetic acid (NAA). Using wild plant aerial parts, callus and microshoots samples, VOCs were extracted successfully using Headspace Solid-Phase Micro-Extraction (HS-SPME) and analyzed by gas chromatography–mass spectrometry (GC-MS). In wild plant extracts, sesquiterpene hydrocarbons were found to be predominant with the following principal components: Alloaromadendrene (11.92%), trans-Cadina-1(6),4-diene (7.54%), and α-caryophyllene (6.77%). The analysis of in vitro microshoots revealed high levels of oxygenated monoterpenes with cis-Myrtanol (16.62%), and β-Cyclocitral (14.3%) as the main components. Callus extract was dominated by monoterpene hydrocarbons and the main compounds identified were (Z)-β-Ocimene (22.27%), p-Cymene (15.13%), and α-pinene (13.78%). In conclusion, an efficient in vitro production system of VOCs in C. montanus was established that can be used in the future for boosting their production without endangering wild plants.Callus and microshoot cultures were established for  Chiliadenus is a small genus that belongs to the Asteraceae family and it consists of ten species distributed along the Mediterranean Sea [Chiliadenus montanus (Vahl.) Brullo  Boiss.) is an aromatic medicinal plant that is mainly distributed in rocks, steppes, and desert areas in the eastern parts of the Mediterranean region [Chiliadenus montanus is described as an herbaceous plant, almost 50 cm in height with yellow discoid heads, hermaphrodite flowers, and hairy alternate leaves found on small lateral shoots [The family Asteraceae is one of the largest families of flowering plants and it includes over 1600 genera and 25,000 species of herbs, shrubs, and trees [nean Sea . Chiliadn region . ChiliadChiliadenus montanus is commonly used as a traditional medicinal plant, popularly known as Haneida, and it is used in folk medicine to treat several diseases and disorders [C. montanus have been widely used by Bedouins as an herbal tea to treat renal troubles, diarrhea, stomachaches, and chest diseases [C. montanus identified monoterpenes, sesquiterpenes, diterpenes, triterpenes, sterols, and flavonoids as major compounds in its natural extracts [C. montanus is considered one of the most valuable medicinal plants in the region and it has great potential as a therapeutic agent against multiple diseases [isorders . The dridiseases ,9. Phytoextracts ,11,12,13extracts . Furtherextracts ,16, antiextracts , antichoextracts , and antextracts  as well extracts  and antiextracts ,22. Therdiseases .C. montanus plant is considered a vulnerable neglected herb that grows in marginal habitats and it is subjected to threats of extinction due to overgrazing, urbanization, and climate change [C. montanus was reported previously and was associated with limited seed viability and production because it only blooms for two months during the growing season [C. montanus plant and the utilization of such systems for the production of secondary metabolites have not been reported. Hence, a micropropagation protocol for the C. montanus plant through in vitro techniques is considered a promising approach for its multiplication and conservation and the ex-situ production of volatile organic compounds (VOCs). The e change ,24. Addie change . Sexual g season . To dateC. montanus using different explant types, media compositions, and growth conditions and to identify VOCs produced from C. montanus callus and microshoot cultures. Moreover, in vitro cultures of C. montanus were evaluated for their VOCs contents and compositions using the headspace solid-phase microextraction (HS-SPME) technique coupled with gas chromatography-mass spectrometry (GC-MS) analysis.The main objectives of this study were to establish in vitro tissue cultures for C. montanus wild plant was identified in the Al-Kamsheh region in Zarqa governorate, Jordan  (A 22.1″ E) . The whoC. montanus were excised from greenhouse plants and used as explants. Initially, the explants were washed under running tap water to remove dirt particles and then treated with 70% (v/v) ethanol for 30 seconds under aseptic conditions before washing thoroughly three times with sterilized distilled water. The explants were then washed with 1% (v/v) sodium hypochlorite solution containing a few drops of Tween® 20 for one minute and then rinsed six times with sterilized distilled water and dried on sterile paper before their use. For callus induction, the disinfected explants were cultured on Murashige and Skoog (MS) media [−1 myo-inositol, and different concentrations of either naphthalene acetic acid (NAA)  or 2,4-dichlorophenoxy acetic acid   as auxin sources in combination with either 6-Benzylaminopurine (BAP)  or kinetin (Kin)  as cytokinin sources and the pH of the media was adjusted to 5.8 before adding 6 g/lagar  as described previously [−2 and 25 mL was dispensed into Petri dishes. The disinfected explants were placed horizontally on the media and incubated for eight weeks at 25 ± 2 °C under complete darkness. For the establishment of callus culture, leaves of S) media  supplemeeviously . The medA Complete Randomized Design (CRD) was used and each treatment was replicated five times (each replicate consisted of two explants in a single Petri dish), and the cultures monitored for callus development. At the end of the incubation period, data were collected for callus diameter, fresh weight, texture, and color. Data were analyzed using SAS ; an analysis of variance (ANOVA) was obtained for each experiment, and mean separation at a probability level of 0.05 was performed according to Tukey’s HSD test. The source of all growth regulators used in this study was Sigma-Aldrich Chemical Co., St. Louis, MO, USA. The best callus induction media was used for callus maintenance that was based on the best callus growth, in terms of increase in fresh weight and morphological changes, and was named thereafter as the callus maintenance medium with a subculture event every four weeks.C. montanus microshoots, nodal segments were excised from in vivo shoots and used as explants. For this purpose, shoots were cut into small segments (3 cm in length) that contained two buds per segment. The nodal explants were then sterilized as described above. The disinfected explants were then cultured on different media including full-strength MS media supplemented with 0.1 M sucrose and 100 mg·L−1 myo-inositol, half-strength White media [−1 myo-inositol and half-strength MS media supplemented with 0.05 M sucrose, 100 mg·L−1 myo-inositol and 4.4 µM BAP in combination with 1.4 µM Gibberellic Acid (GA3). The explants were placed vertically inside an Erlenmeyer flask (250 mL) containing 150 mL of media and then incubated for four weeks at 25 ± 2 °C under a 16 h photoperiod and irradiance of 56 μmol·m−2·s−1 provided by cool white fluorescent lamps . Each treatment was replicated 10 times (each replicate was represented by one explant in a flask) and at the end of the incubation period, data were collected for the number of leaves per shoot and final shoot length.For the direct regeneration of te media  supplemeFor rooting, microshoots (~1 cm length) regenerated directly from nodal explants were cut off and transferred into half-strength MS media supplemented with different concentrations of auxins: indole acetic acid (IAA) , NAA , or indole butyric acid (IBA) . The treatments were arranged in CRD with 10 replicates per treatment with one microshoot per replicate. The percentage of root induction, root numbers, and the growth state of the roots were recorded after four weeks of culturing.−1 myo-inositol, and a combination of 8.8 μM BAP, 2.2 μM Zeatin, and 1.4 μM GA3. In vitro microshoots were regenerated from calli after four weeks of culture and the microshoots were allowed to grow until they reached a length of one cm. Thereafter, individual microshoots were excised and cultured on MS media supplemented with 0.1 M sucrose and 100 mg·L−1 myo-inositol and different levels of BAP  or Kin  in combination with 0.5 μM NAA and the cultures were incubated for another four weeks at 25 ± 2 °C under a 16 h photoperiod. A CRD was used, and each treatment was replicated 10 times (each replicate was represented by a single microshoot), and at the end of the incubation period, data were collected for the number of leaves per shoot and shoot length. Data were analyzed statistically as described in callus culture experiments.For the establishment of microshoot cultures using callus pieces as explant, 0.5 g of callus was cultured on MS media supplemented with 0.1 M sucrose, 100 mg·LC. montanus were used for VOC analysis that included shoots  collected in September 2020 from the wild plant, one-month-old microshoots that were grown on half-strength MS media supplemented with 0.1 M sucrose, mg·L−1 myo-inositol, 4.4 µM BAP in combination with 1.4 µM GA3 and 0.5 µM NAA, and calli that were grown on callus maintenance medium under complete darkness for one month. Volatile organic compounds were extracted from the three samples by using the headspace solid-phase microextraction (HS-SPME) technique coupled with gas chromatography-mass spectrometry (GC-MS), as described previously [n-hexane  with sonication  for 10 min. The extracts were then filtered and dried with sodium sulfate anhydrous (Na2SO4)  and then transferred to a 10 mL uncapped headspace glass vial  and left for 30 min to fully evaporate the n-hexane. Thereafter, the vials were capped and incubated at 50.0 °C for 0.5 min, where a 1 cm 50/30 µm DVB/CAR/PDMS coated SPME fiber  was exposed to the headspace for 30 min to achieve equilibrium. Thereafter, the VOCs were desorbed from the SPME fiber and GC-MS analyses were carried out using Varian chrompack CP-3800 C/MS/MS-200  fitted with a DP-5  GC capillary column (30 m × 0.25 mm × 0.25 μm film thicknesses) as described previously [Three samples of eviously ,31 with eviously . Peak ideviously . The VOCC. montanus. Callus induction in Ruta graveolens L. was successfully achieved on MS media supplemented with 5 µM 2,4-D and 7 µM Kin [Elephantopus scaber plant that belongs to the Asteraceae family [Weak callus induction was observed on MS media lacking plant growth regulators and clear variations were observed between tested media types supplemented with different combinations of auxin and cytokinin types and concentrations . Murashi7 µM Kin  and the e family . 3 proved to be superior when compared to half-strength White media and hormone-free MS media , another on MS media supplemented with 10.7 µM NAA (10%), and the last one on MS media supplemented with 3.9 µM IBA (10%) b. Simila3 , trans-Cadina-1(6),4-diene (7.54%), and α-caryophyllene (6.77%) from the sesquiterpene hydrocarbons group, while from the oxygenated sesquiterpenes group, Muurola-4,10(14)-dien-8.α-ol (22.19%) and δ-amorphene (9.32%) were the most abundant compounds  that was identified in the C. montanus microshoot and callus extracts  of this study. Finally, the qualitative and quantitative variations in VOCs between intact plants and in vitro cultures were reported previously in Artemisia spicigera in vitro cultures [C. montanus samples used in this study could be attributed to environmental factors affecting their production in natural habitats such as light, temperature, water, and nutrients. For most plants, while under in vitro conditions, such factors are controlled continuously and adjusting any individual factor will result in altering their levels [The composition of the microshoot, wild, and callus VOCs showed qualitative and quantitative differences. For instance, the number of different constituents identified in microshoot extract was higher compared to the wild plant and in vitro callus, which produced the lowest number of compounds . In addis plants  and othes plants ,46,47,48lanticus . Ref. [4lanticus  reportedcultures  which isC. montanus were successfully established using different types of explants and media compositions. Callus culture was successfully established using leaf explants cultured on full-strength MS media supplemented with 2.2 µM 2,4-D and 6.9 µM kinetin Kin. Microshoots were successfully regenerated directly from nodal explants cultured onto half-strength MS media supplemented with 1.4 μM GA3 and 4.4 μM BAP, while indirect microshoots were regenerated using callus explant cultured on MS media supplemented with 8.8 μM BAP, 2.2 μM Zeatin, and 1.4 μM GA3. The VOCs from in vivo and in vitro samples were extracted successfully using HS-SPME and analyzed using GC-MS. Clear variations in the qualitative and quantitative aspects of VOCs between the wild plant and in vitro cultures of C. montanus were observed. Callus cultures were found to be rich in monoterpene hydrocarbons, while microshoot cultures were rich in oxygenated monoterpenes when compared to the wild plant extract that was rich in sesquiterpenes. In conclusion, the in vitro cultures of C. montanus reported in this study can be used to boost the production of VOCs under aseptic conditions without affecting wild-grown plants.In this study, efficient micropropagation and in vitro culture systems for


When exposed to varying Na+-containing diets, no changes in Na+ and K+ handling and only minor changes in the expression of Na+ and K+ transporting protein were found in both models. Similarly, the α- or γENaC subunit cleavage pattern did not differ from control mice. On standard and low Na+ diet, cat/+Prss8 and cat/catPrss8 mice exhibited standard plasma aldosterone levels and unchanged amiloride-sensitive rectal potential difference indicating adapted ENaC activity. Upon Na+ deprivation, mice lacking the renal CAP1/Prss8 expression (Prss8PaxLC1) exhibit significantly decreased plasma aldosterone and lower K+ levels but compensate by showing significantly higher plasma renin activity. Our data clearly demonstrated that the catalytic activity of CAP1/Prss8 is dispensable for proteolytic ENaC activation. CAP1/Prss8-deficiency uncoupled ENaC activation from its aldosterone dependence, but Na+ homeostasis is maintained through alternative pathways. The serine protease prostasin  is a confirmed in vitro and in vivo activator of the epithelial sodium channel ENaC. To test whether proteolytic activity or CAP1/Prss8 abundance itself are required for ENaC activation in the kidney, we studied animals either hetero- or homozygous mutant at serine 238 (S238A;  PaxLC1) mice were subjected to a low Na+ diet (0.01%Na+) for 10 days. Following day 3 to day 10, mice received each day 10mg/kgBW of losartan  in drinking water. Blood was collected at the end of the experiment. Plasma aldosterone was measured by the Service of Nephrology, Lausanne University Hospital (CHUV), Lausanne, Switzerland [To block AT1R, control and knockout  was made. For the Prss8 control and knockout mice, quantification of Western blot analyses and plasma electrolytes were analyzed using an unpaired t-test and a one-way ANOVA with a Tukey test to compare +/+Prss8, cat/+Prss8 and cat/catPrss8 mice. Results are represented as mean ± SEM or ± SD and a p value < 0.05 was considered statistically significant; * p < 0.05; ** p < 0.01, *** p < 0.001.All statistical analyses were performed using GraphPad Prism and presented as scatter plot. To evaluate changes in the body weight of the mice, the water and food intake, the urine and feces excretion, the urinary electrolytes, the 24 h Na


The enriched pathways (FDR p < 10−300) included D-arginine and D-ornithine metabolism, glycolysis, oxidation and degradation of branched chain fatty acids, and sphingolipid metabolism. MRS showed good discriminative power between the CHD cases and the controls (AUC = 0.99). In this first study in the Middle East, known and novel circulating metabolites and metabolic pathways associated with CHD were identified. A small panel of metabolites can efficiently discriminate CHD cases and controls and therefore can be used as a diagnostic/predictive tool.Coronary heart disease (CHD) is a major cause of death in Middle Eastern (ME) populations, with current studies of the metabolic fingerprints of CHD lacking in diversity. Identification of specific biomarkers to uncover potential mechanisms for developing predictive models and targeted therapies for CHD is urgently needed for the least-studied ME populations. A case-control study was carried out in a cohort of 1001 CHD patients and 2999 controls. Untargeted metabolomics was used, generating 1159 metabolites. Univariate and pathway enrichment analyses were performed to understand functional changes in CHD. A metabolite risk score (MRS) was developed to assess the predictive performance of CHD using multivariate analysis and machine learning. A total of 511 metabolites were significantly different between the CHD patients and the controls (FDR  Coronary heart disease (CHD) is a major cause of mortality worldwide . Many riChanges in metabolism could both lead to CHD and be a consequence of the disease itself . At the CHD patients usually have comorbidities that accelerate the disease progression. Many of these are diseases of metabolic dysfunction, such as diabetes mellitus, dyslipidemia, and obesity, which impact many body organs and tissues, including the liver, skeletal muscle, vasculature, and myocardium ,8,9. ThePrevious studies have identified novel CHD risk factors using metabolomic data, but this has mainly been in individuals of European ancestries ,11,12, ap < 0.05). Most metabolites  were elevated in the CHD cases, including amino acids, carbohydrates, energy-related metabolites, nucleotides, peptides, and xenobiotics  = 760.0); glycolysis (ER = 572.2); arginine and proline metabolism (ER = 441.3); oxidation of branched chain fatty acids (ER = 429.3); sphingolipid metabolism (ER = 341.8); and valine, leucine, and isoleucine degradation (ER = 328.1). In the following sections, pathways of relevance in CHD have been highlighted.The results of the metabolic pathway enrichment analysis are shown in p = 2.88 × 10−82) and higher concentrations of both ornithine (p = 1.04 × 10−152) and total dimethylarginine   A. L-argi× 10−35) D–G. In t× 10−35) F,G.p = 3.12 × 10−28 and 6.74 × 10−7, respectively; p = 0.72), but female controls had higher concentrations of arginine than male controls (p = 3.38 × 10−11). Females in both the CHD and the control groups had higher concentrations of dimethylarginine than males , most likely because of the high data variability and small sample size. The opposite trend was observed in the controls at statistical significance (p = 2.32 × 10−10). This supports the hypothesis of the lower production of NO in CHD patients with T2D by inhibiting nitric oxide synthase. The OAR was 0.88 in controls without T2D, 0.98 in controls with T2D, 1.96 in CHD cases without T2D, and 1.92 in CHD cases with T2D , we observed that concentrations of arginine and ornithine were significantly lower in subjects with T2D compared with subjects without T2D C. This iwith T2D C. These Increased acylcarnitines and decreased long-chain fatty acids in CHD: All significant acylcarnitines were elevated in the CHD patients: oleoylcarnitine (C18:1) (FDR p = 3.71 × 10−108), stearoylcarnitine (C18) (FDR p = 3.18 × 10−53), propionylcarnitine (C3) (FDR p = 3.31 × 10−44), decanoylcarnitine (10) (FDR p = 3.01 × 10−19), and deoxycarnitine (FDR p = 2.58 × 10−10) (× 10−10) . The mos× 10−10) . This maElevated branched chain amino acids (BCAAs): BCAAs, i.e., leucine, isoleucine, and valine, were significantly elevated in the CHD patients  (FDR p = 4.79 × 10−142) and sphingosine (FDR p = 8.33 × 10−102) ( 10−102) .Sugar metabolism: All six “glycolysis, gluconeogenesis, and pyruvate metabolism” metabolites were significantly different, with FDR p ≤ 8.25 × 10−8 (p = 3.43 × 10−135), pyruvate (p = 3.03 × 10−93), and glucose (p = 6.14 × 10−10) were higher in cases than controls , but the remaining metabolites remained very significant .5 × 10−8 . Lactatecontrols . After ap = 1.53 × 10−24; p = 2.81 × 10−21; The random forest (RF) models on the test set yielded area under the receiver operator curve (AUC) values of 0.97 and 0.98 using the full model, i.e., A (641 metabolites), and the reduced model, i.e., B (17 metabolites), respectively. The accuracies of models A and B were 98.5% and 98.7%, respectively. The mean decrease in accuracy (variable importance) of the random forest model trained on all metabolites is shown in This is the first study that has investigated the metabolic profile and its impact on CHD in a large case-control cohort from the Middle East, using an untargeted metabolomics approach. Our results provide strong evidence that many circulating metabolites and metabolic pathways are altered in CHD patients. The results also showed that a small panel of metabolites can efficiently discriminate CHD cases and controls and can therefore be used as a diagnostic/predictive tool. In this study, we confirm known metabolites that are associated with CHD, identify novel ones, and provide insights into some underlying biological mechanisms for CHD.Ornithine and arginine were significantly associated with CHD. The ornithine concentration in the CHD patients was twice the concentration of arginine, unlike in the controls, in whom arginine was higher. As arginine is the only physiological substrate for NOS-mediated generation of NO ,17, our Our analysis has revealed increased concentrations of acylcarnitines, which is associated with many heart problems such as CHD ,27,28. LBCAAs were higher in CHD patients as well. These are the main source of nitrogen for production of glutamine and alanine in muscles. In myocardial ischemia, an important oxidative energy substrate of the heart may be BCAAs, which are produced by muscle protein mobilization . AccretiOur CHD patients were found to have higher concentrations of sphingolipids than the controls. Sphingolipids were recently identified as cholesterol-independent biomarkers of CHD , and chaSugar metabolism was also increased in the CHD patients. Cardiac contractility requires a constant supply of adenosine triphosphate (ATP). Under normal conditions, the high demand of ATP is primarily satisfied by fatty acid oxidation (FAO), with a small contribution from glucose metabolism . Under sEfforts have been made to develop clinical data-based predictive models, but the accuracy of these models in CHD is <75% . We havep < 2.2 × 10−16; The study cohort comprised 1001 CHD patients and 2999 control subjects . The CHDSerum metabolites for both the cases and the controls were jointly quantified by untargeted, ultrahigh-performance liquid chromatography-tandem mass spectroscopy (UPLC-MS/MS) and curated by Metabolon Inc. Samples were run in analytical plates containing 144 experimental samples per plate (batch). The data were normalized across batches to generate batch-normalized data and to correct for minor instrument technical variation from batch to batch. Each compound was corrected in instrument batch blocks by registering the medians of each batch to equal one and normalizing each data point proportionally. A total of 1159 metabolites were profiled within nine super pathways: lipid (30.1%), xenobiotics (21.1%), amino acid (17.3%), nucleotide (3.1%), peptide (2.9%), cofactors and vitamins (2.6%), carbohydrate (1.9%), partially characterized molecules (1.1%), energy (0.7%), and unnamed metabolites (19.2%). Unnamed metabolites were removed, leaving 937 metabolites for analysis.Quality control steps were conducted to ensure data quality. Samples and metabolites with more than 80% missing data were removed based on the criteria suggested by Wei et al. . This lehttps://www.R-project.org/) adjusting for age, sex, and BMI to mitigate the correlations between metabolites and age, sex, and BMI. FDR corrected p-values (p) from the models were used to identify significantly different metabolites between the CHD cases and the controls, and the effect size was used to identify the direction of the changes in the metabolite concentrations with respect to disease status. A metabolite has a positive effect size if its concentration is higher in CHD patients compared with controls.Individual metabolite differences between the CHD patients and the controls were tested using logistic regression in R . Pathview annotated the KEGG pathway maps [Pathway enrichment analysis provides mechanistic insight into gene lists generated from genome-scale (omics) experiments. This method identifies biological pathways that are more enriched in a metabolite list than would be expected by chance. Pathway enrichment analysis was performed using MetaboanalystR 3.0 . Comma-sway maps  with norrandomforest R package [caret R package [ROCR package in R [glmnet R package [A random forest model was developed using the  package  to asses package . AUCs weage in R . The kne package . We fit Our study has focused on a cohort from an underrepresented population, with subjects representing the Middle East and Gulf region. Similar studies should be carried on new cohorts to replicate our findings and strengthen our conclusions. Overall, our study underscores the value of metabolomics in exploring biomarkers and biological mechanisms to identify potential therapeutic targets for treating CHD.


There were errors made in the original article . The autCorrections have been made as follows:Abstract: “The synergy of tigecycline combined with amikacin or gentamicin was 8.2%” has been corrected to: “Synergistic activity was observed in 8.2% of isolates for tigecycline combined with amikacin or gentamicin”.1. Introduction, paragraph 3: “The dose of antibiotics may be divided into carbapenem-based regimens (when meropenem MICs ≤ 8 µg/mL), colistin-based regimens, and tigecycline-based regimens, whereas aminoglycosides may be often used as adjunctive antibiotics [14]” has been corrected to: “The antibiotic regimens may be divided into carbapenem-based regimens (when meropenem MICs ≤ 8 µg/mL), colistin-based regimens, and tigecycline-based regimens, whereas aminoglycosides may be often used as adjunctive antibiotics [14]”.2. Introduction, paragraph 4: “Therefore, high doses of tigecycline and/or the combination of tigecycline with other active agents may increase the plasma concentrations, leading to adequate bactericidal activity against CRE isolates [2]” has been corrected to: “Therefore, high doses of tigecycline may increase the plasma concentrations, leading to adequate bactericidal activity against CRE isolates [2]”.3. Materials and Methods, Section 2.2.1: “Furthermore, a purified single colony of isolated strains was picked up and emulsified to 0.9% in normal saline” has been corrected to: “Furthermore, a purified single colony of isolated strains was picked up and suspended to 0.9% in normal saline”.4. Materials and Methods, Section 2.2.2: “The tigecycline-based combination regimens consisted of tigecycline-amikacin and tigecycline-gentamicin. The microbroth checkerboard technique was used to evaluate the synergistic activity of each combination. The range of a series of two-fold dilutions was 0.125–8 μg/mL for tigecycline, 0.0625–64 μg/mL for amikacin, and 0.0078–8 μg/mL for gentamicin” has been corrected to: “The synergistic effect of antibiotic combinations including tigecycline-amikacin and tigecycline-gentamicin was determined using the checkerboard technique. The studied antibiotic concentrations of a series of two-fold dilutions were 0.125–8 µg/mL for tigecycline, 0.0625–64 µg/mL for amikacin, and 0.0078–8 µg/mL for gentamicin”.5. Materials and Methods, Section 2.4, paragraph 1: “A two-compartment model with the linear pharmacokinetic behavior of tigecycline, amikacin, and gentamicin was used to generate the relationship between drug concentration levels and time [32–34]” has been corrected to: “The relationship between drug concentration levels and time was generated using a two-compartment model for tigecycline and amikacin and a one-compartment model for gentamicin with the linear pharmacokinetic behavior [32–34]. For amikacin and gentamicin, creatinine clearance (CrCL) was included to simulate the plasma aminoglycoside concentration-time using the equations containing CrCL as covariate factor ”.6. Materials and Methods, Section 2.4, paragraph 3: “Details of the antibiotic dosing regimen are shown in Supplementary Materials Table S4” has been corrected to: “Details of the antibiotic dosing regimen are shown in Supplementary Materials S1 (Table S4)”.7. Materials and Methods, Section 2.4, a new paragraph 5 is added: “MCS is a mathematical technique to simulate 10,000 virtual patients based on mean pharmacokinetic parameters (drug clearance or volume of distribution) and their distribution (standard derivation) in the compartment models (one or two compartments) for computing the plasma concentration and time among individual virtual patients. The Cmax and calculated AUC values from the trapezoidal rule among simulated patients and the probability of target attainment (PTA) of the target PK/PD index were determined”.8. Materials and Methods, Section 2.4, new paragraph 6: “The optimal dosing regimens defined as the dosing regimens reached greater than 90% of PTA for documented therapy and greater than 90% of CFR for empirical therapy” has been corrected to: “The optimal dosing regimens defined as the dosing regimens of each antibiotic MIC or the MIC values from the synergistic study reached greater than 90% of PTA for documented therapy and greater than 90% of CFR for empirical therapy”.9. Results, Section 3.2, paragraph 2: The new sentence “Details of the MIC distribution of single or combined antibiotics are shown in Supplementary Materials S2.” has been added.10. Discussion, paragraph 3: “An earlier study showed aminoglycoside dosage regimens (ranged from 5 to 30 mg/kg) achieved the PTA target at MICs of 0.5 μg/mL with the corresponding target of Cmax/MIC ≥ 10 [45]” has been corrected to: “An earlier study showed aminoglycoside dosage regimens (ranged from 5 to 30 mg/kg) achieved the PTA target at MICs of 0.5 µg/mL with the corresponding target of Cmax/MIC ≥ 10 [33]”.11. Discussion, paragraph 4: “In prior studies, colistin resistance rates of CRKP isolates were only 25% [39], whereas colistin resistance rates of K. pneumoniae isolates ranged from 17.3% to 19% ” has been corrected to: “In prior studies, colistin resistance rates of CRKP isolates were only 25% [39], whereas colistin resistance rates of K. pneumoniae isolates ranged from 17.3% to 76.1% ”.12. Discussion, paragraph 6: “Previous studies also reported tigecycline plus amikacin or gentamicin was effective against K. pneumoniae isolates. For tigecycline plus amikacin, one study showed synergism was 36.4%, whereas another study found synergism was 70% ” has been corrected to: “Related studies also reported tigecycline plus amikacin or gentamicin was effective against K. pneumoniae isolates. For tigecycline plus amikacin, one study showed the synergistic effect was 36.4%, whereas another study found the synergistic effect was 70% ”.13. Discussion, paragraph 8: “After that, mistranslation of amino acids occurs, leading to damage to the bacterial cytoplasmic membrane and interfering with protein synthesis. These protein errors also lead to the reduction in β-lactamase expression. Therefore, both antibiotics likely enhance their target sites, contributing to synergistic effects ” has been corrected to: “Aminoglycosides lead to mistranslated amino acids, in turn leading to damage to the bacterial cytoplasmic membrane and interfering with protein synthesis; these protein errors also lead to reduced β-lactamase expression from aminoglycoside activities. Therefore, aminoglycosides likely enhance combined antibiotics to the target sites, contributing to synergistic effects ”.14. Supplementary Materials: Supplementary Materials S2 were omitted from the previous version. This section has been corrected to: “The following are available online at https://www.mdpi.com/article/10.3390/antibiotics10060736/s1, Supplementary Materials S1: Table S1: A set of parameters of tigecycline; Table S2: A set of parameters and an equation of amikacin; Table S3: A set of parameters and an equation of gentamicin; Table S4: Antibiotic dosing regimens for simulation; Supplementary Materials S2: MIC distribution of single and combined antibiotics”.15. References: Reference 45 was incorrect. It has been replaced with: “45. Eiamphungporn, W.; Yainoy, S.; Jumderm, C.; Tan-Arsuwongkul, R.; Tiengrim, S.; Thamlikitkul, V. Prevalence of the colistin resistance gene mcr-1 in colistin-resistant Escherichia coli and Klebsiella pneumoniae isolated from humans in Thailand. J. Global Antimicrob. Resist.2018, 15, 32–35”.16. The authors apologize for any inconvenience caused and state that the scientific conclusions are unaffected. The original article has been updated.


Acetylcholine is one of the most important endogenous neurotransmitters in a range of organisms spanning different animal phyla. Within pituitary gland it acts as autocrine and paracrine signal. In a current study we assessed expression profile of the different subunits of nicotinic as well as muscarinic acetylcholine receptors in RC-4BC cells, which are derived from rat pituitary gland tumor. Our findings indicate that β2, δ, and M2 subunits are expressed by the cells with the lowest Ct values compared to other tested subunits. The detected Ct values were 26.6±0.16, 27.95±0.5, and 28.8±0.25 for β2, δ, and M2 subunits, respectively. Endogenous neurotransmitter acetylcholine (ACh) acts on nicotinic receptors as well as on muscarinic receptors. Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels, with diverse structure and function. Each subunit bypasses plasma membrane four times, has extracellular N- and C-termini, with ligand-binding site at the N-terminus . There aq/11, with activation of phospholipase C [Muscarinic acetylcholine receptors (mAChRs) are seven-transmembrane domain G-protein coupled receptors. There are five receptor subtypes identified, named M1 –M5. M1, M3, and M5 receptors are coupled to Glipase C . ActivatIn situ hybridization study demonstrated expression of α9 nAChR subunit in pars tuberalis of rat pituitary [Pituitary gland is divided into two functional parts: anterior and posterior pituitary. While posterior pituitary releases hormones into the bloodstream directly, being a continuation of hypothalamus, anterior pituitary is connected with hypothalamus via network of blood vessels, and itself is a gland that contains multiple types of cells releasing multitude of hormones into the bloodstream. The cells within anterior pituitary are specialized into five endocrine cell types differing by the secreted hormone. These cell types include gonadotrophs releasing luteinizing hormone (LH) and follicle stimulating hormone (FSH), thyrotrophs releasing thyroid stimulating hormone (TSH), somatotrophs producing growth hormone (GH), corticotrophs releasing adrenocorticotropic hormone (ACTH), and lactotrophs secreting prolactin. Within anterior pituitary there are also non-endocrine folliculostellate cells, which have glial cell morphology, they affect secretion of pituitary hormones . ACh is ituitary . α10 subituitary . In the ituitary , and it 2 and were cultured in complete medium which contained Dulbecco’s modified Eagle’s medium with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and 4.5 g/L glucose, 45%; alpha minimum essential medium with 1 g/L glucose, 45%; supplemented with the 0.01 mM nonessential amino acids, 15 mM HEPES, 0.2 mg/ml bovine serum albumin, 2.5 ng/ml epidermal growth factor, dialyzed heat-inactivated fetal bovine serum, 10% and penicillin-streptomycin solution , diluted 100x. The complete medium was refreshed every 2 days. Cells used for the experiment were under tenth passages.RC-4B/C cell line was obtained from the American Type Culture Collection (ATCC CRL-1903). The cells were maintained at 37°C in a humidified atmosphere with 5% CO2O. The PCR reaction conditions were initial denaturation at 95°C for 3 min, 30 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, followed by final extension at 72°C for 7 min. Bands were detected by 1.5% agarose gel electrophoresis. As ladder DirectLoad PCR 100 bp Low Ladder  was used. After electrophoresis, sample was analyzed on GelDoc.Total RNA was isolated from RC-4B/C cell line using RNeasy Mini Kit , according to the manufacturer’s protocol. cDNA was synthesized using the Maxima First Strand cDNA Synthesis Kit for RT-qPCR, with dsDNase . 1 μg of RNA was used for the synthesis, synthesis was according to the manufacturer’s protocol. Afterwards, amplification of target fragments was carried out on a PCR thermocycler. 20 μl of PCR reaction included 2 μl template DNA, 1 μl forward primer (10 pmoles), 1 μl reverse primer (10 pmoles), 2 μl dNTP, 2 μl 10x buffer, 0.25 μl Taq DNA polymerase , 11.75 μl nuclease-free HFor RT-qPCR using nAChR and mAChR primers, all polymerase chain reaction primers were purchased from Lumiprobe RUS Ltd.  . qPCR waIn order to evaluate the expression of the acetylcholine receptors in RC-4BC cells, RT-PCR approach were used. The sequence of primers and expected product size are presented in From the initial expression data amplicons of expected size were detected for α2 and δ subunits . In addiThe expression of the subunits was checked by RT-qPCR, and following results for Ct values were obtained . Three tRC-4BC cell line was derived from an aged male rat pituitary adenoma. It contains all types of anterior pituitary cells with majority of cells containing FSH, LH, and prolactin, with many bihormonal cells within cell line . The celIn the current study we detect high expression level of β2 subunit. β2 subunit constitutes part of heteromeric nAChRs. It is worth investigation, whether β2 subunit contributes to the formation of functional receptors within studied cells. Previously it was shown that rat gonadotrophs express functional β2-containing nicotinic receptors 23). α4β2 subunit is the most prevalent receptor type within brain . α4β2 su, who demOur study suggests that α7 and α9 subunits of homomeric receptor subtypes are expressed at low level within RC-4BC cells. It is quite interesting considering that α7 receptor is abundantly present within hippocampal region , while αδ subunit amplified robustly in our experiment. This is quite unexpected as far as δ subunit constitutes the structural subunit of muscle-type nicotinic acetylcholine receptor, and mutations within this particular receptor subunit result in congenital myasthenic disease . While bM2 subunit robustly amplified in our study. Zemkova et al.  that shoAlthough the data obtained may not exactly correlate with the actual number of transcripts due to different sizes of amplicons, nevertheless, the data show that there is expression. For a more accurate number of transcripts, a standard curve can be included in future experiments to avoid misinterpretation of results. It is also important to note that the expression does not indicate the translation of transcripts and the formation of a full-fledged receptor; nevertheless, the data obtained provide a primary picture of expression in cells.Overall, the knowledge gained in this study could be further used in subsequent studies addressing functionality of cholinergic system within pituitary cells. Especially in studies that utilize RC-4BC cells.S1 Raw image(TIF)Click here for additional data file.


The association between miR-27b-3p and circRanGAP1 or NRAS was predicted using bioinformatics analysis. The expression of circRanGAP1, miR-27b-3p, and NRAS were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The potential oncogenic role of circ-RanGAP1 was assessed using CCK-8, colony formation, transwell assays in vitro, subcutaneous tumor mouse model, vein tail metastatic model, and orthotopically implanted intrahepatic HCC model in vivo. Luciferase reporter and RNA immunoprecipitation (RIP) assays were used to explore the binding site between miR-27b-3p and circ-RanGAP1 or NRAS. Protein expression was detected using western blotting. The localization of miR-27b-3p and circ-RanGAP1 was investigated using fluorescence in situ hybridization (FISH). The level of immune infiltration was assessed by bioinformatics analysis, flow cytometry, and orthotopically implanted intrahepatic HCC models.Here, we found elevated circRanGAP1 in the cells and clinical tissues of patients with HCC. Increased circRanGAP1 levels are associated with enlarged tumors and the advanced stage of TNM. CircRanGAP1 promotes the growth, migration, and HCC cell invasion, concurrently with the growth and metastasis of tumors in-vivo. Moreover, circRanGAP1 is mainly located inside the cytoplasm. Mechanistically, circRanGAP1 as an oncogene promotes HCC progression by miR-27b-3p/NRAS/ERK axis, furthermore, affects the infiltration level of tumor-associated macrophages probably by sponging miR-27b-3p. Immune infiltration analysis shows that NRAS is positively correlated with the levels of CD68+ tumor-associated macrophages in HCC samples and that NRAS and CD68 are related to the poor outcome of HCC.These results reveal that circRanGAP1 is a HCC oncogene that function by the miR-27b-3p/NRAS/ERK axis and regulates the infiltration levels of tumor-associated macrophages by sponging miR-27b-3p. Therefore, circRANGAP1/ NRAS axis may be an important potential treatment target against HCC.The online version contains supplementary material available at 10.1186/s40164-022-00342-6. Globally, cancer remains to be the 2nd most common cause of mortality although the death rate has declined significantly. Where liver cancer is ranked 5th most frequent cause of death by cancer in men 6%) and 7th in women 4%) in America . In Chin% in Amer% and 7thCircRNAs differ from conventional linear RNA in that circRNAs are covalently closed loops without downstream 3’- and upstream 5’- splice sites , 9. It wSeveral studies revealed that RanGAP1 plays specific roles in multiple tumors and can affect various physiological processes. Such as, it was reported that one of the important targets for the treatment of brain tumors may be RanGAP1 . DownregTo evaluate the role played by circRanGAP1 in HCC, we utilized a variety of technologies  and observed that circRanGAP1 enhances the metastasis, proliferation, and invasion of HCC cells. KEGG analysis also suggested that the MAPK signaling pathway may be a potential downstream pathway for circRanGAP1. Furthermore, previous studies have identified NRAS as an immune-related gene that induces tumorigenesis by altering the immune microenvironment –22. Ther2 5%.Human liver cells (L02), HCC cells  , MHCC97L, MHCC97H, HCCLM3 , and Huh7 (Japanese Cancer Research Resources Bank) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) which was augmented with fetal bovine serum 10% (Gibco) and penicillin-streptomycin 1% (Invitrogen). Cells were cultured in a thermostatic incubator at 37 °C temperature, humidified atmosphere with 95% air and COEthical approval was obtained from the Research Ethics Committee of Zhongshan Hospital of Fudan University , and each patient was asked for written informed consent. 40 HCCs and 40 normal tissues from patients, who received curative resection treatment at the Liver Cancer Institute of Zhongshan Hospital of Fudan University  in 2015, were obtained. HCC pathological diagnosis was confirmed, and the medical records were checked for further clinicopathological information. Eight pairs of samples were chosen arbitrarily that used for initial gene screening for detecting the level of RanGAP1-derived circRNAs . And the expression of circRanGAP1 (circ_0063513) in these 40 HCC and matched non-tumor tissues were further analyzed.https://portal.gdc.cancer.gov/). Gene expression in fragments per kilobase per million (FPKM) format was converted to transcript per million reads (TPM) and then quantile-normalized.TCGA dataset  was collected from The Cancer Genome Atlas (TCGA) project (https://starbase.sysu.edu.cn/), TarBase, and RegRNA databases. And the miRNAs that targeted the 3-′UTR of NRAS were obtained from the TargetScan_7.1 (http://www.targetscan.org/vert_71/), Starbase, miRDB (http://mirdb.org/), and miRTarbase (http://mirtarbase.mbc.nctu.edu.tw/index.html) database. And the intersecting miRNAs from these databases were included for further study.The potential miRNA targets of circRanGAP1 were predicted utilizing the StarBase . miR-27b-3p mimics, inhibitor, negative control, and plasmids transfection was performed using Lipofectamine 3000 , by following the instructions of the manufacturer.−ΔΔCt method and normalization was done by glyceraldehyde-3-phosphate dehydrogenase (GAPDH). miR-27b-3p and U6 Primers were also purchased from RiboBio . The sequences were then covered by the patents. miRNA expression was normalized to that of the internal control, U6, using the 2−ΔΔCT method. For evaluating PCR product specificity Melting curve analysis was performed. Additional file Total RNA including cells/tissues miRNA was extracted by utilizing TRIzol Reagent (Invitrogen), and synthesis of cDNA from RNA was done through Reverse Transcription Kit (Takara). Subsequently, cDNA was amplified using the Maxinma SYBR Green qPCR Master Mix (Thermo Scientific). We quantified target genes by the 2Radioimmunoprecipitation assay was applied for the cells/tissue proteins extraction using cell lysis buffer (RIPA) with cocktail protease inhibitor and for mitochondrial protein extraction, a mitochondrial isolation kit (Beyotime Biotechnology) was used, in accordance with the manufacturer's directions. Proteins quantification was done by BCA kit, and then 12% gel was used for SDS-PAGE separation of proteins. Proteins were then transferred to PVDF membranes (0.45 μM) . The membrane was then blocked with skimmed milk before primary antibodies incubation overnight at 4 °C, followed by corresponding HRP-conjugated secondary antibody (PeproTech) incubation. Bands were visualized via chemiluminescence. The protein expression intensity was calculated, using Image J software. The antibodies are enlisted in Additional file 4 cells in 200 µL serum-free DMEM were incorporated in the upper chamber ) of the well with DMEM with fetal bovine serum 10%. For the invasion assay, in 24-well Transwell plates  suspended cells (1 × 105) were seeded in the serum-free medium into the 1 µg/µL Matrigel-coated upper chamber , and in the lower chamber DMEM (600 μl) +FBS 10% was added following incubation at the temperature of 37 °C and 5% CO2. The migrating and invading cells on the outer side of the upper chamber membrane were then fixed using 4% paraformaldehyde before staining with crystal violet and counted under a light microscope (100×magnification).The 96 well plate was seeded with 2000 transfected cells/well  at 0, 24, 48, 72, and 96 h. The 10 µL CCK-8 reagent per well was incorporated at each time point following incubation at the temperature of 37 °C for 1–2 h. Wound-healing assay to evaluate cell migration function was performed. To produce a linear wound cell monolayer was disrupted mechanically with the help of a sterile pipette tip (200 μl). The cells’ migratory distance was observed under the microscope. For the transwell assay, 5 × 10Co-infection of HCCLM3 and Huh7 cells was achieved with a mixture of 6 ng pRL-CMV Renilla luciferase reporter, 60 ng luciferase, and miR-27b-3p mimic or inhibitor. Cells were incubated for 48 h, and then the firefly and Renilla luciferase activities were determined using a dual-luciferase reporter assay .RIP assays were carried out by using a RIP RNA-Binding Protein Immunoprecipitation Kit  by following the protocol suggested by the manufacturer. Additional file HCC cells with circRanGAP1 knockdown/overexpression were constructed by utilizing lentivirus. Lentiviral vectors that encoded shRNA targeting circRanGAP1, overexpression of circRanGAP1 or NC shRNA, and mock were obtained from GENECHEM Gene Company . Lentivirus particles were used for transfecting cells with (multiplicity of infection =10) and then selected in a culture medium with 2 μg/ml puromycin. The gene silencing efficiency was assessed using quantitative real-time PCR. The target shRNA sequences are enlisted in Additional file Overnight hybridization was achieved with circRanGAP1 (5’-CY3-ACTCCAGTGGCAGCGTGAAGCTGCCACACG-CY3-3’) and miR-27b-3p (5’-FAM-gCAGAACTTAGCCACTGTGAa-FAM-3) probes  with and without 3 U/µg RNase R was incubated at 37 °C temperature for 20 min. A RNeasy MinElute Cleanup Kit (Qiagen) was used for final RNA purification.Huh7 and HCCLM3 cells were treated with actinomycin D (2 µg/mL) (Sigma) for 4, 8, 12, or 24 h to block transcription. Quantitative real-time PCR was used for analyzing the stability of circRanGAP1 and RanGAP1.2O2. The sections were then microwaved for antigen retrieval and slides were again blocked, this time using 5% BSA. After blocking it was left for overnight incubation at 4°C in primary antibodies followed by secondary antibodies incubation and lastly, visualization with 3,3-diaminobenzidine (DAB) chromogen. Hematoxylin was used for counterstaining. The slide was observed, and images were taken under the light microscope (Olympus), and the intensity of positive staining was measured. Additional file As aforementioned, immunohistochemistry was done by utilizing EnVision two-step visualization system . Briefly, for de-deparaffinization, xylene was used on tumor tissue sections (5 μm), which were then rehydrated with ethanol dilutions, and then blocked using 3% HFrom fresh tumor tissues, we isolated single cells using collagenase IV, which were then stained with relevant monoclonal antibodies (mAbs) for 30 min at 4 °C temperature. Intracellular proteins were first fixed and then permeabilized using the Fixation/Permeabilization Solution Kit (BD Biosciences), in accordance with the kit’s guidelines. Fluorochrome-conjugated CD3, CD45, CD25, CD8, CD4, FoxP3, NK1.1, B220, MHC-II, CD11c, F480, and CD11b monoclonal antibodies (mAbs) were purchased from BioLegend and eBioscience. After staining cells were washed and then resuspended in PBS (0.1%), azide, and bovine serum albumin. FACS caliber flow cytometer  and FlowJo software  were used for flow cytometry analysis. Additional file 3) = [width2 (mm2) × length (mm)]/2. For the in-vivo metastasis model, the mice were randomly allocated to four groups: mock and circRanGAP1 (MHCC97H), NC shRNA, and circRanGAP1 shRNA (HCCLM3). Five mice were recruited for each group. A lung metastatic tumor model was established by the following protocol: cells (2× 107/ml) were suspended in PBS, and 0.2 ml of this suspension was injected into the tail veins of the mouse. At 6 weeks, twenty-four nude mice in each group were euthanized by anesthesia overdose and visceral organs, including the lungs and livers, were collected. Intrahepatic and pulmonary metastatic foci were examined by pathological identification. For the liver orthotopic xenograft implantation model, flank subcutaneous xenografts were modeled by injecting 1 × 107 cells in 100 μl PBS subcutaneously in C57BL/6 mice (5 weeks old), which were kept by following the 3Rs  guideline. Subcutaneous tumors, after 2–3 weeks, were removed and their weight and volume were recorded. Furthermore, they were dissected into sections of 1 mm3 and then implanted into the liver parenchyma. For mice in the miR-27b-3p mimic and miR-NC treatment group, 100OD/kg, miR-27b-3p mimic, and miR-NC (dissolved in DEPC) were administered via tail veins eight times (twice a week) before establishing the metastasis model. 6 weeks post experimentation mice were sacrificed, and tumor size was measured by MRI. Lastly, sections of the tumor, liver, and lung were fixed and stained routinely using hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC). Additional file The approval for using animals during this study was taken from the Committee on Animal Research of Zhongshan Hospital, Fudan University . All the experiments were carried out in accordance with the guidelines mapped out by the Shanghai Medical Experimental Animal Care Commission. For the in vivo growth model, twenty BALB/c nude male mice about 4–6 weeks of age with an approximate weight of 18–20 g were acquired from Shanghai SLAC Laboratory Animal Co., Ltd., China, and maintained in a sterile environment. For the in vivo growth model, mice were divided randomly into 4 sets: a suspension of 2 ×107 MHCC97H cells (mock and circRanGAP1) and HCCLM3 cells (NC shRNA and circRanGAP1 shRNA) was given subcutaneously in the right flank of each mouse. On day 25, 20 nude mice in each group were euthanized by anesthesia, and tumor dimensions were measured by applying the following formula: Volume  in 19 (47.5%) than that in paired normal tissues. The correlation between circRanGAP1 and the clinicopathological manifestations of the 40 HCC specimen was analyzed, and the results (Table. high) in HCC patients had a large-sized tumor   than that in L02 cells, a normal hepatic cell line. HCCLM3 and HepG2 cells have a higher circRanGAP1 expression, whereas MHCC97H and Huh7 cells have lower circRanGAP1 levels  suggests enhanced Ago2, circRanGAP1 region occupancy in the  against NRAS , are known to act as sponge miRNAs, thus reversing their function . For exaNRAS is a RAS family member, that works as a switch regulating GDP/GTP, which encodes GTPase (membrane-bound protein) activity and is a key regulatory factor in multiple biological processes that affect cancer survival and progression . And theTo study the oncogenic role of has_circ_0063513 in HCC, we utilized a bioinformatics tool to perform a KEGG pathway enrichment analysis. Bioinformatic analyses have been widely used in various diseases, including gastric cancer , kidney With the development of immune microenvironment research and technology, a strong link between immune infiltration and tumor progression has been found , 63. ForIn conclusion, we demonstrated that circRanGAP1 (hsa_circ_0063513) is elevated in HCC and that a high level of circRanGAP1 is related to enlarged tumor dimensions and terminal TNM stage in HCC patients. Moreover, we elucidated the potential mechanism by which circRanGAP1 accelerates the HCC progression by modulating the miR-27b-3p/NRAS/ERK axis and influencing the infiltration levels of tumor-associated macrophages. All in all, our study not only elucidated a potential mechanism highlighting how circRNAs affect HCC progression and immune infiltration but also promulgated that circRANGAP1/ NRAS axis may be an important therapeutic target for HCC patients.Additional file 1: Figure S1. Characterization of circRanGAP1 in HCC. Figure S2. circRanGAP1 expression in HCC cells. Figure S3. CircRanGAP1 acted as a sponge of miR-27b-3p in HCC cells. Figure S4. miR-27b-3p plays tumor-suppressive roles in HCC. Figure S5.Figure S6. The effect of NRAS in cirRanGAP1 knockdown cells. Figure S7. Immune infiltration analysis of miR-27b-3p and NRAS. Figure S8. The effects of circRanGAP1 on immunocytes infiltration. Table S1. Sequences of Primers used for qRT-PCR. Table S2. List of Primary Antibodies Used in the Study. Table S3. Target sequences of hsa_circ_0063513 shRNAs. Table S4. circ_0063513 and miR-27b-3p FISH probe sequences. Table S5. The potential targets of RanGAP1.


Brosimum parinarioides (bitter Amapá) and Parahancornia amapa (sweet Amapá) latex as chelating agents. X-ray diffraction (XRD) and electron diffraction patterns (SAED) patterns showed the ZnO nanocrystals were a pure hexagonal wurtzite phase of ZnO. XRD-based spherical harmonics predictions and HRTEM images depicted that the nanocrystallites constitute pitanga-like (~15.8 nm), teetotum-like (~16.8 nm), and cambuci-like (~22.2 nm) shapes for the samples synthesized using bitter Amapá, sweet Amapá, and bitter/sweet Amapá chelating agent, respectively. The band gap luminescence was observed at ~2.67–2.79 eV along with several structural defect-related, blue emissions at 468–474 nm , green emissions positioned at 513.89–515.89 (h-ZnO nanocrystals with three different morphologies have been synthesized via a simple sol-gel-based method using  The morphology-controlled synthesis of metal oxide nanocrystals has gained much scientific interest because of their modulated physical properties induced by intrinsic defects associated with different morphologies ,2. AmongParahancornia amapa Ducke (bitter Amapá) are Amazon rainforest plants that are known for their complex polymeric matrices latex composition [2+ ions and the subsequent formation of ZnO crystals. Nonetheless, these substances have only been commonly used to treat diseases in the traditional Amazon rainforest communities [Recently, substantial effort has also been directed toward the green synthesis of ZnO nanocrystals using sustainable and environmentally friendly methods without expensive, harsh, and toxic chelating. Recent reports have proved several different plant extracts to be efficient chelating or complexing agents to synthesize nanocrystals ,11. Howeposition ,13. The munities .Several other recent reports have also demonstrated that green synthesized ZnO nanocrystals can be used for photocatalytic purposes ,17,18,19ic rings . This gric rings  and is pic rings . The dyeic rings ,24. ThusHerein, we aim to complete the limited literature on synthesized ZnO using plant latex, reporting for the first time the green synthesis of highly crystalline, single-phase, and distinctive-shaped ZnO nanocrystals using sweet Amapá and bitter Amapá latex as effective chelating agents. The impact of these different chelating agents on the purity, size, shape, and optical nature of the ZnO nanocrystal was investigated. Additionally, a typical photocatalytic experiment was performed to evaluate the removal efficiency of methylene blue (MB) dye, a known azo toxic and stable pollutant.Brosimum parinarioides (bitter Amapá) and Parahancornia amapa (sweet Amapá). The sweet Amapá (SA) and bitter Amapá (BA) latex samples were collected in Vitória do Jari, Amapá, Brazil, at 0°52′25″ S and 52°24′06″ W (SA) and 0°57′08″ S and 52°24′45″ W (BA). In a typical experiment, 1 g of Zn(NO3)2·6H2O  was dissolved in 10 mL (BA), 10 mL (SA), and 10 mL  of Amapá latex and stirred for 10 min aimed at obtaining solutions with a concentration of 100 g·L−1. Afterward, the solutions were maintained at 100 °C for 24 h, and the obtained xerogels were calcinated at 400 °C. The calcined samples indicated high productivity of ZnO nanocrystals with yields of about 92% (BA), 98% (SA), and 85% (SBA).This study selected two Amazon rainforest plants, −1 on a Bruker Vertex 70 spectrophotometer. The structural characteristics of the nanocrystals were investigated by X-ray diffraction (XRD) measurements that were performed by using an Empyrean, PANalytical X-ray diffractometer with Co-Kα radiation (λ = 1.78901 Å), in the range of 30°–90° (2θ) range; 40 kV and 40 mA; 0.01° (2θ) step size and 20 s per step. The instrument resolution was assessed using the LaB6 NIST/SRM 660b standard. Rietveld refinement was performed using the FullProf software [The formation of ZnO nanocrystals was confirmed using thermogravimetric and differential thermal analyses . Their functional groups were obtained by a Fourier transform infrared spectrometer (FTIR) operating 4000–400 cmsoftware . The mor−1). The resultant suspension was kept under a stirrer and irradiated using a visible light source  with an irradiance of 7.74 W·m−2 for 120 min. Before this, a similar experiment was performed in the dark as a control experiment. The MB band reduction at λmax = 664 nm was evaluated in a UV–vis spectrophotometer , using 5 mL aliquots collected at intervals of 20 min. The MB dye removal efficiency was determined using a relation between the absorption at 664 nm and equilibrium concentration before (A0 and C0) and after (A and C) irradiation, as represented in Equation (1).In a typical photocatalytic experiment, 10 mg of ZnO nanocrystals were suspended in a stable methylene blue (MB) solution .XRD = 15.8(4) nm (BA), teetotum-like (DXRD = 16.8(3) nm) (SA), and cambuci-like (DXRD = 22.2(5) nm) (SBA) shapes, as shown in 3D representation in the insets of The successful synthesis was confirmed by the high yield (85–98%) productivity of ZnO nanocrystals. Furthermore, the purity and quality of the produced ZnO nanocrystals were confirmed by characterizations performed using different techniques. The TEM analysis consistently showed the reliability of the XRD-based crystal shape predicted results, as shown in 2 and other volatile compounds. Prominent exothermic peaks at 356 (BA), 341 (SA), and 343 °C (SBA) are associated with ZnO crystallization. Additionally, ~5% (weak peaks above 400 °C) weight losses after crystallization are attributed to the release of CO2 from residual latex. These results confirm that 400 °C is assigned as a suitable calcination temperature to obtain stable ZnO nanocrystals.−1 is attributed to the stretching vibration of hydroxyl (O−H) and amines (N−H) groups. The bands positioned at 2877−3056 cm−1 and 2800−2877 cm−1 are due to stretching vibration of C−H related to alkenes and alkanes, respectively. These vibrations are explicitly linked to the presence of −CH3 and −CH2, probably due to lipids and proteins [−1 are associated with the carbonyl group and arise as a result of the existence of ester, aldehydes, carboxylic acids, and amides functional groups, respectively. Moreover, bands related to axial angular deformation of C=C are also observed between 1417 and 1584 cm−1. However, a C=C stretch vibration is only observed for BA latex. The band located at 1250 cm−1 is related to the C−N stretching vibration in the rings, probably associated with alkaloids and proteins present in the latexes. Further, the bands centered at 1180, 1080, and 1040 cm−1 are due to the C−O stretching vibration of esters, carboxylic acids, and secondary alcohol, respectively, probably as a consequence of the presence of mixed chemical structures such as carbohydrates. Finally, the aromatic rings are characterized by weak bands positioned around 880, 825, and 725 cm−1. These bands can be attributed to angular deformation of the aryl group (δArH), O−H, and C−H present in the aromatic rings of volatile compounds of the latex. On the other hand, the nanocrystal’s FTIR spectra reveal a highly significant disappearance of the bands referring to the organic compounds  [i) [i to neutral zinc vacancies (Zn) and negatively charged zinc vacancies (O), VZn, and Zni along with the crystal [The optical properties of ZnO nanocrystals were demonstrated via photoluminescence (PL) using a λ samples a, whose  samples b,c spectples (i)  or zinc  (i) [i) . Blue ems (VZn−)  and sing) [Zni+)  in all s+) [VO+)  is preses (VZn−) . Further crystal  is obserO+ state . The ora++ state .0 ratios of the samples after 120 min, although it has been computed as a remarkable reduction compared to the experiment in the dark (MB). Moreover, the samples exhibited similar photocatalytic reaction rates, but the highest value was recorded for the solution containing teetotum-like ZnO nanocrystals (1.4 × 10−2 min−1). The highest overall removal efficiency was also observed for the sample with teetotum-like ZnO nanocrystals (84%), which is statistically similar to the sample containing cambuci-like ZnO nanocrystals (79%). Comparatively, our teetotum-like ZnO nanocrystals are more efficient and degrade MB molecules faster than the near-spherical activated by −3 min−1 after 120 min of irradiation with visible light. The defective spherical nanocrystals of Aldeen et al. [−3 min−1) was significantly slower than the reported in the present work. Moreover, these authors have used UV illumination, which is a more energetic light source and properly can lead to the destruction of MB molecules. The green synthesized ZnO nanorods obtained by [−2 min−1, which is faster than 1.4 × 10−2 min−2 found in our work. However, the high exposure time of 150 min used by these authors suggests that if our exposure time is increased, the overall degradation can reach 100%. These literature observations prove that the nanocrystals’ shape combined with their structural defects plays a critical role in the degradation of MB dye molecules in an aqueous solution, confirming that our defective distinctive-shaped nanocrystals are effective for the destruction of MB dye molecules.The defective distinctive-shaped ZnO nanocrystals were tested as photocatalysts using a typical photocatalytic experiment, whose results are shown in n et al.  displayeained by  could deLiterally, the photocatalytic performance of ZnO nanocrystals has been associated with their shape, particle size, surface contamination, and structural defects ,42. The 2O2) molecules (Equations (6) and (7)). H2O2 molecules are easily photocatalyzed to generate The overall mechanism responsible for the photocatalytic degradation of MB dye in aqueous solutions containing teetotum-like ZnO nanocrystals is represented in Equation ) and hyd species  , teetotum-like (~16.8 nm), and cambuci-like (~22.2 nm) shaped ZnO nanocrystals, respectively. These morphologies favored the existence of several structural defects such as O