Its larger doses (20 or 40 mg/kg) had no significant emetic effect. We also evaluated the emetic potential of its analogs , all of which exhibited a similar emetic profile. Our observational studies indicated that temsirolimus can reduce the shrew motor activity at 40 mg/kg, and subsequently, we examined the motor effects of its lower doses. At 10 and 20 mg/kg, it did not affect the spontaneous locomotor activity (distance moved) but attenuated the mean rearing frequency in a U-shaped manner at 10 mg/kg (p < 0.05). We then determined the broad-spectrum antiemetic potential of a 20 mg/kg (i.p.) dose of temsirolimus against diverse emetogens, including selective and nonselective agonists of 1) dopaminergic D2/3 receptors (apomorphine and quinpirole); 2) serotonergic 5-HT3 receptors [5-HT (serotonin) and 2-methyl-5-HT]; 3) cholinergic M1 receptors (pilocarpine and McN-A-343); 4) substance P neurokinin NK1 receptors (GR73632); 5) the L-type calcium (Ca2+) channel (LTCC) (FPL64176); 6) the sarcoplasmic endoplasmic reticulum Ca2+ ATPase inhibitor, thapsigargin; 7) the CB1 receptor inverse agonist/antagonist, SR141716A; and 8) the chemotherapeutic cisplatin. Temsirolimus prevented vomiting evoked by the aforementioned emetogens with varying degrees. The mechanisms underlying the pro- and antiemetic effects of temsirolimus evaluated by immunochemistry for c-fos expression demonstrated a c-fos induction in the AP and NTS, but not DMNX with the 10 mg/kg emetic dose of temsirolimus, whereas its larger antiemetic dose (20 mg/kg) had no significant effect. Our study is the first to provide preclinical evidence demonstrating the promising antiemetic potential of high doses of temsirolimus and possibly its analogs in least shrews.Temsirolimus is a prodrug form of sirolimus (rapamycin). With its analogs , it forms a group of anticancer agents that block the activity of one of the two mammalian targets of rapamycin (mTOR) complexes, mTORC1. We investigated the emetic potential of varying doses  of temsirolimus in the least shrew. Temsirolimus caused a bell-shaped and dose-dependent increase in both the mean vomit frequency and the number of shrews vomiting with maximal efficacy at 10 mg/kg ( These e2, serotonin 5-HT3, and neurokinin NK1, are implicated in the pathophysiological mechanisms of chemotherapy-induced vomiting.The functional pathophysiology of nausea and vomiting indicates that emetic processes are controlled by a balance between the gastrointestinal enteric nervous system, the vagus, and the central nervous system (CNS) . In the In recent years, the mammalian target of rapamycin (mTOR) inhibitors, rapamycin, and its analogs  have bec2/3 receptor nonselective agonist apomorphine and the more selective D2 receptor agonist quinpirole  channel agonist FPL64176  in the least shrew model of emesis. Temsirolimus exhibited a bell-shaped dose-response effect, with lower doses (0.5–10 mg/kg) being proemetic and larger doses such as 20–40 mg/kg having little to no emetic activity. These observations also indicated that the 40 mg/kg dose of temsirolimus may reduce locomotor activity. Locomotor tests revealed that although 10 and 20 mg/kg (i.p.) doses of temsirolimus had no significant effect on the total distance moved by shrews, the frequency of rearing behavior was significantly attenuated at its 10 but not 20 mg/kg dose. To determine whether rapamycin’s analogs  the hyl-5-HT ; 3) the cN-A-343 ; 4) the  GR73632 ; 5) the FPL64176 ; 6) the sigargin ; 7) the R141716A ; and 8) R141716A . Our finAdult least shrews between 45 and 60 days old and weighing between 4 and 6 g from the Western University of Health Sciences Animal Facilities were used. The shrews were housed in groups of 5–10 on a 14:10 light/dark cycle and fed an ad libitum diet. Our animal handling protocols were based upon a prior emesis investigation . Animal 3)2)Cl2) were obtained from Sigma/RBI. Apomorphine, quinpirole, serotonin, 2-methy-5-HT, McN-A-343, GR73632, pilocarpine, and cisplatin were dissolved in distilled water. Thapsigargin was dissolved in 10% DMSO (Sigma) in water. mTOR inhibitors and FPL64176 were dissolved in DMSO and then diluted with three volumes of distilled water to a final DMSO concentration of 25%. SR141716A was dissolved in a 1:1:18 solution of ethanol, emulphor , and 0.9% saline. All drugs were administered at a volume of 0.1 ml/10 g of body weight. The doses and routes used for the emetogens were based upon previous publications from our laboratory . Ridaforolimus and rapamycin were acquired from MedChemExpress and Calbiochem, respectively. Quinpirole HCl, serotonin HCl (5-HT), 2-methyl-serotonin maleate salt (2-methyl-5-HT), pilocarpine HCL, McN-A-343, and cisplatin (cis-platinum (II) diamine dichloride (Pt (NHboratory .Tenebrio sp.) every 30 min before the administration of emetogens, to aid in identifying wet vomits as described previously . The number of evoked vomits were counted for the next 30 min. In additional experiments, different groups of shrews were pretreated at 0 min with an i.p. injection of either vehicle  or varying doses  of different analogs of temsirolimus as follows: everolimus (N = 7–9 shrews within a group), ridaforolimus , or rapamycin (N = 6–10 shrews within a group). The number of evoked vomits were counted for the next 30 min as described previously. Each shrew was used once and euthanized using 3% isoflurane at the end of the experiment.To determine the dose-response emetic effect of temsirolimus, different groups of least shrews were pretreated intraperitoneally (i.p.) at 0 min with either the vehicle or varying doses of temsirolimus  or varying doses of temsirolimus . Each shrew was individually placed in a white observation cage of the same dimension, and the two motor parameters were recorded for 30 min by an overhead camera and data were analyzed using the Noldus software. When changing to another shrew, the chamber was thoroughly cleaned with 70% ethanol and dried to eliminate animal odors between test sessions. Each shrew was used once and euthanized using isoflurane at the end of the experiment.Emetic dose-response studies indicated that temsirolimus may attenuate spontaneous locomotor activity at 40 mg/kg dose, and therefore we evaluated its potential motor-suppressive effects  at 10 and 20 mg/kg (i.p.) doses. The total distance moved reflects the ability of a test drug to interfere with the normal motor functioning after administration, whereas rearing correlated to environmental novelty and is consistent with exploratory information-gathering . The loc2/3 receptor agonist, apomorphine , or the more selective D2 receptor agonist quinpirole  (N = 8 within a group) ; or a centrally/peripherally acting and more selective 5-HT3 receptor agonist, 2-methyl-5-HT  (N = 5 within a group) ; 4) an NK1 receptor-selective agonist, GR73632  (2+ channel (LTCC) agonist, FPL64176  (2+ ATPase inhibitor (SERCA), thapsigargin   (N = 7–11 within a group); or 8) cisplatin   or 20 mg/kg temsirolimus. At 30 min, different groups of pretreated shrews received a fully effective emetic dose of one of following emetogens: 1) a nonselective dopamine Da group) ; 2) a pea group) ; 3) a noa group) , or a moa group) ; 5) an La group) ; 6) a saa group) , 7) a caa group) , (Darmanroup)  were treated with either vehicle  or temsirolimus (10 or 20 mg/kg i.p.). After 30 min, the shrews were anesthetized with isoflurane and perfused with ice cold 4% paraformaldehyde in pH 7.4, 0.1 M phosphate-buffered saline (PBS) for ten min. The brains were removed and cryoprotected with 30% sucrose in 0.1 M PBS overnight and sectioned on a freezing microtome  into 20-μm sections. The sections were observed with a light microscope and those harboring the whole DVC were subjected to immunostaining. The sections were blocked with 0.1 M PBS containing 10% donkey serum and 0.3% Triton X-100 after which they were incubated overnight at 4°C with rabbit anti-c-fos polyclonal antibody  in the blocking buffer. The next day, the sections were washed three times (ten min each) in PBS and incubated in Alexa Fluor 594 donkey anti-rabbit IgG  secondary antibodies. The nuclei of the cells were counterstained with DAPI . Images were acquired under a Zeiss confocal microscope with Zen software using ×20 objective. c-fos-immunofluorescent cell nuclei in each region  were counted using ImageJ as described previously . This analysis resulted in a requirement for eight animals in each group. Statistical analysis was performed using GraphPad Prism 6.04. The vomiting frequency data were analyzed using the Kruskal–Wallis (KW) non-parametric one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test and expressed as the mean ± SEM. The percentage of animals vomiting across groups at different doses was compared using the Chi-square test. The total distance moved and rearing were compared with an ordinary ANOVA test followed by Bonferroni’s post hoc test. The comparison of latency values obtained with temsirolimus and rapamycin injected at 5, 10 and 20 mg/kg doses were presented as the fold change of mean latency of temsirolimus (sec) over mean latency of rapamycin (sec).The vomiting frequency between the two groups was analyzed using an unpaired p < 0.001)] with a maximum (3.14 ± 0.74) at 10 mg/kg (p < 0.01)  = 25.34, p = 0.007)]. Significant increases in the percentage of shrews vomiting were observed at 5 and 10 mg/kg doses   . At its  < 0.01) . The chictively) . Howeverctively) , indicatWe then extended our investigation to the emetic potential of temsirolimus analogs, everolimus, ridaforolimus, and rapamycin. A similar picture emerged in that each paralog at 10 mg/kg dose caused significant increases in both the vomiting frequency and number of shrews vomiting, whereas their larger doses generally evoked less vomiting and in fewer animals .p = 0.14; p = 0.03 versus vehicle) (p = 0.28 versus vehicle) (vehicle)  with 10 vehicle) .In the subsequent drug interaction studies, we chose the 20 mg/kg dose of temsirolimus for the evaluation of its antiemetic potential as it did not significantly affect open-field locomotor parameters.In addition, we compared the latency values obtained with either temsirolimus or rapamycin injected at 5, 10, and 20 mg/kg doses. Latency is the period of time taken to record the first vomit for each shrew following the injection of an emetogen. An intraperitoneal injection of temsirolimus or rapamycin at a 5 mg/kg dose had greater latency than that obtained after i.p. administration of either drug at a 10 mg/kg (i.p.) dose . In addip = 0.04) (p = 0.008) (p = 0.01) (p = 0.002)  induced either by 5-HT  or the more selective (2-methyl-5-HT) agonist of 5-HT3 receptors (p = 0.003) in response to 5-HT  but not after the 2-methyl-5-HT challenge  , or the more selective M1R cholinergic agonist McN-A-343 . In fact, the vomiting frequency   (The results are described and summarized in  = 0.04) , and the= 0.008) . Similar = 0.01)  and the = 0.002) . Also, teceptors . Temsiro > 0.05) . Moreovectively)  and the ctively)  were sig1 receptor agonist GR73632 , which significantly reduced both the evoked vomiting frequency  (p < 0.05) (p = 0.001) (p = 0.003) (p < 0.01) (p = 0.006) (p = 0.0001) (p < 0.0001) (p = 0.002)  (p = 0.02)  was tested against the selective substance P neurokinin NK= 0.006)  and the  < 0.05) . Similar= 0.003) . Temsiro < 0.01)  and the = 0.006) . When te 0.0001) . Finally = 0.02) .p = 0.0001 versus vehicle), 58.3 ± 6.9 in NTS (p = 0.0002 vehicle), and 6.2 ± 1.2 in DMNX (p = 0.2 versus vehicle) (p = 0.53 versus vehicle), 28.5 ± 3.3 in NTS (p = 0.14 versus vehicle), and 3.3 ± 0.9 in DMNX (p = 0.99 versus vehicle) . In line with the behavioral results, the 10 mg/kg  but not vehicle) . When 20vehicle) .Over the last decade, many discoveries have shown that the mTOR pathway is activated in a wide variety of cellular processes and is deregulated in human diseases such as cancer . These oThe effects of varying doses of temsirolimus and its analogs are discussed in the following sections:Incremental doses of temsirolimus caused emesis in a dose-dependent and bell-shaped manner, with a maximal efficacy of 3.14 ± 0.74 vomits in 78.6% of shrews at its 10 mg/kg dose. Its 20 mg/kg dose evoked less emesis in 25% of the tested shrews, whereas its 40 mg/kg caused no vomiting albeit at a sedative dose which visibly attenuated shrew motor behavior. Clinically, temsirolimus is available as a concentration of 25 mg/ml  administp < 0.05). From a pharmacological point of view, the dissociation of ambulation from rearing is not unusual  did not significantly affect spontaneous locomotor activity , they attenuated the mean rearing frequency in a U-shaped manner with a significant reduction at 10 mg/kg  and the more selective and potent D2 receptor agonist, quinpirole  (2 receptor-preferring antagonist sulpiride has been shown by our laboratory to completely prevent apomorphine-induced vomiting in least shrews at 2 mg/kg (s.c.) (2 receptor-mediated vomiting (1) the nonselective Dg, i.p.) . The dopg (s.c.) , but sulg (s.c.) . Similarg (s.c.) . Howeverg (s.c.) . In the vomiting . Thus, i3 receptor agonist, 5-HT . Pretreatment with the first-generation selective serotonin 5-HT3 receptor antagonist tropisetron (2.5 mg/kg) can attenuate the frequency of vomiting caused by 2-methyl-5-HT in the least shrew by 79%  the peripherally acting nonselective serotonin 5-HTst, 5-HT  or the mw by 79% . The sect shrews . In the vomiting .1 agonist McN-A-343 used at 2 mg/kg (i.p). The pharmacology of McN-A-343 was initially described in 1961 by Roszowski  subtypes. Because of its relatively high efficacy at the M1 receptors, it is now prevalently utilized to differentiate responses mediated through M1 receptors from those operating through M2 or M3 muscarinic receptor subtypes, especially in the CNS (3) the nonselective cholinergic agonist pilocarpine- and the muscarinic-preferring Moszowski . At firs the CNS . Recentl the CNS . In line1 receptor agonist GR73632 (5 mg/kg) which causes robust vomiting through the activation of substance P neurokinin NK1 receptors in least shrews (1 receptor antagonist netupitant at 10 mg/kg can completely protect shrews from GR73632 (5 mg/kg)-evoked vomiting (4) the selective substance P neurokinin NKt shrews . The selvomiting . Other ivomiting . In the 2+ influx into the cytosol   process by an initial influx of extracellular Ca2+ through LTCCs followed by the release of intracellular-stored Ca2+ from the endoplasmic reticulum via ryanodine receptors (RyRs)  the selective LTCC agonist FPL64176 used at 10 m/kg. LTCC regulates extracellular Ca cytosol . FPL6417/kg dose . In the  (PTHrP) , lends ss (RyRs) . The ind2+-ATPase (SERCA) pump thapsigargin used at 0.5 mg/kg. The SERCA pump transports free cytosolic Ca2+ into Ca2+ stores in the lumen of the endoplasmic reticulum (ER) to counterbalance the cytosolic intracellular Ca2+ released from the ER into the cytoplasm through the inositol trisphosphate (IP3) receptors and ryanodine (RyR) receptors localized on the ER membrane (2+ concentrations through discharge of intracellular Ca2+ stores from the ER into the cytosol (2+ influx implicates various Ca2+ channels present in the cell membrane including store-operated Ca2+ entry (SOCE) and LTCCs (2+ concentration by inhibiting the SERCA as well as releasing Ca2+ from the ER into the cytoplasm via both RyR- and IP3-receptors (IP3Rs). This is followed by an extracellular Ca2+ influx through LTCCs prior to the intracellular activation of emetic signals (CaMKII and ERK1/2) and an increase in the c-fos immunoreactivity in the brainstem emetic nuclei . In the present study, we showed that temsirolimus reduces the frequency of evoked vomiting by 94.14% and protects 80% of the shrews from thapsigargin-induced vomiting. Thus, it is possible that temsirolimus may prevent emesis by countering the intracellular effects of thapsigargin on Ca2+ stores in the ER. However, this hypothesis warrants further testing.6) the specific inhibitor of the sarco/endoplasmic reticulum Camembrane . Thapsig cytosol . Activatnd LTCCs . Thapsignd LTCCs  by trigg1 receptor-selective inverse agonist/antagonist SR141716A . SR141716A can induce emesis in the least shrew at large doses  which is sensitive to cannabinoid CB1 receptor agonists including Δ9-THC (7) the cannabinoid CBg Δ9-THC . In the g Δ9-THC . Based og Δ9-THC , we spec3 receptor antagonist , an NK1 receptor antagonist  (8) the cytotoxic chemotherapeutic agent cisplatin . Cisplatin produces vomiting biphasically in both humans and other vomit-competent species . In humaupitant) , and a gupitant) .2, 5-HT3, CB1R, M1, and NK1), Ca2+ channel regulators, and possibly, SP and 5-HT release in the case of cisplatin.Collectively, our results demonstrated that a high dose of temsirolimus has a broad-spectrum antiemetic efficacy of varying degrees against diverse emetogens. It probably suppresses emesis by blocking the downstream signaling of specific key emetic receptors  of temsirolimus, its larger antiemetic dose  had no significant effect. The limitation of this study was that it was extremely difficult to obtain sufficient blood to determine accurate levels of these drugs from least shrews which were 3–5 g in weight. Moreover, our laboratory was not equipped to determine the concentration levels of these drugs. Despite these limitations, our study clearly demonstrated that the 20 mg/kg dose of temsirolimus exhibited broad-spectrum antiemetic efficacy against an array of centrally and peripherally acting receptor-selective and -nonselective emetogens. As the 20 mg/kg temsirolimus did not affect the locomotor activity or rearing behaviors, it may be also beneficial to expand the pharmacological armamentarium of temsirolimus in cancer patients and possibly its analogs as an antiemetic.


E)-2-(4-bromobenzoyl)-N-(tert-butyl)-4-(4-fluoro benzylidene)-5-oxo-1-propylpyrrolidine-2-carboxamide (RPDPD for short) of 2-aroyl-4-arylidene-5-oxopyrrolidine derivative was synthesized by the one-pot synthesis method and evaluated for its anti-tumor activity in vitro via CCK8 assay and annexin V/propidium iodide (PI) staining of HeLa cells. The results exhibited that compound RPDPD has inhibited the proliferation of HeLa in a dose-dependent manner with an IC50 of 24.23 μmol/L (p < 0.05) and has low hepatotoxicity with an IC50 of 235.6 μmol/L (p < 0.05) to normal hepatocyte LO2 cells. The apoptotic assay demonstrated that compound RPDPD has induced apoptosis in HeLa cells . qRT-PCR results showed that the compound RPDPD could inhibit the expression of oncogene E6/E7 mRNA (p < 0.05) of human papillomavirus (HPV). The results of Western blot showed that the compound RPDPD promoted the expression of TIMP3 protein and inhibited the expression of MMP3 (p < 0.05). In conclusion, the compound RPDPD can inhibit the proliferation of cervical cancer cells and induce the apoptosis of cervical cancer cells, and its mechanism may be related to the inhibition of E6 mRNA and E7 mRNA expressions, and the anticancer effect of the compound RPDPD on cervical cancer is closely related to the TIMP3/MMP3 signaling axis.Heterocyclic compounds were widely used in many domains; pyrrolidone is a derivative of heterocycles that can be used to synthesize anticancer drugs. A new fluorine-containing rhopaladins’ analog( Heterocyclic compounds are extensively used in plenty of fields and are favored by many drug researchers. Pyrrolidone, one of the heterocyclic compounds, can be used as an antiviral , antiepiThe incidence and development of cancer seriously threaten human health. The incidence rate of cervical cancer ranks fourth among cancer in women. In 2018, the incidence of new cases was about 570,000, and the death toll was about 310,000 for cervical cancer in the world . The pat50 of 12.5 and 7.4 μg/ml, respectively) (N cyclization and studied their cytotoxicity to HeLa cells (E)-2-(4-bromobenzoyl)-N-(tert-butyl)-4-(4-fluorobenzylidene) -5-oxo-1-propylpyrrolidine-2-carboxamide (RPDPD for short) of 2-aroyl-4-arylidene-5-oxopyrrolidine derivative was synthesized by a similar method  .. We havctively)  via a taLa cells . AccordiLa cells , in ordethod see . We inveN cyclization method to synthesize 5-oxpyrrolidine -2-carboxamide. The advantages of this synthesis method lie in mild reaction conditions, high yield, and easy availability of raw materials. This new rhopaladins’ analog RPDPD can inhibit the proliferation of the HeLa cell line. Moreover, RPDPD induced HeLa cell apoptosis at early apoptosis. We believed that this synthesis method has potential application prospects in the synthesis of natural products and their analogs, and this compound RPDPD has an important application value as a potential bioactive compound or pharmaceutical.We used a one-pot Ugi 4CC/S−1 was recorded using a PE-983 infrared spectrometer. A Varian mercury 600 spectrometer was used to record NMR in CDCl3, which was related to TMS. Elemental analysis was carried out on the Vario-ELIII element analyzer.The melting point was determined by the x-4 model apparatus without correction. The Finnigan trace MS spectrometer was used to detect the compound structure. The infrared absorption of KBr pellets in cm2 content.The cervical carcinoma cell line HeLa was stemmed from the Chinese Medicine Experiment Center, Affiliated Dongfeng Hospital, Hubei University of Medicine, and maintained in RPMI 1640 medium which contained FBS (10%). The cells were grown in an incubator at 37°C with 5% CO2CO3 during the stirring process. After stirring the reaction system for 12 h, the mixture was cooled and filtered with a paper filter. The resulting solid was recrystallized with ether to obtain rhopaladins’ analog RPDPD.First, 2-(bromomethyl)-3-phenyl-2-acrylic acid  was dissolved in methanol (5 ml), and n-propylamine was added  and was stirred at room temperature for 10 min. Then, 4-bromophenylglyoxal  and tertbutyl thiocyanate  were added. The reaction was carried out at room temperature, and the pH value of the reaction system was adjusted to 6 by Cs4 cells/well, the original medium was removed after the cells adhered the next day, and the compound RPDPD  medium was added and cultured for 24, 48, and 72 h, respectively. With DMSO as the control group, cisplatin treatment was used as a positive control, and normal hepatocytes were used for cytotoxicity experiments, and CCK8  was incubated for 2 h. Also, the plate was evaluated in a luminescence microplate reader  at 450 nm wavelength. The optical density (OD) values were obtained. The inhibition rate and IC50 of each compound on different cells were calculated by GraphPad Prism 8.0.1 software. Cell survival rate (%) = [-A (blank)]/[(A (DMSO)-A (blank)]× 100%.Seeded in a 96-well plate at a density of 2 × 10The effect of RPDPRH on apoptosis was performed using the Annexin V-FITC/PI Apoptosis kit  following the manufacturer’s protocol. The cells were digested, dispersed, and plated into 6-well plates with 2 × 104 cells/well and treated with RPDPRH  at various concentrations for 48 h. Upon completion of the treatment, the cells were harvested and washed with cool phosphate buffered solution   and resuspended in 500 μl binding buffer. Annexin V-FITC (5 μl) and PI (10 μl) were added to the cell suspension, and then the suspension was incubated at room temperature away from light for 5 min. The cells were analyzed using a flow cytometer  immediately, and the results of apoptosis were analyzed by Flow J software . Set 3 hRPDPD  for 48 h. The total RNA was extracted and reverse transcribed into double-stranded cDNA according to the cell processing manual. The amplification procedure for amplifying E6/E7 mRNA was as follows: pre-denaturation at 95°C for 1 min, denaturation at 95°C for 15 s, annealing at 55°C for 15 s, and extension at 72°C for 45 s, a total of 40 cycles. 2-ΔΔCt was used to calculate the relative expression of genes. The primer sets were as follows: E6 F: 5′-TTGCTTTTCGGGATTTATGC-3′, R:5′-CAGGACACAGTGGCTTTTGA-3'; E7 F: 5′-GAACCGGACAGAGCCCATTA-3′, R: 5′-AGAACAGATGGGGCACACAAT-3'. Set 3 has duplicate holes in each group.The HeLa cells were treated with the compound 4/well, and the supernatant medium was removed after the cells adhered the next day, and 2 ml of the compound RPDPD  medium of different concentrations were added to each well and incubated for 48 h. The rabbit anti-TIMP3 antibody  and rabbit anti-MMP3  were used for Western blotting. The gray value of protein was analyzed using ImageJ software, and the relative expression of the protein was calculated. Set 3 has duplicate holes in each group.The cells were inoculated into 6-well plates at a cell density of 2 × 10p < 0.05 indicated that the difference was statistically significant.The statistical software used was SPSS 22.0. The data were expressed as mean ± standard deviation (Mean ± SD), and the data were in line with normal distribution and were compared by one-way ANOVA between groups, and further pairwise comparisons were performed by LSD-t test. N cyclization reaction to obtained rhopaladins’ analog RPDPD, a white solid, with a yield of 0.42 g, 80% in w/w. The spectral data of RPDPD are as follows. Mp: 197–198°C. 1H NMR : δ 7.73 , 7.61 , 7.40 , 7.36 , 7.07 , 6.14 , 3.76 , 3.63–3.55 , 3.28 , 3.18–3.12 , 1.72–1.51 , 1.35 , and 0.87 . 13C NMR : δ 196.1, 169.7, 167.4, 159.4, 132.5, 132.1, 131.5, 130.7, 129.2, 126.1, 124.4, 116.0, 115.8, 74.7, 52.6, 46.1, 35.4, 28.3, 21.3, and 11.6. EI-MS m/z (%): 516 , 514 , 414 (22), 332 (53), 233 (55), and 183 (100). Anal. Calcd for C26H28BrFN2O3: C, 60.59; H, 5.48; N, 5.44. Found: C, 60.45; H, 5.59; and N, 5.31.We used a one-pot Ugi 4CC/SIn the experimental process, the reaction conditions were mild, the yield was high, and the raw materials were easy to obtain. The synthetic route of the new fluorine-containing compound RPDPD is outlined in 50 values of compound RPDPD on HeLa cells for 24, 48, and 72 h were 59.56 μmol/L, 39.64 μmol/L, and 24.23 μmol/L, respectively, and the IC50 of cisplatin on HeLa cells for 48 h was 17.36 μmol/L. The IC50 of compound RPDPD on normal hepatocyte LO2 cells for 48 h was 235.6 μmol/L, but the IC50 of cisplatin on normal hepatocyte LO2 cells for 48 h was just 21.32 μmol/L. The results showed that rhopaladins’ analog RPDPD has good anti-cervical cancer activity and low hepatotoxicity, and the inhibition of RPDPD on cell growth was different with varying concentrations, i.e., the higher the concentration, the higher the inhibition rate of the cells. It can be seen from the experimental result that the inhibitory effect of the compound RPDPD on the growth of HeLa cells was exerted in concentration-dependent and time-dependent manners.The antiproliferative effect of the compound RPDPD was investigated by CCK8 assay against the cervical carcinoma HeLa tumor cell line. The result exhibited that the compound RPDPD at a varying concentration could inhibit cell growth. We can see that the rate of surviving cells decreased with the increasing drug concentrations and also with time . The IC550 of the compound RPDPD was decreased after 48 h. After the cells were treated with the compound RPDPD, the apoptosis rate of the cells was increased. The FACS flow cytometry analysis showed that HeLa cells were treated with the compound RPDPD at its IC50 concentration for 48 h, and the percentage of apoptosis cells was increased in a concentration-dependent manner  play an important role in extracellular matrix (ECM) homeostasis and are involved in tumorigenesis and development, angiogenesis, apoptosis, and tumor metastasis , such asIn conclusion, our study confirmed that rhopaladins’ analog RPDPD affects the proliferation and apoptosis of cervical cancer cells, inhibits the expression of oncogenes E6 mRNA and E7 mRNA, and its anticancer activity may be related to the TIMP3/MMP3 signaling axis. This study is only the first time to explore its anticancer activity, and we will continue to conduct more in-depth research on the mechanism of the compound RPDPD in the future, so as to provide effective experimental data for the development and application of the compound RPDPD in the clinical treatment of cervical cancer.


Background: Intensified training coupled with sufficient recovery is required to improve athletic performance. A stress-recovery imbalance can lead to negative states of overtraining. Hormonal alterations associated with intensified training, such as blunted cortisol, may impair the immune response. Cortisol promotes the maturation and migration of dendritic cells which subsequently stimulate the T cell response. However, there are currently no clear reliable biomarkers to highlight the overtraining syndrome. This systematic review and meta-analysis examined the effect of intensified training on immune cells. Outcomes from this could provide insight into whether these markers may be used as an indicator of negative states of overtraining.Methods: SPORTDiscus, PUBMED, Academic Search Complete, Scopus and Web of Science were searched until June 2022. Included articles reported on immune biomarkers relating to lymphocytes, dendritic cells, and cytokines before and after a period of intensified training, in humans and rodents, at rest and in response to exercise.Results: 164 full texts were screened for eligibility. Across 57 eligible studies, 16 immune biomarkers were assessed. 7 were assessed at rest and in response to a bout of exercise, and 9 assessed at rest only. Included lymphocyte markers were CD3+, CD4+ and CD8+ T cell count, NK cell count, NK Cytolytic activity, lymphocyte proliferation and CD4/CD8 ratio. Dendritic cell markers examined were CD80, CD86, and MHC II expression. Cytokines included IL-1β, IL-2, IL-10, TNF-α and IFN-γ. A period of intensified training significantly decreased resting total lymphocyte  and CD8+ T cell counts , and unstimulated plasma IL-1β levels . Resting dendritic cell CD86 expression significantly increased . All other biomarkers remained unchanged.Conclusion: Although some biomarkers alter after a period of intensified training, definitive immune biomarkers are limited. Specifically, due to low study numbers, further investigation into the dendritic cell response in human models is required. The HPA axis consists of the hypothalamus, pituitary gland and adrenal cortex   when compared to before the training   enduranc O2max)  introduc O2max) . In line O2max) , with a  fatigue . Further fatigue , a reductraining , and redtraining .However, the “window of opportunity” theory is not accepted by all, with suggestions that reductions in immune cell function post-exercise could reflect the lowered number of immune cells in the circulation after exercise, which are redistributed into tissues for enhanced immunosurveillance at sites of infection risk . For exa+ T cells  . Glucoco T cells . Given t T cells .via antigen processing and upregulation of the major histocompatibility complex (MHC), costimulatory molecules (CD80/86) and cytokines   . The MHC, IL-12) . CD80/86, IL-12) . Finally T cells . Cross p T cells . DCs cannd IL-18 . Conversnd TNF-α , and elind TNF-α . To our nd TNF-α , increasnd TNF-α  or unclend TNF-α . DespiteWith evidence that DCs are in part regulated by the HPA axis, and the knowledge that the HPA axis response to exercise stress may be blunted following a period of intensified exercise, it is important to examine the impact that intensified exercise has on DCs. As these cells orchestrate the immune response, specifically, the direct nature of the relationship DCs have with both T lymphocytes and NK cells, it is logical to review evidence surrounding all three immune cells, providing further direction towards a conclusion in the response of the immune system to intensified training.Therefore, the aim of this systematic review is to assess the current literature examining the effects of a period of intensified training on lymphocyte (T cells and NK cells) and DC number and function, in both humans and rodents. This review focuses on the normal impact of high intensity training due to the difficulties surrounding confirmation of NFOR/OTS diagnosis. However, heavy training is a factor involved in the establishment of NFR/OTS, and as such, any highlighted immune biomarkers could potentially indicate NFOR/OTS has occurred. The main purpose being to highlight areas already studied, indicate potential gaps requiring further investigation, and assess if there is scope for the future use of immune biomarkers in the diagnosis of overtraining.This review conforms to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines  and was To develop the inclusion and exclusion criteria for this review a consideration of Population, Intervention, Comparison and Outcome (PICO) was used .2max) of fair or higher (>38.5 ml kg−1. min−1) according to ACSM guidelines for cardiorespiratory fitness  of the change in biomarker from pre-to post-training. A minimum of two studies measuring the same biomarker, using the same measurement units, were required to include that biomarker in the meta-analysis component. Where possible, differing units of measurements were converted into the same “gold standard” units for comparison. If this was not possible, it was excluded from the meta-analysis.A literature search was conducted in the following databases on 26 May 2021: SPORTDiscus, PUBMED, Academic Search Complete, Scopus and Web of Science. Databases were searched from inception up until May 2021 for articles published in English. In addition to database searches, reference lists of relevant studies were screened for eligible studies. The search was re-run in June 2022 to identify any additional articles meeting the inclusion criteria.1) “chronic exercise*” OR “training volume” OR “intensified training” OR “exercise training” OR “overtrain*” OR “endurance training*” OR “physical education and training” OR “high intensity training” OR “chronic exercise training” OR “physical conditioning, animal*” OR “Physical exertion”2) “lymphocyte function” OR “immune response” OR “dendritic cell function” OR “immune function” OR “dendritic cell” OR “myeloid” OR “plasmacytoid” OR “t cell*” OR “cd4*” OR “cd8*” OR “T helper” OR “T cytotoxic” OR “lymphocyte*” OR “NK cell” OR “natural killer cell” OR “cd56*” OR “T regulatory” OR “cd25*” OR “lymphocyte proliferation” OR “T cell proliferation” OR “CD80*” OR “CD86*” OR “cd80*/86*” OR “NK-cell” OR “NKCA” OR “Natural Killer cell cytotoxic activity” OR “killer cells, natural” OR “cytotoxicity, immunologic” OR “lymphocyte activation” OR “antigen presenting cell*” OR “dendritic cells” OR “genes, mhc class i” OR “genes, mhc class ii” OR “interleukin”3) “athlete*” OR “Mice” OR “animals”4) “elderly” OR “Cancer” OR “Elder” OR “older” OR “geriatric” OR “aged”5) AND 2 AND 3 NOT 4.Titles, abstract and keywords were searched using the following search terms:Articles retrieved through the systematic search were exported to ProQuest RefWorks, a reference management software , and further exported to Excel , whereby duplicates were removed and assessment for eligibility began. Two investigators (CB and JH) independently screened articles by title and abstract, and full text when necessary, against the inclusion criteria. Full texts from the eligible studies were then independently screened (CB and JH) for inclusion into the review.2max, age), intensified training period details (mode and duration) and assessed biomarker information . Pre- and post-training values were extracted for each relevant biomarker in the form of mean and standard deviation. Where appropriate data was not presented, the authors were emailed, and were allocated 4 weeks to reply. If no reply was received after 4 weeks, the study was excluded. Any variables included in the search string that did not have sufficient studies to perform a meta-analysis were not included in the results. Where figures were used displaying the mean and standard deviation, data was extracted by eye.Data extraction was conducted by one reviewer (CB) whereby the following data from all eligible articles were extracted into an Excel document: Title, publication details (year and author), participant characteristics  and independently verified by one member of the review team (JH). Three Cochrane Collaboration tools were used for assessing risk of bias; ROBINS-1 for non-randomised controlled trials, ROB-2 for randomised controlled trials and ROB-2 (Crossover) for randomised crossover trials . Specific study components assessed for risk of bias using the ROBINS-1 tool included confounding, selection of participants, classification of intervention, deviations from intended interventions, missing data, measurement of outcomes and reporting of results. Study components assessed using the ROB-2 tool included the randomisation process, deviations from intended interventions, missing outcome data, measurement of outcomes and reporting of results. The ROB-2 crossover tool assessed the same components as the ROB-2 tool with the addition of carryover effects.g standardised mean difference (SMD) was calculated via the RevMan software .Inverse variance, random effects meta-analysis was then conducted on immune biomarker data in Review Manager Software . Hedge’s 2 statistic; 0%–40% indicated non-important (low) heterogeneity, 40%–60% indicted moderate heterogeneity, 50%–75% indicated substantial heterogeneity and 75%–100% indicated considerable heterogeneity . All results were reported as Hedge’s g with 95% confidence intervals (CI). Additional sub-group analysis was conducted on resting immune cell count biomarkers based on the duration of intensified training periods i.e. ≤ 7 days, 8 days-2 weeks, 15 days- 4 weeks or >4 weeks.A separate meta-analysis was conducted for each biomarker where >2 studies measured the same biomarker using the same method and units of measurement. Human and rodent studies were analysed together for all biomarkers, apart from ‘lymphocyte proliferation’ due to human studies measuring peripheral blood lymphocytes, and rodent studies measuring spleenocytes. Effect sizes were classified based on the magnitude of change from pre to post intervention. Classifications included very small (0.01–0.19), small (0.20–0.49), moderate (0.50–0.79), large (0.80–1.19), very large (1.20–1.99) and huge (>2.0) . Statistn = 3) or “Low” (n = 30) because most studies followed a group of athletes over time or assessed the same group of participants before and after a period of intensified training. The bias arising from participant awareness of intervention encapsulated in ROBINS-1 domain 6; bias in measurement of outcome, was judged as being negligible in most studies (n = 24). It is difficult to blind participants from intervention when intensified training is the independent variable and training loads were often monitored or implemented by the investigators themselves, so knowledge of intervention was necessary. It could be argued that as objective immune biomarkers were measured, results are unlikely to be affected by knowledge of intervention, especially in the rodent studies.A complete analysis of ROB is displayed in Despite this, it has been suggested that anticipatory stress may cause alterations to the immune system, such as decreased lymphocyte counts and reduced lymphocyte proliferation . Howevern = 22) or unclear (n = 31) in most studies, mainly because no information regarding excluded participants or reasons for missing data were highlighted. Only one study ) reduced resting lymphocyte number with a moderate effect , I2 = 65%). Subgroup analysis indicated significant decreases in resting lymphocyte count in all exercise durations of >7 days (8 days- 2 weeks (n = 4): Z = 2.29 (p = 0.02), d = −1.36, 95% CI ; 15 days- 4 weeks (n = 7): Z = 3.21 (p = 0.001), d = −0.65, 95% CI ; >4 weeks (n = 15): Z = 2.08 (p = 0.04), d = −0.38, 95% CI ). Exercise durations of ≤7 days (n = 3) did not alter lymphocyte counts at rest (Z = 1.04 (p = 0.30), d = −0.71, 95% CI ).Of the 57 included studies, 29 studies assessed lymphocyte count at rest. Overall, a period of intensified training significantly (Z = 4.07 (p = 0.14), d= −0.64, 95% CI ). There is substantial heterogeneity amongst the studies , I2 = 80%).Of the 57 included studies, 8 studies assessed lymphocyte count in response to exercise. Overall, a period of intensified training did not change the total lymphocyte count immediately post exercise , d= −0.50, 95% CI ; 2 = 77.41, df = 13 (p < 0.00001), I2 = 83%). Subgroup analysis revealed that intensified training periods of 8 days- 2 weeks (n = 3) significantly decreased CD4+ T cell count at rest (Z = 2.14 (p = 0.03), d = −0.80, 95% CI ).Of the 57 included studies, 14 studies assessed CD3+ count immediately post exercise. Overall, a period of intensified training did not change CD3+ count immediately post exercise , d= −1.16, 95% CI ). However, there is considerable heterogeneity amongst the studies , I2 = 94%).Of the 57 included studies, 3 studies assessed CD3+ count at rest. Overall, a period of intensified training did not change CD4+ count at rest , d= −0.30, 95% CI ; 2 = 74.12, df = 18 (p < 0.00001), I2 = 76%). Subgroup analysis revealed that intensified training periods of 8 days- 2 weeks (n = 3) significantly decreased CD4+ T cell count at rest (Z = 2.53 (p = 0.01), d = −1.17, 95% CI ).Of the 57 included studies, 19 studies assessed CD4+ count immediately post exercise. Overall, a period of intensified training did not change CD4+ count immediately post exercise , d = 0.35, 95% CI ). However, there is substantial heterogeneity amongst the studies , I2 = 69%).Of the 57 included studies, 5 studies assessed CD4+ count at rest. Overall, a period of intensified training significantly  reduced CD8+ count at rest with a small effect , I2 = 61%). Subgroup analysis revealed that intensified training periods of 8 days- 2 weeks (n = 4) were the only duration to significantly alter resting CD8+ T cell count (Z = 2.98 (p = 0.003), d = −0.79, 95% CI ).Of the 57 included studies, 18 studies assessed CD8+ count immediately post exercise, before and after a period of overtraining. Overall, a period of intensified training did not change CD8+ count immediately post exercise , d = 0.37, 95% CI ). However, there is considerable heterogeneity amongst the studies , I2 = 89%).Of the 57 included studies, 6 studies assessed CD8p = 0.36), d= −0.15, 95% CI ; 2 = 59.81, df = 18 (p < 0.00001), I2 = 70%).Of the 57 included studies, 19 studies assessed CD+/CD8+ Ratio at rest. Overall, a period of intensified training did not change the resting CD4+/CD8+ ratio , d= -0.04, 95% CI ). There is low heterogeneity amongst the studies , I2 = 16%).Of the 57 included studies, 7 studies assessed the CD4+/CD8+ ratio immediately post exercise. Overall, a period of intensified training did not change the CD4+/CD8+ ratio immediately post exercise , d= −0.25, 95% CI ; 2 = 24.78, df = 10 (p = 0.003), I2 = 64%). Subgroup analysis revealed that resting NK cell count did not alter after a period of intensified training of any duration .Of the 57 included studies, 10 studies assessed NK cell count at rest based on CD56+ expression immediately post exercise. Overall, a period of intensified training did not change NK cell count immediately post exercise , d= 0.04, 95% CI ). However, there is substantial heterogeneity amongst the studies , I2 = 54%).Of the 57 included studies, 3 studies assessed NK cell count based on CD56p = 0.97), d= −0.02, 95% CI ). However, there is considerable heterogeneity amongst the studies , I2 = 91%).Of the 57 included studies, 5 different studies assessed human peripheral blood lymphocyte proliferation at rest, with 3 studies assessing lymphocyte proliferation to more than one stimulant, thus 8 results were entered into the meta-analysis. Overall, a period of intensified training did not change resting lymphocyte proliferation , d = 0.09, 95% CI ). However, there is considerable heterogeneity amongst the studies , I2 = 93%).Of the 57 included studies, 6 studies assessed rodent spleen lymphocyte proliferation at rest with 2 studies assessing lymphocyte proliferation to more than one stimulant, thus 8 results were entered into the meta-analysis. Overall, a period of intensified training did not change resting spleen lymphocyte proliferation , d= −2.38, 95% CI ). However, there is considerable heterogeneity amongst the studies , I2 = 96%).Of the 57 included studies, 3 studies assessed spleen lymphocyte proliferation immediately post exercise. Overall, a period of intensified training did not change spleen lymphocyte proliferation immediately post exercise , d = 1.13, 95% CI ). However, there is considerable heterogeneity amongst the studies , I2 = 88%).Of the 57 included studies, 3 studies assessed NK cytolytic activity as %lysis of K-562 tumour cells. Overall, a period of intensified training did not change NK cell cytolytic activity , d= −0.13, 95% CI ; 2 = 35.96, df = 9 (p < 0.00001), I2 = 75%).Of the 57 included studies, 10 studies assessed unstimulated TNF-α secretion at rest. Overall, a period of intensified training did not change resting TNF- α secretion , d = 0.70, 95% CI ). However, there is considerable heterogeneity amongst the studies , I2 = 79%).Of the 57 included studies, 5 studies assessed unstimulated IFN-γ secretion at rest. Overall, a period of intensified training did not change resting IFN- γ secretion ] decreased resting IL-1 β secretion with a moderate effect , I2 = 1%].Of the 57 included studies, 5 studies assessed unstimulated IL-1β secretion at rest. Overall, a period of intensified training significantly ). However, there is considerable heterogeneity amongst the studies .Of the 57 included studies, 3 studies assessed unstimulated IL-10 secretion at rest. Overall, a period of intensified training did not change resting IL-10 , d= 1.41, 95% CI ). However, there is considerable heterogeneity amongst the studies .Of the 57 included studies, 2 studies assessed unstimulated IL-2 secretion at rest. Overall, a period of intensified training did not change resting IL-2 secretion , d = −0.33, 95% CI ; 2 = 1.64, df = 1 (p = 0.20), I2 = 39%].Of the 57 included studies, 2 studies assessed DC CD80 expression as a percentage of fluorescent intensity. Overall, a period of intensified training did not change rodent bone marrow derived DC CD80 expression , d= −2.30, 95% CI ; 2 = 65.24, df = 1 (p < 0.00001), I2 = 98%].Of the 57 included studies, 2 studies assessed DC MHC II expression. Overall, a period of intensified training did not change rodent DC MHC II expression ] increased DC CD86 expression with a large effect , I2 = 72%].Of the 57 included studies, 2 studies assessed rodent DC CD86 expression as a percentage of fluorescent intensity. Overall, a period of intensified training significantly [Z = 2.99,  and are therefore more susceptible to change with increased exposure to catecholamines across the training period , definitive immune biomarkers indicative of the negative states of overtraining are limited. Incompatibilities in methodologies and units of measurements between studies, as well as low study numbers contributed to the inability to identify more definitive immune biomarkers within the literature. This review highlights the need for further research into biomarkers specifically relating to dendritic cells, especially in human models. Additionally, although this review aimed to include females, no study returned from the systematic search controlled for menstrual cycle meaning only male data could be included. Therefore, future research should aim to conduct a controlled study of immune biomarkers in female subjects in response to a period of intensified training in order to widen the applicability of findings. The inclusion of humoral immunity, such as the measure of salivary immunoglobulin A was not considered for this review, however, future reviews should include this as a possible immune biomarker to provide a more holistic overview of the immune state after a period of intensified training. Overall, a period of intensified training has been shown to significantly decrease resting total lymphocyte counts, resting CD8+ T cell counts and unstimulated IL-1β levels, and significantly increase DC CD86 expression.This review identified numerous immune biomarkers that have been investigated before and after a period of intensified training. Although this review focuses on the normal impact of high intensity training due to the difficulties surrounding confirmation of NFOR/OTS diagnosis, heavy training is a factor involved in the establishment of NFR/OTS, therefore, the results presented could provide evidence that these immune biomarkers are potentially indicative of NFOR/OTS. Results suggest that although some biomarkers indicated significant alterations after a period of intensified training (resting CD8


Planococcus halocryophilus Or1, which is a bacterium metabolically active down to −25°C. P355′s stability and activity at varying pH values, temperatures, and salt concentrations, as well as its temperature-dependent kinetics, were determined and compared to an uncharacterized thermophilic ISP (T0099) from Parageobacillus thermoglucosidasius, a previously characterized ISP (T0034) from Planococcus sp. AW02J18, and Subtilisin Carlsberg (SC). The results showed that P355 was the most heat-labile of these enzymes, closely followed by T0034. P355 and T0034 exhibited catalytic constants (kcat) that were much higher than those of T0099 and SC. Thus, both P355 and T0034 demonstrate the characteristics of the stability-activity trade-off that has been widely observed in cold-adapted proteases.The enzymes of microorganisms that live in cold environments must be able to function at ambient temperatures. Cold-adapted enzymes generally have less ordered structures that convey a higher catalytic rate, but at the cost of lower thermodynamic stability. In this study, we characterized P355, a novel intracellular subtilisin protease (ISP) derived from the genome of  Planococcus halocryophilus Or1, which grows at −15°C and remains metabolically active at −25°C in water containing 18% (3.08 M) NaCl , which usually also results in a higher Michaelis-Menten constant (Km). This is in turn reflected in an increased structural flexibility at the cost of thermodynamic stability  NaCl , 2013. Ttability . This fltability .Bacillus, ranging from the mesophilic Subtilisin Carlsberg (SC)    to a fewcalase™) . Cold-adcalase™) , where tcalase™) . The demcalase™) ; thus, tBacillus subtilis  and intracellular subtilisin proteases (ISPs). ESPs contain a signal peptide that directs their secretion from the cell, as well as a pro-peptide that inhibits activity in their zymogen state and functions as a chaperone during their folding . The prosubtilis  and sharsubtilis . Gamble P. halocryophilus Or1 , as well as two previously characterized serine proteases: the putative mesophilic ISP (T0034) from Planococcus sp. AW02J18  was included in activity assays.All chemicals were purchased from Merck KGaA  with purity >98% unless otherwise noted. A commercial formulation of the ESP Subtilisin Carlsberg (SC) from P. halocryophilus Or1 (GenBank acc. no. ANBV00000000)  and an uncharacterized ISP from Parageobacillus thermoglucosidasius , which is known to be a thermophilic Gram-positive bacterium (The DNA sequence encoding a subtilisin-like intracellular protease was identified by sequence-based mining of the cryophilic permafrost bacterium 0000000) . Isolati0000000) . Annotat0000000)  and pote0000000)  and Sign0000000) , respect AW02J18  . The isp gene was sub-cloned from the delivery vector to the p12 FX-cloning vector (ampicillin resistant) used in E. coli MC1061 for expression.For recombinant cloning and enzyme expression, we used a previously developed screening procedure for subtilisin-like proteases . The P35E. coli. Cells were cultured on lysogeny broth (LB) agar plates containing 100 μg/mL ampicillin overnight at 37°C. A single colony was inoculated in 10 mL LB with 100 μg/mL ampicillin and incubated overnight at 37°C, 200 rpm. Five mL overnight culture was transferred to 100 mL LB with 100 μg/mL ampicillin and incubated for approx. 5 h at 37°C with 200 rpm until an OD600 of 0.5–0.7 before 1 mL sterile 10% L-arabinose was added for induction. The incubation temperature was then adjusted to 20°C, and cultures were incubated overnight at 200 rpm. Cells were harvested by centrifuged at 1000 × g for 10 min and resuspended in 40 mL buffer A1 . Cells were lysed by sonication (Qsonica) using a 12 mm probe in ice water using a cycle of 1-s sonication and 1-s pause for 6 min in total at 13% amplitude. The lysate was centrifuged at 22,000 × g at 4°C to remove cell debris. The following steps were performed at room temperature (RT), but the protein was kept on ice whenever possible. A 5 mL HisTrap high performance (HP) column  was equilibrated in buffer A1 using an ÄKTA purifier system  before lysate was loaded. The column was then washed with 5 column volumes of buffer A1 followed by 5 column volumes of 10% B1 where 100% B1 is 500 mM imidazole, 20 mM sodium phosphate, pH 7.4. Finally, the protein was eluted using 100% B1. Additional impurities were removed by applying eluate from the HisTrap HP column to a 5 mL HiTrap Q high performance  pre-equilibrated in buffer A2 . The column was washed with 5 column volumes of buffer A2 before a linear gradient from 0 to 1 M NaCl was applied. A flow of 5 mL/min was used throughout the FPLC-steps and collected into 1 mL fractions. Purity  and separated using the glycine/2-amino-2-methyl-1,3-propanediol/HCl (ammediol) system in 5–15 or 10–15% acrylamide gradient gels  casted i2, 0.1% (w/w) Triton X-100 (1.7 mM), 100 mM NaCl, and 100 mM glycine, pH 9.5). The proteases were incubated for 10, 30, 60, 90, 120, and 180 min at RT before SDS-PAGE and activity measurement. For SDS-PAGE, 20 μL of each protease solution, each containing approximately 1 μg of the enzyme, was mixed with 12 μL sample buffer (see Section “SDS-PAGE”) and denatured at 95°C for 5 min. Additionally, 10 μL was tested for activity where the final protease concentration of P355, T0034, T0099, and SC were 1.63, 1.75, 2.06, and 0.94 nM, respectively (see Section “Protease activity assay”).Proteases P355, T0034, T0099, and SC  were incubated in maturation buffer  and 0.1 M CAPS pH 11) and electrotransferred to polyvinylidene difluoride (PVDF) membranes . The PVD2, 0.1% (w/w) Triton X-100, 100 mM NaCl, and 100 mM glycine, pH 9.5) as described in “Protease maturation” to mature the proteases before activity assays except for the “Calcium titration” experiment. Matured proteases were kept on ice before experiments. Protease activity was measured in a FLOUstar Omega plate reader (Thermo-Fischer™) in half-area plates (Corning®) at 410 nm and at RT using 0.2 mM N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide (AAPF) as substrate. A 100 mM AAPF stock was made in 100% dimethylsulfoxide (DMSO). The final reaction volume was 100 μL containing 0.2% DMSO and 0.2 mM AAPF.Matured protease stocks were prepared by incubating for 2 h at RT in maturation buffer  Triton X-100, and 100 mM NaCl) unless otherwise stated and carried out at RT. The final protease concentration was approximately 1–4 nM in every activity assay described below unless otherwise stated. This amount of protease produced linear curves with r2 ≥ 0.99 based on linear regression of the initial reaction (0–4 min). Every incubation was carried out at RT unless otherwise stated and activity measurement was done in at least technical triplicates.All protease activity assays contained reaction buffer  and 0–1 mM EDTA and incubated for 60 min. For the Pefabloc® assay, 5 μL matured protease was added to the reaction buffer with calcium in 0–5 mM Pefabloc®. Tris at pH 7.5 was used in the reaction buffer to reduce the auto-hydrolysis of Pefabloc®. The protease was incubated with Pefabloc for 2.5 h RT.Inhibition of activity was assessed by testing the effect of EDTA and PefablocMatured protease was added to a reaction buffer containing the following buffers to determine the pH optimum: glycine at pH 2.5–3.5; sodium acetate at pH 4–5.5; MES at pH 5.5–6.5; HEPES at 7–7.5; Tris at pH 7.5–8.5; and glycine at pH 9–10.5. Buffer concentrations were 0.1 M.r2 > 0.99. CAPS at pH 9.7 was used as a buffer instead of glycine as growth occurred in prolonged storage of glycine buffer (0.4 M glycine). The effect of temperature on pKa of CAPS was compensated by using d(pKa)/dT = −0.009. AAPF stock (100 mM) was diluted with temperature-adjusted water and incubated for 10 min. at either 25 or 45°C in the plate. Finally, 5 μL matured protease was added, and activity was measured immediately.Half-area plates containing reaction buffer were adjusted to 25 or 45°C on an Eppendorf ThermoMixer C. Water filled the gaps between the wells to stabilize the temperature resulting in low standard deviations along with Five μL matured protease was incubated in 95 μL incubation solution . Five μL of the incubation solution was aspirated after 10, 30, 60, 120, and 180 min, and activity was measured in reaction buffer with a final volume of 100 μL. The pH was adjusted to pH 9.5 by the reaction buffer for all measurements. Calcium was omitted in the incubations and reaction steps due to precipitation, except for the carry-over calcium from the stock solutions.Matured proteases were incubated in maturation buffer at different temperatures, and samples were aspirated at different time intervals: 0°C (ice-water) for 24, 48, and 120 h; 25°C for 24, 48, and 120; 45°C for 10, 20, 30 min, 1–4, 24, and 48 h; and 65°C for 10, 20, 30 min, 1–4, 24, and 48 h The remaining activity was measured as described above and normalized to the corresponding matured protease, which had incubated for 0 h at the given temperatures.Five μL matured protease was added to a reaction buffer containing titrated NaCl (1–1000 mM), KCl (1–1000 mM), urea (4–4000 mM), or guanidine hydrochloride (Gnd) (4–4000 mM) and incubated for 30 min before the activity was measured. The activity was normalized to the lowest salt concentration: 0.98 mM for NaCl and KCl and 3.91 mM for urea and Gnd.r2 > 0.99). Absorbance was converted to mM by the slope of the linear standard curve (r2 > 0.99), where the substrate had been hydrolyzed completely. Enzymatic activity was defined as the turnover rate of millimolar substrate pr. hour (mmol/h). The kinetics constants were derived with R (v. 4.0.5) using the add-on package drc (Michaelis-Menten kinetics were determined by measuring the activity at different substrate concentrations ranging from 0.03 to 4 mM (using a 400 mM AAPF stock) in reaction buffer at 25 and 45°C. Protease concentrations were 0.94, 10.02, 4.06, and 2.37 nM for P355, T0034, T0099, and SC, respectively. The concentration of T0034 was lowered to 1.02 nM at 45°C. Initial rate was determined by linear regression . Proteolytic activity against casein was defined as A × h–1 × nM–1 (the slope divided with the protease concentration).Proteolysis of resorufin-labeled casein was assessed by incubating 3 nM matured P355, T0034, and SC, and 60 nM matured T0099 with 0.5 μg/μL resorufin-labeled casein in 100 mM or 1000 mM NaCl. A reaction was quenched every 2.5 min with 2% (v/v) TCA and incubated for 10 min at 37°C, precipitating undigested casein. The solution was filtered using a MultiScreen Solvinert Filter plate (Millipore) by centrifugation at 500 × The BSA was dissolved in 8 M urea (0.27 mM), and the disulfide bridges were reduced using 10 mM DTT for 60 min at RT. Iodoacetamide (30 mM) was added, and the sample was incubated for 60 min at RT in the dark before 35 mM DTT was added to quench the reaction. The reduced and alkylated BSA (raBSA) was dialyzed into 10 mM Tris pH 8 so that [urea] <10 mM and stored at −20°C. Titrated matured protease (0.4–100 nM) incubated with 3.75 μg raBSA (1.88 μM) in reaction buffer containing 100 or 1000 mM NaCl for 2 h in ice-water. Controls were raBSA without protease and protease (highest concentration) without raBSA. The pH shift in the reaction buffer due to the lower temperature was considered by using d(pKa)/dT = −0.025. The reaction was terminated by adding SDS-PAGE sample buffer, DTT (see Section “SDS-PAGE”) along with 42 mM EDTA and heating to 95°C for 10 min. Digestion patterns were examined with SDS-PAGE, and the density of the raBSA band was measured using Gel Doc™ EZ imager (Biorad) software.2O followed by two times incubation of 15 min with 50 μL 50% acetonitrile. Then, 50 μL of 100% acetonitrile was added and incubated for 15 min before the reaction buffer was added to a 1:1 ratio. The liquid was removed after 15 min and lyophilized until the gel plug was dry. Matured protease (600 nM) was added to the dried plugs and incubated for 10 min. Thirty μL reaction buffer was added and incubated for 1.5 h. Triton X-100 was excluded as detergents are not compatible with MS. All incubation was performed at RT except protease digestions, which were allowed to proceed at 37°C. The reaction was quenched with 7.1% formic acid. The resulting peptides from protease digestion were micro-purified using C18 column material (Empore™) in P10 pipette tips (Sarstedt). The column material was activated with solvent B (99.9% acetonitrile and 0.1% formic acid) and then equilibrated with solvent A (0.1% formic acid). Peptides were then loaded and washed in solvent A before being eluted with 70% solvent B and dried for MS. The experiment was carried out in triplicates.A total of 3.8 μg raBSA (BSA digest in ice water) was loaded onto an SDS-PAGE to separate intact raBSA from impurities and nicked raBSA. The raBSA band was cut out and washed twice with 500 μL HNano LC-MS/MS was carried out on an Orbitrap Eclipse Tribrid mass spectrometer connected online to an EASY nanoLC 1200 (both instruments were from Thermo Fisher Scientific). Samples were dissolved in solvent A, desalted on a ReproSil-Pur C18-AQ trap column . The raBSA-peptides from the protease digestion were eluted and separated on a 15-cm analytical column (75 μm inner diameter) packed in-house with ReproSil-Pur C18-AQ 3 μm resin . A flow rate of 250 nL/min was used to elute the peptides with a 50-min gradient from 5 to 35% solvent B.The generated raw files were converted to Mascot generic format. Data was searched in Mascot (version 2.5.0) against the SwissProt Data base . The entire SwissProt database was used as a decoy database (random sequence). The following search parameters were (1) ‘none’ as the protease, (2) minimum 6 residues in a peptide, (3) precursor mass deviation of maximum 10 ppm, (4) fragment mass deviation of maximum 0.02 Da, (5) dynamic modification with oxidation on Met, and (6) fixed modification with alkylation (carbamidomethyl) on Cys. The false discovery rate was set to 1% before data was exported to MS Data Miner . Peptidetidyverse   coupled idyverse , tagger   from niformis  was compn Norway . P355 isrmafrost . T0099 i to 68°C . To the Paenibacillus polymyxa (acc. no. P29139) (64% amino acid similarity) (B. subtilis subsp. subtilis str. 168 (acc. no. P11018) (64% amino acid similarity)   as well ilarity) . P355 shas T0034 , which ias T0034 , the novas T0034 . The catas T0034 .E. coli with their pro-peptide sequences intact. Histidine tags fused to the C-terminal allowed downstream purification by nickel affinity and anion exchange chromatography in the absence of calcium to avoid maturation . Active P355, T0034, and T0099, i.e., 2 h incubation with 25 mM CaCl2 at pH 9.5, lacked an additional 9, 15, and 16 N-terminal residues, respectively  and urea than their meso- and thermophilic counterparts . OverallMicroorganisms that thrive in subzero degrees are usually also halophilic or halotolerant as salt lowers the freezing point of water . Thus, tkcat) than their meso- and thermophilic homologs to compensate for the general decrease in activity at low temperatures  at 25°C of all proteases, while P355 and T0034 displayed the highest at 45°C. The catalytic efficiency increased approximately 1.5 to 2.5-fold for every protease from 25 to 45°C, except for T0034, which quadrupled from 2.09 × 105 to 8.55 × 105 M–1 s–1. The kinetic data . The results are summarized in The goal of this paper was to identify, produce, and characterize a novel protease from the cold-adapted and halo-tolerant bacterium to −15°C . We iden AW02J18 , the preThe ISPs, P355, T0034, and T0099, showed a dependency on calcium for activation , and up Bacillus clausii NN010181 before an exponential increase in active ISP followed by a plateau. This is due to a positive feedback mechanism where protease removes the N-terminal pro-peptide and activates the unprocessed protease , in contrast to what might be expected from a cold-adapted protease. However, it is not unheard of that some cold-active enzymes display a relatively high-temperature optimum . The temt-test indicated no significant difference (p < 0.05) between the lowest Gnd concentration (3.9 mM) and 125 mM Gnd. Overall, SC is the most stable protease in Gnd, while ISPs, and in particular T0034, are less stable.Chemical denaturation is usually correlated with thermal lability . This trkcat) and Km compared to SC and T0099  stability correlates with kcat, i.e., P355 has traded stability for higher activity  and raBSA digestion (0°C) in line with the activity-stability trade-off characteristic in cold adaptation. Altogether, we conclude that P355 along with T0034 are cold-adapted proteases.In summary, we produced and characterized a novel cold-adapted ISP (P355) from the The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/CR and JE: conceptualization. CR and SH: methodology. CR: software, formal analysis, data curation, writing—original draft, and visualization. CR and IT: investigation. JE, ØL, and GB: resources. CR, MT, SH, ØL, GB, PS, and CS: writing—review and editing. JE and MT: supervision and project administration. JE and CS: funding acquisition. All authors contributed to the article and approved the submitted version.


T) was isolated from brine sampled at a soda lake in the Inner Mongolia Autonomous Region, China. Cells of the strain were rod-shaped and motile. Strain IM 1326T was able to grow at 4–42 °C  with 0–13.0 % (w/v) NaCl concentrations (optimum at 4.0–6.0 %) and at pH 7.5–11.0 (optimum at 9.0–10.0). The 16S rRNA gene phylogenetic analysis revealed that the isolate belongs to the genus Aliidiomarina and is closely related to the type strains of Aliidiomarina sanyensis (95.8 % sequence similarity), Aliidiomarina shirensis (95.7 %), Aliidiomarina iranensis (95.4 %) and Aliidiomarina haloalkalitolerans (95.3 %). The whole genome of strain IM 1326T was sequenced, and the genomic DNA G+C content was 49.7 mol%. Average nucleotide identity, average amino acid identity and digital DNA–DNA hybridization values between the isolate and the related Aliidiomarina species were 68.1–84.9 %, 76–78 % and 18.4–20.4 %, respectively. The respiratory quinone was ubiquinone-8. The polar lipid profile included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and one unidentified aminophospholipid. The predominant cellular fatty acids were summed feature 9 , iso-C15 : 0 (16.1 %) and iso-C17 : 0 (13.1 %). Based on the results of phylogenetic analysis, genome relatedness, and the physiological and chemotaxonomic properties of the isolate, strain IM 1326T is considered to represent a novel species of the genus Aliidiomarina, for which the name Aliidiomarina halalkaliphila sp. nov. is proposed (type strain IM 1326T=CGMCC 1.17056T=JCM 34227T).A haloalkaliphilic strain (IM 1326 Aliidiomarina belongs to the family Idiomarinaceae, order Alteromonadales, class Gammaproteobacteria  at 30 °C.The type species of the genus T were examined by using routine cultivation methods on HM medium containing (g l−1): NaCl (30.0), KCl (2.0), MgSO4·7H2O (1.0), NaBr (0.23), CaCl2 (0.27), NaHCO3 (0.06), FeCl3 (trace), tryptone (5.0), yeast extract (10.0) and agar (15.0 for solid medium) [l-arginine and 5.0 % (v/v) DMSO in stoppered tubes. Tests for catalase and oxidase activities and the hydrolysis of starch, gelatin, casein, and Tweens 20, 40, 60, and 80 were carried out using routine cultivation methods on HM medium with 4 % (w/v) NaCl as described by Harrigan et al. [2S formation was detected using a filter-paper strip impregnated with lead acetate [et al. [−1) with various supplementary carbon sources and then comparing the cell density. Acid production from sugars and alcohols was determined, and the substrates used were d-glucose, d-galactose, sucrose, d-sorbitol, mannose, maltose, starch, d-fructose, l-sorbose, ribose, trehalose, d-xylose and glycerol. Production of acid from the above-mentioned carbohydrates and sugar alcohols was tested on the growth medium supplemented with 10.0 g l−1 of the substrate. The pH of the cultures was measured with a glass electrode at the end of the incubation period and compared with the initial pH and the pH of the control tube without substrate. Reduction of nitrate and nitrite were detected by using sulfanilic acid and α-naphthylamine reagent [The physiological and chemotaxonomic characteristics of strain IM 1326 medium)  at 37 °C medium) . Morpholn et al. . H2S for, NaBr 0., CaCl2 . The isolate can move, but no flagellum was observed under the transmission electron microscope . Furthermore, the type of motility was explored. There were no rafts of cells and isolated cells were observed in the microscope image of the edge of colonies , indicating that the cells did not move by gliding; therefore, further exploration is necessary. The colonies were light brown when grown on HM medium (pH 8.5) for 24 h at 37 °C. Strain IM 1326T was able to grow in presence of 0–13.0 % NaCl concentration , at 4–42 °C  and pH 7.5–11.0 . Moreover, it showed more tolerance to alkalinity (up to pH 11.0) than most of the reference species. Strain IM 1326T could hydrolyse gelatin, casein and different kinds of Tweens, e.g. Tween 20, 40 and 60, but could not hydrolyse starch. In addition, strain IM 1326T was positive for nitrate reduction, but negative for nitrite reduction. Cells of strain IM 1326T were oxidase- and catalase-positive. Correspondingly, a respiratory nitrate reductase operon (including four genes FM042_RS01865 to FM042_RS01880), NarK family nitrate/nitrite transporter (FM042_RS01935) and catalase (FM042_RS03320 and FM042_RS09450) were annotated in the genome. However, the draft genome did not contain the gene for amylase and nitrite reduction. Strain IM 1326T could produce H2S from cysteine but did not produce indole from tryptophan. The detailed morphological, physiological and biochemical characteristics of strain IM 1326T are provided in the species description and Strain IM 1326midi protocol . The cellular fatty acid profiles of strain IM 1326T, A. taiwanensis CGMCC 1.15255T and A. haloalkalitolerans DSM 24939T are shown in Table S2. The predominant fatty acids detected in strain IM 1326T were 10-methyl-C16 : 0/iso-C17 : 1 ω9c , iso-C15 : 0 (16.1 %) and iso-C17 : 0 (13.1 %). These are also the dominant fatty acids in the two reference strains. In addition, we observed that, contrary to the original report, the content of C16 : 0 was high (up to 13.2 %) in A. taiwanensis CGMCC 1.15255T [16 : 0 was lower in IM 1326T and A. haloalkalitolerans DSM24939T (Table S2). These data on the fatty acids composition of two reference species were similar to the previous report [Aliidiomarina [Cells grown on HM plates for 2 days at 37 °C were harvested for the cellular fatty acid analysis. The fatty acids were extracted according to the standard T were extracted by a phenol–chloroform system and analysed by using two-dimensional thin-layer chromatography (TLC) as described previously [T were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and one unidentified aminophospholipid . The polar lipid composition of two reference species (including the type species) were identified under identical experimental condition . Most of the Aliidiomarina species contained diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine .2S is produced from cysteine. Production of indole and utilization of urea does not occur. The methyl red reaction is negative, but the Voges–Proskauer test result is positive. Gelatin, casein, DNA and Tweens 20, 40 and 60 are hydrolysed, but Tween 80, aesculin and starch are not hydrolysed. The following substrates are utilized as single carbon and energy sources for growth: l-arginine, l-lactate, l-glutamate, acetate, l-malate, pyruvate, succinate, citrate, l-serine, glycine, valine and histidine. The following substrates are not utilized as sole sources of carbon and energy: d-glucose, d-galactose, sucrose, d-sorbitol, fumarate, l-aspartate, mannose, maltose, starch, l-alanine, l-ornithine, d-fructose, l-sorbose, ribose, trehalose, d-xylose, glycerol and l-lysine. No acids are produced with the following substrates: d-glucose, d-galactose, sucrose, d-sorbitol, mannose, maltose, starch, d-fructose, l-sorbose, ribose, trehalose, d-xylose and glycerol. The respiratory quinone is ubiquinone-8. The major cellular fatty acids of the isolate are 10-methyl-C16 : 0/iso-C17 : 1 ω9c (summed feature 9), iso-C15 : 0 and iso-C17 : 0. The major polar lipids include diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and one unidentified aminophospholipid.Cells are Gram-stain-negative, motile, rod-shaped, approximately 0.3–0.6 µm wide and 0.8–2.1 µm long. Colonies grown for 24 h at 37 °C on HM medium are circular, smooth, convex, light brown and approximately 1.0–3.0 mm in diameter. Oxidase and catalase are positive. Growth occurs at 4–45 °C , at pH 7.5–11.0  and with 0–13.0 % (w/v) NaCl concentration (optimum 4.0–6.0 %). Anaerobic and microaerobic growth does not occur on HM medium with or without nitrate. Nitrate is reduced, but nitrite is not. HT (=CGMCC 1.17056T=JCM 34227T), was isolated from the soda lake Hutong Qagan Lake in Inner Mongolia Autonomous Region, China. The genomic size of the type strain is 2.57 Mb, and DNA G+C content is 49.7 mol% (determined from the genome sequence).The type strain, IM 1326Click here for additional data file.


However, the clinical potential of TP is plagued by its poor aqueous solubility, short half-life, and biotoxicity. Therefore, developing an ideal vehicle to efficiently load TP and achieving targeted delivery to melanoma is a prospective approach for making full use of its antitumor efficacy.Melanoma is the most malignant skin tumor and is difficult to cure with the alternative treatments of chemotherapy, biotherapy, and immunotherapy. Our previous study showed that triptolide We applied exosome (Exo) derived from human umbilical cord mesenchymal stromal cells (hUCMSCs) and engineered them exogenously with a cyclic peptide, arginine-glycine-aspartate (cRGD), to encapsulate TP to establish a bionic-targeted drug delivery system (cRGD-Exo/TP), achieving synergism and toxicity reduction. The average size of cRGD-Exo/TP was 157.34 ± 6.21 nm, with a high drug loading of 10.76 ± 1.21%. The in vitro antitumor results showed that the designed Exo delivery platform could be effectively taken up by targeted cells and performed significantly in antiproliferation, anti-invasion, and proapoptotic activities in A375 cells via the caspase cascade and mitochondrial pathways and cell cycle alteration. Furthermore, the biodistribution and pharmacokinetics results demonstrated that cRGD-Exo/TP possessed superior tumor targetability and prolonged the half-life of TP. Notably, cRGD-Exo/TP significantly inhibited tumor growth and extended survival time with negligible systemic toxicity in tumor-bearing mice.The results indicated that the functionalized Exo platform provides a promising strategy for targeted therapy of malignant melanoma.The online version contains supplementary material available at 10.1186/s12951-022-01597-1. Malignant melanoma (MM) is the most aggressive and fastest-growing skin tumor, accounting for 65% of skin cancer mortality . The 5-yTripterygium wilfordii Hook F, has been widely used in rheumatism, inflammation, immunosuppression, and anticancer treatment -c(RGDfK) (DSPE-PEG2000-cRGD) synthesized by Xi’an ruixi Biological Technology Co., Ltd. ; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (HEPES); CD63 ; CD9 ; TSG101 ; Cell Counting Kit-8 ; Annexin V-APC/7-AAD Apoptosis Detection Kit ; 4′,6-diamidino-2-phenylindole ; PKH67 ; DiR ; Dulbecco’s modified Eagle’s medium (DMEM); Fetal bovine serum (FBS), Trypsin and 1% Pen-Strep . Unless otherwise stated, other reagents and solvents were purchased from commercial sources and can be used without further purification.2 atmosphere at 37 °C.hUCMSCs were derived from the umbilical cord of healthy newborns delivered by cesarean section. The protocols were approved by the Institutional Review Board at Changhai Hospital of Naval Medical University. A375 and HaCaT cells were purchased from the Chinese Academy of Sciences Cell Bank  and cultured in DMEM containing 10% FBS and 1% Pen-Strep under a 5% COMale BALB/c nude mice (18–22 g) provided by Fudan University Shanghai Cancer Center were housed under standard laboratory conditions  with free access to water and standard chow.Culturing of hUMSCs was carried out based on previous methods . Brieflyg for 10 min, 2000× g for 10 min, and 10,000× g for 30 min at 4℃ to remove cells and cell debris. The supernatants were then ultracentrifuged at 120,000× g for 70 min. The pellet was resuspended in PBS and ultracentrifuged again at 120,000× g for another 70 min. Finally, hUCMSC-Exo pellets were resuspended in PBS and stored at −80 °C for further use.Exo was isolated from passages 3–10 hUCMSCs by gradient centrifugation . When hUThe morphology, size, size distribution, and zeta potential of hUCMSCs-Exo were detected using TEM  and dynamic light scattering . In addition, western blotting analysis was implemented to identify the exosomal markers CD9, CD63, and TSG101 of hUCMSCs-Exo. Briefly, the total protein of hUCMSCs-Exo was detected by a BCA kit, separated on a 10% sodium dodecyl sulfate–polyacrylamide gel, and then transferred onto polyvinylidene fluoride membranes. After blocking with 5% skimmed milk, the membranes were incubated with CD9, CD63, and TSG101 primary antibodies overnight and horseradish peroxidase-conjugated (HRP) secondary antibodies. Finally, the immunoblots on the membrane were detected by enhanced chemiluminescence reagents  , 66.DSPE-PEG2000-cRGD was incorporated into Exo by the postinsertion method , 67. BriThe coincubation method was applied to load TP into cRGD-Exo. First, purified cRGD-Exo (600 μg/mL) and TP (40 μg/mL) at different mass ratios (μg protein:μg TP) of 10:1, 5:1, 1:1, 1:5, and 1:10 were mixed and incubated in a shaker at 100 rpm for 1 h at room temperature to prepare drug-encapsulated exosomes, cRGD-Exo/TP. The unloaded drug was quantitated by HPLC to calculate DL and EE by the following Eqs. n represents the TP concentration of the nth sample, L represents the TP content of cRGD-Exo/TP added, and ν1 and ν2 represent the volumes of the sample and receiver medium, respectively.The in vitro drug release behavior of cRGD-Exo/TP was analyzed using the dialysis method . BrieflyStorage stability and serum stability were assessed by monitoring the particle size of cRGD-Exo/TP in PBS  or in 10% Exo-free serum . The size of cRGD-Exo/TP in PBS was detected every day, and that in serum was detected at 2, 4, 6, 8, 10, 12, and 24 h.A375 and HaCaT cells were exposed to PKH67-labeled Exo/TP or cRGD-Exo/TP for 4 h, after which the cells were fixed with 4% paraformaldehyde and stained with DAPI. When the samples were dried at room temperature in the dark, the cellular fluorescence intensity was observed with CLSM . In addition, for semiquantitative analysis, the intracellular MFI of the cells with different treatments was calculated with ImageJ software.Furthermore, TP taken up by A375 cells was also detected. After being treated with TP solution, Exo/TP, and cRGD-Exo/TP (the concentration of TP was 60 ng/mL) for different times, A375 cells (living cells) were counted and lysed. The drug concentration in the lytic cells was analyzed using UHPLC-MS/MS.A375 cells were pretreated with various endocytic inhibitors or cultured at low temperature to probe the internalization mechanisms of cRGD-Exo/TP . A375 ceA375 cells were incubated with different concentrations of TP for 24 h. After that, 10 μL of CCK-8 was added to the treated cells and incubated for another 20 min. The absorbance was detected using a microplate reader. After that, the cytotoxicity of Exo, cRGD-Exo, TP solution, Exo/TP, and cRGD-Exo/TP was also evaluated .A375 cells were pretreated with Exo, cRGD-Exo, TP solution, Exo/TP, and cRGD-Exo/TP for 6 h and then trypsinized and resuspended in DMEM. Then, the differently treated cells were incubated in the apical Transwell insert . Meanwhile, 800 μL of DMEM with 10% FBS used for cell chemotaxis was added to the basolateral champers. After incubation for 24 h, the cells on the top side of the apical chamber were wiped, and then the migrated cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. After that, five random fields under an inverted microscope were visualized, and the migrated cells were counted by ImageJ software.To assess the proapoptotic effect of cRGD-Exo/TP on A375 cells, the cells were incubated with Exo, cRGD-Exo, TP solution, Exo/TP, and cRGD-Exo/TP for 24 h. Then, the cells were trypsinized by pancreatic enzymes without DMSO and resuspended in 500 μL 1× Binding Buffer. The cell suspension was stationarily cultured at room temperature for 15 min in the dark after adding 7-AAD and Annexin V-APC. Subsequently, cell apoptosis and necrosis were detected with a flow cytometer.The cell cycle assay was carried out to explore the correlation between mitosis and cytotoxicity. Briefly, A375 cells were incubated with DMEM, Exo, cRGD-Exo, TP solution, Exo/TP, and cRGD-Exo/TP for 24 h. The treated cells were then fixed with 70% ethanol for 12 h, washed with PBS 3 times, and stained with propidium iodide/RNase A for 30 min. After that, the red fluorescence of differently treated cells was recorded at 488 nm, and the DNA content of cells was also analyzed.c, NF-κB, VEGF, and survivin) of A375 cells incubated with DMEM and cRGD-Exo/TP (70 ng/mL TP) were detected by western blotting as described above.Western blotting was applied to investigate the mechanisms of cell apoptosis. The apoptosis-related proteins  and injected intravenously with 0.2 mL of DiR-labeled Exo/TP or DiR-labeled cRGD-Exo/TP (600 μg/kg of TP). After injection, mice were scanned at interval times  using an IVIS  at 750/780 nm. Finally, mice were euthanized and surgically dissected at 24 h. The fluorescence intensity in the heart, liver, spleen, lung, kidney, blood, and tumors was also recorded.The xenograft tumor model was established by subcutaneously injecting 2–4 × 10n = 21): TP solution, Exo/TP, and cRGD-Exo/TP. The preparations were injected intravenously at drug-equivalent doses of 600 μg/kg TP. After injection, blood was sampled from the orbital vein at 5 min, 10 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, and 24 h. In addition, heart, liver, spleen, lung, kidney, and tumor tissues were collected at 5 min, 30 min, 1 h, 2 h, 4 h, 6 h, and 24 h. Nine mice were sacrificed at each time point (n = 3), whereas at 10 min and 15 min, the mice were only taken for blood and were not killed. The TP concentration in plasma and tissue homogenate was analyzed with UHPLC-MS/MS.The TP concentrations in blood, heart, liver, spleen, lung, kidney, and tumor tissues were also quantified over time. Briefly, 63 tumor-bearing nude mice were randomly divided into 3 groups (n = 6): (1) normal saline, (2) cRGD-Exo, (3) TP solution, (4) Exo/TP, and (5) cRGD-Exo/TP. The dosage of TP in the TP solution, Exo/TP, and cRGD-Exo/TP groups was 600 μg/kg, while that of Exo in the cRGD-Exo, Exo/TP, and cRGD-Exo/TPgroups was also 600 μg/kg. The first day was recorded as 0 d. Before each dose, the length (L) and width (W) of the tumors were measured to calculate tumor volume . After 8 doses (dosing every other day for 14 days), the mice were sacrificed, and then the tumor tissues were acquired, weighed, photographed, and stained for H&E, TUNEL assay, and Ki67 staining. The tumor inhibition rate was calculated using Eq. (n = 6).The xenograft tumor model mice were randomized into 5 groups /bodyweight (g)] and to stain for H&E. In addition, orbital blood was sampled for a routine blood test, hepatic function test and renal function test, including RBC, WBC, ALT, and Cr.2) were 350 °C and 8 L/min, respectively. The pressure of the nebulizer gas (N2) was 40 psi. The capillary voltage was 4000 eV. The positive multiple reaction monitoring (MRM) mode was adopted to determine TP and carbamazepine . The collision energy was 33 eV for both TP and IS. The precursor-to-product transitions were m/z 361.1 → 105.2 for TP and m/z 237.1 → 194.0 for IS.TP concentrations in cells, plasma, primary organs, and tumors were analyzed using UHPLC-MS/MS . The separation was performed on an ACQUITY UPLCTM BEH C18 column . The column temperature was maintained at 40 °C. The mobile phases were 1% acetic acid aqueous solution (A) and methanol (B). Isocratic elution with 40% A and 60% B was adopted at a flow rate of 0.3 mL/mL. The temperature and flow rate of dry gas (NThe results are presented as the mean of at least three experiments with the corresponding standard deviation (SD). Statistical data were analyzed using SPSS software, and a statistically significant difference was denoted by the difference probability level (p < 0.05). The t-test and one-way ANOVA were used to analyze the statistical data.Additional file 1: Fig. S1. Morphology of human umbilical cord mesenchymal stem cells. (A) Primary cells (bar: 100 μm). (B) Passage 3 cells (bar: 200 μm). Fig. S2. Zeta potential of Exo (A), cRGD-Exo (B), and cRGD-Exo/TP (C). Fig. S3. Size change of cRGD-Exo/TP stored at 4°C in PBS for 7 days. Fig. S4. Size change of cRGD-Exo/TP stored at 37°C in 10% Exo-free serum for 24 h. Fig. S5. Viability of A375 cells treated with TP solution at concentrations of 10, 20, 40, 60, 80, and 100 ng/mL. Fig. S6. αvβ3 integrin protein expression in A375 and HaCaT cells. (A) Western blotting analysis of αvβ3 protein expression in A375 and HaCaT cells. (B) The relative expression levels of the protein in the cells. GAPHD was used as a loading control. Fig. S7. TP concentration in the heart, liver, spleen, lung, and kidney of the TP solution, Exo/TP, and cRGD-Exo/TP groups at 0.083, 0.5, 1, 2, 4, 6, and 24 h.