JAZ) family genes. In previous work, we obtained nine SmJAZ genes of Salvia miltiorrhiza and proved that SmJAZ8 was the core repressor of JA-induced tanshinone and phenolic acid biosynthesis. Here, we demonstrate that SmJAZ3 and SmJAZ4 act as repressors of JA-induced biosynthesis of tanshinones and salvianolic acid B . This suggests that SmJAZ3/4 are functionally redundant in tanshinone and SalB biosynthesis. SmJAZ1/2/5/6/9 are activators of JA-induced tanshinone biosynthesis and repressors of JA-induced Sal B biosynthesis. This demonstrates the redundancy and diversity of SmJAZ1/2/5/6/9 functions. Besides, SmJAZ10 inhibited JA-induced Sal B synthesis, but had no effect on the synthesis of tanshinone. Two-hybrid screening (Y2H) showed that SmJAZs formed homologous or heterogeneous dimers. Y2H and firefly luciferase complementation imaging (LCI) assays revealed that SmJAZs also formed a complex regulatory network with SmMYC2a, SmMYC2b, SmMYB39, and SmPAP1. Quantitative reverse transcription-PCR (qRT-PCR) indicated that SmJAZs regulated each other at the transcriptional level. Herein, we prove that SmJAZs have functional pleiotropism, diversity, and redundancy in JA-induced tanshinone and phenolic acid biosynthesis. This study provides an important clue for further understanding the inherent biological significance and molecular mechanisms of the JAZ family as the gene number increases during plant evolution.Jasmonate (JA) signaling regulates plant growth and development, biotic and abiotic stress tolerance, and primary and secondary metabolism biosynthesis. It is extensively modulated by JA-ZIM-domain ( Salvia miltiorrhiza Bunge is a perennial herb. Its dried roots and rhizomes are among the core traditional medicinal herbs in China and have been widely used in the prevention and treatment of coronary heart disease, chronic renal failure, arteriosclerosis, and other diseases. The active components of S. miltiorrhiza are mainly lipid-soluble tanshinones and water-soluble phenolic acids. These include tanshinone IIA (TA IIA), cryptotanshinone (CT), tanshinone I (TA I), dihydrotanshinone I (DT I), salvianolic acid A , salvianolic acid B , rosmarinic acid (RA), protocatechualdehyde and so on  studies on single Arabidopsis JAZ genes indicate that each Arabidopsis JAZ family gene except JAZ5 regulates one or more biological functions  and SmJAZ6-overexpressing line 6 (J6O6) (10.31 ± 0.5 mg/g DW), reaching respectively 9.5 and 1.35 times that present in ATCC3 (7.66 ± 1.97 mg/g DW)   and 2. Hmg/g DW) . Overexpry roots . The linry roots . The accreatment .SmJAZs significantly affected the MeJA-induced accumulation of tanshinones in hairy roots, but different genes produced different effects , contained 2.5 times the concentration found in ATCC3 (0.39 ± 0.02 mg/g DW). The accumulation of CT in transgenic line J5O1 with overexpression of SmJAZ5 and transgenic lines J9O4 and J9O14 with overexpression of SmJAZ9 were significantly higher than that of ATCC3 under MeJA treatment, and the line with the highest concentration, J9O14 (0.84 ± 0.01 mg/g DW), contained levels 3.1 times higher than that of ATCC3 (0.27 ± 0.03 mg/g DW). Overexpression of SmJAZ3 significantly reduced the accumulation of CT in hairy roots induced by MeJA. DT I and CT were not detected in transgenic hairy root lines overexpressing SmJAZ4. The accumulation of TA I in transgenic line J1O20 with overexpression of SmJAZ1, lines J2O2 and J2O6 with overexpression of SmJAZ2, J5O1 and J5O3 with overexpression of SmJAZ5, J6O4 and J6O6 with overexpression of SmJAZ6, and J9O4 and J9O14 with overexpression of SmJAZ9 was significantly higher than that of ATCC3 under MeJA treatment. The strain with the highest concentration, J9O14 (1.81 ± 0.61 mg/g DW), had levels 2.6 times greater than that of ATCC3 (0.69 ± 0.11 mg/g DW). Only the accumulation of TA IIA in transgenic line J9O14 with overexpression of SmJAZ9 (0.58 ± 0.01 mg/g DW) was significantly higher than that in ATCC3 (0.36 ± 0.02 mg DW) under MeJA treatment. The content of total tanshinones  in MeJA-induced transgenic hairy roots overexpressing SmJAZ2/5/6/9 was significantly higher than that in ATCC3, while the accumulation of TTA in MeJA-induced transgenic hairy roots overexpressing SmJAZ4 was significantly lower than that in ATCC3. It was also found that the accumulation of tanshinones in transgenic hairy roots overexpressing SmJAZ10 showed no significant change compared with the control under MeJA treatment. To sum up, under the induction of MeJA overexpression of SmJAZs markedly reduced Sal B accumulation in hairy roots and overexpression of SmJAZ1/2/5/6/9 significantly increased tanshinone accumulation in hairy roots, while overexpression of SmJAZ3 and SmJAZ4 decreased tanshinone accumulation in hairy roots.Overexpression of  effects . The accSmRAS1 and SmCYP98A14 and the tanshinone-related synthase genes SmCPS1 and SmCYP76AH1 in transgenic hairy roots were determined by qRT–PCR. There was no significant difference in the expression of related synthase genes between controls EV6 and ATCC3 , SmJAZ8 and SmJAZ10 (in J1O5) expression  can form homodimers, while 11 JAZs (AtJAZ1/2/3/4/5/6/8/9/10/11/12) can form heterodimers, with the ZIM domain mediating the formation of homodimers and heterodimers in bidopsis , 41, 61.response , 57. Intresponse .SmJAZ1/2/5/6), group III (SmJAZ10), group IV (SmJAZ8), and group V (SmJAZ3/4/9). These JAZ subgroups show different tissue expression characteristics. For example, all nine SmJAZs show different expression characteristics between the seedling and flowering periods. This study demonstrates that these nine SmJAZs have diversity in regulating JA-induced tanshinone and phenolic acid synthesis. However, the nine functional SmJAZs will present greater diversity after protein level interactions, because SmJAZs can form homo- or heterodimers, thus allowing them to regulate each other at the protein level, which is one of the forces driving the functional pleiotropism, diversity, and redundancy of SmJAZs.As the number of JAZs increased with the evolution of plants, they diverged and may be divided into different subgroups according to the characteristics of sequence structure. Clusters found are: group I  or WRKYs (WRKY18 and WRKY40). JAZ8 is resistant to JA-induced degradation along with mutants lacking a Jas domain, which results in loss of the COI1-interacting degron. The increase in expression of stable JAZ constitutes negative regulatory feedback of the JA signal; thus, the sensitivity to JA is reduced [OsbHLH148 in rice can increase the expression levels of OsJAZ1/2/3/4/5/6/10/11, suggesting that OsJAZs in rice control themselves through upregulated expression at the transcriptional level via OsbHLH148 [NtJAZ1/3/7a/10 not only have decreased nicotine content but also show regulation of the upregulated or downregulated expression of the other three JAZs, both with and without MeJA treatment. It is suggested that there is mutual regulation of NtJAZ1/3/7A/10 at the transcriptional level [In this study, overexpression of a specific  reduced , 62, 63.sbHLH148 . In tobaal level .MYC2 acts as an upstream core transcription factor interacting with JAZs to initiate downstream TF expression, JAZs, and other JA-responsive genes. The G-box is a core cis-element of MYC2-targeted JA-responsive genes and plays a key role in initiating downstream gene transcription including that of JAZs [SmMYC2a, a homologous gene of Arabidopsis MYC2 that has been shown to interact with SmJAZs in S. miltiorrhiza, has a positive role in the regulation of tanshinones and phenolic acids induced by JA, and its downstream target genes all contain G-boxes [SmJAZs  were added, respectively. The final concentration of MeJA was ~100 μM. The trichomes were harvested after 0 (harvested before treatment) and 6 days for the determination of active ingredient content, and the trichomes were harvested after 0, 0.5, 1, 3, 6, 12, 24, 48, and 72 hours for the determination of gene expression. The surface water was blotted out three times with distilled water. The trichome roots were baked at 45°C to a constant weight (~4–5 days) to determine the change in active ingredient content, and the hair roots were snap-frozen in liquid nitrogen and stored at −80°C for RNA extraction. The experiment included three biological replicates.MeJA was purchased from Shanghai Yuanye Biotechnology Co. . Hairy roots of http://xfam.org/), and used to extract full-length SmJAZ candidates from the S. miltiorrhiza genome by the HMM algorithm (HMMER) [−6. The Pfam database and the SMART database (http://smart.embl-heidelberg.de/) were used for further analysis to determine if any of the sequences obtained from screening contained the TIFY and Jas domain.Hidden Markov model (HMM) profiles of TIFY (PF06200) and Jas (PF09425) domains were downloaded from Pfam ( (HMMER) , with ann = 500 replications). The gene accession numbers used to build the evolutionary tree are presented in Supplementary Data https://www.ncbi.nlm.nih.gov/).MEGA X was utilized to create phylogenetic trees and multiple sequence alignments , using mhttps://www.ncbi.nlm.nih.gov/sutils/splign/splign.cgi), and GSDS 2.0 (http://gsds.cbi.pku.edu.cn/) and Inkscape v0.92 for mapping.Exon–intron distribution prediction was performed using Splign on NCBI (http://meme-suite.org/doc/meme-format.html).Analysis of conserved structural domains was performed using MEME (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).Gene promoters were analyzed using PlantCARE (S. miltiorrhiza actin gene (SmACT) served as an internal control and was normalized to the control samples [–ΔΔt technique [Total RNA extraction and qRT–PCR were performed using previously established procedures . Primer  samples . The relechnique . Three s−1. The gradient program was as follows: 0–10 minutes, 5–20% B; 10–20 minutes, 25% B; 20–25 minutes, 20% B; 25–40 minutes, 30% B; 40–45 minutes, 45% B; 45–50 minutes, 50% B; 50–58 minutes, 58% B; 58–67 minutes, 50% B; 67–70 minutes, 60% B; 70–80 minutes, 65% B; 80–85 minutes, 100% B; 85–95 minutes, 5% B. The detection wavelengths for salvianolic acids and tanshinones were 288 and 270 nm, respectively.Metabolite analyses followed previously described methods with a Waters 1525 HPLC . The injPCR was used to amplify the entire ORF of the SmJAZs using gene-specific primers .S. miltiorrhiza plants was accomplished using previously published procedures [Transformation of leaf explants from sterile ocedures . To deteSmJAZ plasmids was carried out using the specific primers of rolB, rolC, and the neomycin phosphotransferase II (NPT II) gene of A. rhizogenes ATCC15834, as well as the cauliflower mosaic virus (CaMV) 35S promoter and SmJAZ sequences . In order to verify that the T-DNA had fully integrated into the hairy root line genome, PCR amplification of pK7WG2R-equences .SmMYC2a, SmMYC2b, SmMYB36, SmMYB39, SmPAP1, SmTTG1, SmERF6, and SmWRKY1) were amplified by PCR with gene-specific primers , single transformant colonies were screened for growth on an SD/−Ade/–His/−Leu/−Trp medium. Interactions were observed after incubation for 3 days at 30°C.The full-length coding sequences of transcription factor genes (Agrobacterium was transformed into tobacco (Nicotiana benthamiana) leaves. Two to three days after injection, chemiluminescence images showing fluorescence intensity spectra were measured using a plant in vivo imaging system .To further validate the interaction between SmJAZs and SmTFs, LCI experiments were performed according to a previously described method . BrieflyThis work was funded by the National Natural Science Foundation of China .P.D.M., J.E.D., and Z.S.L. conceived and designed the research. P.D.M., T.L.P., B.B.L., and M.W. performed the experiments. P.D.M. and B.B.L. analyzed the data and wrote the paper.The datasets supporting the results of this article are included within the article and its supplementary file.The authors declare no conflict of interest.Horticulture Research online.Web_Material_uhac166Click here for additional data file.


CcOctβRs) from Cotesia chilonis. CcOctβRs share high similarity with their own orthologous receptors. The transcript levels of CcOctβRs were varied in different tissues. When heterologously expressed in CHO-K1 cells, CcOctβRs induced cAMP production, and were dose-dependently activated by OA, TA and putative octopaminergic agonists. Their activities were inhibited by potential antagonists and were most efficiently blocked by epinastine. Our study offers important information about the molecular and pharmacological properties of β-adrenergic-like OARs from C. chilonis that will provide the basis to reveal the contribution of individual receptors to the physiological processes and behaviors in parasitoids.Octopamine (OA) is structurally and functionally similar to adrenaline/noradrenaline in vertebrates, and OA modulates diverse physiological and behavioral processes in invertebrates. OA exerts its actions by binding to specific octopamine receptors (OARs). Functional and pharmacological characterization of OARs have been investigated in several insects. However, the literature on OARs is scarce for parasitoids. Here we cloned three β-adrenergic-like OARs ( Octopamine (OA) is a biogenic monoamine acting as a neurohormone, neuromodulator, and neurotransmitter in invertebrate physiology and behaviors . Norepin1-adrenergic-like OARs , β-adrenergic-like OARs (OctβR), and α2-adrenergic-like OARs (Octα2R) -x-[R/K]), as well as potential N-glycosylation sites (N-x-[S/T]) , Tribolium castaneum (TcOctβ1-3R), N. lugens (NlOctβ1-3R), D. melanogaster (DmOctβ1-3R), C. suppressalis (CsOctβ1-2R), Priapulus caudatus (PcOctβR), and Saccoglossus kowalevskii (SkOctβR). This clade clustered nicely with D1-like DARs and human β-adrenergic receptors  values for tested compounds were calculated from concentration-response curves for CcOctβ2R and CcOctβ3R  on a non-saturating concentration of OA in the CHO-K1 cells stably expressing each receptor. The reduction in cAMP level was quantified and normalized to the value achieved in the absence of antagonists (=100%). Chlorpromazine, cyproheptadine, metoclopramine, mianserin, phentolamine, methiothepin, epinastine, spiperone, SCH-23390, clozapine, asenapine, amitriptyline and doxepin displayed the antagonistic effects for each receptor, butaclamol, cinanerin and chlorprothixene inhibited the activation for CcOctβ2R and CcOctβ3R .50) were determined from the dose-response curves and shown in 50 values of methiothepin, clozapine and chlorprothixene for CcOctβ1R were not calculated (50s ranging from ~7.34 × 10−9 M (CcOctβ3R) to ~3.76 × 10−8 M (CcOctβ1R)  . AsenapicOctβ1R) . For eaccOctβ1R) .D. melanogaster [Bombyx mori [Periplaneta americana [A. mellifera [N. lugens [P. xylostella [C. chilonis, investigated their expression profiles, and pharmacologically characterized them.The availability of transcriptome and genome information provided an opportunity to study the physiological and behavioral roles of the insect octopaminergic signaling system. To meet this opportunity, determining the molecular and pharmacological data of OAR subtypes is an important step. OARs have been characterized in nogaster ,40,54, Bbyx mori ,56, Perimericana ,58, A. mellifera ,59,60, N. lugens ,49, and lostella ,46,61,623.49RY motif at the cytoplasmic of TM3 and N7.49PLIY in TM7 , acts as an important residue that interacts with the protonated amino group of OA or TA. One of the closely grouped Ser residues (S5.42/5.46) in TM5  can interact with the hydroxyl group of OA or TA [6.44-X-X-C6.47-W6.48-X-P6.50-F6.51-F6.52 is highly conserved in TM6 of CcOctβRs. W6.48 and F6.52 residues were reported to interact with the ligand aromatic ring, which modulates the bend angle of TM6 near the well conserved kink at P6.50, prompting the cytoplasmic end of TM6 to move upon activation [CcOctβRs belong to the class A GPCR family, which are supported by DY in TM7 . Most clOA or TA ,64. The tivation .A. mellifera [Our phylogenetic tree included major insect biogenic amine receptors and vertebrate adrenergic receptors. CcOctβRs were closely related to their orthologous receptors, and they clustered nicely with D1-like DARs and human β-adrenergic receptors , highligellifera , and phyellifera . Additioellifera . Thus, tCcOctβ1R, CcOctβ2R and CcOctβ3R varied in different tissues  and 3.90 × 10−8 M (CcOctβ1R) , 1.82 × 10−9 M (AmOctβ2R), and 3.30 × 10−9 M (AmOctβ3R) [D. melanogaster, EC50 values were 5.56 × 10−9 M (DmOctβ1R), 1.53 × 10−8 M (DmOctβ2R), and 1.40 × 10−8 M (DmOctβ3R) [50s were shifted about two orders of magnitude to higher concentrations  . Thus, amOctβ3R) . In D. mtrations . SimilarB. mori, and DPMF was more potent than amitraz and OA [Varroa destructor, and VdOctβ2R was more sensitive to amitraz and DPMF than AmOctβ2R. Furthermore, behavioral assays using D. melanogaster OAR mutants identified Octβ2R as the sole target of amitraz [As a formamidine acaricide, amitraz is particularly effective against mites and ticks, as well as hemipteran and lepidopteran insects. Amitraz and its main metabolite DPMF act as high-affinity agonists of OARs . For CcOz and OA . For PxOz and OA ,70. The  amitraz .2 serotonin receptor antagonist in mammals but also a potent antagonist of OARs, TARs and DARs in insects [A. mellifera, the physiological and behavioral effects of mianserin have been studied, suggesting that it could affect gustatory and olfactory learning abilities [50 values for CqDOP2 and AaDOP2 [Here, the interactions of three CcOctβRs with a series of potential antagonists were studied. Epinastine is an antagonist of vertebrate histamine H1 receptor, and also acts as an OAR antagonist in insects. Our results indicated epinastine was the most potent on CcOctβRs , and sim insects ,75. Mian insects ,41. In Abilities ,77. Spipbilities ,79, and d AaDOP2 ,57,60, ad AaDOP2 , CsOctβ2d AaDOP2 , and NcOd AaDOP2 .Overall, our study supports the proposition that OA signaling is highly complex in insects. Therefore, establishing pharmacological profiles of molecularly identified and heterologously expressed receptors is a valuable challenge, and by uncovering substances acting through individual receptors, it is possible to facilitate the design of novel in vivo pharmacological experiments which accurately address a specific physiological feature. This detailed pharmacological characterization of three CcOctβRs marks an important step towards precisely understanding the role of OA in this parasitoid.C. chilonis has been continuously reared on its host C. suppressalis. They were initially obtained from rice fields of the experimental farm . They were kept at a temperature of 28 ± 1 °C, a photoperiod of 16:8 h (L:D) and relative humidity of 75 ± 5%. After eclosion, the wasp adults were maintained in the glass containers and fed on 20% (v/v) honey solution [Laboratory colony of solution ,80.C. chilonis was performed [Transcriptome sequencing of erformed  and threC. chilonis adults were sterilized with 75% ethanol, and their heads were then dissected into the TRIzol reagent . Total RNA was isolated according to the manufacturer’s protocol. The NanoDrop 2000 spectrophotometer  was used to determine the purities and concentrations of RNA samples. cDNA was synthesized with 1 μg RNA via TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit . The full lengths of CcOctβRs were cloned by RT-PCR . The putative PKC phosphorylation sites were identified by the NetPhos 3.1 Server . The potential N-glycosylation sites were predicted by the NetNGlyc 1.0 Server . To identify the potential orthologues of the cloned C. chilonis β-adrenergic-like OARs, the phylogenetic analysis of these receptors was performed with other insect biogenic amine receptors and human adrenergic receptors. The phylogenetic tree was constructed with MEGA 7.0 using the neighbor-joining method and 1000 bootstrap tests [The amino acid sequences of β-adrenergic-like OARs from ap tests  and editap tests .CcOctβ1R, CcOctβ2R and CcOctβ3R in various tissues of wasp adults, total RNAs from the heads without antennae, thorax, abdomen, antennae, and legs were extracted using the TRIzol reagent . The qRT-PCR primers were designed with Primer 3   with the following procedure: 95 °C for 30 s, and then 40 cycles of 95 °C for 5 s and 60 °C for 30 s with the melt curve dissociation step. qRT-PCR was done in a 25 μL volume containing 12.5 μL TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) , 1 μL each primer (10 μM), 2 μL cDNA, and 8.5 μL sterile H2O. Reactions for each sample were performed in triplicate. The 28S rRNA was used as the reference gene to calculate the expression levels of CcOctβ1R, CcOctβ2R and CcOctβ3R [−ΔΔCT method [To investigate the relative expression levels of ne 2022) . qRT-PCRCcOctβ3R ,83, and T method .® II One Step Cloning Kit . All expression constructs were finally verified by DNA sequencing . The high-quality expression plasmids were prepared with the EndoFree Plasmid Midi Kit .The eukaryotic expression vectors of CcOctβ1R, CcOctβ2R and CcOctβ3R were generated by RT-PCR using the specific primers , with th2 in an incubator. The culture medium was composed of DMEM/F-12 medium  and 10% fetal bovine serum . To prevent the contaminations of gram-positive and gram-negative bacteria, 100 units/mL penicillin and 100 mg/mL streptomycin  were added to the culture medium, respectively. The plasmids of pcDNA3.0-CcOctβ1R, pcDNA3.0-CcOctβ2R and pcDNA3.0-CcOctβ3R were transfected into CHO-K1 cells at the confluency of 70–90% in Petri dishes (3.5 cm). The Opti-MEM® Reduced Serum Medium (1 mL)  containing 6 μL Lipofectamine® 2000  and 3 μg plasmid was prepared as transfection medium. After incubation for 20 min at room temperature, the transfection medium was supplemented after removing the growth medium. After 4–6 h in the incubator, the transfection medium was substituted with the fresh growth medium. Transfected cells were selected with the antibiotics G418  for 2–3 weeks, and G418-resistant cell clones were trypsin digested and transferred to cell culture plates (12-well) for expansion. The individual cell lines were monitored by immunocytochemistry for receptor expression, and then the stably transfected cells were cultured in the growth medium supplemented with 250 μg/mL G418.Chinese hamster ovary K1 (CHO-K1) cells were cultured at 37 °C with 5% COg for 15 min. The cAMP Parameter Assay Kit  was used to determine cAMP in the supernatant. All tested compounds were dissolved in dimethyl sulfoxide, and further serially diluted with D-PBS.Ligand stimulation of intracellular cAMP production was measured as described previously ,70,78. Tp < 0.05, ** p < 0.01, *** p < 0.001). The EC50 and IC50 values were calculated using a special dose-response, nonlinear regression model.GraphPad Prism version 9.1.1  was used to analyze and display the data. Differences in transcript levels were analyzed by a one-way ANOVA followed by Tukey’s honestly significant difference (HSD) test. To examine whether statistical differences occurred in pharmacological comparisons, a one-way ANOVA followed by Dunnett’s multiple comparison test was performed (*


The structural, morphological and optical properties of as-synthesized composites were characterized by X-ray powder diffraction, scanning electron microscopy and UV-Vis diffuse reflectance spectroscopy, respectively. α-FeOOH–TiO2 composites and TiO2-P25, as reference, were evaluated as photocatalysts for the disinfection of Escherichia coli under UV or visible light in a stirred tank reactor. α-FeOOH–TiO2 exhibited better photocatalytic activity in the visible region than TiO2-P25. The mechanical activation increased the absorption in the visible range of TiO2-P25 and the photocatalytic activity of α-FeOOH–TiO2. In the experiments with UV light and α-FeOOH–TiO2, mechanically activated, a 5.4 log-reduction of bacteria was achieved after 240 min of treatment. Using visible light the α-FeOOH–TiO2 and the TiO2-P25 showed a 3.1 and a 0.7 log-reductions at 240 min, respectively. The disinfection mechanism was studied by ROS detection and scavenger experiments, demonstrating that the main ROS produced in the disinfection process were superoxide radical anion, singlet oxygen and hydroxyl radical.Goethite–titania (α-FeOOH–TiO 2 composite is proposed under UV-Vis light, the FeOOH–TiO2 composite showed higher photocatalytic activity than TiO2-P25.A photocatalytic mechanism for FeOOH–TiO However, this technique can be improved using non-toxic and earth-abundant oxides with photocatalytic activity. TiO2 is a semiconductor employed in disinfection processes.3–9 The disinfection mechanism involves the production of reactive oxygen species (ROS), under UV light, through oxidation and reduction reactions by holes (h+) and electrons (e−) in the valence and conduction bands of TiO2, respectively. The main ROS produced are superoxide radical anion (O2˙−), singlet oxygen (1O2), hydroxyl radical (·OH) and hydrogen peroxide (H2O2). Investigations related to TiO2 have been carried out to decrease the recombination rate of e−–h+ pairs and increase its absorption to the visible range. Goethite (α-FeOOH) is an abundant iron oxyhydroxide,10 with a band gap value of 2.1 eV.11 Few works have been reported using FeOOH–TiO2 composites for E. coli disinfection.12,13 In these works, the route used to obtain the composites was the hydrothermal method. Chowdhury and Mpongwana12 studied the FeOOH–TiO2 composite, FeOOH was akaganeite (β-FeOOH), for E. coli disinfection in presence of H2O2 as electron acceptor; Mangayayam et al. studied the disinfection efficiency of E. coli using Ag–TiO2–FeOx (mainly goethite 37.3%) nanotubes.13 Goethite–titania composites can show enhanced photocatalytic activity due to the related studies reporting the increase of TiO2 light absorption attributed to Fe doping or iron oxides addition, and the photo-Fenton and photocatalytic activity of goethite.11,14–16 Additionally, the mechanical activation, through ball milling, has shown to create defects in materials, which contributes to improving its catalytic properties.17,18In 2017 the WHO/UNICEF reported that 844 million people did not have a drinking water facility. Microbial contamination  0.04 M until pH 13 and kept under constant stirring with air sparging, after 4 h a yellow dark precipitate was obtained. The precipitate was washed with deionized water several times and centrifuged at 11 000 rpm for 10 min . Finally, it was dried at 90 °C for 24 h.Goethite was synthesized by co-precipitation method Fig. S1. A solut2.2in situ  was added to the solution after 4 h of goethite synthesis and kept for 1 h under constant stirring with air sparging. The second technique was by mechanical activation of TiO2-P25 and goethite previously synthesized  with Cu Kα radiation at 40 kV and 30 mA. The morphology was investigated by scanning electron microscopy (FE-SEM JEOL JSM-7041F and FEI Nova Nanosem 200). The UV-visible absorption was analyzed by diffuse reflectance in a UV-visible spectrometer .2.42.4.1.λ < 285 nm). For UV experiments the suspension was irradiated using a Hg–Ne lamp . The visible assays were carried out with a 1.8 W low-intensity LED lamp, with an emission range from 420 nm to 620 nm, as shown in Fig. S5.−9 einstein per cm2 per s) and Reinecke's salt (4.47 × 10−8 einstein per cm2 per s) actinometries were performed for UV and LED lamps, respectively.19The photochemical reactor employed for the disinfection experiments consisted of a borosilicate glass beaker of 0.2 L surrounded by a water-jacket and ports for sampling and air sparging Fig. S4. The lam2.4.2.E. coli K-12 (ATCC 25404) was incubated aerobically at 37 °C in Luria Bertani (LB) broth (Sigma Aldrich). After 18 h of incubation, the bacteria were collected giving a concentration of 108 CFU mL−1. The bacteria suspension was centrifuged at 4500 rpm for 10 min, washed three times with phosphate buffer pH 7. Finally, the bacteria were diluted to 106 CFU mL−1 in saline solution.2.4.3.E. coli (106 CFU mL−1), UV or visible light irradiation, air sparging and kept at 20 °C ± 2. The disinfection of E. coli was evaluated employing TiO2-P25 , goethite and FeOOH–TiO2 composites obtained by in situ and mechanical activation. First, disinfection experiments using TiO2-P25, as reference, at different concentrations  were performed in order to select the concentration with higher disinfection efficiency. The concentration of 300 mg L−1 was selected to compare the efficiency of the materials described in The experiments were performed using 0.2 L of −1 and was achieved inoculating 500 μL of sample. The plates were incubated for 24 h at 37 °C. Control experiments were made without material to evaluate the photolysis and with material in the dark.Aliquots were collected at different times for 300 min and bacteria concentration was determined using the standard plated counting method on LB agar by triplicate. The detection limit was 2 CFU mL2.52.5.1.p-Nitrosodimethylaniline (RNO) and imidazole has been reported for singlet oxygen detection.20–22 TiO2-P25, TiO2-P25-M, G, 1 : 3-M, 1 : 1-M, 3 : 1-M, 1 : 3-I, 1 : 1-I or 3 : 1-I at 300 mg L−1 were suspended in a solution containing RNO (Sigma Aldrich) 45 μM and imidazole (Sigma Aldrich) 8 mM. The experiments were performed under UV or visible light. Aliquots were taken during 24 min and centrifuged at 12 000 rpm for 15 min. The RNO concentration was determined at 440 nm in a Varian Cary 50 UV-Vis spectrometer. Negative controls were made without material.2.5.2.22H16N7NaO13S2) is reduced by O˙−2 giving the product XTT-formazan.23 In 100 mL of XTT 100 μM (Sigma Aldrich), 300 mg L−1 of the corresponding material was placed and kept under magnetic stirring. The solutions were prepared in deionized water. The experiments were performed under UV or visible light. Every 2–3 min samples were collected and centrifuged. The concentration of XTT-formazan was determined at 470 nm. Controls were performed without material.XTT sodium salt , 300 mg L2.5.4.iv) oxysulfate (Sigma Aldrich) 280 mM and sample, material in saline solution previously irradiated with UV or visible light, were mixed and its absorbance was read at 410 nm.26Equal volumes of acidified titanium was used as hydroxyl radical scavenger; KI (10 mM) quenched surface holes and surface bounded ·OH. The reduction pathway was inhibited by introducing nitrogen into the solution.Disinfection experiments were performed with different scavengers. 2.7E. coli disinfection experiments in presence of 1 : 3-M (300 mg L−1) and H2O2 (10 mg L−1), as electron acceptor, under visible light were performed.3.2-P25 spherical particles with a longitude of 126.5 ± 29 nm and 22.1 ± 2 nm, respectively. 2-P25 show the same morphology that 1 : 3-I and longitudes of 86.1 ± 18 nm and 19.4 ± 6 nm, respectively. FE-SEM images of P25, P25-M and G are in Fig. S6.†SEM characterization was carried out to investigate the shape and size of the photocatalysts. et al., the reduction in the band gap value after mechanical activation can be related with the change in bond angles and lengths in the crystal structure by the impact during the milling process.27According to Dannangoda 3.12-P25 at 72, 150, 300 and 500 mg L−1 with UV light is shown. TiO2-P25 at 300 mg L−1 reached a 4.5 log-reduction after 240 min of irradiation. With 72 and 150 mg L−1 of TiO2-P25 log-reductions of 3.7 and 3.8 at 240 min were achieved, respectively. TiO2-P25 at 500 mg L−1 gave the lower disinfection efficiency. The E. coli concentration remained constant during the 300 min in the dark. In the photolytic experiment, a 0.5 log-reduction at 240 min was observed. The following experiments with goethite and the composites under UV or visible irradiation were performed at 300 mg L−1.In 2–FeOOH (1 : 3-M) is more photocatalytic active under UV light than TiO2-P25. With 300 mg L−1 of TiO2–FeOOH (1 : 3-M) a disinfection efficiency of 5.1 log-reduction was reached at 240 min  for the control, P25, P25-M, G, 1 : 3-M and 1 : 3-I are 5 × 10−6, 0.0119, 0.0281, 0.0005, 0.0306 and 0.007, respectively. The data were analyzed by a factorial design and the interaction graphs for each ROS test are in the ESI  for the control, P25, P25-M, G, 1 : 3-M and 1 : 3-I are 0.0006, 0.0241, 0.0394, 0.0084, 0.0327 and 0.0318, respectively.For superoxide detection  the XTT 1O2 generates an intermediate that reacts with RNO.20 The singlet oxygen detection (1O2 under UV irradiation. The reaction rate coefficients (k) for the control, P25, P25-M, G, 1 : 3-M and 1 : 3-I are 0.0003, 0.0411, 0.0561, 0.0006, 0.101 and 0.0197, respectively.According to Kralji and El Mohsni, the reaction of imidazole with etection  showed tiv) ions showed no significant H2O2 formation during 24 min under UV or visible light. The same result was observed using different material loadings (data not shown).The tests between 1 : 3-M and Ti showed a reduction in the disinfection efficiency compared to 1 : 3-M without TBA , a molecular probe that shows more sensitivity than the RNO-imidazole used in the present work.29 Also, the UV-visible absorption spectra of TiO2-P25 and TiO2-P25-M (Goethite did not show appreciable ROS production to determine its photoactivity; however, this does not mean that it does not contribute to the photocatalytic activity observed in the FeOOH–TiOeactions . AccordiO2-P25-M  shows th4.2 composite is a photocatalyst with increased disinfection activity than TiO2-P25. The better disinfection efficiency using UV or visible light was observed with FeOOH–TiO2 treated with mechanical milling. The ROS detected employing UV light were hydroxyl radical, superoxide radical anion and singlet oxygen. A reduction in the disinfection efficiency was detected when hole and hydroxyl radical scavengers, KI and tert-butanol, were added. Under N2 sparging the same effect was observed. A photocatalytic mechanism is proposed based on the band edge positions of TiO2 and FeOOH, and the ROS detected. Where the hydroxyl radical and singlet oxygen are generated after oxidation of H2O and superoxide radical in the valence band, respectively, and the superoxide is produced after oxygen reduction in the conduction band of the TiO2-P25.FeOOH–TiOThere are no conflicts to declare.RA-009-C8RA08412B-s001


We found that the rooting abilities of poplar cuttings with and without leaves are different. By applying different concentrations of IBA and sucrose to the top of cuttings without leaves, we found that 0.2 mg/ml IBA and 2 mg/ml sucrose had the best effect on promoting AR formation. The transcriptome results indicated photosynthesis may influence AR formation in poplar cuttings with leaves and revealed a potential regulatory mechanism of leafy cuttage from poplar cuttings. In addition, we provided a new perspective to resolve rooting difficulties in recalcitrant species.The formation of adventitious roots (ARs) is vital for the vegetative propagation of poplars. However, the relevant mechanisms remain unclear. To reveal the underlying molecular mechanism, we used RNA-seq to investigate the transcriptional alterations of poplar cuttings soaked in water for 0, 2, 4, 6, 8, and 10 d; 3,798 genes were differentially expressed at all the time points, including 2,448 upregulated and 1,350 downregulated genes. Biological processes including “cell cycle,” “photosynthesis,” “regulation of hormone levels,” and “auxin transport” were enriched in the differentially expressed genes (DEGs). KEGG results showed that the common DEGs were most enriched in the pathway of “Carbon fixation in photosynthetic organisms” and “Starch and sucrose metabolism.” We further dissected 38 DEGs related to root and auxin, including two  Cuttage is the most efficient way for vegetative propagation of tree species because it can maintain valuable traits of woody plants . HoweverArabidopsis, AFB2-IAA12/28-ARF5 and TIR1-IAA14-ARF7/19 are involved in the development of lateral roots , LAX (LIKE AUXIN RESISTANT), and SAUR  have also been reported to have mediated the auxin transport signal, and SAURs may be involved in cell expansion at the same time , AIR (auxin-induced in root), and AUR (rooting-associated genes)  . RGF (rod genes) ; RGF1 anbidopsis .P. simonii × P. nigra, also named as P. xiaohei, is a hybrid and commercial tree species , sucrose , and a mixed solution of IBA and sucrose were soaked in water. After different treatments, the cuttings will continue hydroponic rooting in the greenhouse at a temperature of 26 ± 1°C and a light time of 16-h/8-h (light/dark) photoperiod. During the rooting period, different concentrations of IBA or sucrose were added to the absorbent cotton at the top of the cuttings every day, and the slow-release effect of the absorbent cotton was used to make the hormones absorbed by the cuttings. After different treatments, the rooting of the cuttings was observed at 7, 14, and 21 d, respectively.Three-month-old plants of We used different concentrations of IBA and sucrose, and the combinations of IBA, sucrose, and NPA to investigate the effects of auxin and sucrose on promoting AR formation; 0.05, 0.1, 0.2, 0.5, and 1 mg/ml IBA  and 0.5, 1, 1.5, 2, and 5 mg/ml sucrose  were applied to the top of the cuttings, respectively; 0.2 mg/ml IBA + 2 mg/ml sucrose, 0.2 mg/ml IBA + 0.2 mg/ml NPA , 2 mg/ml sucrose + 0.2 mg/ml NPA, and 0.2 mg/ml IBA + 2 mg/ml sucrose + 0.2 mg/ml NPA were used as different combinations; 1 ml of each of the above substances was applied to the top of the cuttings at a 24-h interval.P. xiaohei cuttings with different treatments at 0, 2, 4, 6, and 8 days were collected and the bases were cut into 150–300 μm transverse sections by hand. All sections were treated with the concentration of 3% phloroglucinol  and 30% concentrated hydrochloric acid  for 5 min, and then the rooting of the cutting base was observed by an optical microscope  with 40 times magnification (×40/0.9).https://sciex.com.cn/; MS Applied Biosystems 6500 Triple Quadrupole, https://sciex.com.cn/). The analytical conditions were as follows: LC: column, Waters ACQUITY UPLC HSS T3 C18 (100 mm × 2.1 mm i.d. 1.8 µm); solvent system, water with 0.04% acetic acid (A), acetonitrile with 0.04% acetic acid (B); gradient program, started at 5% B (0–1 min), increased to 95% B (1–8 min), 95% B (8–9 min), finally ramped back to 5% B (9.1–12 min); flow rate, 0.35 ml/min; temperature, 40°C; injection volume, 2 μl  ; 10 μl internal standard mixed solution (100 ng/ml) was added into the extract as internal standards (IS) for the quantification . The mixme, 2 μl .A fresh plant sample (same as hormone measurement) was harvested, immediately frozen in liquid nitrogen, and stored at −80°C; 0.15 g of plant material and 1 ml of water were ground into powder. Both glucose standard solution and anthrone extraction reagent use a soluble sugar kit . Measurement solution was dissolved in 15 ml methanol/water/concentrated sulfuric acid  uL. A spectrophotometer was used to measure the wavelength of the solution at the wavelength of 630 and 645 nm, respectively .The stem bases of cuttings with leaves (0.5–1 cm) were harvested from 0 to 10 d with a 2-day interval for RNA extraction, cDNA library construction, and RNA-sequencing. Total RNAs of the samples were extracted using the RNA pre-Pure Plant Plus Kit . The RNAs were assessed using Qubit Fluorometer and Agilent 2100 Bioanalyzer, and then used for cDNA library construction according to the user manual. The cDNA libraries were sequenced by the MGISEQ-2000 platform at ANOROAD-Beijing . We obtained 12 cDNA libraries, two replicates for each time point. After filtering out low-quality reads, all the clean sequencing data have been submitted to the NCBI SRA (Sequence Read Archive) database (Accession No. PRJNA798213).Populus trichocarpa v4.1) with parameters recommended by RSEM  pipeline. STAR (Spliced Trans Alignment to a Reference) was used to align the sequencing reads to the reference genome . Using the NCBI primer blast platform to design qRT-PCR-specific primers, the detail of the specific primer sequence is shown in 2O .P. xiaohei cuttings, cross sections of the bases were stained with phloroglucinol and observed using an optical microscope. By cross-cutting the bases of P. xiaohei cuttings soaked in water, we found that the xylem, cambium, and epidermis showed a gradual and orderly layered structure within 4 days , and the average reads mapping rate was 82.91%  involved in AR formation. The significantly enriched BPs included “cell cycle,” “photosynthesis,” and “response to an inorganic substance,” which contained 119, 52, and 140 DEGs and were enriched 2.47, 2.56, and 1.8 folds, respectively . The enrt of ARs . Seventet of ARs . The resKEGG enrichment analysis was also performed to identify enriched pathways. The results showed that the common DEGs were significantly enriched in 22 pathways . The sigArabidopsis is the most well-studied plant, we used its annotations to further dissect the common DEGs  showed a continuously raising profile and was increased around 1.8–2 times for all stages. LBDs are mainly regulated by ARFs and are responsible for the development of lateral organs , Potri.008G072800 , and Potri.007G039500  were up-regulated during AR development, and were increased up to 8.5, 13.6, and 2.5 times, respectively  is the largest plant-specific family of auxin-responsive factors, which respond to early auxin signals and regulate auxin transport  showed an elevated expressional profile during the AR formation, and the final expression level was increased 12.7 times  was increased 12 times at 10 d. PIN-Form (PINs) were auxin efflux carrier family protein  and Potri.002G072200  were homologous genes and showed the same increasing tendency. Potri.016G035300  was up-regulated at 2 days and decreased slightly at 8 days. ATP-binding cassette (ABC) transporters positively regulate auxin efflux and influence basipetal auxin transport in the root  exhibited an upregulated expression pattern during AR formation and peaked at 6 d (LIKE AUXIN RESISTANT (LAX) and AUXIN RESISTANT (AUXs) act as auxin transports; only Potri.004G172800  shows an upregulated expression pattern during AR formation  was increased up to 4.9, 20.9, 24.2, 50.7, and 30.4 times on different days, which was a member of the Aux/IAA protein family and involved in lateral root development  increased continuously, which may be located upstream of the auxin regulatory pathway , one root meristem growth factor (RGF9) and one auxin-induced in the root (AIR12) (AUR1 and AUR3) were elevated during the AR formation. The expression level of Potri.009G070800  was increased 4.6, 8.6, 14.2, 16.6, and 11.6 times during the AR formation process  was increased 2.3 times at 2 d. Potri.002G249200  was decreased 23.4 times at 10 days. Potri.018G057700 and Potri.006G235000 , and Potri.006G080500  showed elevated expressional profiles (The DEGs related to root development included two homolog genes of  (AIR12) . Three r process . The expprofiles . The resPnARF5, PnGH3.17, PnIAA14, PnLAX2, PnLBD4, PnLRP1, PnPIN6, PnRGF9, and PnSAUR52. The qRT-PCR results  was applied to the top of the cuttings without leaves . We applex vitro survival  located in the miR390 promoter   which has been identified as a candidate gene by the QTL mapping method was up-regulated during AR formation in our results  positively regulates the expression of promoter . In our bidopsis . We founosum L.) . In our  results .Potri.001G027700, Potri.002G092700, Potri.001G288301, Potri.001G011300, and Potri.019G057500 highly expressed in root cell and related to AR initiation and elongation. Auxin signal transduction has the function of maintaining hormone homeostasis during AR development , a key enzyme involved in the biosynthesis of flavonoids  responds to red and blue lights and co-expressed with BBX32 (Potri.009G140500 is involved in the chloroplast signaling recognition particle pathway targeted by LHCP and response to bright light (P. xiaohei cuttings.In RNA-seq results, we identified some DEGs associated with rooting and auxin signals. Of these genes, ARFs have been reported to mediate the auxin signaling pathway, thereby affecting plant AR formation and development . ARF5 shelopment . Potri.0elopment . Potri.0elopment . Potri.0elopment . Some oravonoids . Flavonoavonoids . The devavonoids . Potri.0elopment . Among tormation . Photosyormation . The BP ng light . The expth BBX32 . Potri.0ht light . We impl


Verasper variegatus. We aimed to identify the optimum diluents, temperatures, dilution ratios, antibiotics, and antioxidants for sperm motility and cell viability. The diluents evaluated were marine fish Ringer’s solution (MFRS), Stein’s solution, 300 mM sucrose, and 300 mM glucose . Neomycin and gentamycin  and antioxidants  were assessed in terms of sperm preservation. The most effective condition for cold storage of spotted halibut sperm was Stein’s solution at a dilution ratio of 1:4 at 2 °C, with a combination of neomycin 800 mg/L and 250 mM trehalose that showed spermatozoa motility of > 43% after 60 days. These storage conditions will be valuable for spotted halibut hatcheries.Intermediate-term preservation of sperm assists the reproductive management of fish spermatozoa; however, no information is available on sperm of the spotted halibut,  Verasper variegatus) is categorized is an endangered species in South Korea, Japan, and China [Paralichthys olivaceus) [Limanda yokohamae), brown sole (Limanda herzensteini) [Platichthys stellatus) [Larimichthys polyactis) [Epinephelus lanceolatus) [The spotted halibut (nd China . Therefond China . Short-tnd China . Such shnd China . In pracivaceus) , marblednsteini) , starry ellatus) , yellow lyactis) , and giaeolatus) . HoweverThe successful storage of fish sperm depends on numerous factors, including extenders, dilution ratio, temperature, and antibiotics ,7. DurinStorage itself and cell metabolism create free radicals, which damage polyunsaturated fatty acids in the cell membrane and disrupt sperm DNA . An antiWe tested the effects of diluents on spotted-halibut sperm quality during short-term storage. We also evaluated the influence of sperm diluent ratio, sperm storage temperature, antibiotics, and antioxidants on sperm quality.The research was performed from January to May 2021. In the Marine Seed Hatchery in Yeosu-si, Jeollanam-do, South Korea, 12 mature male spotted-halibut broodstock of average length 36.06 ± 0.28 cm and average weight 373.4 ± 4.4 g were maintained in seawater at a temperature of 10–12.5 °C, salinity of 30.1 ± 1.4 Practical Salinity Units (PSU), dissolved oxygen of 7.4 mg/L, and pH of 7.8. During the experiment, the fish were twice-daily recipients of commercial floatable feed . For milt collection, the fish were sedated using 50 mg/L 2-phenoxyethanol. Samples of sperm were obtained by stripping and promptly collected using 1.0 mL plastic syringes (without needle). Care was taken to avoid contaminating sperm with water, urine, blood, or feces. The sperm was transferred to 1.5 mL microtubes in an icebox (constant temperature of 4 °C) and immediately transported to the Marine Aquaculture Laboratory of Mokpo National University, South Korea for assessment. Only sperm of motility > 80% by microscopy were used in this study.2, 4.6 g of MgCl2, and 0.5 g of NaHCO3 per liter of distilled water kept at 4 °C), placed into a Leja slide 20 µm , and tested at 10 °C of room temperature. The motion characteristics investigated were the percentage of moving sperm (motility); path velocity (VAP), quantifies the speed of the sperm head along its average spatial path; curvilinear velocity (VCL), the average speed measured along the actual point to point path taken by the cell; straight-line velocity (VSL), the average velocity of a sperm head along the straight line between its initial and final detected places; percentage straightness (STR), calculated as VSL/VAP × 100; and percentage linearity (LIN), which is the linearity of the curvilinear trajectory as VSL/VCL × 100 [Computer-assisted sperm analysis was conducted using the CEROS II instrument  equipped with a Zeiss Axiolab 5 microscope at 10× magnification, including a CM-040GE camera with 0.4-megapixel resolution at 60 frames per second . Each sample was diluted in a ratio of 1:200 with artificial seawater  test  as described previously . At a vo6H12O6 0.10 g, and NaHCO3 0.20 g L−1 distilled water), marine fish Ringer’s solution (MFRS) , 300 mM sucrose, or 300 mM glucose. The solutions were loaded in 1.5 mL microfuge tubes and refrigerated at 4 °C. Sperm motility, kinematic characteristics, duration motility, and survival were assessed at 5-day intervals until spermatozoa ceased movement.To identify the optimum diluent, sperm was diluted 1:2 (sperm: diluent) with Stein’s solution , loaded in 1.5 mL microfuge tubes, and kept at 0, 2, 4, or 6 °C in a refrigerator. Three replicates were performed. Sperm motility, kinematic characteristics, duration motility, and viability were evaluated at 5-day intervals until spermatozoa ceased movement.Sperm were diluted in Stein solution at 1:1, 1:2, 1:4, and 1:10 (sperm: diluent). In triplicate, the solutions were put in 1.5 mL microfuge tubes and refrigerated at 2 °C. Sperm motility, kinematic characteristics, duration motility, and viability were evaluated at 5-day intervals until spermatozoa ceased movement.Sperm were diluted at 1:4 (sperm: Stain’s solution) in triplicate, neomycin and gentamycin  were added, and the samples were stored in a refrigerator at 2 °C. Sperm motility, kinematic parameters, duration motility, and viability were evaluated at 5-day intervals until spermatozoa creased movement.Sperm were diluted 1:4 in Stain’s solution with the optimum neomycin or gentamycin concentration (as determined in p > 0.05) were selected for statistical evaluation. The variables are presented as means and standard errors, and the percentage data was normalized prior to analysis via arcsine square root transformation. For the analysis, the SPSS version 23 software was used. One-way ANOVA and Duncan’s multiple-range test (p < 0.05). Diluent significantly affected sperm motility, kinematic parameters, duration motility, and viability during short-term storage (p < 0.05). The percentage sperm motility in samples stored without diluent (control) decreased significantly after 5-day storage. Sperm stored in Stein’s solution remained motile for 15 days (19.49 ± 0.31%), whereas sperm stored in MFRS, 300 mM sucrose, and 300 mM glucose remained motile for only 10 days. Sperm velocity and cell viability exhibited similar trends. The optimum storage conditions were Stain’s solution—VAP (42.78 ± 1.79 µm/s), VCL (50.51 ± 4.37 µm/s), VSL (41.80 ± 1.32 µm/s), cell viability (31.53 ± 0.92%), and duration motility (1.89 ± 2.49 min)  were > 65% in all treatment groups (.49 min) . Sperm vp < 0.05). Sperm storage in Stein’s solution at a 1:4 sperm: diluent ratio significantly increased (p < 0.05) sperm motility (24.91 ± 2.33%), VAP (25.67 ± 1.10 µm/s), VCL (35.57 ± 1.31 µm/s), VSL (21.68 ± 1.15 µm/s), and duration motility (2.05 ± 2.05 min) compared to a 1:1, 1:2, or 1:10 ratio (p < 0.05) at a 1:2 dilution ratio. Therefore, a 1:4 dilution ratio was used in subsequent experiments.Dilution ratio significantly affected sperm motility, kinematic parameters, viability, and duration motility during short-term storage (10 ratio  for 15 dp < 0.05). Sperm diluted 1:4 in Stein’s solution stored at 2 °C retained their motility (5.04 ± 0.72%), viability (11.24 ± 0.39%), VAP (11.33 ± 1.28 µm/s), VCL (10.95 ± 1.59 µm/s), VSL (13.68 ± 2.77 µm/s), and duration motility (0.85 ± 1.84 min) for 30 days, and for 25 days at 0 °C . Sperm quality increased with increasing gentamicin and neomycin concentrations. The use of antibiotics prolonged sperm viability by up to 60 days. In the antibiotic-free group, sperm motility was lower and immotile on day 35. Gentamicin and neomycin at 800 mg/L increased sperm motility (31.92 ± 2.23 and 37.36 ± 2.91%), VAP (53.86 ± 2.70 and 57.43 ± 1.90 µm/s), VSL (52.14 ± 2.39 and 55.10 ± 2.74 µm/s), VCL (50.60 ± 2.71 and 51.94 ± 3.37 µm/s), and viability (18.64 ± 1.46 and 19.13 ± 2.05%) for 60 days in cold storage . The lowest sperm quality resulted from 25 µM MT in combination with neomycin. In the presence of 800 mg/L gentamicin, maximum sperm motility and viability were obtained using 150 µM MT , followed by 125 µM MT (36.37 ± 2.40% and 26.05 ± 2.23%) (p > 0.05). The lowest sperm quality resulted from 200 µM MT in combination with gentamicin.The effects of the indicated concentrations of Mito-TEMPO (MT) in combination with antibiotics (800 mg/L gentamicin or neomycin) on sperm motility and viability are shown in p > 0.05) , followed by 200 mM trehalose (41.24 ± 2.48% and 27.32 ± 2.17%) ( > 0.05) . In the Sperm motility and viability were maintained during 60 days of storage with 2 mM RG  and 4 mM RG  in combination with gentamicin. Similarly, 2 mM RG  and 4 mM RG  combined with neomycin enhanced sperm motility and viability over 60 days . In thisShort-term storage of sperm at lower temperatures may promote brood stock upgradation at distantly located hatcheries. This is the first study to develop a storage protocol for spotted halibut sperm. The implementation of sperm short-term storage methods for endangered or threatened fish is an effective conservation strategy for these species. In addition, the short storage of sperm can support artificial fertilization in fish. Sperm motility, kinematic parameters, and viability are important aspects of sperm quality, particularly if the fertilization rate cannot be evaluate because of a lack of mature eggs ,18. Paralichthys olivaceus) [Pleuronectes herzensteini) [Limanda yokohamae), and starry flounder (Platichthyes stellatus) [Takifugu rubripes) [Astyanax altiparanae) [Nibea albiflora) [Osmerus mordax) [Argyrosomus regius) [3, and C6H12O6, which exert a buffering effect, tonicity, prevent sperm activation, and protect the membrane, respectively, during cold storage [In sperm storage, the extender, which has a composition similar to seminal plasma, maintains sperm function and extends lifespan . The selivaceus) , brown snsteini) , marbledellatus) . In thisubripes) , yellowtparanae) , yellow biflora) , and Scabiflora) . A sucro mordax)  and gluc regius) . Extende regius) . The ext storage . The medArgyrosomus regius) [Colossoma macropopum) [Clarias macrocephalus) [Platichthys stellatus) [Eleginops maclovinus) [Polyodon spathula) [Oreochromis mossambicus) [Morone saxatilis) [Nibea albiflora) [Salmon salar) [Oncorhynchus mykiss) [Oncorhynchus mykiss) [Oncorhynchus mykiss) [Labeo calbasu) [Perca fluviatilis L.) [The dilution ratio should be selected according to fish species to optimize the initial sperm density and the buffer capacity of the diluent. Some species’ spermatozoa are extremely sensitive to dilution . In this regius) , tambaquropopum) , walkingephalus) , and staellatus) . The optlovinus) , paddlefpathula) , and theambicus) , a 1:1 dxatilis) , yellow biflora) , Atlantin salar) ,33, rain mykiss) , salmon  mykiss) , trout ( mykiss) , and sal mykiss) . A dilut mykiss) , orangefcalbasu) , perch (ilis L.) , and Salilis L.) . For stoilis L.) . In thisilis L.) , the comilis L.) .Platichthys stellatusi) [Astyanax altiparanae) [Ictalurus punctatus) sperm should be stored at 0–4 °C [Low temperatures (approaching 0 °C) are typically advised because they decrease the metabolic rate and energy consumption of cells without altering their structural integrity ,43. Howellatusi)  and 2.5 paranae) . Due to paranae) ,25. Nilet 0–4 °C ,45.Larimichthys polyactis) [Salmo trutta caspius) sperm, penicillin and streptomycin at 500 IU/mL increased sperm motility and viability [Rana temporaria), sperm quality increased with increasing gentamicin concentration [Ictalurus punctatus) sperm because of contamination by a Pseudomonas sp. Therefore, antibiotics may enhance stored sperm quality by suppressing microbial growth and/or activity.Antibiotics—typically penicillin, erythromycin, gentamicin, streptomycin, and neomycin—are added to prevent bacterial contamination of the stored sperm ,47. In tlyactis)  and 500 lyactis) . In the iability . Similarntration . In Atlantration , cold stPagrus major) [Leiarius marmoratus) [Colossoma macropomum) sperm [During the cold storage of sperm, antioxidants inhibit free-radical reactions. In this study, 250 mM trehalose with neomycin increased the motility and viability of spotted halibut sperm during storage for 60 days. Indeed, higher concentrations of trehalose further increased the sperm quality. By contrast, in the presence of gentamicin, a high trehalose concentration reduced sperm motility and viability. Trehalose was useful for the cryopreservation of rainbow trout , whitefis major) , and Amamoratus)  sperm. Tmoratus) . In addimoratus) . MT repomoratus) . MT mainmoratus) . In roosmoratus) . In thism) sperm . RG is am) sperm . The appm) sperm , and 2 am) sperm . RG at 1m) sperm .Optimal conditions for the storage of spotted halibut sperm were Stein’s solution (extender), a 1:4 sperm: extender dilution ratio, 800 mg/L neomycin, 250 mM trehalose, and a temperature of 2 °C.


After 70 h and 100 h of MA, the powder consisted of a mixture of amorphous and nanocrystalline body-centered cubic (bcc) phases. We observed the relaxation in the as-received powder. The relaxation temperature (Tre) increases logarithmically with the annealing time. Annealing above 440 °C results in (1) ordering of L21, (2) dissolution of the residual Ni and Mn, (3) tetragonal MnNi phase formation and (4) γ phases precipitation. The activation energies of the B2 → L21 and Mn (α-Mn) → MnNi (P4/mmm) transformations were calculated.The crystallization kinetics and structure evolution during annealing of the Ni Ni-Mn-X  magnetic shape memory alloys (MSMAs) have been extensively studied in recent years. MSMAs have many advantages, such as a large magnetic-field-induced strain and stress, high working frequency, high adiabatic temperature change in the low field, rare-earth-free and non-toxic elements, large magnetoresistivity and good oxidation resistance. In addition, these materials exhibit multifunctional properties, e.g., magnetic shape memory effect, magnetocaloric, barocaloric and elastocaloric effects. MSMAs are interesting for applications, such as actuators, sensors, and micro-electro-mechanical systems (MEMS). Additionally, their unique properties play an important role in developing alternative refrigeration technology ,2,3,4,5.2MnGa nanoparticles undergo different structural transitions than their corresponding bulk alloy. So, it is important to determine the crystal structure, magnetostructural transformation, and properties of MSMAs nanopowders produced by various techniques. Additionally, MSMAs powders can be processed by the sintering process or additive manufacturing [MSMAs are currently being produced as bulk, rods, thin films, etc., by conventional techniques, such as arc/induction melting, suction casting, directional solidification and magnetron sputtering. There are also several reports on MSMAs powders and nanoparticles ,7,8,9. Wacturing ,11. To iacturing ,13,14,15MSMAs powders are usually manufactured by the following steps: (1) arc/induction melting, (2) mechanical pulverization of bulk/ribbon, and finally, (3) high-energy ball milling. It is worth underlining that ball milling is a simple, cost-effective technique suitable for industrial production . The bal0. Several crystallization mechanisms have been proposed for Ni-Mn-based amorphous powders obtained by high-energy ball milling. Tian et al. [49.8Mn28.5Ga21.7 powder results from the nucleation and growth of ordered-Heusler L21 grains in the disordered bcc matrix. During heating of the ball-milled powders, they also observed the structural transition from fcc to bcc [50Mn34In16 powder, Sánchez-Alarcos [50Mn36.7In13.3 powders produced by high-energy ball milling), showed the one-step transition from the amorphous phase to B2, followed by the L21 ordered structure [Some researchers described the crystallization kinetics of amorphous Ni-Mn or Ni-Mn-Sn thin films produced by the magnetron sputtering technique ,18,19,20-Alarcos  recordedtructure .As mentioned above, in the near future, MSMAs powders can be used in additive manufacturing (AM) or sintering processing of micro to macro actuators/sensors. So, there is a need to develop an appropriate powder production method. One of the most promising is mechanical alloying. MA is a solid-state process during which the elemental powders are repeatedly cold-welded, flattened, fractured and re-welded. One can see that the final MA powder is obtained from pure elemental powders, whereas usually high-energy ball milling process starts from the bulk material of a given (appropriate) crystal structure, which is then crushed until receiving the powder of a defined size, crystal structure and properties. So, the mechanisms and processes are associated with phase formation during the MA and high-energy ball milling, and after thermal treatment of the powders, they are expected to be different. Unlike ball milling, there is a lack of investigation of structure formation and its developing mechanisms of Ni-Co-Mn-In powders obtained by the mechanical alloying technique. Although MA has been widely used for other alloys, the crystallization kinetics of the MSMAs powders obtained by MA technique has not been studied yet. So, the main aim of this paper is to investigate the crystallization kinetics of Ni-Co-Mn-In powders produced by mechanical alloying.45.5Co4.5Mn36.6In13.4 (% at.) powders obtained by mechanical alloying using a high-energy planetary ball mill.In this paper, we described for the first time the crystallization and atomic ordering kinetics of Ni45.5Co4.5Mn36.6In13.4  were produced by mechanical alloying from elemental powders of Ni , Co , Mn , and In  delivered by Strem Chemicals. MA was carried out in a high-energy planetary ball mill Fritsch Pulverisette using the tempered steel vial (80 mL) and tempered steel balls. The ball-to-powder ratio (BPR) was 17:1 and the rotation speed 250 rpm. The total milling time was 70 h and 100 h . To avoid overheating the 10 min rest periods, every 30 min milling was applied. The milling process was carried out under an Ar atmosphere to prevent significant oxidation. The phase transformations were studied by the differential scanning calorimeter  in the temperature between −120 °C and 600 °C with a heating rate of: 10 °C/min, 20 °C/min, 30 °C/min, 40 °C/min, and 50 °C/min. The temperature of the peak was determined by the tangent method  . The powders were annealed in situ (in the DSC chamber) at the temperatures: 150 °C, 300 °C, 330 °C, 340 °C, 350 °C, 360 °C, 380 °C, 400 °C, 440 °C, 500 °C, and 600 °C for 2 min. The heating rate was 10 °C/min. To study the relaxation process, samples after 100 h of MA were annealed at 200 °C for 2 min, 5 min, 30 min, 60 min, 180 min, 50 h, and at 250 °C for 5 min, 30 min, and 45 min. For crystal structure and phase transitions analyses, powders were annealed at 300 °C for 10 min, 2 h, 4 h, 20 h, and 100 h. For the isothermal measurements, samples were heated up to the Tiso with the maximum rate (approximately 300 °C/min) and then held at that temperature for 45 min during recording.Ni3D detector  using Cu anode . XRD diffraction patterns were recorded in a Bragg–Brentano geometry with angular step 0.02° 2θ and a range of 10 to 110 °2θ for as-milled powders and 10–60° 2θ for annealed powder. The (111)L21 superlattice diffraction peak integral intensity was used to analyze the ordering process occurring in the powders during annealing. Microstructure observations were carried out by 300 kV high-resolution transmission electron microscope JEOL JEM-3010  equipped with Gatan 2k × 2k Orius™ 833 SC200D CCD camera. TEM samples were prepared by putting a drop of a powder solution  onto a copper grid with 300 mesh.The XRD measurements were performed using Panalytical Empyrean diffractometer with PIXcell45.5Co4.5Mn36.6In13.4 solid solution formation during MA as a function of milling time (2.5–70 h) was discussed in detail in [bcc peak and Ni and Mn peaks intensities indicate that prolonging milling time from 70 h to 100 h does not significantly influence the structure and phase composition of the received alloy.etail in . The simThe significant broadening of the main reflection , the forward/reverse martensitic transformation peaks were not observed. It is worth emphasizing that the course of the cooling curves looks almost identical in both cycles. While during heating, the difference between the 1st and 2nd cycles is visible just above 100 °C. It may indicate that some other process may also happen . Generally, the as-milled powder may be strongly deformed during the MA because of the high-energy ball-powder collisions. For example, it can be seen as a “jump” in the DSC curve [For the DSC measurements, the as received MA powder was cooled down to −120 °C and then heated up to 600 °C (1st cycle of heating) and subsequently cooled down from 600 °C to 120 °C (1st cycle of cooling) a,c. The SC curve ,27, whicre) increases logarithmically with the annealing time (ta) (re vs. ln(ta) is visible in re) but lower than the crystallization temperature. In our case, Tre ≈ 120 °C, so we chose 200 °C for annealing. The presented results show that prolonging annealing (longer than 1.5 h) at 200 °C does not significantly influence the relaxation process. Only increasing the annealing temperature above 200 °C led to the fully relaxed powder  a,c. The d powder b. The chd powder d. One cad powder d. Then, a) of P1 and P2, the Kissinger approach was applied. According to Kissinger [a means the energy barrier opposing the reaction. If the reaction proceeds at a rate varying with temperature—possesses activation energy—the position of the peak varies with the heating rate. It means that to initiate the process , a specific energy Ea (activation energy) has to be supplied to the system. In a system , each process  is characterized by the separate activation energy. In this study, Ea is calculated based on a variation of the DSC peak temperature (Tp) as a function of the heating rate. The DSC measurements were performed applying the following heating rates: 10 °C/min, 20 °C/min, 30 °C/min, 40 °C/min, and 50 °C/min (a) was estimated from Kissinger Equation (1) as follows:p—peak temperature, A—constant, R—gas constant.As can be seen, several processes may contribute to the exothermic effect observed between 300 °C and 500 °C  divided by gas constant (R)  .As can be seen, the isothermal curves decrease monotonically. It indicates normal grain growth and cannot result from nucleation and growth transformation . It is w1 Heusler-ordered phase and residual Ni, and Mn  were determined . So, one may conclude that the fully ordered Heusler structure is present in the samples, annealed at 600 °C. Moreover, with the increase of annealing temperature, the intensities, angular position and profile of the (110)bcc/B2 ((220)L21) change (bcc/B2 ((220)L21) peak shifts towards lower 2θ angles, which relates to the increase in lattice parameters of that phase. A similar effect for Ni-Mn-In powders annealed at higher temperatures (427–527 °C) obtained by ball milling was observed by Sánchez-Alarcos et al. [1. Additionally, a significant increase in the intensity and decrease in FWHM of the (110)bcc/B2 peak was observed for samples annealed between 330 °C and 360 °C. To analyze that effect, the (110)bcc/B2 peak deconvolution and fitting were done. The peak is a sum of two different profile components: the broad blue , the amorphous phase crystallizes (transforms to bcc). At the same time, the lattice strain of the crystalline fraction reduces, and the bcc/B2 crystallites start to grow. That is why the (110)bcc/B2 FWHM decrease and the intensity increase simultaneously.As previously mentioned, the disordered bcc solid solution was identified in the as-milled sample. For the samples annealed above 330 °C, at approximately 30° 2θ, the (100))bcc/B2 (0L21) chaoad blue , peak 1 oad blue , peak 2,1 and crystallite growth (as mentioned above); and dissolution or formation of the minor phases as: (2) the dissolution of the residual Ni and Mn, (3) MnNi  formation and (4) γ phases formation. During the annealing of the 100MA sample, between 150 °C and 400 °C, no changes in the intensity and profile of the 332Mn, 422Mn, and 200Ni lines were observed (a0 = 2.62 Å c0 = 3.52 Å ICDD PDF 04-006-8085). Additionally, for powder annealed at 600 °C at 43.32° 2θ, one can see the (200)γ the fcc–face-centred cubic γ phase peak  , several processes/transformations occur: (1) further ordering of L2observed —III. Whe17-5179) b—II. The45.5Co4.5Mn36.6In13.4 powders obtained by mechanical alloying, the phase transformations occur in the following order:Taking the above into account, during the annealing of the Ni1 Heusler occurs. The powder annealed between 360 °C and 600 °C consists of the partially-ordered Heusler compound. The fully ordered L21 Heusler structure is observed for powders annealed at 600 °C. Due to the atomic ordering being a time-dependent process, holding a specimen long enough, even just above 360 °C, one may also expect to obtain the fully ordered L21 Heusler structure.It should be clear that the as-milled powder consists of the mixture of the disordered bcc parent phase, the amorphous phase and the residual Mn and Ni. During the annealing between 300 °C and 340 °C, the amorphous phase transforms into the disordered bcc. At approximately 340 °C, the bcc to B2 transformation starts and continues up to 360 °C, where the B2 to ordered L20 [50Mn34In16 powders registered a two-step transformation from amorphous to B2 structure. In contrast, Tian et al. [49.8Mn28.5Ga21.7 powders were observed. They also [1 Heusler phase were formed in the disordered bcc matrix. The volume fraction of the L21 phase increased with the annealing temperature increases. Considering the above, one can expect similar structural evolution during the annealing of the NiCoMnIn powders produced by mechanical alloying (this work). We observed nucleation and growth of the ordered L21 structure when the annealing temperature increased. It was confirmed by the increase of the superlattice reflection intensity ratio (I(111)/I(200)) . The activation energies (Ea) determined for studied Ni45.5Co4.5Mn36.6In13.4 MA alloys are similar to those observed for other magnetic shape memory powders obtained by high-energy-ball-milling  and slightly higher than for NiMn-based films obtained by magnetron sputtering. Despite that, for different alloys, Ea values cannot be directly compared , it may give a rough idea about the expected kinetics parameters of the processes that occur in the sample.Depending on the chemical composition, the manufacturing and the thermal treatment, the transformations in NiMn-based alloys may occur in a different order. 0 ,19. Howe0  in Ni50Mn et al.  fcc into/I(200))  b. Additu et al.  observed45.5Co4.5Mn36.6In13.4 powders obtained by mechanical alloying (MA) were analyzed. We observed that low-temperature annealing led to the relaxation of the stresses accumulated in the powder during the prolonged milling (70 h). Although the process starts at 100 °C, the powder to be fully relaxed, should be annealed at 250 °C for at least 30 min. The relaxation temperature (Tre) logarithmically increases with the annealing time. After 70 h and 100 h of milling, the powder consisted of a mixture of the amorphous phase, nanocrystalline body-centred cubic (bcc) disordered solid solution and minor fractions of non-dissolved Ni and Mn. During annealing between 300 °C and 340 °C, the amorphous phase transforms into the disordered bcc. At 340 °C, the bcc to B2 transformation starts and continues up to 360 °C, where the B2 to L21 Heusler transition occurs. At the higher annealing temperatures (above 440 °C), the following processes/transformations occur:(1)1 phase and its growth ;Further ordering of L2(2)Dissolution of the residual Ni and Mn;(3)MnNi (P4/mmm) phase formation;(4)γ (fcc) phases precipitation.In this work, the crystallization kinetics and structure evolution of the thermally treated Nia) of the transition processes B2 → L21 and Mn (α-Mn) → MnNi (P4/mmm) were calculated as 185(6) kJ/mol and 228(3) kJ/mol, respectively.Using the Kissinger approach, the activation energies (EGaining knowledge about crystallization kinetics, structures of the phases and transformation sequence during thermal treatment of the mechanically alloyed powders is advantageous and may help to design the materials for future applications.


This study was the first to demonstrate the anticancer effects of luteolin (Lut) combined with asiatic acid (AsA) on CaSki and HeLa cervical cancer cells. Lut combined with AsA effectively reduced cervical cancer cell viability by arresting the cell cycle in the sub-G1 phase and inducing caspase-mediated intrinsic apoptosis. Lut combined with AsA treatment downregulated PI3K/AKT signaling , JNK/p38 MAPK signaling and FAK signaling  and upregulated ERK signaling to induce apoptosis and inhibit cancer cell migration. In in vivo study, Lut combined with AsA markedly inhibited cervical cancer cell-derived xenograft tumor growth and the cytotoxic effect was minimal or nonexistent. Collectively, the present study demonstrated that Lut combined with AsA may be used as an anticancer agent in future clinical treatment to improve the prognosis of cervical cancer.Cervical cancer is an important issue globally because it is the second most common gynecological malignant tumor and conventional treatment effects have been shown to be limited. Lut and AsA are plant-derived natural flavonoid and triterpenoid products that have exhibited anticancer activities and can modulate various signaling pathways. Thus, the aim of the present study was to evaluate whether Lut combined with AsA could enhance the anticancer effect to inhibit cervical cancer cell proliferation and examine the underlying molecular mechanisms in vitro and in vivo. The results of a CCK-8 assay showed that Lut combined with AsA more effectively inhibited the proliferation of CaSki and HeLa cells than Lut or AsA treatment alone. Lut combined with AsA caused apoptosis induction and sub-G1-phase arrest in CaSki and HeLa cells, as confirmed by flow cytometry, mitoROS analysis, antioxidant activity measurement and western blot assay. In addition, Lut combined with AsA significantly inhibited the cell migration ability of CaSki and HeLa cells in a wound-healing assay. Furthermore, Lut combined with AsA induced apoptosis and inhibited migration through downregulated PI3K/AKT , JNK/p38 MAPK and FAK  signaling and upregulated ERK signaling. In an in vivo study, Lut combined with AsA markedly inhibited cervical cancer cell-derived xenograft tumor growth. Collectively, the present study showed that Lut combined with AsA may be used as an anticancer agent to improve the prognosis of cervical cancer. Indeed, with additional research to develop standardized dosages, Lut and AsA combination therapy could also be applied in clinical medicine. Cervical cancer is one of the major gynecologic malignancies, with more than 600,000 women diagnosed and 342,000 deaths in 2020 . Convent30H48O5) is a natural triterpenoid compound and the major source extracted from Centella asiatica /2. When the tumor volume reached ~130 mm3 (Day 6), the mice were randomly divided into four groups (six mice per group) and drug treatment was initiated via daily intraperitoneal injection (i.p.): vehicle control group ; AsA group ; Lut group ; and combination group (asiatic acid 100 mg/kg/day plus luteolin 50 mg/kg/day). The mouse tumors were extracted for tissue section preparation and the tumor weight was recorded after sacrifice on day 22. Cancer cell implantation was performed using a 2–3% isoflurane (Panion & BF Biotech) inhalation, and the mice sacrifice was performed using a CO2 chamber. The animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Changhua Christian Hospital, Taiwan .Seven-week-old female BALB/cAnN.Cg-2O for 10 min, then 3% H2O2 for 10 min, 3% BSA  for 1 h and stained for ki67  and cleaved caspase-3 , purchased from Cell Signaling; and integrin β1 , phospho-FAK  and paxillin  purchased from GeneTex were used at 4 °C overnight. Sections were washed with PBS and incubated with OneStep Polymer HRP-conjugated anti-mouse/rat/rabbit IgG secondary antibodies  at RT for 30 min. Sections were then visualized using a colorimetric reagent DAB detection kit . Nuclei were counterstained with hematoxylin  and photographed using an Olympus BX61 microscope (Olympus Corporation). For each animal tissue specimen, four fields at 400× magnification were evaluated using Image-Pro Plus 4.5 software .Fresh tumor tissue was fixed with 10% neutral buffered formalin  and embedded in paraffin. Sections (5-μm-thick) were stained with hematoxylin and eosin  and subjected to immunohistochemical (IHC) analysis. Sections were de-waved, rehydrated, boiled in ddHp value < 0.05 was considered to indicate a statistically significant difference.Statistical analyses were performed using the one-way ANOVA followed by Bonferroni correction’s post-test using GraphPad software . All data representing results are presented as the mean ± standard deviation (SD) from at least three independent experiments. A p < 0.05; and 50 value, of 46.6 ± 2.7 μM for HeLa cells, than CaSki and C33A cells, which had values of 115.4 ± 1.8 μM and 117.0 ± 12.1 μM, respectively. The IC50 values for the AsA treatment were found to be 47.8 ± 0.6 μM for HeLa cells, 71.4 ± 6.2 μM for CaSki cells and 63.2 ± 4.5 μM for C33A cells. Lut was less active than AsA, which showed more strong antiproliferative activity against CaSki cells and C33A cells. In addition, we further investigated cell proliferation under a combination of Lut and AsA. Compared with the control cells (100%), the proliferation of CaSki cells was significantly decreased by 28% and 57% after treatment with 50 and 75 μM AsA (71.6 ± 2.1% vs. 42.8 ± 1.8%) and inhibited by 59% and 71% when treated in combination with 50 μM Lut (40.9 ± 0.3% vs. 28.9 ± 1.5%), respectively. The 75 μM AsA combined with 75 μM Lut (26.7 ± 1.4%) also had approximately 73% inhibition efficiency. Similarly, HeLa cell proliferation significantly decreased by 52% and 91% after treatment with 50 and 75 μM AsA (47.5 ± 2.1% vs. 9.0± 0.6%) and was markedly inhibited, by 89% and 93%, when combined with 50 μM Lut (47.5 ± 2.1% vs. 9.0± 0.6%), respectively. The 75 μM AsA combined with 75 μM Lut (5.1 ± 1.0%) had 95% inhibition efficiency. Additionally, C33A cell proliferation significantly decreased, by 48% and 58%, after treatment with 50 and 75 μM AsA (52.4 ± 0.8% vs. 42.3± 6.9%) and was inhibited by 47% and 49% when combined with 50 μM Lut (53.4 ± 3.1% vs. 50.5 ± 1.0%), respectively. The 75 μM AsA combined with 75 μM Lut (48.8 ± 0.9%) only had 51% inhibition efficiency. Additionally, it can be seen from comparison of CI values, AsA combined with Lut exerted synergistic effects, with the CI being 0.910 and 0.885 for 50 or 75 μM AsA combined with 50 μM Lut for CaSki cells; and 0.894 and 0.941 for 50 or 75 μM AsA combined with 50 μM Lut for HeLa cells; and 1.145 and 1.264 for 50 or 75 μM AsA combined with 50 μM Lut for C33A cells, respectively. Therefore, AsA combined with Lut was more effective in reducing cell proliferation of CaSki and HeLa cells relative to the control cells and to cells treated with Lut or AsA alone, but C33A cells did not show a synergistic effect.To assess the anti-proliferation effects of both Lut and AsA on cervical cancer cells, a CCK-8 assay was performed using CaSki, HeLa and C33A cells with various concentrations of Lut (0–100 μM) with or without AsA (0–75 μM) for 24 h. We observed that Lut and AsA dose-dependently decreased the proliferation of CaSki, HeLa and C33A cells (p < 0.05). In summary, combined Lut and AsA promoted greater apoptosis of CaSki and HeLa cells.To determine whether apoptosis induction was involved in the anti-proliferation activity of Lut and AsA on cervical cancer cells, CaSki and HeLa cells were subjected to Annexin V-FITC and PI staining by flow cytometry. As shown in p < 0.05). Furthermore, we found that treatment with Lut (50 μM) combined with AsA (50 and 75 μM) resulted in significantly higher percentages of sub-G1 phase cells  and lower percentages of G0/G1 phase cells  than in cells treated with Lut or AsA alone (p < 0.05). These results suggested that Lut combined with AsA induced more effective cell cycle arrest in the sub-G1 phase.To understand the anti-proliferation effects of Lut and AsA, cell cycle progression analysis of cervical cancer cells was performed by flow cytometry. In CaSki cells, Lut or AsA treatment significantly increased the sub-G1 phase cells, from 3.9 ± 0.5% (untreated cells) to 13.8 ± 0.2% (50 μM Lut), to 29.5 ± 3.1% (50 μM AsA) and to 70.8 ± 3.6% (75 μM AsA) and significantly decreased the G0/G1 phase cells, from 68.4 ± 1.2% (untreated cells) to 67.2 ± 0.8% (50 μM Lut), to 44.4 ± 1.4% (50 μM AsA) and to 18.0 ± 1.9% (75 μM AsA). Simultaneously, the percentage of sub-G1 phase cells increased from 3.6 ± 0.2% (untreated cells) to 19.3 ± 0.9% (50 μM Lut), to 17.3 ± 3.1% (50 μM AsA) and to 17.4 ± 2.3% (75 μM AsA) and decreased the G0/G1 phase cells from 62.6 ± 0.5% (untreated cells) to 56.4 ± 1.1% (50 μM Lut), to 57.8 ± 2.8% (50 μM AsA), and to 54.5 ± 1.3% (75 μM AsA) in HeLa cells (p < 0.05). In addition, combined treatment with Lut and AsA further significantly increased the MitoSOX Red-derived fluorescence levels of CaSki and HeLa cells  as compared with cells treated with Lut or AsA alone. In addition, when compared with the non-treated control, Lut or AsA monotherapy significantly reduced the GSH level in CaSki and HeLa cells ; Lut in combination with AsA more significantly reduced the GSH level . The catalase expression level did not change in CaSki and HeLa cells. Thus, these data showed that Lut combined with AsA treatment may induce cervical cancer cell apoptosis via upregulating mitoROS and downregulating the GSH level to promote cell death.Excessive mitochondrial ROS has been shown to induce cell injury and death and induce apoptosis or necroptosis. Mitochondrial antioxidants have been reported to be effective in cancer prevention and anticancer therapy. Therefore, we examined mitochondrial ROS and mitochondrial antioxidant expressions using the MitoSOX Red reagent and an enzyme-linked immunosorbent assay (ELISA). The results revealed that the MitoSOX Red-derived fluorescence level was significantly increased at 50 μM Lut,= and 50 or 75 μM AsA in CaSki and HeLa cells  as compared with the non-treated control cells . Furthermore, the combination of Lut and AsA markedly inhibited the migration rate of CaSki and HeLa cells as compared with treatment with Lut or AsA alone at 12 and 24 h, although the combination with a higher dose of AsA in CaSki and HeLa cells did not. As integrin/FAK/paxillin has been reported to cause tumor migration and invasion, we next examined these modulated cell migration-related protein expressions by western blotting. Lut or AsA monotherapy significantly decreased the phospho-FAK (ratio of control: 0.69 ± 0.01 vs. 0.86 ± 0.01), integrin β1 (ratio of control: 0.94 ± 0.01 vs. 0.69 ± 0.07) and paxillin (ratio of control: 0.71 ± 0.01 vs. 0.93 ± 0.04) protein expression levels in CaSki cells, while for HeLa cells, the expressions of phospho-FAK, integrin β1 and paxillin were reduced . Lut and AsA co-treatment more significantly decreased the phospho-FAK, integrin β1 and paxillin protein expressions . These results clearly suggested that Lut combined with AsA inhibited cancer cell migration by decreasing the integrin/FAK/paxillin signaling pathway.To investigate the cell migration ability of cervical cancer cell lines treated with Lut and AsA, we performed a wound-healing assay. The cells in the untreated control group showed a faster cell migration ability than cells treated with Lut or AsA (p < 0.05). Taken together, these findings showed that Lut and AsA induced pro-apoptotic effects in CaSki and HeLa cells through a mitochondria-related intrinsic signaling pathway.To further confirm our prior results showing that Lut and AsA treatment induced cervical cancer cell death by apoptosis, apoptosis-related proteins expressions were determined by western blotting. The western blotting findings revealed that Lut or AsA treatment significantly increased the expressions of cleaved PARP-1, pro-apoptotic protein Bax and cleaved caspase-3, whereas anti-apoptotic protein Bcl-2 was significantly decreased in expression as compared with the control in CaSki and HeLa cervical cancer cells . Moreover, Lut and AsA co-treatment more significantly upregulated the cleaved PARP-1, Bax and cleaved caspase-3 expressions, and downregulated the Bcl-2 expression  as compared with Lut or AsA monotherapy, although no significant differences were observed in the Bax expression in CaSki cells and Bcl-2 expression in HeLa cells compared with AsA monotherapy (p < 0.05). Moreover, combination treatment with Lut and AsA more significantly decreased the PI3K(p110α), p-AKT, p-p70S6K, p-p38 and p-JNK1/2 expressions, and more synergistically increased the p-ERK1/2 expression level in both CaSki and HeLa cells , as compared with Lut or AsA alone. These results confirmed that Lut- and AsA-induced apoptosis in cervical cancer cells might involve the PI3K/AKT/p70S6K and p38/ERK1/2/JNK1/2 pathways.To further to understand the molecular mechanism of Lut- and AsA-induced apoptosis in cervical cancer cells, the phosphorylation statuses of PI3K(p110α), AKT, p70S6K, p38, ERK1/2 and JNK1/2 were determined by western blotting. In CaSki cells, Lut or AsA monotherapy significantly decreased phosphorylation of PI3K(p110α), AKT, p70S6K, p38 and JNK1/2, while significantly increasing phosphorylation of ERK1/2, as compared with the control . Similar results were observed for HeLa cells, with Lut or AsA monotherapy significantly decreasing PI3K(p110α), p-AKT, p-p70S6K, p-p38 and p-JNK1/2 and significantly increasing the p-ERK1/2 protein expression level , although no significant difference were observed the p-JNK1/2 expression (ratio of control: 0.90 ± 0.03) in HeLa cells and p-ERK1/2 expression in both CaSki and HeLa cells after Lut monotherapy . These results clearly indicated that the anti-proliferation effect of Lut combined with AsA in cervical cancer cells acted through a caspase-3-mediated intrinsic apoptosis pathway.To further confirm the involvement of a caspase-dependent apoptotic pathway, CaSki and HeLa cells were pretreated with pan-caspase inhibitor z-VAD-fmk (2.5 μM) for 2 h, followed by combined treatment with Lut and AsA. The results showed that z-VAD-fmk significantly reduced the anti-proliferation effects of Lut and AsA on cervical cancer cells . The western blot findings revealed that co-treated cells had significantly lower expressions of cleaved PARP-1 and cleaved caspase-3 proteins  and higher expressions of Bcl-2 and p-p38 proteins , as compared with cells treated with Lut and AsA in combination . No significant difference in body weight between groups was observed during the experimental period  and the experimental animal survival rate was 100%, suggesting that treatment with Lut and AsA alone or in combination did not cause host drug toxicity (p < 0.05). Moreover, in performing H&E staining, we found that the Lut plus AsA treatment more effectively reduced the tumor cellularity and apoptosis in the CaSki xenografts than AsA or Lut monotherapy. Similarly, consistent with the in vitro findings in this study, IHC study demonstrated more significantly downregulated expressions of ki67 (4.1 ± 1.8 positive cells), integrin β1 (2.9 ± 1.2 positive cells), p-FAK (4.0 ± 1.5 positive cells) and paxillin (4.0 ± 2.0 positive cells) and an upregulated cleaved caspase-3 (13.0 ± 4.5 positive cells) protein expression in tumors treated with Lut plus AsA as compared with the AsA-, Lut- and control- groups . These results further suggested that Lut plus AsA combined treatment acted as an anti-proliferative, anti-migratory and pro-apoptotic agent in vivo.To further confirm the in vitro anti-proliferation effects of Lut and AsA, a CaSki xenograft tumor model was constructed in BALB/c nude mice. The schematic timeline of this experiment was as shown in toxicity C. The avCervical cancer remains a leading cause of high mortality and morbidity in women worldwide. Although chemotherapy can effectively to improve the survival of cervical cancer patients, it is also prone to increase drug resistance, toxic side effect and complications, causing great suffering to patients. Therefore, a new oncology therapy for cervical cancer by plant-derived natural products  has attracted attention. AsA and Lut themselves are rich in natural triterpenoids and flavonoids, and their therapeutic effects on multiple cancers’ proliferation and metastasis have been studied. Based on this, this study aimed to assess the anticancer effect of AsA and Lut in human cervical cancer. Simultaneously, we also evaluated whether Lut combined with AsA more notably enhanced the anticancer effect than Lut or AsA alone in vitro and in vivo. Our study results showed that AsA or Lut alone can reduce the cell viability of human cervical cancer cells  [19 Ren 2016] or S-G2/M-phases arrest  ,56 and LPrevious research revealed that the Lut or AsA induced apoptosis through the inactivation of the PI3K/AKT/mTOR/p70S6K pathway in some common cancers, including glioblastoma , lung 6, ovarianDrug resistance and side effects are frequently developed in patients undergoing cervical cancer chemotherapy. A novel anticancer agent, especially derived from natural sources, is required to address these issues. Our study is the first to have demonstrated an anticancer effect of combined Lut plus AsA treatment on cervical cancer in vitro and in vivo. Lut combined with AsA effectively reduced cervical cancer cell viability by arresting the cell cycle in the sub-G1 phase and inducing caspase-mediated intrinsic apoptosis. Lut combined with AsA treatment downregulated PI3K/AKT signaling , JNK/p38 MAPK signaling and FAK signaling  and upregulated ERK signaling to induce apoptosis and inhibit cancer cell migration, resulting in an anticancer effect on cervical cancer . Further


To investigate the effect of camrelizumab + apatinib combined with radiotherapy on the expression of TRIM27, SCC-Ag, and CYFRA21-1 in advanced oligometastatic non-small-cell lung cancer (NSCLC).  A retrospective analysis of patients with oligometastatic NSCLC who were treated at our hospital from January 1, 2021, to March 31, 2022. Patients who met the inclusion criteria were summarized into an observation group (camrelizumab on the basis of the control group), or a control group . The disease control rate, immune function, changes in the levels of TRIM27, SCC-Ag, CYFRA21-1, and the occurrence of adverse effects were compared between the two groups. p > 0.05). After treatment, the levels of CD3+, CD4+, CD4+/CD8+t cells, and NK cells in the observation group were higher , while the level of CD8+t cells was lower (p < 0.001). There were no significant differences in TRIM27, SCC-Ag, or CYFRA21-1 between the two groups before treatment (p > 0.05). After treatment, the observation group had lower levels of TRIM27 (p=0.035), SCC-Ag (p=0.045), and CYFRA21-1 (p=0.003). There was no significant difference in the occurrence of adverse events between the two groups (p < 0.05).  There were 86 patients who met the inclusion criteria, with 53 cases in the observation group and 33 cases in the control group. There were significant differences in complete remission , partial remission , disease control , disease progression , and disease control rate (49/53 vs. 26/33) between the observation group and the control group. There was no significant difference in immune function between the two groups before treatment ( Treatment of camrelizumab + apatinib combined with radiotherapy is effective for advanced oligometastatic NSCLC, with mild adverse effects. Non-small-cell lung cancer (NSCLC) is a malignant tumor with high mortality, accounting for about 85% of all lung cancer patients in the United States and Europe. Most patients have distant metastases at the time of diagnosis, thus losing the window of complete cure –4. The iA retrospective analysis of patients with oligometastatic NSCLC who were treated in our hospital from January 1, 2021, to March 31, 2022. This study was approved by the Institutional Ethical Committee of Shandong Provincial Third Hospital. All patients in this study signed the informed consent forms. The inclusion criteria were as follows: (1) pathologically confirmed stage VI NSCLC; (2) signed informed consents; and (3) with complete data. The exclusion criteria were as follows: (1) patients with mental disorders; or (2) severe organ failure; or (3) lost to follow-up.After 4 cycles of treatment, the curative effect of patients was evaluated according to the World Health Organization tumor curative effect evaluation standard . Complet+, CD4+, CD8+, CD4+/CD8+, and natural killer (NK) cell levels;Before treatment and after 4 cycles of treatment, an American FACS-type flow cytometer was used to detect immune function indicators, including CD3Enzyme-linked immunosorbent assay (ELISA) was used to detect serum SCC-Ag and CYFRA21-1 levels; TRIM27 was detected by the RT-PCR method .Record the occurrence of adverse effects during the treatment, including gastrointestinal discomfort, abnormal liver and kidney function, leukopenia, and thrombocytopenia.Patients in the control group were given radiotherapy combined with oral apatinib , with an initial dose of 250 mg/d. If no adverse effects, it will be increased to 500 mg/d, 28 days as a cycle, and the treatment will be continued for 4 cycles. Patients in the observation group were treated with camrelizumab  on the basis of the observation group, given by iv., 200 mg each time, once every 3 weeks, for continuous treatment of 4 cycles.χ2 test, and the data by measurement were tested by t-test. p < 0.05 (2-tailed) was used as the threshold for statistical significance.The data in this experiment need to be verified by SPSS21.0  software. The data by count were tested by the p > 0.05, In total, 489 patients with stage IV NSCLC were treated in our hospital from January 1, 2021, to March 31, 2022, including 86 patients with oligometastatic NSCLC who met the inclusion criteria and were summarized into 2 groups according to the treatment method. The observation group  and the control group . There were no significant differences in gender, age, and BMI between the two groups (p < 0.001). See There were significant differences in CR (25/53 vs. 10/33), PR (17/53 vs. 12/33), DC (7/53 vs. 4/33), DP (4/53 vs. 7/33), and disease control rate (49/53 vs. 26/33) between the observation group and the control group (p > 0.05). After treatment, there were significantly higher CD3+t cells (59.29 ± 3.31 vs. 51.89 ± 2.41), CD4+t cells (32.79 ± 2.81 vs. 25.23 ± 2.66), CD4+/CD8+t cell (1.41 ± 0.59 vs. 1.14 ± 0.52), and NK cells (18.26 ± 3.51 vs. 14.26 ± 3.14) and lower CD8+t cell (23.63 ± 1.31 vs. 23.97 ± 1.36) in the observation group. See There was no significant difference in immune function between the two groups before treatment (p > 0.05). After treatment, there were lower TRIM27 (0.35 ± 0.03 vs. 0.51 ± 0.04), SCC-Ag (1.29 ± 0.34 vs. 1.51 ± 0.36), CYFRA21-1 (1.93 ± 0.31 vs. 2.39 ± 0.47) . See There was no significant difference in the index levels between the two groups before treatment (p < 0.05). See There was no significant difference in the occurrence of adverse effects between the two groups , which has the advantages of simple use, small side effects, safety, and reliability. Due to more and more relevant clinical trials, apatinib is gradually being applied to the clinical treatment of advanced NSCLC. Camrelizumab is a new type of NSCLC drug newly developed in China. It can be combined with PD-1 to inhibit its interaction with apoptosis receptors and improve the body's immunity , 20. In In general, camrelizumab + apatinib combined with radiotherapy for advanced oligometastatic non-small-cell lung cancer has good efficacy, mild adverse effects, high safety, and can be widely used.