These linear polymers demonstrated high glass transition temperatures (Tg up to 201 °C) and thermal stability (Tonset up to 230 °C). They were further applied as reinforcing agents in the preparation of the vulcanized rubber compositions based on poly(styrene butadiene rubber) (SBR). The influence of the PIBOMA content and molar mass on the cure characteristics, rheological and mechanical properties of rubber compounds were studied in detail. Moving die rheometry revealed that all rubber compounds filled with PIBOMA demonstrated higher torque increase values ΔS in comparison with rubber compositions without filler, independent of PIBOMA content or molar mass, thus confirming its reinforcing effect. Reinforcement via PIBOMA addition was also observed for vulcanized rubbers in the viscoelastic region and the rubbery plateau, i.e. from −20 to 180 °C, by dynamic mechanical thermal analysis. Notably, while at temperatures above ~125 °C, ultra-high-molecular-weight polyethylene (UHMWPE) rapidly loses its ability to provide reinforcement due to softening/melting, all PIBOMA resins maintained their ability to reinforce rubber matrix up to 180 °C. For rubber compositions containing 20 phr of PIBOMA, both tensile strength and elongation at break decreased with increasing PIBOMA molecular weight. In summary, PIBOMA, with its outstanding high Tg among known poly(methacrylates), may be used in the preparation of advanced high-stiffness rubber compositions, where it provides reinforcement above 120 °C and gives properties appropriate for a range of applications.A set of poly(isobornyl methacrylate)s (PIBOMA) having molar mass in the range of 26,000–283,000 g mol Natural and synthetic rubbers have been used in a wide range of applications such as tires, elevator belts, hoses, tubes, adhesives, gloves, gaskets, coatings, antivibrational flooring, etc. ,2,3. HowTm) (Tm (www.ube.com (accessed on 12 May 2021) for UBEPOL VCR412 and VCR617 products) meaning its use necessitates increasing the cis-1,4-polybutadiene content in a given rubber compound, which may be undesirable.While increasing the content of inorganic filler leads to more pronounced reinforcing effects in vulcanized rubber compounds, it can also lead to unwanted increases in tire mass and hysteresis ,21,22. ATm) , above ~Tm) , ultra-high molecular weight polyethylene , carbon black , diphenyl guanidine , oiled sulphur , stearic acid , N--N′-phenyl-p-phenylenediamine , zinc oxide , treated distillate aromatic extracted oil , N-cyclohexyl-2-benzothiazolesulfenamide , 4-methoxyphenol , N,N-dimethylformamide , 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid , 1,1′-azobis(cyclohexanecarbonitrile)  and 2,2′-azobis(N-butyl-2-methylpropionamide) , wax1  and wax2  were used without further purification. Tetrahydrofuran (THF), cyclohexane and toluene  were purified through the SPS solvent purification system . 1,1,2-Trichloroethane  and 1-methylnaphthalene  were purified by distillation over CaH2. Isobornyl methacrylate  was distilled over CaH2 under reduced pressure (bp = 120–125 °C/1 mm Hg) prior to use. 2,2′-Azobis(2-methylpropionitrile)  and 4,4′-azobis  were crystallized from methanol before utilization.Solution-Styrene Butadiene Rubber (S-SBR) ; measured in this study: 1H) and −20 to 220 ppm (13C) and are listed in ppm. The signal corresponding to the residual protons of the deuterated solvent was used as an internal standard for 1H and 13C NMR. Signal assignment was performed using 2D NMR techniques: heteronuclear single quantum coherence (HSQC), heteronuclear multiple bond correlation (HMBC), H-H correlation spectroscopy (H-H COSY). IR spectra were acquired on Brucker Tensor 27 Fourier IR-spectrometer  using ATR technology  and Spectragryph optical spectroscopy software [NMR spectra were recorded on AMX-600 spectrometer  at 25 °C in the indicated deuterated solvents over a range of −1 to 20 ppm . THF was used as an eluent with a flow rate of 1.0 mL min−1 at 40 °C. Polymethylmethacrylate  and polystyrene standards  were used to perform calibration for the PIBOMA and SBR studies, respectively.A 1260 Infinity II gel permeation chromatograph  was used to determine −1. The onset weight loss temperature (Tonset) was determined as the point in the TGA curve at which a significant deviation from the horizontal was observed. The resulting temperature was then rounded to the nearest 5 °C. Thermal mechanical analysis (TMA) of PIBOMA samples was performed under inert atmosphere (He) using a DIL 402 select Expedis dilatometer  at a heating rate of 5 °C min−1 and a constant load of 0.3 N. Dynamic Mechanical Thermal Analysis (DMTA) measurements were carried out for cured compounds on bars ) with a DMA 242 C model  operating in tension mode . Experiments were performed at 1 Hz frequency with a heating rate of 2 °C min−1 from −180 to 180 °C. The set up provided the storage and loss moduli (E′ and E″). The damping parameter or loss factor (tan δ) was defined as the ratio tan δ = E″/E′.Thermal gravimetric analysis (TGA) was carried out in air on a TGA2 STARe System , applying a heating rate of 5 °C min−1 and resonance frequency of 300 KHz. The domain periodicity was evaluated from three different 1 × 1 μm2 images. On each image, two profiles were taken and for each, the distance over ten consecutive periods was recorded. The images were recorded in the so-called ‘soft tapping mode’, to avoid deformation and indentation of the polymer surface by the tip. All the images were collected with the maximum available number of pixels (512) in each direction. The general procedure for the preparation of the samples for AFM was as follows: cryo-cut of rubber was performed at −120 °C using N2 as a cryogenic liquid and a LEICA EM UC6 ultramicrotome  as cutting instrument. The blocks with a typical size (length × width × thickness = 10 × 5 × 5 (mm)) were trimmed to obtained a top surface of 500 × 200 microns which was gently polished with a diamond knife for further AFM analysis.Atomic force microscopy (AFM) images were recorded with MFP-3D Infinity microscope  in tapping mode . AC160TS-R3 (Olympus) cantilevers were applied with a stiffness of 26 N mt—tensile strength (kPa), Et—tensile modulus (MPa) and ε—elongation (%) were measured at room temperature using a universal test machine Instron 5967  equipped with a load cell of 30 kN. The measurements were achieved at a cross-head speed of 5 mm/min. Before tests, samples were dumbbell-shaped by punching die machine according to ASTM standard D638-14 . Post-mortem analysis of the fractured tensile samples was carried out with an EasyTom 160 . Images were acquired at a tube voltage of 45 kV, current of 80 µA and a power of 3.6 W. These conditions provided a good phase contrast between the thermoplastic particles and the rubber matrix. Images were taken at 2 frames per second, using 5 average frames per projection. During the acquisition, samples were rotated 360° at angular steps of 0.25° while a flat panel recorded each projection. Source-to-object-distance (SOD) and source-to-detector-distance (SDD) were set to 10 mm and 210 mm, respectively, keeping a constant voxel size of 6 µm. The 3D volume reconstruction was carried out with the Xact64 software , which enabled us to perform the inherent geometrical corrections, ring artefact removal and contrast enhancing.−1 , CDTPA  and AIBN  were dissolved in 14 mL of TCA in the Schlenk flask. The solution was deoxygenated by three freeze-pump-thaw cycles and sealed under inert atmosphere (Ar). The further synthesis was conducted at 60 °C for 120 h. Polymerization was quenched by the addition of 4-methoxyphenol (inhibitor), and the reaction viscous solution was diluted with dichloromethane. The resultant polymer containing the RAFT agent residue was isolated by double precipitation into the excess of methanol and dried at 55 °C/1 mm Hg for 12 h. Yield: 16.0 g (80%).1H and 13C NMR was performed using numbering depicted in 1H NMR : δ = 4.62–4.16 , 2.4–1.6 , 1.54 , 1.25 , 1.02–0.98 , 1.10–0.90 , 0.90–0.60 ; 13C NMR : δ = 177.4, 176.5 , 83.1 (C18), 55.1 , 44.9 (C26), 47.1 (C28), 45.3 (C21), 38.5 (C19), 37.5 (CH2S = Alk-5), 35.1 , 34.5 (C24), 29.8 (Alk-2b), 27.2 (C22), 26.8 , 29.5 (Alk-2), 25.1 , 24.2 (Alk-2a), 20.3 (C27 & C29), 19.2–17.1 , 14.3 (Alk-1); IR (ATR-mode): 2952 , 2877 , 1722 , 1472 , 1454 , 1390 , 1370 (m), 1311 (w), 1295 (w), 1237 m, asC–C), 1146 , 1109 (s), 1082 (m), 1051 , 1002 (s), 969 (s), 944 (m), 911 (w), 886 (m), 860 (w), 843 (m), 789 (w), 753 , 704 (w), 629 (w), 585 (w), 547 (w), 515 (m). cm−1; Mn theo = 80,000 g mol−1; Mn SEC = 46,500 g mol−1; Mw/Mn = 1.2 (SEC); Tg = 197 °C (TMA); Tonset = 230 °C (TGA).The assignment of 1H NMR spectroscopy in CDCl3 and gel permeation chromatography (GPC) in THF were used to determine the monomer conversion and the product molecular weights, respectively. Monomer conversion was calculated by relative integration of vinyl protons of residual IBOMA monomer and protons of the methyl group from 1-methylnaphthalene .For kinetics studies the polymerization was performed as described above, however, with the exception that 1-methylnaphthalene  was added in the reaction solution as internal reference for the determination of the conversion. Aliquots were removed from the reaction flask under argon atmosphere using a syringe at predetermined time intervals throughout the polymerization. Mn > 200,000 g mol−1, the free radical polymerization of IBOMA was performed. Typical polymerization procedure is given below by an example of PIBOMA283K synthesis:To overcome the limitations of RAFT polymerization and with the aim to prepare PIBOMA with Mn SEC = 283,000 g mol−1; Mw/Mn = 1.9 (SEC); Tg = 199 °C (TMA); Tonset = 225 °C (TGA).IBOMA  and Vam-110  were dissolved in 14 mL of TCA in the Schlenk flask. The solution was deoxygenated by three freeze-pump-thaw cycles and sealed under argon. Polymerization was carried out at 110 °C for 6 h. The reaction was quenched by the addition of 4-methoxyphenol (inhibitor) and the very viscous solution was diluted with dichloromethane. The resultant polymer was isolated by double precipitation from dichloromethane solution into the excess of methanol and dried at 70 °C/1 mm Hg for 12 h. Yield: 14.4 g (72%); The compounding of SBR rubber and thermoplastics was performed in accordance with the procedure published previously , althougIn the first stage, also referred to as the non-productive (NP) stage, rubber; filler and other ingredients such as antioxidants, activators and waxes  stage, the resultant rubber compounds were further mixed with curatives and accelerators (90) were calculated from the curves of each compound as the time required for the torque to reach the value given by the Equation (1):Smax is the maximum achieved torque, and Smin is the minimum torque on the curve.The curing characteristics of the rubber compounds were investigated using a moving die rheometer . The MDR was preheated at 150 °C for approximately 30 min. After that, the test was performed at 150 °C for 60 min with an oscillation of amplitude of 0.5° (~7% strain) and a frequency of 1.667 Hz. The optimum cure times (tS) was determined as the difference between the Smax and Smin according to Equation (2):The torque increase , respectively). Vulcanization was carried out applying a hydraulic pressure of 150 bars using the LP-S-50 press  for 18 min at 150 °C. The chosen cure time was for all crosslinked samples slightly above tTo examine the influence of the molecular weight of PIBOMA on the mechanical properties of rubber composition, the reversible addition−fragmentation chain-transfer polymerization (RAFT) technique was chosen for the synthesis of PIBOMA samples with precise molar masses . 4-CyanoMn theo) of 80,000 g mol−1  was obtained after 72 h. It was found that IBOMA is polymerizing at 60 °C in a controlled manner, i.e., a linear increase in number average molecular weight (Mn) with the rise in conversion and satisfactory low dispersity indices Mw/Mn < 1.35  stayed satisfactorily low and were in the range 1.16–1.21  were absent, proving the purity of the obtained polymers. The broad signals at 2.39–1.64 and 1.00–0.84 ppm were assigned to CH2 and CH3 groups of the polymer backbone, respectively  stretching vibration. The bands at 1237 and 1146 cm−1 are assigned to asymmetric and symmetric C–O–C stretching vibrations of the ester groups, respectively , while all other candidate solvents provided polymers with the similar molar masses varying in the range of 59,500–73,600 g mol−1 (−1) and the highest yield (80%)  and moderate dispersity (Mw/Mn = 1.90).Due to the limitations of the RAFT method for the controlled synthesis of high molar mass polymers ( g mol−1 ), the deld (80%) , entry 8N,N-dimethylformamide (DMF) and cyclohexane upon heating to 80 °C. PIBOMA was found to be insoluble in acetonitrile, acetone, alcohols  and in N-methyl-2-pyrrolidone (NMP).The solubility of the obtained PIBOMA samples was studied in a variety of solvents . It was Tg varying in the range of 194–201 °C, and depending on their molar masses, they can be arranged in the following increasing order:Polymer thermal properties were investigated by thermomechanical (TMA) and thermogravimetric analysis (TGA). According to TMA , the PIBTg PIBOMA 12 K (194 °C) < Tg PIBOMA 26 K (197 °C) = Tg PIBOMA 46 K (197 °C) ≤ Tg PIBOMA 283 K (199 °C) < Tg PIBOMA 59 K (201 °C).Tg of PIBOMA was found to be practically independent on its molecular weight.It can be concluded, that in the studied range of molar masses the Tonset) of the neat PIBOMA samples evaluated by TGA  < Tonset PIBOMA 26 K (225 °C) = Tonset PIBOMA 46 K (225 °C) = Tonset PIBOMA 283 K (225 °C) < Tonset PIBOMA 59 K (230 °C).The compounding of SBR rubber and thermoplastic was performed in accordance to the procedures published previously . The det−1 were investigated by moving die rheometry (MDR) (S) obtained from the difference between maximum torque (Smax) and minimum torque (Smin) were used for the estimation of the reinforcing effect. Relative to the CB control compound, all samples filled with CB and PIBOMA displayed higher ΔS values, irrespective of PIBOMA molecular weight, confirming the high temperature reinforcing effect of PIBOMA. In contrast, the sample filled with CB and UHMWPE displayed a ΔS value close to the CB control in accordance with the softening of UHMWPE at the MDR measurement temperature (150 °C). The increase in the PIBOMA’s molecular weight led to the rise in ΔS values of the respective rubber compositions (The cure characteristics of rubber compounds filled with 50 phr of carbon black (CB) and 20 phr of PIBOMA samples having molecular weight in the range from 20,000 to 283,000 g molry (MDR) a. The tery (MDR) a. The toositions a. The inS SBR/CB (15.2 dNm) < ΔS UHMWPE/CB (15.5 dNm) < ΔS PIBOMA 26 K/CB (18.1 dNm) < ΔS PIBOMA 46 K/CB (18.8 dNm) < ΔS PIBOMA 59 K/CB (19.5 dNm) < ΔS PIBOMA 283 K/CB (19.7 dNm).ΔMn = 283,000 g mol−1, and thus, the PIBOMA 283 K was further selected to study the effect of thermoplastic content on the rheological properties of rubber compounds (The highest reinforcement was shown by a sample containing 20 phr of PIBOMA having ompounds b. ComparS PIBOMA 283 K 5 phr/CB (15.9 dNm) < ΔS PIBOMA 283 K 10 phr/CB (18.2 dNm) < ΔS PIBOMA 283 K 20 phr/CB (19.7 dNm).ΔS value was demonstrated by the respective rubber composition, indicating higher reinforcement.Thus, the higher the content of PIBOMA 283 K was, the higher Δ−1 and UHMWPE is presented in Tα more exactly). Comparing now these two polymer reinforcement systems, several observations can be made. From ~0–30 °C, PIBOMA 283 K provides competitive levels of reinforcement vs. UHMWPE  > σt PIBOMA 46 K/CB (11.7 MPa) > σt PIBOMA 59 K/CB (9.8 MPa) > σt PIBOMA 283 K/CB (9.1 MPa).εt PIBOMA 26 K/CB (390%) > εt PIBOMA 46 K/CB (354%) > εt PIBOMA 59 K/CB (345%) > εt PIBOMA 283 K/CB (280%).In addition, while the moduli of these vulcanized rubber compounds show typical strain-sensitive behavior, it is also evident that compound stiffness increases as PIBOMA molecular weight increases, consistent with what was previously observed via DMTA in the rubbery regime. One possibility here is that reductions in PIBOMA molecular weight favor uptake of small molecules  by the PIBOMA domains through a diffusion mechanism, resulting in increased plasticization. It may also be that variations in the size of the PIBOMA domains with molecular weight and their changes in debonding behavior at the low strains play a role as well see .Additionally, when the concentration of the highest molecular weight PIBOMA 283 K was varied, the following trends in tensile strength and elongation at break were observed :σt 5 phr of PIBOMA 283 K/CB (15.0 MPa) > σt 10 phr of PIBOMA 283 K/CB (14.7 MPa) > σt 20 phr of PIBOMA 283 K/CB (9.1 MPa).t 5 phr of PIBOMA 283 K/CB (365%) > εt 10 phr of PIBOMA 283 K/CB (330%) > εt 20 phr of PIBOMA 283 K/CB (280%).εAs illustrated in c ~ 55–60% typically [Comparing rubber compositions filled with PIBOMA and the control UHMWPE, it can be seen that the UHMWPE containing composition provides higher stiffness than all other rubber compositions and higher tensile strength than all but the thermoplastic-free composition . The facypically ,52). AddHighlighting the enhanced thermomechanical stability provided by PIBOMA in comparison with UHMWPE, the results of tensile testing at 140 °C are shown in The morphology of PIBOMA- and UHMWPE-filled vulcanized rubber compounds was investigated by AFM. Due to immiscibility between rubber and the thermoplastic polymers, phase separation was observed in macro-scale . While tUHMWPE (55 μm) ≈ PIBOMA 26 K (54 μm) < PIBOMA 46 K (45–80 μm) < PIBOMA 59 K (50–50 μm) < PIBOMA 283 K (65–90 μm).As shown in The vulcanized rubber compound filled with PIBOMA 283 K was studied by X-ray computed tomography (CT) before and after tensile testing . Due to Tg thermoplastic, is for the first time evaluated as a reinforcing polymer for vulcanized rubber compounds. For this purpose, a set of PIBOMAs with Mn = 26,000 ÷ 59,500 g mol−1 and low dispersity (Mw/Mn < 1.2) were synthesized via RAFT polymerization. In order to enlarge the range of molar masses studied, an additional PIBOMA sample with Mn = 283,000 g mol−1 was prepared by free radical polymerization. The structure and purity of these polymers was confirmed by IR and NMR spectroscopy, while the study of their properties revealed that they possess good thermal stability (Tonset up to 230 °C) and high glass transition temperatures (Tg ~ 197–201 °C).The conclusions stated herein are derived from, and applicable to, the specific parameters implemented for this unique work. PIBOMA, a high S values relative to the CB control compound, irrespective of PIBOMA molecular weight, confirming the high temperature reinforcing effect of PIBOMA. Similar effects were observed in DMTA, where for vulcanized rubbers in the range of ~30–125 °C, the reinforcing efficiency of low molecular weight PIBOMAs was competitive with that of UHMWPE and PIBOMA 283 K exceeded the performance of UHMWPE throughout this range. At temperatures above ~125 °C the UHMWPE rapidly loses its ability to provide reinforcement due to softening/melting, whereas all PIBOMA resins maintain their capacity to reinforce the rubber matrix up to 180 °C.The synthesized PIBOMA samples were further tested as polymeric fillers in the preparation of vulcanized rubber compositions based on poly(styrene butadiene rubber) (SBR) and containing 50 phr of carbon black. The effect of the content and molar mass of PIBOMA on the properties of the filled rubber compositions was discussed in detail and compared with CB control compound. In MDR measurements all samples filled with CB and PIBOMA displayed higher Δ−1. Moreover, X-ray computed tomography revealed that fracture arose from either debonding at the rubber-PIBOMA interface at high strains, with cracks observed where the PIBOMA domains were concentrated or from the cracks happened inside the PIBOMA domains. This indicates that PIBOMA is likely stiffer but weaker and more brittle than the cured CB-filled rubber. It was further observed that UHMWPE provided higher stiffness than PIBOMA in tensile testing at room temperature as well. However, this is not inconsistent with the DMTA data, which shows that UHMWPE does provide the highest level of reinforcement under these conditions. In contrast to PIBOMA containing compositions, the size of the UHMWPE domains is consistently smaller (~55 μm), and the observation of higher tensile strength in the UHMWPE containing compounds implies a reduced tendency for interfacial debonding, consistent with the possibility that the UHMWPE undergoes partial melting during rubber compound processing and curing. This, coupled with its greater anticipated compatibility with the relatively non-polar rubber matrix and its susceptibility to radical attack, may tend to favor the formation of a stronger interface in the UHMWPE case under the parameters of this work. Nevertheless, the heat generation typically observed in high temperature vulcanized rubber compound applications results in a need for stiffness at temperatures significantly higher than room temperature, where our high temperature stress–strain and viscoelastic data tends to show that the performance of UHMWPE may suffer more than PIBOMA. In this specific context, beyond the high temperature reinforcement reported here, we emphasize the potential of PIBOMA to provide additional levels of performance following potential, new improvements in interfacial strength and process optimization in the future.Finally, with respect to the tensile testing results, it was observed that, for rubber compositions filled with 20 phr of PIBOMA, both tensile strength and elongation at break decreased with increasing PIBOMA molecular weight. Likewise, increasing PIBOMA 283 K content also gave decreases in tensile strength and elongation at break in tandem with increased stiffness. To better understand this result, AFM and X-ray computed tomography were used to characterize rubber compositions before and after tensile testing. The AFM results revealed that the size of the PIBOMA domains in the rubber compositions increased from 65 to 90 μm as the PIBOMA molar mass increased from 26,000 to 283,000 g mol


Cranial nerve (CN) palsy due to cancer involvement has been considered as an unfavorable prognostic factor for patients with nasopharyngeal carcinoma (NPC). We assessed the role of IMRT based treatment on the recovery of CN palsy and investigated the prognostic value of complete recovery of CN palsy.A total of 115 NPC patients with cancer-related CN palsy were included in the study. We referred CTCAE version 5.0 to evaluate the grade of CN palsy.P = 0.001). There were no significant differences for PFS, OS, DMFS and LRFS between patients with complete recovery and non-complete recovery from CN palsy after receiving IMRT based comprehensive treatment.All patients with grade 1 CN palsy recovered completely during the 2 years of follow-up after definite treatment. Most grade 2 palsy could change gradually to grade 1 palsy or complete recovery during 2 years of follow-up. Patients with more than 2 symptoms of CN palsy had poor 3-year disease-free survival (DFS) than these with 1 or 2 symptoms (60.3% vs. 84.9%, HR 0.25, 95% CI 0.07–0.89, IMRT based comprehensive treatment could effectively promote the recovery of tumor-related CN palsy for NPC patient. More than 2 symptoms of CN palsy was a poor prognostic factor for DFS of NPC patients. The prognostic role of complete recovery of CN palsy was not identified in our study.The online version contains supplementary material available at 10.1186/s13014-021-01846-x. Nasopharyngeal carcinoma (NPC) is a cancer arising from the nasopharyngeal mucosal lining, from where the tumor can invade adjacent anatomical organs. Hence, depending on the involvement of anatomical structures, the clinical symptoms varies considerably from person to person, including epistaxis, nasal obstruction, hearing impairments, headache and cranial nerve (CN) palsy. The incidence of CN palsy is approximate 8 to 12.4% in NPC . SeveralUndoubtedly, IMRT is a “Game-Changing” radiation technique for NPC due to the improved dosimetric properties. Locoregional control and survival have been improved by IMRT, and toxicity has been reduced , 7. CompWe reviewed the NPC database from cancer-specific database platform in XiJing Hospital between January 1, 2012 and December 31, 2016 and 514 patients with histologically proved nasopharyngeal squamous cell carcinoma treated with radiotherapy were identified. Of these patients, 121 (22.4%) had CN palsy symptoms. Six patients were excluded: 2 received radiotherapy alone, 3 did not complete the treatment course, and 1 received palliative treatment. The remaining 115 NPC patients with CN palsy were included in the study. This retrospective review was approved by our institutional review board and was in accord with the Helsinki Declaration of 1975 as revised in 1983.The diagnosis of CN palsy depended on clinical symptoms physical examination, and magnetic resonance imaging (MRI) with or without thin slice computerized tomography imaging (CT) of skull base. CN invasion was detected on MRI that met one of the following criteria: (1) enhancing soft-tissue tumor along the course of the ipsilateral-related nerve, replacing the normal structures of the CN on gadolinium-enhanced T1-weighted images; or (2) perineural spread, defined as an enlargement or abnormal enhancement of the nerve, obliteration of the neural fat pads adjacent to the neurovascular foramina, and neuroforaminal enlargement , 11. PhyAll patients were treated with definitive radiotherapy. The detail of radiotherapy process was described in our previous studies , 13. The2, cisplatin 75 mg/m2), PF regimen , GP regimen , and TPF regimen  given every 3 weeks for 2–3 cycles. Concurrent chemotherapy consisted of cisplatin (100 mg/m2 every 3 weeks or 40 mg/m2 weekly).Of these patients, 63 (54.8%) were treated with IC followed by CCRT, and 52 patients (45.2%) received CCRT alone. IC was administrated to patients with high risk metastatic lymph nodes, such as N2 to N3, extracapsular nodal spread, central nodal necrosis, ≥ 3 cm in greatest dimension, and nodal grouping metastasis. Patients with N0 or low risk N1 received CCRT alone. The induction chemotherapy included TP regimen , progression-free survival (PFS), distant metastasis-free survival (DMFS) and locoregional recurrence-free survival (LRFS). OS was measured from the date of treatment completion to death; PFS was measured from the date of treatment completion to the date of disease progression or death from any causes; DMFS was defined as the time from end of treatment to first detection of distant metastasis; LRFS was defined as the time from end of treatment to first detection of local or lymph node region relapse.2 test. Numerical variable was transformed to categorical variable using median as cut-off if it was not Gaussian distribution, such as tumor volume. The Kaplan–Meier method was used to estimate the endpoints, survival curves were compared using the log-rank test. Cox proportional hazard model was used to identify potentially independent prognostic factors, and the proportional hazards assumption was tested with Schoenfeld residuals. The hazard ratio (HR) and its 95% confidence interval (95% CI) were used to indicate the prognostic value of risk factors. A two-sided P value of less than 0.05 was considered significant.The variation of measurement data in groups were compared by means of Student’s t test, and data was presented as means ± standard error of the mean (SEM) values. Differences in proportions between groups were evaluated by χPatient characteristics are summarized in Table Trigeminal nerve  disorder was the most common CN palsy symptom (90.4%), followed by abducens nerve  disorder (36.5%), oculomotor nerve  disorder (22.6%), hypoglossal nerve  disorder (6.1%) and facial nerve  disorder (3.5%). Of all patients, 68 patients (59.1%) had one symptom of CN palsy, 30 patients (26.1%) had two symptoms of CN palsy and 17 patients (14.8%) had more than three symptoms of CN palsy. Because the incidences of HN disorder and FN disorder were very low, we just focused on the recovery of TN disorder, AN disorder and ON disorder in this study.According to the grade definition of CN palsy, no patients had grade 3 symptom in this cohort. Patients with grade 2 CN palsy had significantly longer duration of symptom before clinical diagnosis. The mean duration time were 4.22 ± 0.29 months versus 1.32 ± 0.19 months between patients with grade 2 and grade 1 TN disorder, 3.72 ± 0.41 months versus 1.86 ± 0.37 months between grade 2 and grade 1 AN disorder, 3.14 ± 0.57 months versus 1.41 ± 0.35 months between grade 2 and grade 1 ON disorder, with significantly statistical differences . For grade 2 of AN palsy, there were more patients received radiotherapy with low radiation dose than those received high radiation dose . For grade 2 of ON palsy, the proportions of patients received low radiation dose and high radiation dose were not different . After comparison of recovery rate between the groups of radiation dose, there were no differences of complete recovery rates of TN palsy and ON palsy between two radiation dose groups  as dichotomy, patients were divided into two groups: low radiation dose groups (≤ 72.62 Gy) and high radiation does group (> 72.62 Gy). There were no significant differences in number of CN palsy symptom between the groups of radiation dose (data was not shown). For grade 2 of TN palsy, there were more patients received radiotherapy with low radiation dose than those received high radiation dose . For the whole cohort, the 3-year and 4-year DFS, OS, DMFS and LRFS were 80.9%, 88%, 89.4%, 97.1% and 78.6%, 84%, 88.2%, 94.4%, respectively.P = 0.001). Patients in group 1 had a trend in achieving a better 3-year OS compared with those in group 2, but without statistically significant differences . Patients in group 1 had significantly better 3-year DMFS than those in group 2    in patients with pretreatment duration within 2 months of TN palsy compared with those with longer than 2 months , DMFS (P = 0.09) and LRFS (P = 0.052) between two groups  and DMFS . N stage and age were independent prognostic factors for OS . For LRFS, multivariate analysis failed to detect any positive prognostic factor , N stage (N0-1 vs. N2-3), histological type, treatment modalities, radiation dose (≤ 72.62 Gy vs. > 72.62 Gy), tumor volume (≤ 55.5 ml vs. > 55.5 ml), number of CN palsy symptom (≤ 2 symptoms vs. > 2 symptoms), initial CN palsy (yes vs. no) and 1-year complete recovery rate of CN palsy (yes vs. no). N stage was a positive prognostic factor for DFS , abducens nerve palsy (36.5%), and oculomotor nerve palsy (22.6%) in local advanced NPC . We founWe showed that the pretreatment grade of CN palsy was significantly associated with recovery process of CN palsy. All grade 1 palsy achieved complete recovery within 2 years after treatment. However, only 70% of patients with grade 2 palsy had complete recovery, although all of them had some improvement. The radical radiation dose of NPC was routinely up to 70 Gy in the tumor target for patients received IMRT , 19. EvePrevious study reported multiple CN palsy and long duration of pretreatment symptom were worse prognostic factors for OS in locoregionally advanced NPC in the era of conventional radiotherapy . In thisIn multivariate analysis, consistent with previous studies, N stage was found to be a significant prognostic factor for DFS, DMFS and OS. We failed to detect prognostic value of CN palsy parameters for patient survival in multivariate analysis. Moreover, we could not find association of tumor volume with the number of CN palsy in this study ; OS, overall survival (b); DMFS, distant metastasis-free survival (c); LRFS, locoregional recurrence-free survival (d).Additional file 3: Fig. S3. Survival analysis of patients with pretreatment duration within 2 months and more than 2 months of abducens nerve palsy. DFS, disease-free survival (a); OS, overall survival (b); DMFS, distant metastasis-free survival (c); LRFS, locoregional recurrence-free survival (d).Additional file 4: Fig. S4. Survival analysis of patients with pretreatment duration within 2 months and more than 2 months of oculomotor nerve palsy. DFS, disease-free survival (a); OS, overall survival (b); DMFS, distant metastasis-free survival (c); LRFS, locoregional recurrence-free survival (d).Additional file 5: Fig. S5. Survival analysis of patients with complete recovery and non-complete recovery from cranial nerve palsy. DFS, disease-free (a); OS, overall survival (b); DMFS, distant metastasis-free survival (c); LRFS, locoregional survival recurrence-free survival (d).Additional file 6: Fig. S6. Correlation between tumor volume and number of cranial nerve palsy.


PPARα, -β and -γ) are currently recognized in vertebrate genomes. PPARγ is known to modulate nutrition, adipogenesis and immunity in vertebrates. Natural ligands of PPARγ have been proposed; however, the receptor also binds synthetic ligands such as endocrine disruptors. Two paralogues of PPARα and PPARβ have been documented in teleost species, a consequence of the 3R WGD. Recently, two PPARγ paralogue genes were also identified in Astyanax mexicanus. We aimed to determine whether the presence of two PPARγ paralogues is prevalent in other teleost genomes, through genomic and phylogenetic analysis. Our results showed that besides Characiformes, two PPARγ paralogous genes were also identified in other teleost taxa, coinciding with the teleost-specific, whole-genome duplication and with the retention of both genes prior to the separation of the Clupeocephala. To functionally characterize these genes, we used the European sardine (Sardina pilchardus) as a model. PPARγA and PPARγB display a different tissue distribution, despite the similarity of their functional profiles: they are unresponsive to tested fatty acids and other human PPARγ ligands yet yield a transcriptional response in the presence of tributyltin (TBT). This observation puts forward the relevance of comparative analysis to decipher alternative binding architectures and broadens the disruptive potential of man-made chemicals for aquatic species.Three peroxisome proliferator-activated receptor paralogues ( PPARα, -β and -γ), which control multiple aspects of lipid metabolism and have been described in vertebrate and invertebrate species [PPARα, PPARβ and PPARγ are classical examples of paralogous genes originated from whole-genome duplication (WGD) events that occurred in the vertebrate ancestor. In some invertebrates, such as Saccoglossus kowalevskii (Hemichordata) [PPAR orthologous gene is found. On the other hand, in some teleost species, PPARα and PPARβ genes duplicated during the teleost-specific third round of WGD (3R WGD) event [PPARγ, a second gene was reported in Astyanax mexicanus (Mexican tetra) [Nuclear receptors (NRs) are metazoan-specific transcription factors that modulate the expression of target genes involved in multiple aspects of physiological homeostasis e.g., ,2. Among species ,4. PPARαhordata) , nonverthordata) ,6, and phordata) , a singlD) event ,10,11,12n tetra) .PPAR paralogues display a characteristic gene expression pattern, modulating different metabolic processes; specifically, PPARγ is highly expressed in tissues with active lipid metabolism [PPARγ-related processes have also been described. With respect to teleosts, PPARγ has been associated with the regulation of nutrition [In general, tabolism , mostly tabolism . In addiutrition ,17, adiputrition ,18, immuutrition ,20 and wutrition ,16,21.Danio rerio (zebrafish) PPARγ, disrupting lipid homeostasis [Pleuronectes platessa (European plaice), D. rerio (zebrafish), or Pantodon buchholzi (butterflyfish) [Proposed endogenous ligands of PPARγ include polyunsaturated fatty acids (PUFA), such as docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), arachidonic acid (ARA) and linoleic acid (ALA), as well as some of their derivatives, particularly oxygenated metabolites such as oxylipins; in addition, some monounsaturated fatty acids (MUFA) may also act as PPARγ ligands ,24,25,26eostasis ,28,29. Tflyfish) ,31.PPARγ gene repertoire in different teleost fish species. Through comparative genomic and phylogenetic analyses, we show that the PPARγ gene duplicated in the ancestor of teleosts, and that both paralogues were retained in Salmoniformes, Clupeiformes, Siluriformes, Characiformes and Esociformes taxa. Furthermore, expression profiles and functional characterization of European sardine’s (Sardina pilchardus) PPARγA and PPARγB is provided. Despite the differences in PPARγA and PPARγB gene expression patterns across several tissues of sardine, similar ligand-binding profiles were found, including activation by TBT, previously unreported within teleosts.In the present work, we provide an exhaustive examination of PPARγ sequences. Amino acid sequences from teleosts belonging to several taxonomic orders were collected from GenBank and Ensembl. In addition, PPARα and PPARβ amino acid sequences of a reptile (Anolis carolinensis), an amphibian , two Actinopterigii (D. rerio and Lepisosteus oculatus), a bird  and two mammals (Homo sapiens and Mus musculus) were used to root the phylogenetic tree. The gathered amino acid sequences were aligned to identify and exclude partial and/or low-quality sequences for further analyses, resulting in a final dataset of 68 PPAR amino acid sequences .A phylogenetic analysis was performed to establish the orthology of novel Clupea harengus) PPARγA and PPARγB genes were localized in the scaffolds NW_012218338.1 (890.5kb) and NW_012221131.1 (264.7kb), respectively. Nine neighboring genes from both sides of these target genes were retrieved from the GenBank database to assemble synteny maps. Atlantic herring loci (GCA_000966335.1) were further used as a reference to BLAST against the genome of the remaining 10 species: European sardine , northern pike , channel catfish , Mexican tetra , red-bellied piranha , Atlantic salmon , zebrafish , Amazon molly , barramundi perch  and spotted gar .The genomic regions containing Atlantic herring (S. pilchardus genome (SP_G) assembly and 11 RNA-Seq datasets , brain + pituitary gland and white muscle) were downloaded from NCBI, while the .gtf annotation file was retrieved from ORCAE . The RGE was accessed using a reference-based approach with Hisat2 V.2.2.0 [PPARγA (Spil_000003g0143.1) and PPARγB (Spil_000027g0044.1) genes were identified via blast in the ORCAE database . Next, Hisat2 was used to align the RNA-Seq datasets to the genome, and StringTie was used to perform gene abundance quantification, under the defaults, following the protocol of [The genome assembly and RNA-Seq datasets of the European sardine  were use V.2.2.0  and Stri V.2.2.0  softwaretocol of . Gene abTM cDNA Synthesis Kit , considering the manufacturer’s recommendations.Liver tissue from a single specimen of European sardine was collected and preserved in RNAlater at −20 °C. Total RNA was then extracted using Illustra RNAspin Mini RNA Isolation Kit , following manufacturer’s instructions. Isolated RNA was treated with RNase-free DNase I and eluted with RNase-free water. After RNA quality and quantity assessment using a microplate spectrophotometer , 500 ng of liver RNA was used for cDNA synthesis with iScriptPPARγ-like were identified in a liver transcriptome dataset of S. pilchardus [Two gene sequences of lchardus . Two setPPARγ gene sequence, featuring the DNA binding domain (DBD) of GAL4 and the LBD of one of the PPARγ paralogues. The produced hybrid protein will conditionally bind to the upstream activation sequences (UAS) of a second experimental vector containing a luciferase reporter gene . Besides the hybrid protein, the pBIND vector also expresses Renilla luciferase, used to normalize firefly luciferase activity. The plasmid sequences were confirmed by Sanger sequencing .PCR products and the cloning vector pBIND  were then digested with XbaI and KpnI (NZYTech) and ligated with T4 ligase (Promega) to produce two GAL4 DBD/PPARγ LBD hybrid proteins, one for each sardine 2.COS-1 cells were cultured in DMEM with phenol red , supplemented with 10% fetal bovine serum (PAN-Biotec) and 1% penicillin/streptomycin (PAN-Biotec), and maintained at 37 °C in a humidified atmosphere with 5% CO5 live cells/mL in DMEM with phenol red supplemented as described before. The next day, cells were transfected with 500 ng of pBIND and 1000 ng of pGL4.31, using Lipofectamine 2000 reagent (Thermo Fisher Scientific), in Opti-MEM reduced-serum medium , following the manufacturer’s instructions. After 5 h of incubation, transfection medium was removed and cells were exposed to 1 mL of DMEM without phenol red (PAN-Biotec), previously supplemented with 10% of charcoal-treated fetal bovine serum (PAN-Biotec) and 1% of penicillin/streptomycin, containing 1 µL of each test compound (explained in detail below). Dimethyl sulfoxide  was used as solvent control, at a final concentration of 0.1% per well. After 24 h of exposure, cells were lysed and firefly luciferase (pGL4.31) and Renilla luciferase (pBIND) activities were determined using a Dual Luciferase Assay System kit , considering manufacturer’s recommendations, in a Synergy HT Multi-Mode Microplate Reader. For luciferase-based reporter transactivation assays, COS-1 cells were seeded 24 h before transfection in 24-well culture plates at a density of 2 × 10All assays were repeated three times independently, with two technical replicates per condition being performed each time. Beyond the European sardine PPARγA and PPARγB, these assays were also performed with human PPARγ (kindly provided by Ana Capitão) for comparison purposes, and to guarantee the reliability of the results.COS-1 cells were exposed to three different concentrations of PPARγ agonist tributyltin chloride ; to rosiglitazone  and T0070909 , human PPARγ agonist and antagonist, respectively; and to fatty acids , arachidonic acid , docosahexaenoic acid  and eicosapentaenoic acid . Two dilutions (50% and 25%) of a mixture composed of seven FAs  were also tested. All the stock solutions and respective dilutions were prepared in DMSO, with its final concentration being 0.1% in each well.Renilla luciferase activity . Final results were presented as fold-induction variations with respect to the solvent control (DMSO). Normalized values of the different replicates were Ln(Log e)-transformed, and a one-way ANOVA followed by Tukey’s post hoc test were performed, using Sigma Stat , to compare the results. A p-value (p) ≤ 0.05 was considered statistically significant. Results are presented as mean ± standard error of the mean (SEM).To determine the statistical significance of the transactivation results, firefly luciferase activity (reported by pGL4.31) was first normalized using The tridimensional models of the LBD of the European sardine PPARγA and PPARγB were deduced using a Swiss-Model homology modelling workspace in alignment mode ,41. HumaL. oculatus) branched off from the teleost PPARγ clade. In agreement with the 3R WGD (third Round of Whole-Genome Duplication), two branches diverged in the teleost PPARγ group, corresponding to the PPARγ gene duplication in the last common ancestor of teleosts. Hence, both PPARγA and PPARγB paralogous genes are identified in some teleost orders, namely, Salmoniformes , Clupeiformes (C. harengus and S. pilchardus), Siluriformes (I. punctatus), Characiformes (A. mexicanus and P. nattereri) and Esociformes (E. lucius). Furthermore, duplicate PPARγB genes (PPARγB1 and PPARγB2) were observed in the salmonids as result of the salmonid-specific fourth round (4R) of WGD  of WGD .PPARγ paralogous genes was also confirmed by examining PPARγA and PPARγB loci composition and neighborhood in 10 teleosts and in a non-teleost (L. oculatus) fish species  of the two  tissues . Our anapression .We next examined the ligand binding profile of European sardine PPARγA and PPARγB in the presence of proposed PPARγ ligands . TributyD. rerio (zebrafish) and P. buchholzi (butterflyfish) [To further address the TBT activation profile, previously unreported in teleost fish, we deduced European sardine PPARγA and PPARγB tridimensional structures and examined the conservation of LBD residues reported to interact with TBT, through structural and sequence alignment, using the human PPARγ crystal structure (3WJ4) as a template . Overallflyfish) . The obsflyfish) , similarflyfish) . Curiousflyfish) . Despiteflyfish) .PPAR-like sequences present in all vertebrate lineages result from WGD events that occurred before gnathostome radiation and after tunicate divergence, yielding the three PPAR paralogous genes. The consequences of the 3R WGD on PPAR gene numbers has also been previously established for both PPARα and PPARβ genes [PPARγ paralogous genes in Mexican tetra (A. mexicanus), suggesting a pronounced loss of duplicated PPARγ in other teleost lineages. In the present work, by means of comparative genomics and phylogenetic analyses, we investigate the presence of PPARγ paralogous genes in an ample collection of ray-finned fish lineages. As expected, spotted gar (L. oculatus) presented a single PPARγ copy, since this species diverged prior to the 3R WGD event , coho salmon (O. kisutch) and rainbow trout (O. mykiss), which resulted of the 4R WGD. The presence of both PPARγ paralogues in such lineages and the retention of a single PPARγ paralogue in the early-diverging teleost lineages such as Elopomorpha  and Osteoglossomorpha , allows us to estimate that the retention of PPARγ duplicated genes occurred at the time of Clupeocephala divergence, approximately 185-190 Million years ago [PPARα and PPARβ, PPARγ duplication resulted from 3R WGD, with episodes of gene loss shaping teleost PPARγ gene repertoire.During the evolution of vertebrates, polyploidization events have been proposed to provide the new genetic material for the emergence of novelties ,49,50,51Rβ genes ,59,60,61Rβ genes  identifiGD event . Moreoveus genes  were retus genes . These oicipated . Additioears ago . Thus, sPPARγ paralogues. This agrees with previous reports with plaice (P. platessa) and gilthead sea bream (Sparus aurata) PPARγs, also yielding no activation upon exposure to FAs [Leucoraja erinacea (little skate) on the other hand, FAs were shown to be potent inducers of PPARγ [Paracentrotus lividus, PPARγ is unresponsive to TBT despite the conservation of H3 cysteine, whereas the cysteine-to-tyrosine substitutions observed in teleosts such as zebrafish and in the mollusk Patella depressa yield null or repressive responses, respectively [Human PPARs can be activated by both saturated and unsaturated dietary FAs, as well as lipids involved in intracellular signaling pathways ,64,65. We to FAs . In the of PPARγ . Sardineof PPARγ ,68,69. Iof PPARγ ,71 and fof PPARγ ,73. TBT of PPARγ ,75. Crysof PPARγ . Cysteinof PPARγ ,76. In aof PPARγ . Furtherof PPARγ . Yet, boof PPARγ . In spitectively . Thus, fPPARγA displaying ubiquitous expression in all analyzed tissues, whereas PPARγB is only expressed in midgut, kidney, liver, head kidney, brain and pituitary gland, and white muscle , liver, kidney, heart, adipose tissues, skeletal muscle, placenta and lung. Further promoter usage and alternative splicing lead to the differential expression of PPARγ splice variants in each of these tissues reviewed in [Although no differences were found in the ligand-binding profile of sardine PPARγA and PPARγB, relative gene expression analysis shows a distinct tissue distribution pattern, with the European sardine e muscle . In mammiewed in ,79,80. TPPARγ gene duplication, as previously described for PPARα and PPARβ paralogues. PPARγ paralogue retention was first reported in A. mexicanus (Characiformes), however, we suggest that PPARγ paralogue retention affected many other teleost lineages. In spite of the differential tissue distributions and expression patterns of sardine PPARγA and PPARγB, indicating tissue-specific functions, both PPARγ genes presented similar ligand preferences. Nevertheless, the modulation of sardine PPARγ paralogues by TBT challenges current paradigms and emphasizes the need to further address the mechanisms of ligand-binding and the susceptibility of teleost lineages towards organotin exposure.In conclusion, this study indicates that 3R WGD led to


Assisted reproductive technologies such as sperm cryopreservation and in vitro fertilization therefore represent critically-needed tools to manage these populations. However, the motility of post-thaw red wolf sperm rapidly declines during in vitro incubation, hindering the ability to develop these technologies. In this study, we evaluated the influence of several culture media (a modified canine capacitation medium (mCCM), a modified North Carolina State University-23 medium (mNCSU-23), a synthetic oviductal fluid (SOF), a fertilization Tyrode’s medium base or Fert-TALP (FERT), and a TRIS-based buffer (TRIS)) on the survival and capacitation of red wolf sperm during extended (18 h) incubation at 38.5°C and 5% CO2. Red wolf sperm motility averaged (±s.e.m.) 73.8 ± 7.1% at the time of collection, and was better maintained over 4 h incubation in mCCM (55.0 ± 9.8%) and mNCSU-23 (54.7 ± 10.4), compared to mSOF (43.8 ± 8.3%), FERT (30 ± 10.5), and TRIS (16.4 ± 4.1%) solutions. Patterns of tyrosine phosphorylation signal, as assessed via immunocytochemistry, indicated induction of capacitation between 2 and 4 h in vitro culture. Tyrosine phosphorylation signal was particularly robust in mCCM and mNCSU-23 incubated sperm, although significant acrosome exocytosis was not observed in response to progesterone supplementation after 3 h incubation in any of the media. In sum, results indicate mCCM and mNCSU-23 are promising base media for the in vitro incubation and capacitation of red wolf sperm, for assisted reproduction applications.The red wolf is a critically endangered canid, with ~250 and ~20 individuals in the in vitro fertilization and artificial insemination is of high importance to the genetic management of critically endangered species such as the red wolf (Canis rufus). However, these technologies require the ability to maintain sperm viability and function during extended incubation, which has not been successful for the red wolf thus far. In this study, various culture media developed for sperm/egg/embryo culture in large mammalian species were evaluated for their ability to maintain red wolf sperm motility under physiological incubation conditions. Media and conditions previously utilized for domestic dog sperm were found to best support sperm incubation and capacitation (process of becoming competent to fertilize an egg) in the red wolf, representing a key step for future development of assisted reproductive technologies for the species.Development of assisted reproductive technologies such as  The red wolf is a critically endangered American canid, with fewer than 20 individuals in the reintroduced population and approximately 250 managed individuals in the Association of Zoos and Aquariums (AZA) Species Survival Plan population . Specifiin vitro fertilization technologies for this species requires long-term sperm incubation capacity.Previous assisted reproduction research in red wolves has characterized semen and optimized sperm cryopreservation and thawing protocols  . Synthetic oviductal fluid (SOF), was originally developed for ovine embryo work, but has also been utilized in domestic dog oocyte culture  . Fert-TAm (mCCM) . A medium (mCCM) ), has al culture . Finallyad libitum. All procedures were done in accordance with the AZA SSP and USFWS guidelines and Smithsonian Institution International Animal Care and Use Committee approval (IACUC #18-05).Adult (ages 2–9 years old) male red wolves housed at AZA Species Survival Plan facilities in the Eastern United States were selected for collection based on health status, mean kinship value, and geographic location. Animals were exposed to natural photoperiod, fed diets consisting of commercially available dry dog food and/or whole carcass, with water provided A single collection attempt was made per animal in the 2018 and 2019 breeding seasons (January–March), for a total of eight ejaculates collected from six individuals . Wolves were fasted 24 h prior to semen collection. On the day of semen collection, animals were captured, transferred to a crate and transported a short distance from their outdoor enclosure to an indoor treatment room, wherein wolves were anesthetized by veterinary staff. Typically, red wolves were anesthetized using butorphanol (0.4 mg/kg body weight IM) and medetomidine (0.4 mg/kg IM). Prior to the start of electroejaculation, the rectum was evacuated using a well-lubricated gloved hand and the penis carefully exteriorized, washed thoroughly with sterile saline and wiped dry.Red wolf semen was collected via electroejaculation as previously described  prior to in vitro incubation and capacitation evaluations.Semen fractions with no evidence of urine contamination (based on color and pH 6–8) and presence of sperm from all stimulation rounds were pooled per individual, then centrifuged at 500 6 sperm/mL at 38.5°C and 5% CO2. The five media included: mCCM , and Tred: mCCM ) was notn = 5), sperm were additionally incubated in either mNCSU-23 or in mNCSU-23 containing only 1 mg/mL BSA without sodium bicarbonate, with osmolarity returned to 300 mOsm with NaCl, to reduce capacitation-stimulation . After 3 h incubation, sperm in each medium were either exposed to 6 µg/mL progesterone (P4) or DMSO vehicle control. Sperm motility, acrosome status, and tyrosine phosphorylation patterns were evaluated as described earlier at hours 1, 2, 4, and 18 of incubation at 38.5°C in 5% CO2.In a subset of the animals , tyrosine phosphorylation , and nuclear  evaluations. Briefly, fixed sperm samples were smeared on glass slides and dried at room temperature. Following three PBS washes, slides were permeabilized with 0.1% Triton-X (Sigma-Aldrich X100) in PBS then blocked in 1% BSA in 0.1% Triton-X PBS for 1 h. Slides were incubated with 2 µg/mL anti-phosphotyrosine antibody in a humidified chamber, either overnight at 4°C or for 4 h at room temperature. Samples were incubated with 1:200 dilution of Alexa rabbit anti-mouse 568, 10 µg/mL FITC-PNA and 5 µg/mL Hoechst 33342 at room temperature for 1 h. After three PBS washes, glass coverslips were mounted with Prolong Anti-fade mountant (ThermoFisher P36980) and slides were observed at 40× magnification with an EVOS Fl Auto 2 (ThermoFisher).At least 90 sperm were counted for each time point and treatment, per animal. Acrosome status was recorded as intact, partially-intact, or reacted. Localization of phosphorylated tyrosine signal was recorded as in the head, midpiece, tail, tail and midpiece, all, or none of the sperm .P < 0.05. Motility data (% motile sperm) were evaluated with a nonparametric Wilcoxon test. Tyrosine phosphorylation pattern data were subjected to linear transformation then evaluated via generalized linear mixed effect model with gamma distribution (link log) in package ‘lme4’ (ver. 1.1-23) , with significance set at 6 sperm/mL and 30–90% total motile (s.e.) averaged 55.0 ± 9.8 in mCCM and 54.7 ± 10.4 mNCSU-23, respectively, compared with 30 ± 10.5 in FERT and 16.4 ± 4.1% in TRIS. While sperm attachment to glass slides prevented quantification of sperm hyperactive motility, characteristic star-spin movement and lateral head displacement with rapid flagellar beating were observed at the 4 h time point only for mCCM, mNCSU-23 (Supplementary Video 1), and mSOF-treated sperm. By 18 h incubation, mNCSU-23 incubated sperm displayed higher motility than those incubated in TRIS (P < 0.05), but was not significantly different from mCCM, mSOF, or FERT incubated sample (P > 0.05).Semen collected via electroejaculation from red wolves ranged from 1.1 to 10.3 mL with a concentration of 18 to 198 × 10l motile . Over thl motile . For exaP < 0.01), not treatment group, was significantly based on the model. Overall, the proportion of sperm with variable patterns of tyrosine phosphorylation (PY) immunofluorescence increased from 0 to 4 h for all treatments, then reduced from hours 4 to 18. PY signal in the ‘midpiece’ rose from 1 to 2 h of incubation, followed by a more robust increase in ‘tail’ and ‘midpiece + tail’ signal at 2 and 4 h  were incubated under NC-mNCSU-23 compared with normal, capacitating mNCSU-23. Motility of sperm in NC-mNCSU-23 was significantly reduced compared with mNCSU-23 incubated sperm at hours 1, 2, and 4 of incubation . Progesterone supplementation had no influence on percent motility for either media. Sperm in both media displayed a similar loss in acrosome integrity over 18 h culture (P < 0.05), with no significant differences between progesterone treatment conditions (P < 0.05), but not progesterone or its interaction with treatment. For example, for midpiece tyrosine phosphorylation, the signal rose in sperm exposed to both treatment conditions at 1–2 h of incubation but was significantly higher in NC-mNCSU-23 incubated sperm compared with mNCSU-23  conditions. A subset of samples  identify a base medium supportive of red wolf sperm incubation, and (2) develop a capacitation protocol for red wolf sperm. We found that red wolf sperm motility and acrosome integrity is better maintained in mCCM, mNCSU-23, and mSOF in vitro compared with FERT and TRIS solutions. Induction of capacitation, based on patterns of tyrosine phosphorylation signal, occurred between 2 and 4 h in vitro. Results indicate that both mNCSU-23 and mCCM are promising sperm handling media, based on their ability to maintain sperm motility and acrosome integrity, and stimulate tyrosine phosphorylation. Our subsequent evaluation of mNCSU-23 indicates additional optimizations are necessary to improve capacitated red wolf sperm’s ability to undergo acrosome exocytosis. Nevertheless, these results represent a key first step toward the recapitulation of capacitation in vitro, necessary to produce fertilization-competent sperm for IVF work and to evaluate the quality of post-thaw semen in the future.The ability to maintain red wolf sperm in vitro in Tyrode’s albumin lactate pyruvate at room temperature or 0°C   might reveal a delayed effect of progesterone, which should be evaluated in future studies. Alternatively, the red wolf may be similar to the bovine, wherein P4 has been shown to stimulate acrosome reaction but not capacitation in fresh epididymal sperm  has reported that sperm require 5–8 h to capacitate and subsequently successfully penetrate fox ova  progesterone after 3 h incubation , despitein vitro , compared with mSOF and FERT . Glucose supplementation has been demonstrated to extend the survivability of human sperm , and the J B N, M d A M M F and N S conceived the study. J B N, M d A M M F, and S H K performed the experiments. J B N analyzed the data. J B N, M d A M M F, S H K, and N S wrote the paper.


Juglans regia L.  leaves extract on osteogenesis of human bone marrow mesenchymal stem cells (hBMSCs).The present study investigates the effects of hBMSCs were incubated with different concentrations of JRL extract . Cell proliferation was evaluated by Cell Counting Kit-8 assay (CCK-8) assay. ALP activity and Alizarin Red staining were used to assess the osteogenesis of BMSCs. Western blot was performed to measure the levels of proteins.Our results showed all concentrations of JRL extract had no significant effect on cell proliferation. JRL extract concentration-dependently promoted osteoblastic differentiation and cell autophagy of hBMSCs, characterized by the increased expression of pro-osteogenic markers alkaline phosphatase (ALP), osteocalcin (BGLAP), osterin, and osteoprotegerin (OPG) and autophagy marker proteins . Furthermore, JRL extract stimulated the activation BMP2/Smad/Runx2 and Wnt/β-catenin signaling pathways in hBMSCs, which play key roles in osteogenesis differentiation. Meanwhile, BMP inhibitor (Noggin) and Wnt antagonist Dickkopf-1 (DKK1) both reversed the increases of BGLAP, osterin, and OPG expression induced by JRL extract.Our findings indicate that JRL extract regulated osteogenic differentiation and cell autophagy of hBMSCs through the BMP2/Smad/Runx2 and Wnt/β-catenin pathways. Osteoporosis (OP) is a common senile disease, and its pathological features are characterized by decreased bone mass and destruction of bone micro-structure, resulting in decreased bone strength, increased fragility and increased risk of fracture –3. PatieThe dynamic balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption is the basis for maintaining the stability of human bone metabolism and the integrity of the body's bones. When the balance is disturbed, bone loss, bone density, and bone microstructure are destroyed, leading to increased bone fragility, and ultimately osteoporosis . Of noteJuglans regia L.  is an annual herbal that belongs to the Juglandaceae family [The treatment of OP is mainly based on drugs. The commonly used bone resorption inhibitors in clinical practice include bisphosphonates, selective estrogen receptor modulators, calcitonin, and estrogen . Althouge family . Its frue family . The exte family , inflamme family , and cane family . Notablye family , implyine family . HoweverIn the present study, we aimed to determine the promoting effects of JRL leaves extract on osteogenesis and cell autophagy of hBMSCs by regulating BMP2/Smad/Runx2 and Wnt/β-catenin pathway, which play a role in osteoblast differentiation .Juglans regia L. were collected from the city of Luzhou in Sichuan province (China). The leaves were shade dried and grind into powder. Then, the material was extracted with 500 mL of ethanol for 2 h using the Soxhlet apparatus. The supernatant was filtered and vacuum evaporated under pressure at 50 °C until a solid extract was obtained (18.2% yield). The resulting solid extract was stored at 4 °C for future use.The leaves of The chemical composition of JRL was analyzed by UPLC-MS. The UPLC-MS analysis was carried out with a hybrid Quadrupole-TOF LC/MS/MS Mass Spectrometer  using a Waters BEH C18 column  with the column temperature at 40 °C. The mobile phase was 0.1% HCOOH-H2O (A)-acetonitrile (B) at a flow rate of 0.3 mL/min in gradient elution as follows: 0–2 min, 95% A; 2–13 min, 5% A; 13–15 min, 95% A. Electrospray ionization (ESI) positive and negative ion modes were used for detection. The ESI source conditions were set as follows: Ion Source Gas1 (Gas 1): 50, Ion Source Gas2 (Gas 2): 50, Curtain Gas (CUR): 25, Source Temperature: 500 ℃/450 ℃ (positive ion/negative ion), Ion Sapary Voltage Floating (ISVF) 5500 V/4400 V(positive ion/negative ion), TOF MS scan range: 100–1200 Da, product ion scan range: 50–1000 Da, TOF MS scan accumulation time 0.2 s, product ion scan accumulation time 0.01 s. The secondary mass spectrum was obtained by information-dependent acquisition (IDA) with high sensitivity mode.2 in a humidified incubator.Human bone marrow mesenchymal stem cells (hBMSCs) were purchased from ScienCell . The cells were cultured in DMEM medium supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin sulfate , which is called as complete medium (CM), and then incubated in an incubator at 37 °C with 5% COJuglans regia L.  extract in the presence of OIM  to identify the optimum concentration of JRL extract that induced the osteogenic differentiation of hBMSCs. Subsequently, cells were separated into 4 groups: PBS and OIM cultured group; JRL extract and OIM cultured group; OIM, JRL extract and 100 ng/mL BMP-inhibitory protein Noggin  cultured group; OIM, LRL extract and 2 mg/mL Wnt antagonist Dickkopf-1  cultured group. The grouping of experiments is shown in Table Cells were treated with one of different concentrations of 4/mL) and seeded in 96-well plate (1.0 × 103 cells/well). Then, the seeded cells were incubated at 37 °C and 5% CO2 for 48 h. Finally, the supernatant was removed. 100 μL CCK-8 solutions were added to each well and further incubated for 1 h at 37 °C. The absorbance values were read at a wavelength of 450 nm.Cell viability was assessed using the Cell Counting Kit-8 assay  according to the manufacturer’s protocol. To be brief, cells were digested with trypsin . The membranes were blocked with 5% nonfat milk and incubated with corresponding protein antibodies or a rabbit anti-β-actin monoclonal antibody. Then, the membranes were subsequently incubated with a HRP Goat anti-Rabbit IgG . The net optical density was analyzed with the gel Image processing system (Image-pro Plus 6.0). Primary antibodies were purchase from Abcam  under the following item No: Osteocalcin , Osterix , OPG , BMP2 , Runx2 , Smad1 , p-Smad1 , Wnt3a , β-catenin , p-β-catenin , TCF-7 , Lef1 , DLX5 , LC3II/LC3I, , Beclin-1, and 962 , and β-actin . The bands were visualized using the chemiluminescence (ECL) system .2. After reaching 80% confluence, hBMSCs were treated with OIM or OIM containing different concentration of JRL  extract for 7 days. ALP activity was determined by the ALP colorimetric assay kit .hBMSCs were cultured in 48-well plates under the optimal conditions of 37 °C and 5% COP < 0.05.The data were represented as means ± standard deviation (SD). The SPSS software  was used to calculate all the values. One-way analysis of variance (ANOVA) followed by Tukey's-B post hoc tests were used for comparison between groups. The statistical significance was We confirmed the chemical composition of JRL using ultra-performance liquid chromatography–mass spectrometry (UPLC-MS). In the chromatographic profile of JRL, melibiose, melezitose, ganolactone B, methylophiopogonanone A, tubuloside A, and 2-Acetylacteoside were detected in positive ionization scan mode Fig. a, whereaFirstly, hBMSCs were incubated in OIM with or without different concentrations of JRL  extract for 1–14 days to ascertain the effect of JRL extract on cell proliferation. According to the results of the CCK-8 assay, different concentrations of JRL extract had no significant effect on cell proliferation at different time points  and Runx2 are two downstream transcription factors of BMP2 pathway. Our data showed that the expressions of BMP2, DLX5, and Runx2 were also increased in OIM and JRL extract-cultured hBMSCs. Moreover, these increased levels were substantially decreased by Noggin (Fig. Finally, we examined whether the BMP2/Smad/Runx2 and Wnt/β-catenin signaling pathways mediate the regulation of osteogenic gene expression and cell autophagy induced by JRL extract. Runx2 is an essential transcription factor for osteoblast differentiation, which was regulated by BMP2/Smad and DLX5 . MeanwhiJRL extract mainly contains naphthoquinone, tannin, flavone, terpene, tocopherols, steroids, and other components , 29. TheMechanically, we found that JRL extract stimulated BMP and Wnt/β-catenin signaling pathways in hBMSCs. The BMP2/Smad signaling pathway plays an important role in the osteogenic differentiation of BMSCs . BMP2 biIn this study, we firstly explored the effects and mechanisms of JRL extracts on osteogenesis of BMSCs. We concluded that JRL extract had a positive effect for the expression of osteogenic genes in hBMSCs. Meanwhile, BMP2/Smad/Runx2 and Wnt/β-catenin signaling pathways mediated osteogenesis differentiation and cell autophagy stimulated by JRL extract. Our study describes a promising therapeutic agent for patients with OP.


Experimental results demonstrated that compound 12 not only mitigated total body irradiation-induced hematopoietic injury by increasing the frequencies of hematopoietic stem and progenitor cells but also prevented abdominal irradiation-induced intestinal injury by increasing the survival of Lgr5+ intestinal cells, lysozyme+ Paneth cells, and Ki67+ cells. In addition, compound 12 decreased oxidative stress by upregulating the expression of Nrf2 and NQO1 and downregulating the expression of NOX1. Further, compound 12 inhibited γ-irradiation-induced DNA damage and alleviated G2/M phase arrest. Moreover, compound 12 decreased the levels of p53 and Bax and increased the level of Bcl-2, demonstrating that it may suppress radiation-induced apoptosis via the p53 pathway. These results indicate that compound 12 has the possibility of preventing radiation injury and can be a potential radioprotector for clinical applications.The risk of radiation damage has increased with the rapid development of nuclear technology and radiotherapy. Hence, research on radioprotective agents is of utmost importance. In the present study, a novel aminothiol compound 12, containing a linear alkylamino backbone and three terminal thiols, was synthesized. Owing to the appropriate capped groups in the chains, it has an improved permeability and oral bioavailability compared to other radioprotective agents. Oral administration of compound 12 improved the survival of mice that received lethal doses of  Humans may be exposed to ionizing radiation (IR) during radiological attacks and accidents, exploration of the universe, and radiation therapy –3. UnderRadioprotectors are compounds used before or during radiation exposure to prevent radiation damage . Over thIn the current study, we designed and synthesized a novel aminothiol compound. It contains a linear alkylamino backbone and three thiols which are capped at the ends of the alkyl side chains. Compared with the aminothiol compounds reported in previous studies , 13, 17,Compound 12 was synthesized as described in Guide for the Care and Use of Laboratory Animals and were permitted by the Institutional Animal Care and Use Committee of the IRM, CAMS.Male C57BL/6 mice (6–8 weeks old) were obtained from HFK Bioscience Co. Inc. (Beijing) and bred in a pathogen-free environment at the laboratory animal center of the Institute of Radiation Medicine (IRM), Chinese Academy of Medical Sciences (CAMS). All animal experiments were performed according to the NIH 137Cs γ-ray source placed in the Exposure Instrument Gammacell-40  at the dose rate of 0.99 Gy/min. For the TBI study, mice were randomly divided into five groups in the survival experiment: control, 8 Gy + vehicle, 8 Gy + 12 (900 mg/kg), 8 Gy + 12 (1200 mg/kg), and 8 Gy + 12 (1500 mg/kg) (n = 10 each). In the remaining TBI studies, mice were randomly divided into four groups: control, 12 (1200 mg/kg), 4 Gy, and 4 Gy + 12 (1200 mg/kg) (n = 5 each). For the ABI experiments, a lead shield was used such that the entire abdomen was irradiated and other parts were shielded, and the mice were assigned to three groups: control, 15 Gy + vehicle, and 15 Gy + 12 (1200 mg/kg) (n = 10 or n = 5). The mice in the IR + 12 groups received compound 12 via oral gavage 1 h before irradiation. The mice in other groups were treated with vehicle or compound 12 in the same way.Irradiation was carried out using a Mice were exposed to TBI or ABI at the indicated doses. The status of the mice was monitored for 30 days following radiation, and data are presented as survival rates.γ-irradiation and were sacrificed on day 14 after irradiation. Peripheral blood was collected from the orbital sinus. White blood cells (WBCs), red blood cells (RBCs), platelets (PLTs), hemoglobin (HGB), the percentage of lymphocytes (LY%), and the percentage of neutrophils (NE%) were analyzed using a hematology analyzer .For hematopoietic damage experiments, the mice in the TBI groups received 4.0 Gy −Sca1+c-kit+ (LSK) was employed to define HSCs as suggested in earlier studies [6) were first labeled with biotin-conjugated antibodies recognizing Gr1, CD4, CD8, CD11b, B220, and Ter119  and then incubated with streptavidin-PerCP (BioLegend), sca1-PE, and c-kit-APC antibodies . The frequencies of HPCs and HSCs were determined via flow cytometry and analyzed by using BD Accuri C6 software .For analysis of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs), bone marrow cells were isolated from mouse femurs with sterile PBS and filtered. Lineage studies , 18, 19.6 bone marrow cells were incubated with LSK and HPC antibodies as described above and then stained with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) at a concentration of 10 μM for 25 min at 37°C. Intracellular ROS levels were evaluated by determining the mean fluorescence intensity (MFI) of DCFH-DA using flow cytometry.To evaluate ROS levels, 5 × 10μm thick sections. The paraffin sections were deparaffinized, rehydrated, and stained with hematoxylin and eosin (HE). Images were acquired using a microscope . For histological analysis, five circular fields were analyzed per mouse in a blind fashion to count the number of crypts and measure the length of the villi.For intestinal injury experiments, mice in ABI groups received irradiation (15.0 Gy dose), and all mice were sacrificed on day 5 after irradiation. The small intestine of each mouse was collected, fixed in 4% formaldehyde, dehydrated, and embedded in paraffin. Next, the blocks were cut into 4 μm thick) were deparaffinized and rehydrated. To block endogenous peroxidase, deparaffinized sections were incubated in 3% hydrogen peroxide for 15 min at 37°C. Then, antigen retrieval was performed by boiling the sections in a citrate buffer solution, and the sections were blocked with goat serum. Next, these sections were incubated with anti-Villin antibody , anti-Lgr5 antibody , anti-lysozyme antibody , or anti-Ki67 antibody  overnight at 4°C. The sections were then stained with the corresponding secondary antibodies for 1 h at 37°C. Finally, the DAB kit was used to detect the cells, and the sections were stained with hematoxylin. Images were acquired using the microscope (Olympus), and immunoreactive cells were analyzed in a blind fashion using the ImageJ software.Sections of the small intestine .The 4 μm thick small intestine sections were deparaffinized and rehydrated, and antigen retrieval was performed as described above. The sections were blocked with goat serum at 37°C for 1 h and incubated with anti-γ-H2AX antibody , anti-p53 antibody , anti-caspase-9 antibody , or anti-caspase-3 antibody  overnight at 4°C. The sections were then incubated with the corresponding secondary antibodies for 40 min at 37°C in darkness. Ultimately, the sections were sealed with DAPI-containing mounting medium, and the fluorescence microscope (Olympus) was used to capture images.The 4 μg/mL), and bovine insulin (10 μg/mL). The cells were kept in a humid atmosphere containing 5% CO2 at 37°C. The 137Cs γ-ray source was used for subsequent studies at the dose rate of 0.99 Gy/min.Rat intestinal epithelial cell 6 (IEC-6) was cultured in DMEM medium, which contains 10% fetal bovine serum, penicillin (100 U/mL), streptomycin (100 5 cells per well) and treated with compound 12 (100 μM) for 2 h before irradiation (8.0 Gy dose). After culturing for 12 or 24 h, the cells were harvested, and the nuclear and cytoplasmic proteins were extracted using a nuclear/cytoplasmic protein extraction kit . Proteins were isolated on SDS–PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 10% skim milk, the membranes were incubated at 4°C overnight with antibodies against p53 , nuclear factor erythroid 2-related factor 2 , Bax , Bcl-2 , p21 , NADPH quinone oxidoreductase 1 , NADPH oxidase 1 , Lamin B1 , and GAPDH . Next, the membranes were stained with the appropriate secondary antibodies at room temperature for 1 h. The bands were developed using enhanced chemiluminescence reagent and analyzed by using Image Lab™ software .IEC-6 cells were seeded in 6-well plates (4 × 105 cells per well and treated with compound 12 (100 μM) for 2 h before irradiation (8.0 Gy dose). After incubation for 12 h, the cells were collected and washed with precooled PBS. Next, the cells were fixed with 70% ethanol at 4°C overnight. After washing with PBS, the cells were stained with propidium iodide (PI) staining buffer  for 30 min at 37°C. Finally, the samples were analyzed using flow cytometry, and the cell cycle distribution was analyzed using ModFit LT 4.1 software.IEC-6 cells were seeded in 6-well plates at 3 × 104 cells per well). The cells were pretreated with compound 12 (100 μM) for 2 h before irradiation (8.0 Gy dose). After incubation for 24 h, the cells were fixed with 4% paraformaldehyde for 20 min and permeabilized in 0.3% Triton X-100 for 30 min. The slides were then blocked with 2% BSA, stained with antibody recognizing Nrf2  for 1.5 h at room temperature, and incubated with PE-labeled secondary antibody  for 1.5 h. Finally, the slides were sealed with DAPI-containing mounting medium, and images were obtained using the fluorescence microscope (Olympus).IEC-6 cells were plated in 12-well plates with cell slides (5 × 10t-test (two-tailed) was employed for comparisons of the mean, and data are expressed as the mean ± SEM. Statistical analysis was carried out using GraphPad Prism 5 software. And statistical significance was set at p < 0.05.For the analysis of survival rates, the Kaplan-Meier method and log-rank test were employed in the present study. An unpaired 1H NMR : the survival rate of the mice in the 1200 mg/kg group was 80%, while that in the 1500 mg/kg group was 50%. We speculated that a low dose of compound 12 would not be effective, and a high dose of compound 12 might exert toxic effects. Therefore, compound 12 was administered at a dose of 1200 mg/kg in the following animal studies.To determine the effect of compound 12 on TBI-induced fatal damage in mice, we investigated the survival rate of mice receiving a lethal dose (8.0 Gy) of irradiation . As demop = 0.0007) compared to the ABI+vehicle group. These results demonstrate that compound 12 can improve the survival of mice receiving a lethal dose of ABI.To assess the effect of compound 12 on ABI-induced fatal intestinal damage in mice, we studied their survival rate after a lethal dose (15.0 Gy) of irradiation . As demop < 0.05). Nevertheless, treatment with compound 12 markedly increased the abundance of WBCs, RBCs, PLTs, HGB, and LY and decreased that of NE in the peripheral blood, suggesting that it could effectively protect against hematopoietic injury in TBI-exposed mice.Myelosuppression, which manifests as a remarkable reduction of peripheral blood cells, has been considered a classical symptom of TBI-induced hematopoietic damage . Lymphoip < 0.001). Nevertheless, compound 12 notably increased the frequencies of LSKs and HPCs. These findings demonstrated that compound 12 could protect mice from TBI-induced HSPC damage.Exposure to IR may induce hematopoietic system damage and HSPC depletions. To determine the role of compound 12 in the changes of bone marrow hematopoietic cells, we explored the effects of compound 12 on the proportions of LSKs and HPCs in the mouse bone marrow . As illup < 0.05), and compound 12 significantly reduced the ROS levels in HPCs and LSKs (p < 0.05). These findings indicate that compound 12 might mitigate radiation-induced oxidative stress damage by reducing ROS levels in HSPCs.Oxidative stress induced by ROS accumulation after radiation exposure can contribute to severe tissue damage . As showp < 0.05). In comparison to the mice in the ABI+vehicle group, mice in the ABI + 12 group exhibited more crypts and increased villi length (p < 0.05). In addition, IR notably reduced the expression of Villin in intestinal cells, whereas compound 12 significantly increased its expression , but the numbers of these cells in the ABI+12 group were obviously increased (p < 0.05). These results clearly demonstrate that compound 12 is helpful in improving the regenerative and differentiative capacity of ISCs in irradiated mice.The intestinal epithelium undergoes continual self-renewal to maintain homeostasis and has a considerable capacity for regeneration after injuries –28. Lgr5re to IR –31. Panere to IR . Furtherre to IR –35. We iγ-H2AX) has been regarded as a marker of DSBs [γ-H2AX in the small intestine of mice. As shown in Figures γ-H2AX in the mouse small intestine increased significantly after ABI, and treatment with compound 12 obviously reduced its expression (p < 0.01). This result indicated that compound 12 could alleviate irradiation-induced DNA damage in mice.Ionizing radiation can cause DNA damage, especially DNA double-strand breaks (DSBs). Phosphorylation of histone H2AX at serine 139 ( of DSBs . Therefop < 0.001), while compound 12 markedly reduced the number of apoptotic cells (p < 0.01).IR can induce tissue damage by promoting apoptosis. We assessed apoptosis in mouse small intestines using the TUNEL assay. As illustrated in Figures p < 0.05). Furthermore, as described in p < 0.01). In conclusion, our findings indicate that compound 12 could inhibit radiation-induced apoptosis by modulating the p53 pathway.Furthermore, we explored the role of compound 12 in IR-induced cell apoptosis using immunofluorescence staining and Western blotting. As displayed in Figures p < 0.001), and compound 12 effectively reduced G2/M arrest (p < 0.05). As reported in previous studies, p53 and p21 are required for sustaining G2/M arrest after IR-induced DNA injury [Since we observed that the expression of p53 in mouse small intestines could be downregulated by compound 12, we further investigated whether compound 12 can affect the cell cycle distribution of irradiated cells by regulating the expression of p53. We first analyzed the cell cycle distribution of IEC-6 cells using flow cytometry. As illustrated in A injury . Hence, p < 0.05). In addition, treatment with compound 12 alone could also increase the level of Nrf2 (p < 0.05).When activated under stress, the transcription factor of Nrf2 can enter the nucleus and promote the expression of antioxidant proteins, thereby regulating redox homeostasis in cells . We firsp < 0.05). Furthermore, the expression of NQO1 in the IR + 12 group increased significantly compared with the IR group (p < 0.05). NADPH oxidase (NOX) is one of the most important sources of ROS in the body, while NOX1 is a ubiquitous NADPH oxidase in epithelial cells [p < 0.001), while compound 12 reduced its expression (p < 0.05). These data indicate that compound 12 can decrease oxidative stress damage by upregulating the expression of Nrf2 and NQO1 and downregulating the expression of NOX1 in irradiated cells.NQO1 is an antioxidant enzyme that can be upregulated by Nrf2 . We asseal cells . We alsoin vitro and in vivo than the uncapped compound.In the current study, we synthesized a novel aminothiol compound 12 as a radioprotector. Since there is a higher number of thiol groups in this molecule compared to other radioprotectors, it can exert higher ROS scavenging and radioprotective activities. In addition, the thiols in the molecule are located away from the alkylamino backbone, thus eliminating ROS before they attack the DNA frame structure. Moreover, the amine and thiol groups in the molecule are capped, so its cell permeability and oral bioavailability can be increased compared with the compound previously reported by our laboratory . We specBone marrow hematopoietic tissue is the most radiosensitive tissue in the body . Myelosu+ ISCs, Paneth cells, and Ki67+ cells in the small intestines of IR-exposed mice. Overall, these results indicate that compound 12 can protect against radiation-induced intestinal injury by maintaining intestinal homeostasis and improving the regenerative capacity of ISCs.Radiation therapy for malignant tumors in the abdomen can damage normal tissues, and small intestine is especially sensitive to ionizing radiation , 43. We γ-H2AX is an early marker of DSBs in the body. We found that compound 12 significantly decreased the expression of γ-H2AX, which suggested that it might reduce radiation-induced DSBs by eliminating ROS in irradiated cells. DNA strand breaks can induce the upregulation of p53 expression, thereby causing cell cycle arrest and promoting DNA repair or leading to cell apoptosis [Ionizing radiation can increase the levels of ROS in irradiated cells, thus inducing DNA injuries, of which DSB is the most lethal. And poptosis , 45. Stupoptosis . Our resL), thus inducing the release of cytochrome c (Cyt c) [Mitochondria play an important role in apoptosis. Studies have shown that p53 can change the permeability of the mitochondrial outer membrane by regulating the expression of the Bcl-2 protein family members  , 47. Cyt (Cyt c) , 48. We We believe that compound 12 can directly eliminate ROS in cells. In addition, we also investigated whether compound 12 could alleviate oxidative injury in radiated cells through other pathways. Nrf2 is an important transcription factor that can adjust redox homeostasis . When acIn summary, a new aminothiol compound 12 was designed and synthesized in this study. It could be administered orally to mice and displayed potent radioprotective activity. Our results demonstrated that compound 12 could improve the survival rates of mice that received fatal doses of irradiation. We showed that compound 12 effectively prevented TBI-induced hematopoietic damage and ABI-induced intestinal damage in mice. Furthermore, we proved that compound 12 could reduce oxidative stress and inhibit DNA damage, thereby alleviating IR-induced cell cycle arrest. Our experimental results also suggested that compound 12 might suppress radiation-induced apoptosis through the p53 pathway. Taken together, compound 12 can be a potential radioprotective agent, and the precise mechanisms underlying its radioprotective activities warrant further investigation in the future.


Nramp gene family in Spirodela polyrhiza has not been previously reported in detail. To identify the Nramp genes in S. polyrhiza, we performed genome-wide identification, characterization, classification, and cis-elements analysis among 22 species with 138 amino acid sequences. We also conducted chromosomal localization and analyzed the synteny relationship, promoter, subcellular localization, and expression patterns in S. polyrhiza. β-Glucuronidase staining indicated that SpNramp1 and SpNramp3 mainly accumulated in the root and joint between mother and daughter frond. Moreover, SpNramp1 was also widely displayed in the frond. SpNramp2 was intensively distributed in the root and frond. Quantitative real-time PCR results proved that the SpNramp gene expression level was influenced by Cd stress, especially in response to Fe or Mn deficiency. The study provides detailed information on the SpNramp gene family and their distribution and expression, laying a beneficial foundation for functional research.Natural resistance-associated macrophage proteins (Nramps) are specific metal transporters in plants with different functions among various species. The evolutionary and functional information of the  Nramp transporter family play important roles in the transport of ions, such as Cd, Fe, Mn, and Zn , 0.5 mM K4[Fe (CN)6], 5 mM ethylene diamine tetraacetic acid (EDTA), and 100 mM KPO4 buffer (pH 7.0) for 40–60 min, vacuum-infiltrated, and incubated for 16–24 h in the dark at 37 °C. Then, the chlorophyll was removed from fronds using 75% ethanol, and the samples were photographed using Leica Z16 microscope (Germany).A series of primer 5 . All ampprimer 5 i by homow method . Consequications . Fronds S. polyrhiza strain 5543 was collected from East Lake  at the city of Wuhan, Hubei Province, China. The plants were cultured in 1/2 MS medium under the conditions described in Murashige and Skoog. (1962) with 16 h/8 h photoperiod (day/night) and temperature of 25 °C/15 °C (day/night) [SpNramp genes under different abiotic stresses, we have set the four following groups: (1) control, (2) Fe2+ starvation and 50 μM Cd2+, (3) Mn2+ starvation and 50 μM Cd2+, and (4) 50 μM Cd2+ under the same conditions described above. Each group included three biological replicates. All plants were sampled at each time point  and then immediately frozen in liquid nitrogen and stored at −80 °C for further analysis.y/night) . To dispSpNramp genes were designed by primer–primer 5 . Bio-Rad CFX96 touch real time PCR system  was used to run qRT-PCR with the TB Green® Premix Ex TaqTM kit . The qPCR program under the following conditions: 10 min at 95 °C, 40 cycles of 95 °C for 15 s, 59 °C for 30 s, and 72 °C for 30 s. A melting process at 60–95 °C was designed to generate the melting curve. The Actin (ACT) gene was used as an internal control. The 2−ΔΔCt method was employed to calculate the SpNramp genes’ relative expression [The oligonucleotide primers of primer 5 . The Omipression . Data weNramp gene family information in genome-wide in S. polyrhiza. A series of promoter analysis indicated that there exists many stress and hormone response elements, which play a vital role in metal stress responses. GUS staining results indicate that SpNramp1, SpNramp2, and SpNramp3 were highly expressed in the root, indicating that the roots are involved in ions uptake. Furthermore, expression patterns demonstrate that SpNramp1, SpNramp2, and SpNramp3 were significantly induced by Fe or Mn starvation, but suppressed by single Cd treatment. Collectively, the study provided a foundation for the mechanisms research of heavy metal element absorption and transport in S. polyrhiza.Notably, the study was the first to elaborate in detail about the


These ionophores were used in liquid membrane carrierfacilitated transport of main group metal cations across supportedliquid membrane (SLM). Cellulose nitrate membrane was used asmembrane support. Effect of various parameters such as variationin concentration of metal as well as ionophore, effect of chainlength and end group of ionophore have been studied. The sequenceof metal ions transported by ionophore 1M is+Na > +Li > +K >2+Ca > 2+Mgand the order of metal ions transported by ionophores(2M–6M) is+Li >+Na >+K >2+Ca >2+Mg.Ionophore 1M is selective for +Na,+Li, and +K and ionophores(2M–6M) are selective for +Li and+Na.A new class of redox-switched anthraquinone derived lariat ethers1-(1-anthraquinonyloxy) 3, 6, 9 trioxaundecane 11-ol( The molecular architecture of lariat ether side arm holds the metalions, and selectivity and carrier ability of redox-switched lariatethers will be helpful in constructing ion-selective electrodes[Carrier-assisted transport through supported liquid membranes isone of the important applications of supramolecular chemistry. Thedesigns of redox-switched crown ethers and lariatethers have been achieved by researchers  in90s.ectrodes, redox-sectrodes, and spe1M–6M) as shown in We have synthesized ionophores  in THF andthe mixture was refluxed for 30 minutes. Then a solution of1-chloroanthraquinone  was addedto it and refluxed at 80°C for 10 hours with stirring. Thisreaction was performed under nitrogen atmosphere. The reactionmixture was concentrated and the residue was mixed with2Cl2CH and then washed with water (twice) followed bybrine. The organic phase was separated and dried (over MgSO4),filtered, and concentrated. Column chromatography  followed by recrystallization(2Cl2CH/hexane then EtOH) gave 2.73 g (80%) ofionophore 1M as a yellow solid.Melting point is 52°C.ν – 3565(OH), 2940 cm−1(2CH),2865 cm−1, 1685 cm−1(C=O),1320 cm−1, 1270 cm−1(2ArOCH),1140 cm−1.IR (KBr) 1H NMR (δ in ppm) –3.25 –4.45 , 7.20–8.35.A solution of tetraethylene glycol (2.89 mL) in THF(10 mL) was added to vigorously stirred suspension of 2M, 3M, 4M, 5M,and 6M were prepared in the same manner by taking triethylene glycol, diethylene glycol diethylene glycol monobutylether, and triethylene glycol monomethyl ether, diethylene glycolmonomethyl ether [Ionophores yl ether , respect1M–6M) were 1-chloroanthraquinone (Lancaster), sodium hydride , and tetraethylene glycol, triethylene glycol,diethylene glycol, diethylene glycol monobutyl ether, andtriethylene glycol monomethyl ether, diethylene glycol monomethylether . The solvents3CHCl, 2Cl2CH, THF  were used as it is.Metal salts as metal picrate (MPic) were prepared as reportedearlier . The reaμm.Membranes were impregnated with redox-switched ionophores (1M–6M),dipped overnight, and used as a membrane support. These impregnatedmembranes were used for carrier-facilitated transport studies of alkali andalkaline-metal cations. Electron microscope studies are under process has beenused as a support in SLM studies. The membrane pore size was 0.2  process.−1 and the other cell contained the receiving phase(50 mL) double distilled water separated by membrane having aneffective diameter of 1 cm. Both phases were stirred withmagnetic stirrer at 120 rpm at room temperature, the samplewas withdrawn from the receiving phase after 24 hours and analyzedfor sample using Systronics flame photometer andUV-V is a spectrophotometer for 2+Mg. Cation flux (MJ)values were calculated by using the relation [C is the concentration of cation in receiving phase in mol/dm3, V is the volume of receiving phase in dm3, A is the effective area of membrane in m2, and t is the time in seconds.relation (1)JM=C(1M–6M) using cellulose nitratemembrane as a support. Blank experiments were also carried out fortransport studies of metal salts in which membrane was devoid ofcarrier. No leakage of cation in the membrane was noted. Theoptimum concentration of metal ion and ionophore was found to be1× 10−1 M and 1×10−4 M, respectively cations. The trend for ionophore2M is +Li >+Na >+K.Ionophore 2M having triethylene glycol chain shows strong binding affinity with high-charge-density cations, so itforms stable complexes with lithium. Therefore, it shows lesstransport for +K, +Na, and no transport for2+Ca, 2+Mg. Ionophore 3M havingsmall diethylene chain shows transport for+Li >+Na only because small flexible side armforms small pseudocyclic cavity by hydrogen bonding [4M having butyl group and 5M,6M having methyl group shows order of transport formetal ion as +Li >+Na >+K.The trend for the transport of cations with ionophore  bonding .Ionopho4M binds lithium in comparison to simple –OH group because conformational rigidity ofthe supporting framework does play a crucial role in binding. Theconformation of side chain is such as to enclose the metal cationand there is interaction between coordinating site of theionophore and the metal ion, as the end methyl group is not toostrong. From the results, it is clear that 2+Mg cation is not transported in sufficient amount by this ionophore(1M–6M) due to its highest charge density.Selectivity of ionophore 4M is shown at the bottom of 6M having diethylene glycol monomethyl ether shows selectivity for +Li due to cavity fit concept. Ionophore(1M–6M) shows selectivity towards +Li due to smallsize and higher charge density of +Li accounts for self-encapsulation [Cation selectivity depends on the particular end group, when theend group is butyl, the ionophore sulation .The results inform us that the metal ion transport mainly depends upon thestructure of the ionophores like number of donor sites, flexibility of chainlength, ionophore concentration, and also on the concentration, chargedensity, and size of metal cation; and this molecular designing helps infabrication of redox-switchable devices, molecular wires, as well as chemicalsensors.