Background: Idebenone is an antioxidant and a coenzyme Q10 analog that has been used to treat neurodegeneration disease. Some studies show idebenone exerts anti-inflammatory effects. However, whether idebenone can be used to reduce the neuroinflammation in Parkinson’s disease (PD) has been little studied.Methods: The study investigated the potential anti-inflammatory effects of idebenone in vitro and in vivo, using cell models of Lipopolysaccharide (LPS)-simulated BV2 cells and animal models of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD with or without idebenone. To verify how idebenone exerts its effects on the BV2 cell activation and PD model, we performed the mechanistic studies in vitro and in vivo.Results:In vitro study showed that pretreatment with idebenone could attenuate the production of pro-inflammatory factors in LPS-stimulated BV2 cells and promoted a phenotypic switch from the M1 state to the M2 state. Mechanistically, idebenone reduced the activation of the MAPK and NF-κB signaling pathway upon LPS stimulation. Furthermore, in vivo experiments confirmed that pretreatment with idebenone could ameliorate MPTP-induced neurodegeneration and modulate microglia phenotypes through inhibition of the MAPK and NF-κB signaling pathway in the SN.Conclusion: These results suggest that idebenone ameliorates the neurological deficits related to PD and this effect is partly mediated by inhibiting the neuroinflammation and modulating microglia phenotypes. Parkinson’s disease (PD) is a neurodegenerative disease that is caused by progressive loss of dopaminergic neurons, Lewy body formation, and mitochondrial dysfunction . In addiMicroglia cells are the main glial cell type that participate in the neuroinflammatory response in the central nervous system (CNS) . Under pIdebenone -1,4-benzoquinone), a synthetic analog of coenzyme Q10, is an important antioxidant for cell membrane and an elementary constituent of the ATP-producing mitochondrial electron transport chain . One of Taking these factors into consideration, we hypothesize that idebenone may have an inhibitory effect on neuro-inflammatory processes and microglial activation in PD. To test this hypothesis, we will investigate the anti-neuroinflammation and microglia modulating effects of idebenone in the BV2 microglial cell line and examine idebenone’s efficacy on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurodegeneration and neuroinflammatory response in PD mice.Idebenone was purchased from Qilu Pharmaceutical Co., Ltd., MPTP, dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) standards, dimethyl sulfoxide (DMSO), and Lipopolysaccharide (LPS) were purchased from Sigma-Aldrich .Male C57BL/6 mice  were purchased from Shanghai SLAG Laboratory Animal Corporation . The mice were individually housed at a temperature of 22 ± 1°C and relative humidity 50–60% under a 12-h/12-h light–dark cycle, with free access to food and water. Animals received four intraperitoneal injection of MPTP (20 mg/kg) at 2 h intervals, whereas control mice were treated with the same volume of saline. Idebenone was resuspended in corn oil and applied daily by oral gavage. 100 mg/kg idebenone or vehicle (corn oil) was administered daily starting at day 7 before MPTP administration . These mThis study was carried out according to the guidelines of the National Institutes of Health and was approved by the Ethics Committee of Xin Hua Hospital affiliated to Shanghai Jiao Tong University School of Medicine.Motor behavior was analyzed in an open-field test after 3 and 7 days of administration of MPTP. Open-field test is an important method for assessing spontaneous locomotor activity in mice model of PD . Mice weThe pole test is an available method to evaluate bradykinesia in PD mice model. We performed the pole test at 3 and 7 days after the last MPTP injection according to a previously described protocol . In brie1. The SH-SY5Y cells were presented by Institute of Neurology, Rui Jin Hospital . The BV2 microglial cells and SH-SY5Y cells were cultured in Dulbecco’s-modified Eagle’s medium (DMEM)  supplemented with 10% fetal bovine serum (FBS)  and 1% penicillin/streptomycin at 37°C with an atmosphere of 5% CO2. Idebenone was dissolved in DMSO as a 1 M stock solution, and dilutions were made in DMEM. The final concentration of DMSO in the DMEM was less than 0.05% which had no effect on cell growth.The BV2 microglial cell line was purchased from China Center for Type Culture Collection3 cells/ml) and treated with idebenone concentrations  for 24 h. Cell viability was examined by a CCK-8 assay kit  according to the manufacturer’s instructions  and treated with 5 μM idebenone for 2 h, followed by incubation with 100 ng/ml LPS for 24 h. Then, conditioned media of the BV2 cells was collected and applied to SH-SY5Y cells that has been seeded in 96-well plates. CCK-8 assay kit was used to assess the changes in SH-SY5Y cell viability after 24 h. Morphological change in the SH-SY5Y cells were examined by phase-contrast microscopy.BV2 cells were seeded in 6-well plates (4.0 × 105 cells/ml) and treated with various idebenone concentrations  for 2 h, followed by 100 ng/ml LPS for 24 h. After 24 h, culture supernatant was collected, and the NO concentration in the supernatant was measured by an NO assay kit  according to the manufacturer’s instructions  at 4°C. Staining was carried out using the ABC method (Vector Laboratories), with 3,3′-diaminobenzidine (DAB) as the peroxidase substrate. TH-positive DA neurons from the substantia nigra (SN) region were examined by light microscopy. Stereological counting of TH-positive neurons was used the method described in previous studies ; goat anti-Arg-1 ; mouse anti-CD16/32 antibody . After washing three times with PBS, brain sections were incubated with corresponding donkey anti-rabbit, donkey anti-goat, or donkey anti-mouse secondary antibodies conjugated to Alexa Fluor-488 and Alexa Fluor-594 fluorochrome . 4,6-diamidino-2-phenylindole (DAPI)  was used to stain the nuclei. The brain sections were examined by a microscope .BV2 microglial cells were fixed with 4% PFA and stained overnight with rabbit anti-NF-κB antibody  at 4°C, followed by incubation with the fluorescent conjugated secondary antibodies.g (4°C) for 10 min and the collected supernatants were then assayed for dopamine and DOPAC content through HPLC. The content of each neurotransmitter was expressed as pg/mg equivalent striatal tissue.The levels of dopamine and its metabolites (DOPAC) in the striatum were measured using an HPLC apparatus . BrieflyBV2 cells, seeded in 12-well plates, were pretreated with 5 μM idebenone for 2 h, followed by treatment with 100 ng/ml LPS for 12 h. Total RNA was extracted from BV2 microglial cells with Trizol reagent  and was reverse transcribed to cDNA using the PrimeScript RT Reagent kit , as previously described . RT-qPCRIL-6 ; IL-1β ; TNF-α ; iNOS ; CD 16 ;CD32 ; CD86 ; Arg-1 ; CD206 ;YM ; Rplp0 .TM XRS+ imaging system .Proteins were extracted from the BV2 cells and brain by homogenization in standard lysis buffer. The concentration of protein was measured using a BCA kit  according to the manufacturer’s instructions . 40 μg ot-test, while comparison among several groups was evaluated using one-way ANOVA followed by a Tukey’s multiple comparisons test. Data were expressed as the mean ± standard error of the mean (SEM). p-values < 0.05 was considered statistically significant.All data were analyzed by GraphPad Prism software, v6.0 . Comparison between the two groups was assessed with an unpaired p < 0.01; Figure p < 0.05; Figure To detect the potential cytotoxicity of idebenone, we analyzed the dose-dependent effects of idebenone  on the survival of BV2 microglial cells. The result demonstrated that idebenone was not cytotoxic to BV2 microglial cells at 1, 2.5, or 5 μM Figure . Activatp < 0.05; Figure p < 0.05; Figure Lipopolysaccharide is regard as a classically M1 microglia inducer, which causes M1 phenotype to express pro-inflammatory cytokines . In our Conditioned medium from LPS-stimulated BV2 cells should contain proinflammatory cytokines and other toxic molecules . We desip < 0.05; Figure Lipopolysaccharide, an inducer of M1 microglia , especiap < 0.01; Figure The NF-κB signaling pathway is also a significant mediator in LPS-activated BV2 microglial cells . Thus, Np < 0.01; 7 days: p < 0.001; Figures p < 0.001; 7 days: p < 0.001; Figures p < 0.01; 7 days: p < 0.01; Figures p < 0.001; 7 days: p < 0.01; Figure p < 0.01; Figure p < 0.05; Figure We sought to identify whether idebenone treatment affected MPTP-induced dopaminergic neuronal death and neurobehavioral deficits. According to previous studies, idebenone was treated at a dose of 100 mg/kg body weight . Mice rep < 0.01; Figures p < 0.001; Figure p < 0.01; Figure To determine whether idebenone in the PD model is beneficial or harmful to TH+ neurons in the SN, we compared the idebenone-MPTP group with its corn oil-MPTP group. The results showed that only 38% of TH+ neurons survived in the corn oil-MPTP group, while 66% of TH+ neurons survived in the idebenone-MPTP group  was significantly higher in the corn oil-MPTP treated mice than the corn oil-saline treated mice at 3 and 7 days after the last MPTP administration  and decreased mRNA expression of M2 markers  were observed at 3 and 7 days after the last MPTP administration  or M2 marker (Arg-1) were analyzed by double immunofluorescent staining with the microglia marker (Iba-1) in the SN and in the striatum. Consistent with the RT-qPCR results, expression of the M1 marker CD16 was lower in Iba-1+ microglia cells in idebenone-MPTP treated group compared with the corn oil-MPTP treated group at 3 days after MPTP injection , and its therapeutic potential has been reported in many diseases, including cerebrovascular disorders and neurodegenerative disorders ,b. A groAccumulating evidence from human and animal studies suggests that the microglia-mediated neuroinflammatory response is a hallmark shared by PD . Microglin vitro. Furthermore, conditioned medium from the LPS-stimulated BV2 cells decreased SH-SY5Y cell viability, while pretreatment with idebenone significantly attenuated the effect of LPS. Our experiments indicated that LPS could induce proinflammatory cytokines released in BV2 cells, which may explain the reduction in SH-SY5Y cell survival in conditioned medium from activated BV2 cells.Previously, some studies showed that idebenone had anti-inflammatory abilities. To further investigate the potential mechanisms of inhibition of inflammatory factors release and modulation of microglia phenotypes by idebenone, different signal pathways activated by LPS were evaluated. NF-κB and MAPKs signaling pathways are significant regulators of LPS-induced expression of proinflammatory cytokines in microglia . Inhibitin vivo experiments, idebenone was treated at a dose of 100 mg/kg body weight. In previous studies, 100 mg/kg of idebenone improved neurological deficits and energy metabolism in animal with cerebral ischemia (in vitro studies implicating the protective role of idebenone, we found that idebenone protected against MPTP-induced motor dysfunction and reduced the neuronal death in the SN and in the striatum. Idebenone also lessened the decline of dopamine levels and its metabolites in the striatum of MPTP-induced PD mice. As the dopamine level is a marker of dopaminergic synaptic function, these results indicate that idebenone exerts neuroprotective effects on MPTP-induced PD mice. This effect may be related to the inhibition of proinflammatory cytokines release, as evidenced by the decreased expression of IL-6, IL-1β, and TNF-α in the SN and in the striatum of MPTP-treated animals.For ischemia ,b and waischemia . 100 mg/ischemia . Thus, iin vivo. Consistent with in vitro study, idebenone suppressed the phosphorylation of ERK, p38, JNK, and NF-κB in the SN of MPTP-treated animals. These data suggested that idebenone could inhibit M1 polarization and promotes M2 polarization by modulating the MAPKs and NF-κB signaling pathway in vivo.Conversion of M1 state to M2 state prevented death of TH+ neurons in the MPTP induced PD model . FasudilOur work reveals a new insight into idebenone neuroprotection. Idebenone could attenuate the proinflammatory cytokines expression, and promote the phenotypic shift of microglia from the M1 to M2 phenotype in LPS-activated BV2 cells and the MPTP induced PD model. Mechanistically, these effects of idebenone may be mediated by inhibition of the MAPK and NK-κB pathway. Taken together, the present data suggested that the preventive potential of idebenone as a novel anti-inflammatory and microglia-modulating drug for neurodegenerative diseases associated with neuroinflammation, such as PD.ZgL and AY designed the experiments. AY, ZhL, LS, XW, YZ, NW, JL, and YL performed the experiments. AY and ZhL analyzed the results. AY wrote the manuscript with contributions from ZgL. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Drought stress is one of the major factors that affects wheat yield. Glyceraldehyde-3-Phosphate dehydrogenase (GAPDH) is a multifunctional enzyme that plays the important role in abiotic stress and plant development. However, in wheat, limited information about drought-responsive GAPC genes has been reported, and the mechanism underlying the regulation of the GAPC protein is unknown.GAPC1 in drought stress in wheat and Arabidopsis. We found that the overexpression of TaGAPC1 could enhance the tolerance to drought stress in transgenic Arabidopsis. Yeast one-hybrid library screening and EMSA showed that TaWRKY40 acts as a direct regulator of the TaGAPC1 gene. A dual luciferase reporter assay indicated that TaWRKY40 improved the TaGAPC1 promoter activity. The results of qRT-PCR in wheat protoplast cells with instantaneous overexpression of TaWRKY40 indicated that the expression level of TaGAPC1 induced by abiotic stress was upregulated by TaWRKY40. Moreover, TaGAPC1 promoted H2O2 detoxification in response to drought.In this study, we evaluated the potential role of TaGAPC1 gene, thereby increasing the tolerance of plants to drought stress.These results demonstrate that the inducible transcription factor TaWRKY40 could activate the transcription of the  Plant environmental stresses include drought, salt, extreme climate and oxidative stress, which seriously threaten food security and agricultural production. Due to global climate change, the environmental stress on plants will be increased over the next few years . However2O2 [GAPC1 is vital to normal fertility in Arabidopsis plants [For a long time, GAPDH has been used as a housekeeping gene in gene expression analysis . The GAP2O2 . In Arab2O2 . GAPC1 r2O2 . Further2O2 . In Arab2O2 , 17. A ds plants .GAPDH genes were identified, and qRT-PCR results indicated that GAPDHs showed different expression levels under the conditions of several abiotic stresses [GAPC genes in response to abiotic stress including drought have not been evaluated. Here, we showed that TaGAPC1 positively regulated drought stress tolerance in Arabidopsis. A WRKY transcription factor TaWRKY40 in wheat was screened by a yeast one-hybrid system. An electrophoretic mobility shift assay (EMSA) confirmed that TaWRKY40 could directly bind to the TaGAPC1 promoter. The expression level of TaGAPC1 was significantly higher under drought stress in wheat protoplast cells with instantaneous overexpression of TaWRKY40 than in wild type wheat protoplast cells. These results demonstrated that TaWRKY40 may positively regulate the TaGAPC1 expression. The high expression level exhibited by TaWRKY40 may be an important clue to investigate the secondary functions of TaGAPC1.In wheat, four types of GAPDHs  including 22 stresses . HoweverTaGAPC1 to various abiotic stress conditions were analyzed by qRT-PCR  occurring after 4 h of treatment. TaGAPC1 showed response to H2O2 stress, peaking at more than 7-fold. These results indicated that the transcriptional level of TaGAPC1 was significantly affected by abiotic stress. In addition to seeds, TaGAPC1 was highly expressed in almost all tissues including roots, stems and leaves  under the control of the strong 35S promoter. Arabidopsis plants overexpressing TaGAPC1 gene showed TaGAPC1 expression using pr1 and pr2 primer pair, and TaGAPC1-GFP fusion genes using the pr1 and pr3 primer pair, but no expression was observed in wild type  after 25 days of withholding water. The contents of RWC and chlorophyll in the OE1–3 and OE1–10 lines were also higher than Col-0 after 15 days of drought stress  radical into H2O2, also showed a similar pattern in transgenic plants after withholding water for 15 days  is toxic to cellular processes and can disrupt the electron transport chain . Thus, wsis Fig. . Consistays Fig. . These rTaGAPC1 under abiotic stress, a 1500 bp promoter was cloned and analyzed by PLANTCARE and NEWPLACE. Bioinformatics analysis indicated that there were multiple cis-acting elements related to stress tolerance, such as MYB, W-BOX and other components  in yeast, as evidenced by the growth of the transformant clone in on selective SD/-Leu + AbA medium  using a promoter fragment of approximately 40 bp as a probe. The results showed that TaWRKY40 could specifically bind to these W box in TaGAPC1 promoter fragments  in vitro  of the TaGAPC1promoter.To further determine whether the TaWRKY40 protein directly bound to the TaGAPC1 under PEG treatment involves the H2O2 signaling pathway, DMTU was chosen to inhibit of H2O2 [2O2, followed by PEG8000 treatment for 6 h. Figure TaGAPC1 was induced by PEG (4.61-fold) and H2O2 (6.93-fold) treatment, consistent with the results in Fig. TaGAPC1  and ABA (6.53-fold)  Fig. b, which ngs Fig. b. There ate Fig. a. These 2O2 and the expression of TaGAPC1 in the ABA signaling pathway, wheat plants were pretreated with DMTU, a scavenger for H2O2, for 6 h, and then exposed to ABA treatment for 6 h. The experimental results showed that pretreatment with DMTU dramatically abolished the expression of TaGAPC1 induced by ABA   plants [TaGAPC1 was significantly induced under PEG, H2O2, and ABA stress  plants . Consist) plants , 31, the2O2 [2O2 and O2− under drought stress in transgenic plants compared with WT plants implied that the ROS scavenging systems of transgenic plants might be more efficient than those of WT plants. Antioxidant enzymes were analyzed, and the activities of SOD and POD were higher in the transgenic lines than in the WT line under drought stress. These results suggested that the overexpression of the TaGAPC1 gene protected plants against ROS injury by integrating with H2O2 and enhancing the activation of the antioxidant defence system. When plants suffer from environmental stress, bacterial infection or hormone stimulation, the guard cell signal transduction process is rapidly activated to optimize the ability to absorb CO2 and to reduce moisture loss by adjusting stomatal movement [TaGAPC1 is smaller than that of wild type under drought condition. , superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) . It has 2O2 . In thismovement . Oxidizemovement . Consiston. Fig. .TaWRKY40, a member of the WRKY family, was identified to interact with the TaGAPC1 promoter by the yeast one-hybrid assay. The WRKY transcription factor is one of the largest families of transcription factors in plants [AtWRKY25 and AtWRKY26. The overexpression of AtWRKY25 and AtWRKY26 enhanced tolerance to heat stress [TaWRKY70 was significantly increased under high temperature conditions, as well as under ethylene, salicylic acid and cold (4 °C) stress conditions. In addition, TaWRKY33 plays a key role in ABA- and drought-responsive signaling networks [TaWRKY40 was also increased when wheat was exposed to abiotic stress. The results in Fig. TaWRKY40 is crucial for the ABA-induced upregulation in TaGAPC1 expression. Therefore, when plants are subjected to abiotic stress, TaWRKY40 bound to the TaGAPC1 promoter and enhanced promoter activity to increase the TaGAPC1gene expression level in the ABA signaling pathway.Many plant regulators are involved in signal transduction networks during plant growth and development, such as the WRKY transcription factor . TaWRKY4n plants . When pln plants , 36. In t stress . In wheanetworks . ConsistTaGAPC1 gene was involved in resistance to abiotic stress and probably the encoded protein acts as a multifunctional protein in addition to its pivotal role in glycolysis. When wheat was subjected to abiotic stress, the inducible transcription factor TaWRKY40 could bind to the TaGAPC1 promoter to positively regulate the expression level of TaGAPC1gene via the ABA signaling pathway, thereby increasing the stress tolerance of plants under abiotic stress. Future studies investigating the activation mechanism and characterizing TaGAPC1 in wheat will improve our understanding of this intricate regulatory network and the molecular mechanisms underlying plant abiotic stress, in which GAPCs are involved , tobacco  and Arabidopsis thaliana  were used in this study. All seeds were provided by the laboratory of Professor Xi ping Deng of Northwest A&F University. Seeds were grown in a phytotron, which was maintained at a photosynthetic photon flux density (PPFD) of 600 μM m− 2·s− 1 and 22/23 °C day/night temperature with 16 h light/8 h dark cycles.Wheat  for 6 h and then exposed to 20% PEG8000 for 6 h. The plants were pretreated with 5 mM dimethyl thiourea (DMTU) for 6 h, and then exposed to 100 μM ABA treatment for 6 h. Treatment with tungstate or DMTU alone was also used as controls in the experiment. Samples from treated or control plants were frozen in liquid nitrogen and stored at − 80 °C until total RNA extraction and qRT-PCR assay. All of the experiments were repeated at least three times.The seedling roots were dipped into a solution of 20% PEG8000, 10 mM H∆∆Ct method. Experiments were repeated at least three times with biologically independent samples. The primers used in this assay were listed in Additional file Total RNA was isolated from different wheat tissues using the RNAiso plus reagent , Realtime PCR and first-strand cDNA synthesis were performed with PrimeScript™ RT-PCR Kit , respectively, according to the manufacturer’s instruction. Thereafter, Realtime PCR was performed in optical 96-well plates  with CFX96 Touch Real-Time PCR Detection System  using the SYBR Green method. The expression data was analyzed using the 2-TaGAPC1 was amplified from wheat cDNA by PCR and cloned into the vector pCAMBIA1302. Arabidopsis plants were transformed using the flower dip method and selected on 1/2 Murashige & Skoog (MS) medium containing 30 μg/mL hygromycin. The hygromycin-resistant T1 seedlings were verified by PCR analysis using specific primers  acetone, at pH 7.8) for 12 h at 4 °C. The absorbance was read at 663 nm, 647 nm and 537 nm. MDA content was measured by a thiobarbituric acid (TBA) assay as described by Heath & Packer . Samples2− [− 1 NBT) for 8 h in the dark. Then, chlorophyll was removed using 70% ethanol. NBT polymerization at the site of O2− accumulation produced in a dark blue polymer. H2O2 content was measured by a KI assay [3PO4 solution and 800 μL 1 M KI solution were added to the 400 μL supernatant. The content of H2O2 was calculated from the absorbance at 390 nm.Nitrotetrazolium blue chloride (NBT) was used to detect O2− . BrieflyKI assay . Samples2O2). The absorbance was read at 470 nm.Enzyme extraction and measurement were performed as Polle described . For SODFor the drought-induced stomatal aperture measurements, leaves in the same position on the plant grown for 15 days without water were sampled, and stomata on the leaf were immediately photographed. Stomata of each exfoliated epidermis were photographed using a light EX30 microscope, and the stomatal aperture was measured by Image J software. Stomatal aperture values are presented as means from at least 50 stomata in one repetition.TaGAPC1 promoter sequence was inserted into the pC0390-GUS vector and PC0390-RUC to generate recombinant plasmids and then the plasmids were transformed into tobacco leaves by Agrobacterium. The GUS activity was measured with histochemical assays as previously described [The escribed , 44. A lescribed , 46.TaGAPC1 promoter were cloned into plasmid pAbAi to screen the wheat cDNA library. The Yeast One-Hybrid system was performed using the Matchmaker Gold Yeast One-Hybrid Library Screening System from Clontech as recommended by the manufacturer.Fragments of the An electrophoretic mobility shift assay (EMSA) was performed as previously described with modifications , 48. A tThe results of physiological measurements presented are the means of three independent experiments  analyzed by the SPSS software . Student’s t-test was performed to determine their significance by LSD at 0.05 probability level.Additional file 1: Figure S1. Detection of transgenic plants. (A) Diagram of the 35S:TaGAPC1 constructs. The primers, pr1, pr2 and pr3, used to analyze TaGAPC1 in transgenic Arabidopsis plants were shown.  PCR analyze of TaGAPC1 overexpressing transgenic Arabidopsis.Additional file 2: Figure S2.TaWRKY40 expression in response to abiotic stresses in wheat. Error bars indicate ±SD . Significant differences were assessed with one-sided paired t-tests . Three biological replicates were performed. Data were derived from experiments that were performed at least three times with similar results, and representative data from one repetition were shown.Additional file 3: Table S1. Primer and probe sequences used in this study.Additional file 4: Table S2. Basic local alignment search tool (BLAST) result for potential candidate interacting proteins with TaGAPC1 promoter in cDNA wheat library.


As a common athletics injury in orthopedics clinic, ankle injury may affect a person’s daily life and ankle injury rehabilitation has gained increasing interests from the medical and robotic societies. A novel hybrid ankle rehabilitation robot is proposed, which composing of a serial and a parallel part. In order to analyze its kinematic performances, the parallel part of the robot is simplified as a constrained 3-PSP parallel mechanism. A mathematical model for the parallel part of the robot is established based on the screw theory. Then the inverse kinematics is obtained, and the reciprocal twists, Jacobian matrices and the singularity of the robot are analyzed. Finally the workspace of the central point on the moving platform is predicted. The kinematic analyses manifest that the proposed hybrid rehabilitation robot not only can realize three kinds of ankle rehabilitation motions, but also can eliminate singularity with enhanced workspace. The workspace of the central point reveals that the hybrid robot can fully meet the demanded rehabilitation space by comparing with the clinic demands. Our results reveals the characteristic structure of the hybrid rehabilitation robot and its superiority, it offers some basis data for the future enhancement of the device. As one of the most complex joints in human skeleton, the ankle joint plays an important role in maintaining the balance of the body  rotate on the rotation orbit to ensure the relative position between the three pushrods and the moving platform is constant. The parallel part of the robot consists of a base platform, a moving platform, a pedal and three pushrods . The pushrods  are actuated by the stepper motor . Thus the plantar/dorsal flexion and the inversion/eversion motions of the platform along Y axis and X axis can be realized. Figure As shown in Fig. From the above descriptions, one may find that the proposed hybrid robot can perform plantar/dorsal flexion, inversion/eversion and abduction/adduction motions for ankle rehabilitation. Also, this robot claims the merits of compact volume, light weight and good portability.Since the structure of the serial part of the hybrid robot is comparatively simple, the following will focus on the kinematic analyses of the parallel part. As shown in Fig. As shown in Fig. A, B and C) and the following settings are defined.Due to the complicated topological structure of the proposed ankle rehabilitation device, the screw theory is adopted to derive more simple kinematic formulations. To facilitate the mathematical modeling, the three pushrods  are represented by the letters  as an example, the following definitions are made:X axis; Y axis.As shown in Fig. h, the distance between the central point of the base platform and the three pushrods  is a. Therefore, it can be judged from Fig. A, B and C) measured in the global coordinate frame S-XYZ areAssuming that the distance between the end effector and the base platform is A′, B′ and C′) at the initial stage areSimilarly, the coordinates of the spherical joint center  an an17) anS-XYZ, if the coordinates of each pushrod related to the S-XYZ can be expressed as followsAccording to Eq. , the disThe inverse kinematics of the parallel part of the novel ankle rehabilitation robot can be obtained as followsBased on the above discussion, the twists of branch 1 are calculated in Eq. . Similari, and we haveThe reciprocal twists are the force constraint of the rigid body in the process of the motions, and it is also the structural constraints of the mechanism  = 3.From the above equation, it can be found that there exists no constrained singularity of the parallel mechanism because rank  a.In this section, the workspace of the moving platform is predicted. The parameters of the robot mainly include the length of pushrods L = 150 mm, R = 150 mm, h = 165 mm and a = 50 mm. Because when robot only performs an inversion/eversion motion, the angle range of the motion only related to the displacements of the pushrod (6′) p2 and the pushrod (6″) p3. Moreover, p2 = − p3. In order to obtain the maximum range of the motion, the extension of the pushrods is set to be 75 mm at the initial stage, the displacements of the actuated prismatic joints In the present study, With Eqs.  and 30)30), the In order to predict the angle range of the plantar/dorsal flexion motion, we estimate the range of the moving platform and set the ranges of the plantar/dorsal flexion as  and  respectively. Then we analyze the trajectory of these two motion patterns with constraints and without constraints of the structure of the mechanism. The results are shown in Fig. 5As shown in Fig. According to Wang et al. , Aman etY axis) and inversion/eversion  are set as β ∊  and α ∊ . The workspace of the central point on the moving platform under the combined motion of plantar/dorsal flexion and inversion/eversion is depicted in Fig. According to the angle range shown in Table X axis, Y axis and Z axis are located in the intervals of 17.7–25.0 mm, −21.9 to 18.9 and 141.9–200 mm, respectively. In summary, the robot proposed can fully meet the demands of ankle rehabilitation exercise. The above work offers some basis data for the future enhancement of the ankle rehabilitation device.As shown in Fig. In this paper, a novel hybrid ankle rehabilitation robot is proposed, which is composed of a serial part and a parallel part. The proposed hybrid robot can achieve three motion patterns, i.e., plantar/dorsal flexion, inversion/eversion, abduction/adduction. The structure of the hybrid robot is analyzed and the parallel part of the robot is simplified as a 3-PSP parallel mechanism. A mathematical model is established for the 3-PSP parallel mechanism. On this basis, an inverse kinematic analysis is carried out, and the Jacobian matrices and singularity of the parallel mechanism are deduced, the results reveal that there exists no constrained and kinematic singularity of the robot. The workspace of the robot is predicted. The comparison reveals that the hybrid robot can fully meet the demanded rehabilitation space.The present study provides some basis for further investigations such as trajectory planning, rehabilitation strategies and so on.


LV weight was also significantly and similarly increased in both AR groups (non-Ovx and Ovx). Increase in stroke volume or cardiac output and loss of systolic function were similar between AR intact and AR Ovx groups compared to sham. We then investigated what were the effects of 17beta-estradiol  treatment on the parameters studied in Ovx rats. Ovx reduced uterus weight by 85% and E2 treatment restored up to 65% of the normal weight. E2 also helped normalize heart size to normal values. On the other hand, it did not influence the extent of the hypertrophic response to AR. In fact, E2 treatment further reduced LV hypertrophy in AR Ovx rats (41% over Sham Ovx + E2). Systolic and diastolic functions parameters in AR Ovx + E2 were similar to intact AR animals. Ovx in sham rats had a significant effect on the LV gene expression of several hypertrophy markers. Atrial natriuretic peptide (Nppa) gene expression was reduced by Ovx in sham-operated females whereas brain natriuretic peptide (Nppb) expression was increased. Alpha (Myh6) and beta (Myh7) myosin heavy chain genes were also significantly modulated by Ovx in sham females. In AR rats, LV expression of both Nppa and Nppb genes were increased as expected. Ovx further increased it of AR rats for Nppa and did the opposite for Nppb. Interestingly, AR in Ovx rats had only minimal effects on Myh6 and Myh7 genes whereas they were modulated as expected for intact AR animals. In summary, loss of estrogens by Ovx in AR rats was not accompanied by a worsening of hypertrophy or cardiac function. Normal cardiac growth was reduced by Ovx in sham females but not the hypertrophic response to AR. On the other hand, Ovx had important effects on LV gene expression both in sham and AR female rats.Aortic valve regurgitation (AR) can result in heart failure from chronic overloading of the left ventricle (LV). Little is known of the role of estrogens in the LV responses to this condition. The aim of the study was to compare LV remodeling in female rats with severe AR in absence of estrogens by ovariectomy (Ovx). In a first study, we investigated over 6 months the development of hypertrophy in four groups of female Wistar rats: AR or sham-operated (sham) and Ovx or not. Ovx reduced normal heart growth. As expected, volume overload (VO) from AR resulted in significant LV dilation (42% and 32% increase LV end-diastolic diameter in intact and Ovx groups vs. their respective sham group;  Severe aortic valve regurgitation (AR) is a chronic disease that results in progressive left ventricular (LV) dilatation and eccentric hypertrophy. Although it is not the most frequent valvular disease in the Western world, it is estimated, based on the Framingham study, that 13% of the population suffers from some degree of AR . ModeratIn prior studies using a rat model of chronic and severe AR, we showed that females had a hypertrophic response similar or stronger to LV VO than males . The maiIt is believed that estrogens can provide a protection against cardiac hypertrophy (CH) development and evolution towards heart failure (HF). This has been demonstrated in animal models of pressure overload in female rats . In the In the present study, we tested if LV remodeling and hypertrophy were influenced by estrogens in female rats suffering of chronic and severe AR. The effect of ovariectomy (Ovx) in females was assessed in order to answer this question. We also wanted to evaluate if Ovx was associated with decreased LV function and potentially worse survival in AR rats. In a second protocol, we studied the effects of 17beta-estradiol (E2) supplementation in AR Ovx female rats to investigate if it was associated with a reversal of the effects associated with the loss of estrogens.n = 11), Ovx sham-operated , AR; (n = 12) and Ovx AR . AR was induced at the age of 10 weeks as previously described by perforation of one or two aortic valve leaflets using a catheter via the right carotid and under echocardiographic guidance (n = 6) and AROvx + E2 (n = 8). E2 (17beta-estradiol) was administered using a subcutaneous pellet implanted in the neck of the animal liberating 0.03 mg/kg/day for 3 months . After this period, a second pellet was implanted to complete the protocol. The protocol was approved by the Université Laval’s Animal Protection Committee and followed the recommendations of the Canadian Council on Laboratory Animal Care.Fifty-eight female Wistar rats (225 to 250 g)  were studied. Ovx females were purchased at the age of 9 weeks, one week after the procedure. Experimental groups were as followed: Sham-operated ) was performed under isoflurane anesthesia (2%) two weeks after surgery to confirm AR severity and at the end of the protocol 26 weeks later, as previously described . The regEchs1) . We tested three different genes as possible housekeeping gene as control for this study. Cyclophilin A gene was chosen since it had the one most stable expression among the different groups.LV gene expression was quantified for 6 animals per group by quantitative RT-PCR as described elsewhere . Pre-optt-test was used when only two groups were compared. A p-value lower than 0.05 was considered significant.Results are presented as the mean and the standard error of the mean (SEM). Two-way ANOVA analysis was performed and Holm-Sidak’s post-test was used for comparison between the groups . A Student’s AR was surgically induced in intact (non-Ovx) and Ovx Wistar female rats at the age of 10 weeks. The protocol had a duration of 26 weeks (6 months). All animals survived the duration of the protocol. In As for the animal and heart characteristics described above, most echocardiographic parameters were significantly changed by AR . AR seveIn We then studied the effects of E2 treatment in both Sham Ovx (ShOvx) and AR Ovx (ArOvx) rats. As summarized in As illustrated in Nppa or Anp) and brain natriuretic (Nppb or Bnp) mRNA levels were both modulated by the loss of estrogens in Sham Ovx animals  gene expression was reduced by Ovx whereas myosin heavy chain beta (Myh7) was increased compared to non-Ovx Sham animals. Nppa mRNA levels were strongly increased in AR animals compared to corresponding Sham group; this raise being stronger in the AROvx group  and procollagen 3 (Col3) gene expression in sham-operated rats  in AR groups. CTGF gene expression levels were unchanged by Ovx and were significantly increased by AR . Among the genes related to ROS metabolism, we observed that NADPH oxidase 4 (Nox4) expression was significantly reduced by Ovx. This was reversed by AR. AR up-regulated Nox2 in both intact and Ovx females.We reported previously that female AR rats unlike males, kept a relatively normal transcriptional profile of many genes related to myocardial energetics . Here, wPdk4  and 4B. Nppb or Bnp) and myosin heavy chains (Myh6 and Myh7) gene expression. Interestingly, E2 helped normalize Nppa and Myh7 expression in Sham animals  two weeks before AR induction had relatively little effects on the extent of the cardiac response to a LV VO, at least at the macroscopic level. Ovx resulted in slower cardiac growth in Sham female rats during the 6 months that lasted the protocol. This was partly reversed by 17beta-estradiol treatment. On the other hand, LV hypertrophy caused by severe VO from AR was similar in all AR groups, non-Ovx, Ovx and Ovx receiving E2. We had previously shown that LV remodeling from AR in this model involved similar LV dilation in rats of both sexes but more wall thickening in females. This resulted in AR females in maintained LV relative wall thickness (RWT) although significant hypertrophy was present The roles of estrogens in pathological cardiac hypertrophy has been studied mostly in pressure overload animal models. It received less attention in VO situations such as in valve regurgitation models or in the aortocaval fistula (ACF) model. ACF is a model of global cardiac VO model. It is less relevant from a clinical standpoint but it remains the most studied pre-clinical VO model. Female ACF rats were shown to develop less hypertrophy, to evolve more slowly towards heart failure and to display better overall survival than males . This adMore than a decade ago, we had reported that Ovx was not associated with major effects in AR females . More reThe steroid hormone 17beta-estradiol is a key player in many biological processes, such as reproduction, development, metabolism, cell proliferation and differentiation . EstrogeNppa and Nppb), contractile protein beta-myosin heavy chain (beta-MHC or Myh7) and early response genes such as c-myc and c-fos among many others. This reactivation of the fetal gene program in the stressed heart is accompanied with the down-regulation of the adult gene program (Nppa) and contractile protein beta-myosin heavy chain (Myh6 and Myh7) were all significantly modulated by the loss of estrogens. This was also true for other genes often modulated in cardiac hypertrophy such as collagen genes (Col1a1 and Col3), ERRalpha, Pdh4 and Nox4. This modulation was not associated directly with an inhibition or a reactivation of the fetal gene program. For instance, Myh6 and Myh7 LV genes in ShamOvx rats followed the usual pattern associated with pathological hypertrophy. On the other hand, Nppa was down-regulated and Nppb expression remained unchanged. If Nppa and Myh7 gene expression was normalized by E2, it was not the case for Myh6.Pathological cardiac hypertrophy is associated with an important remodelling of the myocardial structure, a consequence of cardiomyocyte size increase and extracellular matrix rearrangement. Neurohormonal factors as well as mechanic stress cause alterations in myocardial gene expression including the reactivation of the fetal gene program . This fe program . Here, wWe reported previously that genes associated with myocardial energetics were strongly modulated in AR male rats and that a clear sex dimorphism was present when compared to females . PatholoMyh6 and Myh7) was in part normalized by Ovx in AR females as well as for mRNA levels of Nppb, Err alpha and Err gamma. Again, loss of androgens seemed to provide some benefits to males on this aspect whereas estrogens are mainly neutral in females  and so was the hypertrophy relative to the respective sham group (41% in AR Ovx + E2 vs. around 70–75% for AR and AR Ovx groups). On the other hand, if one considers the indexed heart weight gain between AR Ovx rats receiving or not E2, the hypertrophic response was similar. We did not measure circulating E2 levels and we used uterine weight as a surrogate of estrogen action. The dosage of E2 reversed about two thirds of the expected uterus weight suggesting that it was probably a little low or that type of delivery could not reproduce the natural situation. This could explain in part, why cardiac growth was not restored to normal levels. Obviously, continuous release of E2 does not reproduce naturally occurring circadian rhythm of production and release of sex hormones in the body. In addition, E2 treatment alone cannot restore completely other possible hormonal imbalances created by Ovx. They too, may probably contribute to observations made in this study. Finally, Ovx was performed in young animals, which does not translate well to the situation of post-menopausal and older patients.In conclusion, we showed that loss of estrogens was not associated with important effects on the hypertrophic response to severe and chronic aortic valve regurgitation in female Wistar rats.10.7717/peerj.7924/supp-1Supplemental Information 1Click here for additional data file.


Most epidemiologic studies reporting prevalence of allergic rhinitis (AR) and nonallergic rhinitis (NAR) have assessed solely self-reported prevalence, without confirmation by objective measures. Furthermore, reports of prevalence of NAR in Chinese subjects are scarce. Thus, we aimed to explore the prevalence and risk factors of AR and NAR in a Chinese, based on both clinical manifestation and allergic status.We conducted a population-based cross-sectional survey, involving 1084 local residents from a rural area of Beijing, China. Participants were enrolled using a stratified two-stage cluster sampling method. All adult participants or the guardians of children completed standardized questionnaires to provide relevant demographic and clinical information. Skin prick tests were also performed to determine sensitization to specific aeroallergens. AR/NAR was classified according to Allergic Rhinitis and its Impact on Asthma criteria.P < 0.05 for all). The three most common aeroallergens in self-reported AR group were Blattella germanica (16.6%), Dermatophagoides farinae (14.6%), and Dermatophagoides pteronyssinus (13.9%). Family history of AR and atopic dermatitis were significantly associated with AR , whereas family history of AR and asthma were significantly associated with NAR . Similarly, comorbid asthma, CRS, and atopic dermatitis were significant risk factors for both AR and NAR.Prevalence of self-reported AR was 46.80%. Based on SPT results, the confirmed standardized prevalence of AR and NAR were 16.78% and 24.60%, respectively. Severity scores for nasal itching, sneezing, rhinorrhea and congestion were significantly higher in subjects with AR, than subjects with NAR (Combination of standardized questionnaires and specific allergen tests may provide more accurate estimates of prevalence of AR and NAR and associated risk factors. Traditionally, rhinitis is divided into two types according to its etiology; allergic rhinitis (AR) and nonallergic rhinitis (NAR). Nowadays, most epidemiologic studies of AR are based on mostly self-reported prevalence of symptoms and often lack objective measures. Moreover, epidemiologic evidence of NAR is scant. Thus, we aimed to perform a population-based cross-sectional study in a rural community of northern China, to assess the prevalence of clinical AR and NAR, based on both subjective and objective measures.As an inflammatory disease of nasal mucosa, rhinitis is often defined by its clinical manifestation, such as rhinorrhea, sneezing, nasal congestion and itching. Although sometimes considered as a trivial disease, because it is not associated with mortality, it can lead to a great financial burden and tremendously impair a patient’s quality of life; negatively impacting many aspects of life such as sleeping, working performance, emotion, socializing, etc.According to the latest Allergic Rhinitis and its Impact on Asthma (ARIA) guidelines , classicGlobally, AR affects 10–40% adults and 2–25% children , 12–30% NAR, on the other hand, is a group of heterogeneous diseases, characterized by clinical nasal symptoms with negative specific allergen tests . It is bFew epidemiologic studies have been reported on NAR, and most of the studies have concentrated solely on the self-reported prevalence. The aim of the present study was therefore to assess the prevalence of clinical AR and NAR, using both subjective and objective tests, and the associated risk factors in Chinese subjects from a rural community in Beijing, China.This was a population-based cross-sectional observational study, conducted from November 2011 to December 2011. With a computer-generated list, the participants were recruited from four randomly selected communities in a randomly selected town in Huairou district, a northern rural district of Beijing, China, and directed to assemble at the regional medical care center. All volunteers, who formed half of the population in the selected region, completed a specifically designed questionnaire, as detailed below, under supervision of a group of experienced interviewers, and were also subjected to SPT for assessment of sensitization to specific inhalant allergens. All the children’s questionnaires and informed consent forms were completed by their guardians.The study protocol, as shown as Fig. In order to assure sufficient statistical power for the calculation of clinical prevalence and better evaluation of epidemiological features, the sample size N was estimated before the study according to the formula The questionnaire comprised 24 questions to provide information on a subject’s general demographic characteristics , as well as information on self-reported rhinitis, duration and severity of rhinitis, ocular symptoms, nasal symptoms, duration and severity, self-reported allergens, other allergen-related diseases , smoking history and family history of allergic disease. The severity of symptoms and quality of life (QoL) were assessed by validated Chinese version visual analogue scale (VAS) and the rhinoconjunctivitis quality of life questionnaire (RQLQ) .AR was diagnosed in accordance with the criteria of the 2010 ARIA guideline , and subChenopodium album, humulus, locus, Blattella germanica, pine, plantain, Candida albicans, Penicillium notatum, Alternaria tenuis, Aspergillus fumigatus, Dermatophagoides farina (Der f) and Dermatophagoides pteronyssinus (Der p). Histamine and saline were used as a positive and a negative control, respectively. The skin reaction towards each allergen was measured as the diameter of the wheal produced after 15 min. The result was based on skin index  [SPT were performed by a group of specialized technicians, according to a standardized protocol. All the participants attended the study without taking any antihistamines or topical/systemic steroids for at least 72 h prior to SPTs; using a panel of 20 standardized aeroallergen extracts . The 20 aeroallergens tested included a mixture of animal hair , tree, grasses, grass/cereals, mugwort, dandelion, giant ragweed, e wheal) . In the P < 0.05 was considered to be significant.Data were analyzed using SPSS V.22 software package . The regional study populations were standardized by age and gender of the reference population, based on the sixth China population census in 2010. Descriptive statistics were used for demographic and general information of the study population. Chi-square analysis was performed to analyze the clinical evaluation and the common sensitized allergens evidenced by SPT. Univariate analysis was used, followed by the multivariate logistic regression, to evaluate associated factors of clinical AR and NAR. Both the OR and the 95% CI were measured. A value of The demographic characteristics of the study population are shown in Table Table The majority of clinical AR patients belonged to minimum income household  Table . Asthma P = 0.091 and 0.215, respectively).More subjects were classified as “intermittent” than “persistent” in both self-reported AR group (57.67% vs 42.33%) and clinical AR group (51.95% vs 48.05%). Similarly, patients with moderate-severe AR were more common in both self-reported and clinical diagnosed AR groups . With no statistical significance observed between the two groups, both the self-reported and clinical AR groups shared the similar pattern of the duration and severity of the disease with no statistical differences (P < 0.05).Both AR and NAR patients tended to suffer from severe nasal symptoms nasal itching, congestion and rhinorrhea, and moderate sneezing. However, as shown in Table All the participants finished the RQLQ. The QoL of patients in both clinical AR and clinical NAR groups was impaired in all aspects; however, the clinical AR group showed significantly greater negative impact of all aspects, except disturbance from practical problems, which impaired the QoL of these patients.Blattella germanica , Der f , Der p , respectively. Significantly greater numbers of subjects in self-reported AR group were sensitized to Der f and Der p, as well as to mugwort, Penicillium notatum, animal dander, dandelion, ragweed, and Curvularia lunata, than subjects in asymptomatic group. In the self-reported AR group, 9.5, 7.3, and 16.0% of the subjects were sensitized to one, two and ≥ three allergens. Similarly, in the asymptomatic group 9.8, 6.6, and 10.7% of the subjects were sensitized to one, two and ≥ three allergens.Table P < 0.05), were further analyzed using logistic regression test and multivariate analysis. In this regard family history of AR and atopic dermatitis were significantly found to be associated with clinical AR  and family history of AR and asthma significantly associated with NAR   were found to be the most prevalent allergens responsible for clinical AR, which is consistent with the findings of Lou and colleagues [Blattella germanica, Der p, Der f, as well as Altenaria tenuis were the most common sensitizing allergens in a cohort of over 10,000 AR out-patients [Sensitization patterns for AR have been shown to vary from place to place, and are influenced by climate change , 30, genlleagues . Indeed,patients ; and theBlattella germanica has been suggested to be an important component in the screening panels for aeroallergens in self-reported AR in most regions of China [Blattella germanica and clinical manifestation of AR. Furthermore, although it is possible that the high sensitization rates to Blattella germanica noted in the present study may possibly be a result of cross reactivity with IgE for Der p or Der f; a study by Sun and colleagues [Der p, an IgE cross-inhibition study showed that only a small number of patients appeared to have Blattella germanica and/or Der p as primary sensitizing source, which could false cause false positive SPT reactions against cockroach. However, the finding from the present study that sensitization rates for Blattella germanica in self-reported AR and NAR subjects were comparable (16.6% vs 14.9%) suggests that clinical manifestation of disease, especially the symptoms, should be compared with the specific allergen tests, rather than base a diagnosis of AR purely on tests in the clinical practice.Whilst of China , there illeagues  investigIn this study, we found that family history of AR, and comorbid asthma, CRS, or atopic dermatitis, were all associated with increased risk of both AR and NAR. Although, it is somewhat surprising that a comorbid atopic disease, such asthma or atopic dermatitis, may confer a risk for development of a noninfectious, nonallergic condition such as NAR, this finding is nevertheless in accordance with the findings of Håkansson and colleagues  in adultHowever, the findings of the present study are somewhat limited and thus need to be confirmed in further studies in consideration of these limitations, as well as in centers in other regions across China. In particular, in the present study NAR was differentiated AR, based only on the presence of typical rhinitis symptoms in the absence of negative SPT results, without data of IgE. In this respect, it is likely that the observed prevalence of NAR was higher than the actual prevalence because under the defined criteria for NAR in the present study patients with LAR would have been undistinguishable. Indeed, a systemic review of studies on AR or NAR patients subjected to diagnostic local nasal provocation has recently demonstrated that 26.5% of patients previously considered non-allergic demonstrate local allergen reactivity . MoreoveIn summary, the confirmed standardized prevalence of AR and NAR were 16.78% and 24.60%, respectively. Combination of standardized questionnaires and specific allergen tests may provide more accurate estimates of prevalence of AR and NAR and associated risk factors.


The synthesis of saccharin  derivatives substituted on the benzene ring has seen limited development despite the longevity of this compound’s use as an artificial sweetener. This type of saccharin derivative would however present attractive properties for the development of new bioactive, drug-like small molecule compounds. Here we report the derivatisation of the benzene ring of saccharin using Cu(I)-catalyzed azide alkyne cycloaddition (CuAAC) to synthesise a diverse library of novel saccharin-1,2,3-triazole conjugates. All library compounds retain the capability for interactions with biomolecules via the unmodified sulfonamide and lactam groups of the parent saccharin core heterocycle. The compounds also encompass alternate orientations of the 1,2,3-triazole heterocycle, thus further adding diversity to the potential hydrogen bonding interactions of these compounds with biomolecules of therapeutic interest. Our findings demonstrate that specifically functionalized derivatives of saccharin may be prepared from either saccharin azide or saccharin alkyne building blocks in high yield using CuAAC. On. On1 is ur cells  [10]. Inresidues . Here wecharin 3 . The buig blocks . Derivatoperties . All comN-t-butyl protected 6-azidosaccharin 3, followed by removal of the t-butyl protecting group, over the more direct synthetic approach of using 6-azidosaccharin 2 + Calcd. for C13H13NNaO3S: 286.0508. Found: 286.0529.TBAF  was added to a solution of N-t-butyl-6-amino-1,2-benzisothiazole-3-one-1,1-dioxide 7 + Calcd. for C17H18N2NaO3S: 353.0930. Found: 353.0944.To a solution of ioxide 7   in CH3CN (20 mL) was added N-t-butyl-6-amino-1,2-benzisothiazole-3-one-1,1-dioxide (7) + Calcd. for C11H12INNaO3S: 387.9475. Found: 387.951.To a solution of xide (7)   , Pd(PPh3)Cl2  and CuI  were dried together under high vacuum and then flushed with argon. Triethylamine (20 mL) was added and the reaction mixture was stirred and heated to 40 °C. To this was added ethynyltrimethylsilane l  and the solution was stirred for 2 h. The reaction mixture was filtered through a pad of Celite and washed with EtOAc (100 mL). The residue was purified by column chromatography on silica gel (EtOAc/hexane = 1:19 to 1:9) to give the title compound 9  as a light brown solid. m.p. 114–115 °C (EtOAc/hexane); 1H-NMR  δ 0.27 3), 1.76 , 7.78 , 7.87 , 7.90 ; 13C-NMR  δ −0.3 (Si(CH3)3), 27.9 (C(CH3)3), 61.5 (C(CH3)3), 101.7 (Si(CH3)3C), 101.8 (Si(CH3)3CC), 123.4 (Ar-CH), 124.5 (Ar-CH), 126.3 (Ar-C), 130.2 (Ar-C), 137.2 (Ar-CH), 138.1 (Ar-C), 159.6 (C=O); HRMS-ESI [M + Na]+ Calcd. for C16H21NNaO3SSi: 358.0904. Found: 358.0899.N-t-Butyl protected 24  was refluxed in TFA (3 mL) for 18 h. The solvent was removed in vacuo. EtOAc was added to the residue and the solid was collected by filtration to give the title compound 10  as a white solid. m.p. greater than 300 °C (EtOAc); 1H-NMR 2SO) δ 7.55 , 7.66 , 7.94 , 8.13 , 8.50 , 8.60 , 9.64 ; 13C-NMR 2SO) δ 117.2, 120.1 (Ar-CH), 122.2 (CHtriazole), 125.8 (Ar-CH), 126.8 (Ar-C), 129.1, 130.1, 130.5 (Ar-CH), 136.3, 137.0, 140.7 (Ar-C), 145.0 (Ctriazole), 160.7 (C=O); HRMS-ESI [M − H]− Calcd. for C15H9N4O3S: 325.0389. Found: 325.0378.N-t-Butyl protected 25  was refluxed in TFA (3 mL) for 18 h. The solvent was removed in vacuo and the residue was purified by column chromatography on silica gel (MeOH/CH2Cl2 = 1:4) and gave the title compound 11  as a white solid. m.p. 247–249 °C (MeOH/CH2Cl2); 1H-NMR 2SO) δ 5.67 , 7.33–7.43 , 7.66 , 8.09–8.12 , 8.87 ; 13C-NMR 2SO) δ 53.2 (CH2), 115.7, (Ar-CH), 122.9 (CHtriazole), 123.3, 128.0, 128.2, 128.8 (Ar-CH), 133.5, 133.6, 135.7, 145.5 (Ar-C), 146.0 (Ctriazole), 167.1 (C=O); HRMS-ESI [M − H]− Calcd. for C16H11N4O3S: 339.0546. Found: 339.0534.N-t-Butyl protected 26  was refluxed in TFA (3 mL) for 18 h. The solvent was removed in vacuo and the residue purified by reverse phase (RP-18) column chromatography  to give the title compound 12  as a colourless oil which solidified to a white gum upon standing. 1H-NMR 2SO) δ 3.34–3.37 , 3.42–3.52 , 3.54–3.57 , 3.88 , 4.61 , 7.94 , 8.32 , 8.40 , 8.86 ; 13C-NMR 2SO) δ 49.9, 60.2, 68.5, 69.60, 69.64, 69.7, 69.8, 72.3 (CH2), 116.7, (Ar-CH), 124.0 (CHtriazole), 125.2 (Ar-CH), 128.0 (Ar-C), 129.9 (Ar-CH), 136.7, 141.9 (Ar-C), 144.2 (Ctriazole), 162.2 (C=O); HRMS-ESI [M + Na]+ Calcd. for C17H22N4NaO7S: 425.1136. Found: 425.1132.N-t-Butyl protected 28  was refluxed in TFA (1.5 mL) for 18 h. The solvent was removed in vacuo and the residue was purified by RP-18 column chromatography  to give the title compound 13  as a white solid. m.p. 227–229 °C (MeOH/H2O); 1H-NMR 2SO) δ 3.26 , 3.42–3.49 , 3.50–3.55 , 3.71–3.78 , 5.64 , 8.08 , 8.45 , 8.57 , 9.23 ; 13C-NMR 2SO) δ 60.7 (C-6), 69.6 (C-4), 72.5 (C-2), 76.6 (C-3), 80.0 (C-5), 87.8 (C-1), 117.0, (Ar-CH), 123.0 (CHtriazole), 125.7 (Ar-CH), 126.9 (Ar-C), 130.3 (Ar-CH), 137.2, 140.9 (Ar-C), 144.2 (Ctriazole), 161.0 (C=O); HRMS-ESI [M − H]− Calcd. for C15H15N4O8S: 411.0616. Found: 411.0601.N-t-Butyl protected 29  was refluxed in TFA (1.5 mL) for 18 h. The solvent was removed in vacuo and the residue purified by RP-18 column chromatography  to give the title compound 14  as a white solid. m.p. 274–276 °C (MeOH/H2O); 1H-NMR 2SO) δ 3.35 , 3.49 , 3.67 , 3.73 , 3.82 , 4.80 , 6.20 , 8.09 , 8.43 , 8.55 , 9.35 ; 13C-NMR 2SO) δ 60.3 (C-6), 69.3 , 74.1 , 79.9 (C-5), 84.2 , 91.2 , 117.2 (Ar-H), 123.1 (CHtriazole), 125.7 (Ar-CH), 127.1 (Ar-C), 130.1 (Ar-CH), 136.7, 140.1 (Ar-C), 144.7 (Ctriazole), 160 (C=O); 19F-NMR 2SO) δ -193.6 ; HRMS-ESI [M − H]− Calcd. for C15H14FN4O7S: 413.0573. Found: 413.0563.N-t-Butyl protected 30  was refluxed in TFA (1.5 mL) for 18 h. The solvent was removed in vacuo and the residue was purified by RP-18 column chromatography  to give the title compound 15  as a white solid. m.p. 232–235 °C (MeOH/H2O); 1H-NMR 2SO) δ 3.50–3.58 , 3.60 , 3.76–3.82 , 4.09 , 5.57 , 7.95 , 8.30 , 8.49 , 9.16 ; 13C-NMR 2SO) δ 60.5 (C-6), 68.4 (C-4), 69.4 (C-2), 73.5 (C-3), 78.5 (C-5), 88.4 (C-1), 116.7 (Ar-CH), 122.5 (CHtriazole), 124.9 (Ar-CH), 128.9 (Ar-C), 129.7 (Ar-CH), 136.1, 142.5 (Ar-C), 144.6 (Ctriazole), 162.9 (C=O); HRMS-ESI [M − H]− Calcd. for C15H15N4O8S: 411.0616. Found: 411.0608.N-t-Butyl protected 34  was refluxed in TFA (2.0 mL) for 18 h. The solvent was removed in vacuo and the residue was purified by RP-18 column chromatography  to give the title compound 17  as a white solid. m.p. 184–186 °C (MeOH/H2O); 1H-NMR 2SO) δ 3.22 , 3.38 , 3.41–3.47 , 3.66–3.71 , 3.75 , 4.48–4.72 , 4.80–4.89 , 5.03–5.46 , 5.53 , 7.30 , 7.54 , 7.68 , 8.38 ; 13C-NMR 2SO) δ 45.2 (NCH2), 60.7 (C-6), 69.6 (C-4), 72.1 (C-2), 76.9 (C-3), 80.0 (C-5), 87.5 (C-1), 102.8, (Ar-CH), 114.6 (Ar-C), 116.6 (Ar-C), 122.6 (CHtriazole), 125.7 (Ar-CH), 142.4 (Ar-C), 142.8 (Ctriazole), 152.8 (Ar-C), 161.4 (C=O); HRMS-ESI [M + Na]+ Calcd. for C25H32N8NaO13S: 707.1702. Found: 707.1756.N-t-Butyl protected 35  was refluxed in TFA (3 mL) for 18 h. The solvent was removed in vacuo and the residue was purified by column chromatography on silica gel (MeOH/CH2Cl2 = 1:9 to 1:4) to give the title compound 19  as a light brown solid. m.p. 177–179 °C (MeOH/CH2Cl2); 1H-NMR 2SO) δ 4.12 , 7.21–7.26 , 7.31–7.34 , 8.10 , 8.43 , 8.65 , 8.86 ; 13C-NMR 2SO) δ 31.2 (CH2) 111.9, (Ar-CH), 121.5 (CHtriazole), 124.8, 126.3, 126.4 (Ar-CH), 128.0 (Ar-C), 128.5, 128.6 (Ar-CH), 138.8, 140.8, 142.3 (Ar-C), 147.8 (Ctriazole), 161.4 (C=O); HRMS-ESI [M − H]− Calcd. for C16H11N4O3S: 339.0546. Found: 339.0532.N-t-Butyl protected 36  was refluxed in TFA (3 mL) for 18 h. The solvent was removed in vacuo and the residue was purified by RP-18 column chromatography  to give the title compound 20  as a colourless oil. 1H-NMR 2SO) δ 3.38–3.41 , 3.45–3.53 , 3.55–3.58 , 3.61–3.64 , 4.58 , 4.63 , 7.78 , 8.18 , 8.23 , 8.98 ; 13C-NMR 2SO) δ 60.2, 63.3, 69.1, 69.7, 69.74, 69.79, 69.8, 72.3 (CH2), 110.8, (Ar-CH), 122.7 (CHtriazole), 123.3, 124.2 (Ar-CH), 134.3, 138.5, 145.4 (Ar-C), 147.1 (Ctriazole), 166.7 (C=O); HRMS-ESI [M + Na]+ Calcd. for C18H24N4NaO8S: 479.1207. Found: 479.1202.N-t-Butyl protected 40  was refluxed in TFA (1.5 mL) for 18 h. The solvent was removed in vacuo and the residue was purified by RP-18 column chromatography  to give the title compound 21  as a white solid. m.p. 176–178 °C (MeOH/H2O); 1H-NMR 2SO) δ 3.03–3.10 , 3.16 , 3.20 , 3.46 , 3.73 , 3.95 , 4.09 , 4.34 , 7.99 , 8.31 , 8.47 , 8.92 ; 13C-NMR 2SO) δ 22.9 (SCH2), 61.3 (C-6), 70.1 (C-4), 73.2 (C-2), 78.1 (C-3), 81.0 (C-5), 84.0 (C-1), 111.5, (Ar-CH), 122.0 (CHtriazole), 124.3, 125.6 (Ar-CH), 130.3, 140.0, 143.9 (Ar-C), 146.2 (Ctriazole), 163.2 (C=O); HRMS-ESI [M + H]+ Calcd. for C16H19N4O8S2: 459.0638. Found: 459.0665.N-t-Butyl protected 42  was refluxed in TFA (1.5 mL) for 18 h. The solvent was removed in vacuo and the residue was purified by RP-18 column chromatography  to give the title compound 22  as a white solid. m.p. 186–189 °C (MeOH/H2O); 1H-NMR 2SO) δ 3.08 , 3.29 , 3.43 , 3.51 , 3.59 , 3.80 , 4.51 , 4.70 , 4.85 , 8.14 , 8.42 , 8.65 , 9.15 ; 13C-NMR 2SO) δ 48.3 (SCH2), 61.1 (C-6), 69.2 (C-2), 69.5 (C-4), 77.5 (C-3), 81.6 (C-5), 88.6 (C-1), 112.4, (Ar-CH), 124.7 (CHtriazole), 125.3, 126.4 (Ar-CH), 128.4, 136.9, 140.7 (Ar-C), 142.2 (Ctriazole), 161.3 (C=O); HRMS-ESI [M + Na]+ Calcd. for C16H18N4NaO10S2: 513.0356. Found: 513.0404.24 was prepared from N-t-butyl-6-ethynyl-1,2-benzisothiazole-3-one-1,1-dioxide 5  and azidobenzene a + Calcd. for C19H18N4NaO3S: 405.0992. Found: 405.0993.The title compound enzene a   and benzylazide b + Calcd. for C20H20N4NaO3S: 419.1148. Found: 419.1141.The title compound lazide b   and PEG azide c + Calcd. for C21H30N4NaO7S: 505.1727. Found: 505.1739.The title compound  azide c   and 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl azide d′ + Calcd. for C27H33N4O12S: 637.1810. Found: 637.1811.The title compound azide d′   in methanol (9.2 mL) was added HCl (0.8 mL). The reaction was stirred at rt for 90 h and the solvent was removed in vacuo. The residue was purified by column chromatography on silica gel (MeOH/CH2Cl2 = 0:1 to 1:9) to give the title compound 28  as a white solid. m.p. 169–170 °C (MeOH/CH2Cl2); 1H-NMR 2SO) δ 1.70 , 3.26 , 3.45 , 3.47–3.55 , 3.70–3.78 , 5.65 , 8.13 , 8.49 , 8.61 , 9.26 ; 13C-NMR 2SO) δ 27.3 (C(CH3)3), 60.5 (C(CH3)3), 60.7 (C-6), 69.6 (C-4), 72.5 (C-2), 76.6 (C-3), 80.0 (C-5), 87.8 (C-1), 116.6 (Ar-CH), 123.1 (CHtriazole), 125.0 (Ar-C), 125.6, 130.7 (Ar-CH), 137.5, 138.1 (Ar-C), 144.1 (Ctriazole), 159.3 (C=O); HRMS-ESI [M + H]+ Calcd. for C19H25N4O8S: 469.1388. Found: 469.1397.To a solution of 29 was prepared from N-t-butyl-6-ethynyl-1,2-benzisothiazole-3-one-1,1-dioxide 5  and 2-deoxy-2-fluoro-β-d-glucopyranosyl azide e + Calcd. for C19H23FN4NaO7S: 493.1164. Found: 493.1166.The title compound  azide e   and β-d-galactopyranosyl azide f + Calcd. for C19H25N4O8S: 469.1388. Found: 469.1340.The title compound  azide f   and azidotrimethylsilane g  were dissolved in tert-butyl alcohol/H2O . To the reaction mixture was added a solution of sodium ascorbate  in water (0.25 mL) followed by a solution of CuSO4·5H2O  in water (0.25 mL). The suspension was stirred vigorously at 45 °C overnight. The solvent was removed in vacuo and the residue was purified by column chromatography on silica gel (EtOAc/hexane = 2:3) to give the title compound 31  as a white solid. m.p. greater than 300 °C (EtOAc/hexane); 1H-NMR 2SO) δ 1.70 , 8.10 , 8.46 , 8.62 , 8.75 , 15.5 ; 13C-NMR 2SO) δ 27.3 (C(CH3)3), 60.5 (C(CH3)3), 116.9, (Ar-CH), 125.0 (Ar-C), 125.5 (Ar-CH), 127.3 (CHtriazole), 131.1 (Ar-CH), 137.7, 138.1 (Ar-C), 143.5 (Ctriazole), 159.3 (C=O); HRMS-ESI [M − H]− Calcd. for C13H13N4O3S: 305.0703. Found: 305.0690.N-t-Butyl protected 31  was refluxed in TFA (3 mL) for 18 h. The solvent was removed in vacuo and the residue was purified by column chromatography on silica gel (MeOH/CH2Cl2 = 1:9) to give the title compounds 32a and 32b  as white solids. m.p. 203–204 °C (MeOH/CH2Cl2); 1H-NMR 2SO) δ1.67 , 7.97 , 8.00 , 8.30 , 8.39 , 8.47–8.50 , 8.53 , 9.08 ; 13C-NMR 2SO) δ 29.1, 29.4 (C(CH3)3), 59.6, 63.2 (C(CH3)3), 116.7, 117.2 (Ar-CH), 121.5 (CHtriazole), 125.1, 125.2 (Ar-CH), 127.6, 128.6 (Ar-C), 129.7, 130.4 (Ar-CH), 132.3 (CHtriazole), 136.1, 137.1, 141.7, 142.0 (Ar-C), 143.9, 144.2 (Ctriazole), 162.0, 162.3 (C=O); HRMS-ESI [M − H]− Calcd. for C13H13N4O3S: 305.0714. Found: 305.0715.33 was prepared from saccharin bis-alkyne 6  and 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl azide d′ + Calcd. for C45H56N8NaO21S: 1099.3173. Found: 1099.3144.The title compound azide d′   in methanol (9.2 mL) was added HCl (0.8 mL). The reaction was stirred at rt for 90 h and the solvent was removed in vacuo. The residue was purified by RP-18 column chromatography  to give the title compound 34  as a white solid. m.p. 203–205 °C (MeOH/CH2Cl2); 1H-NMR 2SO) δ 1.65 , 3.19–3.25 , 3.35–3.47 , 3.66–3.77 , 4.61 , 4.80–4.89 , 5.15 , 5.27 , 5.36 , 5.53 , 7.34 , 7.56 , 7.73 , 8.36 ; 13C-NMR 2SO) δ 27.5 (C(CH3)3), 45.2 (NCH2), 59.6 (C(CH3)3), 60.7 (C-6), 69.6 (C-4), 72.1 (C-2), 76.9 (C-3), 79.9 (C-5), 87.5 (C-1), 102.2 (Ar-CH), 112.7 (Ar-C), 117.2 (Ar-CH), 122.6 (CHtriazole), 125.7 (Ar-CH), 139.7 (Ar-C), 142.7 (Ctriazole), 153.0 (Ar-C), 159.9 (C=O); HRMS-ESI [M + Na]+ Calcd. for C29H40N8NaO13S: 763.2328. Found: 763.2366.To a solution of 35 was prepared from N-t-butyl-6-azido-1,2-benzisothiazole-3-one-1,1-dioxide 3 + Calcd. for C20H20N4NaO3S: 419.1148. Found: 419.1144.The title compound ioxide 3   gave the title compound 36  as a pale yellow oil. 1H-NMR 2SO) δ 1.71 , 3.38–3.41 , 3.44–3.54 , 3.56–3.59 , 3.63–3.66 , 4.55 , 4.66 , 8.23 , 8.55 , 8.82 , 9.09 ; 13C-NMR 2SO) δ 27.3 (C(CH3)3), 60.2 (CH2), 60.8 (C(CH3)3), 63.3, 69.2, 69.68, 69.73, 69.8, 72.3 (CH2), 111.9, (Ar-CH), 122.9 (CHtriazole), 125.3 (Ar-C), 125.8, 126.7 (Ar-CH), 138.6, 141.4 (Ar-C), 145.8 (Ctriazole), 158.7 (C=O); HRMS-ESI [M + Na]+ Calcd. for C22H32N4NaO8S: 535.1833. Found: 535.1845.The title compound ioxide 3   and ethynyltrimethylsilane l  were dissolved in tert-butyl alcohol/H2O . To the reaction mixture was added a solution of sodium ascorbate  in water (0.25 mL) followed by a solution of CuSO4.5H2O  in water (0.25 mL). The suspension was stirred vigorously at 45 °C overnight. The solvent was removed in vacuo and the residue was purified by column chromatography on silica gel (EtOAc/hexane = 1:4 to 1:1) to give the title compound 37  as a pale yellow solid. m.p. 187–188 °C (EtOAc/hexane); 1H-NMR 2SO) δ 1.71 , 8.09 , 8.24 , 8.56 , 8.82 , 9.11 ; 13C-NMR 2SO) δ 27.3 (C(CH3)3), 60.8 (C(CH3)3), 116.9, (Ar-CH), 124.0 (CHtriazole), 125.3 (Ar-C), 125.9, 126.7 (Ar-CH), 135.1 (CHtriazole), 138.6, 141.4 (Ar-C), 158.7 (C=O); HRMS-ESI [M − H]− Calcd. for C13H13N4O3S: 305.0714. Found: 305.0712.N-t-Butyl protected 37  was refluxed in TFA (3 mL) for 18 h. The solvent was removed in vacuo. EtOAc was added to the residue and the solid collected by filtration to give the title compound 38  as an off white solid. m.p. 166–168 °C (EtOAc); 1H-NMR 2SO) δ 1.71 , 7.93 , 8.26 , 8.45 , 9.39 , 9.72 ; 13C-NMR 2SO) δ 28.5 (C(CH3)3), 66.6 (C(CH3)3), 113.3, 124.4, 125.4 (Ar-CH), 129.2, 129.8 (CHtriazole), 136.7, 136.8, 147.0 (Ar-C), 165.9 (C=O); HRMS-ESI [M + H]+ Calcd. for C13H13N4O3S: 305.0714. Found: 305.0702.39 was prepared from N-t-butyl-6-amino-1,2-benzisothiazole-3-one-1,1-dioxide 3 + Calcd. for C28H34N4NaO12S2: 705.1507. Found: 705.1551.The title compound ioxide 3   + Calcd. for C20H27N4O8S2: 515.1265. Found: 515.1269.To a solution of The title compound 39  in anhydrous CH2Cl2 (5 mL) at 0 °C was added mCPBA  in anhydrous CH2Cl2 (2 mL) dropwise. The solution was allowed to warm to r.t. over 2 h. The reaction mixture was diluted with CH2Cl2 (50 mL), washed with H2O (30 mL), brine (30 mL), dried (MgSO4), and the solvent was removed in vacuo. The residue was purified by column chromatography on silica gel (EtOAc/hexane = 1:1 to 3:2) to give the title compound 41  as a white solid. m.p. 172–174 °C (EtOAc/hexane); 1H-NMR 2SO) δ 1.72 , 1.94 , 1.95 , 2.00 , 2.03 , 4.17–4.28 , 4.78–4.87 , 5.01–5.06 , 5.14–5.20 , 5.37–5.45 , 8.26 , 8.59 , 8.89 , 9.14 ; 13C-NMR 2SO) δ 20.2, 20.29, 20.33, 20.5 (OCOCH3), 27.3 (C(CH3)3), 46.9 (SCH2), 60.9 (C(CH3)3), 61.4 (C-6), 65.7 (C-2), 67.2 (C-4), 72.5 (C-3), 74.9 (C-5), 84.9 (C-1), 112.3, (Ar-CH), 124.8 (CHtriazole), 125.6 (Ar-C), 126.1, 126.7 (Ar-CH), 135.8, 138.6 (Ar-C), 141.2 (Ctriazole), 158.7 (C=O), 168.6, 169.2, 169.5, 170.1 (OCOCH3); HRMS-ESI [M + Na]+ Calcd. for C28H34N4NaO14S2: 737.1405. Found: 737.1486.To a stirred solution of 42 was prepared using two different synthetic routes, A and B.40  in anhydrous CH2Cl2 (5 mL) at 0 °C was added mCPBA  in anhydrous CH2Cl2 (2 mL) dropwise. The solution was allowed to warm to rt over 2 h and the solvent was removed in vacuo. The residue was purified by column chromatography on silica gel (MeOH/CH2Cl2 = 1:9 to 3:17) to give the title compound 42  as a white solid.To a stirred solution of 41  in methanol (9.2 mL) was added HCl (0.8 mL). The reaction was stirred at rt for 90 h and the solvent was removed in vacuo. The residue was purified by column chromatography on silica gel (MeOH/CH2Cl2 = 0:1 to 1:9) to give the title compound 42  as a white solid. m.p. 145–146 °C (MeOH/CH2Cl2); 1H-NMR 2SO) δ 1.72 , 3.08 , 3.28 , 3.42 , 3.51 , 3.60 , 3.80 , 4.51 , 4.73 , 4.85 , 4.99 , 5.17 , 5.24 , 5.55 , 8.25 , 8.51 , 8.81 , 9.18 ; 13C-NMR 2SO) δ 27.3 (C(CH3)3), 48.2 (SCH2), 60.9 (C(CH3)3), 61.0 (C-6), 69.2 (C-2), 69.5 (C-4), 77.5 (C-3), 81.6 (C-5), 88.7 (C-1), 112.2, (Ar-CH), 124.8 (CHtriazole), 125.5 (Ar-C), 126.0, 126.8 (Ar-CH), 137.0, 138.6 (Ar-C), 141.3 (Ctriazole), 158.7 (C=O); HRMS-ESI [M + Na]+ Calcd. for C20H26N4NaO10S2: 569.0983. Found: 569.0994.To a solution of The title compound 1 may be prepared from either saccharin azide or saccharin alkyne building blocks in high yield using CuAAC. The application of novel alkyne building blocks 5 and 6 and azide building block 3 proved remarkably straightforward to handle. Moreover, the novel target compounds all retain the capability for interactions with the metal centres of metalloenzymes via the sulfonamide anion. The compounds also present two orientations of the 1,2,3-triazole, thus further adding diversity to the potential hydrogen bonding interactions of these compounds with biomolecules of therapeutic interest. Collectively the novel compounds described here represent desirable attributes not found in the more usual N-alkylated saccharin derivatives.In summary, we have demonstrated that specifically functionalized derivatives of


Neuropilin-1 and Neuropilin-2 form a small family of plasma membrane spanning receptors originally identified by the binding of semaphorin and vascular endothelial growth factor. Having no cytosolic protein kinase domain, they function predominantly as co-receptors of other receptors for various ligands. As such, they critically modulate the signaling of various receptor tyrosine kinases, integrins, and other molecules involved in the regulation of physiological and pathological angiogenic processes. This review highlights the diverse neuropilin ligands and interacting partners on endothelial cells, which are relevant in the context of the tumor vasculature and the tumor microenvironment. In addition to tumor cells, the latter contains cancer-associated fibroblasts, immune cells, and endothelial cells. Based on the prevalent neuropilin-mediated interactions, the suitability of various neuropilin-targeted substances for influencing tumor angiogenesis as a possible building block of a tumor therapy is discussed. Cancer, one of the most common causes of death, appears in many ways, and is individually different in almost every patient. Although the causes of cancer are complex and elusive, it actually always arises in only three ways, i.e., by accumulation of genetic defects, by carcinogenic environmental factors, or by innate hereditary defects. Essentially, three key processes can be affected: (i) cell fate, (ii) cell survival, and (iii) genome maintenance. In their regulation, twelve signaling pathways are involved using approximately 140 genes . ViabiliNRPs are involved in a wide variety of signaling pathways and have pleiotropic effects on axon guidance and remyelination, immune response, angiogenesis, cell survival, migration and invasion ,8,9,10. NRPs are evolutionarily conserved cell surface proteins that are expressed by all vertebrates and are widely distributed in adult tissues ,16. TheyBoth NRP proteins can be glycosylated to varying degrees in different cell types ,30,31. MTwo NRP molecules non-covalently associate into a homodimer, presumably via their MAM domain and via motifs within their α-helical transmembrane domain ,36. In phttps://www.uniprot.org/uniprot/O14786, accessed on: 23 January 2019).NRP1 and NRP2 are differently distributed in tissues. First identified on neuronal cells of the central nervous system, especially in the developing embryo, NRP1 was also found to be expressed in blood vessels of different tissues, especially in arterial vessels. In the earliest stages of development, NRP1 and NRP2 are differentially expressed in arteries and veins, respectively . Later iNRP2, on the other hand, is expressed in neural crest-derived cells, such as hepatocytes and epithelial cells of proximal and distal renal tubules . In the reg) and basophilic granulocytes express NRP1, dendritic cells, macrophages and microglial cells express both NRP1 and NRP2, . Th. Th95]. Among the ligands of NRP1-containing signal complexes, VEGF-A is the best-understood binding partner of NRP1 and forms the molecular basis for the development of pharmaceutical compounds that affect VEGF-A binding to the NRP1/VEGFR2 complex ,101. Suc165 and VEGF-A189, but not the smaller variants with 145, 121, or 120 amino acids in length [145 in addition to VEGF-A165, but not VEGF-A121 [165 is of particular importance as it binds to a binding pocket within the NRP1-b1 domain formed by the side chains of residues Y297, Y353, D320, S346 [VEGF-A, encoded in a gene locus on chromosome 6p21.1, occurs in six different splice variants . All VEGn length ,107,108.EGF-A121 . Two VEGEGF-A121 , which cEGF-A121 ,111. FouEGF-A121 ,102,112.20, S346 ,101. Sev20, S346 .165) was first identified to be a ligand for NRP1 [121, dimeric VEGF-A165, binds with its C-termini to the NRP1 tandem domain b1/b2 and concurrently to the ectodomain of the VEGF receptor-2 (VEGFR2) [165/VEGFR2/NRP1 signaling complex [121 can also bind directly to NRP1, it is incapable to induce the formation of a NRP1/VEGFR2 holoreceptor [The 165 amino acid long variant of the vascular endothelial growth factor (VEGF-Afor NRP1 . Unlike (VEGFR2) ,106,107. complex . This co complex . Althougreceptor .145 and VEGF-C. Upon binding its ligand, NRP2 forms a ternary complex with VEGF-C and VEGFR3 on lymphatic ECs and is involved in lymphangiogenesis [The repertoire of NRP2 ligands within the VEGF family comprises VEGF-Aogenesis ,116.Recently, other soluble ligands and other receptor partners of NRP1 have been reported, demonstrating an amazing versatility of NRP1 in this respect. Interestingly, by binding to several growth factors other than VEGF-A, NRP1 becomes a coreceptor for the respective growth factor receptor, thus promoting the formation of a ternary signal complex. In such a way, the following growth factors and receptors may interact with NRP1: Placenta growth factor (PlGF), which is a member of the VEGF protein family and its receptor PlGFR ,118, hepWith the exception of the receptor-type serine kinases TGF-βRI and TGF-βRII that activate SMAD2 and SMAD3, all NRP1-associated growth factor receptors are receptor-type tyrosine kinases that autophosphorylate upon agonistic stimulation and recruit adapter proteins to trigger an intracellular signaling cascade . As partFurthermore, several members of the semaphorin family also bind to NRPs ,129. As The seven class 3 semaphorins, SEMA3A-G, are secreted members of a large family of guidance factors that regulate processes, such as developmental and tumor angiogenesis, by signaling through receptors composed of NRP1 or NRP2 and plexinA or plexinD, respectively . ECs expSemaphorin SEMA3A binds to the a1/a2 tandem domain of NRP1 and causes collapse and retraction of the nerve growth cone . The intNRP2 has been originally described as receptor for SEMA3F (at that time referred to as sema IV) that mediates its repulsive effect on growing neurons and is able to form heterooligomers with NRP1 . In compIntegrins have also been reported to interact with NRPs ,141,142.Another coreceptor of NRP1 is the L1 cell adhesion molecule (L1CAM) which belongs to the immunoglobulin superfamily (IgSF), mediates intercellular contacts between neurons, and interacts with NRPs in both cis- and trans-cellular manner ,158. The165 and PlGF-2, to the b1b2 domain of NRP1 when its chain length is at least 20–24 monosaccharides [165 and PlGF-2 interaction with NRP1 on ECs [Heparin and heparan sulfate have been described as binding partners of neuropilins . Heparincharides . By phys1 on ECs . As NRP11 on ECs . Heparan1 on ECs ,164,165.Calloselasma rhodostoma). Rhodocetin αβ (RCαβ) has been identified as the first non-enzymatic component of a snake venom that recognizes NRP1 on ECs [NRP1 is also a target of soluble toxins, such as the αβ subunit of rhodocetin, a venom component of the Malayan pit viper  vasculature is still unclear, as NRP1 may bind several FGFs  but doesThe interaction of NRP1 with PDGF-A and -B might be relevant for EMT ,189. StiNRP1 and NRP2 can also interact as coreceptors for HGF with the hepatocyte growth factor receptor/scatter factor receptor MET on ECs ,197. HGFOften, oncogenes switch signal pathways in such a way that they become essential regulators of proliferation and survival of tumor cells, termed oncogene addiction . DownregNRP1 can function as a coreceptor for TGFβ by forming holoreceptors with TGFβ-receptors I, II, and III ,201. By NRP1 has a negatively charged cleft in its b1 domain which allows binding of various ligands, such as TGF-β ,125. NRPThe binding and signaling of VEGF and related growth factors seem to be antagonized by class 3 semaphorins . AdditioAlthough NRP1 is the specific SEMA3A ligand, NRP1, with its short intracellular domain, cannot transduce SEMA3A signals on its own, but relies on complex formation with plexinA receptors . By bindBy binding to NRP1 and plexinD1 in ECs, SEMA3C induces internalization of VE-cadherin and shutdown of VEGF-induced signaling via AKT, FAK, as well as p38MAPK, which then causes disassembly of EC junctions and focal adhesions and related cytoskeletal rearrangement . Thus, SSEMA3F overexpressing melanoma cells form poorly vascularized tumors because Nrp2 inhibits tumor development and metastasis by a strong antiangiogenic cascade ,212. In Recruitment of pericytes to nascent vessels is essential for development, stabilization and maturation of the vasculature ,216,217,NRPs interact with specific integrins and activate them to bind to ECM molecules ,156,222.In a similar way, NRP2-mediated VEGF signaling in breast cancer and prostate carcinoma cells is subject to modulation by integrin α6β1 ,227. NRPGalectins are β-galactoside-binding proteins with mostly angiostimulatory activity, possibly by modulating receptor endocytosis ,231. In Binding of the snake venom component, rhodocetin αβ (RCαβ) to NRP1 induces the formation of a ternary complex with MET on endothelial and tumor cell membranes . This leUpon binding of VEGF, NRP1 triggers RAS activation and consecutively phosphorylation of ERK1/2 and AKT . NotablyBinding of GIPC1 to the cytoplasmic SEA (Ser-Glu-Ala) motif of NRP stimulates internalization of integrin α5β1 in Rab5-positive early endosomes . MoreoveMembrane trafficking also plays a key role in regulating signaling pathways . EndocytNRP1 and NRP2 can form docking sites for the endocytic adapter protein GIPC1  ,239,240.NRPs can activate signaling pathways that protect cancer cells from cytotoxic drugs and apoptosis. NRP1 and NRP2 are major positive regulators of the Hedgehog (HH) signaling pathway, which is relevant to angiogenesis and wound healing. It promotes EMT, cancer stem cell (CSC) survival, and tumor growth ,244,245.In medulloblastoma, tumor derived Sonic hedgehog (SHH) induces PlGF production in the cerebellar stroma, which promotes tumor cell survival through NRP1 independent of VEGFR1 . The majIn contrast, NRP1 expression is induced by Wnt/β-catenin signaling in mammary stem cells and in mouse mammary tumor virus (MMTV)-Wnt1 tumor xenografts . In breaNRP2 has been implicated in metastatic progression, although it usually occurs in carcinomas just in small amounts . Presuma165 and inhibiting its interaction with membrane-bound receptors [165 to VEGFR2 expressing ECs promoting angiogenesis [The binding of VEGF and related growth factors to NRPs and corresponding signaling seem to be antagonized by class 3 semaphorins . Additioeceptors . Accordieceptors . Artificogenesis .A soluble splice variant of NRP2, s9NRP2, scavenges VEGF-C and inhibits VEGF-C/NRP2 signaling in prostate cancer. This, suggests that s9NRP2 may be used therapeutically in the treatment of tumors that strongly depend on the VEGF-C/NRP2 axis for survival .Expression of NRP1 is induced by growth factors via the RAS/MAPK signal pathway ,250,258.NRP1 is instrumental in microRNA-based intercellular communication, because it efficiently binds Argonaute2 (AGO2) and AGO2/miR complexes without any involvement of its VEGF binding site, and it promotes their internalization into cells, whereupon they can perform their function and promote, for example, proliferation, migration and angiogenic tube formation . MicroRNThe transcription factor SOX10 induces among others the expression of miR338-p, which in gastric cancer and oral squamous carcinoma, inhibits the expression of NRP1 via phosphorylation of ERK1/2, MAPK, and AKT ,267. WhiSimilar to miR-338 in melanoma cells, in medulloblastoma cells, miR148a  and in nIn glioblastoma multiforme, miR-124-3p acts as a suppressor of NRP1, which promotes tumor cell proliferation and migration as well as tumor angiogenesis via PI3K/AKT/NFκB signaling . In contNRP expression levels correlate with tumor growth, invasiveness, angiogenesis, and poor prognosis . Cancer Furthermore, Nrp1 promotes EMT via TGF-β, HH, and HGF/Met signaling , therebyTumor cells have been in the scientific and therapeutic focus for long . Doubtleregs (CD4+CD25+Foxp3+ T-cells). In the tumor microenvironment, TAMs express SEMA4D on their surface, which forms a cell-cell contact with NRP1 on Tregs and activates them and other cells, such as ECs [regs characteristically express NRPs, via which they crosstalk with the different cells of the innate and adaptive immunity. Thus, they orchestrate immunosuppressive processes [reg cells express abundant NRP1, and the increased expression of NRP in Tregs correlates with poor prognosis, presumably because of its immunosuppressive properties against other CD4+-Thelper cells, CD8+ cytotoxic T-cells and natural killer cells [Dendritic cells and other antigen presenting cells, which surveil the tumor tissue, express NRPs ,57. Evenh as ECs ,300,301.h as ECs ,92,184. rocesses . Conspicer cells ,170. Bluer cells .Another cell type within solid tumors are ingrowing ECs. Their diverse interactions with each other and with tumor cells involving NRPs are highlighted in the following sections. In tumors, NRP1 is involved in evading the control by the immune system and also in angiogenesis and formation of tumor-characteristic vasculogenic mimicry (VM) vessels that significantly contribute to resistance to antiangiogenic therapy .NRPs are involved in the (patho) physiological regulation of (lymph) angiogenesis . InitialThe multifaceted tumor vasculature can arise in various ways : EssentiA variety of factors, especially the tumor microenvironment, determines the type of vascularization in the tumor mass ,325. ConNRP1 is essential for VGFA-triggered and angiogenesis-inducing signaling in ECs and in tumor cells . In partIn developmental angiogenesis of mice, plexinD1 mediates signals from Sema3E, –A, and –F . NRP/SEMSEMA3C is released by pericytes and SMCs and counteracts VEGF-triggered angiogenic signaling in neighboring ECs and, thus, attacks immature vessel sprouts in pathological angiogenesis, rather than quiescent ECs in established vessels ,332. ThiWhile SEMA3C inhibits tumor lymphangiogenesis and metastasis, its furin-cleaved form p65-SEMA3C has tumor-promoting properties in NRP2-expressing cancer cells, which was found at least in vitro, however, not yet in vivo ,336,337.An important aim in tumor treatment is a normalization of the tumor vasculature, rather than inhibition of angiogenesis, so that hypoxia is reduced by improved perfusion and drugs can reach tumor cells better ,340. SEMIn tumors, NRP enhances angiogenesis probably by blocking receptor endoytosis via trans-cellular interactions and thereby stabilizing receptor signaling . In thisIndependent of VEGFR1, PlGF promotes invasion and VM in melanoma via NRP1 . Unlike In cancer cells, signaling cascades are often activated by the constitutive activation of an oncogene, upon which they become dependent (‘oncogene addiction’), and on which a therapy inhibiting this signaling can be based . UnfortuSince NRPs can associate with diverse receptors into holoreceptors, they are promising targets for tumor therapies, and development of highly specific and highly potent NRP1 inhibitors is of outstanding interest ,350. In NRP1 is generally considered as a tumor-promoting coreceptor, but the situation is not entirely clear . In coloThe use of soluble sNRP as a decoy receptor has been investigated in animal models in tumor therapy. sNRP1 caused extensive hemorrhage, damaged vessels, and apoptotic tumor cells in tumors of rat prostate carcinoma cells and inhibited tumor angiogenesis and growth in murine granulocytic sarcoma (chloroma) ,358. LikBlocking NRP1 with antibodies selectively inhibits angiogenesis and in combination with anti-VEGF therapy, it further reduces tumor growth, suggesting that NRP1 antibodies may render tumor vessels more responsive to anti-VEGF therapy .NRP1 is overexpressed by angiogenic ECs of the tumor vasculature and in diverse tumor cells ,360,361.A monoclonal antibody against NRP1 inhibits EC migration and tumorigenesis in mouse tumor models ,131. TarIn renal cancer, angiogenesis inhibition by blocking NRP1/VEGFR2-mediated signaling with bevacizumab in combination with the RTK inhibitor sunitinib had toxic hematologic and vascular side effects and caused hypertension .A phase I trial with a human monoclonal IgG1 antibody (MNRP1685A) targeting NRP1’s VEGF-binding domain was promising, but another phase Ib trial with concomitant inhibition of NRP1 and VEGF in combination with chemotherapy showed an unexpectedly high proteinuria and toxicity ,365. ThuDCLPRC), DG1 (CRRPRMLTC) and DG2 (CRSRRIRLC) have suppressive effects on angiogenesis, tumorigenesis, and invasion, respectively, in preclinical mouse and primate models of human retinal diseases [The first NRP-specific small molecule peptide inhibitor is EG00229 (HY-10799) . Similardiseases  and in adiseases ,371. Thediseases .reg cell activation with good stability in vivo [Distinguishing between the two NRP isoforms, the small molecule inhibitor EG01377 selectively binds to the arginine-binding pocket of NRP1 without binding to NRP2, and it functionally inhibits VEGF-A-induced angiogenesis, cell migration, melanoma cell invasiveness, and T in vivo .165 with NRP1 [Based on the inhibitory peptide KPPR, branched pentapeptides in which the K side chain has been extended by an additional homoarginine have been developed, which are up to 30 times more active than ATWLPPR with respect to their inhibition of the interaction of VEGF-Aith NRP1 ,368,374.Targeting the transmembrane domain of NRP1, a peptide corresponding to the NRP1 transmembrane domain, termed pTM-NRP1, shows antiangiogenic activity in a xenograft mouse model of glioma and inhibits breast cancer growth and metastasis ,376.The NRP1-binding motif has been harnessed to generated peptides that are directed to and penetrate into the tumor. Such NRP1-specific tumor targeting peptides effectively enhance the uptake of various therapeutic agents . These pDual targeting compounds, such as the peptide RGD-ATWLPPR, encompassing ligands of NRP1 and integrin αvβ3 that are both overexpressed in the tumor microenvironment accumulate with higher specificity in the tumor and, unlike uncoupled NRP1 and integrin ligands, reduce turnover and/or surface internalization of NRP1 .A phase 1 clinical trial of the integrin-binding, tumor tissue-homing peptide CEND-1  in combination with nanoparticle albumin bound (nab)-paclitaxel and gemcitabine in metastatic exocrine pancreatic cancer has started this year LD22-4 is an N-terminal fragment of FGF2 that binds with its CendR motif-corresponding C-terminus (KDPKR) to NRP1 and inhibits in vitro migration of ECs, tumor cells, and fibroblasts, and in vivo, it suppresses tumor angiogenesis and growth in animal models of breast, prostate, and lung carcinoma without being cytotoxic, inducing apoptosis, or affecting cell proliferation rates ,386,387.An immunoglobulin FC-fused tumor tissue penetrating peptide, Fc-TPP11 (HTPGNSKPTRTPRR), binds to the VEGF-binding site of NRP1 with 1000-fold higher affinity as compared to NRP2 . After bThe disulfide-bridged TT1 (CKRGARSTC) and its linear analogon LinTT1 (AKRGARSTA) home to breast cancer in mouse models and enhance the antitumor potency of coadministered therapeutics ,390. AftDocetaxel-loaded nanoparticles that dually target NRP1 and CD44 with the tLyP-1 peptide (CGNKRTR) and hyaluronic acid, respectively, home in on metastatic tumor cells and metastasis-supporting neovasculature and suppress tumor cell invasion and inhibit lung metastasis in three mouse models of triple-negative breast cancer .Diagnostically, lipid microbubbles with conjugated NRP1-targeted peptides CRPPR and ATWLPPR specifically bind to NRP1-expressing cells and allow ultrasound imaging of angiogenic tumors .Theranostically, polysiloxane nanoparticles combining a magnetic resonance imaging (MRI) contrast agent, a photosensitizer and the NRP1-targeting motif KDKPPR can be employed to destroy tumor neovessels in glioblastoma, because they accumulate in the tumor vessel wall and show no cytotoxicity unless after exposure to light during photodynamic therapy .CendR peptides also bind to NRP2 and are subsequently internalized . A truncThe risk that tumor-penetrating peptides might also promote the spread of metastases by inducing increased vascular permeability at the tumor site, in addition to improved drug permeation, appears to be limited, as they not only not increased metastasis but also reduced them ,395,396.In addition to reducing intratumoral hypoxia and elevated oncotic pressure and improving the accessibility with chemotherapeutic agents by normalization of the tumor vasculature, as a further possible approach, a tumor-specific vessel disruption in combination with chemotherapeutic agents is also conceivable for some cancers. In xenograft fibrosarcoma and epidermoid carcinoma mouse models, intravenously injected NRP1-specific rhodocetin-αβ selectively destroys the vasculature only in tumors and results in hemorrhage, but not in other tissues .As in normal arteries, NRP1 occurs on the basolateral side of TEC of intrinsically leaky tumor vessels . MoreoveBinding to the b1/b2 domain of NRP1, rhodocetin-αβ triggers NRP1/MET signaling only in cells that expose NRP1 to the bloodstream ,167. TheMacrovipera lebetina inhibits tumor angiogenesis by reducing the expression of NRP1 and VEGF in a quail embryonic chorio-allantoic membrane system as well as in a human colon adenocarcinoma xenograft mouse model [Lebein, a snake venom disintegrin from se model .NRPs, as coreceptors of important RTKs, integrins, and other receptors, are of paramount importance for formation and functioning of the tumor vasculature. In this context, NRPs modulate cellular responses by capturing ligands, regulating growth factor expression, endocytosis and recycling, and by signaling independently. The complex interplay of different cell types within the tumor microenvironment causes dysregulated angiogenic signaling resulting in pathological tumor angiogenesis. The highly irregular shape and comparatively poor functionality of the tumor vasculature complicates treatment with drugs administered via the bloodstream. To promote tumor therapy with cytostatic drugs, vessel normalization is sought. NRPs represent a potential therapeutic target due to their multifaceted roles and the fact that they are highly expressed on tumor ECs and tumor cells. As NRP also plays a key role in the uptake of nutrients by cells, NRP appears to be particularly suitable for introducing drugs into both TECs and tumor cells.


The World Confederation for Physical Therapy defines the profession of physiotherapists as follows WCPT :Physical therapists provide services for a wide range of people to optimise their physical activity, from elite athletes to aging adults seeking to remain active as they age. More than any other profession, they prevent chronic disease by helping people become more active. Exercise is a principal component of physical therapy programmes designed to increase physical activity. (p. x1x)For physiotherapists to deliver the high-quality and varied interventions implied in the above statement, they require [The above statement is linked to the fact that the physiotherapy profession can now be defined as done by Carol Richards in her There are insufficient numbers of physiotherapists  to manage the healthcare and rehabilitation requirements of the South African population of 58 780 000) HPCSA ; StatistWe are now at a stage where many of our universities have developed their researchers, to the point that we have increasing numbers of internationally recognised researchers at post-doctoral levels who can make an impact with large, innovative and appropriate intervention studies for those who currently have little or no access to rehabilitation. These researchers should work collaboratively with experienced clinicians to develop management strategies that can be adapted to the needs of the many people who are not able to access rehabilitation. We need to consider adapting that which we do so well at an individual patient level, in a way that provides good-quality rehabilitation care that is affordable, accessible and realistic for those patients who do not have the means to access one-on-one care. We need to test and develop new strategies in a collaborative and multi-centred way to provide appropriate rehabilitation for the public healthcare system.Already there are some useful studies in the international literature, and some published South African studies with innovative rehabilitation ideas, that could be tailored for use in our communities. These studies now need to be considered for translation into practice and this is where the researchers and clinicians really need to work together to introduce these new ways of practicing to the physiotherapy community working in the public healthcare sector.The South African physiotherapy profession has the ability to become a leader in these types of innovative studies. Given the dire need of appropriate rehabilitation for the majority of people with chronic diseases and long-term disability in low- and middle-income countries worldwide, let us become involved in innovative rehabilitation studies.‘I can do things you cannot, you can do things I cannot; together we can do great things.’– Mother Theresa