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C(sp3) centers adjacent to (hetero)aryl groups are widely present in physiologically active molecules. Metal-hydride-catalyzed hydroalkylation of alkenes represents an efficient means of forging C(sp3)-C(sp3) bonds, boasting advantages as a wide source of substrates, mild reaction conditions, and facile selectivity manipulation. Nevertheless, the hydroalkylation of vinylarenes encounters constraints in terms of substrate scope, necessitating the employment of activated alkyl halides or alkenes containing chelating groups, remains a challenge. In this context, we report a general nickel-hydride-catalyzed hydroalkylation protocol for vinylarenes. Remarkably, this system enables α-selective hydroalkylation of both aryl and heteroaryl alkenes under an extra ligand-free condition, demonstrating excellent coupling efficiency and selectivity. Furthermore, through the incorporation of chiral bisoxazoline ligands, we have achieved regio- and enantioselective hydroalkylation of vinylpyrroles, thereby facilitating the synthesis of α-branched alkylated pyrrole derivatives.
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Site- and enantio-selective alkyl-alkyl bond formation is privileged in the retrosynthetic analysis due to the universality of sp3-hybridized carbon atoms in organic molecules. Herein, we report a nickel-catalyzed remote asymmetric hydroalkylation of alkenyl ethers via synchronous implementation of alkene isomerization and enantioselective C(sp3)-C(sp3) bond formation. Regression analysis of catalyst structure-activity relationships accelerates the rational ligand modification through modular regulation. This reaction has several advantages for synthesizing chiral dialkyl carbinols and their ether derivatives, including the broad substrate scope, good functional group tolerance, excellent regioselectivity (>20:1 regioisomeric ratio), and high enantioselectivity (up to 95% enantiomeric excess).
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Cyclopropene hydrofunctionalization has been a promising strategy for accessing multi-substituted cyclopropanes; however, cyclopropene hydroalkylation remains underdeveloped. Herein, we report a low-valent CoH-catalyzed facial-selective cyclopropene hydroalkylation to access multi-substituted cyclopropanes. This reaction exhibits a broad substrate scope of alkyl halides and cyclopropenes and tolerates many functional groups. Moderate-to-good facial-selectivity is obtained without any directing groups. Mechanism studies provide evidence that alkyl radicals are generated from alkyl halides and irreversible CoH insertion is responsible for the facial-selectivity. Our preliminary exploration demonstrates that asymmetric cyclopropene hydroalkylation can be realized without conspicuous auxiliary groups.
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Regiodivergent alkene functionalization that produces either regioisomer starting from the same raw materials is desirable. Herein, we report a nickel-catalyzed switchable site-selective alkene hydroalkylation. The selection of reaction temperatures leads to protocols that provide regiodivergent hydroalkylated products starting from a single alkene substrate. This protocol allows the convenient synthesis of α- and ß-branched protected amines, both of which are important to the fields of pharmaceutical chemistry and biochemistry. In addition, enantioenriched ß-branched alkylamines can be accessed in a catalytic asymmetric variant. Preliminary mechanistic studies indicate that the formation of a more stable nickelacycle provides the driving force of migration. The thermodynamic and kinetic properties of different reduction elimination intermediates are responsible for the switchable site-selectivity.
Assuntos
Alcenos , Níquel , Alcenos/química , Aminas/química , Catálise , Níquel/química , TemperaturaRESUMO
Substantial advances in enantioconvergent C(sp3)-C(sp3) bond formation reactions have been made in recent years through the use of transition-metal-catalyzed cross-coupling reactions of racemic secondary alkyl electrophiles with organometallic reagents. Herein, we report a general process for the asymmetric construction of alkyl-alkyl bonds adjacent to heteroatoms, namely, a nickel-catalyzed enantioconvergent reductive hydroalkylation of olefins with α-heteroatom phosphorus or sulfur alkyl electrophiles. Including the use of readily available olefins, this reaction has considerable advantages, such as mild reaction conditions, a broad substrate scope, and good functional group compatibility, making it a desirable alternative to traditional electrophile-nucleophile cross-coupling reactions.
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MicroRNAs (miRs) are widely reported to be novel biomarkers involved in the process of coronary atherosclerosis (CAS). Hence, this study aims to explore the function of miR-383-3p targeting IL1R2 on inflammatory injury of coronary artery endothelial cells (CAECs) in CAS. The underlying regulatory mechanisms of miR-383-3p were analyzed in concert with the treatment of miR-383-3p mimics, miR-383-3p inhibitors, and the combination of miR-383-3p inhibitors and siRNA against IL1R2 in homocysteine (HCY)-induced CAECs. MTT, Hoechst 33258 staining, and tube formation assay were employed in order to measure cell viability, apoptosis, and tube formation, respectively. The levels of IL-1ß, IL-6, IL-10, and IL-18 were determined by ELISA. IL1R2 was verified as the target gene of miR-383-3p by dual-luciferase reporter gene assay. MiR-383-3p was down-regulated in myocardial tissues of AS rats while IL1R2 was the reciprocal. The up-regulation of miR-383-3p decreased the levels of IL1R2, caspase-1, IL-1ß, IL-6, and IL-18 expressions, as well as cell apoptosis rate in the HCY-induced CAECs, while IL-10 expression, cell viability, and tube formation ability were increased. These results were contraindicated in the HCY-induced CAECs treated by miR-383-3p inhibitors. In conclusion, miR-383-3p mediating IL1R2 prevents HCY-induced apoptosis and inflammation injury in CAECs through the inhibition of the activation of inflammasome signaling pathway. These findings highly indicate that miR-383-3p may be beneficial in the prevention of CAS and other cardiovascular diseases.
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Doença da Artéria Coronariana/metabolismo , Vasos Coronários/metabolismo , Endotélio Vascular/lesões , Homocisteína/efeitos adversos , MicroRNAs/metabolismo , Receptores Tipo II de Interleucina-1/metabolismo , Animais , Apoptose/efeitos dos fármacos , Doença da Artéria Coronariana/induzido quimicamente , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/patologia , Vasos Coronários/patologia , Citocinas/genética , Citocinas/metabolismo , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Homocisteína/farmacologia , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Masculino , MicroRNAs/genética , Ratos , Ratos Sprague-Dawley , Receptores Tipo II de Interleucina-1/genéticaRESUMO
BACKGROUND: Atrial natriuretic peptide (ANP) is an important endogenous hormone that controls inflammation and immunity by acting on dendritic cells (DCs); however, the mechanism remains unclear. OBJECTIVE: We analyzed the downstream signaling events resulting from the binding of ANP to its receptor, NPRA, and sought to determine what aspects of this signaling modulate DC function. METHODS: We utilized the inhibitory peptide, NP73-102, to block NPRA signaling in human monocyte-derived DCs (hmDCs) and examined the effect on DC maturation and induced immune responses. The potential downstream molecules and interactions among these molecules involved in NPRA signaling were identified by immunoprecipitation and immunoblotting. Changes in T cell phenotype and function were determined by flow cytometry and BrdU proliferation ELISA. To determine if adoptively transferred DCs could alter the in vivo immune response, bone marrow-derived DCs from wild-type C57BL/6 mice were incubated with ovalbumin (OVA) and injected i.v. into C57BL/6 NPRA-/- knockout mice sensitized and challenged with OVA. Lung sections were stained and examined for inflammation and cytokines were measured in bronchoalveolar lavage fluid collected from parallel groups of mice. RESULTS: Inhibition of NPRA signaling in DCs primes them to induce regulatory T cells. Adoptive transfer of wild type DCs into NPRA-/- mice reverses the attenuation of lung inflammation seen in the NPRA-knockout model. NPRA is associated with TLR-2, SOCS3 and STAT3, and inhibiting NPRA alters expression of IL-6, IL-10 and TGF-ß, but not IL-12. CONCLUSIONS: Modulation of NPRA signaling in DCs leads to immune tolerance and TLR2 and SOCS3 are involved in this induction.
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PURPOSE: To make the capacity model of SMART new-type multi-functional ventilator achieve the capability that the flow can be stable and adjusted accurately. METHODS: To analysis the problems in the course of development with fluid mechanics principle, find the deficiencies of original design and improve it. RESULTS: The inspiratory phase waveform of IPPV. SIMV,etc, breathing pattern presented square wave, achieved the goal of adjustable flow. CONCLUSIONS: Using the fluid mechanics principle guiding the design of ventilator gas circuit can get twice the result with half the effort.
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Capacidade Inspiratória , Mecânica Respiratória , Ventiladores MecânicosRESUMO
To increase the reliability and success rate of drug discovery, efforts have been made to increase the C(sp3) fraction and avoid flat molecules. sp3-Rich enantiopure amines are most frequently encountered as chiral auxiliaries, synthetic intermediates for pharmaceutical agents and bioactive natural products. Streamlined construction of chiral aliphatic amines has long been regarded as a paramount challenge. Mainstream approaches, including hydrogenation of enamines and imines, C-H amination, and alkylation of imines, were applied for the synthesis of chiral amines with circumscribed skeleton structures; typically, the chiral carbon centre was adjacent to an auxiliary aryl or ester group. Herein, we report a mild and general nickel-catalysed asymmetric reductive hydroalkylation to effectively convert enamides and enecarbamates into drug-like α-branched chiral amines and derivatives. This reaction involves the regio- and stereoselective hydrometallation of an enamide or enecarbamate to generate a catalytic amount of enantioenriched alkylnickel intermediate, followed by C-C bond formation via alkyl electrophiles.
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Here we describe a simple protocol that uses positively charged nylon membrane dot blot to profile miRNA expression. A library of 515 antisense oligodeoxynucleotides of human and mouse mature miRNAs was synthesized and spotted on GeneScreen Plus membrane using a dot-blot equipment. Total RNA or enriched small molecular weight RNAs (smwRNAs) were enzymatically radiolabeled by poly (A) polymerase and then hybridized to the nylon membrane oligo arrays. The spot signal intensity on the membrane was analyzed using phosphorimaging. This method offers a convenient and economic way to simultaneously detect the expression of hundreds of miRNAs.
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Perfilação da Expressão Gênica/métodos , MicroRNAs/genética , Animais , Southern Blotting/métodos , Técnicas de Cultura de Células , Humanos , MicroRNAs/isolamento & purificação , Hibridização de Ácido Nucleico , Nylons/química , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Coloração e Rotulagem , Coleta de Tecidos e ÓrgãosRESUMO
The PH-BEACH-WD40 (PBW) protein family members play a role in coordinating receptor signaling and intracellular vesicle trafficking. LPS-Responsive-Beige-like Anchor (LRBA) is a PBW protein whose immune function remains elusive. Here we show that LRBA-null mice are viable, but exhibit compromised rejection of allogeneic, xenogeneic and missing self bone-marrow grafts. Further, we demonstrate that LRBA-null Natural Killer (NK) cells exhibit impaired signaling by the key NK activating receptors, NKp46 and NKG2D. However, induction of IFN-γ by cytokines remains intact, indicating LRBA selectively facilitates signals by receptors for ligands expressed on the surface of NK targets. Surprisingly, LRBA limits immunoregulatory cell numbers in tissues where GvHD is primed or initiated, and consistent with this LRBA-null mice also demonstrate resistance to lethal GvHD. These findings demonstrate that LRBA is redundant for host longevity while being essential for both host and donor-mediated immune responses and thus represents a unique and novel molecular target in transplant immunology.
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Proteínas Adaptadoras de Transdução de Sinal/imunologia , Transplante de Medula Óssea , Doença Enxerto-Hospedeiro/imunologia , Transdução de Sinais/imunologia , Imunologia de Transplantes , Proteínas Adaptadoras de Transdução de Sinal/genética , Aloenxertos , Animais , Antígenos Ly/genética , Antígenos Ly/imunologia , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/patologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Mutantes , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Receptor 1 Desencadeador da Citotoxicidade Natural/genética , Receptor 1 Desencadeador da Citotoxicidade Natural/imunologia , Transdução de Sinais/genéticaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Zanthoxylum bungeanum (ZB), a Chinese herb medicine, has been shown to possess a wide range of biological activities including anti-tumor, anti-inflammatory, and anti-microbial activity and has long been used to treat a variety of skin diseases including psoriasis. However, the underlying mechanism of action has not been systematically elucidated. AIM OF THE STUDY: to analyze the chemical composition of the hydro-distilled Zanthoxylum bungeanum essential oil (ZBEO), and to investigate its anti-proliferative activity on HaCaT cells as well as the underlying anti-psoriasis mechanisms. MATERIALS AND METHODS: The chemical composition of ZBEO was analyzed with gas chromatography coupled to mass spectrometry (GC-MS). HaCaT cells was exposed to different dose of ZBEO added in medium prior to morphologic features analysis as well as cell cycle arrest examination with Flow cytometry. Western blot analysis was employed to estimate the expression level of proteins including caspase-8/9/3, PARP, Bax and Bcl-2. RESULTS: Thirty-nine compounds of the ZBEO were identified GC-MS. ZBEO-treated HaCaT cells showed typical apoptotic morphologic features by DAPI staining assay. The ZBEO significantly inhibited proliferation of HaCaT cells in a dose- and time-dependent manner and induced S phase arrest apoptosis in HaCaT cells. Furthermore, western blot analysis revealed that the ZBEO increased expression of cleaved caspase-8/9/3, PARP, and Bax, decreased Bcl-2 levels. CONCLUSION: ZBEO inhibits the proliferation of HaCaT cells, resulting from the induction of cellular apoptosis through both intrinsic and extrinsic pathways. ZBEO is a potential candidate that may be considered for development into an anti-psoriasis drug.
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Óleos Voláteis/farmacologia , Zanthoxylum , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Queratinócitos , Óleos Voláteis/química , Compostos Fitoquímicos/análiseRESUMO
LRBA expression is induced by mitogens in lymphoid and myeloid cells. The Drosophila LRBA orthologue rugose/DAKAP550 is involved in Notch, Ras and EGFR pathways. These findings suggest that LRBA could play a role in cell types that have increased proliferative and survival capacity. Here, we show by microarray and real-time PCR analyses that LRBA is overexpressed in several different cancers relative to their normal tissue controls. We also show that LRBA promoter activity and endogenous LRBA mRNA levels are reduced by p53 and increased by E2F1, indicating that mutations in the tumor suppressors p53 and Rb could contribute to the deregulation of LRBA. Furthermore, inhibition of LRBA expression by RNA interference, or inhibition of its function by a dominant-negative mutant, leads to significant growth inhibition of cancer cells, demonstrating that deregulated expression of LRBA contributes to the altered growth properties of a cancer cell. Finally, we show that the phosphorylation of EGFR is affected by the dominant-negative mutant, suggesting LRBA plays a role in the mammalian EGFR pathway. These findings demonstrate that LRBA facilitates cancer cell growth and thus LRBA may represent a novel molecular target for cancer therapy.
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Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Neoplasias/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Bases , Neoplasias da Mama/metabolismo , Proteínas de Transporte/genética , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Feminino , Células HeLa , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Interferência de RNA/fisiologia , Receptores de Estrogênio/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Restriction enzymes have two major limitations for cloning: they cannot cleave at any desired location in a DNA sequence and may not cleave uniquely within a DNA sequence. In contrast, the clustered regularly interspaced short palindromic repeat (CRISPR)-associated enzyme 9 (Cas9), when coupled with single guide RNAs (sgRNA), has been used in vivo to cleave the genomes of many species at a single site, enabling generation of mutated cell lines and animals. The Cas9/sgRNA complex recognizes a 17-20 base target site, which can be of any sequence as long as it is located 5' of the protospacer adjacent motif (PAM; sequence 5'-NRG, where R = G or A). Thus, it can be programmed to cleave almost anywhere with a stringency higher than that of one cleavage in a sequence of human genome size. Here, the Cas9 enzyme and a specific sgRNA were used to linearize a 22 kb plasmid in vitro. A DNA fragment was then inserted into the linearized vector seamlessly through Gibson assembly. Our technique can be used to directly, and seamlessly, clone fragments into vectors of any size as well as to modify existing constructs where no other methods are available.
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Proteínas de Bactérias/química , Proteínas Associadas a CRISPR/química , Sistemas CRISPR-Cas , Clonagem Molecular/métodos , Clivagem do DNA , Endonucleases/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteína 9 Associada à CRISPR , Genoma , Camundongos , Plasmídeos/genética , RNA Guia de Cinetoplastídeos/genéticaRESUMO
The clustered regularly interspaced short palindromic repeat (CRISPR) gene editing technique, based on the non-homologous end-joining (NHEJ) repair pathway, has been used to generate gene knock-outs with variable sizes of small insertion/deletions with high efficiency. More precise genome editing, either the insertion or deletion of a desired fragment, can be done by combining the homology-directed-repair (HDR) pathway with CRISPR cleavage. However, HDR-mediated gene knock-in experiments are typically inefficient, and there have been no reports of successful gene knock-in with DNA fragments larger than 4 kb. Here, we describe the targeted insertion of large DNA fragments (7.4 and 5.8 kb) into the genomes of mouse embryonic stem (ES) cells and zygotes, respectively, using the CRISPR/HDR technique without NHEJ inhibitors. Our data show that CRISPR/HDR without NHEJ inhibitors can result in highly efficient gene knock-in, equivalent to CRISPR/HDR with NHEJ inhibitors. Although NHEJ is the dominant repair pathway associated with CRISPR-mediated double-strand breaks (DSBs), and biallelic gene knock-ins are common, NHEJ and biallelic gene knock-ins were not detected. Our results demonstrate that efficient targeted insertion of large DNA fragments without NHEJ inhibitors is possible, a result that should stimulate interest in understanding the mechanisms of high efficiency CRISPR targeting in general.
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Sistemas CRISPR-Cas/genética , Reparo do DNA por Junção de Extremidades/genética , DNA/genética , Reparo de DNA por Recombinação/genética , Animais , Técnicas de Introdução de Genes , Engenharia Genética , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Edição de RNA/genética , Zigoto/metabolismoRESUMO
PURPOSE: This analysis was conducted to evaluate cardiovascular toxicity of commonly used anti-VEGF therapeutic agent, bevacizumab, in treating patients with cancer. METHODS: Clinical studies evaluating the efficacy and safety of bevacizumab-based regimens on response and safety for patients with cancer were identified using a predefined search strategy, allowing cardiovascular toxicity and other side effects of treatment to be estimated. RESULTS: In bevacizumab based regimens, 4 clinical studies including 282 patients with advanced cancer (including gliomas, cervical, breast and ovarian cancer) were considered eligible for inclusion. These bevacizumab-based regimens included docetaxel, irinitecan and carboplatin. Systematic analysis suggested that, of 282 patients treated by bevacizumab based regimens, hypertension and thrombo-embolism occurred in 2.5% (7/282), while only 3 patients reported cardiovascular events (1.1%). No treatment related death occurred in bevacizumab based treatment. CONCLUSION: This systemic analysis suggests that bevacizumab based regimens are associated with reasonable and accepted cardiovascular toxicity when treating patients with gliomas, cervical, breast and ovarian cancer.
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Inibidores da Angiogênese/efeitos adversos , Anticorpos Monoclonais Humanizados/efeitos adversos , Doenças Cardiovasculares/induzido quimicamente , Recidiva Local de Neoplasia/tratamento farmacológico , Neoplasias/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Bevacizumab , Seguimentos , Humanos , Metanálise como Assunto , Recidiva Local de Neoplasia/patologia , Estadiamento de Neoplasias , Neoplasias/patologia , PrognósticoRESUMO
One obstacle to developing an effective therapeutic strategy to treat or prevent asthma is that the fundamental causes of asthma are not totally understood. Asthma is thought to be a chronic TH2 immune-mediated inflammatory disease. Epigenetic changes are recognized to play a role in the initiation and maintenance of a TH2 response. MicroRNAs (miRNAs) are key epigenetic regulators of gene expression, and their expression is highly regulated, therefore, deregulation of miRNAs may play an important role in the pathogenesis of asthma. Profiling circulating miRNA might provide the highest specificity and sensitivity to diagnose asthma; similarly, correcting potential defects in the miRNA regulation network may lead to new therapeutic modalities to treat this disease.
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Objective@#To investigate the effects of backpackloads on forward cervical spine posture of school-age children and to provide a reference for healthy body shape.@*Methods@#Forty children aged 4-6, 7-9, 10-12 and 13-15 years were selected and randomly assigned into four groups receiving different backpack loads. Vicon infrared was used the high-speed motion capture system collects spatial coordinate data.@*Results@#The results showed that the mean value and variance of shoulder balance, hip balance and cervical spine forward angle were all higher than that of no-load state, there are significant differences among different backpack loads(P<0.05). In addition, with the increase of age, the impact of backpack on the shoulder balance of children in walking state decreases gradually,Kindergarten[(0.15±0.35)rad] was higher than the young age of primary school[(0.07±0.01)rad], the advanced age of primary school[(0.03±0.00)rad] and junior middle school[(0.01±0.00)rad], while the hip balance ability does not show a decreasing trend with the increase of age, junior middle school[(0.10±0.11)rad] was higher than the young age of primary schooll[(0.10±0.06)rad],Kindergarten[(0.10±0.01)rad] and the advanced age of primary school[(0.10±0.00)rad]. The impoct on neck posture depended on the young age of primary schooll and the advanced age of primary school, Kindergarten[(0.32±0.26)rad] was higher than junior middle school [(0.24±0.12)rad], showing a decreasing trend, but the affected degree of neck posture is obviously higher than the other two.@*Conclusion@#The primary goal of children’s backpack carrying health is to protect cervical vertebra by changing the design of children’s backpack.