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Introduction: As effective growth-promoters and immune-modulators, yeast fermented products have shown positive effects in ruminants. To explore the mechanisms of yeast culture promoting growth and regulating immunity, this study investigated the effects of yeast culture, and ß-glucan as one of its main active ingredients, on the growth performance, immune function, antioxidant capacity and hormonal profile in Mongolian ram lambs. Methods: One hundred and five Mongolian ram lambs were randomly assigned to 3 groups, with 35 replicates in each group. The dietary treatments were: total mixed ration (TMR) as the control group, TMR supplemented with 50-70 g/kg yeast culture (YC) or 75 mg/kg ß-glucan. The test period was 137 days. All the sheep were weighed and 6 serum samples were collected in each group on days 0, 30, 60, 90 and 130, respectively. Results: The results showed that both YC and ß-glucan could promote the growth performance with increased average daily gain and decreased feed to weight gain ratio. Moreover, these two feed additives facilitated the immune function by selectively increasing the serum levels of lysozyme, IgG, IgM, INF-γ, TNF-α and some interleukins (IL-1ß, IL-2, IL-6 and IL-8); ameliorated the antioxidant capacity with higher total antioxidant capacity and enzyme activities of catalase and glutathione peroxidase; altered the metabolism-associated hormone levels with higher growth hormone and thyroid hormone T3 but lower cortisol and insulin. Discussion: In conclusion, both YC and ß-glucan could improve the growth performance, immune function and antioxidant capacity, and regulate the serum levels of metabolism-associated hormones, thus exerting effects of promoting growth and improving immune function. Therefore, YC could be considered as a suitable potential alternative strategy to antibiotics and be used as an animal feed additive. This article provides a theoretical basis for the clinical application of such yeast fermented preparations in mutton sheep husbandry.
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Dwarfing rootstocks enhance planting density, lower tree height, and reduce both labor in peach production. Cerasus humilis is distinguished by its dwarf stature, rapid growth, and robust fruiting capabilities, presenting substantial potential for further development. In this study, Ruipan 4 was used as the scion and grafted onto Amygdalus persica and Cerasus humilis, respectively. The results indicate that compared to grafting combination R/M (Ruipan 4/Amygdalus persica), grafting combination R/O (Ruipan 4/Cerasus humilis) plants show a significant reduction in height and a significant increase in flower buds. RNA-seq indicates that genes related to gibberellin (GA) and auxin metabolism are involved in the dwarfing process of scions mediated by C. humilis. The expression levels of the GA metabolism-related gene PpGA2ox7 significantly increased in R/O and are strongly correlated with plant height, branch length, and internode length. Furthermore, GA levels were significantly reduced in R/O. The transcription factor PpGATA21 was identified through yeast one-hybrid screening of the PpGA2ox7 promoter. Yeast one-hybrid (Y1H) and dual-luciferase reporter (DLR) demonstrate that PpGATA21 can bind to the promoter of PpGA2ox7 and activate its expression. Overall, PpGATA21 activates the expression of the GA-related gene PpGA2ox7, resulting in reduced GA levels and consequent dwarfing of plants mediated by C. humilis. This study provides new insights into the mechanisms of C. humilis and offers a scientific foundation for the dwarfing and high-density cultivation of peach trees.
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Regulação da Expressão Gênica de Plantas , Giberelinas , Proteínas de Plantas , Prunus persica , Prunus persica/genética , Prunus persica/crescimento & desenvolvimento , Prunus persica/metabolismo , Giberelinas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Regiões Promotoras Genéticas , Árvores/genética , Árvores/crescimento & desenvolvimento , Ácidos Indolacéticos/metabolismoRESUMO
Microorganisms inhabit the gastrointestinal tract of ruminants and regulate body metabolism by maintaining intestinal health. The state of gastrointestinal health is influenced not only by the macro-level factors of optimal development and the physiological structure integrity but also by the delicate equilibrium between the intestinal flora and immune status at the micro-level. Abrupt weaning in young ruminants causes incomplete development of the intestinal tract resulting in an unstable and unformed microbiota. Abrupt weaning also induced damages to the microecological homeostasis of the intestinal tract, resulting in the intestinal infections and diseases, such as diarrhea. Recently, nutritional and functional yeast culture has been researched to tackle these problems. Herein, we summarized current known interactions between intestinal microorganisms and the body of young ruminants, then we discussed the regulatory effects of using yeast culture as a feed supplement. Yeast culture is a microecological preparation that contains yeast, enriched with yeast metabolites and other nutrient-active components, including ß-glucan, mannan, digestive enzymes, amino acids, minerals, vitamins, and some other unknown growth factors. It stimulates the proliferation of intestinal mucosal epithelial cells and the reproduction of intestinal microorganisms by providing special nutrient substrates to support the intestinal function. Additionally, the ß-glucan and mannan effectively stimulate intestinal mucosal immunity, promote immune response, activate macrophages, and increase acid phosphatase levels, thereby improving the body's resistance to several disease. The incorporation of yeast culture into young ruminants' diet significantly alleviated the damage caused by weaning stress to the gastrointestinal tract which also acts an effective strategy to promote the balance of intestinal flora, development of intestinal tissue, and establishment of mucosal immune system. Our review provides a theoretical basis for the application of yeast culture in the diet of young ruminants.
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Scheduling is significant in improving the production efficiency and reducing delivery delays for manufacturing enterprises. Unlike the flexible job-shop scheduling problem, two special constraints are encountered in real-world power supply manufacturing systems: 1) periodic maintenance and 2) mandatory outsourcing. As the characteristics of these constraints are not considered in existing scheduling algorithms, schedules generated by most existing approaches are not optimal or even conflict with these constraints. In this article, a self-organizing neural scheduler (SoNS) is proposed to overcome this limitation. A long short-term memory encoder is developed to transform the variable-length structural information into fixed-length feature vectors. Moreover, the reinforcement learning model is proposed to automatically select policies for improving candidate schedules. To validate the effectiveness of the proposed algorithm, extensive experiments are conducted on over 300 problem instances. The nonparametric Kruskal-Wallis tests confirm that the proposed algorithm outperforms several state-of-the-art methods in terms of effectiveness and robustness within a limited computational budget. It demonstrates that the proposed SoNS can solve scheduling problems with the periodic maintenance and mandatory outsourcing constraints effectively.
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The seasonal variations of biofilm communities in a municipal wastewater treatment plant were investigated using multi-omics techniques. The abundance of the main phyla of microorganisms varied with summer (July 2019) and winter (January 2019) samples considerably, the Bacteroidetes enriched in winter and Chloroflexi in summer. The results of metaproteomic and metagenomic showed that most of the functional microorganisms belonged to the Betaproteobacteria class, and the enrichment of Flavobacteria class in winter guaranteed the stability of denitrification performance to some extent. Seasonal variations affected the proteomic expression profiling, a total of 2835 differentially expressed proteins identified were significantly enriched in quorum sensing, two-component system, ribosome, benzoate degradation, butanoate metabolism, tricarboxylic acid cycle (TCA cycle), and cysteine and methionine metabolism pathways. With the expression of nitrogen metabolic proteins decreases in winter, the overall expression of denitrification-related enzymes in winter was much lower than that in summer, the nitrogen metabolism pathway varied significantly. Seasonal variations also induced the alteration of the biofilm metabolite profile; a total of 66 differential metabolites, 8 potential biomarkers, and 8 perturbed metabolic pathways such as TCA cycle were detected. It was found that most of the perturbed pathways are directly related to nitrogen metabolism, and several amino acids and organic acids associated with the TCA cycle were significantly perturbed, the accumulation of TCA cycle intermediates, ornithine, and L-histidine in winter might be conducive to resisting cold temperatures. Furthermore, the correlation between biofilm microbial communities and metabolites was identified by the combined analysis of metabolomic and metaproteomic. The differences of microbial community structure, function, and metabolism between winter and summer in a full-scale pre-denitrification biofilter were revealed for the first time, strengthening our understanding of the microbial ecology of biofilm communities.
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Desnitrificação , Microbiota , Reatores Biológicos/microbiologia , Estações do Ano , Multiômica , Proteômica , Biofilmes , NitrogênioRESUMO
To improve localization and pose precision of visual-inertial simultaneous localization and mapping (viSLAM) in complex scenarios, it is necessary to tune the weights of the visual and inertial inputs during sensor fusion. To this end, we propose a resilient viSLAM algorithm based on covariance tuning. During back-end optimization of the viSLAM process, the unit-weight root-mean-square error (RMSE) of the visual reprojection and IMU preintegration in each optimization is computed to construct a covariance tuning function, producing a new covariance matrix. This is used to perform another round of nonlinear optimization, effectively improving pose and localization precision without closed-loop detection. In the validation experiment, our algorithm outperformed the OKVIS, R-VIO, and VINS-Mono open-source viSLAM frameworks in pose and localization precision on the EuRoc dataset, at all difficulty levels.
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AlgoritmosRESUMO
The protective arm of the renin-angiotensin system (RAS), the ACE 2/Ang-(1-7)/MasR axis, has become a new anti-inflammatory target. As a specific activator of ACE2, diminazene aceturate (DA) can promote anti-inflammatory effects by regulating the ACE2/Ang-(1-7)/MasR axis. However, due to the reported toxicity of DA, its application has been limited. In the current study, we synthesized a low toxicity DA derivative 3 (DAD3) and sought to determine whether DAD3 can also activate ACE2 in bovine mammary epithelial cells (BMEC) and regulate the RAS system to inhibit inflammation. We found that both DA and DAD3 can activate and promote ACE2 expression in BMEC. iRNA-mediated knockdown of ACE2 demonstrated that DAD3 activates the ACE2/Ang-(1-7)/MasR axis and plays an anti-inflammatory role in BMEC. Furthermore, the inhibitory effects of DA and DAD3 on the protein phosphorylation of MAPK and NF-κB pathways were reduced in ACE2-silenced BMEC. Our findings show that ACE2 is a target of DAD3, which leads to inhibition of the MAPK and NF-κB signalling pathways and protects against LPS-induced inflammation in BMEC. Thus, DAD3 may provide a new strategy to treat dairy cow mastitis.
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Células Epiteliais , NF-kappa B , Bovinos , Animais , Feminino , Anti-Inflamatórios/farmacologiaRESUMO
A series of novel artemisinin-piperazine-phosphoramide mustard (PPM) hybrids were designed and synthesized by incorporating phosphoramide mustard (PM) into dihydroartemisinin (DHA) via an efficient, catalyst-free two-step sequential substitution. Artemisinin-PPM hybrids showed better cytotoxic potency against HepG2 cells than both the parent DHA and the reference, vincristine (VCR). Structure-activity relationship (SAR) studies showed that the cytotoxicity was significantly enhanced by the introduction of a thiazole moiety. Hybrid 7 h, the most potent compound with the highest selectivity index IC50 (HEK-293T)/IC50 (HepG2)=16, displayed 7.4-fold stronger potency than VCR against HepG2 cells. In addition, hybrid 7 h was substantially more cytotoxic on all human cancer cells tested than on the corresponding non-cancerous cells. Flow cytometric analysis showed that 7 h significantly blocked the cell cycle in the G0/G1 phase and induced apoptosis in a concentration-dependent manner.
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Antineoplásicos , Artemisininas , Antineoplásicos/farmacologia , Apoptose , Artemisininas/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Mostardas de Fosforamida/farmacologia , Piperazina/farmacologia , Relação Estrutura-AtividadeRESUMO
The phagocytosis and destruction of pathogens in lysosomes constitute central elements of innate immune defense. Here, we show that Brucella, the causative agent of brucellosis, the most prevalent bacterial zoonosis globally, subverts this immune defense pathway by activating regulated IRE1α-dependent decay (RIDD) of Bloc1s1 mRNA encoding BLOS1, a protein that promotes endosome-lysosome fusion. RIDD-deficient cells and mice harboring a RIDD-incompetent variant of IRE1α were resistant to infection. Inactivation of the Bloc1s1 gene impaired the ability to assemble BLOC-1-related complex (BORC), resulting in differential recruitment of BORC-related lysosome trafficking components, perinuclear trafficking of Brucella-containing vacuoles (BCVs), and enhanced susceptibility to infection. The RIDD-resistant Bloc1s1 variant maintains the integrity of BORC and a higher-level association of BORC-related components that promote centrifugal lysosome trafficking, resulting in enhanced BCV peripheral trafficking and lysosomal destruction, and resistance to infection. These findings demonstrate that host RIDD activity on BLOS1 regulates Brucella intracellular parasitism by disrupting BORC-directed lysosomal trafficking. Notably, coronavirus murine hepatitis virus also subverted the RIDD-BLOS1 axis to promote intracellular replication. Our work establishes BLOS1 as a novel immune defense factor whose activity is hijacked by diverse pathogens.
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Brucella , Brucelose , Animais , Brucelose/metabolismo , Brucelose/microbiologia , Endorribonucleases/metabolismo , Endossomos/metabolismo , Camundongos , Proteínas Serina-Treonina QuinasesRESUMO
Two-echelon vehicle routing problem (2E-VRP) is an NP-hard combinatorial optimization problem and a basic mathematical model of modern city logistics. While it is difficult to obtain the optimal solution of 2E-VRP, this study finds a breakthrough that the structure of the optimal route planning for 2E-VRP is usually an embedded Hamiltonian graph. In the graph, routes can be drawn in a planar graph as Hamiltonian circuits without intersections. Based on this finding, an embedded Hamiltonian graph-guided heuristic algorithm is proposed to solve 2E-VRP. As a crucial part of the algorithm, an initialization scheme is designed to search for the farthest vertices from each route and insert the rest of the vertices. In the satellite-adjustment process, a dynamic adjustment for satellites scheme is proposed to adjust the state of satellites. The two schemes aim to construct Hamiltonian circuits with few intersections. Experiments have been conducted on 207 instances to demonstrate the effect of the proposed algorithm on solving 2E-VRP. Experimental results show that the proposed algorithm can obtain more solutions of 2E-VRP with significantly smaller objective-function values. Furthermore, the number of intersections in routes generated by the proposed algorithm is much less than those obtained by the compared algorithms. With the use of the two schemes, the embedded Hamiltonian graph-guided heuristic algorithm significantly outperforms the compared algorithms for 2E-VRP.
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Diminazene aceturate (DA) has been used in the treatment of infections of trypanosomes in animals. Interestingly, its anti-inflammatory effect has recently gained increased interests. However, DA has been reported to have toxic side effects that limit its application. Therefore, we synthesized and screened a novel low-toxic DA derivative, namely the DA derivative 3 (DAD3). In the present study, anti-inflammatory effect of DAD3 was evaluated bovine mammary epithelial cells (BMECs) in vitro model. The results demonstrated that DAD3 had less cytotoxicity, and had a stronger effect in inhibiting secretion of inflammatory factors in BMECs, compared to DA. Mechanistically, DAD3 was able to inhibit the production of pro-inflammatory factors in part by suppressing the generation of mitochondrial reactive oxygen species (ROS) in BMECs upon LPS stimulation. Molecular analysis further indicated that DAD3 was capable of resolving inflammation in BMECs through a mechanism by preventing nuclear translocation of NF-p65, subsequently inhibiting transcription of inflammatory factors. In this context, DAD3 inhibited the phosphorylation of IκB, ERK, JNK and P-38 proteins of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. These results suggested the DAD3 was a novel DA derivative with low toxicity and strong anti-inflammatory effects in BMECs exposed to LPS, through a mechanism by blocking the NF-κB and MAPK signaling pathways. This study also provides an evidence that the DAD3 may be a novel anti-inflammatory agents warranted for further investigation in treatment of mastitis in cows.
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Anti-Inflamatórios/uso terapêutico , Diminazena/análogos & derivados , Animais , Bovinos , Diminazena/uso terapêutico , Células Epiteliais/metabolismo , Feminino , Inflamação/tratamento farmacológico , Inflamação/veterinária , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Glândulas Mamárias Animais/efeitos dos fármacos , NF-kappa B/antagonistas & inibidores , FosforilaçãoRESUMO
For the first time, eight novel artemisinin-piperazine-furane ether hybrids (5a-h) were efficiently synthesized and investigated for their in vitro cytotoxic activity against some human cancer and benign cells. The absolute configuration of hybrid 5c was determined by X-ray crystallographic analysis. Hybrids 5a-h exhibited more pronounced growth-inhibiting action on hepatocarcinoma cell lines than their parent dihydroartemisinin (DHA) and the reference cytosine arabinoside (ARA). The hybrid 5a showed the best cytotoxic activity against human hepatocarcinoma cells SMMC-7721 (IC50 = 0.26 ± 0.03 µM) after 24 h. Furthermore, hybrid 5a also showed good cytotoxic activity against human breast cancer cells MCF-7 and low cytotoxicity against human breast benign cells MCF-10A in vitro. We found the cytotoxicity of hybrid 5a did not change when tumour cells absorb iron sulfate (FeSO4); thus, we conclude the anti-tumour mechanism induced by iron ions (Fe2+) is unclear.
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Antineoplásicos/farmacologia , Artemisininas/farmacologia , Furanos/farmacologia , Piperazinas/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Artemisininas/síntese química , Artemisininas/toxicidade , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Furanos/síntese química , Furanos/toxicidade , Humanos , Células MCF-7 , Piperazinas/síntese química , Piperazinas/toxicidadeRESUMO
For the first time, six novel artemisone-piperazine-tetronamide hybrids (12a-f) were efficiently synthesised from dihydroartemisinin (DHA) and investigated for their in vitro cytotoxicity against some human cancer cells and benign cells. All the targets showed good cytotoxic activity in vitro. Hybrid 12a exhibited much better inhibitory activity against human liver cancer cell line SMMC-7721 (IC50 = 0.03 ± 0.04 µM for 24 h) than the parent DHA (IC50 > 0.7 µM), and two references, vincristine (VCR; IC50 = 0.27 ± 0.03 µM) & cytosine arabinoside (ARA; IC50 = 0.63 ± 0.04 µM). Furthermore, hybrid 12a had low toxicity against human benign liver cell line LO2 (IC50 = 0.70 ± 0.02 µM for 24 h) compared with VCR, ARA, and DHA in vitro. Moreover, the inhibitory activity of hybrid 12a was obviously enhanced when human liver cancer cell line MHCC97H absorbed Fe2+ in vitro.
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Sensors capable of detecting different types of biomolecules have widespread applications in the field of biomedical research, but despite many years of research, the development of biosensors suitable for point-of-care (POC) applications in resource-limited areas is still extremely challenging. Sensors based on photonic crystal hydrogels (PCHs) hold much promise in this regard because of their numerous advantages over other existing bioanalytical methods. All current PCH biosensors are however restricted in the types of analytes they can detect sensitively with good selectivity. By taking advantage of the powerful and ubiquitous antibody-antigen interaction, we report herein the first-ever competition-based PCH biosensors capable of naked-eye detection of various biomolecules (e.g., proteins, peptides, and small molecules) with high sensitivity and selectivity and minimal background and excellent reversibility. We showed such PCH designs could be extended to the fabrication of different enzyme-detecting biosensors. The universal feature of these novel biosensors thus enables future development of POC biosensors in disease diagnostics for other bioanalytes.
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Técnicas Biossensoriais , Fótons , Polímeros/química , Semicondutores , Energia Solar , Cristalização , Imidas/química , Luminescência , Naftalenos/química , Espalhamento de Radiação , Espectrofotometria UltravioletaRESUMO
Twelve derivatives of artemisinin-piperazine-dithiocarbamate have been synthesised, and some of them showing good in vitro cytotoxic activity. Compound 3g exhibits the best inhibitory activity against SMMC-7721â¯cell lines with an IC50 of 0.0025⯱â¯0.04⯵M for 72â¯h, but the toxicity was lower against LO2 cell lines with an IC50 of 0.18⯱â¯0.04⯵M for 72â¯h. The results indicate that compound 3g is more cytotoxic towards cancer cell lines than towards benign cell lines compared with vincristine in vitro. And compound 3g also has good inhibitory activity against colon, breast and prostate cancer cells. Meanwhile, we have also proposed the six-member ring mechanism of DMSO in catalysing the esterification of hydroxyl and acyl chloride. Instead of using the hydroxyl, we can obtain the nucleophilic substitution production simply and efficiently without a Lewis acid, which has not been reported previously.
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Antineoplásicos/farmacologia , Artemisininas/farmacologia , Piperazina/farmacologia , Tiocarbamatos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Artemisininas/química , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Piperazina/química , Relação Estrutura-Atividade , Tiocarbamatos/química , Células Tumorais CultivadasRESUMO
A series of new dithiocarbamates containing a 2(5H)-furanone-piperazine group was synthesized. These compounds show good in vitro cytoxic activity. Among them, compound 6c exhibits the best inhibitory activity against HeLa cell lines with an IC50 of 0.06⯱â¯0.01⯵M for 72â¯h, and it has good inhibitory activity against SMMC-7721â¯cell lines with an IC50 of 0.006⯱â¯0.04⯵M for 72â¯h, but the toxicity was lower against LO2 cell lines with an IC50 of 45.76⯱â¯0.01⯵M. The result showed that compound 6c is far more cytoxic towards cancer cell lines than towards benign cell lines compared with cytosine arabinoside (ARA) in vitro.
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Furanos/farmacologia , Piperazinas/farmacologia , Tiocarbamatos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Relação Dose-Resposta a Droga , Furanos/química , Humanos , Estrutura Molecular , Piperazinas/química , Relação Estrutura-Atividade , Tiocarbamatos/síntese química , Tiocarbamatos/químicaRESUMO
A series of 2-aminopyrimidine derivatives were designed and synthesized as highly selective FGFR4 inhibitors. One of the most promising compounds 2n tightly bound FGFR4 with a Kd value of 3.3 nM and potently inhibited its enzymatic activity with an IC50 value of 2.6 nM, but completely spared FGFR1/2/3. The compound selectively suppressed proliferation of breast cancer cells harboring dysregulated FGFR4 signaling with an IC50 value of 0.38 µM. Furthermore, 2n exhibited extraordinary target specificity in a Kinome-wide screen against 468 kinases, with S(35) and S(10) selectivity scores of 0.01 and 0.007 at 1.0 µM, respectively.
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A series of 2-oxo-3, 4-dihydropyrimido[4,5-d]-pyrimidinyl derivatives were designed and synthesized as new irreversible inhibitors of the FGFR family. One of the most promising compounds 2l potently inhibited FGFR1/2/3 with IC50 values of 1.06, 0.84 and 5.38 nM, respectively, whereas its potency against FGFR4 was diminished by an order of magnitude. Compound 2l strongly suppresses the proliferation of FGFR1-amplified H520 non-small cell lung cancer cells, FGFR2-amplified SUM52 breast cancer cells and FGFR3-amplified SW780 bladder cancer cells with low nanomolar IC50 values, but was significantly less potent against four FGFR-negative cancer cell lines, with low micromolar IC50 values. Biological investigation also confirmed the irreversible binding of the molecule with the FGFR1-3 target kinases. Compound 2l may serve as a promising new lead for further anticancer drug discovery.
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Antineoplásicos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Pirimidinas/síntese química , Pirimidinas/química , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/metabolismo , Relação Estrutura-AtividadeRESUMO
Objective: To clone the full-length cDNA of actin gene of Taenia pisiformis (Tp-actin), and analyze the gene structure, phylogenetic evolution and its use as an internal control. Methods: Tp-actin was amplified by RT-PCR and the cDNA of 3' and 5' ends were obtained through RACE-PCR. After sequencing, these segments were linked to produce full-length cDNA of Tp-actin. The gene structure and phylogenetic evolution were analyzed using bioinformatics software. Primers for Tp-actin and cysteine peptidase (TpCP) were designed using Primer Express software. Primer specificity and amplification efficiency were analyzed with real-time fluorescence quantitative PCR (qRT-PCR). In addition, by using Tp-actin as an internal control, the expression of TpCP in T. pisiformis at various developmental stages was analyzed. Results: As expected, sequencing results showed that the Tp-actin fragment was 1 048 bp in length, and the 3' and 5' ends were 428 bp and 945 bp, respectively. The full-length cDNA of Tp-actin generated from the 3 segments (submitted to GenBank with accession No. JX624787) was 1 279 bp, containing a 30-bp 5'-untranslated regionï¼5'-UTRï¼, a 118-bp 3'-UTR, and a 1 131-bp open reading frame (ORF). Bioinformatics analysis showed that the Tp-actin encoded a protein of 356 amino acids, with a predicted relative molecular weight of 41 749 and a PI value of 5.29. This protein was predicted to contain 6 functional sites and 3 typical signatures of the actin family. Phylogenetic analysis showed that the Tp-actin was 100% and 99.7% homologous in amino acid sequence to those of Taenia solium and Diphyllobothrium dendriticum. qRT-PCR resulted in specific products of 82 bp and 108 bp from Tp-actin and TpCP, respectively, melting curves of which both showed a single signal peak, verifying the high specificity of primers. The linear correlation coefficientï¼R2ï¼ in standard curve of Tp-actin was 0.999, showing high amplification efficiency. Using Tp-actin as the internal control, the relative expression ratio of TpCP gene in gravid proglottid of T. pisiformisï¼1.65ï¼ was significantly higher than that in oncospheres ï¼1.00ï¼, mature proglottids ï¼0.87ï¼ and cysticercus (0.62) (P<0.05). Conclusion: Tp-actin gene is highly conserved and can be used as a reliable internal control.
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Clonagem Molecular , Taenia , Actinas , Sequência de Aminoácidos , Animais , Biologia Computacional , DNA Complementar , FilogeniaRESUMO
OBJECTIVE: To investigate the effects of inhibiting TIM-4 function in Kupffer cells (KCs) on liver graft rejection in mice and explore the underlying mechanism. METHODS: Mouse models of orthotopic liver transplantation were treated with a control mAb group and TIM-4 mAb. The activated KCs were assayed with immunohistochemistry after operation. The expression of TIM-4 in KCs were assayed with Western blotting and RT-PCR and the levels of AST, ALT, TBIL, TNF-α, IFN-γ and CCL2 were assayed detected. The expression of TIM-4 in KCs was observed with laser confocal microscopy. HE staining was used to observe the microstructure of the liver tissues, and the number of CD25+Foxp3+T cells was determined using with flow cytometry; the proteins levels of p-P65and p-P38 were assayed with Western blotting. The donor mice were treated with clodronate liposomes to destroy the KCs in the liver before transplantation, and the liver grafts were examined for graft rejection. RESULTS: The number of activated KCs in the liver graft increased progressively over time. Compared with the sham-operated group, the liver graft showed significantly increased TIM-4 protein and mRNA levels at 1, 3, and 7 days after transplantation (P<0.05) and increased levels of AST, ALT, TBIL, TNF-α, IFN-γ and CCL2 at 7 days (P<0.05). The graft in TIM-4 mAb group showed mild pathological changes with a mean RAI score of 2.67∓0.75, which was significantly lower than that in control mAb group (P<0.05). The mean survival time of the recipient mice was 53.8∓6.4 days in TIM-4 mAb group, significantly longer than that in the control mAB group (14.5∓2.9 days, P<0.05). Donor treatment with clodronate liposomes resulted in comparable RAI scores in TIM-4 mAb and control mAb groups (8.01∓0.64 vs 7.93∓0.56, P>0.05). The protein levels of p-P65 and p-P38 in TIM-4 mAb group were significantly lower than those in control mAb group (P<0.05), and CD25+Foxp3+T cells in the liver graft increased significantly in TIM-4 mAb group. CONCLUSION: Inhibition of TIM-4 function in KCs reduces the production of inflammatory factors after liver transplantation possibly by inhibiting the NF-κB and MAPK signaling pathways and promoting the proliferation of Foxp3+Treg cells to induce allograft tolerance.