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1.
Mol Metab ; : 101045, 2020 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-32599076

RESUMO

OBJECTIVE: Salt induced kinase 1 (SIK1) acts as a key modulator in many physiological processes. However, the effects of SIK1 on gluconeogenesis and the underlying mechanisms have not been fully elucidated. In this study, we found a natural compound phanginin A could activate SIK1 and further inhibit gluconeogenesis. The mechanisms by which phanginin A activates SIK1 and inhibits gluconeogenesis were explored in primary mouse hepatocytes, and the effects of phanginin A on glucose homeostasis were investigated in ob/ob mice. METHODS: Effects of phanginin A on gluconeogenesis and SIK1 phosphorylation were examined in primary mouse hepatocytes. Pan-SIK inhibitor and siRNA-mediated knockdown were used to elucidate the involvement of SIK1 activation in the phanginin A reduced gluconeogenesis. LKB1 knockdown was used to explore how phanginin A activated SIK1. SIK1 overexpression was performed to evaluate its effect on gluconeogenesis, PDE4 activity and cAMP pathway. The acute and chronic effects of phanginin A on metabolic abnormalities were observed in ob/ob mice. RESULTS: Phanginin A significantly increased SIK1 phosphorylation through LKB1 and further suppressed gluconeogenesis by increasing PDE4 activity and inhibiting cAMP/PKA/CREB pathway in primary mouse hepatocytes, and this effect was blocked by the pan-SIK inhibitor HG-9-91-01 or siRNA-mediated knockdown of SIK1. Overexpression of SIK1 in hepatocytes increased PDE4 activity, reduced cAMP accumulation and thereby inhibited gluconeogenesis. Acute treatment of phanginin A reduced gluconeogenesis in vivo, accompanied by increased SIK1 phosphorylation and PDE4 activity in the liver. Long-term treatment of phanginin A profoundly reduced blood glucose levels, and improved glucose tolerance and dyslipidemia in ob/ob mice. CONCLUSION: Our study discovered an unrecognized effect of phanginin A in suppressing hepatic gluconeogenesis, and revealed a novel mechanism that activation of SIK1 by phanginin A could inhibit gluconeogenesis by increasing PDE4 activity and suppressing the cAMP/PKA/CREB pathway in the liver. Moreover, we also highlighted the potential value of phanginin A as a lead compound for the treatment of type 2 diabetes.

2.
Fitoterapia ; 146: 104672, 2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-32553887

RESUMO

Six new rearranged neoclerodane diterpenoids (1-6), as well as three known ones, were obtained from the aerial part of Salvia hispanica L. Their structures were elucidated by extensive analysis of spectroscopic data (1D, 2D NMR, and HRESIMS) and Mosher's method. The absolute configurations of 1, 2, and 4 were determined by single-crystal X-ray diffraction analysis. All isolated compounds were evaluated for their cardioprotective effects against H2O2-induced cardiomyocytes injury, and compound 5 showed statistically significant cardioprotective effect in vitro assays.

3.
Oral Dis ; 2020 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-32564447

RESUMO

OBJECTIVE: To compare the biological effect of PRP and liquid-PRF on human periodontal ligament cells (hPDLCs) in vitro. METHODS: The liquid-PRF was processed with centrifugation at 700g for 3 minutes and PRP was processed according to Curasan's protocol. Migration and proliferation assay were performed by a scratch/transwell assay and a CCK-8 assay respectively. To investigate hPDLCs differentiation, alkaline phosphatase (ALP) assay, alizarin red S staining and gene expression level detection of Runx2, Col1a1, and OCN were conducted. Furthermore, cells cultured with lipopolysaccharide (LPS) to induce an inflammation condition were utilized to investigate the impact of liquid-PRF on inflammatory resolution. RESULTS: Either PRP or liquid-PRF can promote proliferation, migration of hPDLCs, and osteogenic differentiation of hPDLCs. It was noteworthy that liquid-PRF demonstrated a significantly higher ability to promote the biological differentiation and mineralization of hPDLCs compared with PRP. Lastly, when hPDLCs were incubated with LPS, cells cultured with liquid-PRF showed significantly lower mRNA expression levels of inflammatory genes. CONCLUSIONS: Liquid-PRF notably promoted hPDLCs activity and attenuated the inflammatory state induced by LPS.

4.
Acta Trop ; 210: 105580, 2020 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-32533936

RESUMO

Oncomelania hupensis is the intermediate host of Schistosoma japonicum, one of the Schistosoma species that can cause human schistosomiasis. Molluscicidal treatment remains the primary means to control snail. Niclosamide is the only molluscicide recommended by the World Health Organization, and it has been used throughout schistosomiasis-endemic areas in China for almost 30 years. In our previous studies on transcriptomics, morphology, and enzymology of snails after molluscicidal treatment, two effective molluscicides were used, 50% wettable powder of niclosamide ethanolamine salt (WPN) and a new molluscicide derived from niclosamide, the salt of quinoid-2', 5-dichloro-4'-nitro-salicylanilide (LDS, simplified for Liu Dai Shui Yang An). Genes involved in cell structure mintenance, inhibition of neurohumoral transmission, and energy metabolism showed significant differential expression after molluscicide treatments. Damages in the structure of liver and muscle cells were accompanied by inhibited activities of enzymes related to carbohydrate metabolism and energy supply. This study was designed to clarify the dynamic metabolic process by metabonomics, together with the previous transcriptomic and enzymological profiles, to identify potential metabolite markers and metabolism pathways that related to the toxic mechanism of the molluscicide. In total, 56 metabolites were identified for O. hupensis, and 75% of these metabolites consisted of amino acids and derivatives, organic acids, and nucleic acid components. The concentration of glucose, maltose, succinate, choline, and alanine changed significantly after molluscicide treatments. These changes in metabolites mainly occurred in the process of carbohydrate metabolism, energy metabolism, and amino acid metabolism, primarily related to glycolysis/gluconeogenesis, oxidative phosphorylation, and transamination by KEGG pathway identification. Most of the identified pathways were also related to those differentially expressed unigenes and observed enzymes from our previous studies. Inhibited aerobic respiration and oxidative phosphorylation, and energy deficiency were implied further to be the leading causes of the final death of snails after molluscicide treatments. The hypothesised mathematical model in this study identified the rational hysteresis to explain the inconsistency of responses of unigenes, enzymes, and metabolites to molluscicide treatments. This study contributes to the comprehensive understanding of the molluscicidal mechanism in the metabolic process and this could assist in improving existing molluscicide formulations or development of new molluscicides.

6.
Inorg Chem ; 2020 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-32453561

RESUMO

"Coordination-driven self-assembly" offers us an efficient method to fabricate 3D metallacages and 2D metallacycles with controllable shape and size via simple building blocks. Herein, a new discrete platinum(II) amphiphile (AOM), which contains hydrophilic tris(ethylene oxide) chains and a hydrophobic porphyrin unit, was constructed successfully by using "coordination-driven self-assembly". From various characterization methods, such as UV-vis spectroscopy, dynamic light scattering, transmission electron microscopy, scanning electron microscopy, and small-angle X-ray scattering, we found that AOM can self-assemble into vesicles, curved vesicles with open ends, and at last stable bilayer nanotubes in aqueous solution at room temperature and flexible cross-linked structures at about 60 °C. In contrast, AOM formed rigid bilayer nanosheets of micrometers in width and millimeters in length in n-hexane. We hope this investigation will pave the way for the fabrication of controllable soft materials.

7.
Exp Lung Res ; : 1-9, 2020 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-32372722

RESUMO

Purpose: Impaired mucociliary clearance is an initial characteristic of recurrent cough, respiratory infection and chronic respiratory diseases. It has been demonstrated that prolonged inhalation of respirable silica particles results in a variety of pulmonary diseases, but whether the mucociliary system is involved in this process is unclear. This study aims to evaluate the effects of silica particles on mucociliary structure and MUC5B production in respiratory tract.Materials and Methods: C57BL/6 mice were administered with 2.5 mg silica particles through a single intratracheal instillation. The changes of mucociliary structure and MUC5B expression in trachea was evaluated by HE and AB-PAS staining, transmission electron microscopy and immunohistochemistry on days 1, 7, 28 and 84 post-exposure.Results: The mucociliary structure of airway epithelium was obviously impaired by silica particles, showing disordered, shortened or partially lost cilia on the surface, increased mucus in mucous layer and submucosal glands from day 7 to day 84. A variety of ultrastructural abnormalities were discovered in silica-exposed airway cilia, including absence of central pair microtubules, disorganized microtubules and clusters of axoneme on day 1 and 7. The numbers of ciliary axonemes and basal bodies in ciliated epithelial cells were significantly decreased, whereas the proportion of abnormal axonemes was gradually increased with exposure to silica particles (P < 0.05). In addition, silica particles significantly decreased MUC5B expression on the surface of airway epithelium on day 28 and 84, but obviously increased its production in submucosal glands from day 1 to day 84 (P < 0.01).Conclusions: Silica particles could lead to ultrastructural defects in airway cilia, mucus hypersecretion and altered MUC5B expression in trachea, indicating that impaired mucociliary structure and altered MUC5B production might participate in the development of silica-related respiratory diseases.

8.
Vet Microbiol ; 245: 108688, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32456826

RESUMO

Hepatitis E virus (HEV) is a public health concern because of its zoonotic potential; however, the host species spectrum and the genetic diversity of HEV in many birds are unknown. In the present study, a novel genotype avian HEV was isolated from a bird, silkie fowl, and designated CHN-GS-aHEV (GenBank No. MN562265). The genome of CHN-GS-aHEV was analyzed in comparison with other avian HEVs' and the pathogenicity in silkie fowl was characterized. The results show that the CHN-GS-aHEV shares about 81 % identity with known avian HEV in chickens, ORF3 shares the highest identity (85.1 %-88.0 %) at the nucleotide level, while ORF2 shares the highest identity (96.5 %-98.0 %) at the amino acid level, indicating that the CHN-GS-aHEV belongs to a new genotype avian HEV. The pathogenicity study showed that silkie fowl experimentally infected with the CHN-GS-aHEV demonstrated seroconversion, viremia, fecal virus shedding, liver lesions, and increased ALT level. Furthermore, ultrastructural changes in hepatocyte cells by transmission electron microscopy were characterized by the loss of mitochondrial cristae and swollen mitochondria and endoplasmic reticulum in the infected birds, suggesting that these two organelles may play a significant role in HEV replication. Overall, this study reports the complete genome characterization of a novel avian HEV and successful experimental infection in silkie fowl, and may be serving as a prominent indicator for additional avian HEV detection in other species.

9.
Phytopathology ; : PHYTO11190410R, 2020 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-32301678

RESUMO

Osmotin and osmotin-like proteins (OLPs) play important roles in plant defense responses. The full-length cDNA sequence of an OLP gene was cloned from Panax notoginseng using rapid amplification of cDNA-end technology and named PnOLP1. A quantitative reverse transcription-PCR analysis showed that the signaling molecules methyl jasmonate, salicylic acid, ethylene, and hydrogen peroxide induced PnOLP1 expression to different degrees. In addition, the expression level of PnOLP1 rapidly increased within 48 h of inoculating P. notoginseng with the root rot pathogen Fusarium solani. Subcellular localization revealed that PnOLP1 localized to the cell wall. A prokaryotic expression vector containing PnOLP1 was constructed and transformed into Escherichia coli BL21 (DE3), and in vitro antifungal assays were performed using the purified recombinant PnOLP1 protein. The recombinant PnOLP1 protein had strong inhibitory effects on the mycelial growth of F. oxysporum, F. graminearum, and F. solani. A plant PnOLP1-overexpression vector was constructed and transfected into tobacco, and the resistance of T2 transgenic tobacco against F. solani was significantly enhanced compared with wild-type tobacco. Moreover, a PnOLP1 RNAi vector was constructed and transferred to the P. notoginseng leaves for transient expression, and the decrease of PnOLP1 expression level in P. notoginseng leaves increased the susceptibility to F. solani. Thus, PnOLP1 is an important disease resistance gene involved in the defense responses of P. notoginseng to F. solani.

10.
JAMA Cardiol ; 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32236489

RESUMO

Importance: Idiopathic pulmonary arterial hypertension (IPAH) is a fatal disease with high heritability; however, the bone morphogenetic protein receptor 2 (BMPR2) gene only accounts for 17% of IPAH. The genetic basis of IPAH needs further investigation. Objective: To identify novel IPAH susceptibility genes other than BMPR2. Design, Setting, and Participants: This 2-stage, case-control genetic association study enrolled 230 patients with IPAH from 2 referral pulmonary hypertension centers in China. Eligible patients had no BMPR2 variants and were compared with 968 healthy control participants. Data were collected from January 1, 2000, to July 31, 2015, and analyzed from August 1, 2015, to May 30, 2018. Exposures: PTGIS rare variants. Main Outcomes and Measures: Whole-genome sequencing was performed to identify putative IPAH genes in a discovery cohort, with validation in an independent referral cohort. Correlation of genotype and hemodynamic characteristics was then evaluated at baseline and after pulmonary vasodilator testing. Functional assessments were conducted to analyze the effects of identified genetic variants on transcript splicing, enzymatic activity, and endothelial cell phenotypes. Results: Among 230 patients with IPAH (164 female [71.3%]; mean [SD] age, 34 [18] years), an enrichment of rare variants in a gene encoding prostacyclin synthase (PTGIS) was identified in the discovery cohort. The association of PTGIS rare variants with IPAH was confirmed in the replication cohort. In the combined data set, PTGIS rare variants were found in 14 of 230 cases (6.1%) and 8 of 968 controls (0.8%) (odds ratio, 7.8; 95% CI, 3.2-18.8; P = 5 × 10-6, logistic regression). Compared with patients without PTGIS variants, inhaled iloprost induced a more significant decrease of pulmonary vascular resistance (difference in the least square mean, -21.7%; 95% CI, -31.4% to -12.0%; P < .001, linear regression model) and an increase of cardiac index (difference in the least square mean, 18.3%; 95% CI, 8.8%-27.8%; P < .001, linear regression model) in patients with PTGIS variants. The minigene assay indicated that the c.521 + 1G>A variant resulted in aberrant messenger RNA transcripts. The functional studies showed that the 2 missense rare variants (R252Q and A447T) resulted in a decrease in prostacyclin production and increased cell death of pulmonary microvascular endothelial cells. Conclusions and Relevance: This study identified 3 rare loss-of-function variants in the PTGIS gene from 2 independent cohorts with IPAH. The genetic variants of PTGIS predispose pulmonary vascular responses to the iloprost stimulation. These findings suggest that PTGIS variants may be involved in the pathogenesis of IPAH.

11.
Eur Radiol ; 2020 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-32318844

RESUMO

Radiotherapy (RT) is an effective method for treating head and neck cancer (HNC). However, RT may cause side effects during and after treatment. Radiation-induced brainstem injury (BSI) is often neglected due to its low incidence and short survival time and because it is indistinguishable from intracranial tumor progression. It is currently believed that the possible mechanism of radiation-induced BSI includes increased expression of vascular endothelial growth factor and damage of vascular endothelial cells, neurons, and glial cells as well as an inflammatory response and oxidative stress. At present, it is still difficult to avoid BSI even with several advanced RT techniques. Intensity-modulated radiotherapy (IMRT) is the most commonly used therapeutic technique in the field of RT. Compared with early conformal therapy, it has greatly reduced the injury to normal tissues. Proton beam radiotherapy (PBT) and heavy ion radiotherapy (HIT) have good dose distribution due to the presence of a Bragg peak, which not only results in better control of the tumor but also minimizes the dose to the surrounding normal tissues. There are many clinical studies on BSI caused by IMRT, PBT, and HIT. In this paper, we review the mechanism, dosimetry, and other aspects of BSI caused by IMRT, PBT, and HIT. Key Points • Enhanced MRI imaging can better detect radiation-induced BSI early. • This article summarized the dose constraints of brainstem toxicity in clinical studies using different techniques including IMRT, PBT, and HIT and recommended better dose constraints pattern to clinicians. • The latest pathological mechanism of radiation-induced BSI and the corresponding advanced treatment methods will be discussed.

12.
Int J Mol Sci ; 21(7)2020 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-32276369

RESUMO

Sex determination and differentiation are nearly universal to all eukaryotic organisms, encompassing diverse systems and mechanisms. Here, we identified a spliceosomal protein gene BmSPX involved in sex determination of the lepidopeteran insect, Bombyx mori. In a transgenic silkworm line that overexpressed the BmSPX gene, transgenic silkworm males exhibited differences in their external genitalia compared to wild-type males, but normal internal genitalia. Additionally, transgenic silkworm females exhibited a developmental disorder of the reproductive organs. Upregulation of BmSPX significantly increased the expression levels of sex-determining genes (BmMasc and BmIMP) and reduced the female-type splice isoform of Bmdsx, which is a key switch gene downstream of the sex-determination pathway. Additionally, co-immunoprecipitation assays confirmed an interaction between the BmSPX protein and BmPSI, an upstream regulatory factor of Bmdsx. Quantitative real-time PCR showed that BmSPX over-expression upregulated the expression of the Hox gene abdominal-B (Adb-B), which is required for specification of the posterior abdomen, external genitalia, and gonads of insects, as well as the genes in the Receptor Tyrosine Kinase (RTK) signaling pathway. In conclusion, our study suggested the involvement of BmSPX, identified as a novel regulatory factor, in the sex-determination pathway and regulation of reproductive organ development in silkworms.


Assuntos
Bombyx/fisiologia , Genitália/metabolismo , Proteínas de Insetos/metabolismo , Processos de Determinação Sexual , Animais , Animais Geneticamente Modificados , Bombyx/genética , Regulação da Expressão Gênica , Gônadas/metabolismo , Proteínas de Insetos/fisiologia , Masculino , Processamento de RNA , Spliceossomos
13.
J Org Chem ; 85(10): 6803-6807, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32295348

RESUMO

Huperserratines A (1) and B (2), two Lycopodium alkaloids with an unprecedented 5-aza-bicyclo[10.4.0]hexadecane skeleton and an oxime function, were isolated from Huperzia serrata. Their structures including absolute configurations were determined by extensive NMR spectroscopic and X-ray diffraction analysis. Compounds 1 and 2 were the first examples of macrocyclic Lycopodium alkaloids with an aza-12-membered ring. A plausible biogenetic pathway of these compounds was also proposed. Compound 1 exhibited moderate anti-HIV-1 activity with an EC50 of 52.91 µg/mL and a therapy index greater than 3.78.

14.
Poult Sci ; 99(3): 1287-1296, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32115022

RESUMO

Hens of a commercial Hy-line brown layer flock in China exhibited increased mortality and decreased egg production at 47 wk of age. From 47 to 57 wk, average weekly mortality increased from 0.11 to 3.0%, and egg production decreased from 10 to 30%, with a peak mortality rate (3.0%) observed at 54 wk of age. Necropsy of 11 birds demonstrated tissue damage that included hepatitis, liver hemorrhage, rupture, and/or enlarged livers. Microscopic liver lesions exhibited hepatocytic necrosis, lymphocytic periphlebitis, and myeloid leukosis. While no bacteria were recovered from liver and spleen samples, avian hepatitis E virus (HEV) RNA was detected in all 11 tested hens by nested reverse transcription-polymerase chain reaction. Of these, subgroup J avian leukosis virus (ALV-J) proviral DNA was detected in 5 hens by PCR. Alignments of partial ORF2 gene sequences obtained here demonstrated shared identity (76 to 97%) with corresponding sequences of other known avian HEV isolates. Env sequences of ALV-J isolates obtained here shared 50.1 to 55% identity with other ALV subgroups and 91.8 to 95.5% identity with other known ALV-J isolates. Phylogenetic tree analysis of selected sequences obtained here grouped an avian HEV sequence with genotype 3 HEV and assigned an ALV-J sequence to a branch separate from known ALV-J subgroups. Immunohistochemical results confirmed the presence of avian HEV and ALV-J in livers. Therefore, these results suggest that avian HEV and ALV-J co-infection caused the outbreak of hepatitis and liver hemorrhagic syndrome observed in the layer hen flock analyzed in this study.

16.
Viruses ; 12(3)2020 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-32192159

RESUMO

The family Hepeviridae includes several positive-stranded RNA viruses, which infect a wide range of mammalian species, chicken, and trout. However, few hepatitis E viruses (HEVs) have been characterized from invertebrates. In this study, a hepevirus, tentatively named Crustacea hepe-like virus 1 (CHEV1), from the economically important crustacean, the giant freshwater prawn Macrobrachium rosenbergii, was characterized. The complete genome consisted of 7750 nucleotides and had a similar structure to known hepatitis E virus genomes. Phylogenetic analyses suggested it might be a novel hepe-like virus within the family Hepeviridae. To our knowledge, this is the first hepe-like virus characterized from crustaceans.

17.
Crit Rev Oncol Hematol ; 149: 102924, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32172225

RESUMO

Neuroblastoma is the most common extracranial solid tumor, arising from primitive sympathetic ganglion cells, in pediatric patients. The unique features of neuroblastoma include variable clinical behaviors, such as rapid progression to death and maturation to benign ganglioneuroma, followed by regression. Radiation therapy (RT) is usually administered to both the primary tumor bed and persistent metastatic sites after induction chemotherapy for high-risk neuroblastoma. RT to the tumor bed after surgical resection contributes significantly to local disease control and prevention of local relapse, confirming the role of RT. Palliative radiotherapy for metastatic neuroblastoma is also effective and safe and mainly provides symptomatic relief. The late side effects of RT in neuroblastoma patients include growth and developmental failure, hypothyroidism, gastrointestinal dysfunction, neurocognitive defects, pulmonary and cardiac abnormalities, infertility, and secondary cancers. In this article, we reviewed the role and toxicity of RT in neuroblastoma patients.


Assuntos
Quimioterapia de Indução/métodos , Neuroblastoma/radioterapia , Cuidados Paliativos/métodos , Neoplasias de Tecidos Moles/radioterapia , Criança , Irradiação Craniana , Humanos , Lactente , Recidiva Local de Neoplasia , Neuroblastoma/patologia , Neuroblastoma/secundário , Neoplasias de Tecidos Moles/patologia , Neoplasias de Tecidos Moles/secundário , Resultado do Tratamento
18.
Parasitol Res ; 119(5): 1619-1628, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32185481

RESUMO

Schistosomiasis is still prevalent and seriously endangering the health of people and livestock in many countries. There have been great efforts to develop vaccines against schistosomiasis for prolonged protection in epidemic areas. Molecules from lung-stage schistosomula have been regarded as potential vaccine candidates against schistosomiasis. Our previous work has shown that cathepsin L3 from Schistosoma japonicum (SjCL3) is expressed in lung-stage schistosomula, but its role is not well known. In the present study, we characterized SjCL3 and detected its effect as a possible vaccine in vivo and in vitro. From the results of quantitative PCR (qPCR) and western blot, SjCL3 was present throughout the lifecycle of the worm, and its relative expressed level was higher in the liver eggs and adult worms than other stages. Additionally, immunofluorescence assay showed that SjCL3 was mainly concentrated in the eggshell, alimentary canal, and musculature of worms. Compared with the adjuvant group, the immunization of SjCL3 in mice resulted in a 28.9% decrease in worm burden and a 29.2% reduction in egg number in the host liver. In antibody-dependent cell-mediated cytotoxicity (ADCC) insecticidal experiments in vitro, the existence of SjCL3 could in part suppress adherence between macrophages and worm. The above results indicated that the immunization of SjCL3 could induce limited immune protection against S. japonicum infection in mice, and this protease played a role in breaking the process of ADCC, which was beneficial to the survival of worms.


Assuntos
Catepsinas/imunologia , Vacinas Protozoárias/imunologia , Schistosoma japonicum/imunologia , Esquistossomose Japônica/prevenção & controle , Adjuvantes Imunológicos , Animais , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Western Blotting , Clonagem Molecular , Feminino , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Schistosoma japonicum/metabolismo , Esquistossomose Japônica/imunologia , Vacinação
19.
PLoS Genet ; 16(3): e1008561, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32134924

RESUMO

Intraflagellar transport (IFT) is required for ciliary assembly and maintenance. While disruption of IFT may trigger ciliary disassembly, we show here that IFT mediated transport of a CDK-like kinase ensures proper ciliary disassembly. Mutations in flagellar shortening 2 (FLS2), encoding a CDK-like kinase, lead to retardation of cilia resorption and delay of cell cycle progression. Stimulation for ciliary disassembly induces gradual dephosphorylation of FLS2 accompanied with gradual inactivation. Loss of FLS2 or its kinase activity induces early onset of kinesin13 phosphorylation in cilia. FLS2 is predominantly localized in the cell body, however, it is transported to cilia upon induction of ciliary disassembly. FLS2 directly interacts with IFT70 and loss of this interaction inhibits its ciliary transport, leading to dysregulation of kinesin13 phosphorylation and retardation of ciliary disassembly. Thus, this work demonstrates that IFT plays active roles in controlling proper ciliary disassembly by transporting a protein kinase to cilia to regulate a microtubule depolymerizer.


Assuntos
Proteínas de Arabidopsis/metabolismo , Proteína Quinase CDC2/metabolismo , Chlamydomonas/metabolismo , Cílios/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Quinases/metabolismo , Transporte Biológico/fisiologia , Ciclo Celular/fisiologia , Flagelos/metabolismo , Fosforilação/fisiologia , Plantas Geneticamente Modificadas/metabolismo , Transdução de Sinais/fisiologia
20.
J Nanobiotechnology ; 18(1): 44, 2020 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-32169061

RESUMO

BACKGROUND: Traditional sandwich enzyme-linked immunosorbent assay (ELISA) using polyclonal and monoclonal antibodies as reagents presents several drawbacks, including limited amounts, difficulty in permanent storage, and required use of a secondary antibody. Nanobodies can be easily expressed with different systems and fused with several tags in their tertiary structure by recombinant technology, thus offering an effective detection method for diagnostic purposes. Recently, the fenobody (ferritin-fused nanobody) and RANbody (nanobody-fused reporter) have been designed and derived from the nanobody for developing the diagnostic immunoassays. However, there was no report about developing the sandwich ELISA using the fenobody and RANbody as pairing reagents. RESULTS: A platform for developing a sandwich ELISA utilizing fenobody as the capture antibody and RANbody as the detection antibody was firstly designed in the study. Newcastle disease virus (NDV) was selected as the antigen, from which 13 NDV-specific nanobodies were screened from an immunized Bactrian camel. Then, 5 nanobodies were selected to produce fenobodies and RANbodies. The best pairing of fenobodies (NDV-fenobody-4, 800 ng/well) and RANbodies (NDV-RANbody-49, 1:10) was determined to develop the sandwich ELISA for detecting NDV. The detection limits of the assay were determined to be 22 of hemagglutination (HA) titers and 10 ng of purified NDV particles. Compared with two commercial assays, the developed assay shows higher sensitivity and specificity. Meanwhile, it exhibits 98.7% agreement with the HA test and can detect the reference NDV strains belonging to Class II but not Class I. CONCLUSIONS: In the presented study, the 13 anti-NDV nanobodies binding the NDV particles were first produced. Then, for the first time, the sandwich ELISA to detect the NDV in the different samples has been developed using the fenobody and RANbody as reagents derived from the nanobodies. Considering the rapidly increasing generation of nanobodies, the platform can reduce the cost of production for the sandwich ELISA and be universally used to develop assays for detecting other antigens.

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