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1.
Nanoscale ; 13(30): 12848-12853, 2021 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-34477769

RESUMO

Nucleic acid nanostructures are promising biomaterials for the delivery of homologous gene therapy drugs. Herein, we report a facile strategy for the construction of target mRNA (scaffold) and antisense (staple strands) co-assembled RNA/DNA hybrid "origami" for efficient gene therapy. In our design, the mRNA was folded into a chemically well-defined nanostructure through RNA-DNA hybridization with high yield. After the incorporation of an active cell-targeting aptamer, the tailored RNA/DNA hybrid origami demonstrated efficient cellular uptake and controllable release of antisenses in response to intracellular RNase H digestion. The biocompatible RNA/DNA origami (RDO) elicited a noticeable inhibition of cell proliferation based on the silencing of the tumor-associated gene polo-like kinase 1 (PLK1). This RDO-based nanoplatform provides a novel strategy for the further development of gene therapy.


Assuntos
Nanoestruturas , RNA , DNA/genética , Terapia Genética , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , RNA/genética
2.
Cancer Biomark ; 2021 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-34511489

RESUMO

PURPOSE: To explore the exact molecular mechanisms underline osteosarcoma (OS) patients with lung metastases. METHODS: The differentially expressed gene (DEG) as well as differentially expressed miRNAs (DEMs) for OS lung metastases were deeply investigated with two independent sources of databases (GEO dataset and clinical participants); The enriched biological processes and signaling pathways were explored; the miRNAs-mRNAs network was constructed; the functions of potential DEGs and DEMs were also verified with external analysis. RESULTS: The OS patients with lung metastases displayed 323 DEGs as C-C motif chemokine ligand 3 (CCL3), sorting nexin 10 (SNX10), alpha-2-macroglobulin (A2M), carboxypeptidase E (CPE), Rap guanine nucleotide exchange factor 4 (RAPGEF4), PDZ domain containing 2 (PDZD2), calpain 10 (CAPN10), four and a half LIM domains 2 (FHL2), alkaline phosphatase, biomineralization associated (ALPL), interleukin 6 (IL6), solute carrier family 26 member 1 (SLC26A1) as well as smoothened, frizzled class receptor (SMO) were significant differentially expressed. At the same time, 21 DEMs were potential for the progress of OS lung metastasis with hsa-miR-638, hsa-miR-451, hsa-miR-486-5p, hsa-miR-134 and hsa-miR-648 were significant distinct. It could been shown that hsa-miR-638 manipulated the largest number of target genes. The functions of hsa-miR-638 and target mRNAs for the development of lung metastasis in OS could be confirmed by quantitative Real-time PCR analysis. CONCLUSION: This integrated study hypothesized several miRNA dependent signaling pathway for OS patients with lung metastases and initiated a potential strategy for better understanding the lung metastases in clinic.

3.
ACS Appl Mater Interfaces ; 13(18): 20974-20981, 2021 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-33909408

RESUMO

The CRISPR/Cas9 gene-editing system has become a promising strategy for tumor therapy with its powerful oncogene-editing ability. However, the efficient delivery of sgRNA/Cas9 complex into target tumor cells remains a challenge. Herein, we report a facile strategy for the construction of an sgRNA/Cas9 complex co-assembled nanoplatform for targeted gene editing and combined tumor therapy. In our design, the TAT peptide and thiolated DNA linker functionalized gold nanorod can efficiently load the sgRNA/Cas9 complex through the hybridization between the 3' overhang of sgRNA and the DNA linker. Due to the integration of an active cell targeting group (aptamer) and nuclear targeting peptide (TAT), the multifunctional nanoplatform can elicit the targeted cellular internalization and efficient nuclear targeting transportation to realize endogenous RNase H activated gene editing of the tumor-associated gene polo-like kinase 1 (PLK1). With mild photothermal treatment, this sgRNA/Cas9 complex loaded nanoplatform achieved efficient inhibition of tumor cell proliferation. This multifunctional nanocarrier provides a new strategy for the development of combined tumor therapy.


Assuntos
Edição de Genes , Ouro/química , Nanotubos/química , Neoplasias/terapia , Ácidos Nucleicos/química , Sistemas CRISPR-Cas , Proliferação de Células , Terapia Combinada , Humanos , Células MCF-7 , Microscopia Confocal , Neoplasias/patologia
4.
Nano Lett ; 21(8): 3573-3580, 2021 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-33830773

RESUMO

The exploitation of strong light-matter interactions in chiral plasmonic nanocavities may enable exceptional physical phenomena and lead to potential applications in nanophotonics, information communication, etc. Therefore, a deep understanding of strong light-matter interactions in chiral plasmonic-excitonic (plexcitonic) systems constructed by a chiral plasmonic nanocavity and molecular excitons is urgently needed. Herein, we systematically studied the strong light-matter interactions in gold nanorod-based chiral plexcitonic systems assembled on DNA origami. Rabi splitting and anticrossing behavior were observed in circular dichroism spectra, manifesting chiroptical characteristic hybridization. The bisignate line shape of the circular dichroism (CD) signal allows the accurate discrimination of hybrid modes. A large Rabi splitting of ∼205/∼199 meV for left-handed/right-handed plexcitonic nanosystems meets the criterion of strong coupling. Our work deepens the understanding of light-matter interactions in chiral plexcitonic nanosystems and will facilitate the development of chiral quantum optics and chiroptical devices.


Assuntos
Nanopartículas Metálicas , Nanotubos , DNA , Ouro , Fenômenos Físicos
5.
ACS Omega ; 6(14): 9500-9508, 2021 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-33869930

RESUMO

Hyperuricemia (HUA), a chronic disease caused by metabolic disorders of purine, is often accompanied by other diseases such as gout, type 2 diabetes mellitus (T2DM), and hyperlipidemia. However, little is known about the relationship between HUA and these diseases on the protein level. We performed label-free liquid chromatography MS/MS spectrometry analysis of urine samples from 26 HUA patients and 25 healthy controls, attempting to establish the possible protein links between HUA and these diseases by profiling urine proteome. A total of 2119 proteins were characterized in sample proteomes. Among them, 11 were found decreased and 2 were found increased in HUA samples. Plausible pathways found by enrichment analysis of these differentially expressed proteins (DEPs) include the processes for insulin receptor recycling and lipid metabolism, suggesting potential links between HUA and T2DM and hyperlipidemia. The abundance changes of three key proteins (VATB1, CFAD, and APOC3) involved in these processes were validated by enzyme-linked immunosorbent assay (ELISA). In conclusion, our result provides proteomic evidence, for the first time, that the aberrant pathways enriched by described key DEPs are closely related to the incidence of HUA and its concomitant diseases.

6.
Angew Chem Int Ed Engl ; 60(5): 2594-2598, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33089613

RESUMO

Using the DNA origami technique, we constructed a DNA nanodevice functionalized with small interfering RNA (siRNA) within its inner cavity and the chemotherapeutic drug doxorubicin (DOX), intercalated in the DNA duplexes. The incorporation of disulfide bonds allows the triggered mechanical opening and release of siRNA in response to intracellular glutathione (GSH) in tumors to knockdown genes key to cancer progression. Combining RNA interference and chemotherapy, the nanodevice induced potent cytotoxicity and tumor growth inhibition, without observable systematic toxicity. Given its autonomous behavior, exceptional designability, potent antitumor activity and marked biocompatibility, this DNA nanodevice represents a promising strategy for precise drug design for cancer therapy.


Assuntos
Terapia Combinada/métodos , DNA/química , Nanopartículas/química , Neoplasias/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Humanos
7.
J Hazard Mater ; 404(Pt B): 124030, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33045484

RESUMO

Butyl benzyl phthalate (BBP) is widely used as a plasticizer to increase the plasticity and flexibility of plastic products. Although the potential health hazards of BBP have recently received extensive attention, its toxicological properties and mechanisms remain largely undefined. In the present work, growth, reproductive and developmental toxicity of BBP to Daphnia magna were evaluated, and the transcriptomic alteration of early embryos upon BBP exposure was analyzed. In a 21-day chronic toxicity test, reduced survival ratio, decreased body length, increased abnormal ratio, advanced time to first brood, and reduced offspring of D. magna were observed. BBP exposure inhibited expression of the vitellogenin gene. In addition, embryotoxicity of BBP was observed, which showed not only in the induction of abnormal neonates, but also in the shortened embryonic development cycle. RNA-Seq of early embryo treated with 0.1 mg/L BBP indicated that the pathways involved in signal transduction, cell communication, and embryonic development were significantly down-regulated, while those of biosynthesis, metabolism, cell homeostasis, redox homeostasis were remarkably up-regulated upon BBP exposure, which was consistent with the above phenotypic results. Taken together, our results highlight the toxic effects of BBP on the embryonic development and larval growth of D. magna.


Assuntos
Daphnia , Ácidos Ftálicos , Animais , Daphnia/genética , Desenvolvimento Embrionário , Humanos , Recém-Nascido , Ácidos Ftálicos/toxicidade , Reprodução , Transcriptoma
8.
Zebrafish ; 17(6): 382-393, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33232637

RESUMO

This study was aimed to assess effects of three strains of probiotics Lactobacillus acidophilus NCFM, Lactobacillus rhamnosus HN001, and Bifidobacterium animalis subsp. lactis Bi-07 on the intestinal motility and inflammation in the zebrafish models. The intestinal motility model was established using 5 days postfertilization (dpf) zebrafish administered with a fluorescent dye Nile red at 10 ng/mL for 16 h, followed by probiotics treatment for 24 h and the intestinal motility was inversely proportional to the intestinal fluorescence intensity that was quantitatively measured by image analysis. The intestinal inflammation was induced by treating 3 dpf neutrophil fluorescent zebrafish with 0.0125% of trinitrobenzenesulfonic acid for 48 h. Probiotics were administered at low, moderate, and high concentrations determined based on maximum tolerable concentration through soaking. All three strains of probiotics promoted intestinal movement, of which B. animalis subsp. lactis Bi-07 was most potent at lower concentrations. L. rhamnosus HN001 and B. animalis subsp. lactis Bi-07 had the therapeutic effects on the intestinal inflammation and the inflammation-associated mucosal damage recovery. The anti-inflammatory mechanisms of L. rhamnosus HN001 was related to both reduce inflammatory factor interleukin-6 (IL-6) and restored tissue repair factor transforming growth factor-ß-1 (TGFß-1); whereas B. animalis subsp. lactis Bi-07 was probably only associated with TGFß-1 elevation. Using larval zebrafish models for probiotics screening and assessment would speed up product research and development and improve products' efficacy and quality.


Assuntos
Bifidobacterium animalis/química , Motilidade Gastrointestinal/efeitos dos fármacos , Inflamação/tratamento farmacológico , Lactobacillus acidophilus/química , Lactobacillus rhamnosus/química , Probióticos/farmacologia , Peixe-Zebra , Animais , Inflamação/fisiopatologia
9.
J Biol Chem ; 295(30): 10468-10477, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32532819

RESUMO

The single von Willebrand factor C-domain proteins (SVWCs) are mainly found in arthropods. Their expression may be regulated by several environmental stresses, including nutritional status and bacterial and viral infections. However, the underlying regulatory mechanism is unclear. In the present study, we identified a member of the SVWC family from the river prawn Macrobrachium nipponense as a soluble and bacteria-inducible pattern-recognition receptor (designated MnSVWC). In vitro, recombinant MnSVWC exhibited pronounced binding and Ca2+-dependent agglutinating abilities against diverse microbes, including Gram-negative bacteria (i.e. Escherichia coli and Aeromonas victoria), Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis), and yeast (Pichia pastoris). ELISA assays revealed that recombinant MnSVWC recognizes a broad range of various pathogen-associated molecular patterns (PAMPs) and has high affinity to lipopolysaccharide and lysine-type and diaminopimelic acid-type peptidylglycan and d-galactose and low affinity to d-mannan and ß-1,3-glucan. Mutant MnSVWCP57A with an impaired Glu-Pro-Asn (EPN) motif displayed reduced affinity to all these PAMPs to varying extent. Moreover, MnSVWC bound to the surface of hemocytes and promoted their phagocytic activity and clearance of invasive bacteria. RNAi-mediated MnSVWC knockdown in prawn reduced the ability to clear invading bacteria, but did not block the activities of the Toll pathway or the arthropod immune deficiency (IMD) pathway, or the expression of antimicrobial peptide genes. These results indicate that MnSVWC functions as an extracellular pattern-recognition receptor in M. nipponense that mediates cellular immune responses by recognizing PAMPs, agglutinating invasive microbes, and promoting phagocytosis in hemocytes.


Assuntos
Proteínas de Artrópodes , Hemócitos/imunologia , Palaemonidae , Fagocitose , Receptores de Reconhecimento de Padrão , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Bactérias/imunologia , Palaemonidae/genética , Palaemonidae/imunologia , Pichia/genética , Pichia/imunologia , Receptores de Reconhecimento de Padrão/genética , Receptores de Reconhecimento de Padrão/imunologia
10.
J Insect Physiol ; 124: 104073, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32526234

RESUMO

Ferritin is a ubiquitous multi-subunit iron storage protein, made up of heavy chain and light chain subunits. In recent years, invertebrate ferritins have emerged as an important, yet largely underappreciated, component of host defense and antioxidant system. Here, two alternatively spliced transcripts encoding for a unique ferritin heavy chain homolog (MdFerH), and a transcript encoding for a light chain homolog (MdFerL) are cloned and characterized from Musca domestica. Comparing with MdFerH1, a fragment is absent at the 5' untranslated region of MdFerH2, where a putative iron response element is present. Amino acid sequence analysis shows that MdFerH possesses a strictly conserved ferroxidase site, while MdFerL has a putative atypical active center. Tissue distribution analysis indicates that MdFers are enriched expressed in gut. When the larvae receive diverse stimulations, including challenge by bacteria, exposure to excess Fe2+, doxorubicin or ultraviolet, the expression of MdFers is positively up-regulated in different degrees and different temporal patterns, indicating their potential roles in oxidative stress. The two mRNA isoforms of MdFerH appear to be differentially expressed in different tissues, but seem to show the similar expression patterns under diverse stress conditions. Further investigation reveals that silencing MdFers can alter the redox homeostasis, leading elevated mortalities of larvae following bacterial infection. Inspiringly, recombinant MdFerL produced in Pichia pastoris shows significant iron-chelating activity in vitro. These results suggest a pivotal role of ferritins from housefly in iron homeostasis, antibacterial immunity and redox balance.


Assuntos
Apoferritinas/genética , Moscas Domésticas/fisiologia , Imunidade Inata , Proteínas de Insetos/genética , Ferro/fisiologia , Estresse Oxidativo , Sequência de Aminoácidos , Animais , Apoferritinas/química , Apoferritinas/imunologia , Sequência de Bases , Homeostase , Moscas Domésticas/imunologia , Proteínas de Insetos/química , Proteínas de Insetos/imunologia , Filogenia , Alinhamento de Sequência
11.
Int J Biol Macromol ; 155: 524-534, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32229201

RESUMO

Mitochondrial transcription factor A (TFAM) plays a key role in regulating the transcription, replication, and maintenance of mitochondrial DNA. In the present study, a Musca domestica TFAM (MdTFAM) gene was identified and characterized. MdTFAM gene encodes 253 amino acid residues, and the protein possesses two conserved motifs of HMG (High Mobility Group) box. Expression of MdTFAM was investigated based on the qRT-PCR (quantitative real-time polymerase chain reaction) in response to three model oxidative stress-inducing agents, cadmium chloride (Cd), doxorubicin hydrochloride (DOX) and ultraviolet (UV), respectively. Results showed that Cd exposure not only generated oxidative stress and mitochondrial dysfunctions in M. domestica larvae, with a significant increase in malondialdehyde and reactive oxygen species levels, but also induced a dose-dependent increase in the expression of MdTFAM. In addition, either DOX or UV exposure also significantly up-regulated the expression of MdTFAM in M. domestica larvae. These results suggest that MdTFAM play a vital role in maintaining the redox balance and its expression may serve as a useful biomarker for monitoring the oxidative stress induced by Cd, DOX or UV.


Assuntos
Cloreto de Cádmio/toxicidade , Proteínas de Ligação a DNA/metabolismo , Doxorrubicina/toxicidade , Poluentes Ambientais/toxicidade , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Estresse Oxidativo/fisiologia , Fatores de Transcrição/metabolismo , Animais , Antibióticos Antineoplásicos/toxicidade , Biomarcadores/análise , Proteínas de Ligação a DNA/genética , Moscas Domésticas , Malondialdeído/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/efeitos da radiação , Proteínas Mitocondriais/genética , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição/genética , Raios Ultravioleta/efeitos adversos
12.
Chembiochem ; 21(17): 2408-2418, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32227615

RESUMO

Nanomaterials with enzyme-mimicking behavior (nanozymes) have attracted a lot of research interest recently. In comparison to natural enzymes, nanozymes hold many advantages, such as good stability, ease of production and surface functionalization. As the catalytic mechanism of nanozymes is gradually revealed, the application fields of nanozymes are also broadly explored. Beyond traditional colorimetric detection assays, nanozymes have been found to hold great potential in a variety of biomedical fields, such as tumor theranostics, antibacterial, antioxidation and bioorthogonal reactions. In this review, we summarize nanozymes consisting of different nanomaterials. In addition, we focus on the catalytic performance of nanozymes in biomedical applications. The prospects and challenges in the practical use of nanozymes are discussed at the end of this Minireview.

13.
Fish Shellfish Immunol ; 100: 272-282, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32142875

RESUMO

Fibrinogen-related proteins (FREPs) are widely found in both vertebrates as well as invertebrates, and they play a crucial role in host immunity. In this study, we isolated a novel ficolin gene (Mnfico3) from the oriental river prawn Macrobrachium nipponense. The complete cDNA sequence of Mnfico3 was 1133 bp long, containing an open reading frame of 765 bp coding for Mnfico3, a protein consisting of 254 amino acids. The Mnfico3 protein contained a putative N-terminal signal peptide and a fibrinogen-related protein domain present at the C-terminal. Phylogenetic analysis indicated that Mnfico3 had a closer evolutionary relationship with vertebrate ficolins than with its invertebrate homologues. Tissue distribution analysis indicated that Mnfico3 was predominantly expressed in muscle, in which its transcription was increased following bacterial challenge by Aeromonas veronii. Function analysis using recombinant protein revealed that rMnFico3 had broad-spectrum binding capacity to a variety of microorganisms and pathogen-associated molecular pattern (PAMP) ligands. Furthermore, rMnFico3 exhibited Ca2+-dependent agglutinating activity against microbes in vitro, and ability to attach to the hemocyte surface which promoted phagocytosis and subsequent clearance of invasive bacteria in vivo. Silencing rMnFico3 in prawn through RNAi did not alter the expression of antimicrobial peptide genes (ALF and Crustin). These results manifested that MnFico3 functioned as a potential pattern recognition receptor (PPR) to mediate cellular immune response by recognizing PAMPs, agglutinating invasive microbes, and promoting phagocytosis of hemocytes.


Assuntos
Proteínas de Artrópodes/genética , Lectinas/genética , Palaemonidae/genética , Palaemonidae/imunologia , Receptores de Reconhecimento de Padrão/genética , Animais , Proteínas de Artrópodes/imunologia , Clonagem Molecular , Fibrinogênio/genética , Imunidade Celular , Imunidade Inata , Lectinas/imunologia , Filogenia , Receptores de Reconhecimento de Padrão/imunologia
14.
Int J Biol Macromol ; 150: 16-22, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32007549

RESUMO

Environmentally friendly and biodegradable hybrid composites of starch/enteromorpha/nano-clay were developed. Enteromorpha was used as cheaper filler since it is a waste from marine pollution, while nano-clay acted as a reinforcing agent. The microstructures and performance of these composites were investigated by SEM, DMA, XRD, TGA and tensile testing. Enteromorpha has a hollow tubular thallus structure with very weak mechanical properties, so it is not expected to have the ability to reinforce the starch matrix even though they have very a good interface. However, the granulated fine particles of enteromorpha can mix well with the starch matrix and reduce weak points. Furthermore, the delaminated clay by water and ultrasonic treatment reinforced the mechanical properties of the starch-based materials. The results showed that the hybrid composite containing up to 40% enteromorpha reinforced with nano-clay still has similar or even slightly better mechanical properties compared with pure starch-based materials. Since all components are hydrophilic natural materials, the interfaces between them are very good, and the composites are environmentally friendly and biodegradable.


Assuntos
Clorófitas/química , Argila/química , Nanocompostos/química , Amido/química , Fenômenos Químicos , Solubilidade , Análise Espectral , Resistência à Tração , Termogravimetria
15.
Chemosphere ; 248: 126009, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32000039

RESUMO

Cadmium (Cd) is a widespread environment contaminant due to the development of electroplating and metallurgical industry. Cd can be enriched by organisms via food chain, causing the enlarged environmental problems and posing threats to the health of humans. Polydatin (PD), a natural stilbenoid compound derived from Polygonum cuspidatum, shows pronouncedly curative effect on oxidative damage. In this work, the protective effects of PD on oxidative damage induced by Cd in Musca domestica (housefly) larvae were evaluated. The larvae were exposed to Cd and/or PD, subsequently, the oxidative stress status, mitochondria activity, oxidative phosphorylation efficiency, and survival rate were assessed. Cd exposure generated significant increases of malondialdehyde (MDA), reactive oxygen species (ROS) and 8-hydroxy-2-deoxyguanosine (8-oxoG) in the housefly larvae, causing mitochondrial dysfunction and survival rate decline. Interestingly, pretreatment with PD exhibited obviously mitochondrial protective effects in the Cd-exposed larvae, as evidenced by reduced MDA, ROS and 8-oxoG levels, and increased activities of superoxide dismutase (SOD), mitochondrial electron transfer chain, and mitochondrial membrane potential, as well as respiratory control ratio. These results suggested that PD could attenuate Cd-induced damage via maintaining redox balance, stimulating SOD activity, and regulating mitochondria activity in housefly larvae. As a natural polyphenolic chemical, PD can act as a potential candidate compounds to relieve Cd injury.


Assuntos
Cádmio/toxicidade , Moscas Domésticas/fisiologia , Superóxido Dismutase/metabolismo , Animais , Glucosídeos , Moscas Domésticas/efeitos dos fármacos , Humanos , Larva/metabolismo , Malondialdeído/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Estilbenos
16.
Fish Shellfish Immunol ; 98: 414-419, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31962148

RESUMO

Anti-lipopolysaccharide factors (ALFs), as main host-defense molecules of crustaceans, form a unique family of antimicrobial peptides (AMPs). MnALF4 is one isoform of ALFs isolated from the freshwater prawn Macrobrachium nipponense. In the present study, MnALF4 gene was successfully expressed in the yeast Pichia pastoris and the recombinant MnALF4 protein exhibited efficient and broad-spectrum antimicrobial activities against both Gram-positive bacteria and Gram-negative bacteria in vitro. When prawns were injected with rMnALF4 before bacterial challenge with E. coli, the recombinant protein effectively promote the elimination of bacteria by the host. It manifested that rMnALF4 could effectively kill the invading bacteria in vivo. Treatment with rMnALF4 led to remarkable changes in bacterial morphology, such as spheroidization, oversized bacteria, and cell lysis. In addition, rMnALF4 showed weak hemolysis activity to the rabbit red blood cells. Our work suggests that MnALF4 plays an important role in Macrobrachium immunity and is worthy of further investigation as a potential antibacterial agent with high efficacy against bacterial infection and low toxicity to host cells.


Assuntos
Proteínas de Artrópodes/metabolismo , Lipopolissacarídeos/imunologia , Palaemonidae/metabolismo , Pichia/metabolismo , Proteínas Recombinantes/metabolismo , Animais , Anti-Infecciosos/metabolismo , Anti-Infecciosos/farmacologia , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Bactérias/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Regulação da Expressão Gênica , Hemólise/efeitos dos fármacos , Pichia/genética , Coelhos , Proteínas Recombinantes/genética
17.
Int J Biol Macromol ; 153: 1262-1271, 2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-31770559

RESUMO

The tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is a key cytoplasm signaling adaptor that mediates signals activated by TNFR superfamily and the interleukin-1/Toll-like receptor (IL-1/TLR) superfamily. In the present research, a housefly Musca domestica TRAF6 (MdTRAF6) gene is identified and characterized, with a 51.7-kDa protein possessing a RING domain and a conserved C-terminal TRAF homology MATH domain encoded. MdTRAF6 is widely expressed in diverse tissues with high expression levels in gut and fat body, which is of the highest levels in adult in all growth stages. The expression of MdTRAF6 could be remarkably induced by bacterial challenge, and the silencing MdTRAF6 could alter the expressions of NF-κB-like genes (relish and dorsal) and antimicrobial peptide genes (cecropin, diptericin, attacin, muscin), thus leading elevated mortalities of larvae followed by bacterial infection. Inspiringly. MdTRAF6-depleted adult flies display higher mortality, lower fertility and reduced survival of offspring than the controls. Further investigation reveals that knockdown of MdTRAF6 disturbs the ovarian development and impaires the expressions of vitellogenin and vitellogenin receptor genes in the adult females. All these phenotypes show crucial roles of MdTRAF6 in innate immunity via positive regulation of the Toll pathway and negative regulation of the Imd pathway, and in reproduction by maintaining ovarian development.


Assuntos
Moscas Domésticas/crescimento & desenvolvimento , Moscas Domésticas/imunologia , Proteínas de Insetos/metabolismo , Ovário/crescimento & desenvolvimento , Fator 6 Associado a Receptor de TNF/metabolismo , Sequência de Aminoácidos , Animais , Feminino , Inativação Gênica , Moscas Domésticas/genética , Moscas Domésticas/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/deficiência , Proteínas de Insetos/genética , Fator 6 Associado a Receptor de TNF/química , Fator 6 Associado a Receptor de TNF/deficiência , Fator 6 Associado a Receptor de TNF/genética , Regulação para Cima
18.
Fish Shellfish Immunol ; 95: 635-643, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31678183

RESUMO

Small heat shock proteins (sHSPs) are ATP-independent chaperones and involved into various physiological and stress processes. In the present study, a 28.6-kD sHSP coding gene, MnHSP28.6, was cloned and characterized from the oriental river prawn Macrobrachium nipponense. Tissue distribution analysis via qPCR and western blot revealed that MnHSP28.6 predominantly expressed in muscle. The temporal transcription of MnHSP28.6 in muscle after bacterial challenge, heavy metal exposure and doxorubicin (DOX) injection was investigated by qPCR. The results showed that the expression of MnHSP28.6 were strongly enhanced by both Cd2+ and Cu2+ exposure, as well as DOX injection, but not by bacterial infection. Aggregation assays showed that recombinant MnHSP28.6 could effectively prevent temperature-induced aggregation of citrate synthase, and reduction-induced aggregation of insulin in vitro. MnHSP28.6 also could protect muscle extracts from heat-induced protein denaturation and superoxide dismutase (SOD) inactivation. Expressing MnHSP28.6 in E. coli conferred host cell impressive protection against H2O2 compared to control. These results suggest a protective role of MnHSP28.6 in maintaining protein homeostasis, preventing aggregation, promoting resistance to heavy metal and keeping redox balance.


Assuntos
Proteínas de Artrópodes/metabolismo , Proteínas de Choque Térmico Pequenas/metabolismo , Intoxicação por Metais Pesados/veterinária , Estresse Oxidativo , Palaemonidae/genética , Animais , Proteínas de Artrópodes/genética , Proteínas de Choque Térmico Pequenas/genética , Intoxicação por Metais Pesados/prevenção & controle , Temperatura Alta , Chaperonas Moleculares/genética , Músculos/metabolismo , Oxirredução , Substâncias Protetoras , Proteostase
19.
J Proteome Res ; 18(12): 4189-4196, 2019 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-31657219

RESUMO

In recent years, high-throughput technologies have contributed to the development of a more precise picture of the human proteome. However, 2129 proteins remain listed as missing proteins (MPs) in the newest neXtProt release (2019-02). The main reasons for MPs are a low abundance, a low molecular weight, unexpected modifications, membrane characteristics, and so on. Moreover, >50% of the MS/MS data have not been successfully identified in shotgun proteomics. Open-pFind, an efficient open search engine, recently released by the pFind group in China, might provide an opportunity to identify these buried MPs in complex samples. In this study, proteins and potential MPs were identified using Open-pFind and three other search engines to compare their performance and efficiency with three large-scale data sets digested by three enzymes (Glu-C, Lys-C, and trypsin) with specificity on different amino acid (AA) residues. Our results demonstrated that Open-pFind identified 44.7-93.1% more peptide-spectrum matches and 21.3-61.6% more peptide sequences than the second-best search engine. As a result, Open-pFind detected 53.1% more MP candidates than MaxQuant and 8.8% more candidate MPs than Proteome Discoverer. In total, 5 (PE2) of the 124 MP candidates identified by Open-pFind were verified with 2 or 3 unique peptides containing more than 9 AAs by using a spectrum theoretical prediction with pDeep and synthesized peptide matching with pBuild after spectrum quality analysis, isobaric post-translational modification, and single amino acid variant filtering. These five verified MPs can be saved as PE1 proteins. In addition, three other MP candidates were verified with two unique peptides (one peptide containing more than 9 AAs and the other containing only 8 AAs), which was slightly lower than the criteria listed by C-HPP and required additional verification information. More importantly, unexpected modifications were detected in these MPs. All MS data sets have been deposited into ProteomeXchange with the identifier PXD015759.


Assuntos
Bases de Dados de Proteínas , Software , Testículo/química , Humanos , Masculino , Espectrometria de Massas , Processamento de Proteína Pós-Traducional , Proteínas/análise , Proteínas/genética , Proteínas/metabolismo , Proteômica/métodos , Ferramenta de Busca
20.
Int J Biol Macromol ; 134: 73-79, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31075328

RESUMO

In vertebrates, tumor necrosis factors (TNFs) are well-known cytokines involved in a diversity of physiological and pathological events. In the present work we identified a novel TNF-like gene (MnTNF) from Macrobrachium nipponense, which codes for a protein sharing detectable sequence identify (26-27%) to the mammalian TNFSF members, TWEAK and EDA. Tissue distribution analysis indicated that MnTNF was predominantly expressed in nervous tissue, and at relatively high level in haemocytes, gill, intestine and muscle, whereas was almost undetectable in hepatopancreas. In gill, MnTNF was significantly up-regulated at both mRNA as well protein levels upon Aeromonas veronii challenge. RNA interference (RNAi) mediated MnTNF silencing led to a significant overexpression of the antimicrobial peptide (AMP) gene crustin, but a non-significant overexpression of another AMP gene anti-lipopolysaccharide factor (ALF), and inhibited the activation of phenoloxidase (PO) significantly following bacterial challenge. Meanwhile, the expression of NF-κB-like factor gene relish was increased while that of dorsal and STAT was uninfluenced in MnTNF-depleted prawn. We suppose that MnTNF be involved in regulating the expression of AMP genes and the capacity of PO by coordinating with the Imd pathway.


Assuntos
Sistema Imunitário/fisiologia , Palaemonidae/fisiologia , Fatores de Necrose Tumoral/metabolismo , Animais , Clonagem Molecular , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Homeostase , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Filogenia , Análise de Sequência de DNA
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