RESUMO
BACKGROUND: Canine distemper virus (CDV) has been shown to have oncolytic activity against primary canine tumors. Previous studies from this laboratory had confirmed that CDV induces apoptosis in canine mammary tumor (CMT) cells, although the molecular mechanism remains unknown. METHODS: The CDV N, P, M, F, H, L, C, and V genes were identified in CDV-L and cloned separately. Mutants with deletions in the 5' region (pCMV-F Lâ³60, pCMV-FLâ³107, and pCMV-FLâ³114) or with site-directed mutagenesis in the 3' region (pCMV-FLA602-610) of the F gene were generated. Late-stage apoptotic cells were detected by Hoechst 33342. Early-stage apoptotic cells were detected by AnnexinV-FITC/PI. Quantitative real-time PCR was performed to detect the mRNA levels of target genes of apoptotic and NF-κB pathway. Western blot analysis was performed to detect the expression or phosphorylation levels of target proteins of apoptotic or NF-κB pathway. Immunofluorescence assay was performed to detect the nuclear translocation of p65 protein. Recombinant viruses (rCDV-FLâ³60 and rCDV-FLA602-610) were rescued by a BHK-T7-based system. 5-week-old female BALB/c nude mice were used to detect the oncolytic activity of recombinant viruses. RESULTS: In this study, it was first confirmed that none of the structural or non-structural proteins of CDV-L, a vaccine strain, was individually able to induce apoptosis in canine mammary tubular adenocarcinoma cells (CIPp) or intraductal papillary carcinoma cells (CMT-7364). However, when CIPp or CMT-7364 cells were co-transfected with glycoprotein fusion (F) and hemagglutinin (H) proteins of CDV-L, nuclear fragmentation was observed and a high proportion of early apoptotic cells were detected, as well as cleaved caspase-3, caspase-8 and poly (ATP ribose) polymerase (PARP). Cleaved caspase-3 and PARP were down-regulated by apoptosis broad-spectrum inhibitor Z-VAD-FMK and caspase-8 pathway inhibitor Z-IETD-FMK, confirming that the F and H proteins coinduced apoptosis in CMT cells via the caspase-8 and caspase-3 pathways. F and H proteins co-induced phosphorylation of p65 and IκBα and nuclear translocation of p65, confirming activation of the NF-κB pathway, inhibition of which down-regulated cleaved caspase-3 and cleaved PARP. Recombinant F protein with enhanced fusion activity and H protein co-induced more cleaved caspase-3 and PARP than parental F protein, while the corresponding recombinant virus exhibited the same properties both in CIPp cells and in a subcutaneous xenograft mouse model. CONCLUSIONS: F and H proteins of CDV-L co-induce apoptosis in CMT cells, while the NF-κB pathway and fusion activity of F protein paly essential roles in the process.
Assuntos
Neoplasias da Mama , Vírus da Cinomose Canina , Feminino , Animais , Cães , Humanos , Camundongos , Caspase 3 , Vírus da Cinomose Canina/genética , Hemaglutininas/genética , Caspase 8 , NF-kappa B , Camundongos Nus , Inibidores de Poli(ADP-Ribose) Polimerases , ApoptoseRESUMO
Newcastle disease (ND) is the most important infectious disease in poultry, which is caused by avian orthoavulavirus type 1 (AOAV-1), previously known as Newcastle disease virus (NDV). In this study, an NDV strain SD19 (GenBank accession number OP797800) was isolated, and phylogenetic analysis suggested the virus belongs to the class II genotype VII. After generating wild-type rescued SD19 (rSD19), the attenuating strain (raSD19) was generated by mutating the F protein cleavage site. To explore the potential role of the transmembrane protease, serine S1 member 2 (TMPRSS2), the TMPRSS2 gene was inserted into the region between the P and M genes of raSD19 to generate raSD19-TMPRSS2. Besides, the coding sequence of the enhanced green fluorescent protein (EGFP) gene was inserted in the same region as a control (rSD19-EGFP and raSD19-EGFP). The Western blot, indirect immunofluorescence assay (IFA), and real-time quantitative PCR were employed to determine the replication activity of these constructs. The results reveal that all the rescued viruses can replicate in chicken embryo fibroblast (DF-1) cells; however, the proliferation of raSD19 and raSD19-EGFP needs additional trypsin. We next evaluated the virulence of these constructs, and our results reveal that the SD19, rSD19, and rSD19-EGFP are velogenic; the raSD19 and raSD19-EGFP are lentogenic; and the raSD19-TMPRSS2 are mesogenic. Moreover, due to the enzymatic hydrolysis of serine protease, the raSD19-TMPRSS2 can support itself to proliferate in the DF-1 cells without adding exogenous trypsin. These results may provide a new method for the NDV cell culture and contribute to ND's vaccine development.
Assuntos
Doença de Newcastle , Doenças das Aves Domésticas , Vacinas Virais , Animais , Embrião de Galinha , Vírus da Doença de Newcastle , Tripsina/genética , Filogenia , Genética Reversa , Galinhas , Genoma Viral/genética , Genótipo , Tropismo , Vacinas Virais/genéticaRESUMO
Mink enteritis virus (MEV) NS1 is a multidomain and multifunctional protein containing origin binding, helicase, and transactivation domains. In particular, parvoviral NS1 proteins are transactivators of the viral capsid protein promoter although the manner by which they exert these transactivation effects remained unclear. In this study, the region of the transactivation domain of the NS1 C-terminal was found located at aa 557 ~ 668 and any deletion within this region reduced the transactivation activity. A dominant negative mutation of the 63 aa deletion in the C-terminal of NS1 protein resulted in loss of ability to activate P38 and VP2-5'UTR in a dual-luciferase reporter assay system, a VP2 protein expression system, and within the whole MEV genome, independent of downstream genes. Additionally, a full-length MEV clone deficient in its NS1 C-terminal failed to rescue the virus, possibly due to the loss of integrity of DNA sequences interacting with NS1 protein, and expression of VP2 was also inhibited even when normal NS1 protein was supplied in trans.
Assuntos
Vírus da Enterite do Vison , Animais , Ativação Transcricional , Vírus da Enterite do Vison/genética , Vírus da Enterite do Vison/metabolismo , Regiões Promotoras Genéticas , Sequência de Bases , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Ligação Proteica , Vison/genéticaRESUMO
Parvoviruses possess a single-stranded DNA genome of about 5 kb, which contains two open reading frames (ORFs), one encoding nonstructural (NS) proteins, the other capsid proteins. The NS1 protein contains an N-terminal origin-binding domain, a helicase domain, and a C-terminal transactive domain, and is essential for effective viral replication and production of infectious virus. We first summarize the developments in the structure of NS1 protein, including the original binding domain and the helicase domain. We discuss the role of different DNA substrates in the oligomerization of these two domains of NS1. During the parvovirus life cycle, the NS1 protein is closely related to the viral gene expression, viral replication, and infection. We provide the current understanding of the impact of parvovirus NS1 protein mutations on its biological properties. Overall, in this review, we focus on the structure and function of the parvoviral NS1 protein.
Assuntos
Replicação do DNA , Proteínas , Proteínas/metabolismo , Replicação Viral/genética , Ligação Proteica , Mutação , Proteínas não Estruturais Virais/metabolismoRESUMO
The NS1 protein of mink enteritis virus (MEV) is a multidomain and multifunctional protein that plays a critical role in viral replication, with predicted nuclease, helicase and transactivation activities. The nuclease and helicase domains of NS1 protein are involved in interaction with viral DNA. Herein, potential amino acids critical for DNA binding in the MEV NS1 were mutated, all of which resulted in a termination of viral production from an infectious MEV clone. Although E121, H129/131, Y212 and K470/472 mutants retained their P38 and 5'UTR transactivation activity, K196/197 and K406 mutations eliminated this. Interestingly, VP2 protein was produced following transfection of F81 cells with pMEV-NS1-196K2G (K196G and K197G) and pMEV-NS1-K406G when pNS1 was co-transfected in trans, indicating that the substitutions did not affect the integrity of the DNA sequence that bound to NS1 protein but inhibited the biological properties of NS1 protein itself. The ability of NS1 protein to interact with SP1 was inhibited by both 196K2G and K406G substitutions, while 196K2G resulted in failure to bind to the DNA-binding sites in the P38 promoter, and the oligomerization of K406G was inhibited. All of these could explain the transcriptional repression.
RESUMO
Canine parvovirus type 2 (CPV-2) and feline panleukopenia virus (FPV) cause severe disease in young animals, pups, and kittens. CPV-2 evolved from FPV by altering the species-specific binding of the viral capsid to the host receptor, i.e., the transferrin receptor (TfR), and CPV-2 genetic variants have been identified by specific VP2 amino acid residues (297, 426). Early studies focused on the main capsid protein VP2; however, there have been limited studies on the non-structural protein NS1. In this study, we identified the genetic variants of clinical samples in dogs and cats in northern China during 2019-2020. The genetic characterization and phylogenetic analyses of VP2 and NS1 gene were also conducted. The results revealed that the CPV-2c was identified as the major genetic variant. One new CPV-2b and two CPV-2c strains were collected from cats. Four mutation sites (60, 630, 443, and 545 amino acid residues) were located in the functional domains of the NS1 protein. The phylogenetic analysis of VP2 and NS1 genes showed that they were clustered by geographical regions and genotypes. The gene mutation rate of CPV-2 was increasing in recent years, resulting in a complex pattern of gene evolution in terms of host preference, geographical selection, and new genetic variants. This study emphasizes that continuous molecular epidemiological surveillance is required to understand the genetic diversity of FPV and CPV-2 strains.
RESUMO
A novel neurological disorder, shaking mink syndrome (SMS), emerged in Denmark and Sweden in 2000. SMS has seldom been reported in China, but the causative agent has not been detected in the country. SMS outbreaks occurred in multiple provinces in 2020. A total of 44 brain samples from minks associated with SMS were collected from Heilongjiang, Liaoning and Shandong provinces of which 28 samples (63.3%) were SMS-astrovirus (SMS-AstV)-positive by reverse transcription PCR. Histopathological examination revealed non-suppurative encephalitis in three minks. Moreover, the complete coding region sequences (CDSs, 6559 bp) of a sample collected from a 2-month-old mink (termed SMS-AstV-H1, GSA accession No. SAMC816786) were amplified by PCR and Sanger sequencing. The complete CDS and open reading frame 2 sequences of SMS-AstV-H1 were 94.3% and 96.4% identical to an SMS-AstV strain (GenBank accession number: GU985458). Phylogenetically, SMS-AstV-H1 was closely related to an SMS-AstV strain (GU985458). Based on the above results, we describe SMS-AstV-associated encephalitis in farmed minks in China. Future studies need to focus on epidemiology, virus isolation and potential interspecies transmission of SMS-AstV.
Assuntos
Infecções por Astroviridae , Encefalite , Vison , Animais , Infecções por Astroviridae/veterinária , Infecções por Astroviridae/virologia , China/epidemiologia , Encefalite/veterinária , Encefalite/virologia , Mamastrovirus/classificação , Mamastrovirus/genética , FilogeniaRESUMO
MicroRNAs (miRNAs) are vital post-transcriptional regulators that participate in host-pathogen interactions by modulating the expression of cellular factors. Previous studies have demonstrated that feline panleukopenia virus (FPV) alters miRNA expression levels within host cells. However, the relationship between FPV replication and host miRNAs remains unclear. Here, we demonstrated that FPV infection significantly altered cellular miR-92a-1-5p expression in F81 cells by upregulating the expression of specificity protein 1 (SP1). Furthermore, we observed that miR-92a-1-5p enhanced interferon (IFN-α/ß) expression by targeting the suppressors of cytokine signaling 5 (SOCS5) that negatively regulates NF-κB signaling and inhibits FPV replication in host cells. These findings revealed that miR-92a-1-5p plays a crucial role in host defense against FPV infection.
Assuntos
MicroRNAs , Replicação Viral , Animais , Gatos , Vírus da Panleucopenia Felina/genética , Interações Hospedeiro-Patógeno/genética , Interferon beta , MicroRNAs/genética , MicroRNAs/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Replicação Viral/genéticaRESUMO
Feline calicivirus (FCV) is an important and highly prevalent pathogen of cats that causes acute infectious respiratory disease. Here it is shown in vitro that FCV induces the production of cyclooxygenase-2 (COX-2) through the MEK1-ERK1/2 signaling pathway. Screening of FCV proteins revealed that FCV non-structural protein VPg enhanced COX-2 mRNA expression and protein production in CRFK cells in a concentration-dependent manner. Regions 24-54aa and 84-111aa in FCV VPg were essential for up-regulation. In vivo, COX-2 and IL-6 production caused by FCV infection of kittens was significantly suppressed by the MEK1 inhibitor AZD6244 (selumetinib) and lung inflammation and injury were practically eliminated, with body temperature being returned to normal. AZD6244 may therefore find application as an effective therapeutic agent for the treatment of FCV infection.
Assuntos
Infecções por Caliciviridae , Calicivirus Felino , Pneumonia , Animais , Benzimidazóis , Infecções por Caliciviridae/tratamento farmacológico , Infecções por Caliciviridae/metabolismo , Infecções por Caliciviridae/veterinária , Gatos , Ciclo-Oxigenase 2/metabolismo , Feminino , Sistema de Sinalização das MAP QuinasesRESUMO
PURPOSE: Nanoparticles (NPs) decorated with functional ligands are promising candidates for cancer diagnosis and treatment. However, numerous studies have shown that chemically coupled targeting moieties on NPs lose their targeting capability in the biological milieu because they are shielded or covered by a "protein corona". Herein, we construct a functional magnetosome that recognizes and targets cancer cells even in the presence of protein corona. METHODS: Magnetosomes (BMPs) were extracted from magnetotactic bacteria, M. gryphiswaldense (MSR-1), and decorated with trastuzumab (TZ) via affibody (RA) and glutaraldehyde (GA). The engineered BMPs are referred to as BMP-RA-TZ and BMP-GA-TZ. Their capacities to combine HER2 were detected by ELISA, the quantity of plasma corona proteins was analyzed using LC-MS. The efficiencies of targeting SK-BR-3 were demonstrated by confocal laser scanning microscopy and flow cytometry. RESULTS: Both engineered BMPs contain up to ~0.2 mg TZ per mg of BMP, while the quantity of HER2 binding to BMP-RA-TZ is three times higher than that binding to BMP-GA-TZ. After incubation with normal human plasma or IgG-supplemented plasma, GA-TZ-containing BMPs have larger hydrated radii and more surface proteins in comparison with RA-TZ-containing BMPs. The TZ-containing BMPs all can be targeted to and internalized in the HER2-overexpressing breast cancer cell line SK-BR-3; however, their targeting efficiencies vary considerably: 50-75% for RA-TZ-containing BMPs and 9-19% for GA-TZ-containing BMPs. BMPs were incubated with plasma (100%) and cancer cells to simulate human in vivo environment. In this milieu, BMP-RA-TZ uptake efficiency of SK-BR-3 reaches nearly 80% (slightly lower than for direct interaction with BMP-RA-TZ), whereas the BMP-GA-TZ uptake efficiency is <17%. CONCLUSION: Application of the RA scaffold promotes and orients the arrangement of targeting ligands and reduces the shielding effect of corona proteins. This strategy improves the targeting capability and drug delivery of NP in a simulated in vivo milieu.
Assuntos
Magnetossomos , Preparações Farmacêuticas , Coroa de Proteína , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Humanos , Magnetossomos/metabolismo , Coroa de Proteína/metabolismo , Receptor ErbB-2/metabolismo , Trastuzumab/farmacologiaRESUMO
BACKGROUND AND OBJECTIVE: Deep convolutional networks are powerful tools for single-modality medical image segmentation, whereas generally require semantic labelling or annotation that is laborious and time-consuming. However, domain shift among various modalities critically deteriorates the performance of deep convolutional networks if only trained by single-modality labelling data. METHODS: In this paper, we propose an end-to-end unsupervised cross-modality segmentation network, DDA-Net, for accurate medical image segmentation without semantic annotation or labelling on the target domain. To close the domain gap, different images with domain shift are mapped into a shared domain-invariant representation space. In addition, spatial position information, which benefits the spatial structure consistency for semantic information, is preserved by an introduced cross-modality auto-encoder. RESULTS: We validated the proposed DDA-Net method on cross-modality medical image datasets of brain images and heart images. The experimental results show that DDA-Net effectively alleviates domain shift and suppresses model degradation. CONCLUSIONS: The proposed DDA-Net successfully closes the domain gap between different modalities of medical image, and achieves state-of-the-art performance in cross-modality medical image segmentation. It also can be generalized for other semi-supervised or unsupervised segmentation tasks in some other field.
Assuntos
Processamento de Imagem Assistida por Computador , Imageamento por Ressonância Magnética , Encéfalo/diagnóstico por imagem , CoraçãoRESUMO
An essential task for 3D visual world understanding is 3D object detection in lidar point clouds. To predict directly bounding box parameters from point clouds, existing voting-based methods use Hough voting to obtain the centroid of each object. However, it may be difficult for the inaccurately voted centers to regress boxes accurately, leading to the generation of redundant bounding boxes. For objects in indoor scenes, there are several co-occurrence patterns for objects in indoor scenes. Concurrently, semantic relations between object layouts and scenes can be used as prior context to guide object detection. We propose a simple, yet effective network, RSFF-Net, which adds refined voting and scene feature fusion for indoor 3D object detection. The RSFF-Net consists of three modules: geometric function, refined voting, and scene constraint. First, a geometric function module is used to capture the geometric features of the nearest object of the voted points. Then, the coarse votes are revoted by a refined voting module, which is based on the fused feature between the coarse votes and geometric features. Finally, a scene constraint module is used to add the association information between candidate objects and scenes. RSFF-Net achieves competitive results on indoor 3D object detection benchmarks: ScanNet V2 and SUN RGB-D.
Assuntos
Benchmarking , SemânticaRESUMO
OBJECTIVES: To investigate the airway management equipment and clinical practice in emergency department wards in China, and to explore the factors that influenced the nurses' airway management practice. DESIGN: Cross-sectional study. SETTING: A nationwide survey covering the seven administrative regions of China (North China, Northeast China, East China, Central China, South China, Southwest China and Northwest China). PARTICIPANTS: The nurses had to be registered nurses who worked in adult emergency department wards of the selected hospitals. MEASURES: An online survey was designed, piloted and distributed to the members of the Emergency Medicine Committee of the Chinese Nursing Association, and the nurses from the members' hospitals were invited to participate. The questionnaire was used to determine nurses' clinical practice scores of airway management in emergency wards. RESULTS: Finally, we collected 995 valid questionnaires from 31 provinces and 143 districts in China. Among them, 361 (36.28%) nurses responded that their departments used open suction system (OSS) in clinical work, the major barrier for closed suction system (CSS) reported by 630 respondents (63.32%) was cost. Significant differences in all three scores were found in age, nursing experience years, technical title, airway management training experience and nursing specialist (all p<0.05). Correlations were found among airway management attitude, practice of sputum aspiration and practice of ventilator care bundles (r=0.655, r=0.543 and r=0.763, all p<0.001). CONCLUSIONS: Chinese emergency department managers need to identify better methods for assessing equipment availability in OSS. CSS can be a choice when costs, status of the individual patient and severity of disease are comprehensively considered. Emergency department nurses' scores of airway management practice were affected by demographic and job-related characteristics; regular training should be encouraged, and equipment and resources should be guaranteed to improve airway management quality and optimise patient outcomes.
Assuntos
Serviço Hospitalar de Emergência , Recursos Humanos de Enfermagem Hospitalar , Adulto , Manuseio das Vias Aéreas , China , Estudos Transversais , Hospitais , Humanos , Inquéritos e QuestionáriosRESUMO
Organoid, an in vitro 3D culture, has extremely high similarity with its source organ or tissue, which creates a model in vitro that simulates the in vivo environment. Organoids have been extensively studied in cell biology, precision medicine, drug toxicity, efficacy tests, etc., which have been proven to have high research value. Periodic observation of organoids in microscopic images to obtain morphological or growth characteristics is essential for organoid research. It is difficult and time-consuming to perform manual screens for organoids, but there is no better solution in the prior art. In this paper, we established the first high-throughput organoid image dataset for organoids detection and tracking, which experienced experts annotate in detail. Moreover, we propose a novel deep neural network (DNN) that effectively detects organoids and dynamically tracks them throughout the entire culture. We divided our solution into two steps: First, the high-throughput sequential images are processed frame by frame to detect all organoids; Second, the similarities of the organoids in the adjacent frames are computed, and the organoids on the adjacent frames are matched in pairs. With the help of our proposed dataset, our model achieves organoids detection and tracking with fast speed and high accuracy, effectively reducing the burden on researchers. To our knowledge, this is the first exploration of applying deep learning to organoid tracking tasks. Experiments have demonstrated that our proposed method achieved satisfactory results on organoid detection and tracking, verifying the great potential of deep learning technology in this field.
Assuntos
Aprendizado Profundo , Organoides , Redes Neurais de Computação , Medicina de PrecisãoRESUMO
BACKGROUND AND OBJECTIVE: Glaucoma, a worldwide eye disease, may cause irreversible vision damage. If not treated properly at an early stage, glaucoma eventually deteriorates into blindness. Various glaucoma screening methods, e.g. Ultrasound Biomicroscopy (UBM), Optical Coherence Tomography (OCT), and Heidelberg Retinal Scanner (HRT), are available. However, retinal fundus image photography examination, because of its low cost, is one of the most common solutions used to diagnose glaucoma. Clinically, the cup-to-disk ratio is an important indicator in glaucoma diagnosis. Therefore, precise fundus image segmentation to calculate the cup-to-disk ratio is the basis for screening glaucoma. METHODS: In this paper, we propose a deep neural network that uses anatomical knowledge to guide the segmentation of fundus images, which accurately segments the optic cup and the optic disc in a fundus image to accurately calculate the cup-to-disk ratio. Optic disc and optic cup segmentation are typical small target segmentation problems in biomedical images. We propose to use an attention-based cascade network to effectively accelerate the convergence of small target segmentation during training and accurately reserve detailed contours of small targets. RESULTS: Our method, which was validated in the MICCAI REFUGE fundus image segmentation competition, achieves 93.31% dice score in optic disc segmentation and 88.04% dice score in optic cup segmentation. Moreover, we win a high CDR evaluation score, which is useful for glaucoma screening. CONCLUSIONS: The proposed method successfully introduce anatomical knowledge into segmentation task, and achieve state-of-the-art performance in fundus image segmentation. It also can be used for both automatic segmentation and semiautomatic segmentation with human interaction.
Assuntos
Glaucoma , Disco Óptico , Técnicas de Diagnóstico Oftalmológico , Fundo de Olho , Glaucoma/diagnóstico por imagem , Humanos , Redes Neurais de Computação , Disco Óptico/diagnóstico por imagemRESUMO
Canine parvovirus type 2 (CPV-2) is currently circulating in domestic and wild animals, but our knowledge about CPV-2 infections in raccoon dogs is limited. In this study, VP2 gene sequences of CPV-2 were amplified from rectal swabs of 14 diarrhetic raccoon dogs (Nyctereutes procyonoides) in Hebei province, China, in 2016 and 2017. Phylogenetic analysis of the VP2 gene sequences revealed that most of these sequences (11 of 14) belonged to the same subclade as raccoon dog strain CPV-2/Raccoon_Dog/China/DP-1/16 isolated from Shandong province in 2016. A comparison of deduced amino acid sequences revealed presence of the substitutions S297A and S27T in 11 of those 14 sequences. I418T was observed in a minority of the sequences (4 of 14). In addition, A300D and T301I, P13S and I219V, and N419K were found in three of the sequences. This study shows that CPV-2 strains with different substitutions in their VP2 amino acid sequences were spreading among raccoon dogs in Hebei during 2016 and 2017 and suggests that further studies are needed to monitor the distribution of these strains in China.
Assuntos
Infecções por Parvoviridae/veterinária , Parvovirus Canino/classificação , Cães Guaxinins/virologia , Sequência de Aminoácidos , Animais , Proteínas do Capsídeo/genética , China/epidemiologia , Infecções por Parvoviridae/epidemiologia , Infecções por Parvoviridae/virologia , Parvovirus Canino/isolamento & purificaçãoRESUMO
Ostrich diseases characterized by paralysis have been breaking out in broad areas of China since 2015, causing major damage to the ostrich breeding industry in China. This report describes a parvovirus detected in ostriches from four different regions. The entire genomes of four parvovirus strains were sequenced following amplification by PCR, and we conducted comprehensive analysis of the ostrich parvovirus genome. Results showed that the length genomes of the parvovirus contained two open reading frames. Ostrich parvovirus (OsPV) is a branch of goose parvovirus (GPV). Genetic distance analysis revealed a close relationship between the parvovirus and goose parvovirus strains from China, with the closest being the 2016 goose parvovirus RC16 strain from Chongqing. This is the first report of a parvovirus in ostriches. However, whether OsPV is the pathogen of ostrich paralysis remains uncertain. This study contributes new information about the evolution and epidemiology of parvovirus in China, which provides a new way for the study of paralysis in ostriches.
Assuntos
Evolução Molecular , Genoma Viral , Infecções por Parvoviridae/virologia , Parvovirus/fisiologia , Struthioniformes/virologia , Animais , Sequência de Bases , Testes Genéticos , Genômica/métodos , Infecções por Parvoviridae/diagnóstico , Filogenia , Reação em Cadeia da PolimeraseRESUMO
Trichoderma reesei is the major filamentous fungus used to produce cellulase and there is huge interest in promoting its ability to produce higher titers of cellulase. Among the many factors affecting cellulase production in T. reesei, the mycelial phenotype is important but seldom studied. Herein, a close homolog of the Neurospora crassa COT1 kinase was discovered in T. reesei and designated TrCOT1, which is of 83.3% amino acid sequence identity. Functional disruption of Trcot1 in T. reesei by RNAi-mediated gene silencing resulted in retarded sporulation on potato dextrose agar and dwarfed colonies on minimal medium agar plates containing glucose, xylan, lactose, xylose, or glycerol as the sole carbon source. The representative mutant strain, SUS2/Trcot1i, also displayed reduced mycelia accumulation but hyperbranching in the MM glucose liquid medium, with hyphal growth unit length values decreased to 73.0 µm/tip compared to 239.8 µm/tip for the parent strain SUS2. The hyperbranching phenotype led to slightly but significantly increased cellulase secretion from 24 to 72 h in a batch culture. However, the cellulase production per unit of mycelial biomass was much more profoundly improved from 24 to 96 h.
Assuntos
Proteínas Fúngicas/genética , Inativação Gênica , Fenótipo , Interferência de RNA , Trichoderma/crescimento & desenvolvimento , Trichoderma/genética , Sequência de Aminoácidos , Celulase/genética , Celulase/metabolismo , Ativação Enzimática , Proteínas Fúngicas/química , Regulação Bacteriana da Expressão Gênica , Ordem dos Genes , Plasmídeos/genética , Regiões Promotoras Genéticas , Transformação Bacteriana , Trichoderma/citologiaRESUMO
High metastatic rate and recurrence of tumor because of tumor circulating cells are seriously hinders for clinical tumor therapy. Herein, we develop a novel, active-targeting nanotherapeutic by simultaneously loading doxorubicin (DOX) and transferrin (Tf) onto bacterial magnetosomes (Tf-BMs-DOX) and investigate its antitumor efficacy in vitro and in vivo. Drug release profiles indicated that Tf-BMs/BMs loaded with DOX were capable of sustained drug release, suggesting that reduce drugs required frequency of administration and enhance their therapeutic effect. The results of cellular uptake revealed that Tf-BMs-DOX recognized hepatocellular carcinoma HepG2 cells more specifically compared to HL-7702 normal hepatocytes because of high expression of transferrin receptor (TfR) on the surface of HepG2 cells. Tf-BMs-DOX increased tumor cytotoxicity and apoptosis more significantly than free DOX or BMs-DOX by regulating the expression of tumor-related and apoptosis-related genes. Following intravenous injection in HepG2 cell-bearing mice, Tf-BMs-DOX displayed tumor suppression rate of 56.78%, significantly higher than that of the BMs-DOX (41.53%) and free DOX (31.26%) groups. These results suggest that Tf-BMs-DOX have the potential to actively target to tumor sites, as well as the ability to kill circulating tumor cells via intravenous injection. Our findings provide a promising candidate for the clinical treatment of metastatic cancer.
Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Doxorrubicina/uso terapêutico , Neoplasias Hepáticas/tratamento farmacológico , Magnetossomos/química , Terapia de Alvo Molecular , Transferrina/uso terapêutico , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/patologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Doxorrubicina/farmacologia , Endocitose/efeitos dos fármacos , Humanos , Ferro/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Receptores da Transferrina/metabolismoRESUMO
Aim: This study aimed to develop anthracycline-loaded bacterial magnetosomes (BMs) with enhanced anticancer efficiency and elucidate their endocytosis mechanism. Methods: Drug-loaded BMs (DBMs) were successfully prepared and characterized. DBMs endocytosis was investigated within HepG2 cells. The anticancer effect of DBMs was studied both in vitro and in vivo. Results: Doxorubicin-BMs and daunorubicin-BMs showed enhanced growth inhibitory effect in vitro and in vivo with no notable toxicity to normal tissues. The DBMs were internalized into cells through caveolae-mediated endocytosis and macropinocytosis. The loaded drugs were released from DBMs in cytoplasm and entered the nucleus to exert their activity. Conclusion: Our findings offer promising candidates for improved cancer therapy with a clear mechanism of DBMs endocytosis and working principle.