Exosomes derived from programmed cell death: mechanism and biological significance.

Exosomes are nanoscale extracellular vesicles present in bodily fluids that mediate intercellular communication by transferring bioactive molecules, thereby regulating a range of physiological and pathological processes. Exosomes can be secreted from nearly all cell types, and the biological function of exosomes is heterogeneous and depends on the donor cell type and state. Recent research has revealed that the levels of exosomes released from the endosomal system increase in cells undergoing programmed cell death. These exosomes play crucial roles in diseases, such as inflammation, tumors, and autoimmune diseases. However, there is currently a lack of systematic research on the differences in the biogenesis, secretion mechanisms, and composition of exosomes under different programmed cell death modalities. This review underscores the potential of exosomes as vital mediators of programmed cell death processes, highlighting the interconnection between exosome biosynthesis and the regulatory mechanisms governing cell death processes. Furthermore, we accentuate the prospect of leveraging exosomes for the development of innovative biomarkers and therapeutic strategies across various diseases.

The impact of anxiety on gait impairments in Parkinson's disease: insights from sensor-based gait analysis.

BACKGROUND: Sensor-based gait analysis provides a robust quantitative tool for assessing gait impairments and their associated factors in Parkinson's disease (PD). Anxiety is observed to interfere with gait clinically, but this has been poorly investigated. Our purpose is to utilize gait analysis to uncover the effect of anxiety on gait in patients with PD. METHODS: We enrolled 38 and 106 PD patients with and without anxiety, respectively. Gait parameters were quantitively examined and compared between two groups both in single-task (ST) and dual-task (DT) walking tests. Multiple linear regression was applied to evaluate whether anxiety independently contributed to gait impairments. RESULTS: During ST, PD patients with anxiety presented significantly shorter stride length, lower gait velocity, longer stride time and stance time, longer stance phase, smaller toe-off (TO) and heel-strike (HS) angles than those without anxiety. While under DT status, the differences were diminished. Multiple linear regression analysis demonstrated that anxiety was an independent factor to a serials of gait parameters, particularly ST-TO (B = -2.599, (-4.82, -0.38)), ST-HS (B = -2.532, (-4.71, -0.35)), ST-TO-CV (B = 4.627, (1.71, 7.64)), ST-HS-CV(B = 4.597, (1.66, 7.53)), ST stance phase (B = 1.4, (0.22, 2.58)), and DT stance phase (B = 1.749, (0.56, 2.94)). CONCLUSION: Our study discovered that anxiety has a significant impact on gait impairments in PD patients, especially exacerbating shuffling steps and prolonging stance phase. These findings highlight the importance of addressing anxiety in PD precision therapy to achieve better treatment outcomes.

Distinct histological and clinical features associated with pure uterine serous carcinoma: A single institution experience.

AIM: To ascertain the clinicopathological features, survival, and prognostic factors of pure uterine serous carcinoma (pUSC) and compare its clinicopathological characteristics with those of serous-like grade-3 endometrioid endometrial carcinoma (G3-EEC). METHOD: Consecutive patients with pUSC and p53 abnormal (p53abn) G3-EEC were retrospectively selected between 2014 and 2022. Histological and immunohistochemical features were reviewed, clinical information was collected, and survival analyses were performed. RESULTS: Eighty-five pUSC patients (mean age: 61.6 years) were included. Histologically, pUSC showed a predominantly glandular growth pattern (80.0 %) with high-grade nuclear atypia and obvious nucleoli and 53 cases showed admixtures of architectural patterns. The p53 aberrant expression rate was 98.8 %. 41.5 %, 53.7 %, and 67.5 % of cases were classified as negative for ER, PR, and WT1, respectively. Six (12.3 %) of 49 cases had a HER2 score of 3+ by immunohistochemistry (IHC). The overall survival and progression-free survival rates were 72.9 % and 63.5 %, respectively. Advanced stage, no adjuvant therapy, and lymph node metastasis were independent risk factors for poor survival in pUSC. Twenty-five p53abn G3-EEC patients were assessed. Women with p53abn G3-EEC were on average, younger than those with pUSC (53.4 vs. 61.6 years, P < 0.001). Papillary structures were observed more commonly in pUSC (16 % vs. 36.5 %, P = 0.042). Positive PR expression was significantly associated with p53abn G3-EEC (P = 0.009). Survival did not differ significantly between the subgroups in univariate and multivariate analyses. CONCLUSION: In this contemporary series, we affirm the suboptimal prognosis associated with pUSC, and that the survival associated with pUSC and p53abn G3-EEC are not significantly different. pUSC and p53abn G3-EEC have distinct morphological and immunohistochemical characteristics.

A New Segmented Virus Associated with Human Febrile Illness in China.

BACKGROUND: In 2017, surveillance for tickborne diseases in China led to the identification of a patient who presented to a hospital in Inner Mongolia with a febrile illness that had an unknown cause. The clinical manifestation of the illness was similar to that of tickborne encephalitis virus (TBEV) infection, but neither TBEV RNA nor antibodies against the virus were detected. METHODS: We obtained a blood specimen from the index patient and attempted to isolate and identify a causative pathogen, using genome sequence analysis and electron microscopy. We also initiated a heightened surveillance program in the same hospital to screen for other patients who presented with fever, headache, and a history of tick bites. We used reverse-transcriptase-polymerase-chain-reaction (RT-PCR) and cell-culture assays to detect the pathogen and immunofluorescence and neutralization assays to determine the levels of virus-specific antibodies in serum specimens from the patients. RESULTS: We found that the index patient was infected with a previously unknown segmented RNA virus, which we designated Alongshan virus (ALSV) and which belongs to the jingmenvirus group of the family Flaviviridae. ALSV infection was confirmed by RT-PCR assay in 86 patients from Inner Mongolia and Heilongjiang who presented with fever, headache, and a history of tick bites. Serologic assays showed that seroconversion had occurred in all 19 patients for whom specimens were available from the acute phase and the convalescent phase of the illness. CONCLUSIONS: A newly discovered segmented virus was found to be associated with a febrile illness in northeastern China. (Funded by the National Key Research and Development Program of China and the National Natural Science Foundation of China.).

Clinicopathological Features of Inflammatory Myofibroblastic Tumor in the Breast.

Inflammatory myofibroblastic tumor (IMT) is a mesenchymal spindle cell tumour with low malignant potential which is extremely rare in breasts. Because of the lack of typical imaging and clinical characteristics of IMT, it is easy to misdiagnose before operation. We now report a case of a 37-year-old woman presenting with a mass in her left breast. Ultrasound showed a well-circumscribed lesion in the lower outer quadrant. The patient underwent lumpectomy, and histopathology revealed a tumor which was composed of fusiform cells and inflammatory cells. Immunohistochemistry (IHC) showed tumor cells are positive for vimentin, ALK, BCL2, and SMA. The FISH test demonstrated ALK (2p23) chromosomal translocation (ALK positive). The final diagnosis of breast IMT was rendered with nonclassical morphology. Postoperative 30-month follow-up no evidence showed residual tumor or recurrence. As a very rare tumor, breast IMT could be easily misdiagnosed clinically and pathologically. Complete surgical resection of the tumor is preferred, and it has the risk of recurrence and metastasis.

Metformin alleviates crystalline silica-induced pulmonary fibrosis by remodeling endothelial cells to mesenchymal transition via autophagy signaling.

Silicosis is a severe progressive lung disease without effective treatment methods. Previous evidence has demonstrated that endothelial cell to mesenchymal transition (EndoMT) plays an essential role in pulmonary fibrosis, and pulmonary fibrosis is associated with dysregulation of autophagy, while the relationship between autophagy and EndoMT has not yet been adequately studied. Herein, we established a mouse model of silicosis, and we found that the pharmacological induction of the AMPK/mTOR-dependent pathway using 100 mg/kg Metformin (Met) enhanced autophagy in vivo, and results of the Western blot showed that autophagy-related proteins, LC3 II/I ratio, and Beclin-1 increased while p62 decreased. In addition, Met treatment attenuated silica-induced pulmonary inflammation and decreased collagen deposition by suppressing EndoMT, and the proliferation of human umbilical vein endothelial cells (HUVECs) was also inhibited. Notably, the tube forming assay showed that Met also protected the vascular endothelial cells from silica-induced morphological damage. In conclusion, Met can alleviate inflammatory response and collagen deposition in the process of pulmonary fibrosis induced by silica via suppressing EndoMT through the AMPK/mTOR signaling pathway.

Silica nanoparticle exposure inhibits surfactant protein A and B in A549 cells through ROS-mediated JNK/c-Jun signaling pathway.

Exposure to silica nanoparticles (SiNPs) is related to the dysregulation of pulmonary surfactant that maintains lung stability and function. Nevertheless, there are limited studies concerning the interaction and influence between SiNPs and pulmonary surfactant, and the damage and mechanism are still unclear. Herein, we used A549 cells to develop an in vitro model, with which we investigated the effect of SiNPs exposure on the expression of pulmonary surfactant and the potential regulatory mechanism. The results showed that SiNPs were of cytotoxicity in regarding of reduced cell viability and promoted the production of excessive reactive oxygen species (ROS). Additionally, the JNK/c-Jun signaling pathway was activated, and the expression of surfactant protein A (SP-A) and surfactant protein B (SP-B) was decreased. After the cells being treated with N-acetyl-L-cysteine (NAC), we found that the ROS content was effectively downregulated, and the expression of proteins related to JNK and c-Jun signaling pathways was suppressed. In contrast, the expression of SP-A and SP-B was enhanced. Furthermore, we treated the cells with JNK inhibitor and c-Jun-siRNA and found that the expression of protein related to JNK and c-Jun signaling pathways, as well as SP-A and SP-B, changed in line with that of NAC treatment. These findings suggest that SiNPs exposure can upregulate ROS and activate the JNK/c-Jun signaling pathway in A549 cells, thereby inhibiting the expression of SP-A and SP-B proteins.

NF-κB mediates silica-induced pulmonary inflammation by promoting the release of IL-1ß in macrophages.

Long-term exposure to respirable silica particles causes pulmonary inflammation and fibrosis primarily promoted by cytokines released from alveolar macrophages, yet the underlying mechanism is still unclear. From the perspective of nuclear factor kappa B (NF-κB), we studied the mechanism of IL-1ß biosynthesis and release. Utilizing BAY 11-7082, an NF-κB specific inhibitor, we showed the alteration of macrophage viability and examined the expression of both IL-1ß and NF-κB in vitro. We found that silica nanoparticles (SiNPs) were internalized by macrophages and caused damage to cell integrity. The immunofluorescence assay showed that SiNPs exposure enhanced the expression of IL-1ß and NF-κB, which could be effectively suppressed by BAY 11-7082. Besides, we built silica exposure mouse model by intratracheally instilling 5 mg of SiNPs and checked the effect of silica exposure on pulmonary pathological changes. Consistently, we found an upregulation of IL-1ß and NF-κB after SiNPs exposure, along with the aggravated inflammatory cell infiltration, thickened alveolar wall, and enhanced expression of collagens. In conclusion, SiNPs exposure causes pulmonary inflammation and fibrosis that is regulated by NK-κB through upregulating IL-1ß in alveolar macrophages.

The serine-48 residue of nucleolar phosphoprotein nucleophosmin-1 plays critical role in subcellular localization and interaction with porcine circovirus type 3 capsid protein.

The transport of circovirus capsid protein into nucleus is essential for viral replication in infected cell. However, the role of nucleolar shuttle proteins during porcine circovirus 3 capsid protein (PCV3 Cap) import is still not understood. Here, we report a previously unidentified nucleolar localization signal (NoLS) of PCV3 Cap, which hijacks the nucleolar phosphoprotein nucleophosmin-1 (NPM1) to facilitate nucleolar localization of PCV3 Cap. The NoLS of PCV3 Cap and serine-48 residue of N-terminal oligomerization domain of NPM1 are essential for PCV3 Cap/NPM1 interaction. In addition, charge property of serine-48 residue of NPM1 is critical for nucleolar localization and interaction with PCV3 Cap. Taken together, our findings demonstrate for the first time that NPM1 interacts with PCV3 Cap and is responsible for its nucleolar localization.

Comparison of properties of dust in alveolar of rats and the workplace.

OBJECTIVES: The purpose of this study was to analyze the α-SiO2 content, composition, dispersion, morphology, and free radical content of dust between the alveolar and the workplace, to explore the possible changes in the properties (especially the pathogenicity) of dust after it enters the lung. METHODS: We collected the dust in the workplace in HANDAN Coal mine. They were selected by a 400 mesh sieve and was made a suspension of 50 mg/ml, which would be used to perfuse into the trachea of rats. When one week, four weeks, eight weeks, fourteen weeks, twenty weeks after perfusing, we harvested dust in rats alveolar through lung lavage for further processing. RESULTS: In the animal test, typical fibrous nodules appeared 20 weeks after dust exposure. No inflammatory reaction was observed in the saline group. The results of animal experiments showed that there was no significant difference in the content of α-SiO2 between dust in the workplace and the lung lavage (P > 0.05). The content of the Fe element gradually increased with dust exposure time. The 12 elements of Al, Mg, Si, Pb, Mn, Ni, Zn, Cu, Cr, Sb, Cd, and AS were reduced in the experiment group compared with the workplace group. The shape of the dust in the workplace was mostly spherical. The shape of the dust extracted from the lung lavage fluid was mostly blocky and angular, and a few dust edges were sharp, and more than 80% of the particle size was smaller than 5 µm, while less than 1% of the particle size was larger than 10 µm. The amount of hydroxyl radical released by lung lavage dust in phosphate buffer was higher than that of the workplace dust. CONCLUSIONS: After the dust entered the alveoli, the content of α-SiO2 in the dust did not change with dust exposure time, while the content of elements in the dust, the morphology, and dispersion of the dust changed. The ability of dust in alveoli to produce hydroxyl radicals in phosphate buffer was higher than that in the workplace.

Silica dust exposure induces pulmonary fibrosis through autophagy signaling.

Silicosis is a well-acknowledged occupational lung disease caused by inhalation of a large amount of free silica dust during the production period and eventually a considerable negative impact on the patients' quality of life. Autophagy exerts a critical influence on immune and inflammatory responses during the pathogenesis of pulmonary fibrosis. In this study, we sought to determine whether autophagy is involved in silicosis's pathogenesis and how it may affect pulmonary cellular physiology. In the animal experiments, we found persistent activation of autophagy in the development of pulmonary fibrosis, which was also accompanied by tumor necrosis factor and transforming growth factor expression increased. Therefore, the autophagy signaling pathway may regulate the inflammatory response and affect the progression of fibrosis. Further, in vitro experiments, we used LY294002, RAPA, and N-acetylcysteine (NAC) intervened autophagy. Our results showed that PI3K/Akt/mTOR signaling pathway is involved in the autophagy changed mediated by SiO2 exposed, and autophagy might play a protective role in the progression of pulmonary fibrosis. Additionally, NAC's effect is not apparent on SiO2 -mediated autophagy through the PI3K/Akt/mTOR signaling pathway, but it can reduce the inflammatory response on NR8383 cells mediated by SiO2-exposed. Nevertheless, it's interesting that NAC can reduce the inflammatory response on NR8383 cells mediated by SiO2 -exposed. Taken together, our data demonstrated that SiO2 -exposed can induce pulmonary fibrosis along with autophagy both in vivo and in vitro, NAC could alleviate the inflammatory response NR8383 cells by SiO2 -exposed through non PI3K/Akt/mTOR signaling pathway, and the specific mechanism of its action needs further studying.

Genome-wide data mining to construct a competing endogenous RNA network and reveal the pivotal therapeutic targets of Parkinson's disease.

Parkinson's disease (PD) is one of the most common neurodegenerative movement disorders, for which there has been no effective treatments. To clarify the pathogenesis of PD, we constructed a competing endogenous RNA (ceRNA) network based on the genome-wide RNA sequencing data. It was found that 92 RNAs were differentially expressed, including 50 mRNAs, 25 miRNAs and 17 lncRNAs, based on which a ceRNA network was constructed and evaluated from 4 aspects of number of nodes, topological coefficients, closeness centrality and betweenness centrality. The functional annotation and enrichment analysis suggested that 6 functional modules, particularly the peripheral nervous system development and toxin metabolic process, dominated the development of PD. To validate the assumption, the gene set enrichment analysis (GSEA) was conducted basing on the genome-wide RNAs regardless whether they were differentially expressed or not. Consistently, the results revealed that dysregulation of MAG, HOXB3, MYRF and PLP1 led to metabolic disorders of sphingolipid and glutathione, which contributed to the pathogenesis of PD. Also, in-depth mining of previous literature confirmed a pivotal role of these dysregulated RNAs, which had been indicated to be potential diagnostic and therapeutic biomarkers of PD. Overall, we constructed a ceRNA network based on the dysregulated mRNAs, lncRNAs and miRNAs in PD, and the aberrant expression of MAG, HOXB3, MYRF and PLP1 caused metabolism disorder of sphingolipid and glutathione, and these genes are of great significance for the diagnosis and treatment of PD.

Conformational Changes and Nuclear Entry of Porcine Circovirus without Disassembly.

A relatively stable and flexible capsid is critical to the viral life cycle. However, the capsid dynamics and cytosol trafficking of porcine circovirus type 2 (PCV2) during its infectious cycle are poorly understood. Here, we report the structural stability and conformation flexibility of PCV2 virions by genome labeling and the use of three monoclonal antibodies (MAbs) against the native capsid of PCV2. Genome labeling showed that the infectivity of the PCV2 virion was not affected by conjugation with deoxy-5-ethynylcytidine (EdC). Heat stability experiments indicated that PCV2 capsids started to disassemble at 65°C, causing binding incompetence for all antibodies, and the viral genome was released without capsid disassembly upon heating at 60°C. Antibody binding experiments with PCV2 showed that residues 186 to 192 were concealed in the early endosomes of epithelial PK-15 and monocytic 3D4/31 cells with or without chloroquine treatment and then exposed in PK-15 cytosol and the 3D4/31 nucleus. Viral propagation and localization experiments showed that PCV2 replication and cytosol trafficking were not significantly affected by microtubule depolymerization in monocytic 3D4/31 cells treated with nocodazole. These findings demonstrated that nuclear targeting of viral capsids involved conformational changes, the PCV2 genome was released from the assembled capsid, and the transit of PCV2 particles was independent of microtubules in 3D4/31 cells.IMPORTANCE Circovirus is the smallest virus known to replicate autonomously. Knowledge of viral genome release may provide understanding of viral replication and a method to artificially inactivate viral particles. Currently, little is known about the release model of porcine circovirus type 2 (PCV2). Here, we report the release of the PCV2 genome from assembled capsid and the intracellular trafficking of infectious PCV2 by alterations in the capsid conformation. Knowledge of PCV2 capsid stability and dynamics is essential to understanding its infectious cycle and lays the foundation for discovering powerful targets for therapeutic and prophylactic intervention.

Heterozygous/dispermic complete mole confers a significantly higher risk for post-molar gestational trophoblastic disease.

Hydatidiform moles are classified at the genetic level as androgenetic complete mole and diandric-monogynic partial mole. Conflicting data exist whether heterozygous complete moles are more aggressive clinically than homozygous complete moles. We investigated clinical outcome in a large cohort of hydatidiform moles in Chinese patients with an emphasis on genotypical correlation with post-molar gestational trophoblastic disease. Consecutive products of conceptions undergoing DNA genotyping and p57 immunohistochemistry to rule out molar gestations were included from a 5-year period at Beijing Obstetrics and Gynecology Hospital. Patient demographics and clinical follow-up information were obtained. Post-molar gestational trophoblastic disease or gestational trophoblastic neoplasia was determined by the 2002 WHO/FIGO criteria. A total of 1245 products of conceptions were classified based on genotyping results into 219 complete moles, 250 partial moles, and 776 non-molar gestations. Among 219 complete moles, 186 were homozygous/monospermic and 33 were heterozygous/dispermic. Among 250 partial moles, 246 were triploid dispermic, 2 were triploid monospermic, and 2 were tetraploid heterozygous partial moles. Among 776 non-molar gestations, 644 were diploid without chromosomal aneuploidies detectable by STR genotyping and 132 had various genetic abnormalities including 122 cases of various trisomies, 2 triploid digynic-monoandric non-molar gestations, 7 cases of possible chromosomal monosomy or uniparental disomy. Successful follow-up was achieved in 165 complete moles: post-molar gestational trophoblastic disease developed in 11.6% (16/138 cases) of homozygous complete moles and 37.0% (10/27 cases) of heterozygous complete moles. The difference between the two groups was highly significant (p = 0.0009, chi-square). None of the 218 partial moles and 367 non-molar gestations developed post-molar gestational trophoblastic disease. In conclusion, heterozygous/dispermic complete moles are clinically more aggressive with a significantly higher risk for development of post-molar gestational trophoblastic disease compared with homozygous/monospermic complete moles. Therefore, precise genotyping classification of complete moles is important for clinical prognosis and patient management.

Silica dust exposure induces autophagy in alveolar macrophages through switching Beclin1 affinity from Bcl-2 to PIK3C3.

Increased deposition of silica dust in pulmonary interstitial tissues leads to silicosis, in which autophagy plays a defensive role in silica dust-associated stress response and cell death. Our previous studies revealed that silica dust exposure contributed to autophagy in pulmonary macrophages in vivo, while the specific regulatory mechanism is still unclear. This study aimed to figure out the regulatory mechanism as well as the role of autophagy in the pathogenesis of experimental silicosis. We used 3-methyladenine (3-MA) and ABT-737 to suppress the expression of phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3) and B cell leukemia/lymphoma 2 (Bcl-2), two critical initiators of autophagy, and detected and evaluated the autophagy in NR8383 cells with or without silica dust exposure. We found that exposure of silica dust increased autophagy in NR8383 cells and elevated the expression of Beclin1 and PIK3C3, but it reduced the expression of Bcl-2. The relationship among Beclin1, PIK3C3, and Bcl-2 were then investigated using immunoprecipitation analysis, and we found that suppression of PIK3C3 and/or Bcl-2 using 3-MA and/or ABT-737 could alter the autophagy induced by silica dust in NR8383 cells, and the complexes of Beclin1/PIK3C3 and Beclin1/Bcl-2 were both downregulated, which may be that inhibition of PIK3C3 and Bcl-2 altered the affinity of Beclin1 with PIK3C3 and Bcl-2 and lead to the silence of PIK3C3 signaling. These findings indicate that silica dust exposure induces autophagy via changing the connectivity of Beclin1 from Bcl-2 to PIK3C3.

Caspase-Dependent Apoptosis Induction via Viral Protein ORF4 of Porcine Circovirus 2 Binding to Mitochondrial Adenine Nucleotide Translocase 3.

Apoptosis is an essential strategy of host defense responses and is used by viruses to maintain their life cycles. However, the apoptotic signals involved in virus replication are poorly known. In the present study, we report the molecular mechanism of apoptotic induction by the viral protein ORF4, a newly identified viral protein of porcine circovirus type 2 (PCV2). Apoptosis detection revealed not only that the activity of caspase-3 and -9 is increased in PCV2-infected and ORF4-transfected cells but also that cytochrome c release from the mitochondria to the cytosol is upregulated. Subsequently, ORF4 protein colocalization with adenine nucleotide translocase 3 (ANT3) was observed using structured illumination microscopy. Moreover, coimmunoprecipitation and pulldown analyses confirmed that the ORF4 protein interacts directly with mitochondrial ANT3 (mtANT3). Binding domain analysis further confirmed that N-terminal residues 1 to 30 of the ORF4 protein, comprising a mitochondrial targeting signal, are essential for the interaction with ANT3. Knockdown of ANT3 markedly inhibited the apoptotic induction of both ORF4 protein and PCV2, indicating that ANT3 plays an important role in ORF4 protein-induced apoptosis during PCV2 infection. Taken together, these data indicate that the ORF4 protein is a mitochondrial targeting protein that induces apoptosis by interacting with ANT3 through the mitochondrial pathway.IMPORTANCE The porcine circovirus type 2 (PCV2) protein ORF4 is a newly identified viral protein; however, little is known about its functions. Apoptosis is an essential strategy of the host defense response and is used by viruses to maintain their life cycles. In the present study, we report the molecular mechanism of the apoptosis induced by the ORF4 protein. The ORF4 protein contains a mitochondrial targeting signal and is an unstable protein that is degraded by the proteasome-dependent pathway. Viral protein ORF4 triggers caspase-3- and -9-dependent cellular apoptosis in mitochondria by directly binding to ANT3. We conclude that the ORF4 protein is a mitochondrial targeting protein and reveal a mechanism whereby circovirus recruits ANT3 to induce apoptosis.

Cellular proteomic analysis of porcine circovirus type 2 and classical swine fever virus coinfection in porcine kidney-15 cells using isobaric tags for relative and absolute quantitation-coupled LC-MS/MS.

Viral coinfection or superinfection in host has caused public health concern and huge economic losses of farming industry. The influence of viral coinfection on cellular protein abundance is essential for viral pathogenesis. Based on a coinfection model for porcine circovirus type 2 (PCV2) and classical swine fever virus (CSFV) developed previously by our laboratory, isobaric tags for relative and absolute quantitation (iTRAQ)-coupled LC-MS/MS proteomic profiling was performed to explore the host cell responses to PCV2-CSFV coinfection. Totally, 3932 proteins were identified in three independent mass spectrometry analyses. Compared with uninfected cells, 304 proteins increased (fold change >1.2) and 198 decreased (fold change <0.833) their abundance in PCV2-infected cells (p < 0.05), 60 and 61 were more and less abundant in CSFV-infected cells, and 196 and 158 were more and less abundant, respectively in cells coinfected with PCV2 and CSFV. Representative differentially abundant proteins were validated by quantitative real-time PCR, Western blotting and confocal laser scanning microscopy. Bioinformatic analyses confirmed the dominant role of PCV2, and indicated that mitochondrial dysfunction, nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated oxidative stress response and apoptosis signaling pathways might be the specifical targets during PCV2-CSFV coinfection.

Dynamics of microbial community during the extremely long-term fermentation process of a traditional soy sauce.

BACKGROUND: Soy sauce produced by long-term natural fermentation is a traditional specialty in Asia, with a reputation for superior quality and rich flavour. In this study, both culture-dependent and culture-independent approaches were used to investigate the microbial diversity and community dynamics during an extremely long-term (up to 4 years) natural fermentation of Xianshi Soy Sauce, a national intangible cultural heritage. RESULTS: Genera of Bacillus, Aspergillus and Cladosporium were detected by both methods above. The relative abundance of the genera Bacillus and Weissella was significantly higher in the late stage than in the early one, while the genera Klebsiella and Shimwellia were opposite (P < 0.05). For microbial community structure, subsequent analyses showed that obvious changes occurred with fermentation time, while there was a fair homogeneousness among samples of the same year, especially during the late fermentation stage. CONCLUSIONS: The clustering analysis tended to separate the fermented mashes of the 4th year from the earlier stages, suggesting the necessity of the long fermentation period for developing distinctive microbiota and characteristic quality-related compounds. This is the first report to explore the temporal changes in microbial dynamics over a period of 4 years in traditional fermentation of soy sauce, and this work illustrated the importance of isolation of appropriate strains to be used as starter cultures in brewing processes. © 2016 Society of Chemical Industry.

Circovirus transport proceeds via direct interaction of the cytoplasmic dynein IC1 subunit with the viral capsid protein.

UNLABELLED: Microtubule transport of circovirus from the periphery of the cell to the nucleus is essential for viral replication in early infection. How the microtubule is recruited to the viral cargo remains unclear. In this study, we observed that circovirus trafficking is dependent on microtubule polymerization and that incoming circovirus particles colocalize with cytoplasmic dynein and endosomes. However, circovirus binding to dynein was independent of the presence of microtubular α-tubulin and translocation of cytoplasmic dynein into the nucleus. The circovirus capsid (Cap) subunit enhanced microtubular acetylation and directly interacted with intermediate chain 1 (IC1) of dynein. N-terminal residues 42 to 100 of the Cap viral protein were required for efficient binding to the dynein IC1 subunit and for retrograde transport. Knockdown of IC1 decreased virus transport and replication. These results demonstrate that Cap is a direct ligand of the cytoplasmic dynein IC1 subunit and an inducer of microtubule α-tubulin acetylation. Furthermore, Cap recruits the host dynein/microtubule machinery to facilitate transport toward the nucleus by an endosomal mechanism distinct from that used for physiological dynein cargo. IMPORTANCE: Incoming viral particles hijack the intracellular trafficking machinery of the host in order to migrate from the cell surface to the replication sites. Better knowledge of the interaction between viruses and virus proteins and the intracellular trafficking machinery may provide new targets for antiviral therapies. Currently, little is known about the molecular mechanisms of circovirus transport. Here, we report that circovirus particles enter early endosomes and utilize the microtubule-associated molecular motor dynein to travel along microtubules. The circovirus capsid subunit enhances microtubular acetylation, and N-terminal residues 42 to 100 directly interact with the dynein IC1 subunit during retrograde transport. These findings highlight a mechanism whereby circoviruses recruit dynein for transport to the nucleus via the dynein/microtubule machinery.

Class III ß-Tubulin in Colorectal Cancer: Tissue Distribution and Clinical Analysis of Chinese Patients.

BACKGROUND Class III ß-tubulin (ßIII-tubulin) has been reported to express at the invasive margin of colorectal cancer. The present study aimed to investigate the clinical implication of ßIII-tubulin expression at the invasive margin of colorectal cancer. MATERIAL AND METHODS We recruited 111 patients with surgically resected colorectal carcinoma for bIII-tubulin expression analysis. The cases with bIII-tubulin-positive tumor cells found only in the invasive front tumor area were assigned to the invasive front group, while the remaining cases were all assigned to the non-invasive front group. Clinical analysis of ßIII-tubulin and other clinical data was performed. RESULTS The positive staining rates and staining intensity of bIII-tubulin were significantly different between the invasive and non-invasive front groups (p=0.001 and p=0.006), and there was a significant difference in tumor differentiation between the 2 groups (p=0.032). In the non-invasive front group, staining intensity of bIII-tubulin was significantly associated with positive staining rates and lymphatic metastasis (p<0.001 and p=0.048). CONCLUSIONS Our data showed the tissue distribution of bIII-tubulin expression at invasive margin or diffuse distribution. Expression of bIII-tubulin was correlated with tumor differentiation and lymphatic metastasis, suggesting a potential role of bIII-tubulin in tumor differentiation and metastasis. This study may shed light on bIII-tubulin as a novel potential molecular target for a new anti-cancer drug.